Documentos de Académico
Documentos de Profesional
Documentos de Cultura
ACTINOMYCETES
Siili-l i:ili' ;;in\\ili ;iiiil aerial iiiyccliuiu of a typical .-I ic'|)t(iniyc('>, N. (///.'
b
THE ACTINOMYCETES
Vol. I
by
Selman A. Waksman
.^\T"L'7>
^^I^V°
BALTIMORE
THE WILLIAMS & W I L KIN S COMPANY
1959
THE ACTIXOMYCETES
Vol. I: Nature, Occurrence and Activities
Copyright ©, 1959
The Williams & Wilkins Companj^
Made in the United States of America
Library of Congress
Catalog Card Number
59-9962
PREFACE
III any altcnij)! lo classity and dixidc li\- at monthly in(er\ais, se\-erai dilTerent .soil
in^ systems, nay, cvcmi li\ini>; rrrsus non- types. Sami)les taken undei- sterile condi-
luing systems, certain hoiderline bodies are tions were obtainetl from \arious depths
encountered which ma>' he considered as (fiom the surface to 30 inches). These I
transition forms from ont* j2;r()up to another. brought to the laboratory and plated out,
This was recojj;nized l)y tiie eai"ly students using suital)le dilutions and proper culture
of the microscopic forms of life, wiio con- media. After \arving periods of incubation,
sidered the bacteria and similar orji;anisms I counted the colonies of bacteria developing
as "protista" or primiti\-e bodies, i-elatcd, on on the plates. I was soon struck \}y the fact
the one hand, to the i)lants and, on the that a fairly large number of the colonies
other hand, to animals. Recently accumu- that 1 could ob.serve did not look exactly
lated information points also to viruses as like the majority of the others, more typical
transitory between nonliving and living of bacteria. These particular colonies were
bodies. compact and leathery in nature, pyramidal
The actinomj^cetes form sucli a l)orderline in structure, penetrating deep into the agar
system, but on a much more speciahzed medium, freciuently covered with a surface
scale. Considered by some as bacteria fuzz that W'as distinct from the substrate
("higher bacteria"), or Eubacteriales, and growth. On examination of such colonies
bj' others as fungi ("lower fungi"), or even with a low-power microscope, the fuzzy
Hyphomyeetes, actinomycetes are often growth proved to be made up of an aerial,
placed in a group by themselves, with some branching mycelium that resembled that of
of the properties of both. There are found, fungus colonies.
among the actinomycetes, certain forms When I brought the plates to my professor,
that are more closely related to the bacteria he shook his head, smiled, and said, "Yes, I
and others that are nearer to the fungi. have been aware of the occurrence of these
M}^ personal attention was first directed types of bacteria. Freciuently they are desig-
to the actinomycetes about 45 years ago. In nated as a special group, under the name
1914, as a senior in college, specializing in actinomyces. You had better go and see our
soil microbiology, or, as was designated
it botanist. Professor M. T. Cook. He may be
at that time, "soil bacteriology," I was as- able to tell you more about them." Professor
signed b}' my professor, Jacob G. Lipman, Cook was indeed familiar wdth the group,
the task of making a comparati\-e monthly but merely as causative agents of potato
study of the bacterial population of certain scab. He considered them, not as bacteria
soil types located on the experimental but as fungi. He referred me to various
grounds of the college. The results obtained papei's inwhich further information could
in this study were used for a thesis which I be obtained on this group of organisms. I
presented the following June for my B.Sc. decided in my ^•ery early studies, that the
degree.* organisms could be differentiated from both
Throughout the year 1 9 1 4- 1 9 1 o , I sampled bacteria and fungi. To my great satisfaction,
* "Bacteria, Actinomyces and Fungi in the Soil." I learned later that similar suggestions had
Selman A. Waksman, Thesis, Rutgers College, New already been made previously by others.
Brunswick, X.J., 1915 (Abstract published in J.
Bacteriol. 1: 101, 1916). Thus, at the \-ery threshold of my scien-
VI THE ACTINOMYCETES, VOL. I
as scientific assistant and finally as micro- drawn from the various theses sub-
freely
biologist at the New Jersey Agricultural mitted by candidates for their Ph.D. degrees,
Experiment Station. working under my direct or indirect super-
The following treatise is, in part, a sum- vision. I wish to acknowledge the assistance
mary of these investigations carried out for of my colleagues and collaborators, notably
nearly half a century, mostly in the labora- Dr. Ruth E. Gordon, Dr. Hubert A. Le-
tories of Rutgers University, first at the chevalier, ]\Ir. Robert A. Day, and Mrs.
College of Agriculture and Experiment Sta- Herminie B. Kitchen. I also wish to thank
tion, and more recently at the Institute of Dr. C. W. Emmons, of the National Insti-
Microbiology. In a larger sense, however, I tutes of Health, for reading Chapter 17, and
lias been in recent yeai's the t'oeus of so much been isolated, and a niunber of genera and
attention In' the invest ij^ator, especially the hundreds of species have been described.
microbiologist, the chemist, and the medical These include organisms causing animal and
scientist, and hy the pharmaceutical manu- plant diseases and numerous saprophytes
facturer, as the actinomycetes. Only 20 occurring in soils, in dust, in water basins,
years ago scarcely a dozen laboratories in and in other natural substrates.
the whole world were de\-oting much atten- Because of the above two generic names
tion to this group of organisms, and they and for other reasons, the systematic posi-
were concerned largely with either disease- tion of actinomycetes became highly con-
producing or soil-inhabiting forms. Today, fused. Animal and plant pathologists, bota-
literally thousands of investigators in numer- nists, zoologists, mycologists, bacteriologists,
ous laboratories throughout the world are and biochemists were eager to introduce new
isolating cultures of actinomycetes from names in describing as new species freshly
soils and other substrates and studying isolated cultures of actinomycetes. New
their physiological and biochemical activi- genera and new species were thus created,
ties. This increased attention is due primaril}' without due regard to previously established
to the discovery that the actinomycetes names or even previous descriptions. This
comprise many forms that ha\'e the capacity tended to complicate greatly our knowledge
to produce a large number of chemical sub- of the taxonomy and classification of the
stances capable of inhibiting the growth of actinomycetes.
microorganisms, especially disease-producing A number of subsequent milestones in the
forms. These substances have come to be history of actinomycetes should be noted.
known as antibiotics. The discovery that Among them were the isolation by Israel of
certain actinomycetes can produce growth- a pure culture of an anaerobic organism, for
promoting substances or vitamins and cer- which the generic name Actinomyces was re-
tain potent enzyme systems has added served; the introduction of synthetic media
greatly to this interest. ]\Iany of the anti- by Krainsky and by Waksman and Curtis;
biotics produced by the actinomycetes have the recognition of the sporulating mecha-
found extensive practical application in the nisms of actinomycetes by 0rskov; the clas-
control of infectious diseases of man, ani- sification systems of Waksman and Henrici
mals, and plants; also in animal nutrition; and of Krassilniko\' the isolation of anti-
;
and in the preservation of biological prod- biotics from cultures of actinomycetes; and
ucts, including virus preparations, and of finally the studj^ of the cell walls of actino-
nition that they represent a large and highly tremendous interest in these organisms, their
important microbial group of universal dis- distribution in nature, their growth and nu-
tribution, possessing numerous biochemical trition under controlled conditions, and
activities, and of great practical potenti- finally their biochemical activities. Among
alities. the earlier treatises devoted to the subject
From an ecological point of \'ie\v, the of actinomycetes, note should be taken of
interest in the actinomycetes has centered the work of Lieske (1919), Duche (1935),
largely upon the study of their occurrence in Kriss (1937), Krassilnikov (1938), and Cope
soils, in composts, in water basins, in the (1938). have personally contributed to
I
atmosphere, and in the infected tissues of many phases of the study of actinomycetes.
living systems. Their role as causative agents Following my work on "The cultural prop-
of human, animal, and plant diseases at first erties of actinomycetes," published in 1919,
attracted wide attention, but more recently I edited the section on actinomycetes in the
this interest became of limited significance. \^arious editions of Bergey's Manual, begin-
Under some conditions, however, the actino- ning with the first in 1923 and including the
mycetes may play a highly important role seventh in 1958. My more recent books in-
in the causation of certain plant diseases, clude a l)Ook on The Actinomycetes published
such as potato scab. in 1950 and various volumes and papers on
From a biochemical point of view, interest the antibiotics of actinomycetes.
in the actinomycetes has centered largely The rapid accumulation of basic knowl-
upon their role in the transformation of or- edge concerning the actinomycetes justifies
ganic matter in the soil and their ability to a comprehensive treatise at this time. In this
form antibiotics, and enzymes.
vitamins, work, have made no attempt to review or
I
The produced by
interest in the antibiotics even to list the extensive literature on this
actinomycetes has been phenomenal. It all subject. Only certain pertinent references
began with the isolation of actinomycin in have been selected. In view of the fact that
1940. This was followed by the isolation of more than GOOO references on the subject of
streptothricin in 1942 and of streptomycin a single antibiotic, streptomycin, had been
in 1943, and later of chloramphenicol, the collected (as of 1952!) one can readily
tetracyclines, the erythromycins, the neo- imagine the extensive literature covering the
mycins, novobiocin, oleandomycin, nystatin, other antibiotics that have found practical
and numerous others. To date, more than 500 application in the treatment of numerous
different antibiotics have been isolated from human and animal diseases, in animal feed-
cultures of actinomycetes. Many of them ing, and in the preservation of various bio-
have been obtained in the form of pure com- logical preparations and food materials. And
pounds, the chemical nature of which has all of tlu^se antibiotic references, of course,
been determined. Others are still of unknown would be in addition to the thousands of
composition. Nearly 25 of these antibiotics papers that haxe been published relating to
have already found extensive practical appli- the organisms themseh'cs.
cation as chemotherapeutic agents. Of the This treatise is limited to a review of our
total 2,400,000 pounds of antibiotics pro- knowledge of the true actinomycetes. It does
duced in the United States in 1955, valued at not concern itself with the various bacterial
more than a half a billion dollars, at least forms frequently included among the Actino-
two-thirds have been obtained from cultures mycetales, namely, the mycobacteria, coryne-
of actinomycetes. l)acteria, and mycococci.
The interest in the antibiotics evoked In view of the fre(iuent references to mem-
. '
FNTHODrCTom' IX
bors of tlic \;iri()iis <j;(mum-;i of the actiiio- xcniaculai' (•\i)i'ession only for nicnibors of
myceU's hy xcrnacular tlcsi^uations, the this genus, both in singular and in j)lural
followuiji; coninuMits nia>' bo inado h(Mv: senses. 'I'he term "st reptomyces" will be
The terms "actuioniyeete" and "actino-
used as a \-ernacular expression of the genus
mycetes" will be used in tiiis treatise as inclu-
Slrcptomijce^, both in singular and i)lural
sive terms for any or all of the organisms
senses. The terms "nocardia" and "no-
now included in the Actinomycetales, exclu-
cardias" will be used in the vernacular for
siN'e of the mycobacteria and corynebacteria.
The term "'Actinomyces" will be used only membeis of the genus Nocardia, and "micro-
when referring to the single genus of that monospora" and "micromonosporas" for
lAULi: Ol COMKMS
r le
I Acti n ( )i ny cetes
\'<)i,rMK I
Preface v
Introductory vii
1 Historical Back^iound 1
References 278
c QTO
.
C IT A P T K K 1
Historical Background
,1
Figure 1. The first illustrat ion of ;iii ;icl iiKiuiNcct v ever pulilisluMl, Strcplollirix Fdcrshri (Reproduced
from: Cohii, F. Uiitorsuchiiiifien iil)er Bacterieii II. Beitr. Biol. Pfiaiizeu 1: 141-207, 1875).
organism foiiiul in concretions of the iachiy- nation. Cohn says: "On April 15, 1874, he
nial ducts and which he named Sireptothrix transmitted to me a mass which was whitish,
Foersteri. The concretions were transmitted Hke tallow, easily broken down and still
to Cohn by Foerster for microscopic exami- consisting of fine, very thin colorless branch-
iiisToincAi. I'.ACKCHorxn
inji tlirc'ids niiiiiiii<;' parallel to ()ii(> aiiollicr claims to ha\c isolated in ISS."), ;ilso from
or in xarious directions, curNinj;" and in human casi s, an aei'obic, lilamentous, spcn'e-
places also \\a\y." This type of <i;r()\vlli re- forming culture. Xocard isolated an aerol)ic
niindiHl C'ohn of the cui\al ui'es of spii'illa cnhure in ISSS from an animal infection.
and spirochaetes, althon^li it was nioi-e irre<;- This was followed bSS'Jj by the isolation
(
iilar. The threails were fonnd to break up from a human infection of an aerobic cultui'e
into frajj;nuMits, some of which reached a l)yAfanassiev. In ]8!)(), I'lppinger isolated a
lonp;th of 50 ju. The branching (ilanients weiv nonsporulating aerobic or-ganism, and Wolff
surrounded with masses of micrococci, filling and .]. Israel isolated, the same year, a non-
the spaces between llu> threads. These fila- spoi'ulating niicioaerophilic form.
ments were distinctly dilTerent fi"om the These cultures came fi'om dilfei'ent sources
straight, thick, and unbranched (false- and, l:)ecause of their lilamentous nature,
branching) Lcptothrix bitccalis commonh^ were considered to represent the isolates of
found in the mouth. The photographs of the Cohn and Harz. None of the above isola-
organism published by Cohn leave no doubt tions were, however, the cause of as much
that this was a true actinomycete. Cohn con- confusion as tlu^ report made b}' Bostroem
sidered this organism to be a bacterial form of his isolation in 18i)() of a pure aerobic cul-
with branching myceUum, though all at- ture of an actinomycete from a case of ac-
tempts to cultivate the organism failed. tinomycosis. This culture, now known to be
Two years later, Harz examined a patho- a Streptomyces, rapidly found a place among
logic specimen, obtained from "lumpy jaw" the various collections and was believed at
of cattle and submitted to him by Bollinger. first to be the true cause of actinomycosis.
He gave to the organism observed in this The general consensus now is that this cul-
specimen the generic name Actinomyces and ture did not represent the causative agent
the specific name bovis. Xo pure culture was of the disease but was merely an air con-
obtained. The masses of filaments were found taminant. Unfortunately, this error remained
to be arranged radially, which suggested the to plague the subsequent literature of the
name "actino-myces" or "ray-fungus." actinomycetes and became a cause of much
Neither of these two generic descriptions confusion. First, the claim that Actinomyces
was universally accepted, largely because bovis was an aerobe rather than an anaerobe
the first (Strcptothrix) had been preempted was wrong; second, the wide distribution of
in 1839 by Corda for a true fungus and the the contaminant led many to assume that
second (Actinomyces) had been meeting actinomycosis w'as caused by an aerobic or-
with much criticism, because the descrip- ganism similar to the group now designated
tion of the organism was based on its etiol- as Streptomyces.
ogy rather than on its morphology and cul- For many years, investigators continued
tural characteristics. to believe either that the causative agent of
The first isolations of pure cultures of ac- actinomycosis was an aerobe or that there
tinomycetes from human and animal infec- were two forms, one an aerobe and the other
tions involved some difficult problems in an anaerobe. There is no doubt now that Bos-
ecology and taxonomy. They were the pri- troem never succeeded in growing the true
mary causes of much confusion in the history etiologic agent of actinomj^cosis but that
of actinomycetes. O. Israel claims to have some of his attempted isolates became con-
isolated in 1884, from a human infection, an taminated with saprophytic actinomycetes
aerobic filamentous organism, the hyphae from the dust in the air, and thus resulted in
undergoing ready fragmentation. Bostroem the mistaken isolation. Topley and Wilson
THE ACTINOMYCETES, Vol. I
(1929) proposed that this isolate be named Animportant cause of confusion was the
Actinomyces graminis. Vuillemin (1931) con- fact that the actinomycetes were grown on
sidered it to be identical with Actinomyces nitrogen rich organic media, now known to
sulphureus Gasperini (1894). be totally unsuitable for them to form a char-
In the absence of pure cultures of the acteristic growth, essential for comparative
causative agent of the disease for compara- studies and As a
for proper identification.
tive studies,some of the early workers on result, a highly complex terminology was
actinomycetes had only a limited concept of developed for the designation of actinomy-
the growth and life cycle of these organisms. cetes; numerous descriptions of "new" spe-
This is illustrated, for example, in the de- cies soon began to appear. This is illustrated
scription by MacFayden (1889) of the his- by the summary made, as early as 1892 to
tory of an actinomycete colony: 1894, by Gasperini (Table 1). There is no
"It has its starting point in one or more wonder, therefore, that the nature and classi-
cocci transported by the plasma currents or fication of the actinomycetes soon appeared
by the agency of a carrier cell (leucocyte). hopeless.
The cocci multiply by elongation and subse- The adoption of the name "actinomy-
ciuent fission. By elongation some of the cocci cetes"was suggested by Gasperini and Lach-
give rise directly to short bacillary forms, ner-Sandoval. Sanfelice, impressed by the
and through these to long filaments. The analogy of the biological properties of the
further extension of the colony is effected by actinomycetes and those of the tuberculosis
the growth and multiplication of both organism, suggested that the relationship of
threads and cocci. The majority of the the actinomycetes to the bacteria was closer
threads tend to develop clubs at their outer than to the fungi. Gasperini emphasized that
ends (involution forms)." For more phan- the species or varieties belonging to the ac-
tasy and inaccuracy, one would have to tinomycetes, included under one genus Ac-
search widely in microbiological literature. tinomyces, show great variations in form and
Not much progress in the general under- in behavior, especially in their ability to
standing of these organisms seems to have produce aerial spores and soluble pigments.
l)een made during the next 20 years, as illus- Some of these properties were recognized to
trated by reference to them in the Second be inconstant and were found to depend on
Edition of H. W. Conn's (1909) Agricultural the conditions of culture and the composition
Bacteriology. In speaking of the actinomy- of the medium; minor variations of the latter
cetes, he says: could bring about marked changes in growth
"Under this head are included a few forms
and pigmentation.
of fungi which resemble other bacteria in
some respects, but differ in others. They are Historical Periods
composed of threads which are commonly
Before we consider in detail the historical
larger than the threads of bacteria, and
backgroimd of our knowledge of the actino-
which may show freciuent branching, a char-
They mycetes, we must recognize certain distinct
acteristic not usual in bacteria. also
periods in which the ^'arious concepts con-
ha\'e a peculiar method of forming reproduc-
ing l)odies. The group is not one of xevy great cerning the nature of these organisms and
importance. One type of Streptothrix is ex- their importance in the cj^cle of life became
tremely abundant in soil and appears as crystallized. There is, of coiu'se, considerable
round, white opac^ue colonies with an exten- ()\erlapping of the \-arious periods, since no
sive brown halo upon the plates." one period came to an end ])efore another
HISTOIUCAI, KACKCKorXI)
Tahi.k 1
Name
THE ACTINOMYCETES, Vol. I
et al.), and the importance of actinomycetes This resulted in extensive investigations de-
in natiu'al pr-ocesses were given ever-growing voted to the nutrition of actinomycetes, the
consideration. biosynthesis by them of various chemical
4. The biochemical or, more preciselij, the compounds, their chemical structure, life
antibiotic period. A new era in the study of cycle, and numerous biochemical activities.
actinomycetes })egan about 1940. Then, it
was estabhshed that a large number of these 1. Actinomfjcetes as Caumtive Agents
variety of chemical substances that have the The initial work on the actinomycetes was
capacity to inhibit the growth of various done by two eminent botanists, F. Cohn in
microorganisms, and that some of these 1874 and C. 0. Harz in 1877. Unfortunately,
substances can find chemotherapeutic appli- two circumstances soon shifted the interest
cations in the treatment of numerous infec- in these organisms from the botanists to
tious diseases of man, animals, and plants. clinicians and veterinarians.
iiisroHicAi- M\('i<(;i{(trNn
1. Colin's woi'k on act iiKiinycclcs, and toi- these diseases and t he complex nonicnclat lire
that niattci" on Itactcria in i>;(>n(M-al, was coni- that soon exolxcd continued to cause con-
plrtoly lu'jilcctcd by ncai-ly all botanists fol- fusion for yeai-s. .\.s late as l!)25, Dresel sug-
lowing him. i'^cn so outstaiidiiiiz; a itotaiiist gested that the term "actinomycosis" bo re-
as Rohind Thaxtcr, who about 1
">
years later ser\-ed for tho.se di.seases that are caused by
studi(Hl another group of actinoniycctos, the anaerobe (Aclinoninccs jsrncli) and that
nanu'ly, the oi'ganisms causing i)otato seal), aiiothei' name be selected foi' the di.seases
called them fungi (Oospora scabies), com- caused by aerolies, in ca.se the name "St rep-
pletely o\-erlooking their close relationship tothrix" should finally l)e dis(|ualified.
Koch, and numerous others. It was but nat- or species. Wolff and Israel i.solated from
ural that diseases caused by actinomycetes human actinomycosis an organism, "a strep-
should also soon begin to attract attention. tothrix fimgus," which differed from "Strep-
In 187(), Bollinger observed branching mj^- tothrix actinomycotica" in that the growth
celium in the diseased jaw of a cow and recog- under anaerobic conditions was A'crj^ free,
nized that a microbe was the causative agent whilst in the presence of oxygen it was very
of the disease. He handed this material to scanty. Levy isolated from five actinomyces
Harz, who examined the granules and ol)- ca.ses in man an organism which i-e.sembled
served the characteristic radiation, with the that of Wolff and Israel in growth its free
result described above. Simultaneously, J. under anaerobic conditions. Kruse recog-
Israel examined granules containing similar nized two species as causing actinomyco.sis:
mycelium in two pathologic specimens of (1) "Streptothri.v actinomyces" of Rossi-Doria,
man; unfortunately, he was confused by the said to be an "aerobic fungus;" and (2)
presence of secondary infections due to '^Streptothrix israeli," an "anaerobic fun-
staphylococci. It was Ponfick, in 1879, who gus." A number of other investigators de-
definitely established the role of actinomy-
cetes as causative agents of human diseases.
Israel's first account appeared in
clinical
1885. Wolff working in collaboration with
Israel soon established the anaerobic nature
of the organism.
These pioneering studies were follow^ed by
the careful work of Gasperini and others who
interpreted clearly the nature of the dis-
ease of actinomycosis and the role of actino-
mycetes in its causation.
The study of diseases caused by aerobic
actinomycetes in animals and in man also
began to receive attention, with the obser- FiGURE 3. Chil) formation by a culture of A.
vations of Xocard and Trevisan. I'nfortu- hovis grown in human blood .serum (Wright, J. H.
nately, the nature of the causative agents of .J. Med. Research 13:349 404, 1905).
—
scribed, according to Foulerton, cases ''which the organism in question to belong to a new
clinically present the features of actinomy- species.
cosis, but which are caused by parasites Thus, the differentiation between aerobic
which differ sufficiently from streptothrix and anaerobic forms as causative agents of
actinomycotica to entitle them to be re- specific diseases gradually became estab-
garded as separate species." Bruns noted a lished, particularlj'- through the work of
culture which he believed to be similar to Foulerton and Price- Jones (1902), Wright
that described by Berestnew as occurring in a (1905), and others.
case of "pseudoactinomycosis." Bruns ob- The first historical period is thus charac-
jected to the use of this term and considered terized by serious difficulties that were a
direct result of the complications involved
~-
I.
in the isolation and identification of the
causative agents of disease conditions in
animals and man, and by the problems of
proper nomenclature, which will be discussed
• ». •
in detail in Chapter 4. Attention has already
been drawn to the confusion introduced by
Bostroem, in 1890, who isolated from in-
fected lesions aerobic air contaminants, which
he designated as the causative agent of the
disease. Another cause of confusion was the
introduction of the term "streptothricosis,"
based on Cohn's original designation, as a
—
synonym not always recognized as such
for "actinomycosis," or a disease caused by
actinomycetes. Later suggestions that such
Figure 4. Appearance of cultures of A. bonis
names as "nocardiosis" and "maduramyco-
in agar tubes (Reproduced from: Wright, J. H.
J. Med. Research 13: 349-404, 1905). sis" be used did not help to straighten out
the ensuing confusion.
The study of the causation of plant dis-
eases by actinomycetes also falls within this
period. As has been noted, Thaxter eluci-
dated, in 1891, the nature of the pathogenic
organism concerned in potato scab. He called
it Oospora scabies. The culture was isolated
and carefully studied. This soon led to ex-
tensive investigations by numerous botanists
and plant pathologists, which continued into
the subsequent periods.
Outstanding work on the occurrence of
actinomycetes, their morphology and sys-
tematic position, was also carried out during
this period. It is sufficient to mention such
hovis in agar (Wright, J. H. J. Med. Research 13: and soon after Neukirch, and various other
349-404, 1905). bacteriologists.
HISTOUKWI. BAC'KC.Korxn 9
2. The Soil rrriixl {1900-1919) transformations. lie pointed out that actino-
Just as tho first period was initiatod and mycetes in general are ()inni\()n)us organ-
organism belonged to the group^•ery large agent. This led him to postulate the signifi-
of actinomycetes now included in the genus cance of these organisms in natural proc-
Streptomyces. Beijerinck attempted to throw esses. Actinomycetes were found to occur
light upon its physiology and its role in soil alnuidantly in the soil to considerable depths,
where they may exceed in numbers other the presence of numerous types of actino-
groups of microorganisms; this was explained mycetes received considerable attention.
by their greater resistance to conditions Highly significant in this connection was
unfavorable to their nutrition. Beijerinck the contril)ution of Krainsky. His paper pub-
thus laid the groundwork for our present may be considered as a classic
lished in 1914
knowledge of the occurrence and physiol- on a with the contril)utions of Cohn,
p.ir
ogy of actinomycetes. He also believed that Harz, Thaxter, and Beijerinck. It opened
these organisms play an important role in new pathways in th(> development of our
the humification of the organic matter in soil. knowledge of the actinomycetes. Krainsky
Among the other fundamental studies on emphasized that the voluminous literature of
the actinomycetes carried out during the the actinomycetes contains little of a physi-
early days of this period, mention should be ological nature; the numerous descriptions
made of the work of Hiltner and Stormer, of different species are so much alike that
who made the first comprehensi\'e study of one gains the impression that there are no
the abundance of actinomycetes in the soil. proper characteristics for distinguishing dif-
They found that the season of year and soil ferent kinds of actinomycetes. He empha-
treatment have a great influence upon the sized that the terminology as well had not
numbers of these organisms. This work was been sufficiently established. Numerous ac-
soon followed by that of numerous other in- tinomycetes have been isolated, without rec-
vestigators, notably that of Fisher (1909), ognition of their role in nature, since they were
Fousek (1913), H. J. Conn (1913-1918), always considered from the point of ^'iew of
Krainsky (1914), and Waksman and Curtis their pathogenicity and hygienic importance.
(1916, 1918). The role of actinomycetes in Krainsky's significant contribution was his
the breakdown of organic residues in the soil, emphasis of the importance of recognizing
methods for determining their presence and the growth characteristics of actinomycetes
abundance in the soil, and the recognition of on synt hetic media. He demonstrated that the
nature and concentration of both the carbon
and nitrogen sources are of great importance
in this connection. The nature of the aerial
mycelium, pigmentation of the colony, and
the formation of soluble pigment are all con-
trolled by the composition of the medium. He
further d'^monst rated that the formation of
chromogenic pigments on organic media
(tyrosinase reaction) is characteristic not of
one but of seA'eral species. The formation of
invertase and diastase w'as also found to be
characteristic of different species.
Krainsky further established the ability of
certain actinomycetes to decompose proteins
and cellulose and to reduce nitrate and even
suggested the possibility of their being able
to utilize the resistant lignin. As a result of
these studies he came to the conclusion that
tlio size Miul color of colony aiul the color of on the "Morphologic und i^iologie dcr Strah-
the aerial luyccliuni, under diU'ereiil condi- lenpilzc" may be considered as the begimiing
tions of nuti'ilion, arc siilliciciil lo charac- Some of the most outstanding
of this pei'iod.
terize a specii's. |'hysiolo«i;ical proiuMi ies can contributions to our knowledge of the tax-
be used to supplement such chai'acterization. onomy and classification of actinomycetes,
Krainsky thus inliddiicrd a new approach their occiiirciice in iiatui'e, and
theii' antago-
for cliaracterizin«i indi\idual species of uc- nistic properties were recognized dui-ing this
tinomyeetes. Many new organisms were now period. Lieske's work, unfortunately, did not
recognized, and th(> grc^at ahundance of many exert so great an influence upon the subse-
species in the soil was thus estal)lished. Si- (luent developments in the field as it should
nuiltaneously and indepen(l(>ntly, A\'aksman have. This was due piimarily to the fact that
and Curtis l)(\i;an their work in 1!)1."). 'i'licir Lieske was not familiar with the importance
first paper, put)lished early in 19H), treated of synthetic media in charactei-izing actino-
the use of synthetic media for characterizin<>; mycetes, nor did he appreciate the fact that
actinomycetes. l^efore compl(>ting their stud- these oi'ganisms represent munerous species
ies, h()we\-er, they became familiar with widely distributed in nature. This led him to
Krainsk3''s work and took full ad\-antage of doubt the existence of more than a few
it in describing various new species, l^nfor- which thus tended to obsciu-e rather
species,
tunately, because of the prevailing World than to stimulate further developments in
War I, they could not obtain Krainsky's cul- this field.
tures and had to depend
on descrip-
solely The work of 0rskov, in 1923, on the mor-
tions for identification purposes. This led to phology of the actinomycetes, was highly
a degree of confusion in the identification of significant and stimulating, and marked a
some of the species. Incidentally, Krainsky's turning point in the field. It pointed a way
cultures appear to have been destroyed be- toward establishing a new system of classi-
fore anyone else had access to them. The fication of actinomycetes, based on their
work of Waksman and Curtis culminated in morphological properties. The subsequent
a comprehensive study of the cultural prop- investigations of H. L. Jensen, beginning in
erties of actinomycetes by Waksman in 1919. 1930, on growth of actinomycetes in soil,
Attention should also be directed, at this their morphological and cultural characteris-
point, to the work on the mor-
of Drechsler, tics, tended to broaden further our knowl-
members of the genus Streptomyces. the marked variability of their growth on dif-
ferent media and the great complexity of the
3. Biological IWiod (1919-1940)
problem invoh'ed. This work ^^•as soon fol-
During this period, not only the morpho- lowed by the studies of Erikson (1935, 1940)
logical and ecological propcn-ties of the ac- on the pathogenic actinomycetes belonging
tinomycetes were studied, but also their to aerobic and anaei'obic groups and of Kriss
physiological and biochemical activities.The (1937) on the variations of actinomjcetes.
publication in 1921, of Lieske's monograph Considei-al)le emphasis was also laid, dur-
12 THE ACTINOMYCETES, Vol. I
^^ o
O CO
K .
o
IIIS'I'OHICAL HAC'KCHOIM) 13
inu; this pc'riod, on \\w inctaholisin of the ;ic- Information was also gradually accumu-
tiiioinycctcs, l)(><!;iniiiii^ willi the work of lating on the .systematic position and im-
Waksinan and his associates in I'll!) to li)'_M, portance of act inoiuNcetes in natuial proc-
antl kviiliiif;' to nunuM-ous other in\('stiji;a- esses. Sex'eral efforts were made to dexclop
tions in tliis licld. Stuihcs soon fohowcd on a system of cla.ssification of ac^t inomycetes
the antaiionist ic a('1i\ili('s of act inoniycotcs, thai would take into consideration much of
notably tlic work of l^oi'ochihna (l!)oo), Xak- the accumulated information. The earlier
hini()\-skaia (l!)o7), A\'aksnian and Foster in\'(>stigalions on the cultural j)i"operties of
Figure 9. Morphology of aetinomycetes compared to certain l^acteria and fungi with which they
have often been confused; f. to r., starting at top: Lcptothrix. Clndothrix, Streplubacillus, Oospora,
Oidium, Actinomyces (Reproduced from: Lieske, K. Morphologic mid Hiologie der Strahlenpilze. Verlag
von (Jebriider Borntraeger, Leipzig, 1921, p. 6).
14 THK ACTIN()MYCETP:S, Vol. I
aerobic Nocardia species was now becoming biogenesis of antibiotics. Many new species
definitely established. ha\'e been described, and a \'ariety of differ-
Numerous investigations were made, dur- ent metabolic reactions investigated. Since
ing this period, on the actinomycetes con- this field is so new and since the work done
cerned with the causation of plant diseases. in this connection is so extensive, it is diffi-
It is sufficient to mention the work of Mil- cult to summarize or to classify the numer-
lard and Burr, Lutman et al., Schaal, and ous contributions in any sort of chronological
Goss. The organisms concerned in the causa- order. It is much simpler to divide the period
tion of potato scab were found to belong to on the basis of certain antibiotic groups that
the genus Streptomijces. The sail and the have received the greatest consideration or
fresh-water actinomycetes, belonging largely application. These contributions need not
to the genera Streptumijces and Micromono- correspond to any particular date or lab-
spora, were also studied extensively. That oratory, since fretiuently the same active
the importance of actinomycetes as causative substance has been isolated almost simul-
agents in human diseases was not neglected taneously in several different laboratories in
isshown by the publications of Colebrook, different countries of the world.
Henrici (1930), and numerous others. With The writer has contributed a number of
the introduction of the sulfa drugs as anti- reviews dealing with the historical back-
bacterial therapeutic agents, attention was ground of the development of our knowledge
also directed toward the chemotherapy of of the antagonistic effects of microorganisms
diseases caused by actinomycetes. and the production of antibiotics. The first
review was published in 1937; it was largely
4. The Biochemical or the Antibiotic
concerned with the antagonistic activities
Period (1940-1958) of the organisms themselves, especially with
The most recent period in the history of organic matter decomposition and their pos-
actinomycetes has been characterized by a sible role in soil processes. The second and
unique development, that of formation and third reviews were published in 1940 and
utilization of antibiotics and of certain other 1941, and were more concerned with the
biochemical products, notably vitamins. potentialities of the antagonistic microor-
Since the introduction of penicillin as a ganisms as producers of antibiotics. This was
chemotherapeutic agent, no other group of followed (1945, 1947) by a comprehensive
microorganisms have contributed so much survey of organisms and their antibiotics in
to the field of human and animal therapy as a volume entitled Microbial Antag()nis7ns
the actinomycetes. Beginning with actino- and Antibiotic Substances. Later another \'ol-
mycin announced in 1940, a large number ume was published in collaboration with
of chemical compounds ha\'e been isolated Lechevalier on Actinomycetes and Their An-
from cultures of these organisms. Screening tibiotics (1953).
programs, initiated in the previous period by Numerous other reviews and ^'olumes were
a group of Russian investigators and devel- publishetl d(>aling with antibiotics as a whole
oped by the writer and his associates (1942), or with certain chemical forms in particular.
found ext(Misiv(> applications. Only ('(M'tain of these need he mentioned
Considerable attention has also been paid, here: 1, the monumental \-()lumes on .1/)/?'-
during this period, to the general problems biotics by 11. W. Florey, K. Chain, and their
bearing upon the physiology and biochemi- associates; 2, the volume Aclinomijcetes —
cal activities of the a(^tinomycctes, especially Antai/onists and Antibiotic Stil)stances by
metabolic processes, enzymatic systems, and Krassiliiiko\'; 3, the general n^xiews by
lIISroUK'AL HACIvClHOUND 15
Figure 10. The Ki"<)\vth of an actinoinyccte belonging to the gcmis Strvptoinyces, in artificial culture
(Prepared by Grundy of Abbott & Co.).
Benedict and I'cflinan; 4, finally', tlic \ari- ies, the period has witnessed great interest
ous special reviews of particular antibiotics, in the actinomycetes as a whole, their ecol-
as streptomycin, neomycin, the tetracy- ogy and classification, morphology and cytol-
clines, chloramphenicol, erythromj^cin, and ogy, genetics, biochemical activities and role
others. innatural processes. The numerous journals
In addition to the purely antibiotic stud- and \-olumes devoted to antibiotics contain
IQ THE ACTINOMYCETES, Vol. I
and Preservation
Most of the techniques used in the isola- majority of actinomycetes, notably members
tion and cultivation of bacteria and fun^i of the genera Streptomyces and Micromono-
also apply to actinomvcetes. The isolation spora, grow in the form of flakes or spherical
of these organisms from soils and other nat- compact masses, leaving the medium clear.
ural substrates is brought about by first The mass of growth can easily be remo\-ed
plating out such materials in proper dilu- by filtration through ordinary paper. Only
tions on suital)le agar or gelatin media. The when growth undergoes lysis do the cells
plates are incubated at favorable tempera- disintegrate completely and a certain degree
ures, for 2 to 7 days, and the colonies* of turbidity occurs.
picked and transferred to sterile litjuid or
solid media for further development. When Method of Study
the culture is found to be contaminated with In view of the ability of actinomycetes to
other organisms, a second plating may ha\'e produce both substrate or vegetati\-e growth
to be resorted to, to obtain pure cultures. and aerial mycelium that usually forms spe-
Actinomycete colonies can easily be dis- cial reproducti\'e cells, known as spores or
tinguished on the plate from those of fungi, conidia, it is natural that these organisms
on the one hand, and of true bacteria, on the should be found in nature both in the form
other. They are compact, often leathery, of hyphae or masses of more complex myce-
giving a conical appearance, and have a dr}" lium and as spores. The presence of hyphae
surface. They are often covered with aerial or mycelium in the soil or in other materials
mycelium. If the colony is well developed is usually considered as evidence that actino-
and the aerial mycelium abundant, the sur- mycetes lead in such substrates an autoch-
face spores can easily be picked with a sterile thonous or natural existence and that they
needle.If growth is limited, or the aei'ial form a part of the nati\-e microbial popula-
mycelium not fully developed, sharp, razor- tion. The presence of spores alone, without
like needles are recjuired to transfer a part mycelium, may suggest that actinomycetes
of the growth to fresh media. ha\'e been introduced there by air or by wa-
When grown in liquid culture, either in a ter.
Table 2
Classification of streptomyces on the basis of carbon utilization (after Pridham and Gottlieb)
L-Xylose —
D-Mannitol —
Lactose —
Na-acetate ( —
(S. lavendulae)
Na-acetate + Na-acetate —
Na-succinate — Na-succinate -f-
I
D-Mannitol -+-
DL-Inositol +
((S. antibioticus)
ISOLATION', IDI.N'l'IIK A'lMON, ClI/riN' A'lloX , AND I'H IISIIUNATION 21
limitation. The direct examination of undis- tinomycete population of soils and composts.
tiu'bed material offers a distinct advantage, It was first introduced by Rossi and Cho-
however, which consists in presenting a clear lodnv. A slit is made with a sharp knife in
22 THE ACTIXOMYCETES, Vol. I
Figure 15. Growth of actinomycetes in natural substrates, as shown by contact sliile method. Some
definitely belong to the Microtnonospora and others to the Streptomyces types.
the material to be examined. A clean cover organic residues, and stained with a suitable
slide is placed in close contact \vith the fresh dye, such as phenol-erythrosin.
soil, manure, or compost material. The slide The contact slide method offers certain
is allowed to remain undisturbed for 1 to 3 ad\'antages over the direct examination of
weeks, then removed, and cleaned on one soil or other material and over the direct
side. It is dried, fixed over a flame, carefully staining of such material. It permits the
washed to remo\^e coarse particles of soil or development, on (he slide, of organisms pres-
ISOLATION. ll)i:N'ni"l('\llo\, ('ri,ll\ ATloN, AM) I'lM ;Si:in' A'IMoN
(Mil ill tilt' suhslratc niul tlic loriii;il ion of oxer each cylinder so that the corners of the
sponilnl iiin' Ixxlics. This iiKikcs il possible coN'erglass ;ire e(|iiidisl ant from the center
not only io dtMnoiisti'iitc the acliinl ijrcsciicc ot the agar cylinder. The |)etri dish is co\--
of sucli oi'gaiiisnis, hut also Io (lilTcrciit iatc eicd and inciiliated .il "JS (
'. When the cul-
and rrcotiiiizc ('(>rtaiii tu'oad iiioi|)lio|o<;i('al ture has icached the proper stage of devcl-
fj;r()ups. It is also possit)lt' to use this met hod opmeiil, one of the co\'ergIasses is remo\'ed
for llic stud\' of the criccts of \arioiis tical- from the agar cylinder with the >terili/:ed
nuMits, siK'h as the additions of plant and pincette. The growth of the niyceliuni clings
animal residues and hnic to the soil, response to the iintlersurface of the co\-erglass, form-
of actinoniycetes to diffei-ent methods of ing a ring. The microscopic examination of
fertilization and croppiii.ti', and the relation the periphery of this circle makes it possible
between saprophytic and plant paiasitic to observe both aerial and vegetative myce-
forms to the root systems of i)laiits. lium, an undisturbed condition, and also
in
Cholodny observed that the direct micro- to follow the growth of the organisms at
scopic method does not si\e so accurate a every stage of their development.
picture of the abundance of actinoniycetes Permanent slides are stained and pre-
in the soil as does the contact slide method. pared as follows. The ring of the growing
The latter, however, has one distinct disad- mycelium on the undersui'face of cox-erglass
vantage, since it does not permit estimation is fixed by means of 2 oi- 'A drops of abs(jlute
sterilized clean coverglass is pressed down ter, and mounted on slides. This method was
24 THE ACTIXOMYCETES, Vol. I
modified by Giolitti and Bertani and others. to distinguish l)etween the two types of colo-
Excellent differentiation can thu.s be ob- nies.
tained for the vegetative and sporulating At first, organic media were used to enu-
growth. merate the abundance of actinomycetes in
Electron microscope methods. The electron the soil. Alore recently, synthetic media have
microscope has opened a new field for the been employed with only small amounts of
detailed study of the finer structure of cells a protein or polypeptide added, such as 0.005
of actinomycetes. References to the results per cent peptone, 0.025 per cent powdered
thus obtained are presented in Chapters 5 egg albumin, or 0.02 per cent casein. This
and 6, and elsewhere throughout this vol- tends to prevent development of the more
ume. rapidly growing and spreading bacterial and
fungal colonies and allow development of
Plate Methods the more slowly growing actinomycetes. The
The plate methods are the oldest and still \'ariouswater dilutions are so adjusted as
most reliable procedures for determining the to allow a final count of only about 30 to
nature and measuring the abundance of the 100 colonies per ordinary Petri plate.
microbiological, including the actinomycete, If one is interested in obtaining a great
population of such materials as soils, com- variety of actinomycetes for isolation pur-
posts, water, and milk. In making micro- poses, rather than in large numbers of colo-
biological counts of such materials, the nies, synthetic media are best for plating
actinomycetes are usually included with the purposes. With asparagine, glutamic acid, or
bacteria. More recently, selective media fa- sodium nitrate as a source of nitrogen, and
voring the preferential development of glycerol, starch, glucose, or a salt of an or-
actinomycetes have been devised, as by ad- ganic acid, such as malate, as a source of
dition of sodium propionate to the plate carbon, a relatively simple medium can be
(Crook et al.,1950) or by addition of anti- prepared. Such a medium will favor the de-
biotics. Certain purification procedures, such velopment of actinomycetes rather than of
as the use of various concentrations of sul- fungi and bacteria. The colonies of actino-
furic acid, have also been utilized (Fellinger, mycetes produced on such media are cjuite
1931). characteristic and can easily be recognized.
Beginning with the work of Hiltner and Numerous modifications of the plate
Stormer in 1902, actinomycetes came to be methods have been proposed, depending on
recognized as a separate group of organisms, such factors as the treatment of the soil, the
distinct from the bacteria and fungi, and nature of the medium used, and the tem-
were frecjuently so reported. The samples of perature and length of the incubation period
soil, compost, or other material are usually (Rao and Subrahmanyan, 1929). Some meth-
suspended in sterile tap water, and various ods are devised for the purpose of excluding
dilutions with sterile tap water subseciuently most of the bacteria and fungi. In others, an
made. These dilutions are then plated out attempt is made to distinguish l)etween the
on suitable agar or gelatin media. The plates abundance of viable spores and mycelial
are usually incubated at 28-30° C and exam- fragments of the actinomycetes. Skinner
ined after 2 to 7 days. It is sometimes diffi- made use of the fact that, when shaken in
cult to differentiate between bacterial and suspension, vegetative mycelium of actino-
actinomycete colonies, unless one has had mycetes breaks into viable fragments, which
considerable experience^ with actinomycetes are killed when shaking is prolonged (Table
or unless one uses microscopic magnifications 3). He observed further that spores do not
ISOLATION, IDliN rU'lCAIloN, ( l i;i"I\ A TloN , AND I'ln :S1 ;H\ A'llON Zi)
x'orable tor select i\'e de\(>lopment of the or- feliuin 1)1' reptomyce.s colony (Reproduced
a st
j^anisms. Such media may contain nutrients from: Lie.ske, R. Morphologic vnd Biologie der
Strahlenpilze. Verlag von Gebriider Borntraeger,
favorable to actinomvcetes, because of tluMr
Loil)zig, 1921, ]). 168).
specific metabolism, such as paraffin utiliza-
tion, steroid oxidation, or keratin decompo- T.\BLE 3
sition. On the other hand, substances are EjD'cct of shaking upon suspensions of substrate
added to the medium which may discourage actinomycete mycelium (Skinner)
strains was first suggested by Waksnian d methods for the maintenance of cultures in
al. for obtaining highly potent antibiotic- which the organisms will undergo the least
yielding strains from a particular antibi- morphological or biochemical change. The
otic producing organism. It was also used by ability to form spores is an important desir-
Umezawa et al. (1949) for the isolation of able property to be conserved.
chloramphenicol-producing organisms. Various methods are used for the preser-
For the isolation and cultivation of mem- xation of actinomycete cultures. Artificial
bers of the genus Actinomyces, or the meso- media, including both synthetic and organic,
philic anaerobic forms, special methods have have been used for maintaining cultures of
incubated anaerobically in an atmosphere of years' storage undcn- mincM'al oil and that
5 per cent COo for () days. the streptomycin-producing strain of this
organism was \-iable after (> yeai's' storage
Preserving Cultures of Actinomycetes under the same conditions.
The gi'eat variability of actinomycetes, Ilaynes et al. can-ied out extensive inves-
frequently accompanied by loss of desii-able tigations on the preser\-ation of the collec-
properties, makes it ne(;essary to dex'elop tion of more than 2800 cultures at tlu^ Xorth-
.
(M'li l\('u;i()ii;il Ixcscaich Lalior.-ilorics of the tails of the method were lurlher descriix-d
r. S. Department of Aij;ii('iiltiire. Lyopliili- ;is follow s:
zatioii has been iclied upon e\clnsi\ cly, toi- "Soil cullui-es wei-e prepared by inoculat-
so\'(M"al years, tor the preseix at ion of tlie ing 10 ml of biol h in a 25- by 150-mni culture
ha('t(M'ia, includinii the act inoniycetes. \o lube with the ^lowlli from a slant culture
aetinoni>'cele was encouiUered w hicli did not of a |)arl iculai' si ivpt oni>-ces. The inoculated
siif\"i\'e lyophih/.at ion. With few exceptions, tubes were |)lace(l on a lotary shaker and
tlie lyophiHzed cultures lia\e icniained \iahle incubated with shaking foi' 2 days al 28
for as l()n<i as tlie>' ha\'e l)een under ohser\'a- to 80° C
Two ml of the broth culture were
tion, some l)eins 14 years old. pipetted into a lube of sterilized soil and the
Pridham vi al. (19o()) made a detailed suspension thoroughly mixed with the tip of
stuily of the different media fa\-oral)le to the pijK'tte. The tubes were plugged and
the mainteuanee of the desirable properties allowed to air-dry al I'oom lemperattu'e for
of actinomycete.s. A
eomparison was made 2 to :\ weeks. During this period, growth
of 500 [^trains grown on 19 different media. usually occurred in the soil-broth mixture,
Veast-glucose and tomato-paste oatmeal appearing on the surface as a mat that was
agar allowed the growth of 85 to 8(3 per cent often covered with abundant aerial myce-
of the cultures with th(^ formation of abun- lium."
dant aerial mycelium. I'otato-glucose agar, Other (experiences wer(> reported by P'rom-
however, gave only 12 per cent viabilit}'. mer (195G).
Glucose-asparagine agar and synthetic
Additional iNIethods and Media
(known as Czapek's) agar were among the
worst Numerous additional reports are found in
Soil has lieen found very effective in the literature on the methods for the culti-
maintaining actinomycete cultures. Several vation of actinomycetes, and for the study
methods are used in the preparation of the of their morphological and physiological
soil cultures. A good, fertile soil is selected properties. Some of these methods are spe-
and freed from roots and pebbles. One-hun- cialized in nature, as, for example, the meth-
dred gm portions of such soil are placed in ods for the isolation of antibiotic-producing
250-ml Erleiyneyer flasks. If the soil is acid, strains of Streptomyces (Waksman ct al.,
1 gm of ground CaCO.3 is added. If the soil 1940, 1947), and the principles of the screen-
is poor in organic matter, 0.25 gm of dried ing program for antibiotic-producing organ-
by the addition of broth and incubation at Tresner and J^ackus proposed another sim-
room temperature (25 to 80° C) for 1 week. ple method for the preservation of cultures
In no instance did growth of microorganisms of actinon\ycetes. Spores of the organisms
occur in the soil-broth suspensions. The de- are streaked on appropriate agar media in
28 THE ACTINOMYCETES, Vol. I
optimum temperatures. One ml of 40 per their original characteristics for longer than
kept at room temperature with covers in lium, and growth habit remained essentially
unchanged most cultures. In a few cases,
place until all of the formaldehyde has been
in
Distribiilioii in Nature
any group
Tlu' distribution in nature of ent forms of the same general type. It is
they live and multiply; other substrates after the recognition of their existence as a
represent only temporary habitats for ac- separate group of microorganisms. The exact
tinomycetes, where they are distributed by enumeration of the large numbers of species
water and air movements. They are also found in the soil was actually begun, how-
found in the far north and on high moun- ever, during the first years of this century.
tains, in deep layers of soil, and in oil de- For a time after the first designation and
posits. Some genera favor one habitat and description of an actinomycete by Cohn, but
others favor another. Strcptomyces is most little attention was paid to the occurrence of
commonly represented in soils and in com- actinomycetes in nature, aside from the ani-
posts; Micromonospora species are abundant mal pathogens. Globig was among the first
in lake bottoms some members of the genera
; to draw attention, in 1888, to the occurrence
Actinomyces and Nocardia are known to be of actinomycetes in the soil. He isolated a
causative agents of human and animal dis- thermophilic organism, using potato as a
eases; Thermoactinomyces and other ther- medium. Another form was soon isolated
mophilic genera grow abundantly in stable from the air by Rossi-Doria (1891) and des-
manure and in high-temperature composts. ignated Streptothrix alba. In 1900, Beijerinck
Enghusen (1956) explained the wide distri- established that actinomycetes occur in great
bution of Strcptomyces species by the small abundance in the soil. He found them in
size of their spore (1.0-1.5 by 0.5-0.8 ix) and garden soil at a depth of 1 m, in sandy soil
by their resistance to drying (for 3 years or to a depth of 2 m; he also found them in the
more) bottom mud of a river bed. Beijerinck em-
On the basis of their actinomycete popu- phasized that actinomycetes are omnivorous
lations, from both a quantitative and a fiiial- organisms, living and growing under a great
itative point of view, the following natural variet^y of conditions.
substrates may be recognized: The first quantitative enumeration of
1 Soils, comprising virgin and cultivated, actinomycetes in the soil was made by Hilt-
garden, field, and forest soils, as well as ner and Stormer, in 1903. The gelatin plate
drained peat bogs. method was used. The numbers of actino-
Sea waters and sea bottoms.
2. mycetes were found to A'ary between 13 and
Fresh water basins, comprising lake
3. 30 per cent of the total microbial flora of
and river waters and bottoms. the soil capable of developing on the plate.
upon the plant a temporary or permanent crease was ascriV)ed to the addition of fresh
habitat; others are able to cause diseases of undecomposed plant and animal residues.
plants. The introduction of stable manure into soil
8. The bodies of man and animals, espe- also had a marked effect in increasing the
cially the digestive system. number of actinomycetes.
9. Geological formations. Fischer found that actinomycetes com-
prise 15 per cent of the microbial population
Occurrence and Al)un<lance of Actino- of sandy soils. Fousek reported that the
mycetes in the Soil higlu^st numb(M-s of actinomycetes are found
The distribution of actinomycetes in \ari- in the fall of the year (27 to 35 per cent of
ous soil types began to receive attention soon all colonic^s); the lowest numl)er occurred in
nisTiniuTioN IN NA'rrHi'; 31
(0
o
^ E.coli
I B.subtilis
o B.mycoides
Staph, aureus
> 60-
•H
PO
o
-p
c
o
the spring (18 to 2o per cent). He also con- soils contain large numbers of actinomycetes.
chiiled that the addition of fresh organic resi- Although the actual numbers diminished
dues in the fall was largely responsible for with depth of soil, they increased in propor-
the increase in the number of actinomycetes. tion to the bacteria. At the surface of certain
Their presence and on the roots
in forest soils soils, actinomycetes, as measured by the
of grasses and leguminous plants was con- number of colonies produced on agar plates,
sidered as further evidence that they play made up 9 to 15 per cent of the total popu-
an important role in the decomposition of lation, or a total of 743,000 to 933,000 per
plant residues. These observations were con- gram of soil. At a depth of 30 inches, the
firmed by Conn in 1916, who reported that numbers dropped to 240,000 per gram, but
actinomycetes make up as much as 40 per the percentage rose to about 66. In Califor-
cent of the microbial population in soils rich nia soils, the numbers varied from 380,000
in plant roots, as compared to the population to 1,890,000 per gram, and the percentage
of cultivated soils, where the numbers of of the total population from 19 to 45.
actinomycetes were only about 21 per cent. The total and relative numbers of actino-
Extensive studies on the actinomycete popu- mycetes in a number of American soils, as
lation of the soil were also made by Miinter. determined by the use of egg-albinnen agar,
Krainsky obtained much lower numbers, are shown in Table 4. Acid soils, waterlogged
howe\'er, because the synthetic media he soils, and soils poor in organic matter con-
used permitted the growth of only 20,800 tained the lowest numbers of actinomycetes.
actinomycetes per gram of soil. Although Heavy soils and organic matter-rich soils
valuable for the recognition of a greater \a- contained the highest numbers.
riety of species, these special media do not Gillespie and Waksman and Joffe found
allow the development of so great a number that the critical degree of acidity for the
of total colonies of actinomycetes as do or- growth of the majority of actinomycetes in
ganic media. the soil is pH 4.8 to 5.0, the optimum reac-
Waksman and Curtis also reported that tion being pll 7.0 to 8.0. Jensen (1928) iso-
32 THE ACTINOMYCETES, Vol. I
Table 4
Figure 18. Relationshi}) lietween crop yields and numbers of actinomycetes (Hei)r()duced from:
Singh, J. Ann. Appl. Biol. 21: 163, 1937).
10,000 to 4,000,000 per gram. But here again The effect of partial sterilization of soil
the bacterial numbers were considerably upon the changes in itsactinomycete popula-
lower, or 1,000 to 160,000 per gram. tion was studied by Waksman and Starkey
Krassilnikov (1938) recorded much lower (1923). The.y found that this treatment of
numbers of actinomycetes for ^'arious Rus- the soil brought about a considerable change
made up only 5 to 7 per cent of
sian soils they
: in its microbiological population, the actino-
the total number of colonies developing on mycetes behaving differently from the fungi
the plate. The soils of the dry steppes of and bacteria, depending upon the nature of
Northern Kasakhstan were characterized the treatment and the organic matter con-
(Tepliakova and Maximova) by a significant soil. In general, the actinomycetes
tent of the
number of actinomycetes. They were most were among the more persistent forms.
abundant in the dark chestnut and carl)on- In a study of the survi\'al and growth of
ate soil. Their numbers increased with the Nocardia in the soil, Brown (1958) found
depth of soil, whereas the number of species that in partially sterilized soil wide fluctua-
diminished or remained unchanged. Halo- tions occurred during the fii'st month, fol-
phylic and facultative halophylic actinomy- lowed by a steadying in count, and at the
cetes were isolated from salt meadows; the end of the year .V. cdhdans was still present
former develop better with l.o to 3 per cent in high numbers. In the untreated soil N.
NaCl or Na2S04 in the medium, and the lat- celiulans disappeared in G months. A regular
ter Avithout additional salt. cyclical morphological do\-(>lopmont of the
34 THE ACTIXOMYCETES, Vol. I
increasing order.
The streptomyces isolated from soil at
depths of 8 and 16 inches were the same as
Table 6
Date
(1930-1931)
;
nisTHim'TioN IN XA'rrHK 35
techni(iue, demonstrated the presenc(> of my- treatment of forest soil with formalin (1 per
celial filaments in the soil. On continued cent) does not destroy the actinomycete
shaking, the number of colonies obtained population. Warren ct al. found that soils in
from a given cjuantity of soil was increased which the plants were sprayed with 2,4-D
to a certain maximum; then the number di- allowed extensive growth of actinomycetes
minished as a result of destruction of the that possessed strong antifimgal }5roj)erties.
able nutrients in the form of bacterial cells. numbers of spores in the soil and not of ac-
An examination of soils enriched with tive organisms, the evidence presented here
streptomyces spores, using the Rossi-Cho- proves the contrary. By separating the no-
lodny contact slide method, revealed that cardias (designated as proactinomycetes)
those spores remained in the soil largely in from the streptomyces (designated as actino-
an ungerminated state. They grew only on mycetes). Topping failed completely to rec-
the rim of the soil adhering to the glass and ognize the nature of these organisms,
in the dead bodies of soil amoebae (Pfennig). namely, their vegetative versus sporulating
Nature of the soil actinomycete population. growth, and the possible confusion between
No attempt will be made to review here in them; he did recognize, however, the nocar-
detail the numerous other investigations on dias and nocardia-like organisms as members
the abundance of actinomycetes in the soil, of the native or ''autochthonous" microflora
as determined by the plate method. Suffice of the soil. Further information on the occur-
to say that all these studies established the rence of nocardias in the soil is found in the
fact that actinomycetes form an essential work of Frey and Hagan (1931) and Gordon
constituent part of the soil microbiological and Hagan (1937).
200-
<
O
150 -
Od
LJ
Q.
(/)
z 100 -
o
INCUBATION- DAYS
Figure 19. Comi)aris()ii bctwccii donsity of vegetative inyeolium and ])late counts of actinomycetes
in soil. Continuous line: density of myceliiun; l)rok(>n line: platecounts (Reproduced from: Jensen, H. L.
I'roc. Linnean Soc. N. S. Wales 68: 69, 1943^.
DISTinHrTIoN I\ NA'ITHI': 37
'The addition of orj^auic matter to the soil ai'c found inii\ rtsally, the spccilic substrate
lias a inarkiHl st iimilatiii<«; (^I'tVct upon the and enxironment greatly influence their na-
d('\'(>lopnuMit of actinoniycctcs, as illustrated tui'e. This is true, foi' example, of soils in
\)\ Waksinaii and Stai'kcy (l!)24). which suc('essi\-e crops of ])otatoes ha\'e been
One nioiv prol)l(Mn pcM'taininfi; to (he sur- grown.
\-i\-al of actinoniycctcs in the soil must ho Further studies on the wide disi libution
considci'c'd, namely, the sui\i\al of or<i;an- of actinomyeetes in soil have been made by
isnis addotl to the soil. Fousek reported that Xegre (Sahara soils), Jensen (19:^0, 1931,
addition of cultures of actinomyeetes to soils HKU, 193()), Waksman (1932), Adachi and
rich in oriianic matt(M- hastencMl decomposi- Imanuu-a (1933), Ilopf, Strutz (1952), Jag-
tion of the humus and inci'eased liberation now (1957), and others. Detaiknl studies of
of the nutrients. It is a well established fact the occurrence in soil of actinomyeetes pos-
that wluMi lowmoor jx'at soils are freshly sessing antagonistic properties against other
draintMl and culti\'ated, actinomyeetes will microorganisms and capable of producing
(kn'clop at a rapid rate in such soils (Waks- antibiotic substances are presented in Chap-
man and Purxis, 19o2). If the potato scab ters 14 and 15.
fouml i)('i- milliliter of lake imul, ninount iiiii Tulilinaexamined the aliimdance of (hese
to 4") jH'i" ci'iit ol (he tt)l;il iniciohial popula- organisms in the waters and sediments of
tion. \\'\\h an iiuTcaso in the depth of the the Don basin. Their numl)ers in the bottom
l)ottoni inatcM'ial there was an increase in material wei-e 10 to lOOO limes gi'eater than
tlie nunilnTs and percentaj^es (up to (>() to those in the water. They were belie\-ed to
70 per cent) of actinoniycetes. Tlie conclu- l)e responsible for the unpalatable odors im-
sion was readied that the ,a;iMuis M irromoim- parted to the ])oii waters. When the organ-
sporo represents a tiiil\' inthgenous ji;i-oup of isms were isolated in pure culture, similar
microbial inhabitants of waters and bottom odors were j)roduce(l in artificial media.
deposits of inland lakes. Since thes(> organ- l']gorova and Issatchenko also found an ex-
isms are aerobic and grow very slowly, it was tensive population of actinoniycetes in river
at first iH'Heved that their role in the decom- bottom deposits. The numbers varicxl from
position of organic matter in water basins 30, 585 to more than a million per gram of
was only a minor one. Their ability to attack dry material. Larg(^ luiintx-rs were r(>coi(led
resistant organic materials, such as hgnins, even at a depth of 20 cm. ( )nly few organisms
suggested, however, a potentially important were found in the water itself. When some
function for these organisms. of thewater and a layei- of bottom material
Erikson isolated 10 strains of Micromono- were placed in cylinders, sterilized, and inoc-
spora from lake mud and lake water. These ulated with pure cultures of actinomycetes,
were capable of growing on a large variety excellent growth was obtained in a short
of resistant organic compounds, such as time. The earthy smell and the unpleasant
chitin, cellulose, and, to a lesser degree, lig- fla\'or of the Moscow ri\'er water was as-
nin. It was suggested that these organisms cribed to the multiplication of actinomycetes
play a significant part in lacustrine ecology, in the bottom muds,
especially during the
being adapted to life under aquatic condi- summer and months. They were largely
fall
tions and being able to utilize resistant sub- of the streptomyces type. The sandy soils
stances of the type found in lake mud. along the river banks contained a larger
Actinoniycetes are also found in river wa- number of actinomycetes. The odor pro-
ters and in river bottoms. As a consequence, duced by them is washed out by rains and
a serious problem may arise, namely, the im- carried into the river. This takes place espe-
parting to the water of the odor character- cially after the soil undergoes a spell of dry
istic of cultures of actinoniycetes. This odor or freezing w-eather. The numbers of actino-
renders the water unsuitable for drinking mycetes increase particularly in autumn.
purposes. Adams drew attention to the fact The passage of the odoriferous substance
that the ''earthy" taste of the water of the into the w^ater depends also on the nature
rWer Nile was due to actinoniycetes that in- and adsorbing capacity of the bottom mate-
habit or "contaminate" the water. Burger rial sandy bottoms give a marked odor, and
:
and Thomas suggested that the peculiar clay bottoms only little odor because of its
taste of the water was due to the decomposi- adsorption on the clay (Issatchenko). Thay-
tion of plants growing in the waters, the ac- sen has also made a study of the relation of
tinoniycetes forming merely a small part of actinomycetes to the odor of river water, as
the total microbiological population. shown later in this chapter. Numerous other
The role of the actinoniycetes in the pro- studies have been made on the occurrence of
duction of odoriferous substances in the wa- actinoniycetes in difTerent kinds of waters,
ter has received considerable attention. to which they can impart odors and tastes
40 THE ACTINOMYCETES, Vol. I
Table 8
Depth of
peat, cm
Dis'nniu'nox i\ nature 41
tiiu't typ(>s were recognized, l)('l()ii,ii;iiii;' lo the also largely I'esponsible for I heir wide dislri-
j>;('iiiM';i ThcnntKuiindniiircs and M icroniono- but ion.
spora. The presence of microscopic; particles of
Tliat the intestinal canal is the source of dust in the air was known to the ancients.
thermophilic actinoniycetes was demon- The Roman poet first observed
I.ucretius
strateilby TsikHnsky and ]-iruini. 'I'he oc- such particles by passing a ray of sunlight
currence of thermophilic forms in soil has through a darkened room. With the birth of
fienerally been correlated witii the ai)plica- modern microbiology, ever-gi'owing atten-
tion of stable manures (Alishustin). Ilenssen tion was paid to the microbes found in the
recently recorded the isolation from manures dust. Leeuwenhoek observed them in rain
and composts of 1 1 thermophilic species of drops; Ehrenburg found them in rain water
actinomycctcs placed in fi\'e <i;en(M'a, as and in snow; J. Tyndall saw them directly
shown hi detail in Chapter 28, \'()1. II. in the dust. The whole (juestion of sponta-
Sewage is frequently found to contain ac- neous generation, which aroused so much
tinoniycetes. As pointed out previously, attention and which was settled largely
Kedzior was the first to demonstrate the through the work of Pasteur, had a great
presence of thermophilic actinoniycetes in deal to do with the interest in the microbial
sewage. Brussoff isolated an organism, desig- population of the atmosphere. The question
nated as A. cloacae, definitely a strepto- of contagion and the problems
of epidemics
myces, from the slime of the Aachen purifi- appeared to be at that time closely related
cation This organism was found
system. to the air as a carrier of microbial agents.
capalile of decomposing cellulose. Brussoff More recently, the bearing of the dust micro-
accepted Beijerinck's and Krainsky's con- flora upon the problem of allergy has begun
cepts of the omnivorous nature of the actino- to receive considerable attention.
niycetes. Still he believed that his organism Foulertoii and I'rice-Jones spoke of the
was capable of fixing nitrogen, a fact not isolation of actinoniycetes (Sfrcptothrix crijth-
usually accepted at present, although it grew rea) as air contaminants. Tli(\v also men-
better on nitrogen-containing media. tioned the isolation of actinomycete cultures
(»S/. leucea and .S7. leucen saprophijiica) from
Occurrence of Actinoniycetes in the At- sewage and from drinking water.
mosphere Lidwell suggested that the problem of
sampling air for microlies comprises collec-
Actinoniycetes occur abundantly in the
tion ()f the sample from the atmosphere and
atmosphere, both in the form of mycelium
the determination of the microbial cells in
and as spores. This can easily be illustrated
the sample, their enumeration, and their
by exposing agar or gelatin plates to the air
classification. Methods of air sampHng in-
for a few minutes or by collecting some of
clude: (a) sedimentation, usually into open
the du.st and analyzing it. Actinoniycetes
dishes; (b) filtration, through wool, sand, or
are also unix'ersaily found on rocks, plants,
other materials; (c) cent I'ifugat ion; (d) col-
animals, clothing, food, and other surfaces
lection in li(iui(l by bubbling air through it;
exposed to the atmosphere. Because of the (e) electrostatic pi'eci])itation.
ability of actinoniycetes to withstand desic- Special apparatus was constructed for col-
cation, their presence on such surfaces re- lecting atmospheric dust. I']xp()sures were
sults from the breaking up of fine soil par- made at different altitudes and under dif-
ticles suspended in the atmosphere and the ferent conditions. The first comprehensive
drying of water drops. \\'iii(l currents are studv was undei-takeii 1)V Miciuel in 1883.
42 THE ACTINOMYCETES, Vol. I
Among the many organisms that he found undesiral)le musty odors the organisms im-
in the atmosphere, some, frequently desig- part to the food. The characteristic odors
nated as branching bacilli and cladothrix, produced by actinomycetes have attracted a
no doubt belonged to the actinomycetes. great deal of attention. Rullmann first be-
Rossi-Doria, in 1891, made a detailed in- lie\-ed that the odor was characteristic only
\'estigation of the actinomycetes in the air. of a certain species that he designated as A.
He was the first to use the specific name alba odorifer. The odor itself was referred to fre-
for actinomycete species. Since the genus to quently as "earthy," since it is similar to
which this species belongs is now know^n as that of well-aerated soil.
Stirptomyces, the Streptothrix alba of Rossi- Actinomycetes usually develop upon food-
Doria is now recognized as the type species stuffs under conditions not very favorable
rich in carbon monoxide. They suggested Actinomycetes are responsible for unde-
that the culture used CO gas as a source of sirable odorsand flavors produced in milk.
energy, the CO being oxidized thereby to Barthel was primarily concerned with the
COl' . The organism was described as a Ba- occurrence of microorganisms in fresh milk
cillus, under the name of B. oligocarbophilus. and in the cow's udder. He isolated two cul-
Lantzsch repeated these studies later and tures designated as A. albus and .1. chro-
found that the organism was actually a true mogcncs, both typical Strcptomyccs species,
actinomycete; it could assimilate not only and came to the conclusion that these come
CO) but also higher aliphatic hydrocarbons, from the air. Fellers directed attention to
except benzol and xylol. He, therefore, the possible damage to milk due to the un-
changed its name to .4 . oligocarbophilus. desirable odors and flavors. Hla\'ackova
Numerous other investigators observed (1951) reported that as many as 16.3 per
the occurrence of actinomycetes in the air. cent of samples of raw milk may contain
Caminiti found the organisms in hospital actinomycetes. Their presence was consid-
air, Barthel observed them in the dust of ered as an indicator of the degree of contam-
stables, Bellisari reported their occurrence ination of the milk with dust and excreta.
on the dust covering cereals. These organisms may be pigmented and
may thus cause additional damage to milk
Occurrence of Actinomycetes on Food and butter. If the milk is insufficiently or
Products improperly pasteurized, the actinomycetes
Actinomycetes are found extensively on will survive. Gratz and Vas isolated two
and in various food products. In some cases streptomyces cultures from Littauer cheese.
they are able to produce extensive growth Jensen recorded their occurrence in butter.
and have, therefore, been recognized as the Chatterjee found an actinomyces in fer-
was believed to be duo not so much to the The earthy oi' "nuiddy taint" occasionally
actual destruction of the foodstuffs as to the t'ouud ill fish was studied in detail by Thay-
niS'I'IMIU'IMoX I\ NA'ITHK 43
so noticeable when the fish were boiled that Left bank of river, odor Right bank of river,
strong odor slight
they were inedible. The odoriferous sub-
stance was soluble in water and volatile in Yards bolow tidal limit
steam. The river water itself where the i)ar-
ticular fish were caught also had this ll'(» 200 220 600 1100 120 220 000 1100
\egetation removed from the dry bank of true, for example, of potatoes. Some of these
the river. None of these cultures was of the organisms are saprophytic in nature and
odoriferous type. In the contaminated river, others are pathogenic, being responsible for
the actinomycetes represented "an abnor- the causation of specific plant diseases, as
mally high proportion in the microflora." pointed out in Chapter 18.
Among the other food products subject to Various theories have been proposed con-
considerable damage from the occurrence of cerning the function of the actinomycetes in
actinomycetes is the cacao bean. In 1927 the plants, since they are known to occur in
Ciferriundertook a study of the causative the outer layers of roots and tubers. Bei-
agents of the musty odor of these beans in jerinck found various plants to be superfi-
the Dominican Republic. He found this odor cially infected with actinomycetes. In 1903,
to be associated with the occurrence of ac- Petri isolated an actinomycete culture from
tinomycetes; the commonest form was a the rootsstrawberry plants; although
of
variety of S. albus. Bunting, in 1932, isolated this organism could be inoculated into fresh
three cultures of actinomycetes, described plants, it was considered to be a saprophyte,
under a species name S. cacaoi. The pungent since the plants were in a healthy state even
musty odor of the cacao was found to be after 6 months. Lutman observed actino-
caused by these organisms. mycete filaments growing along the cell
According to Haines (1932), actinomy- walls of potatoes and other plants; the fila-
cetes occur commonly in commercial cold ments were branching and were passing into
stores. They were isolated from the walls the cell lumen, twisting and bending in tor-
and especially from the sti'aw on the floor tuous paths; the stems above ground, the
of the stores. leaves, and the flowers were also completely
Among the other food products that may infected. Lutman postulated the theory that
be contaminated with actinomycetes, it is the cells of the actinomycetes take part in
44 THE ACTINOMYCETES, Vol. I
the synthesis of alkaloids and proteins in since evidences of it have been found in fos-
has been studied in detail by Peklo, Millard in the mouth, pharynx, intestine, and actino-
and Burr, and numerous others concerned mycotic lesions. They are considered as strict
with the causation of plant diseases by ac- parasites of man and many animals. The sep-
tinomycetes. The earlier work of Arzberger aration between the smooth A. bovis found
and Peklo on "plant actinomycoses," and in cattle and rough A. israeli in mtui may not
the possible role of actinomycetes in the be tenable.
tuberization of species of Myrica, appeared The further assumption that the etiologic
also to have a bearing upon this problem. agent A. bovis propagates in the soil and
that cattle become infected while grazing
Occurrence of Actinomycetes on and in upon grass has been another source of error.
Animal Bodies Emmons pointed out that the true etiologic
agent A. bovis has not yet been isolated
The etiology of actinomycotic infections
from a natural habitat outside the animal
in man and in animals is closely bound with
body. It has been assumed that this organ-
the occurrence of specific organisms at the
ism has both a saprophytic and parasitic
sight of infection. Whether actinomycosis is
existence within the body itself. Emmons
a result of exogenous infection due to the
obtained pui'e cultures of the microaerophilic
consumption, by animals, of grasses and
Actinomyces from the surface of discolored
foodstuffs containing actinomyces spores and
teeth, from carious teeth, and from tonsilar
mycelium, or of endogenous infection due to
crypts. When first isolated, these cultures
the presence of these spores and mycelium
grew more rapidly and the hyphae were
as regular inhabitants of the healthy mouth
coarser than those of the strains isolated
aroused, in the past, a great deal of discus-
from clinical actinomycosis, but upon re-
sion (Lentze). The history of the causative
peated subculture, they became similar to
agent of actinomycosis is closely boimd with
.4. bovis in morphology. Lord demonstrated
the occurrence of actinomycetes at the sight
the presence of actinomycetes in the contents
of infection, since considerable difficulty has
of carious teeth and in the crypts of tonsils;
been experienced in the isolation of the caus-
he considered the buccal cavity as the source
ative agent of the disease and in bringing
of infection. Further studies of the occur-
about experimental infections by these or-
rence of actinomycetes in the oral cavity
ganisms.
have been made by Xa(»slund and by Ludwig
Bollinger demonstrated that the common
and Sullivan.
types of b()\'in(^ actinomycosis are due to an
Actinomycetes have also been found exten-
organism named by Plarz in 1877 Actino-
si\ely in numerous othei' organs and excreta
myces bovis. Wolff and Israel are credited
of the human and animal body, such as the
with the first isolation from maxillary actino- fresh ex(;reta of suckling animals (Aloro) and
mycosis of cattle of a mic^-oaerophilic strain the intestinal canal animals
of hcnilthy
of A. hnvh, which is now considered as the (Hopffe). Fischer (1915) actinomy-
fovnid
true etiologic agent of the disease. Api)ar- cetes (»S. albus) to occur abundantly in the
ently, actinomycosis is an ancient disease. caecum, in tlu^ large intestine, in the rumen,
)|SI'KIIU"I"1()\ l\ N AITHI'] 45
animal experiments were carried out, in most nificant concentrations in such an artificial
obtained tends to establish the fact that in a idly. Although this was originally ascribed
natural soil, in the presence of the numerous to the presence in the soil or in the sea of
competing forms of life, no antibiotic is pro- antibacterial substances, comparable to an-
duced; were the reverse to be the case, the tibiotics, the actual facts have not substan-
antibiotics would have played an important tiated this explanation. Such substances are
part in the distribution of various groups of usually removed on sterilization, whereas
microorganisms in the soil. Only among the many of the antibiotics are resistant to heat.
actinomycetes do we find a large proportion It may, of course, be argued that the disap-
of organisms capable of producing antibiot- pearance of cultures of organisms added to
One could have expected a much greater
ics. soil or water is due not so much to the pres-
due to various plant products left in the soil are the effects of products of metabolism of
as a result of the decomposition of plant actinomycetes, such as streptomycin, chlor-
residues, such as tannins, oils, resins, or lig- amphenicol, and the tetracyclines, upon the
nins and lignin derivatives. intestinal population of animals. Streptomy-
5. Enrichment of a fresh natural soil with cin is known to favor the rapid development
living cultures of microorganisms has failed of resistance among the sensitive bacteria.
to stimulate or favor the selective develop- Baumgartel succeeded in extracting desoxy-
ment of other organisms that would be capa-
ribonucleic acid from resistant strains and
ble of producing antibiotics active against
transplanting it to sensitive strains. Sensi-
the organisms introduced. Antibiotics are
tivity and resistance of an organism to a
not enzymatic systems that are stimulated
given antibiotic thus depend upon the chemi-
by the addition of special nutrients. Earlier
cal composition and the metabolic mecha-
claims of the favorable effects of additions
nisms of the organisms concerned rather
of microbial cultures in enriching the de-
velopment of antibiotic-producing organisms than upon a dialectic concept of "struggle
have not been confirmed on further study. for existence." The possibility that the lo-
fresh sea water, they do not nniltipl}^ in the not (>xcluded, however, as shown by Brian
new enviromnent, but tend to die out rap- (1057).
i H A i» I i: i{ I
mycetes that was over recorded aiul to whicli ism. The use of the generic name Ardnomijcrs
both a generic and a specific name were was finally limited to a grouj) of anaerobic
gi\-en was described by Cohn in 1875 as
actinomycetes. This limitation was first
longer be considered as ^'alid. There was not He emphasized this as follows: "be-
for this.
cause the use of the generic term Actino-
even general agreement, subsecjuently,
whether the organism studied by Cohn was myces for them logically leads to giving the
pathogenic or nonpathogenic. In spite of all
name actinomycosis to those cases of sup-
these objections, the name Streptothrix con- purative processes due to infection with
tinued to be used for many years, until it was certain members of the group."
on the basis of lack of \'al id- In 1878, Rivolta suggested the name
finally discarded
accordance with the rules of botanical Discomyces for the causative agent of a
it}', in
nomenclature.
disease, known as l)otryomycosis, and now
recognized to be due to a true bacterium.
The second actinomycete was studied and
described by Harz in 1877 as Actinomyces
The organism was considered, however, to
at that time. It is now definitely established cause the name Actinomycc (without the
that the causative agent of actinomycosis is terminal "s") was gi\'en by Meyen in 1827
an anaerobe. This was not fully recognized to a tru(^ fungus, Actinomycc horkclii. How-
at first, and subsequent isolations of aerobi(t e\'er, according to the Intei-national Rules of
47
48 THE ACTIXOMYCETES, Vol. I
Nomenclature, two generic names, if differ- to the filamentous "higher bacteria," or the
ing even by one letter, are to be regarded as Chlamydobacteriaceae he remarked quite
;
distinct. It was finally decided that the correctly that, "On endeavoring to place the
generic name Nocardia should not be applied organism in its proper genus, we found our-
to the group of actinomycetes as a whole, as selves confronted by one of the most perplex-
suggested by Trevisan, but should be limited ing problems of botanical nomenclature,
to only one of the genera of the group, as which promises a rich harvest to those who
will be shown later. are fond of such study." Even prior to that,
Sauvageau and Radais, in 1892, placed the namely in 1898, Lachner-Sandoval pointed
actinomycetes among the true fungi, namely, out the marked morphological differences
the Hyphomycetes in the genus Oospora. between Oospora and the actinomycetes.
Only a year before, Thaxter (1891) described In 1897, Migula considered the name
the causative agent of potato scab, which is Cladothrix Cohn as a proper designation for
a true actinomycete, as Oospora scabies. the actinomycetes. However, the cladothrix
Thaxter himself, however, used this name group includes certain higher bacteria (C.
only provisionally, since he expressed doubt dichotoma) definitely distinct from the acti-
as to the correctness of this designation. nomycetes; it represents organisms consist-
Oospora, as described by Saccardo, is now ing of chains of cells surrounded by a sheath
recognized as standing for a true fungus, and showing false branching. Earlier, Ep-
(liissow, who analyzed this name in detail, pinger (1891) discovered an acid-fast actino-
looked upon the actinomycetes as belonging mycete, which he believed to exhibit false
1 ,
•iiMiera, Ixised upon the pathojj;enicity of the 1 'oksmatt ia, Tlurmomonospora, Thermopoly-
former and tlie formation of aerial mycelium sporn, wei'e suggested.
l)y tin- latter. Krainsky (1!)1 1) did not con-
sider this difference sufficient to justify the
Recognition of Species of Actinomycetes
creation of two separate genera; he con- l]\(ni greater problems in the classifica-
sider(>d the actinomycetes to be closely re- tion of actinomycetes appeared in the recog-
lated to the mycobacteria. Lehmann ami nition of specific names, for two important
Neumann (1912), however, suggested plac- and obvious reasons: 1. No genera can be
ing the actinomycetes, as an independent established without the proper recognition
group, between the hyphomycetes and the of species. 2. There are so many more species
schizomycetes. than genera. H. J. Conn said, in 1917, that
WoUenweber (1921) suggested division of "no species described in the past can be
the genus Actinomyces into two subgenera: regarded as characterized sufficiently for
J'ionnothrix, comprising the forms lacking recognition except those that are said to be
aerial mycelium and Aerothrix, forming a the cause of some definite disease." This
true aerial mycelium. The name Mijcococcus becomes particularly evident when one con-
was proposed by Bokor, in 1930, for an siders such forms as Actinomyces chromo-
aerial-mycelium-producing actinomycete genus Gasperini, one of the more readily
that was able to decompose cellulose. Neither recognized saprophytic forms. Its character-
the biochemical characteristics of the culture istic property is the production of a brown
nor its morphology justified the creation of pigment on gelatin or peptone media, a
this new genus. property ascribed by Beijerinck to the pro-
In 1936, Brumpt placed the actinomy- duction of a quinone. In 1914, Krainsky
cetes, under the name, Microsiphonales, reported four distinct organisms that agreed
among the Hyphomycetes. The forms para- fully with the original description of Gasp-
sitic to man were classified as one genus, erini. Conn mentioned at least 30 forms the
Actinomyces = Nocardia. published descriptions of which agreed with
In addition to the above names, \arious the .1. chromogenus. The conclusion was
others were suggested as generic designations reached that this name should not be recog-
for the actinomycetes as a whole or for some nized even for a group of organisms suffi-
of its constituent groups. Among them were ciently different from other groups.
Conidiomyces Perroncito (1875), Actino- Similar confusion was found to exist as
cladothrix Afanassiev (1889), Micromyces regards other "species" described earlier,
G ruber (1891), Actinobacillus Lignieres and such as Actinomyces alb us or A. odorifer.
Spitz (1904), Actinobacterinm Haas (190()), Descriptions of these organisms were based
Cohnistreptothrix Pinoy (1911), Actinococcus on the white color of the aerial mycelium or
Beijerinck (1914),Anaeromyces Castellani on the formation of an odor, both being
et al. (1921 ), Euactinomyces Langeron (1922), properties characteristic of many distinct
Brei'istreptothrix Lignieres (1924), Proactin- forms of actinomycetes. As a result, none of
omyces Jensen (1931), Asteroides Puntoni the earlier identifications can now be suffi
and Leonardi (1935), and a number of others, ciently recognized.
:
Slreptothrix
The nanu' Sin plolhri.r was proposed l)y
formation of aerial hyphae. Without endo- the lack of A-alidity for this name. Buchanan
spores, but by a multiple segmentation of a (1925) finally considered the name Strcpto-
52 THE ACTINOMYCETES, Vol. I
thrix as a generic designation among the bac- since all the others had been preempted. He
teria as invalid. classified the genus into seven species: A.
We hoped that Buchanan's final decision bovis, A. asteroides, A. israeli, A. nocardi, A.
would stand. Unfortunately, this was not madurae, A. caprae, A. vesicae.
the case. As recently as 193G, Woytek still As pointed out above, Wright proposed
considered Strcptothrix as the proper designa- limiting the name Actinomyces to the para-
tion for the anaerobic pathogens and still sitic forms which produce rays in tissues;
reserved the name Actinomyces for the he used the name Nocardia for the forms
aerobic saprophytes. Erikson (19-10) was producing aerial mycelium and spores. The
most emphatic in directing attention to this same concept was adopted by Jordan.
historical misinterpretation. Petruschky accepted the separation of the
actinomycetes into two groups, although he
Actinomyces preferred the name Strcptothrix to that of
The name Actinomijces is the most com- Nocardia for the second group. Pinoy also
mon one used to designate a group of actino- divided the actinomycetes into two groups:
mycetes. As pointed out above, it was first Nocardia comprising the aerobic, spore-
used by Harz for a genus of thread-forming producing forms, and Cohnistreptothrix
bacteria observed in the pus of cattle affected (rather than Actinomyces) for the anaerobic,
by "lumpy jaw." The pus consisted of nonsporulating forms.
granules made up of "slender filaments, ir- Dresel (1925) finally disposed completely
regularly branched, radiating from the of the confusion between (a) the anaerobic
center, and with the ends of the filaments in organism, as clearly elucidated by the work
the form of refractive swellings." The Greek of Wolff-Israel, causing actinomycosis and
word Actinoynyces means ray-fungus. Harz producing sulfur granules, for which the
never grew this organism in pure culture, generic name Actinomyces Harz must be
but he noted that it is the causati\'e agent of retained, and (b) the aerobic organisms,
actinomycotic infections in animals. comprising some that are widely distributed
Various subsequent investigators adopted in nature as saprophytes and others that are
this name for the actinomycetes as a whole. able to cause infections. He stated emphati-
Trevisan, however, recognized the relation- cally that if the name Strcptothrix cannot be
ship between organisms described as Strcp- retained for the second group, clearty a new
tothrix and and suggested
Actinomyces, name must be found. This lack of recogni-
discarding both these names in favor of tion of the distinction among the anaerobes
Nocardia. Among those who used the name and aerobes has been responsible for much
Actinomyces for actinomycetes as a whole, of the confusion in the designation and classi-
should be mentioned Gasperini (1895), fication of actinomycetes, beginning with
Lachner-Sandoval (1898), Levy (1899), Bostroem's isolation from an actinomycotic
Berestnew (1899), and later Lehmann and infection of a contaminating aerobe and
Neumann (in the editions of their book continuing through the work of numerous
subsequent to 1896), Orla-Jensen, Waksman subsequent investigators. These included
(in his early work and in the early editions of such eminent contributors to the subject as
the Bergey Manual), and Krassilnikov. Licske, who kept emi)hasizing the change of
Stokes (1904) examined the \^ari()us ge- the anaerobe into an aerobic form, and Kras-
neric names proposed for the pathogenic silnikov, who refused to conceive of purely
actinomycetes and came to the conclusion anaerobic forms, in spite of the impressive
that the name Actinom,yces should be used, and clear-cut evidence to that effect.
:
111 liiiic, h()\V('\('r, llic iiciicric ii.iiur Acli- 'J. Many act iiioniyceles rc|)ro(liice l»y
noini/cis cniiu' to Ix' recognized lur itie MiKiei'- fragmcnls or oidia that arc similar in size
oliir tiroiip III ;i('l iuoiiiN'cel es coneenied with and in shape to the rod sha|)cd and s|)lieri(^al
that they are closely related to the fila- points to the relationship of the actinomy-
mentous Almquist himself isolated
fungi. cetes to the bacteria I'ather than to the fungi.
in organisms, one as a
pvn-e cultnre three When the spores are liV)erated as a I'esult of
dust contaminant, another from water, and the break-up of the sporophores, the empty
a third from the body of a person who re- shells become A'isible; the cell wall is soluble
cently had died from cerebrospinal meningi- in 10 per cent KOH solution and in anti-
tis. All appeared to be forms that would now formin. When the cells of Streptomijces were
be considered as members of the genus treated with lysozyme, the cell walls were
Streptomyces, although the third form did found to be mucoid in nature and were
not produce any aerial mycelium. hydrolyzed to glucosamine.
Since then, consideral)le discussion has 7. Cells of actinomycetes do not contain
taken place concerning the relationship of chitin oi- cellulose*, characteristic of true
the actinomycetes to the bacteria, on the fungi.
one hand, and the fmigi, on the othcM'. The 8. Acthiomycetes, like true bacteria, are
information that has been gradually accunui- procaryotes, whereas fungi are eucaryotes.
lating tends to emphasize that actually the 9. The phenomena of lysis and phage
actinomj^cetes are more closely related to the sensitivity among the actinomycetes point
true bacteria. further to their relationship to the bacteria
Relationship to bacteria. The following i-ather than to the fungi.
facts may be presented to substantiate this 10. I'inally, the recent evidence concern-
relationship ing the sensitivity of the actinomycetes to
1 . The diameters of the hyphae and spores antibiotics ])laces them definitely with the
of actinomycetes (usually O.o-l.O m) :^1'p l)acteria and not with the true fungi.
similar to those of bacteria but not of fungi. These facts fully justify the conclusion
54 THE ACTIXOMYCETES, Vol. I
that the actiiiomycetes should be classified can, under certain conditions, give branching
with the bacteria, under the Schizomycetes. forms. Further, the cytological structure of
No wonder that Afanassiev suggested in the actinomycetes, characterized by the ab-
1889 that the filament of an actinomycete sence of well-defined morphological nuclei,
is a gigantically elongated bacterial cell. definitely places them with the bacteria. He
Among the other earlier investigators, the concluded that the Actinomycetales are
following considered actinomycetes as bac- closely related to the Mycobacteriales, as
teria: Cohn, Bostroem, Wolff and Israel, characterized by the evidence of bacillary
Berestnew, and Lieske. ^^arious subsequent
and filamentous forms taking the Gram
investigators emphasized the particular re-
stain, sometimes by acid resistance, and by
lationship of certain actinomycetes, notably
capability of branching.
the Nocardia group, to the mycobacteria.
Relationship to Jnngi. The relation of the
Grootten, for example, (19:U) analyzed the
actinomycetes to fungi, especially the Fungi
relationship between the actinomycetes and
Imperfect!, was based upon the following
the hyphomycetes, on the one hand, and the
similarities:
bacteria, on the other. He emphasized that
the fact that actinomycetes are often 1. The production and maimer of branch-
branched does not justify in itself their clas- ing of the aerial mycelium and the manner
sification with the fungi, since certain bac- of spore formation, especially in the genera
teria {M ycobacU-rium, Corynebactcrium, etc.) Strcptotnyces, M icromonospora, Walsmania.
N().Mi:\("i,A'i"ri{i; and (;i:\i:i{ ai, s^•s|'l:^ls oi' ci-assii'icatiox
1875) . Some of the more significant systems, H. W. Conn, in his Agricultural Bacteriol-
in which the actinomycetes were given im- ogy (1909), divided the higher bacteria into
portant consideration, may he summarized four genera: Cladothrix, Leptothrix, Strepto-
here. thrix, and Actinomyces. Orla-Jensen (1909)
Earlier Systems of Classijication created a family ^
sporos. Without uraiHiU's of t'roo siilt'ur and willioiil I '.)().')); 2. M firohdctiriacKU, witlidiil ;i iiiyce-
l)Mct(>rioi)uri)uriii. N'cvcr producing a psciido-
liinn.
plasmodiwm. Al\\a\s iKniinotilc.
order Actinoniycotalos coutaiiis
riic .\()c(tii/i(ic< (K were di\i(l('(l ;is lollows:
Ttic a single
tainilv , AcHnoiin/cctaceae. I ( Irow ;i('r(il»ic;illy, easy to cull i\;it(', ;ind
produce ;irt hrospores.
I''amil\ I. Aclinoiiii/ccldCKii
(lenus 1. Xorardid )e 'j'oni and Tre-
A. No ('\id('iit aerial tlireads or eoiiidia t'orined.
I
mycelium.
3. Micromonospora, with single spores
borne at the ends of branches.
Jensen (1931) modified the above system
by retaining the last genus unchanged, and
by designating the first genus as Actinomyces
and the second as Proactinomyces, as shown
Figure 25. S. rubrireticuli, a verticil-forming
streptomyces (Reproduced from: Shinobu, R.
here
Mem. Osaka Univ. No. 4B: 74, 1955). A. No spores are formed.
Family Proactinom ycetaceae
genera, Corynebaderium and Mycobacterium; I. No mycelium is formed.
Actinomycetaceae, with only one genus Acti- a. Acid-fast organisms.
nomyces. Kluyver and van Xiel suggested Genus Mycobacterium
the removal of the family M ycobacteriaceae b. Nonacid-fast organisms.
from the Actinomycetales altogether. Genus Corynebacterium
Langeron suggested the following system
II. Mycelium is formed.
of classification of actinomycetes:
Genus Proactin om yces
I. Euactinomyces, aerobes.
B. Spores are formed.
II. Cohnistreptothrix, anaerobes, grow
Family .4 ctinom ycetaceae
poorly on artificial media.
I. Spores in aerial mycelium.
Genus Actinomyces
II. Spores terminally on branches of
vegetative mycelium
Genus Micromonospora
Lignieres (1922) divided the actinomy-
cetes into three groups:
1. Actinomyces. Aerobes; mycelial hyphae
long, not Ijreaking up into rods.
2. Brevistreptothrix. Anaerobes; mycelial
hyphae short, l)reak up into long rods.
3. Actinobacillus. No mycelium. Cells rod-
shaped.
I'untoni and Leonardi (1935) divided the
actinomycetes into three genera:
1. Actinomyces Ilarz, a,er()l)ic, producing
Figure type verticil-forming strep-
26. Anitclla
tomyces (Rei)rodvu'ed from: Shinobu, R. Mem. aerial mycelium and arthroconidia.
Osaka Univ. No. 4B: 75, 1955). 2. Actinobacterium Haas (Cohnistrepto-
N()Mi:\('i.A'rrin: axd ci'A'krai, s^stiims oi' ci.AssiFirATiox 59
y P.
X
^
(hri.r Pinoy), anaerobic, without aerial or isolated rod-shaped elements; (3) wavy
hyphae. or straight filaments; (4) presence or absence
3. Asteroides, aerobic, without aerial of conidial spores; (5) aerobic or anaerobic
mycelium, fragmenting into bacillary frac- manner of growth; (6) saprophytic or "ani-
tions, partially acid-fast, little adherent mal-pathogenic" forms. No importance was
colonies. attached to club-formation, presence of
These three genera corresjjoud fairly radiate granules, acid-fast character, pres-
closely to the presently recognized Strepto- ence of "wavy" filaments or dissimilarities
myces, Actinomyces, and N^ocardi'a, respec- in metabolism. Puntoni (1940) suggested
tiveh'. division of the actinomycetes on the basis
In 1989, there were presented before the outlined by Puntoni and Leonard!. Erikson
Third International Congress for Micro- emphasized the justification of distinguish-
biology, a series of papers on the classifica- ing the two anaerobic forms, namely the
tion of actinomycetes that can be briefly "Israeli," or human type, and the "bovis,"
summarized here. or microaerophilic forms found in cattle.
Xaeslund (1940) emphasized llint ihc Krassilniko\- and Waksman and Umbreit
following characteristics should be con- presented their systems which are now dis-
sidered: (1) branching or nonbranching of cussed in detail.
the hyphae; (2) filamentous, mycelial forms Krassilniko\' at first accepted the classifi-
:
STRAIGHT FASCICLED
OPEN LOOPS
PRIMITIVE SPIRALS
HOOKS SPIRALS
mONOVERTICILLATE
NO SPIRALS
cation proposed for the order Actiiiomy- hadcriaceae, with two genera, Mycohade-
cetales by Buchanan. He divided it into two rinm and M ycococcus.
families: Adinomycetaceae, with four genera, In 1940, Waksnian and Unihreit, and later
Adinomijces, Proadinomyces, Mycobacte- Waksinan, proposed the following system of
rium, and M ycococcus; and Micromono- classification of the Actinomycetales:
sporaceae, with one genus Micromonospora. A. Mycelium ludiineiitary or al)sent : Family
Later, he suggested (1941, 1949) division of M ycobacteiiaceae Chester.
the actinomycetes into three faniiHes: Adi- I. Acid-fast organisms: M ycolxwleriiDii L & N
II. Nonacid-fast organisms: ('orynchartcriuni
nomycetaceae, with two genera, Actinomyces.
L & N
and Proadinomyces^; Micromonosporaceae, B. Mycelium produced
with one genus, Micromonospora; and Myco- I. Substrate mycelium divides by segmenta-
.
Vmmly I'rodclinonn/cctaccdc I, iV N
1. Aiiaorol)ic or microarropliilic. usually
parasitic, notiacid fast organisms:
( \)hn ii^tnptoth ri.r 1 'i tio\ .
species was described, but not named, by described as Streptothrix alba Rossi-Doria
Nocard. Trevisan regarded it as the type and later known as Actinomyces albus
species of the new genus. N . actinomyces Krainsky. This is one of the commonest and
Trevisan (Syn. Actinomijces hovis Harz) best known species of the genus, and though
was given as the second species. This was it may now be recognized as a group and be
followed by A^. foersteri (Cohn) Trevisan subdivided into several species, it was con-
(Syn. Streptothrix foersteri Cohn). In con- sidered, for the time being, as definite a
sidering these facts. Breed and Conn con- species as any others. It produces colorless
cluded that "There appears to be no justifi- vegetative growth, with white aerial myce-
cation for the use of the term Nocardia lium, and forms ovoidal spores in coiled
Trevisan for the entire group of organisms chains on lateral branches of the aerial
included in the Actinomycetaceae. It may, hyphae. It is proteolytic, licjuefying gelatin
however, he properly used for a subdivision and peptonizing milk with the production
of the genus Actinomijces, provided, how- of an alkaline reaction in the latter. It does
ever, N . farcinica is retained in the genus not produce any soluble pigment either on
Nocardia and is established as the type of organic or synthetic media, and forms a
the genus." characteristic earthy or musty odor, as de-
Waksman and Henrici further considered scribed in detail later.
the need of a valid name for the aerobic The generic name Micromonospora 0rskov
sporulating species. They concluded that was applied to those forms which produce
the only solution for this problem was to single spores on lateral liranches. Although
coin a new generic name, and proposed it was recognized that Tsiklinsky had pre-
Streptomyces, a word derived from the first \'i()usly applied the name Thermoactino-
two names given to the actinomycetes as a myces to similar cultures, her description of
whole (Strepto-thrix and Actino-rtiyces). the genus, in which she also included thermo-
Since the Botanical Code recommended that species with catenulate spores, was
])hili('
family names be derived from generic names, based on temperature relations rather than
the new family name Streptomycctaceac was on morphology.
proposed for the spore-forming actinomy- On the l)asis of these and other considera-
cetes. tions, Waksman and Henrici proposed a
Streptoynycetaeeae included actinomycetes system and classification of
of nomenclatvu'c
with branched, slender substrate mycelium. the Actinomycetales which has been used as
NOMKNTLATrHK AND (IKNKHAL SVSTi;.MS OK CLASSIFICATION 63
:
name Microhispora, with the type species tions; they may, therefore, be designated as
M. rosea. These investigators proposed divi- facultative aerobes. The preference for
sion of the family Streptomycetaceae as fol- anaerobic conditions varies with the species,
lows : especially in the case of freshly isolated cul-
I. Spores formed in chains from aerial tures. When originally isolated under exclu-
hyphae Streptomyces sion of oxygen, cultures grow well later also
II. Spores formed in pairs on aerial in presence of oxygen, but only at optimum
hyphae Microhispora temperature. Production of aerial mycelium
III. Spores formed singly on sporophores. is closely bound to optimum temperature.
1. Mesophilic Micromonospora For example, Thermopolyspora bispora grows
2. Thermophilic. .Thermoactinomyces and produces aerial mycelium at 50 to 60°C
IV. Sclerotic granules produced (conidial under aerobic and anaerobic conditions;
forms unknown) Chainia below that temperature, growth takes place
According to Henssen (1957), the majority only under anaerobic conditions; at 70°C,
of thermophilic actinomycetes thrive better growth is good but aerial mycelium and
under anaerobic than under aerobic condi- spores are no longer produced.
Henssen proposed division of the family
Streptomycetaceae into the following genera,
taking into consideration the thermophilic
forms
A. Substrate mycelium nonseptate, spores pro-
duced on aerial or substrate mycelium.
I. Spores produced on substrate mycelium,
mesophilic forms
Micromonospora 0rskov
II. Spores produced on aerial mycelium.
1. Aerial hyphae develop only as side or
the various efforts to subdivide the genera cils into a separate group was suggested by
into species, were justified. Others were not, the writer 40 years ago, and was discarded
especially when new genera were added with as hardly advisable. More recently Pridham
insufficient j ustification. et at. (1957) created four sections in the
Gulliermond and Mangenot (1946) sug- genus Streptomyces, under the names Mono-
gested division of the Actinomycetales into verticillus and Biverticillus, each comprising
two groups: (a) acid-resistant forms made either straight or spiral-shaped sporophores.
up of two genera, Actinomyces and Myco- Other investigators attempted to bring these
bacterium; (b) nonacid-resistant forms with forms together into a distinct genus but were
two genera, Corynebacterium and PJeifferella. dissuaded by the writer. The fact that the
Bisset and Moore suggested the creation composition of the medium greatly influences
of two separate orders, Streptomycetales this manner of sporulation was among the
and Actinomycetales, in order to distinguish, many reasons advanced against the recogni-
by the type of branching, the aerobic sporu- tion of a separate genus.
lating genus Streptomyces from the parasitic Evidence is gradually accumulating to
anaeroljicgenus Actinomyces. The second justify, substantiate, or even enlarge upon
order was subdivided, on the basis of type the previously established systems of genetic
and arrangement of the component cells, and specific classification. Such evidence is
into two families: 1. Actinomycetaccae, in- based upon the following four criteria: (a)
cluding the anaerobic Actinomyces, and a cell morphology, (b) cell genetics, (c) cell
new genus, Jensenia, comprising the "soil composition, and (d) biochemical activities
diphtheroids"; M ycobacteriaceae includhig
2. , of the organisms.
the genera M ycobacterium Corynebacterium,
, Pridham and Gottlieb (1948) suggested
and Nocardia. division of the actinomycetes on the basis
Thirumalachar (1955) proposed creation of their carbon utilization. All the strepto-
of a new genus Chainia, with a type species, mycetes tested were able to utilize D-glucose,
C. antibiotica, on the basis of formation of D-mannose, starch, dextrin, and glycerol,
spherical sclerotic granules, in large aggre- but not erythritol, phenol, the cresols, Na-
gate masses, from the mycelium. This formate, Na-oxalate, and Na-tartrate. The
phenomenon is frecjuently observed among conclusion was reached that L-rhamnose,
Streptomyces species, and hardly warrants raffinose, L-xylose, D-fructose, L-arabinose,
creation of a separate genus. Gattani (1957), and D-mannitol are best for characterizing
for example, reported the formation of Streptomyces species, as brought out in Chap-
sclerotic granules by various strains of S. ter 2. This property was utilized by Kuro-
grisevs when grown on certain media. No sawa, Gordon and Smith, Zahner and Ett-
aerial mycelium is produced under such con- linger, and others. In spite of the valuable
ditions. A hypha thickens; other hyphae sur- information thus obtained, however, the
round it, resulting in brownish green masses, conclusion may be reached that in no case
eventually becoming black. Some of the can the utilization of carbon sources serve
granules coalesce in pairs or in threes, giving alone as a major criterion for characterizing
rise to a larger granule up to 75 /x in diameter. actinomycetes. The information is only sup-
Baldacci recently proposed (1958) that all plementary in nature.
the verticil-forming organisms be united Gordon and Mihm dixided a total of 676
into one genus under the name Strepto- cultures received imder the generic names
rerticillium. The idea of separating all the Streptomyces, Nocardia, and M ycobacterium
forms producing primary or secondary verti- into groups on the basis of sporulation, as
\()Mi;\(M.ATri{i'. AM) (;i;\i;i{ \i, s^s'^l:^Is of ci-assificatiox ()7
Tami.k 10
Distrihiition of cnloniiil chararlcrs of strai»!< inilialh/ dcHiijualcd aft Slrcptoniyccs, XocMrdia,
or Mycohiicteriuin ((Sonloii jiiul Milim)
68 THE ACTINOMYCETES, Vol. I
Table 12
Slide
Gross culture Physiologic and biochemical properties
culture
Starch
Color of agar
Starch
substrate
agar
growth Surface
growth
Strain
o-o
'ri\i> strains of Nocardia studied were scp- antagonism exp(>riments, the heterogeneity
rated, on the basis of th(> cultuiai data pre- of many species which heretofore were con-
sented, into "tnie" Xocdnhd and "strepto- sidered as homogeneous.
myees-like" Xocardia. Tlu^ foiiuer liave l)ocn Wak.sman and Lechevalier found that, on
fiirtlier tentatively diviiled into the follow- the basis of sensitivity to isoniazide, the
ing groups: (a) X. astcroidcs group (all strains phylogenic j)()sition of the various Actino-
of N. astcroidcs plus N. blackweltii, N. cunic- mycetales was indicated as follows: The
uli, X. caprac, N'. sijlvodorifcra, N. pohj- pathogenic forms of the genus Mycobac-
chromogcncs, X. minima, and iV. paraffinac); terium are susceptible to less than 1 mg of
(b) .V. corallina {X globcnda, X. crijthrop-
. isoniazide per milliliter, the avian form l)eing
olis, and X. convohita); and (c) .V. opaca least susceptible; the saprophytic forms were
group (.V. opaca and .V. rubra) (Table 12). less susceptible, some being resistant to 100
\'arioiis other criteria ha\'e been suggested and even 1000 mg per milliliter, most of them
as aids in the classification of the actino- being susceptible to than 5 and 10 mg
less
in the struggle for life between rival organ- tion;some were more susceptible (10 mg per
isms. They manifest their activity toward and others were resistant (more
milliliter),
competing organisms only, but never against than 1000 mg per milliliter). The strepto-
cultures of the same species. He believed that mycetes were most resistant, requiring as a
strains of one species produce antibiotics rule more than 1000 mg of isoniazide per
inhibiting the growth of all the strains of milliliter for inhibition. The various genera
rival species. This antagonism may be uni- of the Actinomycetales could thus be listed
lateral or bilaterial. He suggested that the in the following order of increasing resistance
specificity of antibiotics produced by various to isoniazide: pathogenic mycobacteria —
species be used in taxonomy for the differ- saprophytic mycobacteria —> Micromono-
entiation of these species. He reported suc- spora -^ Nocardia —* Strcptoinyces.
cess in demonstrating, by means of cross Among the new approaches to the tax-
Table 13
Classification of the actinoinyccles based on cell wall composition (Commins and Harri.s)
70 THE ACTINOMYCETES, Vol. I
onomy of actinomycetes, that based upon 1. Streptothrix alba. Colonies opaque to white,
(II A I' r i: H
71
72 THE ACTINOMYCETES, Vol. I
filledwith protoplasm; toward the center of the larly in some forms, both in the sterile axial
thallus, the vacuoles increase in size, and may be hyphae, where they may contain either a medium
associated with the presence of metachromatic septum or a number of peripheral metachromatic
granules. The vegetative hj^phae of the mycelium granules, and in the sporogenous hyphae, where
are far larger than those of ordinary bacteria of they are associated with the regularly spaced
the acid-fast group; the hyphae also lack the uni- septa.
formit}' in diameter generally characteristic of the 8. The spores germinate readily in proper
true bacteria. media, producing from one to four germ tubes,
2. The aerial mycelium produced on suitable the approximate number being more or less char-
media usually occurs as a mat of discrete fructi- acteristic of the species.
fications. Each of these represents a well -char-
acterized sporogenous apparatus, consisting of a 0rskov divided the actinomycetes into
sterile axial filament bearing branches in an open three morphological groups:
racemose, or dense capitate, arrangement. The
I. Those that produce an undivided substrate
primary branches may function directly as sporo-
genous hyphae, or they may proliferate branches mycelium and an aerial mycelium which breaks
of the second and of higher order; sporogenesis, in up into bodies that possess the quality of spores.
the latter case, is confined to the terminal ele- II. Those that produce an initially undivided
ments, the hyphal portions below points of attach- substrate mycelium. After having reached a cer-
ment of branches remaining sterile. tain point, it divides by septa into rod-shaped
3. Two tendencies in the development of fructi- elements; these continue to multiply with a char-
fications were recognized: (a) one leading to an acteristic"angular" growth; aerial mycelium may
erect dendroidal type, in which successively pro- or maynot be formed; in the former case its ele-
liferated fertile elements undergo processes of ments resemble those of the vegetative mycelium.
sporogenesis in continuous sequence; (b) the other III. Those that produce a substrate mycelium
leading to a prostrate, racemose type, in which resembling that of group I, but devoid of aerial
sporogenesis is delayed in the older branches until mycelium and producing spores borne singly at
the younger branches have also attained their the distal end of short mycelial branches.
final extension. The majority of species show these
tendencies combined in different ways. According to 0rskov the angular growth
4. The sporogenous hyphae of many species into which the members of group II pass
are coiled in peculiar spirals. These exhibit pro- after the formation of an initial mycelium is
nounced specific characteristics in the number,
similar to the process of cell division in the
diameter, and obliquity of their turns, and es-
mycobacteria and corynebacteria. This simi-
pecially in the direction of rotation, which may be
dextrorse or sinistrorse. This phenomenon was larity makes it impossible to draw a sharp
later found not to be constant, however, but to line of demarcation between true bacteria
vary with the composition of the medium. and actinomycetes.
5. Sporogenesis begins at the tips of the fertile
Jensen emphasized that the nocardias, or
branches and proceeds basipetally. In some species
proactinomycetes, as he designated them,
the process involves the insertion of septa, which
are, in certain cases, relatively very massive, and represent a heterogeneous collection of types,
in others, so thin as to be barely discernible. standing between the mycobacteria and
0. Ciranules which possess the staining prop- corynebacteria, on the one hand, and the
erties and uniformity of size characteristic of
streptomyces, on the other.
nuclei are readily differentiated in the spores of
The morphological structure of actino-
many sj)ecies; they generally occur singlj', but in
the larger spores of a few forms, two are often mycetes depends largely upon (a) the nature
found occupying diagonally opposite positions. of the organism, (b) the composition of the
As in the vegetative metachromatic
thallus, medium, (c) conditions of growth, especially
granules occur in the aerial mycelium, being verj^ and (d) presence of growth-stimu-
aeration,
rarely found in spores or sporogeneous hyphae but
lating and growth-inhibiting factors. The
becoming very abundant in degenerate sterile
thallus consists of a homogeneous cytoplasm
7. Peculiar spherical structures appear regu- which, as it grows older, becomes vacuolated
M()l{lMl()l,()(;^ . ('^T()l,()(;^. and i,ifI'; ("vcm.ms 73
\
Figure 34. S. griseus: j)rimary niNcclium at 40 hours from suhnicrficd culture, showing germinating
initial cell. Visual light, X 2000 (Reproduced from: Dickenson, P. H. aiuLMacdonald, K. I). J. (ien. .Micro-
spore formation takes place within the hy- strated by Flaig ei al. (1952), Enghusen
phal wall. The hyphal contents divide si- (1955), Baldacci and Grein, and others, has
multaneously into fragments, separated by also been reported by Vernon for S.flaveolus,
less dense oartitions, having the appearance for example.
of septa. The spores remain in chains, held Further information on the structure of
together by a sheath-like hyphal wall, which the mycelium and spores of various actino-
undergoes change as the spores mature. In mycetes is found in the work of Elisei (1944),
some cultures, the sheath persists, the spores Carvajal (1946-1947), Scotti and Gocchi,
being liberated by means of a longitudinal Webley (1955), and many others.
split; the old wall remains as a ribbon-like
sheath, with cross markings indicating the Sexuality among Actinomycetes
position of the spores; in other cultures, the The fusion of mycelial threads among ac-
sheath disintegrates to small fragments. The tinomycetes has been observed first by
surface structure appearance of the spores Lieske, then by Korber (1929), and others.
varies for the different organisms and is
According to Krassilnikov (1938), this can
believed by some to be a species characteriza- take place either as the confluence of germi-
tion (Ettlinger et al., 1958). The formation of nating spores, through the germination
spines on the surface of the spores, demon- tubes, which give rise to a single hypha de-
veloping into a mycelium, or as the anasto-
mosis of two hyphal filaments. Krassilnikov
suggested that this actually is or resembles
the sexual process comparable to that which
takes place in many yeasts. He further em-
phasized the great biological significance of
this phenomenon, which possibly explains
the variability of the actinomycetes.
According to Klieneberger- Nobel the
morphological changes in the growth of a
streptomyces on the surface of a medium
take place as follows The spores germinate,
:
FiGiRE 36. Diagrammatic life The primary and secondary mycelia are
cycle of a streptomyce.s.
separated for convenience of illustration though usually the latter originates within the former. At-
tention is called to the gradual shrinkage of endofragments of the primary mycelium to produce
iso-
gametes; the secondary mycelium may he hisporulative (Reproduced from: Roach, A. W. and Silvey,
75
76 THE ACTINOMYCETES, Vol. I
tial cells" are artifacts; she believed that phae and the reproductive spores are pro-
aerial hyphae may arise by budding from duced in the aerial mycelium. Some actino-
any substrate (vegetative) hyphae. Wilkin mycetes form only the substrate mycelium,
and Rhodes observed that the nature of the whereas others produce both types. Some
medium influences the morphological forms aerial mycelium-forming cultures may lose
produced by a Streptomyces: on synthetic this property, and may thus be distinguished
media, the cycle proposed by Klieneberger- from nocardias only by certain physiological
Nobel was confirmed on complex media, the
; properties, as pointed out elsewhere. Some
formation of "initial cells" and "secondary nocardias, on the other hand, also produce a
mycelium" was suppressed, however. The typical aerial mycelium, as shown by Gordon
"primary mycelium" pi'oliferates on com- and Mihm (1958).
plex media to give "chlamydospores," which, According to Henrici (1930), surface colo-
on germination, give rise to typical "pri- nies produced by various actinomycetes are
mary mycelium." Further information on of two general types: (a) One type is char-
the life cycles of actinomycetes is given by form a highly
acteristic of those strains that
Wilkin and Rhodes, and Roach and Silvey. developed, extensi\'ely branching mycelium,
Dickenson and Macdonald made electron notably members of the genus Streptomyces.
microscopic observations on submerged Colonies of this type are very firm, almost
cultures of two species of Streptomyces. The cartilaginous in consistency, and adhere to
evidence obtained tended to confirm the the solid substrate, because the mycelium
theory of Klieneberger-Nobel, that at an grows into that substrate; when touched
early stage in the life history of the organ- with a wire loop, the colony does not break
ism concerned, fusion occurs between por- but separates from the substrate as a unit.
tions of the same or different hyphae, cul- In cross section, such colonies usually have
minating in the formation of "initial cells." a slightly conical form and show marked
Further studies on problems of recombi- radial foldings. At first their surface may be
nation of nuclear material and on the life glossy or matted, but, if aerial spores are
cycles of actinomycetes in general, are dis- de\'eloped, the surface becomes covered with
cussed in Chapter G. chalk-like powder which, as the colony grows
older, may acquire various shades of color.
Colony Formation The powdery spores frequently appear in
The grovvth of an actinomycete on a solid concentric rings, (b) The second type of
or in a liquid medium results in the forma- colony is characteristic of the strains that do
tion of a mass of unicellular mycelium us- not form an extensive mycelium, notably
ually designated as a "colony." This is not members of the genus Nocardia. Their thal-
a colony in a true sense, since it is not an lus has a tendency to break up into hyphae
accumulation of many cells, but rather a of variable length, and in certain strains most
mass of branching hlaments which originated of the growth may consist of short filaments,
from a spore or from a bit of mycelium. The resembling in appearance pleomorphic bac-
two types of mycelium making up a colony terial strains. Colonies of this type are less
of a streptomyces often show fundamental tenacious than those of the first; they often
differences in appearance, composition, and have a mealy consistency and tend to crum-
biological activities. The substrate or \'eg;r- ble wluni touched with a wire loop.
tativcmycelium grows into the medium, Actinomycete colonies are usually round
whereas the aerial mycelium grows on the and smooth, or much folded and lichenoid in
surface; the well-(le\'eIopc(l spomliiting hy- appeai'ance. When examined under the mi-
M()|;i'ii()1,();;n , ('^'l'()l,(»(;^ , and i.iki-: ('^('IJ•:s 77
FlGiRE 37. Sporuliition of .S. r///.s('//.s (Rpprodufed from: Haldacci, K. and (Irciii, A. (Jiorn. Micro-
biol. 1: 34. 1955).
crosc'ope, the edge of the colony shows a pointed out that a similar difference exists in
characteristic picture of radiating hyphae. cultures of the genus Actinomyces, where
Wlien grown in liiiuid niecUa in a stationary most strains show a granular growth V)ut
condition, the colonies may be formed in- some gi\'e a clcjudy growth in broth (Erik-
dividually on the bottom of the container, son, 1940; Holm, 1948). The colonies of the
or they may adhere to the surface of the wall, bovine strains of this anaerobic genus Actino-
or they may form a ring of growth or a pelli- mijcc's are smooth and soft in consistency and
cle on the surface. The colonies may also do not adhere to the medium; the mycelium
grow in the form of flakes, but the medium undergoes fragmentation very rapidly, giv-
is never made turbid, as in the case of l)ac- ing no extensive ramification; .such strains
tei'ial growth, unless the colonies undergo show occasional turl)idity in the medium.
lysis through the action enzymes
of or In contrast, hun;an strains give no stable
phages. When grown in a submerged or in variants and produce no turbidity (Erik.son).
a .shaken condition, actinomycetes produce Various attempts have been made to
small, bead-like masses of growth, some of divide the actinomycetes into groups on the
which may l)e granular in natui'e. basis of the size of the colonies (Krainsky)
According to Jen.sen, strains of Xoranlia or the length of hyphae (Lieske). These
with more persistent mycelium produce firm propei'ties are not recognized now as of pri-
agar colonies and a discretely graiuilar mary significance in classification, since the
growth in licjuid media, which remain clear; nature of the organisms and the conditions
those with small or rapidly dixiding myce- of growth are of prime importance.
lium show a soft growth in solid media and a
growth Staining Properties
diffuse, bacterium-like in liquid
media. Jensen took exception to Lieske's The mycelium of the actinomycetes can
(1921) .statement that "every form of tur- be dried on and stained with ordinary
slides
M(»KlM^()l,()(;^ .
(•^'|()|,()(;^ . and i.ii'h: ('^("L^;s 79
N—
c \
^-^/
HQ
10 zo 20
/
Figure 39. Mycelium and sporulation of a mifromonospora (RoprodiiccMl from: Jcnspii, H. L. Proc.
Linnean Soc. N.S.W. 55: 249, 1930).
celiuin in a nomlried coiidit ioii can also Ix' has been reported. The acid-fast properties
stained with dyes that readily enter the li\- of actinomycetes, notably among the no-
ing cell. There is little difference in the stain- cardias, have aroused considerable attention.
ing properties of the \-ai-ious types of niy- Numerous acid-fast strains have been de-
cehum and the aerial spoi-es. scribed. The microaerophilic and pathogenic
I'ractically all actinomycetes are gram- species of Actinomyces seem devoid of all
positive. Occasionally, a gram-negative form acid-fastness; so are many species of No-
-
cardia. Others are acid-fast to a variable sul^stance was found in the form of grains,
degree that depends both on the oi'ganism which, in Lieske's opinion, pointed to the
and on the cultural conditions. close relationship of these organisms to the
According to Jensen, acid-fastness in bacteria.
pathogenic forms is often stronger in vivo The occurrence of fatty particles in the
than in vitro. Man}^ species can probably cells of actinomycetes was observed by
best be described as potentially acid-fast, \arious investigators. Glycogen and chitin
because this property may be apparent for could not be foimd.
only a very brief period in the organism's
life history and can be induced by special Substrate Mycelium
media, like milk (Jensen, 1981-1934; Um- Actinomycetes produce a substrate or, as
breit, 1939) or synthetic media containing
it is mycelium
often designated, vegetative
paraffin or high concentrations of glycerol
that usually varies in size, shape, and thick-
(Eriekson, 1949).
ness. The color of the substrate growth
Acid-fastness is not a permanent property
ranges from whitish or cream to brownish,
of an organism. On continued cultivation on
yellow, red, pink, orange, green, or black.
ordinary media, the acid-fast characteristic
Water-soluble and water-insoluble pigments
may be on the other hand, the property
lost;
may be produced, depending on the organ-
may be strengthened by growing the organ-
ism and the composition of the medium.
ism in media containing oil or fat or on
Some of the pigments, especially the dark or
animal passage. Acid-fastness cannot be con- chromogenic pigments, are formed upon
sidered as a characteristic for species difTer-
complex organic media and are often a result
entiation.
of the action of certain enzymes of the tyrosin-
Some of the observations of stained prep-
ase type upon proteins and their deriva-
arations of actinomycetes have to do with
tives. Other pigments are synthetic in nature
the presence in the thin cells of a nucleus and
and are formed on simple media.
other particulate constituents. Crystal violet
The spores of actinomycetes germinate in
and thionine SO 2 as well as crystal violet
the medium with the formation of one or
,
CI lib Formation
An iiUeresting morphological i)henonie- Fkure 41. Sclcrotia t'onnation in a .stropto-
nou among certain actinomycetes, deter- inycos (Reproduced from: Tliiruinaiachar, M. ,1.
growth. The nature of the organisms, com- in length and in structure. The aerial myce-
position of medium, and conditions of incu- lium may cover the whole colony, in the
l:)ation are of great importance in this con- form of a cottony mass or as a powdery,
nection. Certain actinomycetes have the chalk-like surface, or as an almost granular
capacity to form, on agar media, colonies layer.
that show concentric rings of sporulating and The sporophores of Streptomijces differ
spore-free zones in their aerial mycelium. greatly in their structure. Some are straight,
One can frequently observe similar zonation long or short. The short hyphae correspond
in the growth of nonsporulating colonies, the to a powdery surface and the long hyphae
zones being thinner or thicker in the sub- to a cottony surface. Some of the sporo-
strate growth of the organisms. Various phores are curved, with various degrees of
theories have been proposed to explain this curvature, ranging from very slight at the
phenomenon, which can be observed also in tip of the sporophore, through "open cork-
the growth of certain bacteria and fungi. screw" shaped bodies, finally to compacted
Among these theories, the following deserve spirals, giving a "fist-like" appearance. The
consideration: (a) that it is due to diffusion, sporophores are arranged individually, mon-
by the growing cultures, of metabolites, opodially, at varying distances from one
which stimulate or inhibit the growth of the another, or are close enough together to
zone next to that producing the metabolite; give a broom-shaped appearance.
FiGURK 42. Types of streptomyces spores (H(>pro(luce(l from: Flai^, W. (I id. Zeiitr. li;ikt(Miol.
Parisitenk. Abt. II. 108: 383, 1955).
.M(tIn'll()l.()(;^, ('VT()l,()(;^•. and i.ii'I': ('^('I-!';s 83
The sjiiral-shnpod sponilatinj; liypliac aic cur\('d throughout or onl>' at the end. The
characteristic in their sti-uclurc and lcii<ith, number of turns wirics in accordance with
the iiiiinlx'f (»!' turns Ixmiiu; I to •_'(), usually the length ol the spiral, as shown al)o\('. Not
.")
to 10, the cui\atui"('s IxMiiji; clockwise all t he aerial hyphae gi\'e ri.se to spores, some
tilext lorse) oi' countorcl()ck\vis(> (siiiisli-orse). of the hyi)liae remaining sterile.
Kutzuei- (>\ainino(l 'AS'2 strains of Slnpli)- .Vnothcr interesting phenomenon obserxcd
Of these, 20o fornied no spirals,
miices. 1
.").';
in the aeiial mycelium of some species of
produced sinistrorse and 2() dexti'orse spirals, Slrcploinucc.s is verticil formation, the sporu-
similar to the prexious ohserxations of lating hyphae lieing arranged in groups of
Dreclisler anil Stapp (l!)")^). .') to 10, eiiuidistant from one another.
The sporulating liyphae consist of sterile Waksman (11)19) first reported this for S.
axial filaments bearing sporulating branches reticuli and N. the second
reticuloruber ,
produced; the sporulating hyphae may be ious strains excreted diffusilile substances
84 THE ACTINOMYCETES, Vol. I
that caused other strains to form aerial They were first described by Lieske. The
m^'cehum. aerial spores of such cultures are no different
from those of other cultures. Krassilnikov
Secondanj Aerial JMijcelium reported the formation of coremia
(1938)
Cultures with a normally gray aerial my- both in the substrate and in the aerial
celium may
be observed to form in spots a mycelium. These coremia are cylindrical or
white or yellowish mycelium. When such cone-shaped, 1 to 2.5 mm in length, the cen-
mycelium is transferred to fresh agar, cul- tral portion being made up of vegetative
tures are obtained that differ from the hyphae and the surface of aerial hyphae.
mother culture only in the color of the aerial
mycelium but in no other property. Oc- Spore Formation
casionally, a "secondary aerial mycelium,"
As pointed out previously, Lachner-San-
cottony in nature and white in color, is doval was the first to describe, in 1898, the
produced from a powdery gray, rose, or blue mechanism of sporulation among the actino-
aerial mycelium. This is usually sterile in
mycetes. The "fragmentation" spores were
nature, as shown, among others, by Jensen
considered to be analogous to the spores pro-
(1931) and Kutzner. Cultures obtained from duced by true fungi. They are formed by the
the secondary aerial mycelium (strains) breaking up of the protoplasm within the
are distinguished from the original cultures wall into particles or fragments, more
cell or
(strains) by the color, sterility, and mor-
less uniform in size. These are later liberated
phology of the aerial hyphae. by the splitting of the cell wall. During the
Coremia contraction of the fragments, empty and
clearer partitions are formed between them,
The sporulating hyphae of certain species
which have been occasionallytaken for
of Streptomijces may be grouped together
cross walls. The spore-bearing threads thus
into clumps reminiscent of the typical core-
assume the appearance of chains of cocci,
mia of fungi. They are formed only at cer-
the spores falling apart readily. This manner
tain spots on the surface of the colonies.
of sporulation begins at the top of the aerial
spore-bearing hyphae antl proceeds toward
the base; it is characteristic of the genus
Streptomyces. Recently, Cordon ;ind Mihm
(1958) have shown that certain strains of
Nocardia {N. asteroidea) ha^'e a similar mode
of sporulation in the aerial mycelium.
In the "segmentation" spores, the sporu-
lating hyphae break up by means of cross
walls. At first the hyphae are imicellular. At
a certain stage of growth, cross walls are
produced and the hyphae break up into
small segments. These are cylindrical in
shape and uniform in size, usually 1 to 2.5
by 0.7 to 0.8 ^l. Neukirch (1902) designated
the segmentation spores also as "fragmenta-
M(>Hi'ii()i,()( ;^ .
('^ 'l'()l,()(;^, and i,ii-i: ('^('ij:s 85
rise to other branches, thus forming struc- spores of Streptomyces has been made by
tures similar to bunches of grapes. Each Jensen (1931) and more recentlj^ by Flaig
branch bears at the end a single spore, pro- et al. (1952, 1954). The latter suggested a
duced by the splitting off of the tip of the system of classification of this group of or-
hypha. Sporulation occurs most abundantly ganisms on the basis of spore structure:
on synthetic media. Recent studies on spor- I. Spores long, oval-shaped
Table 14
Distribution of different spore types among the various groups of streptomyces (Kutzner)
Group No.
MOKI'IIOI.OCN. ( ^TOLOC^'. AM) I,ll'i: ('V("M:s 87
A stroptomycos may lose, hy luiiiarKni or formations in tissues of the host, which at-
l)y natural variation, the properly of toim- tracted so much attention among earlier
inji; aerial niyccliiini; it may thus apjx'ai' to workers and which were also found (Wright
li'row as a typical nocardia. This was found Naeshnid) in sennn or ascitic fluid media,
1o hold true, foi' example, of the strepto- are now recognized to be mechanisms of
luyciu-produciuji strain of N. (irisciis. When I'esistance of the host against invasion.
this occurs, it is accompanied by a change According to Erikson (1953), the com-
in the pliysiology of the organism, hut not bined demands of anaerobiosis, para.sitism,
in the morphologictd properties of the \-ege- and complex luitritive re(|uirements ap-
tative growth. Freciuently such a cultui'e parently leave little scope for mycelial varia-
may revert to its original form and i)r()duce tion among the members of the genus Ac-
typical a(M-ial mycelium. linomt/ces. These organisms exhibit a pattern
To retain the property of proper sporula- of (hn-elopnuMit similar to that of the no-
tion, cultures are usually maintained in soil. cardias, and the chief type of variation re-
A good soil with a fair supply of organic ported is that of "smooth" and "rough"
matter, neutral or slightly alkaline in reac- colonies. The former is associated with
tion, with a moisture content of about GO readily fragmented growths producing tur-
p(>r cent of the water-holding capacity so as bidity in li(iuid cultures; the latter with co-
to favor proper aeration is inoculated with a herent, filamentous, well-branched growths
spore suspension or with substrate mycelium. leaving the liciuid substrate clear in the
The flask is incubated at optimum tempera- manner characteristic of actinomycetes in
ture vmtil good growth is obtained. The flask general. The changes described
are from the
is then thoroughly shaken and further incu- typical, rough, breadcrumb colony to the
bated. After growth has reached a maxi- soft smooth one, not the reverse (Triuss and
nuun, the soil is allowed to desiccate. When Politowa, H«l; Lentze, 1938; Ludwig and
the culture is then transferred from the soil Sullivan, 1952).
to a synthetic agar medium, good sporula- According to Morris, .4. bovis passes
tion is obtained. Excess organic nitrogenous through a complete life cycle, consisting of
nutrients, especially phospholipids, favor two clearly defined generations. The germi-
substrate growth at the expense of the aerial nation of the spores takes place bj- budding.
mycelium, as shown by Erikson (1947). The mycelium, if formed, is less stable than
in the case of streptomyces. Branching is
Morphological Characteristics of Some impermanent in the haploid phase, but is
of the Important Genera of Actino- permanent in the diploid phase. The mech-
mycetes anism for the formation of the initial cell in
Actinumijces the genus Aciinomijccs is less specialized
ing vegetative mycelium. Xonsporing aerial cells. The diploid generation produces a
hyphae are occasionally formed in the firm- haploid spore by reduction division. The
textured .4. israeli. A. hovis, however, pro- club shape of the newly germinated diploid
duces soft, smooth colonies, without aerial form and the altered shape of the haploid
mycelium. The vegetative mycelium divides phase cells represent the pleomorphic forms
into diphtheroid rods (Erikson, 104!)). Club described by various investigators in cer-
;
tain specialized media, such as those con- actinomycetes in a single colony may be so
taining a high concentration of serum. thin as to beyond the limits of the visible
lie
Various reports concerning filterable forms light microscope and may thus yield filter-
of actinomycetes are found in the literature. able forms. More recent studies on the sporu-
It is sufficient to cite a recent contribution lation process of ^4 dmom//ces have been made
by ]\Ional. Cultures of A. bovis were filtered by Bisset, as shown later (Vol. II, Chapter
through a collodion membrane. When the 24).
filtrate was supplemented with various nu-
Nocardia
trients, growth took place. This consisted of
filamentous forms and typical arthrospores According to Jensen, nocardias are char-
there were also a "symplastic stage," diph- acterizedby the early formation (when iso-
theroid forms, streptococcal forms, and a type lated cells are allowed to develop into micro-
of cell similar to a T or a double Y. Suspen- colonies) of an initial mycelium that sooner
sions of .4. hovis obtained from culture col- or later divides into irregular rod-shaped
from human cases were
lections or isolated cells resembling mycobacteria or coryne-
which were intro-
placed in collodion sacs, bacteria and often becoming so short as to
duced into the peritoneum of rabbits. After look like cocci. The initial mycelium varies
the first and second passage, the animals in extent from hardly visible, irregular,
showed no symptoms of actinomycosis. elongated rods with a few short lateral
After the third passage, consisting in the branches, to something approaching that of
inoculation of the rabbit with the product Streptomyces, from which it differs only by a
of the second passage, atypical actinomy- more loose and crumbly consistency when
cosis was produced, in which the parasite the hyphae begin to divide. Swollen cells
was in the form of a symplastic or of a that on fresh medium germinate with one or
diphtheroid bacillus. After the fourth pas- more slender "germ-tubes" are of common
sage, typical actinomycosis was obtained. occurrence. The aerial mycelium of Nocardia
The conclusion w^as reached that cultures of varies to a similar extent. It is often invisible
A. hovis contain growth elements that will to the naked eye, and may
be altogether
pass through a collodion ultrafilter. Erikson absent or may few short fila-
consist of a
(1940) did not confirm the ability of actino- ments, which sometimes look like mere gran-
myces to give rise to filterable forms. ules. At the other extreme, the aerial my-
Erikson emphasized the difficulties en- celium may be abundant but composed of
countered in isolating an actinomyces from elements resembling those of the vegetative
the mixed flora obtaining in most patho- mycelium, usually less branched than in
logical low fertility rate of
material; the Streptomyces, and usually not differentiated
dismembered mycelial fragments and in- into conidia-like spores.
dividual cells; and the need for anaerobic The nocardias are related, on the one
methods of cultivation. Successful isolation hand, to the mycobacteria, and on the other,
depends to a high degree on fresh material to streptomyces. This gradual transition
containing a quantity of viable elements. from one group of microbes to another must,
Although lOrikson stated that her experi- in the words of Erikson (1945), "be accepted
ments "have disposed of the existence of a as another of the innumerable instances in
filterable stage and have yielded no evidence which nature prodigally overlaps manmade
in favor of any hypothetical life cycle in- taxonomic boundaries." In the early stages
volving either anacrolnc or aerobic forms," of de^'elopment, nocardias are characterized
Kriss ct al. (1945) stated that the hyphae of by the formation of an undivided substrate
MoHriioT.ocs', (
'^ 'l'()l,(»(;^ , and Lii'-h; ('\t'les 89
:
celium. According to Krassilnikov (1938) the ]\IcClang (1949) observed that if fragmen-
scant aerial mycelium of the first group is tation follows xevy shortly after germina-
devoid of branches and spirals and divides tion, branching will be sparse and very little
into sharp-ended cylindrical cells. Reference mycelium will be developed. With a delay in
has already been made to the recent obser- fragmentation, there is opportunity for re-
\'ations of Gordon and Mihm (1958) on the peated branching; a larger or smaller my-
sporulation of N. asleroides in a manner sim- celium may result, according to the rate of
ilar to that of streptomyces. growth and the amount of subseciuent cell
Jensen (1931) described filamentous and division. The time of branching and fragmen-
short-celled forms of the saprophytic A^. tation in a gi\'en strain was relatively con-
polychromogcnes, as pointed out in Chapter stant under controlled conditions.
(). Umbreit (1939) also distinguished several McClung divided the n .cardia group into
distinct types among the nocardias a-forms :
three subgroups
with short unstable mycelium, soft colonies,
I. Scant mycelial development, sparse branch-
and diffuse growth in broth; (8-forms with ing, and type 1 fragmentation. Colonial tex-
long stable mycelium, firm streptomyces-like ture soft, pasty and sometimes mucoid, pig-
growth on agar, and colony type of growth ment intracellular and insoluble.
in liquid media, which remains clear. Um-
II. E.xtensive development, straight
mycelial
branches which do not overlap, and type 3
breit considered the growth in broth a more
fragmentation. Colonial texture soft and
definitive characteristic than any other, al-
pasty, pigment intracellular and insoluble.
though he remarked that "borderline cases III. Extensive mj'celial development, no frag-
exist in which the investigator must resort to mentation of hyphae, contorted and profuselj'
other characteristics." Jensen suggested di- produced branches which overlap. Colonial
texture waxy or cartilaginous. (Jenerally both
vision of the genus Nocardia into two genera,
intracellular and solul)Ie pigments are pro-
reserving the last name for the /5-forms and duced.
retaining the name Proactinomyces for the
a-forms, but he added that these names JMorris describes as follows the life cycle
could probably with equal right be used for oi Nocardia: At first, a microcyst is formed,
the potentially acid-fast and the nonacid- which germinates on budding. The bud
fast strains, respectively. A horizontal di- gives rise to a long multinucleate filament,
vision (j3-forms = Nocardia, a-forms = which divides into individual cells by the
Proactinomyces) would give considerable formation of transverse cell walls. The cells
morphological homogeneity, except in re- multiply by simple fission, by complex vege-
spect to motility, but would entail much tative reproduction, and by branching. No
heterogeneity in respect to potential acid- prolonged diploid phase, similar to that of
fastness and the correlated characters of actinomyces, is produced. Heat -fixed gram-
carbon metabolism and proteolytic power. stained preparations of nocardia and actino-
On the other hand a vertical division (acid- myces may resemble each other, but cyto-
fast = Nocardia, nonacid-fast = Proactino- logically they are mark(Hlly different. The
myces) would result in greater biochemical conclusion was reached that there can be no
homogeneity but a corresponding morpho- doubt that the aerobic nocardia constitutes a
logical heterogeneity. The relationship of separate genus from the anaerobic actino-
these nocadial forms to the bacteria, on the myces. The germination of the microcyst of
one hand, and to the streptomyces and nocardia ])y ])udding is compai'able to that
micromonospora on the other, is brought out of the spore in streptomyces, in micromono-
in Table 15. spora, and in actinomyces, but is different
M(tHi'H()i.(»(i\'. (•^'r( )!.()( ;^•. and ijfi; ('^('M•:s 91
Tahi.k 15
Tcnlatirc plu/loi/inctic scheme of the Actinoini/celules (Jensen 1U53)
Nocardia Nocardia
nonacid- acid-
fast a fast a
^ Second stage:
Actinomyces
mvrelium
Nocardia Nocardia
nonacid- acid-
fast /3 fast /3
from that of Eubacteriales. The important By proper staining techniques, they ob-
difference bet\veen uctinomyces and nocardia served nuclear structures comparable to the
consists in the production by the former of nuclei of higher organisms. Fragmentation
true reproductive spores and the occurrence of the hyphae was preceded by cross-wall
of a prolonged diploid phase, in contrast to formation, the first step of fragmentation
the occurrence, in the latter, of a microcyst being the reorganization of the hyphal nuclei.
and a greatly reduced diploid phase. When The formation of the cross walls was pre-
branching occurs in nocardia, it is not of the
ceded by nuclear division.
permanent type but resembles that of an
Further information on the early studies
actinomyces, and is thus differentiated from
of the nocardia group, their relationship to
permanent l)ranching. Both genera have a
the mycobacteria, and their morphological
well-marked haploid generation arising from
changes is found in the work of Vierling,
a spore by Vnidding, and a diploid one arising
from the initial cell. Karwacki, Badian, Bisset, Hagedorn, and
Webl) and Clark (1957) demonstrated the numerous others. The life cycles of nocardias
existence in the growth cycle of N. corallina have been chscussed further by Combes et at.
of a unicellular, uninuclear coccoidal stage. (1957).
92 THE ACTIXOMYCETES, Vol. I
liquid media (Jensen, 1931; 0rskov, 1938). If cal adaptation. This vegetative growth may
such strains should permanently lose their
occur on many substrates rich in nitrogen
such strains, of which the classification seems Erikson (1948, 1953) noted further that
arbitrary.
the absence of readily fermentable carbohy-
favorable characters. She described the most tions arising in unaerated or solid cultures
3. The resistance of the organism to exterior arise at the periphery of established growths
influences. The most valuable property is
on old plates frequently exhibit a distorted
that of producing large numbers of special, and shrunken appearance, with straight
This aerial filaments instead of spirals, colorless
resistant cells (spores). is the great
advantage possessed by the genus Strepto- or only faintly pigmented, and with unusual
myces over the genus Nocardia. modes of vegetative branching. Such cases
The substrate mycelium of streptomyces of unstable reversible modifications com-
develops homogeneously, giving rise to a monly make up the greater part of the het-
The aerial hyphae produce straight or cur\'ed cullur(>s b(>longing to the nocardias, ])ut
sporulating branches, giving rise to conidia. appears only seldom among the strepto-
^r()l{PIl(>l,(>(;^ , (•^'|()l.(>(;^ , and i.iki". cvcj.ks 93
iuy('(Mi>s. ()i-sk()\- :m(l ollicrs Ix-licxcd ili;it emphasized the profound physio-
\('st igaloi'
t\)nuatioii of si^pta is ihc lirsl staj^c in ihc logical between the mesophilic
dil'fei'ences
process ol' tlu- hrcak-iip oi' ilu> mycelium micromonosporas which can utilize cellulose,
into fnigmciils. chit in, htinin, and other I'esistant com-
Cortain swellings of the terminal ends of pounds, and tile lheiinoi)liilic forms, which
hyphae may he obserxed in old cultures. cannot (Mrikson, i'.)H, 1!).")2). The thermo-
They are also foriiKMl uiuUm- al)normaI urow 1 li philic M. ruhioris was said to exhibit con-
conditions, as in conc(>ntrateti media oi' in sistently- a pattern of mycelial de\-elopment
tlu> presence of certain specific sul)stances which differs umnistakably from that of the
like catTeine. These swellings may he con- mesopliilic M
fusnt. Pigment was not pro-
.
\ery slender, profusely branched filaments, aerial mycelium and spores is visibly much
liearing single spores on short lateral higher. These results tend to sui)port the
branches. There is no aerial mycelium; in decision made l)y Henssen and in this treatise
instances where it has been reported, it is to separate the thermophilic and mesophilic
sparse, infreriuent, and rex'ei'sihle. TJiis in- forms into dilTei-ent genera.
94 THE ACTINOMYCETES, Vol. I
into a long filamentous cell, which divides to be diploid. These cells give rise, on germi-
into a number of individual cells by the for- nation, to a "secondary mycelium," multi-
mation of transverse cell walls. The branches cellular in nature with permanent branching.
are permanent, since no transverse w^all is Single spores arise at the end of their stalks,
produced at the junction of the parent cell formed at first as finger-like protrusions of
and the branch. A haploid mycelium is thus the mycelium.
developed; the filamentous outgrowths from The morphological features of the other
the cell show a direct protoplasmic connec- genera of actinomycetes are discussed in
tion between cell and filament. Where two Chapters 26 to 29 (Volume II).
r: IT A V T i; n
An actinomycete culture is made uj) of an to an extent that many of the modern pleo-
extensive mass of mycelium, either of a sub- morphists tend to I'eturn to the older con-
strate or vegetative nature or of both sul)- cepts of Naegcli and other earlici- j)l('oinor-
strate and aerial hyphae. Sooner or later, the phists that any bacterium may traiisnnite to
greatly in size and in shape, and frecjuently two hundred new words to express his ideas.
in certain physiological properties. Largely The life cycle of a bacterium was said to
consist of two simultaneous, paralk^l, and
because of under the influence
this, as well as
of different en^'ironmental and nutritional coordinated processes: (a) a multiplicative
and Robert Koch, many, if nf)t most, bac- The life cycle of bacteria was said to begin
cellas constant in nature and immutable or lowed by progressi\-e changes in cell com-
monomorphic. This attitude tended to dis- plexity; when a maximum, fixed for the
tion and inheritance among microorganisms. the haploid mychit. The cycles may be in-
On the other hand, the pleomorphists were complete, shortened, or completely arrested.
inclined to consider the microbial cells as This confusion was also reflectcnl in the
and the other at another time. S-type — smooth, transparent colonies, con-
Recently, the old problem of morphologic sisting of gram-negati^'e rods, growing an-
95
96 THE ACTINOMYCETES, Vol. I
Figure 46. Colony variants of S. yrisciis (Reproduced from: Didaney, E. L. ct al. Mycologia 41: 390,
1949).
aerobically and nonpathogenic. 2. 0-type aerobic, gram-positi\'e rods. This cyclic de-
—gram-positive, diphtheria-like rods, con- \'elopment was said to be accompanied by
sidered as anaerf)}nc transition forms, capa- changes in the cultural, morphological, and
ble of developing in the human organ- biochemical properties of the organism.
ism. 3. R-type —large colonies, made up of Ilenrici, however, emphasized that the
VAKIATIONS. Ml'TATIOXS. WD AI ).\1TATI()X.S 97
concepts of \hv pleomorphists showed ;i foiinat ion of specific enzymes, power to cause
conAplotc lack of systematic in\'(»stijj;a1ioii. infect ion, jjroduct ion of aerial ni\'ccliinii, and
Ill an attenij)! to patch together th(> lilc maimer of sponilal ioii.
cycles from haphazard obscix atioiis of cul- Some of the variations obtained for cer-
tures in widely difTereut media, no considera- tain organisms may be permanent in nature;
tion was ji;iven to the age of the culture or to others are only temporary. When a culture
the phase of growth. The designation of all is so treated as to result in injurious or
the structuresby names borrowed in part stinuilating effects, some of its properties
from mycology, in part from cytology, or may be lost, whereas others may be gained.
coined for the occasion serA-ed only to ob- Certain characteristics of the culture may not
scure the problem and to make more diffi-
it change as a whole, but undergo only a degree
cult for the reader to follow. Henrici empha- of change, as in the formation of adaptive
sized that the necessity for creating a new enzymes. Such variations are usually (juanti-
terminology had its origin in the oliscurity tative rather than (lualitative in nature.
of the thought which the author was trying When a comparison was made of the prop-
to express. The confusion introduced by this erties of a number of strains of S. griseus,
terminology was all the greater because the whether isolated from different substrates or
same structures were referred to by the obtained from a single culture, all degrees of
various authors under different names, and gradation were obtained. These variations
the same names were used to designate differ- involved the ability of the strains to produce
ent structures. Henrici demonstrated experi- antibiotics or enzymes, the intensity and
mentallj^ that bacteria vary continually in nature of soluble and insoluble pigments,
morphology with increasing age of the cul- and the length of the aerial hyphae.
ture. He correlated morphologic variations The effect of temperature upon the be-
with the rate of growth, and showed that the havior of a given culture is illustrative. It is
transition from one type to another occurs sufficient to cite the classical observations of
at the points of inflection between phases of Pasteur concerning a change in patho-
the growth curve. genicity of the anthrax organism when culti-
vated at 42°C, and the loss by Serratia
Variations Among Microorganisms
grown at 37 °C power to produce its
of its
Among the major factors that influence characteristic pigment. Other illustrations
variability of microorganisms are: (a) the comprise the increased power of infection
previous history of the culture, (b) the na- brought about by the reisolation of an or-
ture of the substrate or nutritional condi- ganism from animal tissues infected with it.
tions, and (c) the environmental factors. Variations among microorganisms include
Different organisms differ in this respect: the following:
some species greatly resist \-ariation, others 1. Xonhereditary modifications brought
readily undergo variation. about by the uncMjual influence of different
One must differentiate between gradual conditions.
variation of an organism growing in a cer- 2. Hereditary continuous variations, char-
tain medium and mutation organism of the acterized by the gradualness of the change
that consists in a complete change of one or through successive generations. These in-
more characters. Mutations are represented clude (a) adaptive variations, in which the
by the appearance or disappearance of cer- nature and direction of the change bear an
tain morphological or physiological prop- adaptive relationship to the conditions under
erties, including production of pigment, which the change appears, (b) nonadaptive
98 THE ACTINOMYCETES, Vol. I
Figure 4S. \';ui;il>ility of aclinoinyccic coioiiics in a jjhite culture i Hc^produccci from: Stanier, R. Y.
J. Bacteriol. U: 557. 1942).
strains into inactive strains that may also morphological, comprising colony structure
be free from aerial mycelium; (e) change of and cell structure, or biochemical, com-
colorless strains into pink variants, accom- prising both (juantitative and
formation,
panied by a change in the nature of the qualitative, of important metabolic products
antibiotic-producing capacity; (f) develop- Kriss recognized four types of variation:
ment, among acid-fast organisms which morphological, cultiu'al, physiological, and
cause infection in animals, of two subtypes, applied. These are illustrated by the varia-
one licjuefying gelatin and the other not bility of a culture of S. coelicolor, as presented
liquefying gelatin. in Table IG.
Tahi.k K)
Colony size
102 THE ACTINOMYCETES, Vol. I
^.^^^^ SURVIVAL
\ AHl vrioNS, Ml'TATloNS, AM) ADAITATIoXS 108
(icd throuiiii the dcnioiist r;it ioii of uciictic (Mil i:il ion of iii;iiiy species hii-^cly on t lie l);(sis
recoinhination ns will !>(> shown lalei'. of (he nature and intensity of the I'oi-nialion
ol' ])i^nionts on different media. Major siib-
Culliinil (iitd riuisioloiiinil Wirialions
(li\-isions of the tieniis Slrcploinnccs, ospe-
\'ariations of tiie piiiuientation of actino- eially into sections oi'species-^roujjs, are also
mycetes an* charactei-istic. 'riu>v are ol' based, most fre(|uently, upon pigmentation.
particulai- siiiniticance because of the differ- The key used in iiei-jroy's system of classi-
fication of actinomycetes, especially the growth and stability of the cultures were
genus Streptomyces may be taken as an illus- obtained on peat soil. Mineral soils tended
tration. This key is based largely on the to retard or inhibit growth and increase
pigments produced on organic and synthetic variability. The more pathogenic races were
media. On continued cultivation of the cul- most stable on most media. These variations
tures under artificial laboratory conditions, led to the question whether the descriptions
the pigment may undergo changes in nature of many species as causative agents of potato
and intensity, or frequently be lost alto- scab represented distinct species or only
gether. When the characters of an organism variants of a single species.
are based on pigmentation, it becomes very In a study of the variability or mutability
difficult to make comparisons even if type of .4. mutabilis, an organism apparently be-
cultures are available. The streptomycin- longing to the streptomycetes, Masumoto
producing strain of S. griseus could hardly showed that in a synthetic medium, with
be recognized when compared with the orig- ammonium chloride as a source of nitrogen,
inal description of the organism made by the nature of the carbon source greatly in-
Waksman and Curtis. The latter was based fluenced the formation of nonaerial myce-
upon cultures that were at first believed to lium from aerial mycelium-producing types.
be similar to one described by Krainsky. Sucrose and lactose were the two sugars most
Since no one ever had an opportunity to favorable for this purpose. Other carbon
compare freshly isolated cultures wdth those sources, notably mannitol, never gave the
of Krainsky, the Waksman and Curtis or- nonaerial mycelial type.
ganism is now usually recognized as the type Attention has already been directed to the
species for S. griseus. marked variations in cultures of S. griseus
S. coelicolor has also been studied exten- (Schatz and Waksman, 1945; Waksman
sively, especially from the point of view of et al., 1948). Formation mycelium,
of aerial
pigmentation and agar-decomposition. Erik- pigmentation of the substrate mycelium,
son observed that the major variations of production of streptomycin, acid formation,
this organism comprise loss of pigmentation, glucose consumption, and autolysis were
loss of capacity to produce aerial mycelium, observed among the qualitative and quanti-
and occasionally loss of ability to liquefy tative variations. The formation of the vari-
agar. Spontaneous formation of variants ants of S. griseus were described as follows:
could be found more readily in the spores of Freshly isolated streptomycin-producing
degenerate colonies, rendered atypical by cultures formed typical aerial mycelium,
artificial methods of cultivation. A strain characteristic of the species. These cultures
that had lost the power of pigmentation gave produced an alkaline reaction in glucose-
a variant which produced sectored colonies, containing media, as well as characteristic
some of which possessed the blue pigment. surfaceand submerged types of growth; they
Thomas made a study of the variability underwent only limited lysis and were mark-
of scabies. He isolated six physiologic races
>S'. edly resistant to the antibiotic action of
that differed in their pathogenicity on 10 streptomycin. They gave rise to two kinds
different potato varieties. An increase in the of variants:
nitrogen, phosphorus, and potash content of 1. A nonsporulating variant that formed
the medium resulted in a delay in the produc- no aerial mycelium and no streptomycin;
mycelium. Nitrogen and phos-
tion of aerial it was sensitive to the antibiotic action of
phorus were generally favorable for growth, streptomycin in a glucose-containing me-
but potash tended to retard it. Maximum dium, and was characterized by a type of
VAHIA'l'loXS. MITATIONS, AND ADAITATIONS 105
were changes of a cultui'al and morpho- cardias, and from nocardias to streptomyces
logical nature; fewer changes of a physio- the reversephenomenon occurs but seldom.
logical and biochemical nature; and no This reasoning led Krassiluikov to the con-
change in the antibiotic nature of the organ- clusion that actinomycetes are present in
isms. This is cjuite at variance with the natural substrates, such as soil, largely in the
above reports on the production of mutants form of micrococci. Kedrovski, however,
from the streptomycin-producing S. griseus emphasized that the reverse is true, actino-
and streptothricin-forming S. lavendulae. mycetes giving rise to rod-shaped forms of
the tuberculosis type.
Mutations
Spontaneous mutations in stored spores of
The concept of "mutation" and the usage streptomyces were studied by Wainright.
of this term has led to a particular contro-
Saltations
\'ersy. The term has been applied to "sudden
changes which are neither the result of a x\mong the mutations, the phenomena of
process d
gradual acclimatization or educa- occupy an important place among
saltations
tion ror of selective isolation." In recent the actinomycetes (Rippel and Witter).
years the subjects of mutations and varia- They are similar in natiu'e to those occurring
tions among microorganisms have gained in colonies of fungi or bacteria. New forms
new impetus from studies on the nutrition appear in a colony either as sectors or as
of the organisms, involvinggrowth factors daughter colonies. These sectors may differ
and metabolites, formation of antibiotics, from the mother colony by the presence or
and development of resistance to a given absence of aerial mycelium, by a change in
antibiotic to which they were originally sen- color of the substrate or aerial mycelium, by
sitive. structure or rate of growth of the colony, by
The formation of mutiuits by actinomy- presence or absence of "fairy rings," etc.
cetes has long been recognized. These were (Lieske, Kriss, Krassiluikov). These sal-
considered as special types of ^^ariants. The tants, upon careful transfer to fresh media,
formation of new strains through the muta- will produce new stable varieties; they differ
tion of a culture, however, is more fimda- from the original species in their morpho-
mental and hereditary. White strains were logical, physiological, or cultural properties.
obtained from blue-pigmented forms; strains Some of these saltants could easily be desig-
free from mycelium, from those pro-
aerial
nated as different species had their origin
ducing such mycelium; red strains, from
not been known.
orange-yellow forms. These mutations were
Schaal found as many as nine sectors in a
accompanied by changes in morphological,
single colony of S. scabies. The cultures ob-
cultural, and physiological charactei's which
tained from these sectors \^aried in the nature
differentiated the new strains from the
of the mycelium, in the rate of growth, and
mother cultui'(\'<. The differences thus ob-
may in the pigmentation. The formation of spirals
tained l)e so distinct as to give the new
strain a characteristic of a species.
and the direction of the turns in the spirals
Krassiluikov and his collaborators made were also variable characters. Nutrition
a detailed study of such stable mutants. exerted a marked effect: production of aerial
They emphtisized that the \'ariations or mycelium was inhibited by a high nitrogen
mutations take place from the simpler to the content of the medium; ])resence of thiamine
more complex forms, as from micrococci to favor(>d rapid growth and the formation of
mycobacteria, fioin mycobacteria to no- sectors. There was no correlation, Iiow(>\-er.
VARIATION'S. MITATIOXS. AND Al )AI'TATJ()XS 107
|T[ f/YCEL'UMl
( GRAV WYiELIUV
ITE SECTOR)
(GRflV MYCELIUM)
(GRAY MYCELIUM)
(GRAY MYCELIUM.
ONE SECTOR)
108 THE ACTINOMYCETES, Vol. I
recovered by illumination with ^dsible light, violet and gamma rays results in hereditary
a phenomenon that came to be known as changes affecting colony morphology and
^'photoreactivation." pigmentation. These changes are largely
A culture of *S'. flaveolus was treated with associated with instabilities that I'esult in
x-rays in doses I'anging from 25,000 to ."^00, further variation during colony growth and
000 roentgen units. In one experiment iS per spore formation. These instabilities persist
cent of the spores survived 100,000 r units, indefinitely, giving rise to new variants hav-
and 0.03 per cent 300,000 r units. The sur- ing their own patterns of instability. These
Vw'A rate was in\'ersely proportional to the changes differ from gene mutations in that
dose. The following mutants were obtained: they can be induced with much greater fre-
(a) biochemically deficient strains which (}uency, and that gamma rays are as effective
grew well on nutrient agar but very poorly as or more effective than ultraviolet irradia-
or not at all on asparagine glucose agar; (b) tion, suggesting chromosomal rearrange-
strains with pigmentation more intense than ments. Mutations are caused by treatment
or different from that of the wild type; and with x-rays, ultraviolet, and cold, a period of
(c) asporogenous strains. About 24 per cent sensitivity during early spore germination
of the survi\'ing spores in a suspension being common to all; in the last two treat-
treated with 200,000 r units were mutants. ments, there is dependence on metabolic
One of the procedures for obtaining highly activity. The phenomenon of photoactiva-
potent antibiotic-producing strains consists tion was studied further by Erokhina and
in combining ultraviolet or chemical treat- Alikhanyan.
ment of spores with single-colony isolation. Horvath et al. observed that when an
Another method takes advantage of the antibiotic-producing strain of S. glohisporus
frequently greater resistance of organisms to was grown in a sterile filtrate of the macer-
the antibiotic they produce, as in the case ated mycelium of another antibiotic-produc-
of streptomycin, in the plating out of cul- ing organism {S. globosus), there occurred,
tures in media containing increasing con- after several repeated transfers, a change in
centrations of the particular antibiotic; the the morphological and physiological prop-
colony that will develop on the plate will erties of the first organism. The newly pro-
tend to represent more potent strains than duced strain retained its properties for a
the original ones. Dulaney et at. presented considerable time. Cultivating actinomy-
details of the first method, as shown in cetes in media containing con\'allamin
Figure 46. The most variable characters in- changed the staining properties of the organ-
cluded color of spores and degree of sporula- isms and the appearance of the colonies.
tion; surface and margin of colony, and When grown in ordinar}^ media, such cul-
colony sectoring; amount and color of exu- tures tended to return slowly to the original
date on colony surface; amount and color of stage (Hassegawa et al.).
soluble pigment released in substrate. There According to Krassilnik()^% the intra-
was also marked variation in amount of strain and intraspeci(\s antagonism among
streptomycin produced, although no com- antinomycetes is not just a bizarre property
plete correlation could be obtained between but is highly significant in nature and can be
the latter and the morphological type. Some utilized for species characterization. This
of the strains retained the capacity to gi\e phenomenon has a hearing upon the whole
high streptomycin yields, and others lost it. problem of the significance of antibiotics in
According to Xewcomhe 49 and 50)(Figs. the part played by actinomycetes in the cycle
exposure of spores of streptomyces to ultra- of natui'c (Scriabin).
WMMA'lIoNS, Ml TAI'loNS. WD A )A
I I' T.VTK ».\S 109
the product i\(' capacity of cultiii'cs ol" Slrcp- Miildflinic (fftrt of Jf lulrofiiinioliiif loxnlc on the
rcrrisr nnilolioii of ijl iiliiiiutlv locus (.Masliiiiiii
tonujccs of low aiitihiotic production, the
and Ikcda)
followiuij; tr(>atnuMits should he rcsoHcd to:
(a) uUraxiolct irradiation; (h) intcMisificatiou
Treat-
of \itaiity l)y frc(|Ucut pa.ssagos; (c) i'cfriiz;ci--
may become not only resistant to, l)ut also Often, colonies of intermediate character are
dependent on, this agent, as was found to be produced. This is particularly true of cases
true for bacterial strains requiring strepto- where the mutation rate is high and where
mycin for their growth. reverse mutation occurs.
medium.
Bradley (1958) exposed a population of a
•y- «»•
single strain of S. griscus, for se\eral grcjwth
cycles, to a sterile culture filtrate of another
strain of 5. griscus. The first accjuired several
genetic characteristics of the .second strain,
namely, streptomycin sensitivity changed to
changed
resistance, bacteriophage .sensitivity
to resistance, absence changed to presence
of soluble pigment, and presence changed
to absence of pigment in the vegetati\'e my-
celium. The filtrate contained a low con-
centration of streptomycin, which did not
inhibit the growth of the first strain, but
streptomj'cin-resistant mutants were se-
Figure 53. J'ormatioii of heterokaryotic col-
lected. The observed morphological changes onics of S. fradiae on minimal agar. Plates 1 and
were coupled with bacteriophage and strep- 3 seeded with 10' spores of strain
6F4-1 (methio-
tomycin su.sceptil)ility. The hybridization nine and isoleucine requiring, and streptomycin
sensitive) and 6FS-16 (histidine and argininc rc-
was .said to be the result of selection of nui-
fiuiring, and streptomycin resistant), respectively.
tants rather than gene transfer. Plate 2 received a mixture of parental spores and
Alikhanian and Alindlin grew biochemical sliows formation of prototrophic colonies (Re-
])roduced from: Braendle, D. H. and Szyi)alski, W.
mutants of ^S. rimosus on suitable agar media,
I'roc. Nat. Acad. Sci. V.\: 947 !)o,5, 1957).
and observed that at the point of contact of
the mutant colonies more abundant growth karyosis and spore production in the aei'ial
and more abundant sporulation occurred. hyphae (llopwood).
Nuclear fusion and reduction occur in *S'. According to Braendle and Szyljalski, aW
coelicolor somewhere l)etween hyphal fusion of the wild-type strains of Streptomyces
in the substrate mycelium, allowing hetero- studied wei'e prototrophic, i.e. they formed
112 THE ACTIXOMYCETES, Vol. I
colonies and sporulated on synthetic agar quiring histidine and arginine but strepto-
with ammonium sulfate and glucose as the mycin-sensitive (Fig. 53). A mixture, con-
only nitrogen and carbon sources. Nutri- sisting of approximately lO'"* spores from each
tionally deficient mutants were developed parent, was incubated for 2 to 6 days on
by ultraviolet irradiation of the parental minimal agar. This mixture yielded several
prototrophic culture, concentrating the mu- hundi'ed prototrophic "recombinant" colo-
tants by the filtration technique, and finally nies, whereas the plates seeded with spore
employing selective media and the replica- suspensions of only one of the parents
plate principle to detect and identify the showed no growth. The formation of hetero-
mutants. Auxotrophic strains were isolated karyons, nutritionally balanced, i.e. able to
which required single or multiple supple- grow in the absence of all the nutritional
ments of various amino acids or alternati\'e reciuirements exhibited by any one of the
requirements of two, three, and even four parents, was demonstrated for S. griseus, S.
amino acids, often as the result of a single fradiae, S. venezuelae, and S. albus. Only *S'.
two types of nutritionally marked conidia only between mutants derived from the same
on a selective medium. A cross was per- parental culture. The interspecific crosses
formed between two strains of S. fradiae, and a limited number of intraspecific crosses
one streptomycin-resistant and requiring between different strains designated as S.
methionine and leucine, and the other re- griseus were unsuccessful.
CM \ r I i; i{
Physiology
113
114 THE ACTIXOMYCETES, Vol. I
different concentrations, will change the 6. Influence of other organisms, with the
growth characteristics and metabolic pat- resultant associative and antagonistic effects
tern of the particular organism. Not the exerted by them and upon them.
least important among these considerations These factors influence the extent of
is the recognition of the strain specificity of growth of the microbial cell, its chemical
an organism, whereby certain reactions are composition, and the nature and concentra-
limited not to a genus or even a species, but tion of specific metabolic products produced.
to a certain race or strain.
Metabolism of Actinoniycetes
The metabolism an organism represents
of
a special phase of its physiology. To com- Any comprehensive discussion of the met-
prehend it, we must understand the food- abolic activities of a group of organisms
stuffs necessary for the maintenance of its must consider their utilization of various nu-
growth and activities; the manner of obtain- trients, decomposition of these nutrients into
ing the required energy the products formed
; simpler compounds, the various mecha-
as a result of such activities and the various
; nisms of transformation of these nutrients,
intermediary reactions through which the involving those concerned with both break-
nutrients passwhen they are used for cell down and synthesis, formation of waste
synthesis.Normal metabolism of an or- products, and a variety of other reactions
ganism, when it grows under natural condi- involved in the life of living cells.
tions similar to those it finds in a natural In the very early studies on the growth of
environment, is often differentiated from ab- actinoniycetes,it was found that these or-
normal metabolism, when the growth of an ganisms vary greatly in their mitrient re-
organism is made to deviate from the nat- quirements. Some were found able to con-
ural path of life to which
it has been accus- sume simple elements and compounds;
tomed. Such a deviation occurs in virtually others required complex organic materials.
all methods used for growing microorgan- Considerable adaptation to various nutri-
isms on artificial media and under controlled ents also w^as observed. The amount of cell
conditions. It is only seldom that a microbe material synthesized depended on the avail-
grows in nature in a pure culture. Once it ability of the nutrients and on the effect of
has been isolated and made to grow in an the accumulated products.
artificial substrate, its metabolism may be Beijerinck first studied an organism he
considerably modified. considered to be a bacillus {B. oligocarbo-
Among the factors influencing the metab- philus), later found to be an actinomycete,
olism of microorganisms, the following ai"e that was capa))k' of deriving its carbon and
l'll\S10l,(HiV 115
Table 19
Metabolic changes characterizing the two phases during the submerged growth of
S. griseus (Dulaney and Perlman)
Phase I Phase II
the culture increasevS, pointing to the pref- lactate also formed during the early stages,
erential utilization of the proteins as a source but this disappeared rapidly (Table 19).
of energy. With the advance in growth, Van Dyck and DeSomer also recognized
sugar utilization and ammonia consumption two stages in the growth of a streptomyces
proceed at a rapid rate, parallel to the in- (S. aureofaciens) . The first stage is char-
crease in dry weight of the organism. acterized by cell synthesis and nutrient up-
Dulaney and Perlman divided the meta- take. The second stage is characterized by a
bolic processes of actinomycetes into two slower increase in cell synthesis protein and
;
phases, crescense and senescence. In the ribonucleic acid decrease, and desoxja-ibo-
first phase, there was uptake of soluble nucleic acid changes but slightly; the de-
nitrogen, carbon, and phosphate into the crease in nucleoprotein cannot be ascribed to
mycelium the oxygen demand was high and
; lysis, since it is not accompanied by a de-
the utilization of glucose was rapid, but crease in weight of mycelium or by an in-
there was very little antibiotic production. crease in the nitrogen in the medium.
In the second phase, mycelial weight de- The course of metabolism of S. venezudae
clined, phosphate and nitrogen were ex- has been studied by Gottlieb and Legator
creted into the medium, oxygen demand (1953). The course of growth and the meta-
fell, and streptomycin was produced. Some bolic processes of an actinomycin-producing
strain of *S. chrysomallus were reported by
Dietzel et al. (1950). Schmidt - Kastner
found, for example, that the addition of DIj-
isoleucine and sarcosine resulted in certain
new actinomycins differing in their peptide
chains. The metabolic processes of neo-
mycin-producing S. fradiae were examined
by Giolitti and LugU (1956).
Numerous other metabolic processes of
actinomycetes, notably of members of the
6 8
TIME', DAYS gxnuis have been described.
Streptomyces,
Sekizawa (1958), for example, found that a
Figure 55. Metabolism of *S. griseus (Repro-
produced from: Cochrane, V. W. and Dimmick, I. culture of Streptomyces produces ethoxy-
J. Bacteriol. .58: 727, 1949). cthene-1,2 dicarboamide, as represented by
^ll^sl()l.()(;^ ir
various
dium, the fei'inentation process falls into 2
T]u> imtiitioM of the or<>;aiiisins
j)hases, and the secondary germination or
and [hv ct'lVn'ts of cortaiii specific cmiroii-
gi'owth is not observed.
nuMital factoi's may be coiisidei-ed in fui'-
The (^o.jvalues showcnl the same tendency
tluT detail.
both in soybean and casein media. In the
I none (19.")S") rei)oi-t(^d a iiiiili lexcl o\vi;-en
lattei', th(> presence or absence of metal salts
demand for N. (/ris(us grown on soylxvm
did not influence the (Joj value, but it played
medium at 24 and 9() hours. \\'ith d(>ficient
an impoiianl I'ole in streptomycin forma-
aeration and in alkaline media, the sec-
tion.
ondary high le\'el oxygen demand inci'eased;
with an excess aeration the latter disap-
CarI>on Nutrition
peared. Streptomycin inhibited the oxygen
uptake of young cells at abo\-e a certain Actinomycetes grow in nature on a wide
concentration this inhibiting action
; is some- variety of substrates. The nutrition of ac-
^vhat pre\ented by the addition of 10 "
M tinomycetes can be considered on the basis
.Mg and 10"' .1/ Mn. of the various essential elements reciuii'ed,
The carbon dioxide output of S. griscus notably, carbon, nitrogen, and certain min-
gave a maximum rate at 24 hours. The Qo_> erals, as well as sources of these elements.
value and QCO2 curve were lower with my- These sources range fi'om complex organic
celium than with spore inoculation. The environments, such as drained peat bogs,
R.Q. value uidicated minimum at 48 hours. high organic soils, and composts of straw or
Both young and old cells were inhibited of stable manures, to fairly simple media,
]\v streptomycin, but the inhibitory action such as poor sandy .soils and simple syn-
was influenced by the relative concentra- thetic substrates.
tions of streptomycin and an unknown fac- Actinomycetes are able to utilize a great
tor in the medium. The 3'oung cells were variety of organic compounds as sources of
more sensitive than the old cells. There was energy. These compounds include organic
believed to exist a difference in the char- acids, sugars, starches, hemicelluloses and
acters (or the enzyme systems) of the young cellulose, proteins, polypeptides and amino
and old cells. The carbon dioxide output acids, nitrogenous bases, and numerous
was inhibited by streptomycin much more other substances. Some actinomycetes can
than the oxygen uptake. also attack fats, hydrocarbons, benzene
Fermentation processes of soybean me- ring compounds, and, more limited de-
to a
dium by two strains of S. griseus fell into gree, lignin, tannin, and rubber. There is
four pha.ses: (a) the amount of mycelium considerable selectivity in the utilization of
increases, glucose consumption is slight, these substances by different kinds of actino-
oxygen demand is high, and streptomycin mycetes. Some of the nutrients, like glucose,
production is very low; (b) the amount of maltose, dextrin, starch, glycerol, amino
mycelium is almost constant or slightly in- acids, and proteins, are consumed \ery
creases, glucose consumption is high, strep- readily; in fact, they are the best sources of
tomycin production increases, and oxygen carbon. Sucrose, xylose, raffinose, and cer-
demand decreases markedly; (c) mycelium taui other sugars, sugar alcohols, and sugar
118 THE ACTINOMYCETES, Vol. I
Table 20
Glucose
1M1^ si(»i.()(;\' 1 1!)
and rathnose-negative, 2. Khaninose- and cornllino (Reader, Peters et al., Lutz). ^Mar-
rafhnosc-positive, o. Rhanuiose-positive and tin and Batt have .-^hown that this organism
raffinose-negative, 4. Rhamnose-negati\-e reciuires the addition of thiamine to synthetic
and raffinose-p()siti\-e. These were further media, especially for the utilization of am-
subdivided on the l)asis of utiUzation of moniiun ions.
xylose, lactose, niannitol, and acetate. The ^'arious organic acids, such as malic and
I 2'a>
0).- 5j
O C -O
E- —
•/:
— +
0)
(1).- X ?^
> S - :^
Carbon source
I'liN sl(»L()(;^ 123
Jagnow reported that even oxalic acid can (7 III. .Vppl. Microbiol. 2: 289, 1954).
T.VBLE 25
Utilizadon of carbon sources bif viriilogrisein- and griseoriridin-producing and related
streptomyces (Anderson et al., 1956)
124 THE ACTIX()AIYCU:TES, Vol. I
c
l'II\SI()L(KiV 125
carbon compounds (Webley and de Kock, Utilization of different amino acids by a strepto-
myces as compared to that of a fungus
1952). This property is usually associated
(Wak.sman and Lomanitz)
with oxidative metabolism, potential acid-
fastness, production of red or orange pig-
ments of the carotenoid type, and lack of
diastatic and proteolytic enzymes. The
saprophytic strains, with yellow, greenish,
or no pigments (Jensen, 1931-1932; Krassil-
nikov, 1938; von Plotho, 1948), seem to be
devoid of acid-fastness and fail to utilize
paraffin, but they are more fermentative
and often show diastatic and proteolytic
effect.
Nitrogen Nutrition
Proteins, peptones, and certain amino
acids form the best sources of nitrogen for
actinomycetes, followed by nitrates, am-
monium salts, and urea. Actinomycetes are
unable to fix nitrogen and have to depend,
like the great majority of fungi and bacteria,
upon fixed compounds of nitrogen for their
cell synthesis.
Table 27
Nitrogen ulilization by different actinomycetes
(Lieske)
Nitrogen source
l'll^'SI()|,()(i\' 127
dinVrcnt amino acids by a sti-(>|)toinyc('s and l)(<()iii/iiistti<ni of phinl prolans In/ ilijfcnnl
microorganisms (Wuksnian and Starkey)
a funiius has Ixhmi made hy W'aksnian and
Lonianitz, as shown in Taljlc 'JS. In sonu*
rroU'in
cases, thi> act inomycotcs arc cx'cn more (>f-
duced by special permission from: Rivett, R. W. this was the mechanism by which members
and Peterson, W. H. J. Am. Chem. Soc. 69: 3007, of the glutamic acid series are utilized via
1947). the tricarboxylic acid cycle.
Certain actinomycetes, when growing in a
DL-threonine best production of neomycin,
; peptone medium without any carbon
with alpha-alanine, L- and DL-aspartic sources, are able to produce urea (Guitton-
acids, L- and D-ghitamic acids, L-histidine, neau). Out of 477 cultures of streptomyces
L-proline, DL-threonine, and N-Z amine. isolated by Stapp, 177 were able to use urea
Some actinomycetes, notably nocardias, readily as a source of nitrogen. Some of the
attack proteins to a rather hmited degree. cultures used as soiu'ces of nitrogen, xanthine,
Casein may not be hydrolyzed. Even gelatin, hypoxanthine, and adenine, in concentrations
which is readily used by the great majority of 0.05 to 0.1 per cent. A large number also
of streptomyces, is attacked by only some used uric acid, but not uracil, p^a-idine,
nocardias, and frequently not ^-ery readity. imidazol, and pyrrhol.
In general, nocardias are unable to utilize According to Moore ( 949) certain species
1 ,
xanthine, tyrosine, and certain other amino of Nncardia are able to utilize pyridine,
acids. The presence of glucose does not have aniline, nicotinic acid, and nitrobenzene as a
the same depressing effect upon the decom- source of nitrogen, and phenol plus am-
lMl^sl()I,()(;^ 129
from the soil a nonmotile coccus and a rod- a "strongly autotrophic" actinomycete.
shaped organism that grew in pure mineral Takamiya and Tubaki (1956) observed
solution and produced, after 2 or 3 weeks, a an actinomycete growing on a phosphate
snow-white, nonwettable surface pellicle. solution. The culture could be grown in a
They suggested that the cultures grew at the pure mineral solution. The culture grew
expense of the traces of volatile organic chemo-autotrophically, utilizing the process
compounds found in the laboratory air. of H2 oxidation in order to assimilate CO 2 .
Later, Beijerinck (1913, 1914) reported that It was named *S'. autoirophicus. Three other
the cultures could also utilize H2 in the at- cultures of chemo-autotrophic actinomy-
mosphere. The two cultures were described cetes were isolated. Hirsch demonstrated
as ActinohaciUus (Bacillus) oligocarhophilus that a culture of Nocardia, N . petroleophila,
and as Actinobacillus ( = Streptothrix Cohn) grew slowly but steadily upon a purely
paulotrophus, a thread-forming organism. In mineral medium in contact with laboratory
1922, Lantzsch cultivated, from a surface air. No growth took place without CO 2 . By
pellicle produced spontaneously on a cjuartz means was established
of tracer technique, it
suspension in water, a culture similar to that the CO2 is assimilated and incorporated
Beijerinck's Bac. oligocarhophilus and found in the cell substance. As energy sources for
80 5
- s-
- 20
LACTIC ACID
96
HOURS
Figure 62. Utilization of glycerol and sodium lactate b\' *S'. venezuelae (Reproduced from: Gottlieb,
IJ. and Legator, M. Mycologia 45: 512, 1953).
I'lIWSlOLOCiV i:il
ami slower C(^-.> exctjution) aiul a iiiji;luM- cell there is an increase in desoxyribonucleic
(irv \v(Mu;lit in tiu- initial staji.^ of iirowth than acid synthesis. Thei'c is also a delay in
tivity. According to Erikson, they do not The introduction of synthetic media for
attack egg or blood serum; they do not clot the growth of actinomyces (Howell and
or hydrolyze milk; they seldom grow on Pine) made possible the study of the ability
gelatin; they have little or no hemolytic of these organisms to produce lactic acid.
action on blood agar. Certain strains iso- According to Erikson and Porteous (1953),
lated from human infec^tions have been A. Israeli has the capacity to convert as
found to show a slight degree of hemolysis much as 30 to 60 per cent of the glucose
on blood-agar plates at different times, but utilized to lactic acid, imder suitable condi-
not consistent^. They do not produce tions of growth.
soluble pigments on protein media or in- Like aerobic actinomycetes, A. hovis is
acid is formed. Maltose, lactose, and sucrose ported that anaerobic forms are highly sensi-
are also utilized by all strains. Positive or tive to drying, being unable to survive even
negative reactions with salicin and mannitol for one day.
have been found of value in differentiating
Influence of Temperature
strains, such as human ve7'sus bovine. A.
hovis was found by Rosebury to have a The range of temperature within which
limited tolerance for oxygen, which varies, many microbes are able to develop is com-
however, among strains. paratively wide. Most microbes begin to
/
/ ..•' r' •'
/
8
k-
NO GLUCOSE
0.1*
0.5*
I*
2*
2 4 6 8 10
DAYS
Figure 64. Effect of glucose concentration on pH (Reproduced from: Knissilnikov, X. A. 1950, p.
206).
nl^ sroLoov 133
90
:
man and to
range. Bacteria pathogenic to Lowest possible temperatures are usually
warm-blooded animals develop within a not sufficient to kill microbes. Even tem-
much narrower range of temperature than peratures of li([uid air w^ere not sufficient to
do saprophytic bacteria. M. tuberculosis, for kill staphylococci and M. tuberculosis. How-
example, has its minimum at about 30° C, ever,some bacteria are rapidly killed at 0°.
its maximum at 42° C, thus giving 12° C as Repeated freezing and thawing have a more
a range of growth. (3n the other hand, many deleterious effect than freezing alone; dif-
saprophytes have a range of growth of al- ferent organisms vary in this respect. Bac-
most 40° C. teria and spores in a dry state are preserved
According to their temperature relations, by liquid air. The mycelium and spores of
microbes are usually divided into three certain fungi were not killed even by — 110°,
groups whereas the mycelium of other fungi was
1. Psychrophilic forms, with a minimum destroyed readily at low temperatures, the
at 0°, an optimum at 15 to 20°, and a maxi- spores being more resistant.
mum at 30° C. Most bacterial cells, except the thermo-
2. Mesophilic types, with a minimum at philic forms, are killed ata temperature of
9 to 30° C, an optimum at 28 to 38°, and a 56° C hour; at 60° C, 10 minutes is
for 1
temperature. This effect is more marked on may not be sufficient to destroy all actinomy-
the upward scale than on the downward cetes. Some actinomycetes can be adapted
scale.Microbes are much more resistant to to grow at higher temperatures. Lieske, for
temperatures below the minimum than to example, was able, after a few transfers, to
those above the maximum. grow his culture Nocardia 74 at 48° C,
I'HVSIOI.OCV 135
whoivas the oi'ininal upp(>r l<MU|>(Matiii(' at lOO'^C foi- I minute enhanced the initial
limit for this culture was 12"^ ('.
g(>rmination rate of the s|)ores within the
Further studies on the elTeet of lenipeia- lirsl ;;-honr period. When the spoics ha\-e
turo and humidity upon the growth and been subjecled to temp<'rat ures of 1()()°(' for
death-rate of spores and mycelium of \aiious more than 1 hour, sectored colonies showing
streptomyces ha\-e been made hy .la^now loss of aerial mycelium and impaired viabil-
(1057). ity fre(|uently developed. iM'ikson (l()55b)
Amouii; the actinomycet(\s, a special u;roup further leported that a pathogenic partially
stands out in relation to temperatui'e condi- acid-fast culture of A'', sebivorans and a
tions. These are the thermophilic actiuo- closely allied form were capable of with-
mj^cetes that are eapal)le of growing at standing exposure to 90°C for 10 minutes
temperatures of ")() to 65° C. These or- when dispersed in phosphate buffer suspen-
ganisms occur abundantly in manure com- sion. Subjection to heat treatment (90°C for
posts, in lieaps of hay, and in pasteurized 1 minute) of a blood culture belonging to the
not at all at 25 and 60° C. The manner of Alicrobes vary greatly in their sensiti\-ity
spore formation of this organism suggests to drying. Some are highly sensitive and
that it was a member of the Micromono- cannot I'esist drying more than a few min-
spora group. Schiitze reported the presence utes or a few hours; others remain ali\'e for
in decomposing clo\er hay of representatives many years. The composition of the me-
of two types of therm()j)hilic actinomyeetes, dium is of great importance in this connec-
one of which was designated as ,1. thenno- tion; the presence of protein in the medium
philus Berestnew and the other as .4. greatly increases the period of resistance to
monosporus Lehmann and Schiitze. The drying. Bacteria remain ali\'e in normal air
latter may also be considered a memfjer of for a much shorter time than indry air,
the Micromonospora type. because of the moisture content of the
Further studies on the germination of former. In dry air, many bacteria remain
heat-resistant spores of M . vulgaris led l''.rik- alixc foi' years. Bacterial spores resist drying
son (1955a) to conclude that heat actixation nnich more I'cadily and for a longei- period
136 THE ACTINOMYCETES, Vol. I
than do vegetative cells. The mycelium of est intensity and are only slightly protected,
most fungi is readily destroyed on drying, are destroyed.
while the spores are killed only after pro- The red and orange rays of the light
longed drying, the period varying with the spectrum, as well as the infrared, or heat
species. The greater resistance of the spores rays, have no upon the growth and
effect
is due to their lower water content; the activities of microbes. Blue and violet, and
mycelium of a Pcnicillium was found to con- especially the ultraviolet, are the most in-
tain 87.6 per cent of water, and that of the jurious rays of the spectrum. \^arious ex-
spores 38.9 per cent. planations of the mechanism of the micro-
Actinomycetes are very abundant in dry bicidal action of ultraviolet radiations have
soils and are, in general, markedly resistant been offered. It is known that microbes ab-
to drying. Acosta kept a culture of an actino- sorb the lethal rays and that the proteins
mycete (.4. invulnerabilis) alive in a fully dry have absorption bands between 2480 A and
state for 9 years. Berestneff inoculated a cul- 2710 A. The effective radiation for steriliza-
ture of aS. violaceus on sterile rye straw and tion is in the region of waxe lengths of 2800
allowed it to grow until sporulated. After A to 2500 A. The capsulated organisms are
being kept m a dry state for 10 years in the most susceptible; the sporulating organisms
laboratory, it was still alive. are most resistant.
Lieske prepared dry cultures (on sterile When a microbial culture is suddenly
paper) of Streptomyces, Nocardia, and
filter brought to light, the protoplasm contracts
Actinomyces in a desiccator, over dry CaCl2 and a partial dehydration of the cell contents
and sulfuric acid. The cultures were alive takes place under the influence of the rise
after 18 months. Different organisms differ in temperature. When the culture is returned
greatly, however, in their ability to survive to darkness, the reverse takes place. Ultra-
drying under ordinary atmospheric condi- violet radiation produces an enormous con-
tions. traction; the vacuoles are reduced and may
Krassilnikov (1938) emphasized the re- even disappear, followed by plasmolysis.
markable ability of actinomycetes to survive, The antibacterial action of radiations,
under most unfavorable conditions, for very ( > 3500
comprising both the long ultraviolet
long periods. A) and short visible (<4900 A) rays, has
recently attracted considerable attention
Influence of Light
from the point of view of destruction of un-
Actinomycetes do not need light for their desirable organisms and development of
activities. In fact, strong light, especially mutants from desirable ones. The region of
M^hen prolonged, has an injurious effect upon 3500 to 4900 A is particularly effective. It
their development. The effect of light de- was concluded that the extended nature of
pends to a great degree upon the medium in the killing curve suggests the production of
which the organisms are grown the cells are ; some toxic sul)stance, or the destruction of
usually more resistant in milk than are those some essential compound in the cell, the
in bouillon. The resistance of the organisms eflect of which, up to a certain limit, does
to light is greater in a dried than in a moist not permanently destroy the ability of the
condition. The injmious action of light in- cell to divide and develop
o
further. Wave
creases with the intensity of the source of lengths shorter than ."JOOO A
most ef- are
light. Sunlight acts only on the surface of ficient at 2650 A, close to the wave lengths
solidmedia or in the air to which the or- at which nucleic acids act as most active
ganisms are exposed. In liquid media, those absorbents. The phenomenon of photoreac-
organisms which are sul)j(M'le(l to the great- tix'ation greatlv influ(Mi('(>s the killing effect
^II^SI()I,()(;^ 1:^7
of ultra\i()l(>( lij;hl (Kcliici-, lMtl('ii<:;('i- and The action of a poison and the resistance
McCoy). of the cell dejK'iui upon the rapidity with
which the sul)stance passes thioiigli the
Action of Stiniulant8 and Poi8on8
l)lasnia membrane and penetrates into the
Poisonous substances usually alTcct niicro- c(41. The fat -soluble substaiutes are, tlierc-
or<!;anisnis inone of the follo\viii<>; ways: 1. fore, moi-e poisonous. The strongest poisons
At certain low concentrations, which ai-e ai-e the metallic salts. AgNO.3 in a concentra-
definite for each substance, there is no aj)- tion of 1 :8()0,()()() most bacteria, and
will kill
preciable action. 2. At somewhat higher con- llgClo is still more toxic; they are most
centrations, these substances may exert a soluble in ether, alcohol, and fats. Iodine
St inudatina; effect u])on growth. A further
'.].
and otluM' substances, such as alcohols,
increase in concentration of th(> substance chloroform, and CS2 behave in a
ether,
produces a bacteriostatic effect upon growth. similar manner. Copper salts are not soluble
4. Still further increases ha\-e a microbicidal in lipoids, l)ut tend to foi'm complex organic
between stages 2 and o, but hardly any be- Of the various microbial poisons, the fol-
tween 3 and 4, because a concentration lowing groups are found to be particularly
which has a bacteriostatic effect may be- effective: salts of certahi heavy metals, in-
come bactericidal on prolonged action. cluding silver, gold, mercury, and copper.
With regard to their antimicrobial effects, The last two have found an extensive ap-
chemical substances are looked upon as in- plication as fungistatic and fungicidal
dil'ferent agents, as stinudants, or as poisons, agents. Iron has little antimicrobial activity,
depending entirely upon the activity of the and lead, nickel, and zinc are not active as
particular substance. poisons. ]\Iineral acids, particularly those of
Microbial stimulants have been classified the halogen group, have a powerful action.
into three groups: (a) chemical stimulants, Of the soaps, only the salts of saturated
(b) effective nutrients, including oxygen, fatty acids are poisonous. Potassium per-
and (c) effective metabolic products. When manganate and peroxides have a strong
])resent in very small doses, toxic substances effect. Formaldehyde is effective. Ethyl
may act as stimulants; fluoride, for ex- alcohol is active only in certain dilutions
ample, increases the production of zymase with water. Of the aromatic compounds,
by yeasts. The chemical stimuli are often phenol and its derivatives, especially the
differentiatedbetween those that affect the chlorinated and brominated compounds, are
germination of spores and those that favor
most important.
growth and reproduction.
A number of other organic compounds, as
Kruse proposed the following rules foi- th(^
substituted ammonium salts, salicylanilide,
toxic action of poisons upon microlx^s: 1.
dichlorodihydroxybenzomethane, and many
The bacteriostatic or l)actei'icidal action of
others, lun-e found extensive use as fungi-
a poison increases with the concentration. 2.
amount of inoculum,
cides. The dyes may be added to the list of
\\\\\\ an increase in the
bactericidal agents.
the toxic effect of the medium decreases or
disappears. 8. The weaker the nutritive In recent years, a new type of antimi-
power of the medium, the greater is the crobial agent has gained universal recogni-
toxicity; it is strongest in water and weakest tion. It comprises the antibiotics, or com-
in protein solutions. 4. The poison has a pounds of microbial origin. Their effect upon
greater bactericidal action at higher tem- ictinomycetes are discussed in detail in
Mineral elements play a highly important in their nutrition. It was soon established
role in thegrowth of microorganisms. They that the great majority of actinomycetes,
function both as essential nutrients for cell like other microorganisms, grow at rather
synthesis and as i-egulatory mechanisms for low concentrations of salts. Some, however,
various transformations that take place in are able to tolerate very high concentrations.
the living systems. The composition of most
Essential Nutrients
of the synthetic media used for the growth of
actinomycetes bears out this fact. Aside Among the organisms studied most ex-
from the required sources of carbon and tensively from the point of view of mineral
nitrogen, actinomycetes require phosphorus, requirements, S. griseus occupies a leading
sulfur, iron, potassium,magnesium, and cer- place. A chemically defined medium is more
tain other inorganic elements. In some cases, desirable than a complex organic medium
as in the production of certain antibiotics, for investigating metal re(iuirements for nu-
pigments, and \itamins, such elements as trition and for antibiotic production.
potassium, calcium, chlorine, manganese, co- In a study of the mineral requirements of
balt, and zinc play most interesting parts in S. griseus by Chesters and Rolinson, a chem-
the biochemical reactions involved. ically definedmedium was used. It was made
In most of the earlier studies on the effect metal-deficient by treatment with chloro-
of inorganic salts on the growth of actino- form solutions of diphenylthiocarbazone at
mycetes, complex organic media were used. pH 7.3 to remove zinc and copper and with
Miinter (1916), for example, used a medium 8-hydroxyquinoline at pH 5.2 to remo\'e iron
containing blood protein, gelatin, and agar. and at 7.3 to remove manganese. Media
He still observed that potassium and sodium from which the metals were omitted singly
salts, when used in 5 per cent concentrations, were compared to media in which they were
are favorabU' for growth but not for sporu- included. When zinc was omitted, the me-
lation. He further noted that the addition of dium supported the synthesis of only 75 mg
small amounts of Ca, Ba, and Sr uere fa- of cvU material per 100 ml of medium. Ad-
vorable for growth and sporulation; higher dition of zinc eciuivalent to 1 part per million
concentrations were injurious. ofmedium permitted maximum growth (550
With the recognition of the important lole mg/100 ml) and maxinuun streptomycin
of actinomycetes as producers of niilibiotics ])r()ducti()n. Further increases in the con-
and vitamins, there has Ixhmi an cvvv grow- centrations of zinc resulted in decreased
ing interest in the role of mineral (>lements antibiotic production, so that only 50 per
138
mini:kal MirrAHoi.isM and iifff-ct oi" sai/fs ox ckowtm \:\9
cent of tlio niaxiniuin antibiotic product ion tassium, magnesium, zinc, and iron were
took j>lac(> with oO parts per nnllion of zinc. needed good streptomycin production
for
Tlic addition o\' ().()') part per niillion of cop- and supported excellent growth. .Manganese
jxT it'sultcd in optimum {!;ro\vtli and anti- sliinulalcd antibiotic production but liad no
biotic production. Iron afTcctcd ii;ro\\lh and citect on growth. Calcium had no effect on
formation of tlio antibiotic at ditfcM'cnt IcncIs: either antibiotic production or growth.
optinnun concentration for f^rowtii was 0.8 Temple found that magnesium and potas-
l)art per million, wliereas ojitinunn antil)i- sium exerted the most noticeable effect on
otic production required 1.0 to 2.0 parts of both growth and antibiotic production. Iron
copper. Xo etTect of manganese could be ob- had a lesser effect on growth but was re-
served o\er a range of O.OOo to 50 parts jicr ((uii-ed for high streptomycin yields. A modi-
million. fication of Thornberry's medium to contain
Thornberry and Anderson developed a annnonium citrate and inositol required ad-
synthetic medium for streptomycin produc- ditional calcium. If tap water were used in
tion which contained, in addition to carbon place of distilletl water, no additional cal-
and nitrogen sources, potassium, magne- cium was needed. Saunders and Sylvester re-
sium, zinc, iron, copper, and manganese. The ported that traces of zinc, copper, iron, mag-
effect of a number of metals was studied bj^ nesium, and manganese were necessary for
adding the metals to, or omitting them from, optimum streptomycin production. Principe
this medium. Growth was estimated by com- and Thornberry found that the addition of
l)arison with that obtained in a complex me- cobalt in a concentration of 0.003 M in-
dium. The conclusion was reach(>d that po- creased streptomycin production by 83 per
140 THE ACTINOMYCETES, Vol. I
cent. Growth was not affected at concentra- stimulation of growth appeared to result
tions lower than 0.009 M but was inhibited from ions of bromine at 2 parts per million,
at that and higher concentrations. zirconium at 10 parts per million, and all
fect at any concentration employed. Slight copper and iron caused an increase in growth,
MlM.KAl. Mi:i'.\U()|.ISM WD 1 ;i'l"i:( "I' Ol" SAI/I'S ON CHoWni
niNciii production. M;in,i;;inese ;ind zinc, Kjffivt of imldl.'i on the {/loirlli, fitrcploini/cia
in antibiotic production.
Streptomycin production by S. hiLinicnsis
magnesium, had no such effect. Lechevalier ein solution. Iron appeared thus to play a
was able show a reciuirement for traces of
to (|uantitative role. Zinc had no effect on
potassium, magnesium, iron, and calcium for grisein production.
optimum neomycin production in media con- Kelner and Morton reported that iron has
taining glutamic acid and glucose. a similar effect on the production of actin-
Dulmage also reported that in a synthetic orubin. In a trypt one-glucose medium sup-
medium containing glutamic acid and glu- plemented with mineral salts, ferrous sulfate
cose to which metal salts were added, it was added in concentrations of 5 mg/1 resulted
necessary to have potassium, magnesium, in increased antibiotic production, whereas
and calcium for both growth and
iron, zinc, concentrations of 20 mg or more caused a
neomycin production. When glutamic acid decrease in yield.
was used as the only source of carbon and In various other studies the production of
nitrogen and the medium treated with 8- antimicrobial activity by three strains of
hydroxyquinoline or ethylenediaminetetra- streptomj^cetes was foimd to be influenced
acetic acid to render it metal-deficient, a re- by manganese, zinc, iron, and copper. Con-
quirement for iron, calcium, magnesium, and centrations lower than 2 mg/l were ineffec-
zinc for neomycin production by S. fradiae tive. Copper was inhibitory at a concentra-
was reported by Mohan and Nickerson. tion of 10 mg/1. A complex medium was used
These investigators were able to show a re- in these investigations.
quirement for calcium and magnesium for The addition of different concentrations of
growth, about a 50 per cent decrease in sea water to a glucose-peptone-yeast-extract
growth when iron was omitted, and no effect medium resulted in an increase in soil iso-
on growth when zinc was omitted from the lates (Jann et al.). Two streptomyces cul-
medium. tures gave increased antibiotic production
Acker and Lechevalier made a study of on addition of sodium, potassium, calcium,
some nutritional requirements of S. griseus and magnesium as chlorides, sulfates, or
for growth and candicidin production. A nitrates. With a third culture all of the above
synthetic medium was used; it was made metals, especially calcium caused an increase
metal-deficient by calcium carbonate co- in production of antibiotic. In the case of a
precipitation. Essential requirements for fourth culture, only calcium and sea salts
potassium, magnesium, iron, and zinc were gave an increase in antibiotic yield.
demonstrated. No effect of manganese on The importance of cobalt in the produc-
either growth or antibiotic production was tion of A'itamin B12 due to the presence of
is
concentrations of iron were increased, gris- limiting factor for \-itamin pi'oduction l)y S.
ein activity increased logarithn\ically. This griseus, e\'en in acomplex medium. Cobalt,
was due to the fact that ii-on is a part of the added as Co(\03)..-6H20, caused a 3-fold
grisein molecule. When iron was added to a increase in vitamin B12 production, compared
.mixi:h.\i, mi:i\\H()I.ism and i'.kkixt of s.\i/rs ox cHowi'if 143
CONTROL
4
DAYS
Figure 68. Effect on streptomyces mycelial gruwth produced by various concentrations of compost
soil extract (Reproduced from: Spicher, G. Zentr. Bakteriol., Abt. 2, 108: 579, 1955).
to mediti from which cohaU was omitted. The necessity for col)alt to gi\'e increased
Levels of 1 to 2 parts pei- milhon cobalt yields of vitamin B12 by S. olivaceus and
yielded maximum results; levels of 20 to 50 other organisms is now well established (Hall
parts per million were toxic. Colialt has et al.). Coppei', at concentrations of 82 parts
since been used routinely in culture media per million, almost completely inhibits vita-
for screening and for production of vitamin min B12 synthesis; at concentrations of 133
Bi-> The form of cobalt and the concenti'a-
. parts per million, it permits only slight
vestigator and the media used. A cobalt -tol- or vitamin production was observed at con-
erant culture of Saccknromijces cercvimie, centi'ations of oO parts per million. Strips of
developed Perlman and O'Brien, added
l)y metallic copper in the medium inhibited
to a soybean meal-glucose mediimi inocu- vitamin B12 synthesis. Strips of stainless
lated with *S. griscus could be utilized as a aluminum, iron, or lead placed in
steel, tin,
source of cobalt for ^•itamin B]-.) production the medium showed no effect.
more efficiently than inorganic cobalt added In addition to its effect upon the produc-
as the nitrate. The increased efficiency was tion of \itamin B12 , cobalt exerts other
noted over a concentiation range of 0.01 to effectsupon the gi-owth of actinomycetes.
0.1 ;ug ml, but not at higher levels. (See Hickey and Tresner reported that the addi-
also Burton and I.ochhead, C'harney, Du- tion of 2 mg of CoChi-filljO to a modified
lancy and Williams, Kojima and Matsuki, Bennett's medium gi'eatly laN'ored the rate
Principe and Thoriiberry, Smith ct ai). and extent of sporulation of S. Jradiav and
Radioactive vitamin B12 was produced l)y of a number of other streptomycetes. Sj)oru-
S. griseus from cobalt^" (Chariet et al.). lation was suppressed when the cobalt was
144 THE ACTINOMYCETES, Vol. I
myces. Zinc sulfate concentrations of 0.001 metals and concentrations were: iron at 0.05
per cent increase the rate of pigment forma- mg per 100, manganese at 0.0005 mg per
tion in strains of red-yellow-pigment -forming 100, and zinc, copper,and molybdenum at
streptomycetes (Chain et al.). Omission of 0.005 mg per 100. Combinations were more
magnesium or calcium from the medium re- effective than single additions, the most
duced the amount of diffusible pigment but effective being iron-zinc, iron-manganese-
omission of zinc or iron did not have any zinc, and iron-manganese-zinc-molybdenum
effect on pigment production by S. fradiae (Fig. 69).
60
§40
<
MIM'.KAI. MI.'I'AKOI.ISM AND IlKl'llCT <»l' SAI/I'S ()\ CHOWTH 145
inyeos cultures was studied. 'I'lic metals wei'e cated a special effect of this elenienl on he t
lluMi added singly and in all possible e(tiu- dilTusion of streptomycin into the culture
l)inati()ns at eoneenl rat ions of ;i parts pei' medium of N. iiris( us. The presence of this
niillion. (Ifowtli was usetl as the eiitefion of element does not ap|)eai' to be a general re-
(>tVeet,and the results wore analyzed statis- (luirement for nutrition of actinomycetes.
tically. Iron and zinc \V(>ro h(>no(ieial foi- the -Manganese is not i-e(|uire(l generally for
iirowth of all eii;;ht cultures iiucst i.<;ated. growth or ant il)i()t ic product i(.)n in some ;
production. Mn could replace Mg in the Stapp has also shown that actinomycetes
growth requirement, but only a Mn con- can tolerate a high salt concentration. Many
centration of 10~- M
allowed any chloram- strains were able to grow in a 10 per cent
phenicol production. In a medium containing concentration of KNO3 NaCl was tolerated
;
optimum concentrations of Zn, Fe, and Mg, by some strains in a maximum 8 per cent
the presence of the other microelements concentration, 6 per cent by others, and
studied, singly or in groups, had little effect only 1 per cent by some strains
still others;
on growth or the production of the antibiotic. tolerated 30 per cent NaS04 and others only
Metals are common contaminants of most 2 per cent. Some strains were able to grow
of the media used for the growth of actino- in a 10 per cent concentration of MgS04
mycetes. Complete removal of some metals, and one grew weakly even in a 50 per cent
such as calcium, copper, and iron, is virtu- concentration of the salt.
have been reported to occur in sea water and per cent NaCl. The fact that actinomycetes
are found in salt-rich substrates, such as
in sea bottoms. According to Kober, S. oligo-
carbophilus, grown in a 0.5 to 0.6 NaCl so- M curative muds (Rubentschik) and in the sea
close to shore, speaks for their adaptation to
lution, produced fewer but larger colonies
salt-rich environments. Krassilnikov re-
than in XaCl-free solutions. Although
MgS04 is not essential for growth, it had a ported on the ability of various actinomy-
cetes (Streptomyces, Nocardia) to grow well
favorable effect even in fairly high concen-
in the presence of 10 per cent NaCl or 20
trations. Calcium was not essential for
per cent Na2S04 . He noted that the major-
growth, but its absence had an injurious
ity of actinomycetes are able to grow in
effect unless magnesium was present. Potas-
higher salt than bacteria.
concentrations
sium, in concentrations of 0.5 per cent KCl,
This was true particularly of the pigmented
was injurious; this effect was neutralized by
organisms.
high concentrations of MgSOi . S. oligocarbo-
These results tend to suggest that the role
philus is noticeably hallophilic^ and could tol- of metallic elements as integral parts of cell
erate 25 percent MgS()4 THoO, butonly 5 per systems and of catalytic processes of en-
cent NaCl. At 15 per cent MgS04-7H20 zymes is of considerable importance. McFJ-
or at 3 per cent NaCl, the colonies gi-ew roy and Nason presented certain patterns
well. which appear to be e\-ol\ing from physico-
MINIIHAI. Ml.l "AHol.ISM AND I'.IM'l.Cr Ol' SAI/I'S ( »\ CIJOW'IMI 147
chemical and nut ril ioiial .^liidics ol' metals Ivxamples of this kind are the iron in the
ill \arinus metabolic I'eactions. .Mdlylidc- antibiotic gi'isein, cobalt in \ itamin li|j , and
iium. coppcM', and iron are said to lie mo,si iron ill a st rept oiiiycete |)igmeiit known as
closely assoi'iated with elect I'ou-t ranst'errinu; ferroxci'din.
systems. ""Phese metals are not reiniired spe- Outward manifestations of the effects of
eilieally t'oi' the comlMnation of substrate to metals u|)on a microorganism can be ob-
protein, l)iit ratht>r t'unction as "el(M'tron ser\-ed as changes in giowth, sporulation,
couplers" from one protein system to an- and ability to utilize ceitain substrates as
otlu'r. Matiuesium and, to some tlej2;r(H', man- \v(»ll as the formation of metabolic products
ijanese function primarily in ij;roup transfer such as pigments, and vitamins.
antil)iotics,
phate. Manganese and, to a lessor dosroo, t lit ion has been studied largel}^ from the
zinc and magnesium, predominate in general point of \iew of antibiotic production. It is
Metals may also play an important role reaction in connection with the effects of
as a structural part of a specific molecule. metals in the actinomycete system.
CHAPTER 9
Biochemical Activities
The manifold aspects of the biochemistry phate, were obtained by Hockenhull in fluo-
of actinomycetes are from elucidated.
still far ride-inhibited systems. Glucose oxidation
Nevertheless, considerable progress has been was depressed by 10~^ M
sodium iodoacetate
made in recent years in our understanding of and by 10~^ M
sodium arsenite, but was
some of the chemical mechanisms involved stimulated by 10~- M
sodium arsenate;
in the growth and activities of these organ- 10-=^ M
2,4-dinitrophenol and 10-« so- M
isms. By far the greatest number of investi- dium azide had no effect. Streptomycin pro-
gations on the activities of actinomycetes duction was decreased by 3 X 10~^ so- M
have been concerned largely with antibiotic dium arsenate but not by 10~- sodium M
production and the mode of action of anti- fluoride or 10~^ M
sodium iodoacetate. *S.
biotics. Limited consideration has also been griseus metabolized members of the tricar-
given to certain other fundamental princi- boxylic acid cycle, although citrate and a-
ples of actinomycete biochemistry. ketoglutarate gave much lower values of
CO2 at pH
than did pyruvate, acetate,
7.3
Respiration succinate, fumarate, or malate. Keto acids
Hockenhull et al. reported that, under were produced, in presence of arsenite, from
highly aerobic conditions, glucose was con- fumarate, malate, glucose, lactate, acetate,
verted by S. griseus mainly to cell material succinate, glutamate, and citrate in descend-
and CO2 Under restricted aeration, lactic
. ing order of yield. Except from fumarate,
acid was also formed. Pyruvic acid was pro- which yielded some material behaving like
duced during the stages of most rapid a-ketoglutarate,the product was chiefly
growth. The metabolism of glucose by acti- pyruvate.
nomycetes was dependent upon the presence According to Cochrane and Hawley, ri-
from 0.62 mg/ml, in the absence of phos- tate. Fructose 1 ,6-diphosphate is dephos-
phate, to 1.64 mg/ml, in presence of 0.01 M phorylated by one or more enzymes, with
potassium phosphate. Further increases in optimal activity at pH 4.5 to 5.0; it also can
phosphate concentration did not affect the be oxidized by triphosphopyridine nucleo-
utilization of the sugar. tide-dependent reactions, involving glucose
Phosphate esters, tentati\'ely identified as 6-phosphate as an intermediate. The con-
glucose 1 -phosphate and glucose 6-phos- clusion was reached that S. coelicolor can
148
HiociiKMicAi, Acii\ i'ni;s 149
nu'taholizc <i;Iii('(»s(' ;i(M'(»liic;ill\- t luouiili cit licr o\i(li/.c(| only at sut)sl coMccnt
i-;ilc i;i t ions
()!• both of twi) p;it liw ;i\'s, the "licxosciiioiio- higher lli.iii iliose normally employed. The
phosphntc"' ;iii(l I he I'lulxlcii-Mcycrhot' cells li;i(l to he preinciih.'ited in older to ob-
sys|(>m. tain oxidation of fum;iiate, nialate, and
According to Douglas aiul San ('Icmciitc, citrate, (lilmour ct al. ha\-e further shown
homogenized suspensions of *S. scabies gi'own that the labeling pattern suggests that glu-
under sul)nierged aerated conditions arc al)lc tamic acid arises from acetate through ex-
to oxidize glucose, succinate, fumaratc, cit- tensi\-e operation of the conventional tricar-
rat(\ and acetate when placed in a Warhuig- boxylic acid cycle. The role of this acid
type nianometric icspironietcr. The high cycle in amino acid synthesis is establi.shed
endogenous respiration is due to oxidation by the incorj>oration of radioactixity into
of nitrogenous substances; the cells also mo.st of the amino acids of the cellular pro-
oxidize glutamate, aspartate, threonine, teinfrom proliferating cells utilizing acetate-
phenylalanine, glycin(\ and A'aline and leu- 1-C".
cine to some extent. It was suggested that Accoi'ding to Peilman, most of the studies
tyrosine is oxidized tiirough dihydroxyph(Mi- on the metaboli.sm of carbohydrates by S.
ylalanine with suliseciuent formation of griseus tended to indicate that carbon di-
melanin. Addition of dinitrophenol increases oxide is the main metabolic product; no
the oxygen uptake l)y cells in contact with accumulation of significant (luantities of
pyruvate and a-ketoglutarate, and decreases intermediates in this oxidation has l)een re-
the uptake in cells metabolizing glucose. ported. Some doubt was expressed whether
As pointed out previously, anaerobic acti- the Krebs tricarboxylic acid cycle operates
nomyces cultures are able to fix CO2 This . in S. griseus metabolism of glucose (Garner
has been confirmed by Pine, using C'*, as and Kofli(>r). The report of succinic acid
brought out inTable 31. Succinic acid pro- production by other species suggested that
duced by this organism had a high percent- at least some of the carbohydrate is metab-
age of its radioactivity in the methylene olized by these organisms by way of ti car-
carbons: "When grown in the presence of boxylic acid cycle (Cochrane et al.). Lactic
oxN^gen, 90 per cent of the total activity of
the succinic acid was found in the methylene T.\BLE 31
carbons as compared to G2 per cent when it .inaerobic fennctilation of glucose plus carbon
was grown anaerobically." dioxide-C^^ by Streptomyces sp. (Pine)
Butterworth et al. (1955) have shown that
Substrate or product
a C3-C1 condensation coupled with tricarbox-
ylic acid cycle activity is a major pathway
of carbon dioxide fixation by S. griseus. The
intramolecular distribution of C'^ in glutamic
acid, produced by the incorporation of la-
l)eled carbon dioxide, confirmed the theory
that the tricarboxylic acid cycle operates
extensively^ in the terminal respiration of
S. griseus.
Gilmour d al. (1955) demonstrated the
oxidation of tricarboxylic acid cycle inter-
mediates l\v nonproliferating cells of S.
griseus. Succinate and a-ketoglutarate were
150 THE ACTINOMYCETES, Vol. I
acid has been reported in and in*S'. griseus S. fradiae cultures where the medium is
100
O RESTING CELLS
A GROWING CELLS
TIME HOURS i
Figure 70. Utilization of acetate-1-C" by growing and resting cells of S. gnseus (Heprodnced from:
Gilmour, C. M. et al. J. Bacteriol. 69: 721, 1955).
luociii.MicAi. \("i"i\ ri"ii;s 151
With resj)iratory ('"( )_ , there was a deci-ease l'\u't her studies on respiration of N. (/risrua
in tiu' in(li\"iilual chemical i-eco\-eries with ha\'e been made by (lotllieb and .\nderson,
an increase in the aji;e of the culture. The ()ginsky (I and Inoue, whose work was
(tl.,
""M
•0
80
LEGEND
TO i
SO-
Q PROLINE
GLUCOSE SO -
f]J
MYCELIUM 40-
^ CARBON DIOXIDE 30 -
STREPTOMYCIN
20-
10 -
T
2 4 5
DAYS OF FERMENTATION
Figure 71. Carbon balance of S. griseus fcrmpiilatioii (H(>i)iii(lue('(l from: WoodrufT, H. B. and
Ruger, M. J. Bacteriol. 56: 318, 1948).
152 THE ACTINOMYCETES, Vol. I
Oxidation of Steroids and Related Com- cellshad no effect on the conversion. The
pounds antibioticshad no effect on the progesterone-
oxidizing ability of washed cell suspensions
It has long been recognized that actino-
of S. lavendulae grown in media supple-
mycetes are capable of decomposing fats
was mented with progesterone.
(Netchaieva) . Until A'ery recently, it
According to Vischer et al., submerged
commonly assumed that the only actino-
cultures of various streptomyces are able to
mycetes capable of oxidizing steroids are
convert cortexone to 1 Go; -hydroxy cortexone.
fonnd largely among the species of Nocardia.
Saburi et al. isolated several streptomyces
Turfitt, for example, demonstrated by means
which utilize cholic acid as the sole source of
of enrichment culture methods, that the
and other steroids carbon. The utilization of these acids de-
breakdown of cholesterol
pends on the nuclear constitution and the
in soils is carried out primarily by nocardias,
length of the side chains of the bile acids. S.
especially N. erythropolis. Among the other
gelaticus was found capable of converting
active species, he listed A^. aquosa, N. glo-
cholic acid in a C-22 acid (C22H28O4). When
berula, N. coeliaca, and N. restricta.
S. gelaticus was cultured in a synthetic me-
More recently, however, different strepto-
dium containing cholic acid as the sole source
myces {S. griseus, S. fradiae, S. venezuelae,
of carbon, it oxidized cholic acid to an acid
and S. aiircofaciens) were found and (Sisler
Herzog with melting point 280 to 282° (decomposes),
Zobell, Perlman et at., Hirsch et al.,
which was presumed to be 7a-hydroxy-3, 12-
et al.) capable of oxidizing steroids and lip-
Some species oxidized lipids completely
When it was
dioxo-A'*-bisnorcholenic acid.
ids.
grown in a medium containing cholic acid
to carbon dioxide, without the formation of
and glucose as the carbon sources, various
any intermediate metabolites.
intermediates such as 7Q:,12a-dihydroxy-3-
Washed cells of S. albns are able to con-
oxocholanic acid, 7Q:-hydroxy-3,12-dioxo-
vert estradiol to estrone (Welsch and Hens-
cholanic acid, 7a-hydroxy-3, 12-dioxo-A''-
ghem); pregnenolone is converted to proges-
and progesterone to cholenic acid were formed (Hayakawa et al.).
terone by S. griseus,
hydroxy-progesterone by various actinomy-
Hayakawa et al. (1958) later demonstrated
that S. rubescens possesses an alternate path-
cetes (Perlman et al.). The conversion of
progesterone-(A'*-pregnene-3 ,20-dione) to
way for the degradation of cholic acid from
that of *S. gelaticus.
Ai'^-androstadiene-3,17-dione by Fried et
According to Collingsworth et al., S.
al. suggested that the enzymes which carry
fradiae is able to convert ll-desoxy-17-hy-
out this transformation are adaptive in
droxycortisone (comp. S) to 17-hydroxy-
origin. The metabolism of progesterone by
corticosterone (comp. F).
washed *S. lavendulae cells grown in media
Webley and deKock studied the oxj^gen
supplemented with progesterone or in un-
uptake by washed suspensions of Nocardia
supplemented media was inhibited by azide,
opaca. The uptake was increased by the
cyanide, or arsenite but not by selenite or
presence of n-dodecane, n-tetradecane, n-
fluoride (Perlman et al. lOo.'S). The conver-
grown un- hexadecane, n-octadecane, and paraffin wax.
sion of progesterone l)y cells in
Decyl, lauryl (dodecyl), and octadecyl alco-
supplemented media was also inhibited by
hols also ga\'e increascnl oxygen uptake, but
the addition to the pi'ogesterone-cell-suspen-
sion of certain antibiotics, such as strepto- amyl, ?',soamyl, ?'.sY>hexyl, and heptyl alcohols
mycin. Addition of any of the antibiotics to were toxic. The long-chain fatty acids (C7-
the steroid-cell-suspension 12 or more hours Cir,) were all metabolized at \-ery low con-
after the steroid was mixed with the washed centrations (0.0012 iM).
. .
follow s: Acids with an odd ininitici' of cail)on annnoni.M, liber.ated in the decomposition of
atoms in the side chain (phenylpropionic, the proteins, was lost by \-olal ilizal ion.
phenyl\-aleric, and phen\lhept>lic acids) A iuimb(>r of amino acids can be utilized
wei'e conx'ei'ted to IxMizoic acid, and cinnaniic by actinomycetes in synthetic media. This
acid was an intei-niechate; o-hych-oxyphenyl- is true of i)roline, glutamic acid, arginine,
acetic acid was identified as a coinnion prod- aspartic acid, and histidine. The fact tliat
uct when acids witli an e\-en inmihei- of car- actinomycetes can utilize anunonia and ni-
hon atoms (plienylacetic, phenyihutyiix, trate as sourcesof nitrogen, and that amino
plienylcaproic,and phenylcapi-yHc) weixuised, acids are degraded to annnonia, suggests
thus support iiii;- tlie theory of /:J-oxidat ion that the carl)()n residue of the amino acids
as a mechanism of l)reakd()wn of short -chain may be more important in the actinomycete
fatty acids by A\ opaca. \\'el)ley ct al. (1957) economy than iwv the nitrogen-containing
later demonstrated that a strain of A^. opaca groups. Problems of deamination and amino
will con\-ert o- and 4-monochlor()phenoxy- acid degradation streptomycetes have
l)y
l)Utyric acids to the corresponding sul)sti- been discu.ssed by Gottlieb and Ciferri.
tuted acetic acids. During these conversions Actinomycetes are characterized by in-
an intermeditite is formed which proved to tense activity in breaking down proteins to
lie fi-hydroxy-7-(4-chlor()pheiio.\y) butyric ammonia. This is true especially when a long
acid. period of incubation is employed. These or-
Adelson et al. found in soil a strain of S. gani.sms are capable of allowing a large accu-
(piseus that had the capacity to bring about mulation of ammonia e\'en in the presence of
demyelination of boA'ine spinal cord in vitro. available carbohydrates.
Just what mechanisms this process involves When washed cell material of S. lavendulae
are still difficult to tell. was shaken with different amino acids at
pH 6.8, deamination could be measured by
Proteolytic Activities of Actiiioniycetes
ammonia formation. Arginine and histichne
Actinomycetes are capable of attacking a were acted upon most readily; /8-alanine was
large number of plant and animal proteins, deaminated only about one-third as readily
as pointed out elsewhere. Mace was among as D-alanine; leucine, isoleucine, and certain
the first to demonstrate their marked pro- other amino acids were deaminated not at
teolytic properties. The proteins are hydro- all or only in mere traces.
lyzed to amino acids, polypeptides, and The formation of proteolytic enzymes are
ammonia. Waksman and Starkey compared discussed in detail in Chapter \ 1
ing both the amount of protein decomposed sues, such ashinnan hair and feathers, or-
and that of cell material svnthesized. When ganisms belonging to the genus Actinoplanes
154 THE ACTINOMYCETES, Vol. I
90-
80-
"m
7D-
60-
LEGEND
50-
[J PROLINE
40- iL
^ HfYCELIUli
iAL
30-
20-
10-
J T
DAYS nr FTRMEN'ATlON
T T
Figure 72. Nitrogen l)alance of &. griseus fermentation (Reproduced from: Woodn.ff, H. B. aiul
Ruger, M. J. Bacteriol. 56: 319, 1948).
al).
Detailed studies of the decomposition of
hoof meal, horn meal, leathers, and similar
materials were made by Xoval and Nicker-
son and Xoval (Table 32). These investiga-
tors succeeded in isolating an enzj^me prep-
aration (keratinase) fi-om a culture of S.
fradiae, as is shown in Chapter 11.
.
ciiitin. Th(> following compounds were from the rumen of cattle. The organism is
to carry out this process. Unfortunately, isolated. They were found capable of utiliz-
little is known of the enzj^matic systems in- ing various salts of organic acids, including
volved in the action of these organisms upon stearate and palmitate, but not formate.
156 THE ACTIXOMYCETES, Vol. I
Kalininko came to the conclusion that the certain media. Gelatin, for example, does
decomposition of rubber in nature is carried not favor the formation of odoriferous sub-
out by molds and, especially, by actinomy- stances.
cetes. Among the latter organisms, three As is shown in Chapter 3, the odor pro-
forms were found to be particularly active, duced by actinom3^cetes is responsible for a
namely, *S'. cocHcolor, S. aurantiacus, and S. certain type of spoilage of fish that absorb
longisporus ruber. Further studies on rubber it into their digestive systems, from which
decomposition ha^'e been made by Spence it spreads throughout the bodies of the fish.
actinomycetes that appear to include species albus, A. roseolus, and .4. verrucosus, all
of both Streptomyces and Nocardia are capa- apparently belonging to the genus Strepto-
ble of attacking paraffin. A film is formed myces. He considered them as members of
upon the paraffin. Most of the lost paraffin the microbiological population active as geo-
is recovered as CO 2 . Oxidation of petroleum logical agents. Molisch (1925) also observed
hydrocarbons by marine organisms was cultures of actinomycetes capable of bring-
studied by Zobell ct al. See also Chapter 7, ing about lime precipitation. Colonies grow-
for hydrocarbon oxidation by nocardias. ing on media containing Ca acetate, 2 per
cent solution, are surrounded by crystals of
Production of Odors calcium carbonate.
Many actinomycetes, especially species of Krassilnikov (Issatchenko, 1948) observed
Streptomyces, are characterized by the pro- the precipitation of CaCOa on roots of plants
duction of a specific odor, which is typical covered with actinomycetes. In two old cul-
of freshly plowed soil. It is musty, or earthy, tures of actinomycetes isolated from salt
and occasionally fruity in nature. Rullmann lakes, Rubentschik (1948) observed the crys-
believed that the odor is characteristic of tallization, in protein media, of CaCOa .
streptomyces that produce cylindrical spores sen (1951) reported that A^. corallina can
do not give rise to any odor. The presence of nitrify up to ()4 per cent of oxime of pyruvic
carbohydrates in the medium favors odor acid in an inorganic salt solution. Glucose
production. The thermophilic actinomycetes inhibited nitrification, by stimulating sjni-
are responsible for the more fruity scents, thesis of cell material, at a C:N ratio above
which arise particularly from young cultures. 20:1. In peptone media, Nocardia converted
The odoriferous substance can be extracted the oxime almost quantitatively to nitrite,
from the culture. It is soluble in ether and the rate of its formation being etjual to that
Nitrogen Fixation
\'arious reports ha\'e been made in the
past of the ability of one or more actino-
mycetes to fix atmospheric nitrogen. Fedonn'
and Kudriasheva (1950) came to the con-
clusion that \arious actinomycetes are capa-
ble of fixing atmospheric nitrogen, thus con-
firming the claims of Emerson, Carter and
Greaves (1928), Kober (1929) and von
IMotho (1940). On the other hand, such
chums were denied by Beijerinck (1900),
Fousek (1912), Miuiter (1916), Waksman
(1920), and Krassilnikov (1938). Repeated
studies by Waksman (1920), Allison et al.
(1984), and Shinobu (1953) failed to confirm
the reports.
Recent evidence .seems to .suggest a nitro-
gen-fixing capacity for certain particular
by Wlich.
species, as reported for .4. spinae
Metcalfe and Brown (1957) described two
nocardias, A', calcarca and N. cdlulans, iso-
lated from grassland lime .soils. These cul-
tures were found to ha\-e the capacity to fix
Acid Production
As pointed out pre\'iously, during the
growth of actinomycetes in media containing
158 THE ACTINOMYCETES, Vol. I
large concentrations in the cultures of the cent glucose, the pH was lowered appreci-
sporulating strains of but the
*S'. griseus, ably, even in buffered media, as a result of
aerial mycelium-free mutants are capable of the formation of organic acids; with 2 per
forming considerable amounts of this acid. cent glucose, especially in unbuffered media,
Lactic acid also appears to be a characteristic the pH levels went down to as low as 3.2 in
metabolic product of various nocardias, as 2 days.
shown by von Plotho and others. Actinomycetes are capable of utilizing
The formation of succinic acid by various readily various organic acids. Some of the
species indicates that at least some of the higher molecular acids are converted in the
carbohydrate is metabolized through the process to lower molecular acids. Martin and
carboxylic acid cycle. During the growth of Batt have shown that the oxidation of pro-
S. fradiae in certain poorly buffered media, pionic acid by cell suspensions of Nocardia
sufficient acetic acid accumulates to inhibit corallina is stimulated by carbon dioxide.
further growthand metabolism of the or- Thiamine-deficient cells convert propionate
ganism. In these cases, the accumulated to pyruvate with a simultaneous accumula-
acetate is metabolized when the pH of the tion of succinic acid. Two oxidation path-
medium is been
raised. Propionic acid has ways to pyruvate were demonstrated; only
reported as a metabolite in the growth of one of these included a symmetrical inter-
micromonospora. mediate (probably succinate).
Cochrane and Dimmick found that, in a According to Masuo and Kondo, various
glucose medium in the presence of an excess streptomyces accumulate a-ketoglutaric
of CaCOs S. coelicolor produced large
, acid, with or without some pyruvic acid
amounts of succinic acid, small amounts of from glucose or glycerol. Some organisms
lactic acid, and traces of fumaric and an un- convert glycerol to dihydroxyacetone, and
identified keto acid. Volatile acids were not D-sorbitol to L-sorbose.
detected. This property of forming acids Among the other metabolic products of
from sugar was believed to be characteristic actinomycetes, it is sufficient to mention the
of the particular species. lactone of (S-hydroxy-Q:,a ,7-trimethyl pi-
Numerous other investigators reported melic acid, a substance found as a degrada-
that actinomycetes are able to produce or- tion product of certain antibiotics (Anliker
ganic acids from carbohydrates. Magnus et al.).
{
bi()C1ii:mi(\i. \c'n\ i'|-ii;s 159
t;iti\-(>ly ;md (iu:iiitit;ili\-cly, .-i .s|,il»l(> and saccharide was a branched struct lU'e of J)-
cliaractcristic properly of the cell under fixed aral)inose and l)-galactose units with soin(^
{'ou(iitions of j^rowlh. Addition of glucose of the arabinose forming nonredncing, ter-
to the niediuin in wliieh N. griscufi wa.s {>;ro\\ n minal residues. This e\idence points further
reiluced the concent ration of some of the to the close taxonomic relationship between
amino acids (arfiinine, histi(hn(>, lysine), hut .\ . (isfcroidrs and M
ycohaclcriiim liihrrculosis
not of otliei's (t ryptoplian). The iiitr()j;en (Bishop and lilank).
content of tlie cuHure was about 10. o per -Vccording to (iuinand d <il. {\\)')H), the
cent, inde])endent of adiHtion of suji;ar. mycelium of X . aslcrnidcs yielded on extrac-
Witter {Wr.VA) was unable to find either tion with 1:1 alcohol-ether and chloroform,
chitiu or a definite nucleus in the species of a mixture of lipoproteids, consisting partly
actinomycetes examined, a characteristic of peptides containing six amino acids and
that (Ustinji;uishes them definitely from the partly of acid lipids.
true funi2;i (See also Schmidt). Hagcdorn Romano and Nickerson made a study of
established an isoelectric point for actino- the cell The cells were first
wall of S. fradiae.
mycetes also similar to that of bacteria. broken in the Mickle disintegrator; the cell
Detailed cell wall studies support the bac- walls were then readily lysed by lysozyme.
terial natun^ of the actinomycetes. Xo evi- On hydrolysis with 2 A'' hydrochloric acid,
dence was found of the presence of polymers, reducing substances, accounted for largely
such as chitin, mannan, glucan, or cellulose, by hexosamine, were liberated. The cell wall,
which are found in yeasts and true fungi. like that of gram-positive bacteria, appears
The cell wall composition in all of the acti- to contain a mucopolysaccharide in associa-
nomycetes studies resembled the cell wall tion with protein.
composition of gram-positive bacteria. The According to Sohler and Honiano, the cell
majority of the streptomycetes are lysed by wall of Strcptomyces mucoid in spcM'ies is
Table 35
Tablk 36
Amino aci(l.s foiiiid in hi/droli/ziitcs of (iclinonn/ccle cell iralla* (Solilcr, Koiiiaiio, and .\"ii-kers(ju)
Amino acid
162 THE ACTINOMYCETES, Vol. I
Table 38
Sugars identified in the cell ivalls of
actinomycetes (Sohler)
Table 39
Nitrogen, sulfur, and phosphorus content of
actinomycete cell walls (Sohler, Romano,
and Nickerson)
Concentration,
per cent
j{i(>('iii:Mi('AL A("i'i\i'ni;s 163
Tahi.k 40
Composition of cell walls of some (jram-posilivc Eiibucleriulcs and AcliiwmyccLalcii (Work)
164 THE ACTINOMYCETES, Vol. I
mj^cetes to antibiotics produced b}^ other production lagged behind the growth peak.
organisms has been used as a method of se- The presence of oxygen was essential for
lecting and identifying such cultures. A cer- streptomycin synthesis (See also Woodruff
tain culture may be sensitive to its own anti- and Ruger, Eiser and ]\IcFarlane, Christen-
be much more sensitive to other antibiotics. The effect of nutrition of S. griseus upon
Some strains of griseus, however, have
*S'. streptomycin production has been studied
been found to be much less sensiti\'e to strep- by Bermett (1947). The effect of different
tomycin than are many other strains. Ex- amino acids on the biosynthesis of strepto-
posure to streptomycin has been used as a mycin is illustrated in Table 41.
method of selection of new cultures, pre- The biosynthesis of chlortetracycline by
sumably higher-yielding types. aureofaciens has been studied by DiMarco
*S'.
An
examination of data on the efficiency (1956), and of erythromycin by Corum et al.
of conversion of substrate to streptomycin (Table 42). The effect of specific nutrients
indicates that approximately 15 per cent of upon the formation of different actinomy-
the carbon added to the medium, either as cins and upon the constituent forms of the
carbohydrate or as glycerol, may be con- same actinomycins has been examined by
verted to streptomycin. The actinomycetes Brockmann and his group, by Schmidt-Kast-
producing chlortetracycline and chloram- ner, and by Katz et al. (1958).
phenicol are also capable of incorporating Hunter et al. have shown, by the use of
into the respective antibiotics substantial C''*02 , that the carbon of the guanidine side
quantities of the chloride ion present in the chains in streptomycin is derived largely, if
medium. It is also probable that the grisein- not entirely, from COo Further information
.
have shown that the peak of streptomycin et al., Petty and Matrishin, Yagashita and
Table 41
Effect of different amino acids on growth and streptomycin production by S. griseus (Spilsbiiry)
Nitrogen source
lilOCllliMlCAl. AC"n\ ITIICS 165
Tabi,e 42
Cheiniciil chiuuns in (hr .'<i/nlhclir medium inoculated with S. crytlireus (C'oruin ct at.)
Days
-
filaments undergo fragmentation. Presence nation reactions; for the other six, aggluti-
of glucose inhibits adaptation to maltose. nation could be demonstrated only with diffi-
The nature of adaptive enzyme seemed to be culty.The spores of the latter appeared to
different from maltase. For a maximum pi'o- contain more of the agglutination receptors
duction of the mycelium, a switch to a slower than did the mycelium.
and more economical metabolism of carbo- Breley (1933) obtained such highly erratic
hydrate, such as that of maltose in the results for complement fixation and precipi-
second growth phase, is essential. tation that he concluded, "Nocardia and
Streptothrix are bad antigens in vivo and
Antigenic Properties of Actinomycetes in vitro. ^^ Lieske's results also tended to ques-
Considerable attention has been centered, tion the significance of the results obtained
in recent years, upon the antigenic proper- by this method in differentiating various
ties of actinomycetes. Biagi (1904), in look- groups of actinomycetes. Lentze (1938), as
ing for a suitable system for classifying ac- well, drew attention to the technical diffi-
tinomycetes, other than their morphology culties involved in carrying out agglutina-
and physiology, decided to study their ser- tion experiments with actinomycetes, be-
ological behavior. He immunized rabbits with cause of frequent spontaneous agglutination.
five aerobic forms and made cross-aggluti- He demonstrated the existence of R and S
nation studies. Homologous and heterolo- strainsamong the cultures isolated from
gous agglutination of low titer was obtained, clinical cases of actinomycosis. Goyal (1937)
but the tubercle bacillus was not aggluti- studied a number of cultures of actinomy-
nated by any of the antisera. Calendoli cetes, representing at least three of the
(1905) was also able to show some cross- genera now recognized ; all of them were des-
agglutination reactions between two actino- ignated, however, as strains of Streptothrix.
mycete cultures. Choukevitch (1909), work- He came to the conclusion that an extract,
ing with organisms that appeared to belong comparable to tuberculin and designated as
largely to the genus Nocardia, obtained an streptothricin, had the same antigenic prop-
antiserum, by intravenous inoculations, that erties as similar extracts of the tuberculosis
"vvould agglutinate \'arious strains. Leao and diphtheria organisms. He suggested the
(1928) used sera from human actinomycosis presence of antigens common to all these
cases and an antigen from one of the isolated groups. This did not agree, howe^'er, with
cultures; positive agglutination was obtained the results of other investigators. Claypole
in a titer of 1 : IGO, complement fixation being (1913), for example, demonstrated that sera
positive, and precipitin tests negative. Holm from aerobic pathogenic nocardia and hu-
(1930) divided a group of strains of the man tubercle bacillus would show, b.y com-
anaerobic Actinomyces into two subgroups plement fixation tests, a certain degree of
on the basis of cross-agglutination. quantitative differentiation between the
Aoki (193G) has shown that complement antigenic substances of these organisms.
binding reactions of actinomycetes cor- Erikson (1940) established that the an-
responded fully with their agglutination aei'obic human (A. israeli) antl l)()\'ine (.4.
lilut illation reactions aiiioni!; the \arioiis nation techni(|Ues olfer promising tools in
li-enera of the act inomycetes. 'I'hey demon- the classification ui Stnptomjiccs species.
strated that tlie mici'oaerophilic foi-ms are \\'o(lehouse and Hackiis suggested an in-
seroIoi;icall_\' related. The j)reseiice of com- genious method for iitili/.ing the antigenic
mon f>;roup antiiiieiis was iiuHcated. Slack r/ properties of actinomycetes for taxonomic
al. (M).").")) (Uvided tlie microaeropliilic forms purj)oses. The double diffusion techniciue on
isolat<Hl from ditf(M-(Mit sources into (wo sero- agar media was used. Precipitation bands
lofj;ical firoujis, tliiis (>stal)lishiii,<>; that habitat ar(> formed wiien the diffusion from antigenic
does not correlate with antigenic composi- sources encroach upon that of antiserum.
tion. U two extracts are alike, their bands join
Solovieva cf al., Yokovama and Hata, and to form arches above the serum source. If
Okami arc among others who studied the they are unlike, they cross each other with-
serological reactions of act inomycetes. Gon- out interference. Various degrees of relation-
zalez-Ochoa and \'as([U(>z-Hoyos (1953) were ship i)etween organisms can thus be ob-
able to di\-ide the pathogenic actinomycetes tained. There was l)ut little overlapping with
into four groups on the basis of their sero- the other genera of the Actinomycetales.
logic relations: (a) the 6or/.s' group, including
also certain Xocardia species; (b) the .s-o-
Other Biochemical Activities
maliensis group; (c) the madurae group; and Because of their special significance, sev-
(d) the paraguaycus'i.s gi'oup, the last show- eral other biochemical properties of actino-
ing serologic relations with the streptomyces mycetes are treated in detail in separate
isolated from soil, namely S. albus, S. griscus, chapters. These include the formation })y
and S. lavendidae. actinomycetes of enzjanes, of \arious lytic
The fact was always emphasized that mechanisms, pigments, vitamins, and anti-
many of the difficulties involved in the analy- biotics. The antibiotics are receiving par-
sis and interpretation of the results of the ticular consideration because of their grow-
antigenic and serological reactions of actino- ing practical importance (Chapter 15, and
mvcetes are due to lack of certaintv as to the Chapters 31-30, Volume III).
CHAPTER 10
Lytic Mechanisms
Lysis of microbial cells in general and of undergo lysis. The animal pathogens were
actinomycete cells in particular comprises a the first to receive consideration for their
nmnber of reactions, some of which fre- capacity to lyse at certain stages of growth.
quently are considered as enzymatic proc- Later, this phenomenon was shown to hold
esses. These can be classified into three true also for plant pathogens. Finally, some
groups: (a) autolytic reactions, in which the of the soil saprophytes were found to pos-
growing cells, at a certain stage of their de- sess the same property.
velopment, undergo lysis; (b) bacteriolytic The mechanisms responsible for the proc-
processes, whereby certain complex mechan- esses of autolysis among actinomycetes
isms involving also enzymatic reactions have been variously called lysins, autolysins,
was clianuicd to alkaline. In hroth cultures, uj^on dead cells. The conclusion was reached
lysis took i)la('(> in 2 to :\ weeks, and was as- that the autolytic substance of actinomy-
sociated with the t'onnalion u[' a nonenzy- cetes isi)roduced in the cells themselves and
niatic and nontransniissible lytic factor. is liberated at the nioniciit of their decompo-
Diniitriev and Soutiex- (I!) 17) su<i;f^ested sition.
that tlu> lysed preparation of patho<;enic ac- Krassilnikoxand Koicniako suggested a
tinoniycetes, desii>;natetl as actinolysate, I'e.semblance of autolysis among actinomy-
could be utilized in the therapy of infectious cetes to phage formation by bacteria. The
caused !>>' these organisms. They empha- lytic factor of actinomycetes was found to be
sized, howe\er, that sucli ]M-eparations must highly specific, since it had no effect upon
first be })urified and concentrated. other species or even upon other .strains of
Krassilniko\' and Koreniako (IDi^S) stud- the same organism; it was thus distinguished
ied the phenomenon of autolysis amonji; fi-om lysozyme. Different strains of an or-
those actinomycetes that are now known to ganism underwent lysis with varying degrees
belong to species of Streptomyccs and .Vo- of rai)idity. Production of the lytic factor or
cardia. In the streptomyces, either the entire its mode of action was believed to be differ-
colony underwent lysis or only partial lysis ent for the different organisms.
occurnnl, usually beginning in the center of Of the 1000 or more freshly isolated cul-
the colony and proceeding to the perijjhery; tures studied by Krassilnikov, only a few
the colony liecame slimy, flat, and trans- were able to undergo autolj'sis. A culture of
parent, finally changing to a viscous consist- an organism that gave, when freshly iso-
ency. In the nocardias, the colonies were lysed lated, heavy compact growth co^'ered with
simultaneously throughout the entire sur- white aerial mycelium, produced, on contin-
face. The phenomenon of autolysis was not uous transfer, flat, smooth, and somewhat
accompanied by the formation of saltants or moist colonies that lost the pi"operty of form-
new races. The lytic substances were pro- ing aerial mycelium. Gradually the growth of
duced by the cells themselves and were not the culture was reduced to a thin slimy film,
of outside origin. Autolysis usually began at and, on repeated transfer, became trans-
the time of aging of the culture; at ()0 to parent, until the culture finally ceased to
70°C, complete lysis occurred in a few min- grow altogether; all attempts to keep it alive
utes. Any factors retarding growth of tlie or- were unsuccessful. ( )ther cultures of actino-
ganism were found to hasten lysis. Thus, a mycetes belonging to different morphological
culture of A^. cifrea underwent lysis under and physiological grcnips showed similar ly-
suboptimal conditions of temperature, or un- sis, although in different degrees.
90
o S. fradia*
o S. gritaut
N. polychromog«n«*
N .rubra
N. a«(«roid«>
30 40 50 60
TIME (MINUTES)
Figure Lysis of cell walls of Streptomyces vs. Nocardia by lysozynie (Reproduced from:
73. Romano
A. H. and Sohler, A. J. Bacteriol. 72: 866, 1956).
at 30 or 37°C, lysis takes place in 4 to 6 Lysis took place when the organism entered
hours. Not all the mycelium is lysed uni- the sporulating stage.
formly, some of the hyphae producing The process of autolysis in cultures of S.
chlamydospores, spherical bodies, or other griseus, the streptomycin-producing organ-
fragmentary material. Under favorable con- ism, has been studied extensively. When the
ditions, these bodies are able to grow and growth of this submerged culture
organism in
develop into fresh colonies. reaches a maximum, lysis sets in and frag-
Katznelson isolated from manure com- mentation of the mycelium occurs. The peak
posts a culture of a thermophilic actinomy- of streptomycin production lags somewhat
cete, whi(!h grew well on organic media. behind the growth peak. The changes in re-
When transferred to a synthetic medium con- action of medium take place in two phases.
taining ammonium sulfate and starch, it un- One occui;s during growth, when the medium
derwent lysis after 24 to 48 hours of incuba- becomes acid, and one is associated with ly-
tion at 50°C. During growth, the culture sis of when the medium becomes
the culture,
became acid. Addition of CaCOs to the cul- alkaline and may reach a pH of 8.6.
ture prev(!ntod lysis. Stanier reported that an Dulaney et al. (1947) differentiated two
agar-decomposing strain of S. coelicolor un- phases of metabolic activity of S. griseus,
derwent rapid autolysis and soon died out. grown inider shaken conditions:
LVl'K" .M1:C1IAN1S.M8 171
1. Tlu' j^rowtli phase, cliaracterizi'd hy for- resistant to the action of this antibiotic.
mation of inyccliuin, ri'ductioii of soluhlo This \ariant is similar to th(! active culture
imlricnts in the nii'dinni, production and in such cultin-al cliaracleristics as lack of
utilization of lactic acid, a lii^h oxyj^cn de- dark pigmentation on organic media, proteo-
mand, and little production of streptomycin. lytics action, and hemolytic capacity. By
2. Till' aulolytic phase, chai-acterized hy a pioper culture and selection, this asporogen-
marked decrease in \\eiij;ht of mycelium, an ous strain can be made to revert to the
increase in inorganic phosphorus and soluble sporulating strain, which will have the ca-
nitrogen in the medium, a droi) in oxygen j)acity of producing streptomycin. Some as-
demand to zero, and production of large porogenous strains are definite mutants, ac-
quantities of streptomycin. cording to Appleby.
Increases in carbohydrate or jjhosphate According to Lvnnb, *S'. griscu.s undergoes
content of the medium have but little effect lysis more rapidly under submerged condi-
on the general changes, although they may tions of growth than under stationary condi-
lengthen the growth phase. \'arious strains ti(jns. The culture tends to become viscous as
show no significant differences in the meta- a result of formation of the lysed material.
bohc changes, although they may show *S'. fradiac, the culture that produces neomy-
marked differences in streptomycin produc- cin, behaves in the same way. Lumb also
tion. observed that the accumulation of the anti-
8chatz and Waksman (1945), studying the biotics corresponds to the beginning of lysis.
production of streptomycin by different Various attempts have been made to cor-
strains of S. griscus, observed that colonies relate the phenomena of autolysis among the
devoid of aerial mycelium formed no strepto- actinomycetes and their bacteriolytic action
mycin (Dulaney found exceptions). Such upon gram-positive bacteria. Although both
colonies gave rise to cultures that underwent seem to involve the hydrolysis of proteins,
much more rapid lysis than the normal cul- bacteriolj'sis involves lower proteolytic ac-
tures with aerial mycelium. In the practical tivit.y, is active at a different pH le\'el, and
production of streptomycin, it is generally proceeds further than autol3^sis.
ob.served that, under submerged conditions Gorjunova considered the process of lysis
of growth, maximum formation or accumula- among actinomycetes as enzymatic in na-
tion of the antibiotic corresponds to the be- ture, since it is accompanied by the break-
ginning of lysis; advanced lysis usually re- down of proteins. According to her, the lytic
sults in a rapid destruction or inactivation of agents consist of two components that can
the streptomycin already produced. be separated by dialysis. Neither of these is
-4 N. polychromoganat
"O S. grium
• S. bobilio«
° N. rubra
80 100 120
TIME (MINUTES)
Figure 74. Liberation of reducing sugars (expressed as per cent of dry weight of cell walls), on 1
A' HCl hydrolysis at 100°C of cultures of Streptomijces and Nocardia (Reproduced from: Romano, A. H.
and Sohler, A. J. Bacteriol. 72: 867, 1956).
Lieske reported in 1921 that certain acti- Strahlenpilze einmal \o\\ therapeutischer Be-
nomycetes have the capacity to dissolve bac- deutung werden konnen, ist fraglich, aber
teria and other microorganisms. A suspen- immerhin nicht ausgeschlossen. Die grossen
sion of dead bacteria, such as staphylococci, Erwartungen, die man auf die Pyocyanase,
was mixed with agar and poured into plates. den entsprechenden Stoff des Bacterium py-
These plates were inoculated with actinomy- ocyaneum gesetzt hat, sind leider bisher
cete cultures and incubated. The actinomy- nicht erfiillt worden. Dass mit den Enzymen
cetes were able to dissolve the bacterial cells, der Strahlenpilze, die jedenfalls eine bedeu-
as shown by the clear zones produced around tende bakterizide Wirkung haben, bei An-
the actinomycete colonies. Lieske demon- wendung geeigneter Methoden bessere Ergeb-
strated further that living bacteria likewise nisse erzielt werden konnen als mit Pyo-
may be dissolved; both spore-forming and cyanase, ist keineswegs unmoglich."
nonspore-forming gram-positive and gram- In 1928, Lieske again compared the phe-
negative bacteria were sensitive to the action nomenon of bacterial lysis brought about l\v
of certain actinomycetes. Lieske considered cultures of actinom3^cetes with the action of
this effect to be due to bacteriolytic mechan- pyocyanase, a bacterial product, not enzy-
isms of an enzymatic nature. It may be of matic in nature. He noted that no further
interest to (juote Lieske, because of the pres- study was made of the actinomycete prepara-
ent great significance of the lytic mechanisms tion (which he considered to be an enzyme)
of actinomycetes. It is of particular interest and that it was not known whether it, like
to note that he questioned, although he did pyocyanase, could be used for curative pur-
not exclude, the therapeutic potentialities of poses.
this phenomenon. He .said: In 1924, Gratia and associates suspended
"Ob die bakteriolytischen Enzyme der dead cells of staphylococci and of other bac-
\
.
teria in ajj;ar and ('X1)()S(h1 the plates (o the nihcanl couclusion that actinomycetes pro-
air. A white culture of an actiiKMnycele. des- duce various substances that act upon bac-
i«j;nate(l as "St reptolhrix," l)ul delinilely ;i teria eithei- in an antagonistic or in a lytic
Stn i>l(>nii/C( s, dexeloped on the plat(>s. W'lien nKinnei'.
this cuhure was transt'en-ed to a suspension The most extensive studies ou the bac-
of dead staphx-loeoeci in sterile saline solu- teriolytic actixities of certain actinomycetes
tion, the bacterial suspension became clari- have been made by Welsch, following in the
fied in 'M\ liours, acconipanicHl by flaky footstei)s of (iratia and his associates. lie-
j;rowth of the actinoniycete. When the lysed ginning in 193(1, he and hiscollaboratorshave
emulsion was Hltered, was found capable
it published a large ninnber of pa))ers on this
of dissolvinj^ a Uvsh suspension of dead bac- lytic principle.
teria. This culture could attack all staphylo- Welsch first obtained a bacteriolytic jirep-
cocci tested, as well as certain other bactoi'ia, aration from a culture of .S. albus. It pos-
such as }^s. aeruginosa; howe\er, it was in- .<essed strong activites and was heat-labile;
acti\"e ui)on .1/. tuhcrcitlosis and K. aili. T\\v it was destroyed at (h')° in 2 hours and at 80°
lysetl material was desiji;nated as a mycolij- in ")
minutes. At low temperatures, it re-
satr. It did not possess the toxicity of a non- mained stable for many months; at pH 4.0,
lysed suspension, l)ut retained its antig(Miic it was destroyed in 24 hours. The active sub-
properties. Gratia later asserted that the stance was precipitated by ammonium sul-
"Streptothrix" culture was also able to at- fate, ethyl alcohol, and acetone. The produc-
tack living cells of bacteria, except E. coli tion of this bacteriolytic substance was
and Ehcrthdla typhosa, which had to be first closely rclatetl to the sporulation of the or-
killed by heat. ganism; there was no relation, however, be-
In 1935, l^orodulina demonstratetl the tween its production and autolysis of culture.
ability of certain actinomycetes to dissohe This substance was designated as actinomy-
various spore-forming bacteria, especially c(tin.
Bacillus subtilis. She emphasized that the Welsch further repoi'ted that some of the
lytic substance of actinomycetes is produced activity of actinomycetin in the filtrate was
in acid media but not in basic. The substance lost on passage through bacterial filters.
was resi.stant to heat, although it was de- Three groups of l)acteria were recognized in
stroyed in the autoclave. Xakhimovskaia their relation to actinomycetin:
(1937) also demonstrated the ability of vari- 1 Those bacteria which were lysed by the
ous actinomycetes to form a lytic substance, aiiueous extract of the agar cultures of S.
which is excreted into the medium. The sub- albufi, namely, pneumococci and hemolytic
stance inhibited the growth of various bac- streptococci.
teria and even dissolved them. 2. Bacteria which were not dis.sohed by
Krassilnikov and Koreniako (1939) con- the most acti\-e soluble suiistance, but which
sidered this property of actinomycetes to lyse were depres.sed by the mycelium of the acti-
certain bacterial cells as due to ly.sozyme, noniycete; these included various sarcinae
which was first described l)y Fleming in 1922. and B. megatherium.
Kriss (1940) isolated from a culture of S. 3. Bticteria which were not acted upon
violaceus, a thermostable substance which either by the mycelium or by the acti\-e sub-
was designat ed a s />ac7f'/v'o///.s/// Thi s subst a nee
. stance. These comprised the colon typhoid-
was soluble in water and was beliexed to be paratyphoid and the pyocyaneus groups.
diflerent from bacteriophage and identical When these bacteria were killed by heat or
with Ivsozvme. Kriss reached the i"ather sig- were placed under conditions unfavorable to
174 THE ACTINOMYCETES, Vol. I
multiplication, they were dissolved by the mechanisms the same preparation may con-
;
lytic substance.For instance, cells of E. coli tain both. The antibiotic-producing organ-
acted upon by radium emanation, which isms form no lytic substances or only very
stops their multiplication, became suscep- limited amounts.
tible to the lytic substance. Welsch and Thuysen (1956) summarized
Actinomycetin was shown to consist of a the bacteriolytic properties of S. alhus strain
protein-enzyme system, active particularly G. Evidence was presented for the existence
upon gram-negative bacteria and upon cer- of several specific systems: 1. A colilytic
tain living gram-positive bacteria. The pre- agent or actinozyme, responsible for the lysis
cipitated preparation was found also to of heat-killed gram-negative bacteria and in-
possess bactericidal properties. This "bac- volved in the dissolution of at least some
tericidin" was believed to exist in the culture heat-killed gram-positive organisms; 2. a
filtrate as a harmless substance, which was streptolytic agent, acting upon heat-killed or
activated on precipitation. living streptococci; 3. two pneumolytic
Further studies brought out the fact that agents, one acting upon either heat-killed or
the lytic principles of actinomycetes are living pneumococci, and the other upon the
rather complex in nature. They frequently living only; 4. a complex staphylolytic sys-
contain as many as four substances that dif- tem, dissolving living staphylococci and
fer in their action and in the organisms acted other bacteria and comprising two compo-
upon. The lysis of living bacteria by actino- nents which, after isolation and purification,
mycetin preparations was considered to be a were found to be specific peptidases acting in
result of two factors: (a) one acting upon the synergy upon some unidentified constituent
living cell, being a bactericidal factor; and of the cell wall. The enzymes of actinomy-
(b) the other acting upon dead cells, being a cetin which are able to dissolve living bac-
bacteriolytic factor. The second factor is terial cells were considered as true antibiotics.
helped along by the process of autolysis. The mode of action of the staphylolytic
Later, Welsch (1947) designated the bacteri- systems was likened to that of lysozyme,
cidal substance as ribonudeinase and the which also acts upon a constituent of the
which is the en-
lytic principle as actinozyme, cell wall of sensitive bacteria. Lysozyme pro-
zymatically active protein. This enzyme is duction by actinomycetes has been reported,
excreted by the organism in the process of but differences of specificity between this en-
sporulation. The presence of carbohydrates zyme and actinomycetin G were pointed out
favors its production in artificial media. previously. The unsusceptilibity of staphylo-
Muggleton and Webb (1952) also demon- cocci to lysozyme, due to the nature of the
strated that the exocellular bacteriolytic cell wall, was considered sufficient evidence
system of streptomyces depends upon an en- for discarding the idea of a lysozyme-like
zyme of the ribonuclease type; a deoxy- nature of the staphylolytic agent. The nature
ribonuclease was also present in the culture of the products liberated from cell walls by
filtrate. McCarty found that the lytic mech- the staphylolytic system definitely estab-
anisms of »S. albus are mucopolysacchar- lishes that the peptidases involved are dif-
U|)()ii ;i (It'filiitc ciiiupoiKMil (if the ti;i('t("ri;il |)rocesses and Iheir relation to true anti-
cytuplasin, access to wliicli is jxissiMc only biotics (DarpouN .Mnd i'"ai\ re-Aniiot ).
after disruption of the cell \v;ill, as seems to Paknla and lye used an act inoniycet in
\)0 tlu> eas(^ t'oi' trypsin. l)reparation for the extraction of deoxyri-
S\V(M"t/, niadi^ a study of the phenomenon lioniicleic acids from bacteria.
of halo t'ormation in the pi'ocess of hactei-io-
lysis hy actinomycetos. When llie actinomy-
l*r<Mlii<-| ion and Arli>ili<>s of A«'lino-
pliaj.M'
cetes were grown on l)aet(M'ial \vat(M- a.uar
plates (usiuo; heat-killed h\ coli and heat- \arious actinomycetes can he attacked by
killed or lix'inu; Staph. (iuf( iis in the a,u;ar), lilterahle \iruses or phages, known as acti-
the me(,liuni was clarilied, the zones of nophages. In this respect, the organisms show
hacterial ly^sis being at times suri-ounded l)y a high degree of specificity. These actino-
a halo of incomplete clarification. This phages occur abundantly in nature, particu-
phenomenon was explained by assum- larly in mamn-es and soils (Gilmour and
ing \hv existence of a staphylococcal factor Butala).
fa\()rable to lysis by actinomycetes. This fac- Wiebols and Wiei'inga were the first to ob-
tor diffuses from the cells heated to 52 to sei'\e, in 19:^0, that cultures of certain acti-
56°, or treated with an antiseptic, or dis- nomycetes, now considered as species of
solved through the action of actinomycetes. Streptomyces, isolated from infected potatoes
Gratia and Dath (1925) also spoke of com- underwent lysis. This was found to be due
plete lysis preceded by a preparatory stage to the production of a specific, transmissible
in which the microbes are agglutinated and phage. Repeated additions of phage-contain-
are swollen. ing culture filtrates to fresh cultures of the
Jones have shown that certain
et al. acti- organism resulted in the development of a
nomycetes possess a system composed of a phage which produced a large number of
lipoidal bactericidal substance, a ribonu- plaques in cultures of the same organism
clease, and a proteolytic enzyme. Killed grouii on solid or in li(|uid media. Phages ac-
gram-negative bacteria as well as gram-nega- tive upon the animal pathogens .4. bewis and
ti\'e forms of gram-positive organisms are .V. farcinica were also obtained. One culture
lysed; living organisms are not affected. produced a polyvalent phage which was also
Gram-positive bacteria are killed only under active upon *S. scabies.
conditions favorable to autolysis. made a study of the mech-
Miilhens (1941)
The lysis of pathogenic bacteria by strep- anism of phage action upon actinomycetes.
tomyces may be of considerable economic The whole colony was found to be attacked,
importance. A culture producing a wine- beginning at the periphery and proceeding to
colored soluble pigment and a white to gray the center. In some cases the culture had to
aerial mycelium exerted a lysogenic effect be repeatedly reinoculated into phage-con-
upon Fhytotnonas, Krwinia, and various taining preparations before lysis was attained.
other gram-positive and gram-negative bac- The interest in the phages of actinomycetes
teria. I'otato extract-glucose agar media received a new stimulus with the discover}'
were particularly fa\'orable to the production of the potentialities of some of these organ-
of the lytic agent. When the streptomyces isms to produce important antibiotics. This
cultur(> was added to soil infected with Phy- first became evident in the pioduction of
2 4
Figure 75. Formation of plaques (Reproduced from: Woodruff, H. B. et at. J. Bacteriol. ol: 536
1947).
some important ecionomic problems. The ly- centration. Streptomycin production was
sis of the culture by phage appeared to be partly or completely stopped by the phage.
quite distinct from that produced by auto- Cultures resistant to the phage action could
lytic factors. Saudek and Colingsworth re- easily be isolated from phage-infected cul-
corded in 1947 that the action of the trans- tures.
missible lytic agent ui)on griseus had all
»S. By exposing suhmerg(>d cultures of S. gri-
the properties of phage. Inyoung cultures seus, in a stationary condition, for 24 hours,
the phage developed rapidly and brought Woodruff et at. (1947) easily demonstrated
about lysis of the mycelium. The plaque the presence of actinophages in the atmos-
method was used for measui'ing phage con- ))here. Tpon transfer of a filtered broth into
I.VrK" MlCll ANISMS //
cation was ohtaiiicd. After six transfers, I'acli l\[licl of (iildilioii of pltiKjv upon pliiujc iiiiill I pi Icu-
lioii (Hid streptoiin/cin prodiiclion htj different
pliaiic particle inci'eased to 7") X 10'"". I'liaf2;e-
slrrplonii/ces in slationary cuUureii (Reilly,
resistant strains \\(>re I'eatiily isolated; sneh
Harris, and Wak.smati)
enltures retained tluMr capacity to pi-oduce
streptomycin Iml wvvv not al)solntel>- fi-ee
from phaii;(\
% V
Figure 76. Phage particles adsorbed on the .surface of a germinating streptomyces spore (Repro-
duced from: Mach. F. Centr. Bakteriol. Parasitenk. Abt. II, 111: 555, 1958).
Table 44
Phage inuUiplication in cultures of actinomyceies and its effect upon the production
of antibiotics (Reilly, Harris, and Waksman)
S. griseus 4
S. griseus 4.
S. griseus 4.
S. griseus 4.
S. griseus 3478.
S. griseus 3478.
S. bikiniensis . .
S. bikiniensis . .
S. lavendulae
S. lavendulae
N. asteroides
N. asteroides
LV'I'K" MIXIIAMSMS 17!)
DilTcriMit ]>h:ijj;os are ahlo to attack the cultui'es contained \-aiMous ])hages with dif-
saincDi'jiaiiisin. Cultures of .S. (jrisciis iikuIc fei'ent lytic i)roperties.
ivsistant to one type ot phat^c may in lime All indicator cult ure was used to i-cxcal the
hocoinc s(Misitivr to anotluM". The tonnation de\-elopment of the phage of the lys(»genic
of lysoticnic strains coutaininfj; a prophage culture. It often influenced the lytic proper-
\vas(l(Mnoiistrat(>(l hy Wclscii (n)o4). Wolsch ties of a gi\('n phage. This infiuence became
(I!>.")7) further cltMuonst rated that se\eral ac- partitailarly significant when the inchcator
tinophages can \)v found in a singh^ natural cultui-e was itself lysc^genic. I'hages which
substrate. They differ in the niorpholojiy of ha\'e the faculty to cause lysis of their own
their jihuiues. their host-ranji;e, and their an- (ailture wei'c often isolated from lysogenic ac-
tigenic projierties. A gi\"en a('tinophag(> may tinomycetes. This suggested that many acti-
he presiMit in its natural hai)itat in a concen- no])hages are able to change their lytic
tration of 1(F to 10^ particles ml. ])i-op(M'ty more or less easily.
.V search was made for truly lysogenic Some phage-resistant variants obtained
streptomyces. Thymol-st(M'iliz(>d cultui'c flu- exi)erimentally differed from their initial cul-
ids of \-arious organisms were tested upon tures in their antibiotic properties. Many of
many indicator strains. About 15 per cent of tliese variants were found to be lysogenic.
the organisms freshly isolated from a natural The possibility was .suggested that accjuisi-
substrate or taken from a collection of cul- tion of new antibiotic properties may be con-
tures actually carried a phage. The true lysog- nected with lysogenization.
eiiy of this phenomenon was demonstrated Uautenstein suggested that the broad dis-
bearing certain cultures on the sujjerficial S\-eshiiiko\a and Pariskaya made a de-
growth layer of others. Some of the lysogenic tailed examination of the occurrence of actin-
180 THE ACTINOMYCETES, Vol. I
Figure 77. Phage particles adsorbed on a streptomyces spore (Reproduced from: Mach, F. Centr.
Bakteriol. Parasitenk. Abt. II; 111: 556, 1958).
ophage in greenhouse soils. In four out of nocardia phages. Of the former, one attacked
fourteen samples of soil, free actinophages a culture designated as A'", paraguayensis, and
were revealed by direct count. In most cases of the latter, three attacked streptomyces.
these actinophages proved to be polyvalent. All other Actinomycetales were resistant to
In another ten samples of greenhouse soils both types of phage, as shown in Table 45.
and in twenty-five samples of forest and On the basis of these results, the authors con-
meadow soils, as well as in filtrates of field cluded that the genera Nocardia and Strepto-
soils, no free actinophage Avas found. Many myces are closely related and should not be
actinomycetes isolated from these soils were
separated into different families. Such gen-
susceptible to the eight different actino-
eralization is hardly justified. The A^. para-
phages previously isolated. Mach (1958) iso-
guayensis used in these tests is not a no-
lated from composts and forest litter three
cardia, but a streptomyces, as will be brought
actinophages of which two were polyvalent
out in Vol. II. The nocardia phages that
and one strongly specific. These three phages
were morphologically distinct from one an- attacked the streptomyces cultures may
other, as shown by the electron microscope. have been polyvalent phages, a potentiality
Bradley and Anderson (1958) isolated indicated in the early studies of Wieringa
from soil three streptomyces phages and five andWeibols (19o(i). The sensitivity to phages
.^ lie Mi;cn ANisMs isi
acterizatioii ot" oi'uanisnis will he discussed in Ejffict of Hcrcrdl (iclinophtigrs upon dijjcrent
detail in XiA. 11. Aclinoiiijiccldlcs (Hradlov and .Vndcrsori)
'IMie iuhihilion ot' the i)lia,iie hy clieiuical
Lysis of a host l)y a standard plia^c sus|)(Mision
is indicated Ijy a plus sign; no lysis is denoted by
ajieiits was studied l)v I'erlinan el al. (1 !).")!).
a minu-s sign.
Cause ct al. (11)57) reported tliat aetiiiomy-
cetes possess the al)ilit.\- to produce antibi-
otics which defend tiu>nisel\-es ajj;ainst
Hemolysin Production
Production of Enzymes
Act iiiomycetes produce a varict.y of extra- enzymes as the actinomycetin complex pos-
cellular and endocellular enzymes. Some of sess only the ability to dissolve isolated l)ac-
these enzymes hav(> beiMi isolated from the terial cell walls; they are wrongly classified,
culture filtrates or {ho mycelium, concen- therefore, with lysozymc. The ability of puri-
trated, and purified. Others have only been fiedenzymes isolated from the actinomycetin
demonstrated in the mycelium of the organ- complex to form amino acids sets them a.side
ism. from lysozyme, which forms reducing groups.
The assertion of Kriss that actinomycetes
L vsozvnie produce "lysozyme" still requires confirma-
The production of lysozj^me systems by tion of the ability of these organisms to form
actinomycetes at first aroused consideralile enzjmies that liberate reducing groups and
attention. These systems were confused, an amino sugar complex of the type relea.sed
h()we\er, with autolytic and bacteriolytic by lysozyme as well as the peptidase type,
mechanisms, on the one hand, and with anti- in accordance with the requirements laid
biotics, on the other. down by Salt on.
In his classical studies of the lytic agents
of B. sKhtilis, Xicolle first suggested that Proteases
bacteriolytic substances produced b}" micro- Miinter, Waksman, and Lieske first estab-
organisms might ha\'e properties in common lished that various actinomycetes, mostly
with enzyme systems. According to Welsch members of the genus Streptomijccs, possess
(1947), the bacteriolytic system of certain strong proteolytic activities. Some cultures
actinomycetes is lysozyme-like in nature and were able to decompose very energetically
is able to digest bacterial cell wall substrates. proteins in gelatin, egg-white, and blood
However, according to (Ihu^'sen and Saltoii serum. This is true of both saprophytic and
and (Ihuysen, such enzyme systems, unlike pathogenic types. They vary greatly, in this
lysozyme, are able to liberate amino acids respect, both (jualitatively and (luantita-
but not reducing substances. tively, as can be simply demonstrated by the
Salton defined the enzymic properties of process of gelatin li(iuefaction or casein de-
lysozyme on the basis of the following de- composition in ordinary plates. As a rule,
terminations: (a) turbidity reduction of iso- s{)ecies of Nocardia are poorly proteolj^tic,
lated cell wall structures or lysis where the whereas certain species of Streptomyces are
wall is in situ as with intact bacterial cells; highly acti\'e in this respect. The degree and
(b) liberation of reducing groups; (cj libera- rapidity of proteol^'sis also varj^ with indi-
tion of an ac(>tylamino sugar complex of vidual species.
glucosamine and the acidic hexosamine. Such Slapp found that out of 477 freshly iso-
1S3
184 THE ACTIXOAIYCETES, Vol. I
lated cultures of streptomyces, only one (1938), many cultures are destroyed upon
failed to liquefy gelatin. The liquefying ac- being heated at 40 to 45°C for a long time,
tion of the others was characterized by vary- but their proteolytic capacity is not affected.
ing degrees of rapidity. Many of the organ- The proteolytic activities of the various
isms produce a soluble brown pigment in species of actinomycetes are so marked that
gelatin, which, according to Beijerinck, is Waksman (1919) suggested the use of this
in the nature of a quinone that tends to property for diagnostic purposes. Lieske,
harden the liciuefied portion of the gelatin. however, stated that proteolysis is not a
The (luantitative ability to secrete pro- constant property and cannot be used for
teolytic enzymes can also be measured by characterization of the organisms. Krassil-
the degree of gelatin liquefaction and of nikov (1938) tested 200 cultures every 8 to
casein hydrolysis. Many species are also able 12 months for 3 to 5 years. Various forms of
to decompose complex vegetable and animal gelatin were used for the test. The results
proteins. Culture filtrates of certain actino- were always identical. A strain that dissolved
mycetes were found to contain at least two gelatin rapidly when first isolated continued
proteolytic enzymes, one capable of digest- to do so after 1, 2, 3, 4, and 5 years. Strains
ing casein and the other of attacking the that failed to liquefy gelatin at first failed
are more resistant to the effect of higher ruber and N. viridis, do not liquefy gelatin
temperatures than are corresponding animal at Some of the yellow species (N. flava)
all.
enzymes; the former enzymes are able to are weak liciuefiers. There are also reports
withstand heating at 70°C for 80 minutes in the literature that pigmented nocardias
though at 80°C they are destroyed. Lieske did not liquefy gelatin. The white {N. alba)
found that the resistance of the enzymes to forms, however, are able to liquefy gelatin.
temperatures is greater than that of the No large scale production of proteolytic
living cells of the organisms, which are killed enzyme preparations has so far been ob-
at ()2 to ()5°C. According to Krassilnikov tained from actinomycetes. Sterile culture
i'K< )i)r("i'i(tN OF i:\ZNMi;s 185
exeiM a niai'ked ( iTect not only upon animal ilmlidii (if (lidsldsr, inrcrldsc, anil ijiolcnse by
proteins hut also upon proteins (leiixcd iVoni ilijft 11 III iicUiKinn/celcs (Joii.s(mi, lIKiO)
the nitrogen of the solubilized wool was ac- this enzyme is formed in a simple synthetic
cumulated in the form of ammonia. medium containing chitin as the only source
The cell-free culture broth of *S. fradiae of carbon and nitrogen. The enzyme is also
3739 was capable of enzymatically digesting produced in the absence of chitin; the pres-
keratins and casein. The enzymes that ence of chitin in the medium was not es-
caused both of these digestions were similar sential for, although it favored, formation of
in their optimal activity at about pH 9 and the enzyme. The presence of glucose tended
in their nonsensitivity to sulfhydryl rea- to repress the formation of chitinase. The
Magnesium appeared to be the metal
enzyme was found to be rather unstable in
gents.
culture filtrates, but the presence of chitin
refiuired for the digestion of wool by the
tended to stabilize it.
culture broths.
Bucherer has shown that ^'arious species
The culture broths of »S. Jradiae were cap-
of Streptomyces, notably *S'. griseolus, S. ex-
able of solubilizing a maximum of 10 to 20
foUatus, S. fradiae, S. aureus, and S. griseus
per cent of several native keratinaceous sub-
are able to break down
According to
chitin.
stances; trypsin and papain could solubilize,
Schmidt-Lange and Bucherer, both patho-
at the most, al)out half as much (5 to 10
genic and saprophytic actinomycetes are
per cent) of each of the same keratins. By capable of producing the enzyme chitinase.
ammonium sulfate precipitation, a product Yamaguchi (1957) found that different
was obtained that had about Hi times as species of streptomyces, such as S. fradiae,
much wool-digesting ac^tivity per niilligi'am ha^'e the capacity to produce a powerful
of protein as did the culture broth. cuticle (of pig Ascaris) digestive substance
.
I hat could he precipitated I'loiu cult lire lil- niko\', the wide dist ribiil ioii of this propei'ty
t tales ami by ethyl alcohol, acetone, and tends to reduce its diagnostic \alue. .V single
other proteiii-precipitatiiij;- a^;eiits. The en- aljjha type amylase was found to be produced
zyme-like siil)staiic(> was distinct from the by (i\-e different st rej)t()myces species (Simp-
easein-diti;est inn; ai^eiit son and McC'oy, 1953). The mechanism of
break(l(»wii of starches by aetinomycetes is
Amylases (liscu.s.«ed further by lioisand Savary (1!)45).
Xumerous actinomyeetos are ab!(> to hy- The amylases of aetinomycetes are al)le to
drolyze starcli rapidly, either to the dextrin withstand the effect of higher temperatures
stage or directly to maltose and glucose. Tlu^ better than are t h(> cells of the organisms
j)roduction of amylolytic enzymes by actino- producing them. Surovaya obtained a potent
mycetes was first recorded by FcMini, who diastatic preparation from a culture of »S'.
capalile of producing such enzymes. These potato medium, and a .sati.sfactory enzyme
results were later confirmed by \arious preparation, designated as "superbiolase,"
investigators, including Caminiti. Some was obtained. This preparation was active
claimed that the starch is hydrolyzed only at 70 to 100°C and had an optimum pH at
partially, not to the sugar stage. ().() to ().7. The starch was converted first
Krainsky made a detailed study of a large into soluble form and then into dextrin.
number of aetinomycetes that were found Saccharification of the dextrin proceeded
cajjable of producing amylase. This phe- much more slowly than the liquefaction of
nomenon was further studied extensively by starch.
W'aksman and Lieske, who observed that The hydrolysis of mannosidostreptomycin
onl>- \er3^ few aetinomycetes lack the ability to streptomycin, by various strains of S.
to produce such enzymes. griseus, is said to be due to an amylase
For the screening of a large number of (Christensen et al., Langlykke and Perlman).
cultures of aetinomycetes, agar media con- Maruta and Tanaka isolated the enzyme
taining starch as the source of carbon are mannosidostreptomycinase by precipitation
used. The plates are streaked and allowed to of the culture broth with 1 to 2 per cent lead
incubate. After 5, 10, 15, and 20 days, the acetate. The optimum pH for the action of
surface of the agar is coA'ered with a solution the enzyme was 6.8 to 7.4 and optimum
of I-KI, and the amount of starch hydro- temperature 37°C. Certain aldohexoses, such
lyzed is measured by the width of the clear as glucose and mannose, inhibited the action
zone around the streak. Formation of zones of the enzyme; D-sorbitol produced an ac-
1.0 to 1.5 cm wide after 10 days' incubation celerative effect. In the regular streptomycin
is an index of good amylase production. For fermentation b}^ S. grisevs, the content of
the production of amylolytic enzjmies, inor- mannosidostreptomycin is high during the
ganic sources of nitrogen, especially nitrates, early stages of fermentation and low in the
appear to be preferable to organic com- older stages. The presence of glucose in the
pounds. early stages represses the action of the en-
Stapp recorded that 83 per cent of all ae- zyme.
tinomycetes isolated from soil and belonging Many aetinomycetes are able to attack
to thegenus Streptomyccs produced amylase. dextrins, glycogen, and and to produce
inulin
Although the amylolytic property is char- the corresponding enzymes. Lieske, who was
acteristic of the species, as noted by Krassil- one of the few to study these enzyme sys-
188 THE ACTIXOMYCETES, Vol. I
terns, reported that the anaerobic pathogenic toward the anode and the other remained
forms, or species of Actinomyces, differ from at the starting point. The two fractions
the aerobic forms in that they form such were found to represent two proteins with
enzymes in mere traces, if at all. No attempt identical enzymatic functions.
has been made to study these enzyme sys-
them for any Invertase
tems in detail or to utilize
practical purposes. Invertase is widely produced by actino-
mycetes, as shown by Caminiti, Krainsky,
Polysaccharidases and Waksman. Lieske was unable to demon-
Various actinomycetes are capable of uti- strate the production of this enzyme l)y the
lizing agar and other polyuronides as sources cultures he investigated, but he did not deny
of energy. The agar is thereby liciuefied. such capacity. The formation of in\'ertase
This is true particularly of such forms as and other saccharidases by actinomycetes
S. coclicolor (Stanier). has also been studied by Hofmann and
A detailed study of the enzymes in\olved Latzko (1950).
in the decomposition of seaweeds and sea- The ability of some actinomycetes to uti-
weed products (laminarin and alginates) b}^ lizesucrose as a source of carbon is largely
actinomycetes has been made by Chesters dependent upon the property of the organ-
et al. (1956). Various nocardias (V. citrea) isms to produce invertase. This capacity has
have been found to be active in l)reaking not been established for all organisms, how-
down calcium alginate and laminarin. Cer- ever. Krassilnikov says that nocardias are
tain streptomycetes were particularly active able to utilize sucrose without prior in\'er-
in decomposing laminarin. In this respect, sion. According to Waksman, only those
they were much more active than bacteria. forms that are able to produce invertase
The enzymes laminarinase and alginase wei'e make abundant growth on media containing
isolated from cultures of these organisms sucrose.
and found to be highly active upon the In view of the constancy of this pr(jperty,
corresponding substrates, as well as upon it has been suggested that invertase produc-
starch and calcium pectate. These enzymes tion be utilized for species differentiation.
were obtained either from the culture fil-
trates of the organisms or by treating the
Cellulolytic Enzymes
mycelium with 15 per cent ethyl alcohol. Although the property of decomposing
Sorensen (1957) found that both *S. alhus cellulose is widely distributed among micro-
and M. chalcea, when grown in a xylan- organisms, neither the cellulolytic mechan-
containing medium, have the capacity to isms nor the cellulases involved in the proc-
produce the enzyme xylanase. This enzyme esses of decomposition are well understood.
is extracellular and can attack the xylan As shown previously, many actinomycetes,
chain at random along its length, yielding a especially streptomyces, are able to grow
mixture of shorter or longer chain fragments. on cellulose as the only source of carbon.
These saccharides, of which xylotriose is the This was established through the work of
shortest, are attacked further by xylanase, Fousek, Krainsky, Waksman, and others.
giving the following end products: xylose, Various actinomycetes capable of decom-
arabinose, xylobiose, and uronic acid or posing cellulose ha\'e been described under
uronic acid-xylose oligosaccharides. The xy- different names, such as Mycococcus cytopha-
lanase produced by the streptomyces con- gus of Bokor, Micrococcus cylopkagxs of
tained two fi'actions, of which one trax'eled Merker, and the organism described by
TKoDi ciioN oi' |•.^/,^ .\ii:s 189
,-^ -^
enzyme with a maximiun acti^'ity at pH i A i I
10 15 20 30 35
4.0 to 4.5. Hydnxjuinone was oxidized most
r//^E IN MINUTES
lapidly, catechol more slowly, and p-phen-
Figure Oxidation processes by cell-free ex-
78.
ylenediamine very slowly. When growing S. tracts of .V. (oraUina: 1. endogenous; 2, thymuie;
antibioticus in submerged cultures on diffei- 3. uracil; 4. l)arl)iturate. (Reproduced from: Lara,
ent media using a rotating shaker, Sevcik F. J. S. J. Bacteriol. 64, 2S1, 1952).
190 THE ACTINOMYCETES, Vol. I
adenosine diphosphate, and diphosphopyri- dation of acetate, malate, and lactate; glu-
dine nucleotide, as well as in its susceptibility cose, pyruvate, oxalacetate, succinate, and
to iodoacetate and fluoride in the breakdown oxalsuccinate were inhibited only slightly.
of fructose 1 ,6-diphosphate. Sodium azide oxalacetate slightly inhibited
Intact cells or extracts were unable to oxidation of succinate and lactate, but not
ferment hexoses under normal conditions. glucose and pyruvate. It was suggested that
It was suggested that obligate aerobiosis of the cytochrome system acts as an electron
S. coelicolor results from a biochemical le- transfer system in S. griseus; the participa-
sion, the inability to regenerate anaerobi- tion of some other systems, such as that of
cally the diphosphopyridine nucleotide re- fiavoprotein, may also be considered.
duced in the oxidation of triose phosphate. According to Musilek and Sevcik (1958),
According to Sato, aerobic actinomycetes the addition of sodium arsenite in a final
possess the respiratory pigments of the a, b, concentration of 4 X 10"^ M to the medium
c, (I, and do cytochromes. On the other hand, of S. erythreus reduced biosynthesis of eryth-
the anaerobic actinomyces of the alkali type romycin by 87 per cent, with a simultaneous
possesses a, b, d cytochromes, and those of the Sodium acetate and
increase in pyruvic acid.
acid type possess no cytochrome at all. Birk sodium propionate in final concentrations of
et demonstrated the production
al. of a b- 0.5 per cent decreased the inhibitory effect
type cytochrome by S>. fradiae. of arsenite on erythromycin biosynthesis.
In the presence of 2,6-dichloroindophenol, Other salts of organic acids did not reduce
crude extracts of S. scabies were found by the effect of arsenite. The latter completely
Douglas and San Clemente capable of cat- inhibited oxidative decarboxylation of pyru-
alyzing the dehydrogenation of succinate, vate and oxidation of acetate by the washed
citrate, malate, and glutamate. Transami- mycelium of S. erythreus, but only partly
nase activity was also demonstrated, since inhibited glucose oxidation. Biosynthesis of
a:-ketoglutarate was converted to glutamate erythromycin depends on uninterrupted oxi-
in the presence of aspartate, leucine, or dati\'e decarboxylation of pyruvic acid to
valine as amino group donors. acetic acid. The authors suggested the prob-
According to Inoue (1958), S. griseus pos- ability of the part played by acetic acid as
sesses the oxidase of the intermediates in the the initial substrate in the biosynthesis of
Krebs cycles. Glucose, pyruvate, acetate, propionic acid, which is assumed to be the
oxalsuccinate, and lactate were oxidized precursor of the lactone nucleus in the eryth-
readily, but the oxidation rate of citrate was romycin molecule.
very low in the given condition. Formate
was not oxidized at all. Glucose oxidation Catalase
was iiihil)ited by monoiodoacetate and so- The relation between the development
dium fluoride. Succinate oxidation was in-
and catalase activity of S. griseus was stud-
hibited by malonate, the latter being oxi-
ied by Kovacs and Matkovics. A close
dized slight ly. Citrate appeared to l)e formed
relation was found between sti-eptomycin
as a result of an oxalacetate-acetate con-
densation reaction, a reaction not inhibited
production and catalase acti^'ity. A high
catalase activity was not necessarily a pre-
by streptomycin.
Inoue further rei)()rte(l that »S. griseus recjuisite for streptomycin production, but
lengths were found at (>()() m/x, 507 m/x, Jmd tomycin. With only little catalase activity,
550 myu, respect i\-ely. Cyanide inliil)it('(l oxi- there was \-ery little str(^i)tomy('in produced.
rHoDicrioN OF i;\z>Mi:s 191
Steroid Oxidation
Tyrosinase
The ability of \-arious actinomycetes to
Tlie protluclion of a brown j)iji;nient by oxidize steroid hormones has recently at-
actinomvcetes grown on protein media has tracted considerable attention, as shown in
usually been associated with the ability of Chapter 9. "Resting cells" of certain strepto-
the organisms to form tyrosinase. According
mycetes are able to transform steroids. Ac-
to Beijerinck, there is involved in the reac- cording to Turfitt (1944), various species of
tion the formation of a ([uinone, which turns Nocardia are capable of attacking steroids,
brown at an alkaline reaction and in the with the possible exception of halogen-sub-
presence of oxygen. The action of ciuinone
stituted derivatives. The oxidation
of cho-
in the presence of iron was found to be simi-
formation of a choles-
lesterol results in the
lar to that of the enzyme tyrosinase. Since
terone, followed by molecular fission, the
an excess of oxygen is required for the for- products of which may be utilized by the
mation of the ciuinone, only limited amounts organisms for their further growth.
are found in deep cultures. The quinone is
Perlman et al. (1957) studied the enzymes
belie^ed to be formed from the peptone in
the medium; although good growth was pro- Table 47
duced on media containing asparagine, Aryl sulfatase activity of rapidly yruwing iiiyco-
KXO.'i and ammonium sulfate as sources
, bacteria, nocardias, streptomyces and corynebac-
of nitrogen, only traces of quinone, if any, teria (Wayne, Juarez, and Nichols)
were found. The tyrosinase reaction is not
Species
involved in the production of all black pig-
ments by actinomycetes; some species pro-
duce such pigments in purely synthetic
media in the complete absence of peptone.
It has always l)een assumed that the po-
tato scab organism is a chromogene, and that
the formation of the black pigment is due to
the tyrosinase reaction. Millard and Burr
reported, however, that some of the plant
pathogens did not give the tyrosinase reac-
tion. Afanassiev could not confirm the patho-
genicity of these cultures. Skinner concluded
that the production of a dark color by the
192 THE ACTIXOMYCETES, Vol. I
that oxidize progesterone. Schiesser studied The production of enzymes of the isoci-
cortisone-oxidizing enzymes. Harris et al. tritasepathway was demonstrated by Bardi
(1957) found that »S'. globisporus, S. virido- et al. (1958) for .V. riigosa.
chromogenes, and other streptomycetes are Lara (1952) reported that enzyme prepa-
capable of deacetyhition and oxidation of rations of the N. corallina group can be ob-
dehych'oepiandrosterone acetate. tained by extracting ahmiina ground cells.
A detailed discussion of the hterature on The activity of these extracts against thy-
steroid oxidation by actinomycetes is found mine and uracil was demonstrated only when
in the re\'iews of Eppstein et al. and Wett- methylene blue was added. These com-
stein (1955). pounds were oxidized to substances having
the over-all composition of 5-methyl bar-
Other Enzymes
bituric acid and barbituric acid, respectively.
Numerous other enzymes and coenzymes
In one experiment uracil was formed from
have been found to be produced by actino-
thymine; the results of many other experi-
mycetes. It is sufficient to mention coen-
ments, however, indicated that the demeth-
zyme A (Gregory et al., 1952). The abil-
ylation of the latter did not normally pro-
ity of various nocardias (N. asteroides, N.
ceed under the conditions used, leading to
pelletieri) to bring about the demyeliniza-
the conclusion that normallj^ uracil is not an
tion of bovine spinal cord may be due to an
intermediate product in the decomposition
enzyme system. Some of these mechanisms
of thymine. Cell-free extracts of N. corallina
produced by Streptomyces and Nocardia
with activity towards thymine, uracil, and
species are thermostable and others are
barbituric acid were obtained only from
thermolabile (Adelson et al.). Anaerobic ac-
cells grown in either thymine, uracil, or bar-
tinomycetes are able to produce acid phos-
bituric acid; enzyme preparations from glu-
phatase (Howell and Fitzgerald, 195.3).
cose grown cells were de^ oid of such activity.
Wayne et al. developed aryl sulfatase tests
This points to the adaptive nature of the
for differentiating saprophytic mycobacteria
enzyme system (Fig. 78).
from the tuberculosis organisms. This study
Barbituric acid was hydroh'zed l)y the
was of particular interest in differentiating
atypical mycobacteria and nocardias. Only
enzyme preparation with the formation of 1
21 per cent of all nocardias tested and none mole of CO2 2 moles of NH3 and 1 mole
, ,
produced the enzyme (Table 47). Almost and uracil degradation according to the fol-
allmycobacteria hydrolyzed demonstrable lowing scheme:
amounts of phenolphthalein disulfate if sufh-
Thymine 5-Methvl barbituric acid
cient inoculum was used and permeability
diffei"ences compensated for. The interesting
conclusion was rea(^hed that if M. rhodo-
Uracil — > Barbituric acid Malonic acid -\- Urea
chrous is mycobacterium, it is the only
a true
member tested which d()(\s not j^roduce aryl ^ i
r. II A r T K H 1 2
\'ari()us actinomycetes and their meta- The formation by ^S. griseus of a growth
bolic products exert a favorable effect upon factor for Leuconostoc citrovorum was demon-
the growth of lower forms of life, including strated by Emery et al. (1950). This factor
fungi and other microorganisms, and upon was not identical with either B12 or B13
higher forms of animal and perhaps plant This factor had no activity upon pernicious
life. This is due, partly at least, to their anaemia, but possessed leucocyte-stimulat-
ability to synthesize several vitamins. ing activity.
Herrick and Alexopoulos grew S. virido- The production of carotenoids by actino-
chromogenes in a liquid medium, then filtered mycetes has also been demonstrated. Pro-
the culture and autoclaved it. The fungus tiva has shown that certain actinomycetes
Sfereum gausapatum inoculated upon this are able to produce riboflavin and fiavopro-
medium produced a heavier growth than tein in synthetic media.
usually obtained on sterile media. Even more
striking results were obtained when Phyco- Formation of Vitamin B12
myces blakesleeanus was used as the test
The announcement, in 1948, by Rickes et
organism; since this culture is used in as-
al. that certain actinomycetes are able to
saying for the presence of thiamine, the con-
produce vitamin B12 resulted in much in-
clusion was reached that actinomycetes
commercial possibilities of these
terest in the
produce this vitamin. In a further study of a
organisms as sources of this vitamin. It has
number of cultures of actinomycetes, it was
now been established that numerous actino-
demonstrated that all were able to produce
mycetes are able to produce this \-itamin if
thiamine or its intermediate or precursor.
cobalt salts are added to the media to serve
Mackinnon observed that various strains
as precursors. The production of B12 can
of S. albushave a marked stimulating effect
be measured by microbiological assays. Var-
upon the growth of Trichophyton discoides.
ious strains of S. griseus, including the
This effect was comparable to that exerted
streptomycin- and grisein-producing forms,
by the presence of thiamine. The same effect
was obtained when P. blakesleeanus was and \'arious Streptomyces species, such as
inoculated on a synthetic medium in which S. fradiae and *S'. aureofaciens, are able to
the streptomyces had previously been grown. produce some vitamin B12 without affecting
Filtered cultures of *S. albiis added to syn- the yields of the corresponding antibiotic
thetic media had the same effect upon the substance. Certain nonantibiotic-producing
growth of thiamino-nMiuiring organisms. actinomycetes, like S. olivaceus, were also
193
. .
found capable of producing this vitamin In addition to vitamin B12 , various other
(Ganguly et al.). biologically activecompounds, designated as
According to Hall et al. (1953), S. olivaceus B121, Bi2c
, and Bi2d are produced by S.
, ,
when grown under submerged aerobic con- griseus (Anslow et al.). The formation of
ditions is capable of producing large amounts vitamin Bi2b by S. aureofaciens was demon-
of vitamin B12 The yields of the vitamin
. strated by Lichtman et al. (1949). The B^b
were influenced by the composition of the was effective, by parenteral administration,
medium. In the presence of proteinaceous in the treatment of Addisonian pernicious
material such as distillers' solubles, glucose, anemia.
CaCOs and cobaltous ion, about 1.5 /ig of
, In a study of the production and purifica-
vitamin B12 was produced per millilter in tion of vitamin B12 by \'arious actinomycetes,
deep tank fermentors, but as much as 3 ng Tarr has shown that Bi2a was formed by S.
of the \-itamin was obtained in some media. griseus and by aureofaciens in aerated
*S'.
Appreciable amounts of the B-complex vita- herring press water containing 2 mg per
mins, niacin, pantothenic acid, biotin, py- milliliter of added cobalt (as cobaltous ni-
ridoxine, thiamine, and riboflavin were pro- trate). Highest recovery of vitamin Bi2a in
duced. these products (about 1.1 mg per milliliter
By selective cultivation, including mutant with S. aureofaciens and 0.8 mg per milli-
formation by ultraviolet light and x-rays, liter with S. griseus) was obtained by chro-
it is possible to increase considerably the matography on filter paper strips treated
yields of vitamin B12 by a given culture. with potassium dihydrogen phosphate, elu-
The effect of cobalt as a limiting factor in tion of the vitamin, and aseptic addition of
the biosynthesis of the acti^T vitamin B12 the eluates to previously sterilized Lactobacil-
by S. griseus was immediately recognized lus leichmannii assay medium. Treatment of
(Hendlin and Ruger, 1950). Shull and Rou- the crude fermentation products with po-
tien (1951), Pridham et al. (1951), Saunders tassium cyanide (2.5 mg. per milliliter)
et al. and Garey et al. (1951) made a
(1951), caused partial resolution of Bi2a to B12
sur\'ey of \'itamin B12 production by different Letunova (1958) made a comprehensive
actinomycetes. Particular attention was paid study of cultures isolated from the lime of
to *S. griseus (Rickes et al., 1948) and »S'. stagnant reservoirs of the biogeochemical
fradiae (Jackson et al., 1951). province enriched in cobalt. Nearly 86 per
Chemical investigations have brought out cent of these cultures, as well as 90 per cent
the fact that there are several forms of of strains isolated from lime deposits of the
vitamin B12 . Possibly not all of them occur Co-impoverished pro\'ince, were able to form
naturally but are formed during the isolation vitamin B12
process. The incorporation of cobalt into
Certain nocardias are also capable of pro-
the actinomycete metabolite has been estab-
ducing B12 . Bardi et al. (1958) studied a
lished l)y the use of radioactive cobalt in
strain of .¥. rugosa that was found to release
fermentation media and the isolation of the
free porphyrins into the medium under suit-
vitamin containing the ial)eled isotope.
able aeration conditions. By ultraviolet ir-
Some of the vitamin B12 produced by ac-
and radiation, mutants were obtained that gave
tinomycetes is ])ound on the cells, ma}'-
ionizal)ie salts, sonic energj', or heat. Sub- tion l)etween vitamin B12 and porphyrin
seciuent to release, the cell-free li(iui(l may ])r()du('ti()ii was demonstrated. The free por-
lie treated with cyanide to convert the vita- l)hyrins consisted mainly of coproporphyrin
min present to the more stable cyanide form. III, and of uroporphyrin, probably III, in
PRODUCTION OK \ ITAMINH AM) OTIIIIU (iUOWTU FACrrollS 195
minor ninount. Tracos of coproporpliyriiis from this insect roared in the lai^oratory.
Iand of uroporphyrin T, and of ot her non-
1 1 , The microorganism is not transmitted
itlcntiliiMl poi-phyrins wvw also n'lcascd in through the egg but is taken up by the young
\\\v nuxliuni. The total fr(>c porphyrin con- nymph fi'om the environment, such as the
tent in l)roths may roach, in som(> strains, contaminated surface of the egg, and more
20 to 40 juii", ml. The mutants producing;" often perhai)s from the dry excreta of other
j2;r(>at(>r amounts of porphyrins ^avv lower meml)(M-s of the species. Insects wore reared
yiokls of \itamin Hp.. than tiic |)arcnt strain. freefrom the actinomycete l)y sterilizing the
Loss aerated cultures pi-oduced more por- and feeding with suitable
surface of the (>gg
])hyrins and less \'itamin Hi_> . The lack of precautions. They grew and moulted nor-
cobalt lowered porphyrin production. Added mally until the 4th or 5th instar. Moulting
5-anunolae\ulic acid (100 to 500 Mg/ml) in- was then delayed or failed entirely in spite
creased porphyrin production, but did not of repeated feedings of blood. Very few in-
exertany effect on vitamin Bio levels. Small sects without the actinomycetes became
amounts of succinic and glyoxylic acids were adult,and those few were almost certainly
detected in suspensions of A''. )-ugosa incu- incapable of reproduction.Normal growth
bated with citrate under anaerobic condi- and moulting and egg production were re-
tions. Since glyoxylic acid can be a precursor sumed when the insects were reinfected with
of glycine, was suggested that this reaction
it the organism.
might have some meaning in porphyrin bio- Little is also known of the role of the
synthesis by this strain. streptomycete that is able to infect nema-
A detailed discussion on the effect of co- todes that grow in the cockroach (Hoffman).
t)alt concentration in the medium upon the Other references in the literature concern
upon the growth of swine has been presented Stimulating Effect of Products of Ac-
by Brande et al., and a detailed analysis of tinoniycetes upon Plants and Bac-
the nature of the growth promoting effect of teria
antibiotics upon animals has been made by Koaze demonstrated that the culture fil-
Porter (1957). Porter summarized the evi- trate of a streptomyces had a strong pro-
dence that their use results in appreciable moting effect on the germination of rice
benefit to growing livestock. Early fears that plants, in a dilution of 10~^ to 10~^ A crys-
their use for breeding stock might lead to the talline substance was obtained by ethyl
establishment of resistant strains of patho- acetate extraction and chromatography on
genic bacteria have been found to be ground- aluminum oxide which was active in con-
less. The feeding of antibiotics to animals centration of 0.1 mg/ml. It was a neutral
was found to result in a healthier environ- substance, soluble in alcohol, benzene,
ment. chloroform, and water. On analysis it gave
The mechanism by which the antibiotics C10H16X2O2 . It was found to be L-prolyl-L-
produce this favorable effect on growth of valine anhydride (diketopiperazine). The
the animals may be due to their action on substance had no antibiotic activity.
the tissues, or primarily upon bacteria. This In a study of the effect of antibiotics upon
may depend partly on the state of health of plant growth Koaze et al. (1957) found that
the animals. Conditions of poor health can certain antibioticsshowed a similar promot-
lead to a state of disease, making it difficult ing effect; neomycin B and sarkomycin had
to draw a distinguishing line between the a marked effect on the growth of broad
therapeutic effect of antibiotics and their beans.
nutritional action. Antibiotics may exert a IVIention may also be made of the growth-
direct antibacterial on pathogenic
action promoting effect of certain actinomycetes
bacteria in the gut or animal tissues. Their upon cellulose-decomposing and nitrogen-
presence in small quantities in both the gut fixing bacteria (Sanborn, 192G). The mech-
and tissues may also act as a prophylactic. anism of this action is still insufficienth'
fiod oithor with ril)ofl:i\iii or its nucleotides st re])tomyces cultures, a pigment was formed
or decomposition pi'odiicts. ()nly spot 2 in optimal media; a considerable intensifica-
react (m1 with DJiiliychiii. When I he spots were tion of I he pigment (lycopersin) occiu'red.
cut oil the chroniatoiiranis and tested in- ('olhlotriclmm atramcnlarium responded to
di\iduall>\ only one spot , ('liaraclei'ized by a the same streptomyces metabolites by an
blue lluorescence, pro\{'d capable ol' sus- acceleration in the maturing and l)y a greater
taininji; the growtli of /.. c(ts( i. density of stromata. Ccratostomella ulmi and
RubcMitsc'hik (7 al. found that \ariou.s strep- Streptomyces scabies responded l\y more
toniyccs, such as »S. g)'iseus, S. c<wlicolor, and rapid sporulation.
»S. ylobisporus, when grown in cultures with The favorable effect of certain strepto-
other organisms, are capable of forming myces upon the sporulation of other cultures
A()latil(> materials which stimulate the of the same genus has been demonstrated by
growth of K. coll, B. subtilis, B. mesentericus, Dondero and Scotti. These results led to the
and other bacteria. Every Streptomyces spe- conclusion that actinomycete metabolites
cies was said to exert a characteristic effect, contain specific stimulatory substances in
S. grisens stimulated the growth of Azoto- addition to inhibitory substances, or that the
bacter. substances may be growth-inhibiting at one
Grossbard reportc^d that A'arious anti- concentration and growth-stimulating at an-
fungal substances produced by actinomy- other.
cetes exert stimulative effects upon the The significance of these phenomena in
growth of fungi, Avithin a concentric circle natural processes in general and in the soil
adjoining the zone of inhibition. Pigmenta- in particular is still to be elucidated. This is
tion was intensified in cultures of Verticil ium also true of the stimulating effect of actino-
dahliac, Helminthosporium vidohae, and mycin upon the growth of some strains of
Fusarium oxysporum. The last usually fails rhizobia and its inhibiting effect upon others,
to produce a pigment on synthetic media, notably the slow growing strains (Trussell
but in response to the metabolites of certain and Sarles).
CHAPTER 13
Production of Pigments
Among the most characteristic properties The members of the red-brown group
biotics.
of actinomycetes is their abihty to produce produce neither true pigments nor active
a great variety of pigments, both on organic antibiotics.
and on synthetic media. This pigment-pro- Only very few of the pigments of actino-
ducing capacity is so characteristic as to mycetes have been studied in detail. Some
serve for the naming of a large number of are soluble in water, others are soluble in
the species, notably those belonging to the alkalies, and still others in organic solvents,
genus Streptomyces. Very few of these pig- such as alcohol or chloroform (Table 48).
ments are pure. Most of them are mixed, These pigments have been divided into three
with gradual transition from one color of groups: anthocyanins, carotenoids, and mela-
the rainbow to another. nins. The red-blue pigments of the actino-
Conn and Conn emphasized the value of mycetes belong to the first group; red-or-
pigmentation in classifying actinomycetes. ange-yellow pigments belong to the second;
Stapp isolated from various soils477 cul- the black and brown pigments belong to the
tures of streptomyces. Their growth on third. Some of the pigments are fluorescent,
glycerol-asparagin agar gave the following and some change in color with a change in
pigmentation: 31 per cent of the colonies reaction of medium. Some are intracellular;
were gray in appearance; 19 per cent yellow others are exocellular and dissolve readily in
(cream to orange); 18 per cent brown; 17 the medium. Some of the pigments are pro-
per cent white; 8.4 per cent red; 4.2 per cent duced only on certain specific media, notably
blue; 1.3 per cent green; and 0.8 per cent synthetic media; others are produced on a
black. variety of media. In many cases, a change
von Plotho (1948) divided the actinomy- in the composition of medium results in a
cetes into four large groups according to the change in the natvu'e of the pigment.
pigments produced: 1. red-yellow; 2. red- The pigments of actinomycetes are usually
blue; 3. red-brown; 4. colorless. Some of the described in terms of \'arious shades of blue,
pigments possess indicator properties: at an violet, red, rose, yellow, green, brown, and
acid reaction, the red pigment predominates; black. The shades of color are also frequently
at an alkaline reaction, the second pigment mouse
indicated, as light gray, deep gray,
prevails. The colorless group have given the gray. The pigments may be concentrated
greatest number of antibiotic-producing either in the A'egetative myceliinn or in the
strains. Members of the red-l^lue group pro- aerial mycelium and spores.
duce strong pigments but weak antibiotics. The pigment-producing property of ac-
Members of the red-yellow group produce tinomycetes is variable, depending upon the
both strong pigments and liighly active anti- nature of the medium, the age of culture,
198
PHODIC'TIOX OF riCMI'lXTS 199
media lias l)(>cn used to designate the clu-o- Red-yellow -(- + -(--)--)__
mogenic st reptomyces. The tyrosinase action
characteristic of tliese
he\-ed l>v Beijerinctc to (>.\plain
organisms was
tiie
iie-
mecha-
Red4)ro\vii
Red-blue
—
—
—
—
—
— ___
__)_ _^
to be protluccnl on synthetic media with tration of the pigment through the cell wall.
starch, sucrose, and other carbon sources. Kriss suggested classification of the pig-
This is the reason for the designation of the ments of actinomycetes into four types:
culture variously as S. violaceoruher and S. I. Pigments soluble in water and in 96
tricolor. Beijerinck (1914) described a cul- per cent alcohol; these are capable of pass-
ture, .4. cijancHs, now classified with the ing through the li\'ing cell plasma. This
nocardias, which produced a pigment similar group has been subdivided into (a) antho-
in its properties to the anthoc.yanins. This cyanins, soluble only in water, and (b) hy-
pigment was recently designated as litmoci- droactinochromes, soluble in water and in
din. alcohol.
Lieske recognized two types of pigments II. Lipoactinochromes, insoluble in water
among the actinomycetes: (a) the chromo- but soluble in alcohol and in other organic
phores or pigments which are not excreted solvents.
from the mycelium into the medium, and III. Pigments insoluble both in water and
(b) the chromopars or pigments which are in organic solvents.
readily excreted. The first group comprises IV. A combination of water-soluble and
the pigments found in the vegetative myce- water-insoluble pigments.
lium grown on synthetic media; these are Lieske isolated a culture of a st reptomyces
yellow, orange, red, blue, violet, brown, that produced a carmine-red pigment that,
black, and green. The aerial mycelium of when boiled in dilute acid, became soluble
these cultures may be white, rose, lavender, in alcohol and in ether. Certain actinomy-
red, yellow, orange, green, or gray. The sol- cetes produce a red pigment that is made
uble pigments are usually yellow, blue, and soluble onlyby the action of concentrated
red; occasionally the}^ are green, orange, or HCl; on treatment with H2SO4 it is changed
brown. to a blue-green pigment. A^. polychromogenes
Kriss established that even the chromo- produces a red pigment, soluble in chloro-
phore pigments are partly dissolved in the form, ether, and acid, but not in alcohol,
medium, possibly because of the lysis of the glycerol water, or dilute alkali; this pigment
mycelium and the spores. Some of these pig- is also changed to blue-green by H0SO4 .
ments are insoluble in water and are bound Certain light yellow pigments produced
to the proteins. Others are dissolved in the by actinomycetes are insoluble in organic
fats and lipoids of the cell. Some maj^ be solvents, but are soluble in dilute KOH so-
200 THE ACTINOMYCETES, Vol. I
lilt ion; they are changed, on treatment with anthocyanin; it was isolated as the crystal-
These pigments are readily soluble in wa- similar but not identical to azolitmin. On the
ter and in aqueous glycerol, alcohol, and basis of this differentiation, she believed that
other water-containing solvents. They are the two organisms represent distinct species.
insoluble in absolute alcohol, chloroform, Oxford and Erikson ct al., however, could
and other organic solvents. They are red in not accept the phenazine or anthocyanin
dilute acid solutions and blue in alkaline so- nature of these pigments.
lutions. The neutral point is about pH 7.5. Tonolo et al. (1954) described the isolation,
The blue pigment of S. coclicolor has been from a culture related to »S. coclicolor, of a
extracted with cold and hot water as well as pigment designated as streptocyanin. The
with alcohol. This pigment became red w^hen course of pigment production in a glycerol
treated with acid, and green when treated medium is shown in Figure 79. The pigment
with 25 per cent alkali solution. Addition of was soluble in acetone, pyridine, and diox-
lead acetate to the aqueous solution of the ane. It colored blue-violet with H2SO4 and
pigment brought about the formation of a decomposed at 290 to 300C°. It gave a band
violet precipitate. Miiller (1908) first sug- in the visible spectrum. A quinoid structure
gested the similarity of this pigment with was postulated for the pigment. It showed
anthocyanin. Kriss confirmed these results antibiotic activity.
fully. Krassilnikov also confirmed the results
Green Pigments
and emphasized that the anthocyanins or
allied pigments are characteristic of several Actinomycetes were found to produce sol-
actinomycetes. He pointed out that all the uble green pigments, which is the reason for
blue pigment-producing actinomycetes be- such species names as S. viridis, S. virido-
long to S. coclicolor Miiller. The cultures all cliromogenes, and S. vernc. Some of these
produce a blue pigment which diffuses into pigments are soluble in glycerol and in al-
the medium, conferring upon the medium kaline solution, but not in organic soh'ents.
the corresponding color. If the reaction of The color of the pigment in water is green;
the medium changes to acid as a result of in glycerol, yellowish green. The composition
the growth of the organism, the color of the of the medium influences the nature of this
culture becomes on alkalinization of the
I'ed; pigment. Green pigments produced by ac-
medium, the color again tuins blue. tinomycetes were also reported by other in-
8r
-
4
O
(T.
UJ"
U
Q.
>
_l
o
2-
5*-
202 THE ACTINOMYCETES, Vol. I
Table 49
Growth (ind soluble pigment pruduction of S. griseus in calcium salts of
alpha-hydroxy and dibasic acids (Benedict and Lindenfelder)
ri{()i)L("rioN oi' im(;.mi;nts 203
in the culture. At 37°C pigment formation in acjueous alcohol when boiled for 30 min-
is greatly diminished. utes, but unstable in dilute acid or alkali. An
alcoholic solution of actinonwcin gives no
Role of Pigments in the Life and Me- shows char-
coloration with ferric chloride; it
tabolism of Actinomycetes acteristic light absorption in the visible re-
Various hypotheses have been proposed to gion (E\'^j^ = 200 at -450 /x) and in the uhra-
explain the role of pigments in the growth violet region (EW, = 216 at 215 m/z). It
of the microbial cell. Their function in the is highly active against certain gram-posi-
mechanisms of the cell has been
respirator}' tive bacteria. Waksman and Tishler found
suggested. Some have claimed for them a that 10 (Jig given intraperitoneally or sub-
role in the defense mechanism of the cell cutaneously, killed 20-gm mice in 2-4 to 48
against the action of foreign cells or against hours.
the effect of sun raj^s. The recent interest Various forms of actinomycin ha\'e since
in the subject of antibiotics has tended to been isolated. Interest in this group of anti-
concentrate attention upon these substances biotics has grown especially since it has been
that are pigmented in nature. demonstrated that they exert a marked effect
Benedict and Lindenfelder (1951) ha\e in the treatment of certain forms of cancer.
shown that the different varieties of *S. (jri- The chemical studies of Dalgleish ct al., of
seus possess characteristic pigment-produc- Brockmann and Grubhofer, and of many
ing capacities on special media. Some of the others added greatly to our understanding
204 THE ACTINOMYCETES, Vol. I
Table 50
Classification of pigmenled antibiotics of actinomycetes (Benedict)
( )x3'tetracycline
e. Circenish yellow weakly basic, C:ioH34N404 , stronglj- antifungal.
F'radicin
2. Insoluble in water, slightly soluble in ether, soluble in other organic solvents.
a. Yellowish substance, active against yeasts and filamentous fungi.
Actinone
b. (Jolden yellow compound, C9H10N2O2S2 .
Aureothricin
c. Brilliant yellow neutral comjjound, C8H8N2O2S2 , active against various bacteria and fungi.
Thiolutin
d. Orange yellow, C14H12N4O4S4 .
Thioaurin
e. Yellowish orange basic substances, C.-)iHs6N408-3HCl (A), highly active and extremely toxic.
Xanthomycins A and B
f. Saffron-yellow weakly acidic, C23H18O6 , active against gram-positive bacteria.
Resistomycin
3. Soluble in water and in organic solvents other than ether.
a. Yellowish orange, active against various bacteria.
Luteomycin
b. Yellowish green neutral, active against gram-positive Ijacteria.
Actinomyceline
c. Yellowish weakly acidic; active against yeasts and filamentous fungi.
Flavacid
4. Insoluble in water and ether, soluble in other organic solvents.
a. Yellow acid substance, acid stable, active against gram-positive bacteria.
Griseolutein
1). Yellow substance; antifungal and trichomonadicidal.
Trichomycin
II. Yellowish red to dark red, blue and purple. With the exception of grisein and the microcins, all are
primarily active against gram-positive bacteria.
1. Practically insoluble in water, slightly soluble in ether, and soluble in other organic solvents.
Act inorhodin
b. Anthrocyanin-likc pigment, s()lul)l(' in \\ater and ether.
Lit niocidin
c. Low nitrogen-containing compound; red form, water-insolul)le, ether-soluble.
T?hodomycet in
d. .'Vinplioteric suhsl it utcd (juinones, low water sohil)ility.
Kliodomycins .\ and 1?
Table 50 —Continued
b. Iinptiro, firiscMii-likc ftictoi', piodnci imi influenced markedly l)y iron, nnriower- sped iinti t linii
firiseiii.
Aiiiihidiie ;{r)i{)
of the cluMiiistrv and activity of this i^roup pyridine, and dimethylformamide; slightly
of aiitil)ioti('s (Waksnian, Katz and \'iiiiiig, soluble in ether, petroleum ether, and ben-
1958). zene; and sparingly soluble in water. It is
very stable in acid solution and withstands
.4 ctinorhodin
heating for 1 hour at 100°C. It is active
Brockniaini and Pini, and von Plotho iso- against a variety of gram-positive, gram-
lated, in 1947, a culture of streptomyce.s negative, and acid-fast organisms, as well
which produced a polyoxyquinone pigment, as fungi, Endarmxha histolytica, and certain
d(\signated as actinorhodin. It was soluble in hcmoflagellatcs.
acetone, pyridine, and dioxane. It gave a Aureothricin also belongs to this group.
l)iue color with H2SO4, decomposing at 270°C. Bolhofer
(t al. obtained bright orange-
This pigment formed two distinct bands in yellow crystals of thioanrin (C14H12N4O4S4)
the \isible spectrum. It was active antibiot- which melted with decomposition at 179 to
ically. The antibiotic is believed to possess 181 °C. Thioanrin is ielati\ely insoluble in
a quinone structure, having three free hy- water and in many oi'ganic soh'ents. It in-
(Iroxyl groups, one carboxyl group, and two hibits the growth of various bacteria, but
hytlroxyl groups adjacent to a carboxyl unlike thiolutin, it shows little activity
group (Brockmann and Hieronymus). against finigi.
Shockman and Waksman found that rhodo-
Coclicolorin
mycetin is similar to actinorhodin. The anti-
biotic shows a blue color in alkaline solution, This is a purplish red powder, m.p. 142 to
dark blue in sulfuric acid, and becomes red- 14()°C, which was found (Hatsuta) to be red
\iolet on addition of boric acid. atpH 5.0, purple at pH to 7, and green (i
biotics may form pigmented compounds. the primary selection of S. rimosus strains
The tetracyclinesgive green compounds by subjecting young cultures to ultraviolet
with copper and nickel and red compounds irradiation on solid media which are favor-
with ferrous and ferric iron (Albert). able for antibiotic production.
C H A V I i: \{ I 1
Antagonistic Properties
Phonoiiiena of Association and Antago- tericidal oi- fungicidal, and lytic effects. 3.
nism Antagonism of function versjis antagonism
Soils and water iiasins arc iiihabilcHl by of growth. 4. Direct and indirect antagonism.
mixed inicrohioloiiical populations. .Vnioiig 5. One-sided or two-sided antagonism. 6. Iso-
tracts, where they may be responsible for Among the xarious types of antagonism,
certain mixed infections. the most definite and best understood are
I'asteiH", the biochemist, and DeBary, the those that result in the formation of anti-
botanist, were the first to emphasize the biotic substances, formerly spoken of as
significance of possible antagonistic relations toxins, lysins, or bacteriolysins. The physi-
among different microorganisms li\'ing in cal, chemical and biological properties of
such mixed populations. When two organ- these substances vary greatly. Some are de-
isms were grown on the same substrate, one stroyed by boiling, others on exposure to
usually was found, sooner or later, to over- light. Some are resistant to heat and to ultra-
Avhelm and e\-en bring about the death of violet rays. Some are soluble in water, others
the ()th(M'. Such antagonistic activities em- in special solvents. Some are highly toxic
brace phenomena other than mere competi- to animals, others are relati^Tly nontoxic.
tion for or exhaustion of nutrients: they were Numerous early investigators observed
found to be due largely to the formation of the depressive effect of fungi upon bacteria
specific chemical substances (antil)iotics) and vice versa. It is sufficient to mention
responsible for these effects. the observations of Tyndall (1876). How-
The terms "antagonism" and "antibiosis" ever, the first well-recognized antibacterial
usually refer to the rexhiction in growth and preparation was that of pyocyanase, pro-
activities of organisms living in association. duced by Ps. aeruginosa, formerly known as
The organisms thus affected may respond B. pyocyaneus. Emmerich and Low (1899)
bj^ exhibiting temporary or permanent modi- considered pyocyanase to be an enzyme sys-
fications in their physiological characteris- tem. Emmerich and Saida (1900) actually
tics; their morphology may l)e changed; a used it to destroy (dissolve) tubercle bacilli.
reduction in virulence may also result. Se^'- Subsequent to these early studies, an ex-
eral tj^pes ofantagonism are now recognized: tensive literature accumulated on the pro-
1. Antagonism in vivo versus antagonism in duction of antibacterial substances by bac-
vitro. 2. Bacteriostatic or fungistatic, bac- teria, culminating in the treatise by
207
208 THi: ACTINOMVCETES, Vol. I
Papacostas and Gate (1928) on "Microbial organisms, but, on the contrary, are able, in
associationsand their therapeutic applica- spite of their slow de^•elopment, to suppress
tion" and the more recent systematic work the growth of almost all bacteria and fungi.
of DuboR on tyrothricin. This was foinid to be true on plates in which
The fungi as producers of antibacterial actinomycetes were seeded after the growth
substances also received considerable atten- of fungi and bacteria had already occurred.
tion (Waksman, 1945). The most spectacular When a culture ofan actinomycete and a
work was that on penicillin^ first recognized staphylococcus were mixed, streaked on agar
by Fleming (1929), and finally established plates, and incubated at 37°C for 24 hours,
by Chain, Florey, d al. (1940) as an impor- the plate became covered exclusively with the
tant chemotherapeutic agent. growth of staphylococci; on further incuba-
The actinomycetes were first recognized tion, however, the actinomycete assumed
by Oasperini (1890) as potential destroyers the upper hand and gradually replaced the
of fungi and bacteria. Lieske reported, in bacteria. The growth of the actinomycetes,
1921, that actinomycetes, especially the under these conditions, may have been bet-
aerobic, spore-forming types (streptomyces), ter than in pure culture, thus giving the im-
are not hindered in their growth by other pression that the bacteria actually served as
a ,iir(t\\ til siiimilanl. On the oilier Imiid, I's. zones. Tliis denionst latcd the fad that the
jii/oci/aiti IIS repressed the .u'rowtli n\' x.iiioiis actinomycete produced a lytic substance;
a('tiii()niyc(>t{>s in mixed eultuic, and cxcn which diffused through the agar and dis-
hrouiiht al)()iit destruction of tlie actino- sohcd I he (lend bactei'ial cells.
al)ilit\- to e.\eri't(> toxic substances that ha\(' The nature of the ant imici-obial effects of
till' capacity to inhibit the f^rowth of other dilTer(>iit microbes is greatly influenced by
orti;aiiisnis oi' exiai to ilestroy them. the energy and nitrogen sources in the me-
M tiller (H)OS) first observed that certain dium. Schiller belie\('d that antagonism
piij;meiit-i)ro(hiciiiij; streptomyces {S. cocli- could be induced bv using microbes as nu-
mlar) ari' able to suppress the (h^velopmeiit trients: in a dilute glucose solution without
of yeast-like fuiiiii. In a study of the assoei- nitrogen, yeasts were said to be "forced" to
atix'e and antaii'onistie etfeets of certain kill and digest bacteria; if the yeasts were
actinomycetes and fungi, Porter (1924) dem- added to a full}^ developed bacterial culture,
onstrated that several organisms now known a bacteriolytic substance was produced
to belong to the genus Streptomyces, namel}' which was also active outside of the yeast
»S. tricolor,S. albus var. ochraccus and S. cells. Howe^•er, when the bacteria were inoc-
nigrificans, produced marked inhibitory ef- ulated into cultures of yeasts suspended in
fects upon the growth of fungi. He suggested distilled water, the yeasts were killed. Vari-
that such inhibitory action may aid in species ous efforts, however, to adapt cultures of
identification. actinomycetes by the process of ''forced
Gratia (1924) definitel}^ established the antagonism" to grow on specific bacteria
potentialities of actinomycetes as powerful failed to yield antibiotics that the organisms
antimicrobial agents. He and his associates w^ould not produce normally when grown
isolated a preparation, designated "myco- upon proper artificial media.
lysate," which was actually used in the treat-
ment of many clinical cases caused by The Soil as a Source of Antagonistic
tion of antibiotics does not appear to be some product secreted by these colonies which is
correlated, therefore, with the stimuhition obnoxious to the spreading colony, whether it be
Bac. rnycuides, Bac. vulgaius, or to spreading
of reactions influencing the breakdown of
moulds such as Pemcillium or Aspergillus. An
nutrients or certain other oxidation and fixa- examination of the colonies producing this toxic
tion processes. effect showed that the majority consisted of Ac-
Greig-Smith, in his studies on the occur- tinomj^ces or Streptothrix as they have been
called. Some of these darkened the medium and
rence of toxic substances in soil, gave a clear
were apparently j4c^ chromogenus Certain of these
.
"In making counts of soil-bacteria, it is not cumulate also as a result of a different ap-
uncommon to find colonies of Bac. mycoides or of proach. IMillard and Taylor succeeded in con-
races of Bac. vuUjatus spreading over the surface of trolling potato scab, caused by S. scabies, by
the nutritive agar. Very often it will be noted that, the use of green manures and grass cuttings.
while the majority of the colonies are covered by
When potatoes were grown in sterilized soil
the spreading growths, there are a few that are
infected with S. scabies, scab was reduced by
untouched. The mycoides-colony may approach to
within two, five, or ten millimetres, and then the simultaneous introduction into the soil
spread out and surround the colony, leaving a ring of S. praecox, a saprophytic organism. By
of clear agar medium. It is evident that there is increasing the proportion of the saprophyte
£. co/i
6. mycoides
S' subfilis
Staph, aureus
= Ps. aeruginosa
to the i);it lioticii. the (l(\<2;i'(>(> of scnhhiiiii; on showed that act inoniycetes exert an antago-
tlio test potatoes was reduced Iroiii 100 per nistic elTect upon the acli\-ities of other
cent to nil. microorganisms. Thus the ground work was
(loss tound, ho\\(>\'er, thai the i;('neral soil laid foi' the systematic study of the antago-
niicrofioi-a ratlier than eei'tain specific or- nistic properties of act inoniycetes and their
«>;anisnis ha<l a conlrollinji; elTect upon tlie ability to pi'oduce antibiotic substances.
development of seal); inoculation of soil
Table 51
60 TO SO SO 100 no 130
R. JAPONICUM
Figure 82. Relative susceptibility of test organisms to antagonistic actinomj-cetes (E. coli = 100)
(Reproduced from: Landerkin, G. B. and Lochhead, A. G. Can. J. Res. 26C: 505, 1948).
procedures, with numerous modifications, spores. Since the capacity to produce anti-
have found extensive apphcations in the bacterial substances was possessed by only
study of the antagonistic properties of ac- certain cultures, it was suggested that this
tinomycetes and have come to be known as property could be utilized for the systemati-
screening methods. zation of species of actinomycetes. It was
In 1935, the first comprehensive survey also suggested that various bacteria could
for the occurrence of antagonistic forms be differentiated on the basis of their sensi-
among actinomycetes, largely soil inhabit- tivity to the actinomycetes.
ants, was begun in Russia. Similar surveys Krassilnikov and Koreniako found that
of the occurrence of antibiotic-producing many species of actinomycetes, members of
Nakhimovskaia. Out of 80 cultures of actino- without subsequent lysis. This chemical na-
mycetes isolated and tested, 47 were able to ture of the active substance was believed to
exert antagonistic effects, but only 27 pro- be similar to that of lysozyme.
duced active substances. (Jram-positive bac- Ki'iss is()lat(>d a chemical agent from cul-
teria were inhibited, but not gram-negative tures of actinomycetes. The substance was
bacteria or fimgi. No relation was observed found to be insoluble^ in ether, petroleum
between antagonism and formation of pig- ether, benzol, and chloroform, and was re-
ments, manner of sporulation, or shape of sistant to the effect of light, air, and high
AN'rAcoMS'i'ic iM{()iM:i{'rii:s 213
Tablk 52 Tahi.k ot
Sid-rcjis of antayoitistic arliiiiniiiicilcs iKolulcil from Dishihulion, of nntibiolic (icliviti) of actinonij/cetes
soils and other naliiral siihstrata (Boiu'dict ) arcordhuj to ycnus (Kniersoii et al.)
214 THE ACTINOMYCETES, Vol. I
Table 55
Antibiotic activity of 600 actinomycetes isolated
from northern Canadian soils (Landerkin,
Smith, and Lochhead)
AN'i\\(;(»Msiic i'i{()i'i:inMi':s 215
species oi h' hi zohiit HI wvvv iiiliil)it(Ml hy iVwcr DiKlnhiititiii (if (uildfinnislic slreplomyces
strains: Rh. Irifolii was inliihitcd liy only !)
(l-iii(liiiT and Wallliiius.ser)
strains, and tlu^ cow pea rhi/.ohia by only I Nature of soil and vegetation Per cent of active strains
strain.
( '(nn|)()sts 8.6
Ill 1952, Poppo and Strntz isolated, tVom
lai'dcii soils
(
7.4
soil and ot her mat tcr, 'J'JO cnlt urcs of act iiio- iMcld soils 14.8
niyci'tcs. ( )t' tiu'sc, (I'J possessed antibacterial l*asture.s 18.6
l)i-opei-ties. 'I'en of the active cultures were Forest soils 17.2
1 5 row II soil 7.4
found to exert a stronj>; eti'ect upon \arious
River muds 3.8
j>;rani-posit i\'e l)acteria. Cdycerol-glycocoll
Virgin soils 22.2
solution pro\ed to be especially useful in the
production of the antibiotic substances.
latter, about one-third produced an antibi-
Lindner and AVallhausser (1955) isohited
oticsubstance when grown in liquid media
2,500 antagonistically active cultures of
under submerged conditions. The pei'centage
streptomyces from -10,000 soil samples. Their
of the upon gram-positive
cultures active
distribution in different soils is shown in
bacteria only was higher than that of strains
Table 5(3. Of these, 77 per cent were active
active upon both gram-positive and gram-
upon gram-positive bacteria {Staph, aureus),
negative bacteria. No culture was found to
40 per cent upon gram-negati\'e bacteria (E.
be active only upon gram-negative bacteria.
coll), 32 per cent upon mycobacteria {M.
Of the cultures active only upon gram-posi-
tuberculosis 607), and 18 per cent upon fungi
tive bacteria, about 20 per cent were active
(.1. niger). Wallhausser (1951) pi'oposed a
upon B. suhtilis, 20 per cent upon Staph,
series of charts for representing the mode of
aureus only, and about GO per cent upon
growth inhibition of one organism by an-
both.
other.
Jarikova et al. (1958) isolated 1,879 actino-
The results obtained in these sur^•eys were
mycete cultures from soils of diff'erent re-
found to depend upon the test organisms
gions of the Soviet Union; of these, 1,2G2
used, the composition of the media upon
cultures, or 67.2 per cent, proved active un-
which the organisms were grown, and various
der the conditions of their experiments. The
experimental conditions. Most of the active
largest number were found in the greenhouse
cultures belonged to the genus Streptomyces.
soil of the Botanical Garden, in meadow-
However, Endo, who tested the activity of
granular soils, and in steppe soils, as well as
1 16 strains of Nocardia upon 10 bacteria and
in light chestnut soils. The greatest number
6 fungi and yeasts, found that 27 per cent
of cultures having activity were found in
of all strains were active upon at least one
soils of eastern and southern regions.
of the test organisms. The conclusion was
Attention must be directed to the fact
i-eached that the Nocardia group has as high
that generalizations concerning the charac-
percentage of active strains as Streptomyces.
terization of certain types of soil by the oc-
The genus Micromonospora as well was found
currence of specific antagonists are hardly
to be capable of exerting antagonistic effects
they are based upon detailed
justified unless
against certain bacteria.
and oft-repeated investigations.
Craveri et al. (1957) isolated 500 cultures
of streptomyces from ^•arious soils in Italy. Antifungal Surveys
Just about a half had the capacity to inhibit The ability of various actinomycetes to
microbial growth on solid media. Among the antagonize the growth of fungi has long
216 THE ACTIXOMYCETES, Vol. I
60- -60
n % CAUSING SLIGHT INHIBITION
-50
I 50- ^ % CAUSING STRONG INHIBITION
§
o
<2 40
^ % CAUSING VERY STRONG INHIBITION
- 40
10-
B. subtilis S. aureus
I
P. vulgaris
flI
E. call
ij
P. aeruginosa
been known. Major attention was directed 2 days but began to undergo lysis on the
toward their activity upon plant pathogenic fifth day, large sections of the mycelium
fungi (Tins, 1932; McCormack, 1935). disappearing. On the seventh day only
Alexopoulos was the first to study, in 1941, chlamydospores were ob.served. In 9 days
the distribution of antagonistic activities the fungus completely disappeared, whereas
among actinomycetes against fungi. Of 80 the actinomycete was making a normal
cultures tested against CoUetotrichum glocos- growth.
porioicles, 17.5 per cent were strong inhibi- Leben and Keitt isolated a streptomyces
were weak inhibitors, and
tors, 38.8 per cent that was antagonistic to various phytopath-
43.7 per cent had no effect at all. Meredith ogenic fungi, but not to most bacteria. The
made a survey of the distribution of organ- active material was heat-labile, soluble in
isms antagonistic to Fusan'um oxysporum various organic .solvents, and in water at pH
ciibense in Jamaica soils. The antagonists 9.3. It inhibited the growth of fungi and of
were not evenly distributed, 10 of the 60 soil only very few bacteria. Of 3,788 actinomycete
samples giving 44 per cent of the antagonistic cultiu'cs isolatetl from soil by Cooper and
organisms. Those actinomycetes that were Chilton and tested against Pythium, 896, or
antagonistic to Fiisarium when grown in 23.() per cent, showed some antagonistic ef-
their own soil-solution agar were not always fect upon the fungus. Certain actinomycetes
antagonistic when tested in soil-solution agar were found to be responsibh^ for the destruc-
prepared from another soil. A culture of an tion, in soil, especially in j)artly sterilized
jictinomycete isolated from a compost pro- soils, of the mycelium ofOpJuOboIus graminis,
(hiccd lysis of the Fiisarium. When spores tlie cause of the take-all disease of wheat.
of l)oth organisms were mixed in an agar The parasitizing and antibiotic effects of
medium, the fungus de\eloped normally for actinomycetes and other soil organisms were
AN'iwcoMS'i'ic iM{()iM:iri'ii;s 217
l)('li('\('(l l(» l)t> rcspoiisiMc I'oi' the check in 200 strains were selected as warranting fur-
t lu'dcxclopiiHMil (i| I lie < )i)h /III Kilns in nat iiral ther study and wei'c grown in a \ariety of
soils. media in shake-flasks, llw cultinc filtrates
Stoss(>l studicMl the (list riluil ion in soil of wei'e tested for antibiotic actixity 1)\' paper-
act iiioniycet(>s acti\"e aj^ainsi |)athoi;(Miic disc assay against nine organisms. Fifty-two
fungi. Dilutions of soil samples wei'e added of th(> 200 strains sIiowcmI pi'omise. Culture
to suitable ai2;ar media, so as to ])eiinit 'M) lilti'ates from eight sti-ains as well as two
to ")() colonies to de\-elop on each plate. The substances isolated from the mycelium of
phites wore incubated for 4 days at 2()°C\ dil'fei-ent strains were then tested in the
Suspensions of spores of four nuitually non- greenhouse against certain plant diseases.
antajionistic phytojiathog-enic fungi were Xin(> of tlie 10 preparations showed broad
tluMi spi-ay(Hl on the plates. After 2 days' antifungal spectra in laboratory tests and
further incubation, 170 cultures of actino- activity against at least one disease in green-
mycetes producing marked inhil)ition zones house tests. Phytotoxic effects were observed
against all the test fungi were isolated. Plate with some of the test materials. The details
cultures of the isolates were sprayed again of the test are brought out in Table 57.
but with separate suspensions of eight fungi
Correlation of Antimicrobial Activity
and two bacteria. Twenty-one cultures,
mostly actinomycetes, were selected on the and Pigmentation
basis of comparative inhibition. These or- von Plotho correlated pigment production
ganisms were grown in fi\'e liquid media in by actinomycetes with their antagonistic
shaken flasks. The culture filtrates were properties. Two hundred and ninety-one cul-
tested for activity against Glomcrella cingn- tures grown in colorless media were classi-
lata, by the use of a modified cylinder plate fied into four groups on the basis of pigments
method. Sixteen cultures produced an anti- produced either in the mycelium or in the
biotic in one or more media. Of the 170 medium. Activity was determined by testing
organisms originally isolated, five appeared against M
ijcobacterium eos. Of the 61 cultures
to produce desirable substances, as deter- (21 per cent) showing activity, 21 were in
mined by the above screening tests. Further the colorless group, 20 were in the red-yellow,
studies on the antagonistic effects of actino- 12 in the red-blue, and 8 in the red-brown
mycetes upon plant pathogenic fungi have group. By using media in which pigments
been made by ]\Iukherjee and Xandi (1955) could be seen readil}^, investigators might
and others. Some actinom3xetes are particu- well try such a correlation to learn whether
larly active against yeasts (Takahashi, 1952). particular groups could be eliminated with-
The activities of soil actinomycetes upon out further testing. A detailed analysis of the
fungi pathogenic to man were examined b}- antibiotic pigments of actinomycetes was
Schatz and Hazen. presented in Chapter 13.
Pridham ct al. (195G) made an extensive
Screening for Specijic Antibiotic-
survey of actinomj^cetes, largely strepto-
yroducing Organisms
myces strains, for their growth-inhibiting
effect againstphytopathogenic bacteria and By using the techniciue reported by Waks-
fungi. About 500 pure cultures were sub- man, Reilly, and Johnstone (19-46) of adding
jected to primary screening by three meth- a particular antibiotic to the medium before
ods against a minimum of 12 test organisms. plating out soil samples, Umezawa et al.
Based on their antimicrobial spectra, some (1949) were able to demonstrate the presence
218 THE ACTIXOMYCETES Vol. I
Table 57
Inhibitor!/ activity of streptoniyces against selected phytopathogemc
bacteria and fungi (Pridham et al., 1956)
Test organism
AxrAcoMS'iMc PH()i'i';i{'rii-;s '21!»
;i\('i';iu;c, "J") |)(M' cent of the i>(>l;ilcs wci'c and Kolano\a reported that
Ki'assihiikov
found to i)ro(lu{'(> an ant il)iotic. Ajjproxi- neai'ly all act inom^'cetes possess
antiphage
matoly !l() p(>r (-(Mil of all lh(> antil)ioti('-pro- properties. Some inhibit many phages and
(lucinii cultures foiincd si rcpiot luiciii oi' others are elTectixc onl\- upon specific phages.
(•los(>iy iTJatcd ('onii)ouiids. A half of the Actinomycetes show characteristic anti-
;i
fcMiiaindcr pfoduccHJ st rcptonixciii, ouc-t liiid l>hage spectrum. There is no relation between
(of the other halfi formed let i-aeyelines. aiiti\irus nnd antiphage antibiotics; there-
Finally, most of thos(> that remained could fore, tlu^ lattei- cannot be used as models for
he identified witli on(> of the x-arious anti- study of antivirus agents.
biotics which ha\'e heeii isolated from cul- All iiitenssting in vitro techniciue utilizing
tiu'es of streptomyc(Hes. In other words, a modified agar plate diffusion method for
2,250 cultures <>;a\-e strei:)tothricin-like anti- the detection of antitumor activity was
liiotics, about 12.") streptomycin, 40 yielded described by Miyamura. Ehrlich's ascites
tetracyclines, oo produced other i)re\-iously tumor cells are incorporated into a ba.sal
described antibiotics, and only oO yielded medium. The materials to be tested are
new antibiotics. placed in cups and allowed to diffuse through
the medium; the cups are then removed and
Test Organisms
the plateis flooded with methylene blue; the
A wide selection of organisms is commonly agar covered with a glass plate and incu-
is
used in testing cultures for antibiotic ac- bated; the diameters of the blue zones are
tivity. Freciuently, a highly sensitive or- measured. Positive results were obtained
ganism like B. subtilis or Staph, anreus is with various anticancer agents including the
u.sed. In a search for a specific antibiotic antibiotics trichomycin and sarkomycin and
against a particular parasite, such as AI. 8-azaguanine. Other antibiotics of actino-
tufx-rrxlosis^ the latter itself or a form closely mycetes (tetracyclines, streptomycin, and
related to it is used as the test organism. In neomycin) gave negative results.
most cases, however, \-arious organisms are Aral and Suzuki published a modification
used, including gram-positive and gram- of the above method. Serial dilutions of the
negative Ixicteria, saprophytic and parasitic materials to be tested are placed in tubes.
fungi. Plate method techni(iues have been The tumor cells are added, the suspension is
developed by Waksman and Reilly (1945), mixed thoroughly, and then buffered glucose
Waksman et al. (1947), and Williston ef al. agar containing methylene blue is added.
(1947). The results are read after 3 hours' incubation.
Recently, actinomycetes possessing ac- All antitumor substances tested, except
tivity against viruses and experimental 8-azaguanine, gave positive results. These
cancers have been investigated. For this substances included sarkomycin, carzinoph-
purpcse, special procedures are used, such as ilin, the gancidin complex, and others.
tho.se involving bacteriophages (Jones and Rombouts (1953), Stevenson (1954), and
Schatz; Schatz and Plager, 1948). Out of Links c/ a/. (1957) i.solated certain cultures of
527 cultures of actinomycetes screened by streptomyces from soil that produced an
Landerkin et al. (1957) for antiphage ac- antibiotic possessing the capacity of causing
tivity, eight inhibited the development of the swelling of hyphae of se\-eral fungi. The
bacteriophages; of these eight, only one had substance responsible for this swelling was
an elTect upon phages of two species of bac- designated as the ''bulging factor." This sub-
teria. None of the cultures tested had any stance, isolated from the medium, proved
effect upon the Xewcastle virus. to be a streptothricin-likc base readily solu-
.
ble ill water, l)ut not in organic solvents. It dissimilar; some strains pnn-ed inhibited and
was stable an acid reaction, but was
in others indifferent
rapidly destroyed in an alkaline reaction and When the antimicrobial properties of ac-
at 100°C. tinomycetes became well recognized, it was
The onl}^ report of a substance active only natural that attempts should be made
upon actinomycetes and not upon true to encourage the development of these or-
bacteria or fungi was made by Szabo and ganisms in the soil, in order to depress the
Marten (1955), who isolated from a culture growth of various organisms capable of
of an actinomycete an "anti-actinomycete causing plant diseases.
factor" that was highly active against ac-
tinomycetes, without any activity upon
Activity of Actinomycetes upon Plant
Pathogenic Fungi
gram-positi\'e and gram-negative bacteria.
An extensive literature has accumulated
Effect of Actinomycetes upon Sapro- upon the antagonistic effects of actinomy-
phytic Soil Bacteria cetes upon fungi, especially upon plant
The effect of actinomycetes upon the pathogens. References to such effects are
growth of soil saprophytes and upon plant reported earlier in this chapter. Winter pre-
pathogens has also received considerable sented (1949) further evidence concerning
attention. Nikolaieva first observed, in 1914, the ability of various actinomycetes to at-
that actinomycetes may exert a repressive tack Ophiobolus graminus, an important
effect upon the growth of Azotohader. Nickell parasite that attacks wheat. Sanford and
and Burkholder (1947) studied the inhibition Cormack tested the effect of eight cultures
of growth of Az. vinelandii by \'arious ac- of actinomycetes upon the disease-producing
tinomycetes isolated from soil. When cul- fungus Helminthosporium sativum. In com-
tures of actinomycetes were mixed with soil parison with a disease rating of 66 per cent
samples and plated out, the nitrogen-fixing for the untreated pathogen, four actino-
bacteria were either greatly reduced in num- mycetes depressed the \'irulence of the
ber or completely eliminated. It was sug- pathogen to 33, 22, 22, and 1 per cent, re-
gested that this serves to illustrate the spectively; two had no marked effect; and
importance of microbial antagonism in eco- the other two appeared to increase the viru-
logical investigations. lence by 12 and 16 per cent, respectively'.
Another antagonistic effect of actinomy- Perrault demonstrated that the growth of
cetes upon nitrogen-fixing bacteria may CoUetotrichum sepedonicum in agar media
prove to be of even greater economic im- was impeded by several microorganisms iso-
portance. Konishi and Fukuchi have shown lated from potato tubers affected with ring
that certain actinomycetes are able to in- rot. Four of these organisms were actino-
hibit the growth of root-nodule bacteria; mycetes and were able to produce antibiotic
some of the (!ultures, like »S'. flavus, were par- substances that diffused readily through the
ticularly effective. medium and prevented all growth of the
Babak (1958) tested the sensitivity of (iO pathogen. One culture produced a lysis of the
Azotobacter cultures to various species of plant pathogen.
Streptomijces and to various antibiotics, McGahen treated soils in the sugar-cane
such as penicillin, streptomycin, gramicidin, belt of Louisiana with bagasse compost, with
and chlortetracycline. They were all found cowpea and soybean trash, and with blood-
to be susceptible to the antibiotics. Their meal, tankage, bonemeal, and ammonium
sensitivity to the S. roelicolor grou]) was sulfate. The actinomycete^ populations were
)
measured hy the dihilioii iiicl liods; the aiili- Are Aniiliiol ics Prudiu-ed in the Soil?
l)i()(if actixitics were dclcnniiUHl hy llic dc- In I!) I."), W'aksman pi'esented e\idence
^\vo of inhibit ion of ri/tltiuni arrhcnonKtncs thai il is highly doubtful that ant ibiot ics are
in cuhurc. Xilroiiicn-rich inalcM-ials, such as produced in the soil itself or that this phe-
rowpca and soybean trash, bloodnical, and nomenon is of any great significance in
tankafiic naxc niai-kcd incrcasos in the ac- modifying the mici'obiological population of
tinoniycctc populal ions. The ant ibiot ic indi'X the soil. The exideiice was based upon the
was increased by the use of l)agasse compost following observations:
and cowpea-soybean trash; a doerease of the (1 The fact that an oi'ganisni produces an
antibiotic index occurred, lio\ve\-ei-, when the antibiotic in aitilicial cultui'e is no evidence
soil was treated with bonemeal, l)loodmeal, that is is capable of doing so in soil, pai'ticu-
or tankage. Annnonium suU'ate did not ma- laily since I'elat iAcly small changes in a
terially affect either the ])oi)ulati()n or the nutri(Mit medium may fundamentally affect
antibiotic index. It was suggested that nitro- the production of antibiotics in pure culture.
gen-rich materials, like bloodmeal and tank- (2) ]\Iany known antibiotics are extremely
age, cause an increase of th(^ nonantagonistic unstal)le and could not be expected to re-
and/or weakly antagonistic actinomycetes in main vmchanged in soil for sufficient time to
favor of the moderately and highly antago- have any effect.
nistic forms. The cellulosic materials cause
(3) There is no evidence that production
an increase of the antagonistic over the of antibiotics affects in any way the survival
nonantagonistic actinomycetes. of organisms producing them.
(4) The fact that the organisms
found in
Antagonistic KlTects of Fungi and Bac-
the soil no greater resistance to
possess
teria upon Actinomycetes than
particular antibiotics comparable
In a natural environment, such as soil, the strains found in other substrates add further
antagonistic properties of actinomycetes, if weight to the non-existence of such antibi-
they develop at all, will be exerted largely in otics in soil, in concentrations sufficient to
an aerobic enviroinnent. Under anaerobic or exert an effect.
microaerophilic conditions, the actinomy- Brian, Krassilnikov, and others argued
cetes themselves may be antagonized; l)ac- against these assumptions. Brian emphasized
teria, like Ps. fluorescens, have been shown that some antibiotics are very stable, that
to exert a marked antagonistic action upon they can be produced locally, where they can
actinomycetes, causing their lysis. B. mcga- have a maximum effect. Siminoff and Gott-
ierium can also be antagonistic to certain lieb (1951, 1952) could demonstrate the for-
species of actinomycetes, but also can be mation of antibiotics, notably streptomycin,
antagonized by others. The effect of bacteria in sterile soil in fresh soil. Pramer
but not
upon the potato scab organism was studied and Starkey established that streptomycin is
in detail h\ Kieszling, who was able to pre- rapidly destroyed in the soil.
vent scab (le\'elopment in the soil by the use Some recent evidence that antil)otics are
of such bacterial cultures. produced in soil has been submitted by
Numerous fungi are also capable of exert- Stevenson (195()). In most cases, llowe^er,
ing a marked destructive effect upon actino- the assumption that the occurrence of
mycetes. This is true, for example, of the ef- streptomyces capable of producing anti-
fect of the fungus antibiotic penicillin upon l)iotics will lead to the formation of such
human and animal diseases caused by actino- antibiotics in soil, and that this will lead to
mj^cetes. the elimination of pathogenic bacteria is far-
222 THE ACTINOMYCETES, Vol. I
fetched.Such assumptions have been made, first believed that the same principle would
for example, for the ehmination of typhus apply to the isolation of antibiotic-producing
abdominahs bacteria in irrigated soils, organisms. Claims were actually made that
merely because several streptomyces have favorable results were obtained. But the in-
been isolated from such soils. troduction into the soil of cultures of micro-
organisms led to their destruction (Katznel-
Soil Enrichment with Pathogenic Or- son, 1940).
ganisms Waksman and Woodruff (1940) attempted
Soil enrichment with various chemical to encourage by soil enrichment the develop-
compounds results in the isolation of organ- ment of antibiotic-producing actinomycetes.
isms capable of bringing about certain spe- They at first believed this had succeeded.
cific reactions. This is true, for example, of They said that the survival of bacteria added
the development of organisms capable of de- to the but not indigenous to it, resulted
soil,
composing cellulose or pectin by the addi- in the rapid dying out of such added bac-
tion of these substances to the soil; it is also teria. This was believed to have been accom-
true for the enrichment of soil with sub- panied by an increase in the numbers of soil
Table 58
Growth inhibition of streptomyces by their respective antibiotics (Waksman, Reilly, and Johnstone)
Dilution units per mg,
Nature Organism
of antibiotic producing antibiotic
AXTA( U )\ rSTIC l'H( )PERTIES 223
to isolate cultures of act inomycetes that Soil s.'iinplcs ;iii(l (;iit hwoniis were du}^
mijiilu poss(>ss specific actixity against the Iroin the }>;i-n\('s, hut the cullurcs ohtaiiicd
orjianisms adde(l to tlie soil. "\\(M'(> disappoint iiifily dcxoid of intorcsling
'I'he writer I'eceixcd numei'ous su.nj^est ions antaiioiiists." Soil samples wei'c ulso taken
that lie use soils from cemetei'ies
in which aloiif^ the i-()ut(' of a scwa^o effluent fi-oui a
sufforors from tuberculosis and other in- saiiatoi'iiun foi' tuberculous patients. "Sam-
fectious diseases or cancer \ictims were re- ples of tlie soil taken alon*;' this chamiel of
cently hui'led. lie can do no better than to ti'eatment from the faw se\vaj;-e to a point .")()
quote from Routi(Mi and Finlay, who fol- yai'ds downiiill yielded no organism of high
lowed up such suiiii'est ions. 'I'hey attempted antagonistic powers."
on two occasit)ns to ist)late antagonistic or-
Cross-resistance of Microorganisms as
ganisms from such sources. Soil from an
Diagnostic Criteria
orthodox Jewish cemetery, wh(M'e bo(h(\s wei'c
interred soon after death without (Mubalming, Waksman, Reilly, and Johnstone (1946)
Avas examined foi' antagonists against the first demonstrated that act inomycetes are,
pathogens introduced into tiie soil with the as a rule, resistant to theantibiotics produced
diseased body by them (Table 58). However, some or-
ganisms tended to be less sensitive to certain
Table 60
antibiotics than to others. These investi-
Cross-resistance of anlibiotic-produriny
gators suggested taking advantage of this
aciinomycetes (Teillon)
phenomenon for the isolation from the soil
of specific organisms producing a particular
antibiotic, and for isolation of more potent
antibiotic-producing strains from a mixed
mother culture. It was later shown (Waks-
man and Lechevalier) that certain organisms,
like S. fradiae, are sensitive to their own
antibiotics. Krassilnikov accepted the phe-
nomenon of cross-resistance within the
species of actinomycetes as offering a de-
224 THE ACTIXOiMYCETES, Vol. I
sirable property for species differentiation modified the effects of the major antibiotics
(Table 59). Umezawa, Oki and Hata, produced by the organisms. Some strains
Kuroya, and others also accepted this con- related to *S'. griseus were able, for example,
cept for classifying actinomycetes. to produce streptothricin as well as strepto-
Teillon (1952), who made a detailed study mycin. Phenomena of autoinhibition are also
of the cross-resistance of actinomycetes pro- not uncommon among actinomycetes, such
ducing antibiotics, found various important as S. aureofaciens and S. fradiae.
exceptions to the principle expostulated by Finally, the quantitative variations in the
Kuroya and Krassilnikov (Table 59) . On the production of antibiotics do not permit the
basis of these results, he expressed doubt
laying down of hard and fast rules concern-
concerning the validity of the claim of the
ing the possible effect of cross-inhibition un-
Japanese investigators that "the cross-inhibi-
der natural conditions. This is brought out in
tion test is a useful and easy method of dif-
Table 61 on the auto- and cross-inhibition
ferentiation." The method of cross-inhibi-
of various neomycin-producing and other
tion did not appear to Teillon as sufficiently
strains of S. fradiae. It is possible to observe
reliable for differentiating streptomyces cul-
and their antibiotics. The organisms occasionally certain self-inhibition of growth
tures
were shown to form a variety of metabolic by various actinomycetes, especially by pig-
products that could readil}^ mask the results. ment-forming types. This has been ascribed
Further, some organisms produced certain by Krassilnikov et al. (1958) to special sub-
other antibiotics that, although in them- stances of an enzymatic nature designated
selves possibl}'- not quantitatively significant. as "necrohormones."
:
c II A p T i: n I .1
Production of Antibiotics
on viruses and phages, and others are ac- of closely related chemical compounds. Only
tive on tumors. Some of the antibiotics are one of the actinomycete antibiotics, chloram-
said to be of a broad spectrum type, able to phenicol, has been synthesized. Others have
repress the growth of both bacteria and been modified chemically to give compounds
fungi, or of bacteria, rickettsiae, and the with slightly different types of activity. This
larger viruses. Some have a narrow antibi- is true of the formation of dihydrostreptomy-
otic spectrum, such as those that are largely cin from streptomycin and of tetracycline
active against gram-positive bacteria or my- from chlortetracycline. Definite chemical
cobacteria, or yeast-like organisms (Waks- structures have been established for a num-
200
DAYS
Figure 85. Utilization of galactose bj' S. growth and actinomycin prodviction:
antibioticus for
A A = dry weight of mycelium in mg per 100 ml of medium; X X = percentage of residual
galactose; O O = actinomycin produced, Mg/n^l of medium (Reproduced by special permission
from: Katz, E., Pienta, P., and Sivak, A. Appl. Microbiol. 6: 238, 1958).
produces oxytetracycline and rimocidin. otic or closely related compounds are found
2. A single strain of an antibiotic-produc- in different soil regions throughout the world.
ing organism may form several chemically This is true, for example, of cultures of strep-
related antibiotic substances. This is true of tomyces producing actinomycin, streptomy-
the production of mannosidostreptomycin cin, chloramphenicol, the tetracyclines, the
and streptomycin by S. griseus; of neomycin erythromj^cins, and others. Since the strains
:
prod lie i 11,l;; tlic siinic antibiotic may \aiy, tlie especially' in search for antixiral or ant it u-
morpli()l()j;icai critci'ia alone cannot he used more complicated procedures arc
moi- agents,
as a basis for identit'vintj; antibiotics, or the employed. Such cultures of actinomycetes
formation of a particular antibiotic I'oi' iden- as are found to j^jossess the desirable anti-
tificat ion of species. microbial pro[)erties are selected for further
4. A ciiauf^'e in the nutrition of the or<j;aii- study.
ism may result in a cliaugo in the nature of 4. The selected cultures are gi'own in
the antibiotic produced. Tliis is true, for ex- various li({uid media, in stationary or under
ample, of the formation of the various actino- submerged conditions, for varying periods
mycins. (usually 3 to 10 days), and the antibiotic
5. As a rule, aiitibiotic-formin<>; organisms spectra of the broths determined.
are resistant to the antibiotics they produce, 5. Suitable media and proper growth con-
as shown in Chapt(>r 14. This phenomenon is ditions are established for each individual
taken advantage of in the isolation of fresh culture. The nature of the medium for the
cultures capable of forming a given antibi- production of the antibiotic is of great im-
otic, and in the selection, from a given cul- portance; each culture may require special
ture, of more potent strains. Not all anti- media for optimiun production of the anti-
biotic-forming organisms, however, behave biotic.
in the same manner. 6. An efi'ort is then made to isolate from
the medium the active substance produced
Methods for Isolating and Testing Antibiotic-
by the culture, concentrate it, and purify it.
producing Organisms
7. The isolated antibiotic is studied for its
200 200
175
150
125
z
100 u
>
o
z
75 h-
u
RESIDUAL <
GLUCOSE 96
50
C 25
^0
10
DAYS
FiGTRE 86. Utilization of glucose by <S. a/i;j6io//fus for growth and actinomycin production: A A =
dry weight of mycelium in mg per 100 ml of medium; X X = i:)ercentage of residual glu-
cose; O O = actinomycin produced, fig/m\ of medium (Reproduced by special permission from:
Katz, E., Pienta, P., and Sivak, A. Appl. Microbiol. 6: 237, 1958.)
Table 62
Comparative atitibiotic spectra uf streptomycin
and streptothricin
B. subtilis
B . mycoides
B. cereus
B. megaterium . . .
Staph, aureus. . . .
Sar. lutea
M. phlei
M. tuberculosis . .
Ph. pruni
E. coli
Serr. marcescens.
Aer. aerogenes. . .
Pr. vulgaris
Ps. fluorescens . .
Ps. aeruginosa . .
CI. butylicum . . . .
230 THE ACTINOMYCETES, Vol. I
Table 63
Antibacterial activities of the various
streptomycins (Rake et al.)
PH()l)r("'ri()\ OK ANTJIUO'IMCS 231
The Actiiioiiiyt'iiis
Streptothricin
Katz, and Vining (1958) proposed a system biotic that had come to occupy an important
for classifying the various actinomycins, place in chemotherapy. Its delayed toxicity
based on their chemical structure. The paper prevented its immediate clinical use (Waks-
chromatographic methods are very conven- man, 1943).
ient for their differentiation. The in vivo activity of streptothricin was
Among the interesting developments in first established by Metzger et al. (1942);
connection with the study of the actinomy- its action upon the tuberculosis organism
cins is their effect upon neoplastic growths. was later demonstrated by Woodruff and
The work of Hackmann, begun in 1952, on Foster (1944). It was later crystallized by
the effect of actinomycin C on experimental Fried and Wintersteiner (1945), and by
tumors and the clinical observations of Kuehl et al. (1945-1946). The search for
Schulte and Lings (1953) opened a new field streptothricin-like substances continued for
for the potential utilization of this group a long time, partly because of their anti-
of antibiotics as chemotherapeutic agents. tuberculosis properties (Weiser et al.) and
These results were soon confirmed by a num- partly because of their intriguing chemistry.
ber of clinical investigators. It is sufficient to Numerous related compounds were described
mention the studies of Trounce et ah, Sigiura under a variety of different names.
and Schmidt, Nitta et al., Yamashita et al., Numerous other preparations were later
and Farber and Burchinal (1958). These isolatedfrom cultures of *S. lavendulae (Bo-
studies brought out the fact that although honos Some were found to be the same
et al.).
(iiffei'ent actinomycins may vary somewhat as streptothricin, and others were either
in their toxicity, they are similar in their closely related or comprised mixtui'es of dif-
cytostatic activity (Pugh et al., 1956). Foley ferent antibiotics.
reported his observations on the mechanism Soon after the isolation of streptothricin,
of the action of actinomycin in bacterial another antibiotic, designated as proactino-
systems (For a review of the literature, see mycin, was isolated (Gardnei- and Chain,
PRODrCTlOX OF ANTIHIOTIC'S 233
I!) 12) tVoin a culture of :ui organism l)elieved Special iiiclhods wcic soon developed for
to l)t> a uocarclia (proacMinomycos) but now \hv isolation of other streptomycin-produc-
recognized as a strrptoniyces. Its antil)ac- ing cultures, as well as for obtaining more
terial and other biological properties wore potent strains from the mother culture, by
by Florey
later established ct al. (1945) and using media containing varying concentra-
by !Marston and Florey. tions of streptomycin (Waksman, Bugie, and
Schatz; Waksman, Reilly,and Schatz),
Streptomijcin
Streptomycin was the first antibiotic pro-
The experience gained in the study oi' the duced by actinomycetes that took a promi-
formation and isolation of streptothrichi nent place in the treatment of mnnerous in-
from culture's of actinomycetes proved to be fectious diseases in man, animals, and plants.
highly important in planning a search for It also proved to be the first drug effective
other antibiotic agents that would possess against the Great White Plague of man, tu-
similar or e\'en more desirable biological and berculosis, as first shown by Schatz and
chemical properties, such as a broad anti- Waksman (1944).
biotic spectrum and a lesser toxicit}^ to the The activity of streptomycin upon bac-
animal body. After further extensive studies terial infections in experimental animals was
of many actinomycetes representing a great first established by Jones et al. (1944), and
variety of species and varieties, two cultures in experimental tuberculosis by Feldman
were found to yield an important antibiotic. and Hinshaw (1944), and later by Youmans
These cultures were isolated from the soil and McCarter (1945), and Smith and Mc-
and from the throat of a chicken. They both Closkey (1945). The effectiveness of strepto-
belonged to a species described in 1916 as mycin was first es-
in clinical tuberculosis
Actinomijces griseus, the first representative tablished by Hinshaw and Feldman (1946),
of which was isolated by Waksman and Cur- followed by Cooke et al. (1946), who treated
tis in1916 from the soil. The generic name the first case of tubercular meningitis with
of the organism was changed by Waksman streptomycin, and soon by numerous others
and Henrici in 1943 from Actinomyces to (Keefer et al.).
Streptomyces. To honor this new generic Streptomycin w^as soon isolated in crys-
name, the new antibiotic was designated as talline form (Carter et al., 1945; Peck et al.,
streptomycin. 1945) and its chemical nature determined.
As previously noted, the two cultures of In standardizing streptomycin (Waksman,
the streptomycin-producing organism were 1945), it was found that 1 unit of the anti-
fu'st isolated in September 1943. Because of biotic, as determined by its activity upon a
the similarity of the new antibiotic to strep- standard strain of E. coli, under standard
tothricin, both in isolation procedures and in conditions of culture, was equivalent to 1 /xg
FiGURK .^'.1. First cxixTiniciit in which the efft'ct of .streptomyfin upon the giuwlii of M . tuberculosis
M-as demoiistnitod (l{opn)(hK-('d from: Schalz, A. and Waksman, S. A. Pr.)c See Exptl Biol Med 57:
246, 1044).
Streptomycin was detected in tli(> solntion imuni was reached between 48 and GO hours
during the first 12 houis of ^lowtfi of the of growth. After this, adecHne in the strep-
organism. The foiinatioii of the antibiotic tomycin coiilent of the sohition occurred.
mcreased steacUly from tiien on until a max- This decrease continued mitil after al)out 9()
rH( )i)i(^'ri()\ OK wriBioTics 235
lioui's, when tlu> slope of the ciirNC (Iccicnscd stance containing at least one guanidine
with :i tciulency to lo\"i'l ot'f. In all cases the group. This compound has not yet been iden-
peak of tlie st reploinyciii eurx'e Iat;i!;e(l l)e- tified l)ut may l)e invoh'cd in the biosynthe-
hiiul the p(\-ik of the t^rowth eur\-e by about leptomycin by the organism.
sis of st
24 hours. 'I'he eoiu-hision was nviched that On catalytic hydrogenat ion of sln^ptomy-
streptomycin prinhiction in \\\c nunliuni is cin, two hydrogen atoms are added to the
not primarily a function of tiie act i\'e ,i;i()\vtli molecule, gix'ing dihydi'ost I'cptomycin.
phase of N. (in'scus. Only al)out 25 to oO per K(>cently it was i-eported that certain or-
cent of the total streptomycin had l)e(>n re- ganisms (S. humiflNs) can produce dihydro-
leasinl into tlie solution hy the time the streptomycin directly in the medium. This
growth peak was reached. product is similar to streptomycin in its anti-
On continued growth in artihcial nuHha, bacterial and pharmacological j^rojK'rties, ex-
S. gi'iscus may become contaminated with a cept that in many cases it exerts a less severe
phage or virus, designated as actinophage. effect upon \'estibular dysfunction, although
The growth of the streptomycin-producing it may give greater autotoxicity. These re-
organism and th(^ prothiction of streptomy- sults so far have not been confirmed.
cin are rapidly cUminished. B}^ the use of Certain organisms belonging to the Strep-
the placiue method, it is possible to measure tomyccs group also produce desoxy strepto-
the actual concentrations of actinophage in mycin, which was found to be more toxic
the culture. The number of particles per mil- than streptomycin.
liliter can reach as high as 10'". Phage-resist- Streptomycin is active against a large
ant strains can be readily obtained from in- number of bacteria found among the gram-
fected cultures. negative, gram-positive, acid-fast, and spi-
Streptomycin is a strong Ixise, belonging rochaetal groups. Many of the bacteria af-
to the glucosides, in which a diguanido-group fected by streptomycin are able to cause a
is linked to a nitrogen-containing disaccha- great variety of human, animal, and plant
ride-like compound. A molecular weight de- diseases. Streptomycin is also active against
termination on the trihydrochloride in water certain plant-disease-producing fungi be-
ga\-e al)out 800 for the free base after the longing to the phycomycetes. It is not active
necessar}' corrections for the chloride ion. against anaerobic bacteria, protozoa, vi-
Investigations carried out by Hunter et al. ruses, and the majority of fungi. The sensi-
on S. griseus with the aid of C^K)2 have ti^'ity of a given organism to streptomycin
shown that the carbons of the guanidine side depends not only upon the species, but also
chains in streptomycin are derivedvery upon the strain, and upon the composition
largely, and possibly entirely, from carbon of the medium in which it is tested. The bac-
dioxide. The maximum incorporation of teriostatic and bactericidal action of strepto-
C^Oo into streptomycin was between 0.4 and mycin upon M. tuberculosis, the causative
O.o per cent. A much lower degree of fixation agent of tuberculosis, is particularly signifi-
of C'^ was obtained when no C^O-j was passed cant. It is actiA'e in a concentration of 0.05 to
for the hrst 72 hours of the fermentation. L- 0.4 Mg/nil. The bactericidal action also varies
arginine possibly functions as an intermedi- with concentration and the length of contact
ate in the biosynthesis of the guanidine side with streptomycin, 0.3 /zg exerting an effect
chains of streptomycin. A variety of com- in 48 hours, and 20 fjig/m\ in 6 hours.
pounds, either containing guanidine groups Sensitive bacteria become more resistant
or readily changed into such compounds, are to streptomycin upon prolonged contact with
converted by *S'. griseus into a further sul)- the antibiotic. This is of considerable theo-
236 THE ACTIXOMYCETES, Vol. I
retical and practical importance. Freshly found to be a mixture of several closelj^ re-
isolated cultures of tubercle bacilli from pa- lated compounds, all exerting antimicrobial
tients with pulmonary tuberculosis are uni- effects, thus suggesting the term "neomycin
formly sensitive to streptomycin. When a complex" (Dutcher and Donin, Ford et al).
culture is exposed to streptomycin in rela- One fraction isolated from the complex, was a
tively low concentrations, growth of the mul- basic, water-soluble, nitrogenous substance,
tiplying cells is inhibited but not that of the designated as neomycin A. It gave 1,700 di-
nonmultiplying cells. This resistance persists lution units/mg against B. subtilis, by plate
for a considerable time and is not accom- assay, but only 50 units/mg against E. coli,
Neomycin Chloramphenicol
Waksman and Leche^'alier first isolated Among the antibic^tics that have found ex-
neomycin, in 1949, from a culture of S. jra- tensive therapeutic applications is chloram-
diae. Neomycin compounds
or closely related phenicol, isolated in 1947 by and by
l^^hrlich
of different oth(>r organisms, such as S. al- myces {S. venezuelac) l^mezawa et al. iso-
.
Novobiocin
This antibiotic is produced by S. niveus
and by S. spheroides. It has been isolated
simultaneously in different laboratories and
described under different names (strepto-
nivicin, cathomycin, cardelmycin, vulcano-
mycin, etc.). It is highly active against Staph,
aureus, as well as against a variety of other
gram-positive and some gram-negative bac-
teria, but it causes allergic dermatitis.
Streptovariciii
2 4 6 8 10 The Polyenes
UNITS OF GRISEIN /ml Most of the previously listed antibiotics
Figure 90. Effect of antibiotic concentration are active primarily on bacteria and have
on development of resistance by E. coli in agar only a limited activity on true fungi. There
media: O, Gri.sein alone; A, Grisein + strepto- are, however, a number of antibiotics pro-
mycin 1 Mg/ml; n Grisein + streptomycin 2/xg/ml. duced by actinomycetes that have a highly
(Reprodnced from: Reynolds, D. M. and Waks-
selective activity upon fungi. As pointed out
man. S. A. J. Bacteriol. 55: 750, 1948).
previously, the first survey of the distribu-
tion of antifungal properties among actino-
The Macrolides
mycetes was made by Alexopoulos. A large
The first of these antibiotics (erythromy- nimiber of compounds ha^'e now been
cin) was isolated l)y JVIcCkiire et al. (1952) isolated and described under the names of
from a culture of »S'. enjthreus. It is a basic actidione, fradicin, thiolutin, nystatin, candi-
compound, soluble in water, alcohol, and cidin, ascosin, candidin, trichomycin, anti-
other organic s()lv(Mits. It has significant ac- mycoin, filipin, amphotericin, and others.
tivity against gram-positive organisms and Most of them belong to the polyenes. Only
some of the more important gram-negati\'e a few of them, notably nystatin and tricho-
bacteria, such as the Neisseria, Ilemophibis, mycin, have found practical application.
and Brucella groups; it is also activeupon Recently, Ball et al. mad(^ a comprehensive
rickettsiae, the larger viruses, and some pro- study of the production of polyene antibiot-
tozoa. ics l)y species of Streptomijees. These polyenes
A number of other ervthromvciu-like com- w(M-e grouped together on the basis of cer-
. : o
>^^-o—AA/^ —o—W—
i— AW '
8 10 12 14 (6 20 40 60
UNITS OF ANTIBIOTIC / mi
tures were characterized by paper chroma- effect in human tumors. The same may be
tography using agar blocks, by behavior on said of another antitumor agent designated
agar plates incorporating ion-exchange res- as carzinophilin. A number of other sub-
ins, and by ultraviolet absorption spectrum stances (DON, mitomycin, sulfocidin) were
(to detect polyene substances). About half, found to exei't marked antitumor activity,
or 195 cultures, produced a mixture of anti- but none of these has as yet found any prac-
biotics of both polyene and nonpolyene type. tical application in the control of tumors in
Figure 92. The use of bacterial strains sensitive and resistant to a given antil)i()tic lor the identifi-
cation of the particular antibiotic. E. coli strains (from right to left): streptomycin-resistant, strejito-
mycin-dependent, and streptomycin-sensitive. The plates were streaked from top to bottom: original
streptomycin-producing iS. c/riseiis; culture producing an unknown antibiotic not of the streptomycin
type; an unknown streijtoinycin-])roducing culture.
I'HODrCTlON OK WTIHIOTICS 241
tialit ics. ( )| her ;iiit i])i(>tt)/,();iii nut ihiol ics pi'o- 'l\\ltl,K (17
iIucihI hy act iiioniycrlcs include coiiiiiocidiii, Release nf (uitihiotic aclivilij from mycelium of S.
griscMi.s bti chemical and physical treatments
ciifocidiii, t'ci'iuicidiii, and \ alinoniNcin.
(Perhnan and Langlykke)
A niiinhcr of antixiral sutistanccs nrv also
Antibiotic
))r(Hlucc(l l)y actinoinycctcs. Sonic of tluMii, potency
'rre;Umi-nt of my< clium suspension* released by
like the tctnicyelines, are actix'c upon the treatment,
Mg/ml
lafiicr \iruses, such as psittacosis, and ha\-e
None 19
already found extensive practical apjilica-
Heated in lioilint;- water l);tlli lor 10 luiii- 38
tion. Others are active^ ujion th(> smaller vi- iites
ruses l)ut are either insullicienl ly active or Exposure to sonic eiiei't^y for 15 minutes 107
are loo toxic to lia\e found practical applica- Addition of sufficient concentrated HCl
tion. Anion<i these, it is sufficient to mention to give:
1)H 5.1 .37
ehrlichin, abikoviromycin, achroviromycin,
pH 4.2 67
noformicin, hygrosporin, and i)rimycin. 3.0
1)H 109
A numberof actinom^Tetes arc also able pH 2.5 188
to produce antiphage agents (Jones and pH 1.8 175
Schatz). Some of these substances have been Addition of sufficient 10 A' NaOH to give:
names hke chr^'somycin.
pH 8.0 45
gi\-en
pH 9.1 109
pH 9.9' 161
Miscellaneous Antibiotics pH 10.8 143
Addition of sodium chloride to give con-
A hu'ge number of other antibiotics pro-
centration:
duced by actinomycetes, chiefly streptomy- 0.003 M 19
ces, ha\-e been described. Some have a wide 0.01 M 19
spectrum. Others have a narrower spectrum. 0.03 M 33
0.1 M
Still others have a verj^ narrow spectrum. A
53
0.3 iM 97
few have found practical apphcations or of-
Addition of sodium sulfate to give con-
fer ])r()mise. centration:
Screening programs for antibiotics are be- 0.003 M 19
ing continued on a large scale. Particular at- 0.01 M 43
0.03 M 97
tention is being directed at present to agents
0.1 M 159
active against virusesand tumors. Although
0.3 M 147
a number of active substances have already Addition of sodium citrate to give con-
been isolated, none has so far given very centration:
promising practical results in the treatment 0.003 M 25
of disease.
0.01 M 64
0.03 M 142
It important to note that various antibi-
0.1 M
is
129
otics of actinomycetes are also active against 0.3 M 130
plant pathogenic bacteria and fungi (Waks-
* Samples from 4-day-old fermentation were
man et al, 1944).
centrifuged and the supernatant liquid assaj-ed.
The great majority of antibiotics are pro- The supernatant liquid contained 108 Mg/mf of
duced by members of the genus Streptomyces. streptomycin. The collected solids were resus-
A few are formed by species of Nccardia and pended water to original volume, and
in distilled
found to exert any antagonistic effects upon nomenon, since addition of streptomycin to
other microorganisms. the mycelium of the organism does not re-
Numerous other compounds were isolated sult in its absorption, and the "binding"
from various cultures of actinomycetes. They power of the mycelium is apparently not a
are described in detail in Chapters 31-40 function of its weight (Perlman and Lan-
(Volume III). glykke).
may be released (Table 67) by treatment of activity. This is not always the case, how-
the cells with acid, alkali, or ionizable salts, ever, as shown for neamine, a degradation
but not by disintegration of the cells by sonic product of the neomycins ; it retains its anti-
energy, bacteriophage, or enzymatic treat- biotic properties, although the spectrum is
ment. Considerable amounts of other anti- changed. Xakamura has shown that an anti-
biotics, including streptothricin, the neomy- biotic of the luteomycin type (C26H33NO12)
cins, chloramphenicol, and chlortetracycline, gave, on acid hydrolysis, a greenish black
have been found to occur bound to the my- substance, designated teomycic acid (C17-
celium of the respective actinomycetes and H23XO11), which retained its antibiotic prop-
may be released by treatment with acid, erties. However, actinomycin treated with
Actinomycetes are capable of attacking a gen in the plant residues was liberated as
great variety of plant and animal products, ammonia, thus pointing to considerable pro-
notalily crop residues; they thus bring about tein decomposition; much of the nitrogen
the partial or complete decomposition of must also have been used by the organisms
these products. Actinomycetes may, there- for the synthesis of their own cell material.
fore, be considered, on a par with the fungi In the decomposition of oat straw, 24.5 per
and the true bacteria, as one of the leading cent of the hemicelluloses were destroyed
groups of microorganisms concerned in the in 50 days only ; little cellulose and some lig-
destruction of organic materials and in trans- nin were attacked. Cornstalks were only
formation and mineralization of organic mat- slightly attacked when no lime and phos-
ter in nature. The literature on the soil ac- phate were added but underwent rapid de-
tinomycetes, beginning with the work of composition when these were introduced. Of
Beijerinck, in the early days of the century, the total dry plant material, the actinomy-
and continuing through recent studies, cetes brought about, on an average, 20 per
abounds in data on the abundance and ac- cent decomposition. They attacked
the wa-
tivities of actinomycetes in composts and in ter-soluble substances most readily (30.5
soils rich in organic matter. The essential role per cent), then the hemicelluloses (16.7 per
of these organisms in the formation and de- cent), and the cellulose least readily (5.4
composition of humus was early recognized. per cent). The most striking point was the
These processes result in bringing about the fact that the actinomycetes decomposed not
liberation of plant nutrients in available only the cellulose and hemicelluloses, but
forms and are thus of great importance in the lignin in these materials as well, even to
plant nutrition and soil fertility. a greater extent than did the fungi, as il-
lustrated in Table 68.
Decomposition of Plant Materials
In a comparative study of the decomposi-
In a study of the decomposition of al- tion of cornstalks by several species of Strep-
falfa by different groups of microorganisms, tomyces, alone or in the presence of a fungus
Waksman and Hutchings found that pure or a bacterium, the streptomycetes were
cultures of actinomycetes were able to de- highly effective in decomposing a consider-
compose, in 39 to 74 days, 33 to 43 per cent able amount
of the cellulose and the hemi-
of the hemicelluloses and 23.2 to 25.3 per celluloses.Although their decomposing ca-
cent of the cellulose, as well as a part of the pacity was less than that of the fungus Hu-
lignin. Nearly 20 per cent of the total nitro- micola, especially in the absence of added
243
244 THE ACTINOMYCETES, Vol. I
T.\BLE 68
Decompostion of alfalfa by pure and mixed cultures
of microorqanisms (Waksman and Hatchings)
Inoculum
1 4
tlu' plant nial(>rials. Mannans and xylans ('<iiii fxiralivc tlrri)}it position o/j't/lnn, in ninu-ohs bij
myceliinn than do fungi, only small (|uanti- organic matter. As shown previously (Chap-
ties of nitrogen are as.similated into conii)lex ter 7), Waksman and Starkey demonstrated
240 THE ACTINOMYCETES, Vol. I
^80 -
o
u
60
I
40 r
H\
V
>2 20
rn
m
3
k:
In Solution n
i
vSan <y
II
,N A/Atuf-l ;na rer/a ^
<N/n Solution medium
Table 73
Annual soil
18 28 42
DAYS OF INCUBATION
protoiiLS, lilx'ratinij; tlu> nit rof^cii as ammonia. I iijliicttrc of dried hlood (I per cent} ii fxni tin
Table 75
Decomposition of sedge and reed peat hy
microorganisms (Waksman and Stevens)
On the basis of 20 gm of dry peat decomposed for
28 davs under favorable moisture conditions
Organism
C
40
28* C
30
20
10 _>
UJ
O
O 15'
20 -
- 10
25 30
INCUBATION - DAYS
Figure 95. Influence of temperature upon growth and CO2 production by actinomycetes. Continu-
ous line: densit}* of mj'celium. Broken line: CO2 evolved (Reproduced from: Jensen, H. L. Proc. Linnean
Soc. N.S. Wales 68: 70, 1943).
•*»^
*'!•
^%i^#.
'.-%
^.•'
•<
Figure 96. Typical growth of actinomycetes in high temperature composts, as illustrated b}' contact
slide method.
249
. :
be established, however, that, under certain on in connection with the deterioration pro-
special conditions, actinomycetes may play gram during the Second World War in the
a far more important role in these processes Pacific, various cultures of actinomycetes
than is commonly supposed. It is sufficient were isolated. Their exact part in causing
to present the following evidence deterioration of service materials has not
1 Certain foodstuffs are known to deteri-
been fully established.
orate as a result of characteristic earthy and References to numerous other forms of
pungent flavors and odors imparted by ac-
potential deterioration of essential materials
tinomycetes, as pointed out previously. This
by actinomycetes are found in the literature.
is true of milk, cacao, potable waters, and
Galli-Vallerioand Reiss pointed out the
fish. The
flesh of fish is tainted through ab-
ability of actinomycetes belonging to the
sorption from the water of the odoriferous
streptomyces to attack photographic paper,
substance produced by actinomycetes. Cacao
both developed and unde\clopcd. They
can be damaged in a similar manner. The
found such cuHures in the wash water used
damage to Brazil nuts by actinomycetes has
been suggested; an organism, described as in photographic work. The ability of actino-
.4 . brasiliensis, a streptomyces, was isolated mycetes to attack rubber, paraffin, and other
by Spencer from the shells of such nuts. complex materials has already been men-
2. Certain fabrics, notably woolens, cot- tioned. The nature of the damage that may
ton goods, and paper, may be stained or thus be caused has not been determined.
<: ir \ I' I i; i{ I 7
obligate or as facultatixe.
Actinomycetes as Causative Agents of
A parasite may also be \irulent, if it has
Disease
the capacity to infect a living organism. Vir-
ulence varies greatly in nature and intensity, Although actinomycetes have been iso-
depending not only upon the species of the lated from various organsand excretions of
infecting organism, but also upon the strain diseased human and animal bodies, they
and its pre\'ious history, as well as upon the have not always been the causes of the dis-
nature of the host. The mode of infection, eases. At present, there are two major dis-
the ability of the parasite to spread through eases with which actinomycetes are usually
the various tissues of the host, its toxic mani-
associated. One is caused by anaerobic or-
festations, the degree of communicability, all
ganisms and is known as actinomycosis. The
contribute to the intensity of virulence.
other is caused by aerobic organisms and is
An organism may be made to increase or
known as nocardiosis. Various other syno-
decrease its \'irulence by
animal pas-
serial
nyms, such as streptothricosis and madura-
sage or by growth of the culture under sap-
Phenomena mycosis were once used to designate these or
rophytic conditions. of dissocia-
and the development similar diseases, but these terms were grad-
tion in the culture of
resistance to a particular treatment also con- ually discarded (Foulerton, 1910).
tribute to the degree of its virulence. Often Cope insisted upon adopting the generic
such changes are accompanied by a change name Actinomijces for the "whole group of
in the morphology of the organism or in its organisms" and actinomycosis for the dis-
immunological properties. ease caused l)y them, since the name "is
251
252 THE ACTIXOAIYCETES, Vol. I
«
:
l)y Wright )
"It ^row l)est uiiiltM' aiia('n)l)ic coiKlitioiis and Figure 98. Stained dental scum (Reproduced
did not grow at room tem])eratvire. On llic surface from: Ndeslund, C. Acta Patliol. Micrcjljioi.
of anaerobic agar slant cultures on the tliirtl, Scand. 2: 140, 1925).
fourth and fifth da.\' numerous minute isolated
dew-drop-like colonies appeared, the largest pin velopment of long filamentous forms forming a
head in size. These gradually became larger and network. The longer filaments were arranged more
formed ball-like, irregularly rounded elevated or less radially, were straight or wavy or spiral and
nodules varying in size up to that of a millet seed, sometimes branched. Cultures on potato or in
exceptionally attaining the size of a lentil or milk are not mentioned. Some twenty guinea-pigs
larger. As a rule the colonies did not become and rabbits were inoculated, most of them in the
confluent, and an apparently homogeneous layer peritoneal cavity with pieces of agar culture.
of growth was seen to be made up of separate Eighteen animals were killed after four to seven-
nodules if examined with a lens. In some instances teen weeks, and four were still alive seven to nine
the colonies presented a prominent center with a months after the inoculation. Seventeen rabbits
lobulated margin and appeared as rosettes. A and one guinea-pig showed at the autopsy tumor
characteristic of the colonies was that they sent growths mostly in the peritoneal cavity and in one
into the agar root -like projections. In aerobic agar instance in the spleen. In the four animals still
slant cultures no growth or a slow and very feeble living tumors were to be felt in the abdominal
growth was obtained. In stab cultures the growth wall. The tumors in the peritoneal cavity were
was sometimes limited to the lower portion of the millet seed to phmi size, and were situated partly
line of inoculation or was more vigorous there. In on the abdominal wall and partly on the intestines,
bouillon, after three to five days, growth appeared the omentum, and mesentery, and in the liver or
as small white flakes, partly floating and partly in adhesions. Frequently small millet-seed-sized
collected at the bottom of the tube. Growth oc- tumors were situated in the neighborhood of the
curred in bouillon under aerobic conditions, but larger tumors. While the surface of the small
was better under anaerobic conditions. The micro- tumors was always smooth, the surface of the
organism in smear preparations from agar cultures larger tumors showed small hemispherical promi-
appeared chiefly as short homogeneous, usually nences, giving them the appearance of conglomer-
straight, but also comma-like or bowed rods, whose ates of smaller tumors. On section the larger
length and breadth varied. In many cultures tumors presented a tough capsule from which
short-clump rods predominated, and in others anastomosing septa extended inward enclosing
longer, thicker, or thinner individuals were more cheesj^ masses. Microscopical examination of the
numerous. The ends of the rods often showed olive tumors showed in all cases but one the presence of
or ball-like swellings. In egg cultures growth oc- typical actinomyees colonies, in most cases with
curred in the form of white opaque graimles, the typical clubs. The general histological appearance
largest about the size of a pin head. Microscop- of the tumors was like that of actinomycotic
ically the growth was characterized by the de- tissue."
:
The micro-organism seems to be all of one spe- which differ profoundly from Actinomijces bovis in
having spore-like reproductive elements. I think
—
cies grows well only in agar and bouillon cultures
that these should be grouped together as a sepa-
and in the incubator; in the other usual culture
media and at room temperature, it grows onlj' rate genus with the name of Nocardia, and that
very little or not at all. It is essentially an an- those cases of undoubted infection by them should
aerobe. It does not form spore-like reproductive be called nocardiosis and not actinomj^cosis. The
elements." term actinomycosis should be used only for those
inflammatory processes the lesions of which con-
Wright added: tain the characteristic granules or 'drusen.'
"Because the microorganism here described
"In cultures its colonies are similar in char-
does not grow well on all the ordinarj- culture
acter to colonies of the microorganism in the
media and practically not at all at room tempera-
lesions of actinomycosis. If colonies of the micro-
ture, I did not believe that it has its usual habitat
organism are immersed in animal fluids, such as
outside of the body. It seems to me very probable
blood serum and serous pleuritic fluid, the fila-
that Actinomyces bovis is a normal inhabitant of
ments of the colonies in immediate contact with
the secretions of the buccal cavity and of the
the fluid may, under certain unknown conditions,
gastro-intestinal tract, both of man and animals,
become invested with a layer of hyaline eosin- but I have no proof of this to offer at the present
staining material of varying thickness, and the
time. In these secretions it should not exist in the
filament may then disappear. Thus structures are
characteristic forms seen in the lesions, but it
produced that seem to be identical with the char-
probably will be found in the form of fragmented
acteristic 'clubs' of actinomyces colonies in the
filaments growing in company with bacteria, and
lesions."
not now differentiated from them. I believe that
the part played by foreign bodies so frequently
found in actinomycotic lesions is not that of the
carrier of the microorganism into the tissues from
without, l)ut that the foreign body, bj' its trau-
matic and irritative effect furnishes a nidus in the
tissues for Actinomyces which enters therein with
the secretions from the buccal cavity and gastro-
intestinal tract, develops into characteristic
colonies, and produces lesions which we call
actinomycosis."
There were numerous other reports of the the microflora of human beings, nota-
occurrence of streptomyces in human or- bly in the sputum, tonsilar crypts, and skin.
gans, as m the ear (Odom ct al.), in tumors This organism exerted an antagonistic effect
(Leyton and Leyton), and in tear ducts and upon \'arious human pathogenic fungi and
glands (Gruter). Mackinnon and Artaga- yeasts. It was suggested that such organisms
veytia-Allende (1956) examined 38 strains may play an important role in keeping the
of aerobic actinomycetes producing locaUzed microbiological e(iuilibrium in the human
mycetoma in various zones of the world. })ody.
They identified these organisms as species Further information, some of which is
Figure 101: My(( iumi dis (Reproduced from: Pijper, A. and Pullinger, 13. 1). J. Trop. Med.
Hyg. p. 2, June 15, 1927).
commonly found in nature, usually produc- originates from the mouth, but affects the soft
tissues and skin of face and neck, and the tongue,
ing reddish or yellowish colonies, is acid-
and secondarily, the maxillary bones. The salivary
fast, and usually forms spores; it comprises glands, larynx, thyroid, and lacrymal glands, the
the forms now included under Nocardia. orbit and even the brain may more rarely be in-
Rosebury isolated foui' strains of .4. hovis volved. The comnioiicst lesions appear on the
C'AISA'I'ION Ol" AN I.MAI, DISIvVSES 261
cluu'k or sul)m;i\illar\ skin, ami aic cliaiaclcii/cd that .V. (islcntidcs |)i'(»(liic('s ;i dilTii.^e perito-
by inthiratoil or (>(l(Muatoiis swellings, hluisli or
nitis ill cxpcriiiiciitnl ;iiiiiii;ils. According to
rodtiisli ill color, with a toii(l(Mic\' to t'orni a scries
H(>iib()\v, Smith, and (ii-inison, about 90 per
of irrof^ular folds separated l)y furrow s, the healing
lesions forming scars as new lcsii)iis dcvcloj). (•('lit of all clinical cases of actinomycosis arc
"Thoracic aclinoinycosis accounts for al)()Ut caused 1)\' .1. lion's; the reiiiaiiiiiig 10 per
fifteen iier c(Mit of liuniaii cases. It is found niainlx cent are caused by A', asteroides, uKJst strains
in the lunjis. witli llic formation of alisccsses and
of which are partially acid-fast. Benbow ct
cavities which are usually small. l']xtonsive lesions
al. excluded from this classificat ion the myce-
may be found in the bronclii, and their rupture
may lead to dissemination of the infection by way tomas which are caused by otluM- species of
causative agent of the disease. These aerobic and McNaught were infected in
(1946) the lungs
twenty-nine and, of these, eleven had metastases
organisms cause specific types of mycetomas.
to the brain. Occasionally the presenting symp-
Infections of the hmgs and of the skin are toms of headache, nausea and vomiting may indi-
frequently produced. The organisms are cate either brain tumor or brain abscess; or, the
culti\'ated much more readily than the ana- symptoms may be those an infectious meningitis
of
(tuberculous) with minimal or no findings in the
erobic types and are pathogenic to labora-
lungs. Symptoms referable to a pulmonary infec-
torj" animals. .V. farcinica,from
isolated
tion include general malaise, fever, productive
cattle, forms a yellowish, wiinkled growth on cough with sputum, night sweats, anorexia and
solid media. A'', caprae, isolated from the loss of weight. Roentgenograms of the lungs
lung of a goat, gives a more whitish growth usually show a progressive infiltrative proce.ss
and greater fragmentation of the mycelium. which may lead to multiple cavity formation.
Hematogenous spread results in metastatic lesions
A^. canis, which produces infection in dogs,
throughout the body. Histologically, such lesions
is similar to A'', caprae. may be of a purulent nature, containing centers
Conant and Rosebury described the clin- composed of polymorphonuclear neutrophils and a
ical features of nocardiosis as follows: few mycelial fragments, or such areas maj^ show a
more advanced granulomatous reaction leading to
"Nocardiosis is a chronic suppurative, purulo- granulation tissue, giant cells and scarring."
granulomatous disease of the subcutaneous tissues
Granules are not formed by N. asteroides.
and bones (mj^cetoma) characterized bj' multiple
tumefactions and draining sinuses from which IVIariat (1957) made a detailed study of
granules (yellowish-white, red, or black) are ex- the causative agents of chronic subcutaneous
pressed in the pus or found in the tissues; or, a lesions, known as mycetomas. Only five
pseudotuberculous infection (systemic) of the
species were recognized:
lungs and pleura with hematogenous spread
throughout the body, especially to the brain and
1 Nocardia asteroides. An infrequent caus-
meninges, inwhich filamentous, bacillar}^ or ati\'e agent of mycetoma. Semi acid-fast or-
coccoid, acid-fast forms may be found in the ganism. Enzymatic reaction reduced or ab-
sputum, spinal fluid, or pus from subcutaneous sent; pathogenic to experimental animals.
abscesses.
2. Nocardia hrasiliensis. Prevalent in
"Mycetoma of the extremities results in the
Central and South America. Semi acid-fast.
clinical picture of Madura foot, although other
subcutaneous tissues of the body also may become High enzymatic potential. Xo agreement on
infected. The characteristic lesion with pain, experimental pathogenicity.
swelling, and sinus formation, and eventual club- 3. Streptomyces madurae. World-wide dis-
bing and marked deformity of the infected member
tribution. Causative agent of mycetoma.
is developed only after months or years. Infection
tritioiial pioportios of these ()i<;;iiiisins \va\v relationship between I lie ael inoniycetes and
bo(Mi pic'S(Mit(>d in Chajifcr 7. the mycobacteria.
Further ;iii;ilysi>s ol' nocarchosis, or the l"u-ther studies of the ailei'gic reactions of
aerobic actiiuMnycotie ditseusesdue to a(Ml)l)i(' actinomycetes ha\'e been made b\' .Mafhie-
actinomy(etes, larj>;ely of tlie Noainhd type, son ft ni. (1035).
are foiuul in the numerous te\tl)ooks on l)ae-
terioh)j>;y (Plehn). 'I'herapy of Actinomycotic Diseases
Gruter made a comprehensix'e stu(l>' ol'
The therapy of actiiujmycosis has been
the occurrence" of actinomycetes in eye in-
gi\en rather limited consideration (Memiing,
fections, especially in tear glands and ducts.
1933; Heuber, 1940).
He came to the conclusion that a nocardia
According to Cope:
was invoh'ed. He designated his culture .1.
discofoliatus. "The i)r()ji;nosis of actinomycosis varies greatly
according to the part of the bodj' affected. It is
According to (Jordon and Hagan, certain
most favourable with those cases which affect the
acid-fast actinomycetes isolated from soils
head and neck, less favourable with abdominal
and plant material are similar to those found cases, and most unpromising with thoracic disease
in lesions of men and animals. The pigments —
"The great majority probably 97 per cent^of
produced hy these organisms range from those cases in which the cheek, jaw, tongue, and
yellow through orange to coral. One of the neck are involved come to a satisfactory issue.
The disease may last for several j^ears but nearly
soil forms, soon after isolation, was found
always ends satisfactorily. The 3 per cent of cases
to be pathogenic to rabbits but not to guinea in which death results are those in which the
pigs. pathological process either extends deeplj' towards
the base of the skull and causes cerebral complica-
Allergic Reactions tions, or extends downwards to the superior and
posterior mediastinum, or more rarely, becomes
^'arious attempts have been made to ex- generalized."
amine the immunological reactions of ac-
tinomycetes. Goyal compared 11 cultures
Treatment of actinomycosis consisted first
obtained from collections and as fresh isola- of radiation therapy and use of \'accines.
]\Iore recently, with the advent of the sulfa
tions. Mo.'^t of them appeared to be members
When drugs (given internally or applied locallj^}
of the genus Xocardia. inoculated into
rabbits, theyproved to be either entirely and especially the antibiotics, chemotherapj^
nonpathogenic or onl.v slightly virulent, ex- of actinomycotic infections took a new turn.
(1951), Fischer and Harvey (1956), Bianco found to be, when tested in vitro, erythro-
et at. (1957), and numerous others. mycin and novobiocin; however, the only
Among the antibiotics used were peni'^,inin therapeutic action in experimental animals
(Dobson and Cutting, 1945; Drake, 1946; was exerted by cycloserine.
Holm, 1948; Nakhimovskaia et al., 1957), Mackinnon et al. made a detailed study
streptomycin (Pemberton and Hunter, 1949; of the effect of \'arious chemotherapeutic
Torrens and Wood, 1949), chloramphenicol agents upon mycetoma and nocardiosis.
(Littman et al., 1952), the tetracyclines They found that strains belonging to the
(Martin et al., 1956; Lane and Kutsaber, same species show sufficiently similar sus-
1956), and various others (Banerjee et al.,
ceptibility as to prove species sensitivity.
1954; Hanf, 1956). Three species of Streptomijces (S. somaliensis,
In a comparative study of the inhibitory
S. pelletieri, and S. madurae) were susceptible
effect of antibiotics upon the growth of the
to at least two of the following antibiotics:
anaerobic A. Israeli, Garrod found penicillin
penicillin, streptomycin, chlortetracycline,
to be active in a concentration of 0.1 unit
and chloramphenicol. These species could be
and streptomycin in 23.7 units, with the
distinguished by their relative sensitiveness
tetracyclines and chloramphenicol falling
to these antibiotics. Diaminophenylsulphone
between (2.2 to 4.2 units). This bears out
is active upon A^. brasiliensis, N. asteroides,
the sensitivity of anaerobes to penicillin and
their relative resistance to streptomycin. and S. somaliensis, but less so upon S. pel-
Frequently an antibiotic is active only in letieri. The pathogenic actinomycetes were
vitro or in vivo. The most effective antibiotics found to show development of resistance to
against A'', asteroides, for example, were streptomycin and to aromatic diamidines.
:
r II A p I i: i{ 1 «
"\'ory l'(>\v forms anionji; the nctinomycetes Because of its historical significance, the
arc capable of causiuii; plaiil diseases. In spite description given by Thaxter is (juoted here
of tlie great ahuiKlance of aetinomycctes in
"Vegetative hyphae brownish, .06 [0.6?] to 1.0 /*
nature, especially in the soil, the numl)er of illdiameter, curving irregularly, septate or pseu-
plants attacked by them, as compared to (loseptate, branching. Aerial hj'phae at first white,
the number of plants attacked b}^ bacteria, then gray, evanescent, breaking up into bacteria-
like segments, after having produced single
fungi,and viruses, is rather limited. Except
terminal spiral spores by the coiling of their free
for two species —
the Irish potato and the
extremities. Forming a firm lichenoid pellicle on
—
sugar beet plants subject to infection by nutrient jellj^ and usually producing a blackish-
actinomycetes do not occupy a \'ery promi- brown discoloration of the substratum on which it
nent place in human economy. grows, causing the disease known as scab on potato
tubers, and a similar disease of beet roots."
265
;
lated cultures. Further, Wollenweber's de- scab each type was found to be caused by a
;
scriptions were incomplete and the exper- different species. All of them are now recog-
imental data presented in support of his nized as belonging to the genus Streptomyces.
assumptions were limited. Since most of the Leach et al., however, could not confirm
cultures isolated from scabby potatoes thus the reports of the multiplicity of scab-pro-
described are similar to the large numbers of ducing actinomycetes. On the basis of their
saprophytic organisms commonly found in studies, they emphasized that scab lesions
the soil and on surfaces of potatoes, the are a result of a reaction between the patho-
pathogenicity of the species belie\'ed to cause gen and the host tissue; they may be influ-
scab is open to (juestion. enced as much by the host as by the patho-
Millard and Burr isolated from potato gen. They recognized, however, the existence
scab a large number of cultures of actinomy- of pathogenic races of the organism. Cocchi's
cetes believed to be responsible for the causa- results, on the other hand, tended to sup-
tion of the disease. Only three of these cul- port, at least partly, Millardand Burr's con-
tures were obtained in duplicate, and only clusions; two of the three strains he isolated
one was found to be identical with Thaxter's were identified as S. flavus and S. clavifer.
original isolate. The latter produced the Taylor and Decker isolated 143 cultures
deep form of scab and was capable of attack- of actinomycetes of which 128 were nonacid-
ing the roots and stolons of the potato plant. fast and 15 partly acid-fast. Starch was hy-
Millard and Burr believed that different drolyzed by all of the nonacid-fast isolates
types of scab were caused by different or- and by none of the partly acid-fast isolates.
ganisms. They used glycerol synthetic solu- The ability of the cultures to produce a dark
tion as the basis for growth of their cultures. brown surface ring of growth on milk was
The mere isolation of an organism from a characteristic of 66 isolates, all nonacid-fast.
scabby potato is no proof, however, that the Of the remaining 77 cultures, 71 produced
organism is capable of causing the disease. on alkaline reaction; four formed a hard,
This has been emphasized by Decker and acid curd; one showed no visible change in
numerous others. reaction in the presence of abundant growth
DeBruyn studied in detail the specific na- one did not grow at all on milk.
ture of scab-producing actinomycetes. He Thirteen of the acid-fast isolates ga\e mi-
employed the method described by Kiess- croaerophilic growth on agar-shake tubes. A
ling. Sap of different varieties of potatoes is dense ring of colonies developed beneath the
added to synthetic media and inoculated surface of the agar, at depths of 5 to 10 mm.
with actinomycetes. Since the pH of the sap As the cultures grew, additional rings de-
changes with the ripeness of the tuber, the veloped beneath the original one. With the
growth of the organisms in such media is nonmicroaerophilic isolates, no growth was
parallel to the pH change. The cultures made produced below a depth of o mm; any sub-
their best growth in the presence of sap of a surface developm(Mit was continuous with
CAISA'I'IOX ()|- I'LAXT DISEASES 267
the surl';ic(> lirout li. (Ichiliii was licnicdcd l)y types of cullurcs was similar on all media
all ol" the noiiacid-t'asl cullurcs and hy the conlaining dilTeicnl nil rogenous compounds,
two nonnuci-Dacrophilic acid-l'ayt forms. 'J'lic with the exception of uica; Ik; parasitic and
I
13 microMcrophilic acid-last isolates ditl not some of the saprophytic cultures failed to
liquefy jj;ela(in. grow on a medium to which 0.5 per cent of
Typical potato scab, as distinguished from urea was added. This was found to be due
superficial russetting of the surface, w as jjro- to the toxicity of the ammonia produced as a
duccd h\- all of the "dark brown ring" iso- result of the decomposition of the urea. On
lates; none of tiie other isolates that were the basis of their physiological and biochemi-
tested caused typical scab on potato tubers. cal properties, namely, ability to utilize su-
Within the first group, ^•ariations occui-nni crose and raffinose, inhibition of growth by
in the amount of scab formed on the tubers, ammonia, and ability to produce a melanin
though all produced some scab infection. The pigment in a tyrosine medium, parasitic cul-
particular cultures did not differ consistently tures could be differentiated from ordinary
in type of scab lesion produced. In many saprophytic actinomycetes.
cases scab lesions \-arying from shallow de- Finally, it may be of interest to report here
pressions to deep pits were found among the the recent work of Hoffmann (1958). Nu-
replicated pots of one isolate,and frequently merous infection experiments were carried
within the same pot or on the same tuber. out with twenty Streptomyces species, using
Superficial russetting was not considered a munber of scab-susceptible potato \'arieties
as typical of potato scab. It was more abun- in the greenhouse and under field conditions.
dant with certain isolates in the nonacid-fast He found that S. scabies was the sole patho-
group. This russeting was not observed in gen of potato scab; the same was true of
the tests of the "dark brown ring" group. beet scab. He regarded the "parasitic" forms
The correlations observed between patho- of scab reported by other investigators as
genicity and the beha\'ior on milk held true saprophvtic organisms accompanying the
for a large number of actinomycetes. The actual pathogen.
conclusion was reached that the ability to
produce typical lesions of potato scab was Environment
correlated perfectly with the production of a The nature of the soil in \vhich the pota-
dark brown ring of surface growth on sepa- toes are grown and the environment play
rated milk. the greatest role in the production of scab.
Afanasiev isolated se\'cn cultures of the The oiganic matter content of the soil, and
scab-producing organism from three differ- its degree of decomposition, the inorganic
ent types of scab: common, deep, and russet. structure of the soil, and the soil reaction
Indi\'idual potatoes were found to show two (Gillespie) are among the most important
or even all three forms of scab. The difference factors in this connection. The environmental
in the kind of scab caused by cultures of ac- conditions comprise moisture, temperature
tinomycetes was believed to be one of degree (Jones et ai), and aeration.
of pathogenicity rather than of type of organ- According to Dippenaar, the optimum
ism involved. Certain marked physiological temperature for scab infection, as deter-
differences were found between the parasitic mined by the average number of lesions per
and the saprophytic cultures. The parasites tuber,was found to be betw^een 17 and 21 °C.
were able to utilize sucrose and raffinose; The optinunn temperature for the develop-
most of the saprophyt(\s were unable to ment of the disease was about the same as
use these two sugars. The gi-owth of both the one best suited for the host, and did not
268 THE ACTINOMYCETES, Vol. I
12
10
7.0
Figure 104. Influence of pH on the growtli of the potato scab organism (Reproduced from: Dip-
penaar, B. J. Union of South Africa Dept. Agr. Sci. Bull. 136: 68, 1933).
vary with the soil moisture or the soil re- Goss (1937) made a detailed study of the
action. Increasing soil moisture decreased environmental factors influencing the cau-
the amount of scab at all soil temperatures sation of potato scab by S. scabies. The soil
and in all types of soil used, and increased was found to be the major source of infec-
the yield of potatoes. Lower moisture was tion.Although crop rotation reduced con-
required to control scab at 13 and at 25 °C siderably the incidence of the disease, the
than at either 17 or 21 °C. The disease on fact that tubers in soil never before planted
actively growing tubers was controlled by to potatoes could still show severe scab sug-
increasing the soil moisture after the tubers gested that various other factors were in-
had developed scab. Relatively scab-free volved which influenced the severity of the
tubers growing in a scab-infested soil with disease.
a high soil moisture content become will Among these factors, high temperature,
scabby if the soils are allowed to become dry. low moisture, alkalinity, abundant aeration,
In greenhouse experiments, Dippenaar and the addition of barnyard manure are
found that a reaction of pli 5.0 and lower generally accepted as fa\'oring the incidence
either controlled or reduced the disease in of the disease. On reading the extensive lit-
severely scab-infested soils bul did not elimi- eratiu'e, one would assume that it should be
nate scab entirely. A reaction of pH 4.8 and possible to correlate the occurrence of the
lower, however, had an adverse effect on the disease in the field with one or more of these
potato plant. factors, rnfortimatel}^ it has often been
CAUSATION OF 1M>A\T DISEASES 269
100
pH
Figure 105. Relation of soil reaction to the occurrence of potato scab (Reproduced from: Dippenaar
B. J. Union of South Africa Dept. Agr. Sci. Bull. 1.36: 52, 1933).
tion. In sterilized soil thei-e was a greater est time and in which the inoculum had ap-
amount of scab. This was due to a lack of parently- become well established before the
270 THE ACTINOMYCETES, Vol. I
Table 76
The effect of the competition of soil microorganisnts
upon the occurrence of scab in
inoculated soils (Goss)
Sterilized soil,
CAlSA'noN ol' PLANT I ) ISIIASIOS 271
toes (pust iilc Ixpci. 'I'lic luiiiiMt ion ol' cliloi'o- increased with increases in the pll. These;
liCiiic :i('i(l ill thccclls of the IciiticcI urea \\;is resultswere said to support the theory that
\hv causal i\(' lucchaiiisin (.loliiisoii and the mechanism of scab resist aiure in {)otato
Schaal, l!).VJ; Schaal and .Idliiison, I!)."),"); tubers iiixolves enzymatic oxidation of chlor-
Schaal rl dl., l!».");;i. They toiiiid thai, in ogenic acid, which produces a (|uinone toxic
colls adjacent to the pcfidcrni, chloroj^-cnic (o the scab organism.
aciil was pccsciit in lai-.tici- (luaiititics in po- Th(> mechanism was believed to depend
tato tul)(M-s of scah-r(>sislant xarictics tlian on the amount of chlorogenic acid in the
ill those of suscoptil)Ic xarictics. A test that periderm and on the presence of tyrosinase
would not re(|uife destruction of the whole in the same tissue. The localization of this
tulter was de\ised so the tested tuber could acid around the lentic(>ls that serve as the
l)e usetl for seed purposes. The test was normal entrance points for the scab organism
based on the fact that cliloi-ogenic acid is was important. A tendency for the acid to
localiz(>d in the cells dii-ectly und(M' the c\n- accumulat(> in cells adjacent to injured areas
derinis and coi'ky co\-ei'ing and in cells was observed. Tyrosinase was found in high
around the lenticels of th{> highly r(>sistant concentration in the tissue contahiing chloro-
\-arieties. Scab resistance was found to be genic acid and was found to become oxidized
associat(Hl with the amounts of chlorogeiiic when the tissue was
The oxidation
injured.
acid present. products formed, such as (juinones, were be-
The test consisted in placing several drops lieved to be toxic to the invading organism.
of 2 per cent aciiieous ferric chloride solution Johnson and Schaal (1957) brought out
on the surface of a tuber and macerating further that the concentrations of chloro-
with a stainless steel knife the tissue covered genic acid and of total o-dihydricphenols
by the test sohition. By pricking this area un- were much higher in the periderm of tubers
der a drop of ferric chloride solution, the of certain scab-resistant varieties than in
pre.sence of chlorogenic acid in or near the tubers of scab-susceptible varieties. The
lenticels could be determined. The distribu- greatest difference between the \'arieties was
tion of chlorogenic acid in the tuber was found at the stage where the tubers were
measured by spreading the ferric chloride growing most rapidly. The tubers were
.solution o\-er the freshly cut surface of a analyzed at three stages of growth and after
half of a tuber. In resistant varieties a green 5 months of storage at 35 to 38°F. The re-
color reaction was found near the surface. sults indicated that the ferric chloride spot
In some highly resistant varieties the chloro- scab resistance could be made on im-
test for
genic acid was present throughout the tubei-, mature or on freshly harvested mature tu-
its greatest concentration being in the cells bers.
directly under the corky co\'ering. The color
reaction was found to l)e greater in the im- Effect of Treatment
mature than in the mature tubers. Different Addition of stable manure to soil has usu-
varieties showed differences in color reaction. ally been found to increase the incidence of
The effect of six phenolic compounds on scab, because of the resulting alkaline reac-
the gi-owth of S. scabies was tested at pH tion and th(> accumulation of humus. Similar
Table 77
Effect of soil sterilization and moisture content on scab following inoculation with S. scabies (Goss)
Set No.
("AISA'I'IOX t>l' I'LANT 1 )ISI':ASI':S 273
iiifcstcd soil ;it pi I .").() lo .").!. 'riicrc \\;is no sidered as doubtful. Although Ti iciiodrrma
not icc.-ihic iii('iH\-is(> ill soil acidily diiriiii;' ;i might be of some assistance in this capacity
.")8-(l;iy period. Jii artificial luodia, howcxcr in poorly aerated soils whicii po.sse.ss low ad-
the lii-ow til of (•(M'taiii haclcria made condi- sorpti\-e capacities, its fum^tion in many
tions uiil'a\()rahl(' tor the ,<;ro\vth oi S. sca- soils was (luestioiied.
hicii. The conclusion was reached that when The physical as well as the biological en-
scab is controlled in some soils hy ii,reeii ma- vironment in many cultivated soils was con-
nure crops, this may he due to the "antibi- sidered a strong barrier against the establish-
otic" (luaiities of eeitain predominant soil ment of the Trichoderma. When introduced
microorj>;anisms. into a r)-(lay-old *S'. scabies culture, Tricho-
Kenlvnight attemi)ted, unsuccessfully, to derma was strongly inhibited by the scab. A
control scab through tlu^ antagonistic ac- soil-inhabiting bacterium was also found to
ti\'itics of other microorganisms. Addition of produce a material that was toxic to Tricho-
organic matter to the tended to aggra- soil derma and to the actinomycete alike. Daines
\ate the development of scab except in ex- argued that "in such complex physical,
treme cases of scab infection. The findings of chemical and biological environments, as are
Millard and Taylor that green manure was afforded by soils, these antagonistic relation-
effective in controlling scab in the presence ships may be modified or even entirely de-
of S. praccox were not confirmed. When this stroyed."
and other species of actinomycetes were in- KenKnight argued that the absence of
troduced into scab-infested soil in green ma- proper biological controls in the abo\'e ex-
nure and in ^•arious other media no control periments may have been due to failure to
was obtained. KenlCnight thus agreed with establish the introduced organisms in the
Ctoss, who also failed to obtain control of soil, or to the failure of these organisms to
scab with S. praecox. show antagonism toward any or all of the
McCormick observed that S. praecox was parasitic actinomycetes under soil condi-
antagonistic on solid media to <S'. viridis and tions. He found an analogy between the in-
S. intermedins, but not to certain other para- troduction of organisms not well suited to
sitic actinomycetes. This observation sug- the soil conditions and efforts to obtain a
gested specific differences in the action of the stand of wheat by planting the seed in un-
antagonist. Xo control of scab was obtained broken prairie sod. He believed that Kies-
with B. megaterium, an organism antago- shng's mixed cultures of bacteria that had
nistic to certain actinonwcetes. Pseudomonas the capacity to control scab, except for one
which was antagonistic to certain
fluoresceus, type of scab caused by a species other than
actinomycetes and to Trichoderma lignorum, S. scabies, suggested that if practical control
reported to be antagonistic to many fungi, of potato scab is ever obtained by biological
exerted no effect on the scab organism. means in soil infested with several parasitic
Daines reported (1937) that Trichoderma actinomycetes, it will be with mixed cultures,
Jignorum produces a diffusible substance because it appears unlikely that a single
which is toxic to S. scabies in an artificial organism will be found that is antagonistic
liquid medium. Because of the rapid destruc- to all strains of the scab organism.
tion of this toxic principle by aeration at the The relation of soil fungi to the de\-elop-
pH of potato soils and because of remo^•al of ment of potato scab has been studied further
the toxic material from solution by charcoal by Pratt (1918) and by others.
and by the soil itself, the eflBcac}^ of this Detailed studies ha\-e been made of the re-
fungus in combating potato scab was con- lation of the soil population to the develop-
.
ment of the .s(;ab organi.sni. The ciuestion was cetes. Under the influence of the generic des-
raised whether actinomycetes diminish in ignation of. the actinomycetes used by Thax-
numbers and whether the parasitic potato- ter, he described several actinomycetes as
scab-producing strains tend to die in soil in species of Oospora, namely, 0. crctacea, 0.
which no potatoes have been grown for a rosella, 0. intermedia, 0. tenax, 0. nigrificans,
long time. The actinomycetes were found to and 0. violacea. All of these were, however,
remain fairly constant in their numbers and typical actinomycetes, belonging to the
in their percentage relation to the total num- genus Streptomyces. Kriiger studied particu-
ber of organisms. The pathogenic types, how- larly the type of scab known as "girdle" scab
ever, were gradually reduced in numbers. of sugar beets. The strains of the organisms
Tests were made b}^ planting a susceptible he isolated were believed not to be identical
potato variety in the soil; if the scab-produc- with the potato scab form of Thaxter.
ing strains were still present, scabbing would Various other isolations of actinomycete
result. The formation of russetted tubers sug- cultures were made from beet scab. Some
gested the possibility that the scab organism, of these cultures were found to be parasitic
after a long period of deprivation of its host, but varying in the degree of virulence. Mil-
was weakened in pathogenicity and produced lard and Beeley recognized two distinct
the true, deep scab only in rare instances. *S'. types of mangel scab, the raised and the pit-
scabies is also capable of causing necrosis of ted forms. The raised scab was subdivided
subterranean stems of potatoes. The stems into the mound and knob types, which were
may become girdled and rotted at the base found to develop particularly on yellow-
with vascular discoloration extending up the skinned varieties of mangels. The pitted scab
stem six to eight internodes (Hooker and was similar to the common scab of potatoes,
Kent) whereas the raised scixh was not formed from
the cambium of the ^'ascular rings, but re-
Sugar Beet and Mangel Scab sulted from the proliferation of the pericycle.
Certain actinomycetes are also capable A culture was isolated from mound scab
of causing scab on various root crops, nota- which reproduced the same type of scab in
bly sugar beets and mangels. Kriiger was the artificial inoculation experiments; it was de-
first to establish, in 1904, that the production scribed as A. tumuli. From pitted scab a
of scab on sugar beets is due to actinomy- culture was isolated which also reproduced
Figure 107. Mangel scab (licproduced from: Millard, W. A. and Beeley, F. Ann. Appl. Biol. 14:
311, 1927).
CAISATIOX OF I'l.Wr l)ISi;.\SKS 275
cullui'c also ;itt;u'k(Hl \\\v loots ;iii(l rootlets tested on seedlings of wheat, garden pea,
ot' the iiiociilntcHl in;iii,H('l plants, on which it soybean, corn, radish, and (aicunibei', and on
pro(hic('(l nunicroiis characteristic dai'k potato sprouts. Six of these cultures caused
hi'owii, nothilar out.iiTow ths. It was identical neither appreciable neci-osis of potato stems
witli N. scahi( s. nor injury to seedling roots. Four cultures
caused sex'ere necrosis of roots as well as a
Swvvl l*<>tat<> Pox or Sofl I?ol reduction in root weight of wheat, pea, soy-
bean, and was most pro-
radish; necrosis
An actiiioniycete, (iesi»;iiate(i I)}' Tauheii-
nounced on loot tips, and the development
haiis as .1. poohnsis, was t'ound to l)e a con-
of secondary roots was almost inhilMted.
t riliiitinu; factor to the causation of soft fot
Corn roots were only slightly necrotic;, but
of sweet potato(\s. It was considered as a
their weight was markedly reduced. Three
suporfic'ial wound parasite, usually follow-
of the four cultures also caused scab of po-
ing the pox spots produced bj'^ a fungus.
tato tubers and necrosis of potato stems.
Other cultures of actinomycetes were la-
Palm described the occurrence in South
ter isolated from sweet potato(\s. One of
Sweden of actinomycotic infections of Beta
them, designated as .4. pox, was believed
vulgaris, Brassica sp., Raphanus sativus, and
to be the cause of the pox disease (Adams).
Daucus carota. He concluded that the infec-
Still another organism causing sweet po-
tion of these plants may be caused by the
tato rot was described as S. ipomoea. It pro-
same pathogen. The disease appears as
duced aerial mycelium and was thus a mem-
sunken spots, delimited by the more promi-
ber of the genus Streptomijces. Sweet potato
nent veins of the bulb scales, and are of a
rot does not develop in soils of pH below 5.2;
greenish mother-of-pearl-like color. The
above that reaction, the disease develops
pathogen lives intracellularly, filling the cells
readily. According to Person and Martin,
completely with its very thin (1-1.2 ii in
sweet potato rot is more serious in dry soils
diameter) mycelium. The mycelium is non-
and in wet seasons than under normal mois-
septate, of a strong yellow color,and spore
ture conditions. The disease has been pro-
formation on the host is not seen. The
in or
duced in the greenhouse and in field inocula-
organism undoubtedly belongs to the chro-
tion experiments with pure cultures of S.
mogenic actinomycetes, of the genus Strep-
ipomoea. The optimum reaction for growth
tomyces.
is pH 5.6 or above, and the optimum tem-
for plant growth has not been fully eluci- cetes are abundant in the roots of plants,
dated. they may take part in the synthesis of alka-
Formation of small tubers on the roots of loids and proteins. Since large numbers of
the oleander has been demonstrated by Ro- soilactinomycetes are pectin-dissolving, the
berg (1934). The organism is similar to A. different varieties found in the various host
alniand was described as A. elaeagni. plants may be only modifications of one large
All three of these actinomycetes definitely species. The walls of higher plants were be-
belong to the genus Nocardia. Only one of lieved to be living, through the presence and
them is included in the classification, namely, action of strands of actinomycetes. The ef-
disinfected seed and in disinfected soil are ments are intercellular and grow within the
found to contain numerous actinomycete fil- middle lamellae. After complete remo\'al of
aments. the paraffin, the sections are strained 4 min-
According to Lutman, potato scab lesions utes in carbon-aiu'amin (distilled water 97
are associated with strands of actinomycetes ml, liquefied phenol 3 ml, certified auramin
extending from the abnormal cells of the 0.1 gm), washed, destained in a 0.5 per cent
cork camljium to the interior of the tuber. solution of XaCl in 70 per cent alcohol with
Similar strands have been found in clean 0.5ml of HCl (cone.) per 100 ml, washed,
tubers grown on land never known to pro- and mounted in glycerol.
duce scabby tubers. The strands found under Voros et al. reported that streptomycin
the scabs seem to be unusually large and exerts a protectiA-e effect upon the potato
numerous, especially those about fi\^e to ten plant, rendering it resistant to Phijthophtora,
cells below the pathological tissue. via the polyphenol-polyphenolase system of
The cell walls of .lerusalcm artichoke tu- the host plant. The fact that polyphenolases
bers and the enlarged roots of beets, carrots, are copper enzjniies, their acti^•ity depend-
parsnips, and turnips contain gram-i)ositiv(! ing upon the copper supply of the plant, and
filaments which seem to he of the same sort the fa(^t that copper and streptomycin were
as those occui-i'ing in the potato plant. The found to exert a synergistic effect may help
suggestion was made that sincc^ actinomy- to explain the aboN'e effect of streptomycin.
EPILOGUE TO VOL. I
These then are the actinomycetes, dismissed only about two decades ago
as a "little-known group of microorganisms," and considered by some as
fungi and by others as bacteria. I have presented in these pages my personal
experiences with the actinomycetes, especially their occurrence in nature,
their structure and functions, and their role in natural processes. I have
attempted to summarize some of the reactions whereby they carry out their
characteristic biochemical activities, their varied biochemical potentiali-
ties,which are at present being taken advantage of for the benefit of the
human race. The subsequent volumes will deal with the problems of how to
recognize them and how to utilize them for the production of valuable drugs
that are saving millions of human lives.
277
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INDEX TO SPECIES OF
ACTINOMYCETES
319
A AA
Nocardia Guignardi, 5
actinomyces, 62 intermedia, 274
alba, 184 M elschnikowi 5 ,
madurae, 67, 68, 124, 127, 181, 191, 261 citreus, 154
minima, 67-69 clavifer, 266
opaca, 67-69, 152, 153 coe/u-o/o/-, 51, 100, 101, 104, 105, 110-112, 115, 121,
paraffinae, 67-69, 160 122, 131, 145. 148. 156, 158, 160, 170. 188-190,
paraguayensis, 180, 181 197, 199, 200, 220, 222, 256
pelletieri, 67, 68, 124, 127, 192 craterifer, 160
pelroleophiln, 130 cyaneus, 181
polychroniogenes, 67-69, 90, 101, 154, 160-162, diastnticus, 125, 179, 187
170, 172, 199 diastalochromogenus , 185
preloriana, 258 erythreus, 127, 160, 165, 190, 238
rangoonensis 67, 68 , erythrochromogenns, 121, 185
refilricla, 152 exfuiialns, 186
rhodnii, 45, 195 flaveolus, 105, lOS, 121
/•»/»'/, 184 flavovirens, 20
/w//>m, 68, 69, 160 162, 170, 172 y?o!'»s, 160. 220, 266
r//.c/<Asa, 192, 194, 195 fragilis, 123
sebivorans, 135 floridae, 120, 122, 236
sornaliensis , 124, 127 fradiae, 54, 110-112, 116, 126-129, 141-145, 150,
sylvodorifera, 68, 69 152, 154, 158-162, 170, 171, 185, 186, 190, 193,
viridis, 184 194, 223, 224, 226, 230, 236, 237
Oospora fulvissimus, 160, 185
cretacea, 21 gardneri, 20
iNi)i:x I'o si'i;(Mi:s ok AciiNOM^cirnis :52l
1
tlclaliciDi, \2\ . 'I'J ruhrircliiiili , 58, 155
(llol)i>iponi>i, lOS, lo7. I'.IJ, 1!I7, •222 scabies, 101, IOC, 1 II, 149, 1.55, 160, 175, 179, 190,
globispontft niliiiiris. \'u 191, 197, L'lO, LMl, 218. 265, 267, 268. 270-273,
globosiis, lOS, 157, ItlO 275
griseoflaviis, 185 soitialiensis, 262, 264
grisculus, 121. 186 speclabilis, 238
griscoviridis, 12.'? sipheroidcs, 238
giiseiis. 20, 66. (IS, 71. 77, 7S, S7, !)(>, '.)8, KM 107, stvrilis, 105
110-112, 115-117. 11!) 12:?, 125, 127, 12!). IM, tricolor, 1!)9, 20!)
138-143. 145. 147 155, 157 162. 164. 167. 170- venezuelae, 68, 112, 116, 130, 131, 145, 1.52, 160,
172, 175, 176-17!), 181, 18,5-187, 18!), 1!)(), 1!)3, 181,236
194. 1!)6. 1!)7. 202, 203, 222 226, 230, 233, 235, verne, 200
240-242 verticillatus, 21
griseus var. pttrpunua, 236 vinaceus, 122, 226
halsledii, 185 violacea, 42
hmnidus, 235 violaceoruber, 199, 200
interniediiis, 273 riulaceus, 105, 136, 155, 160, 173, 185, 200, 222
ipoiiioea, 160, 275 violaceus niger, 201
kanaiin/cetiois, 236 violac ens -ruber, 185
lorendulac. 20, 50, 105. 106, 123. 125. 128, 129, 145, r/nV//s, 200, 273
150, 152, 153, 1.57, 1.58, 160-162, 167, 178, 185, viridochromogcncs, 160, 189, 192, 193, 2C0
222, 225, 232 Sireptothrix
lipmanii, 160 actinomyces , 7, 51
longisporus, 222 a/6a, 30, 42, 62, 70
longisporus ruber, 156 albido-flava, 5
nigrificans, 209 ,4/?>»s, 5
madurae, 261, 262. 264 auraniiaca, 51
marinus, 125 carnea, 5
nigra, 42 chromogena 9 ,
322
( J EN ERA L IM)i;.\ 323
Fermentation
Nocardia, 89, 90, 91
of glucose, 149, 150, 157
Streptomyces, 75
by Streptomyces griseus
Light, influence, 136
carbon balance, 151
Limestone precipitation, 156
nitrogen balance, 154
Lipase, 189
F -forms, 101
Lipoactinochromes, 202
Flavoprotein production, 193
Luminescence, relation to antibiotic activity, 206
Foodstuffs, actinomycetes in, 42
Lysis of Streptomyces fradiae, as affected by
Fungi, relation to actinomycetes, 1, 13, 53, 71
calcium, 145
Lysozyme
Generic interaction in Streptomyces, 110 action on cell walls, 159, 160, 170, 172, 173
Grisein production, 142, 145, 195 enzymatic properties, 183
Growth
as affected by salt concentrations, 146, 147 Macrolides, 238
-promoting effects on Magnamycin, 238
animals, 195, 196 Mangel scab, 275
bacteria, 196 Mannosidostreptomycin, hydrolysis by amylase,
in soil, as affected by rye straw and dried blood, 187
246, 247 Mannosidostreptoniycinase, 187
of various genera Media for growth of actinomycetes, 18
Actinoplanes 62 ,
Melanin, 203
Micromonospora, 61 Mesophilic organisms, 134
Nocardia, 48 Metabolic processes of Streptomyces, phases, 116
Sireptomyces, 50, 51, 54, 55, 58, 59, 138-145 Metabolic reactions, role of metallic elements,
Streptospornndiniii , 63 146, 147
Glucose utilization, effect on Metabolism
acidity, 132 of aerobic actinomycetes, 129, 130
streptomycin production, 133 of anaerobic actinomycetes, 131, 149
carbohydrate, by Streptomyces, 148, 149
Hemicellulose decomposition, 155, 244, 246 fatty acids, by Nocardia opaca, 152, 153
Hemolysin production, 181 historical stiulies, 114
Heterokaryosis in Slrcplomiices, 110 112 role of antil)iotic biosynthesis in, 241, 242
Humvis, formation and ({(M'oniposit ion, 247, 248 Met h\ inycin, 23S
Hvdroactinochromes, 201 Mineral re([uirements of Streptomyces griseus, 138
Cl'.M.IJAL INI)i:.\ 325
M()i|)li(il()iiic;il cliarnclcrisl ics < lilur jinxhicl idii. i:;, !,")(;, i.>.")()
nmtaK«Miic and hMlial ctTcM'ts, 102, lOI), 10.3, 107 \itaniin synthesis, rei.ation to Kiowtli sliniulatinK
I roa i)r()duclion from tiiiani(iin(>, ISO crt'ect \\)'A. li)')