Microbiology
Biogenesis of Bacterial Cellulose has been revealed. Since bacterially synthesized cellulose is
Robert E. Cannon, Ph.D. and Steven M. Anderson,
Ph.D.
ABSTRACT
Cellulose is the most abundant biological polymer on Earth.
It is found in wood and cotton, and forms the basic structural
foundation of the cell wall of almost all eukaryotic plants.
Bacteria are known to secrete cellulose as part of their metab-
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olism of glucose and other sugars. The focus of this review is
upon bacterial cellulose synthesis. We emphasize recent lit-
erature directed primarily upon Acefubucferxylinum, which
has been most widely studied. Our review covers the following
topics relating to cellulose synthesis: genetics, biochemistry,
ultrastructure, growth conditions, and ecological considera-
tions as they relate to the diversity of microbes capable of
synthesizing this abundant, unique polymer - cellulose.
1. INTRODUCTION
Cellulose is the most abundant natural polymer on Earth. It
For personal use only.
forms the cell wall of eukaryotic plants and algae, and is also
found to be the major constituent of the cell wall of fungi.
Cellulose is also synthesized by bacteria, especially by the
Gram-negative bacterium Acetobarer xylinum, which is the
primary focus of this review. This microorganism has been the
most widely studied of the few bacteria that produce cellulose,
although a number of other bacterial cellulose producers are
discussed briefly. The following topics are covered: survey of
cellulose-synthesizing bacteria, growth and cultivation require-
ments, ultrastructure of bacterial cellulose synthesis, biochem-
istry of cellulose synthesis, genetics of biosynthesis, and ecology
of cellulose producers.
Cellulose which is synthesized by bacteria as a secondary
metabolite is a polymer of glucose. The molecules of glucose
are linked together as I+4p-glucan chains. Cellulose synthe-
sized by A. xylinum is polymerized into long microfibrils out-
side of the cell wall and is of the same chemical constituency
as the cellulose made by higher plants. This similarity makes
A. xylinum an ideal model system to study the cellulose bio-
genesis process. The synthetic pathways of many glucose poly-
*
mers are fairly well understood and the synthase enzymes
involved have been well characterized (e.g., chitin synthetase).
The biosynthetic pathway for bacterially produced cellulose is
still not completely understood, thus providing a fertile area
for research. The biosynthetic process is multi-step involving
both the production of cellulose and its crystallization into
extracellular fibrils. Our understanding of these processes is
increasing rapidly as more information concerning the enzyme,
cellulose synthase, and its organization in the cell membrane
1991
chemically identical to that of higher plants and other organ-
isms, this may suggest a probable common evolutionary origin
among cellulose producers. By studying the synthetic process
using microbes, which are easily maintained and manipulated,
it may be easier to uncover the details of genetics and bio-
chemistry of cellulose biosynthesis.
Could the cellulose produced by bacteria like A. xylinum be
put to productive use commercially? Will it replace or su-
percede the cellulose that we now get from trees for paper or
cotton plants for fiber? Probably not, but bacterial cellulose
does have some advantages over that produced by other or-
ganisms. One of the advantages of Acetobucfer cellulose is
that it does not need to be delignified before processing as is
the case with woody plant cellulose that is processed into paper.
A . Xylinm probably has the greatest potential for commer-
cialization since other cellulose-synthesizing microbes do not
product sufficient quantities of the cellulose polymer for in-
dustrial purposes. Acetobucfercellulose holds water well when
left undried and has strong tear resistance. It can assume or be
woven into various shapes and it retains its shape well. One
current example of an application for bacterial cellulose comes
from the S O W Corporation, which uses cellulose produced
by A . xylinum to make sensitive diaphragms for stereo head-
phones. Microbial cellulose is also available as a food product
called Nata in the Philippines.* Weyerhauser and Cetus Cor-
poration have recently developed a strain of Acerubucter that
is capable of large-scale cellulose production under fermen-
tative conditions with agitati~n.~ The fibers produced termed
Cellulon, have a number of potential commercial applica-
tions, such as binders for ceramic powders and minerals, thick-
eners for paint, ink, adhesives, and even foods. Another possible
use for Cellulon is as a paper coating because of its high purity.
Also recently reported was the use of Acefubucrercellulose as
a temporary skin substitute to protect burned tissue as normal
skin regenerate^.^ This is the first example of specific medical
applications of bacterially produced cellulose. The cellulosic
material, identified by the trademark BioFill, has been used
successfully for treatment of severe bums, skin grafting, and
chronic skin ulcers. Advantages of this treatment were pain
relief, good adhesion, an effective barrier to infection, fast
healing, good fluid (water and electrolyte) retention, low cost,
and short treatment time as normal, healthy skin grows to
replace the artificial cellulosic skin substitute. A potential dif-
ficulty to human use of cellulose may be the need to remove
435
 Critical Reviews In

endotoxic components that may contaminate bacterial cellu-
losic products.
Our knowledge of the process of cellulose synthesis carried
out by bacteria exemplified by A . xylinum has increased greatly
over the past decade. This basic research has formed the foun-
dation for future attempts at potential commercialization of
bacterially produced cellulose, which are currently in their
formative stages.
II. SURVEY OF CELLULOSE-PRODUCING
BACTERIA
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producing Acetobacters is production of a floating cellulosic
pellicle when the organisms are cultured statically. The pellicle
forms from the organization of cellulose fibrils synthesized and
secreted by the bacterium. Many strains oxidize ethanol to
acetic acid. They are ubiquitous and commonly found on de-
caying fruits and vegetables, vinegar, fermented beverages,
sugar cane juice, garden soil, etc.
Other bacteria that produce cellulose in lesser quantity than
A . xylinum include Sarcina ventriculi, which is the sole Gram-
positive, cellulose producer.6 Also, a number of bacterial spe-
cies have been found to produce exocellular cellulose fibrils
when isolated from activated sewage sludge. The cellulose

Many Gram-negative bacteria secrete extracellular polysac-
charide material, but only a few have been shown to produce
cellulose (Table 1). A . xylinum, the most studied of bacterial
cellulose producers, is a Gram-negative, aerobic, rod-shaped
organism. It is classified in Bergeys Manual of Systematic
Bacteriology (Volume 1, 1984) in Section 4 -Gram Negative
Aerobic Rods and Cocci5 It is a member of the family Ace-
tobacteraceae. Species within the genus, Acerobacrer include
A . aceti, A . liquifaciens, A . pasteurianis, and A . hansenii. A .
xylinum is treated in scientific literature as a species, but for
the purpose of strict classification, it has been considered a
subspecies of A . aceti. For the purpose of this review, A .
xylinum will be used throughout to indicate the primary, cel-
For personal use only.
fibrils were believed to account for floc formation. The specific
genera isolated included Pseudomonas, Achrombacrer, Al-
caligenes, Aerobacter, Rhizobium, Agrobacterium, and Azo-
tobacter. Cellulose fibril formation apparently plays an important
part in the infection of plants by Agrobacterium tumefaciens
by aiding in attachment of bacteria to plant tissue eventually
leading to gall formation.8 A more detailed discussion of the
possible role(s) of cellulose for the bacteria that produce it is
presented in the ecology section of the review.
111. BACTERIAL GROWTH
CHARACTERISTICS, REQUIREMENTS, AND

lulose producing bacterium. Members of the genus Acetobacter IDENTIFICATION OF CELLULOSE

are chemoorganotrophscommonly producing pale colonies when
grown on solid media. A prominent characteristic of cellulose- The undefined medium that is characteristically used to cul-

Table l
Survey of Bacterial Cellulose Producers

Organism (genera) Cellulose produced Cellulose assay Biological Role

Acetobacter Extracellular pellicle Cellulose To hold in aerobic environment

Cellulose ribbons synthase To colonize natural substrates
X-ray diffraction
Alkali insolubility
Fluorescent
brightener
Degradation by
cellulase
Agrobacterium Short extracellular fibrils X-ray diffraction Attach to plant tissue
Alkali insolubility
Fluorescent
brightener
Degradation by
cellulase
Rhizobium Short extracellular fibrils Same criteria as Attach to host plants
Agrobacterium
Pseudomonas No distinct fibrils Alkali insolubility Flocculation in wastewater
Degradation by
cellulase
Sarcina Amorphous celluloseb Alkali insolubility Unknown

a Cellulose I - highly crystalline, metastable microfibrils, native cell~lose.~

Cellulose 11 - crystalline allomorph of Cellulose I with antiparallel glucan chains, rare in nature.

436 Volume 17, Issue 6

 Microbiology
ture Acetobacter qlinum to maximize both growth and cel-
lulose production was developed by Hestrh and Schramm (Table
2)." Other carbon sources have been shown to be suitable for
growth, but they have not been as useful for production of
cellulose. A . xylinum grows well over a temperature range of
25 to 30"C, but its temperature optimum is 30C. The pH of
the undefined medium is 6. Historically, to maximize cellulose
production, A. xylinum has been cultured statically allowing
for the formation of a pellicle composed of cellulose holding
within it bacterial cells floating on the surface of the medium.
If cultures are aerated by shaking, bacteria grow faster, but
produce less cellulose. Rotary shaking results in production of
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round balls of cellulose rather than the flat, sheet-like pellicle
of static cultivation. Doubling time for A. xylinurn held in static
culture is between 8 and 10 h, while in aerated culture (by
shaking), the organism doubles every 4 to 6 h. When A. xy-
h u m is cultured on solid medium, colonies that are formed
by cellulose-producing strains have a dry, wrinkled appear-
ance. This contrasts to celluloseless strains (cellulose-negative
mutants), which appear as smooth, spreading colonies that are
usually two or three times the size of cellulose-synthesizing
strains after a comparable period of growth.
Table 2
Undefined and Synthetic Media for
For personal use only.
Cultivation of Acetobacter xylinum
Undefined" Synthetic'*
Glucose 2.0% Glucose 1.0%
Yeastextract 0.5% NH.,Cl 0.1%
Peptone 0.5% Citric acid 0.115%
Sodium 0.27% N%HPO, 0.33%
phosphate
Citric acid 0.115% KCI 0.01%
MgSO, * 7H,O 0.025%
Nicotinamide 7.5 mg/I
To be able to manipulate growth and know exact chemical
constituents, we have developed a synthetic minimal medium
for A. xylinurn that permits good growth (Table 2, Figure 1)
and maintains cellulose production.'2 The synthetic minimal
medium is useful for genetic manipulation of A. qlinum be-
cause it can be used to isolate auxotmphic mutants. A culture
grown in minimal medium with 1% glucose added produced
about half as much cellulose as one in the undefined medium,
at a slightly slower growth rate. If any of the ingredients of
the medium were omitted, no growth occurred. The synthetic
medium was used successfully to isolate a number of amino
acid- and nucleotide-requiring auxotrophs after mutagenesis of
A . xylinum 'by N-methyl-N'-nitro-N-nitrosoguanidine.
To insure that the biosynthetic product made by A. xylinurn
is cellulose, a number of different techniques are employed.
The techniques vary in complexity and accuracy, and it may
be necessary to rely on a number of methods to be sure that
1991
the microfibrillar product is cellulose rather than other non-
cellulosic polysaccharides.l3 Fibrils of cellulose are extremely
strong, and insolubility in alkali (2% NaOH) has therefore been
used as a criterion for presence of cellulose. However, other
glucose-based polymers (e.g., p-I ,2-D-glUCan,p-l,3-D-ghl-
can) are also insoluble in base and therefore one should be
careful in using this as the sole criterion for demonstration of
cellulose production. Also, the crystalline structure of the pol-
ymer is resistant to treatment by a combination of acetic and
nitric acid. Cellulose gives a characteristic diffraction pattern
upon X-ray crystallography, which is the ultimate method dem-
onstrating its presence. Several inhibitors have been identified
that interfere with cellulose synthesis.l4 These include cou-
marin and dichlorobenzonitrile, which apparently disrupt gly-
cosylation in the synthesis process. In addition, substances
called fluorescent brighteners, e.g., Calcofluor White ST or
Tinopal LPW, have been used to confim cellulose production.
When these stilbene derivatives react with the glucan chains
of cellulose by hydrogen bonding, they prevent normal micro-
fibril formation and assembly of cellulosic ribbons by A. q-
l i n ~ r nDespite
.~ the disruption of normal ribbon formation, the
binding of these substances causes the bands of cellulose to
fluoresce brightly when viewed under ultraviolet light. It is
possible to incorporate the fluorescent brighteners into the solid
medium. Colonies of A . xylinum and other cellulose-synthes-
izing bacteria, which are actively synthesizing cellulose flu-
oresce, while cellulose-negative strains do not. Unfortunately,
these brighteners bind to other exopolysaccharides, so addi-
tional tests must be done to c o n f m that cellulose has indeed
been synthesized by the bacteria.
IV. ULTRASTRUCTURE AND MODEL FOR
CELLULOSE SYNTHESIS
The ultrastructure of the cellulose synthesis apparatus is best
understood in A. xylinurn. Transmission electron microscopy
and freeze-etching have revealed the presence of a row of pores
on the longitudinal axis of cells. These particles or pores may
contain or be composed of the last enzyme(s) involved in cel-
lulose synthesis, and glucan chains that eventually form into
the composite ribbon of cellulose may be secreted through these
pores.9 Figure 2 is a depiction of this hypothetical model. Each
cell that is actively synthesizing cellulose produces a cellulosic
ribbon ranging in width from 40 to 60 nm, which appears to
be parallel to the longitudinal axis of the bacterial cell. Al-
though A. xylinum is nonmotile, it can be pushed by the for-
mation of the ribbon of cellulose at a rate of 2 pdmin. The
ribbon of cellulose is composed of microfibrils, which are the
form in which cellulose is secreted through extrusion sites in
the outer membrane of the bacterium. These microfibrils appear
to be about 1.5 nm wide and aggregate into 3 to 4 nm micro-
fibrils via crystallization of adjacent glucan chains. Finally,
these microfibrils band together to form the larger cellulosic
437
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I e
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c.l - M a 8 Yedlum (Undellned)

to I --+ - Ylnlmel 111)Olucosa
w - YInImal 211) Olusoae
+

A--+ - Ylnlnel 0.6% OIUCOS.

.=-a - Ylnlmal 0.2611) O I U C O ~ ~

FIGURE 1. Growth curves for A. xylinum in undefined and minimal media at four glucose
concentrations (2, 1, 0.5, and 0.25%).Cells incubated statically at 2 6 T , but shaken briefly
at each time point to release cells consistently from the pellicle.*
For personal use only.

about these processes comes from studies employing fluores-

and Bundle Initiation
LPS envelop.
flGURE 2. Possible model of cellulose assembly. Glucan chain aggregation
requires multienzyme complexes followed by crystallization of microfibrils
through extrusion pores. Ribbons of cellulose form at the cell ~urface.~
ribbon. Figure 3 shows the presence of the extrusion pores on
the side of an A. xylinum cell, a typical ribbon secreted by a
cell, and a more highly magnified view of individual ribbon^.^
Bureau and Brown have demonstrated that cellulose synthesis
occurs in the cytoplasmic membrane of A. xylinum rather than
in the outer membrane as had been previously thought. Final
crystallization of microfibrils may still occur in or near the
outer membrane and be dependent upon the ordered nature of
the extrusion pores that are seen in freeze fracture preparations
of cells.
The enzymes involved in the terminal stages of cellulose
fibril biogenesis are cell-associated, probably within either in-
ner or outer membranes of the Gram-negative bacterium (see
biochemistry section). It is not completely understood how the
individual glucan molecules polymerize and assemble into the
microfibrillar bundles of cellulose. Much of what is known
cent brighteners and cellulosic derivatives that alter ribbon
formation. Fluorescent brighteners such as Calcofluor bind to
extruded glucan chains preventing microfibrils from banding
together into complete ribbons. Carboxymethylcellulose and
other cellulose derivatives affect ribbon assembly at a different
level of organization. I6They commonly stop fasciation of bun-
dles of microfibrils so that individual microfibrils remain sep-
arated, never forming into highly organized fibrils. More
recently, Kai and Kitamura and Haigler and Chanzy* have
used techniques of X-ray and electron diffraction to study the
effects of fluorescent brighteners upon cellulose structure in
A. xylinum. Kai and Kitamura reported that brighteners do not
effect the extrusion of monomolecular layers of cellulose I from
cells. At high concentrations of brightener, a diffraction pattern
resembling neither cellulose I nor II appeared. As brightener
concentration was lowered, the cellulose I pattern returned.
Haigler and Chanzy, using different washing methods than Kai
and Kitamura to prepare cellulose samples prior to diffraction
techniques, did not observe altered cellulose. They speculate
that the harsh treatment with alkali and alcohol used by Kai
and Kitamura might have introduced some form of crystallin-
ity. Haigler and Chanzys results also showed that cellulose I
could be reformed from amorphous cellulosic material that is
produced after dye treatment. In the final analysis, when po-
lymerization and crystallization processes are disrupted, nor-
mal microfibrillar ribbons of cellulose cannot form.
Strong magnetic field strength has also been shown to alter

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For personal use only.

FIGURE 3. (Top) Freeze-etching of cells of A. xylinwr that are synthesizing cellulose. Particles in
the iipopolysaccharide layer are thought to be groups of multienzyme complexes involved in polymer-
ization of cellulose. (Middle) Typical twisting ribbon of cellulose seen by negative staining. (Bottom)
Higher magnification of cellulose ribbons negatively stained showing striation. Four separate ribbons
cross with larger striations corresponding to microfibrillar bundles of c e l l ~ l o s e . ~

ribbon assembly in A. xylinum, although specific mechanisms V. BIOCHEMISTRY OF CELLULOSE

are unknown at present.19
The overall model to account for cellulose ribbon assembly
has been described as "hierarchical" by a number of inves-
tigators. First, small glucan chains aggregate by a self-assem-
bly mechanism into the 3 to 4 nm microfibrils. This event is
followed by banding of the microfibrils into bundles, which
then form into the complete ribbon. The high degree of or-
ganization of the extrusion pores on the surface of the outer
membrane facilitates the coordination of the assembly process.
An even higher degree of organization of cellulose fibrils can
be observed when whole, washed pellicles are viewed under
scanning electron microscopy. Structures resembling tunnels,
which would indicate a considerable degree of regularity in
the organization of microfibrils, appear." These structures pro-
vide an even higher level of fibril orientation than ribbon for-
mation described above and argue that synthesis of cellulose
by A. xylinum is not occumng randomly. The diameter of
tunnels is about 7 pm, with sufficient room for bacteria to
move through them.
SYNTHESIS
Even though cellulose is a relatively simple polymer, we
still do not completely understand the biochemistry of its syn-
thesis. Difficulties in both development of an in v i m assay
for cellulose synthesis and purification of the enzymes involved
in this process (e.g., cellulose synthase) have hindered bio-
chemical analyses. These problems are being overcome through
use of combined genetic and biochemical techniques with the
organism A. xylinum, and a clearer picture of the biochemical
steps in cellulose synthesis is beginning to emerge.
In addition to glucose, other substrates such as hexoses,
hexonates, pyruvate, glycerol, and dihydroxyacetone can be
used in the biosynthesis of cellulose.z'-23Many of these sub-
strates are metabolically associated with either the pentose or
citric acid cycles, indicating that cellulose synthesis may be
tied to oxidative carbohydrate metabolism." However, since
cellulose formation in Acetobucter can occur independently of

1991 439
 Critical Reviews In

h
protein synthesis,25it does not appear to be an indispensable
'OO
feature of carbohydrate metabolism.
Knowledge regarding the proposed biochemical pathway
leading from exogenous glucose to cellulose has come from Cellulose
two sources: (1) studies on the enzymes involved in glucose- . Glucose
carbohydrate metabolism, and (2) labeling studies with isotopic
glucose used to delineate precursor-product relationships. A \ ,
group of enzymes involved in carbohydrate metabolism have
been found in crude and partially purified extracts of A. xy-
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Within this group of enzymes, it has been pro-

linum.14*24*26*27
posed that glucokinase, phosphoglucomutase, and UDF'G-
pyrophosphorylase are most likely to be involved in the path-

-
way leading to cellulose synthesis (Figure 4).
Water soluble
*A-A A
Glucokinase Phosphoglucomutase
Glucose- - - - - - - > Glucose-6-phosphate-- -- -- - -- - - - - - >
Alkali rolubk

Chloroform soluble
--
For personal use only.

-
4 - A

FIGURE 4. The proposed biochemical pathway for cellulose synthesis in

Acetobacter xylinum.

The phosphorylation of hexose sugars provides a common

intermediate for both cellulose synthesis and oxidation of car-
bohydrate via the pentose and citric acid cycles. Phosphoryl-
ation of exogenous sugars is carried out by glucokinase and
fructokinase in A. xylinum.26Glucokinase activity is always
present in cells regardless of which exogenous sugar is pro-
vided, whereas fructokinase activity, like that of other sugar-
metabolizing enzyme systems, appears to be subject to glucose
repression. The flux of phosphorylated sugars through the two
pathways described above will in all likelihood determine the
level of cellulose produced. In the cellulose pathway, phos-
phorylated glucose in the form of glucose-6-phosphate (G6P)
is enzymatically converted to glucose-1-phosphate (G1P) by
phosphoglucomutase.24 UDPG-pyrophophorylase is then re-
1 5 . - 15 30 45 60
sponsible for the synthesis of UDP-glucose from GlP and
UTP.= Even though all four nucleoside sugars may act as Time (minutes)
precursors for cellulose synthesis, there is no evidence for any
other nucleoside diphosphoglucose pyrophosphorylase in A. FIGURE 5. The time course for a pulse-labeling experiment during which
xylin~m.'~ This observation is also consistent with UDP-glu- cellulose is synthesized from "C-glucose by wild-type Acetobocter xylinum
cells. Incorporation of the label into both cellular fractions (e.g., chloro-
cose acting as primary precursor for cellulose synthesis. This
form-,water-, and alkali-soluble fractions) as well as specific cellular com-
proposed pathway has been substantiated through the use of ponents (e.g., UDP-glucose) is demonstrated."
isotopic-labeling studies in which the flow of carbon from I4C-
glucose to celrulose has been examined.28After pulse-labeling
A. xylinum cells, the incorporation of label into glucose-phos- Labeled G6P, GlP, and UDP-glucose appear shortly after cells
phate and UDP-glucose was determined. The results of this are exposed to L4C-glucoseand their subsequent decrease is
study are presented in Figure 5 and support the roles of G6P, linked to an increase in the incorporation of I4C into cellulose,
G1P, and UDP-glucose as precursors for cellulose synthesis. e.g., an alkali insoluble cell fraction.

440 Volume 17,Issue 6

 Microbiology
UDP-glucose is the substrate for cellulose synthase (E.C.
2.4.1.12, UDP-Glucose: 1,4-~-~-glucan4~-D-,olucosyltrans-
ferase) that is believed to be the major enzyme involved in the
last step(s) of cellulose biosynthesis. The development of both
an in vitro assay and a purification protocol for cellulose syn-
thase has provided for the initial characterizationof this enzyme.
Hesmn and Schramm" and G l a ~ e ?initially~ described the
in vitro synthesis of an alkali insoluble polymer from UDP-
glucose by a particulate fraction from A . xylinum. Unfortu-
nately, the rate of synthesis was <O. 1% of the in vivo rate and
therefore did not provide an adequate system to study either
the structure of the cellulose produced or the mechanism of
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synthesis. Aloni et al.30*31were able to achieve a 400-fold
increase in the in vitro rate of synthesis after stabilization of
the particulate/membranous fraction with either PEG 4000 or
calcium ions and upon activation of the system with GTP.
They also showed that is was necessary to add back a regulatory
protein from the soluble fraction to obtain maximal activity.
Recently, Lin and have been able to achieve partial
purification (>90% homogeneity) of cellulose synthase from
detergent solubilized membranes. A 96-fold purification was
obtained after two rounds of a cellulose entrapment purification
protocol. This involves adding substrate and enzyme activators
to a detergent-solubilized membrane preparation and trapping
the enzyme by centrifugation of the synthesized cellulose prod-
For personal use only.
uct. Their preparations contain two major polypeptide products
of 83 and 93 kDa. The 83 kDa polypeptide has been demon-
strated to be a glycoprotein by lectin affiiity chromatography,
Schiff periodate, and fluoroscein isothiocyanate-Concanavalin
A staining. Column chromatography results were consistent
with cellulose synthase being a multimeric enzyme. Lin and
Brown suggest that the 93 kDa polypeptidechain may represent
a second subunit of the multimeric complex. More recently,
Lin et al.33have demonstrated by photoaffinity labeling with
an analog of UDP-glucose that the 83 kDa subunit is the cat-
alytic subunit for cellulose synthase. In a membranous or de-
tergent-solubilized extract this analog, [(P-32P)-5N3-UDP-
glucose], would photoincorporate into both 57 and 83 kDa
polypeptides. Further analysis using competition assays with
UDP-glucose have corroborated that the active site for the
enzyme is contained in the 83 kDa polypeptide. They suggest
that the 57 kDa polypeptide is phosphoglucomutase,which has
been labeled due to contamination of the photoprobe prepa-
ration with trace levels of 32P-Glucose-1-Phosphate.
As mentioned earlier, cellulose synthase activity in in vitro
preparations could be enhanced by the addition of guanosine
nucleotides or analogs of guanosine. The work of Ross et al."
has further demonstrated that the specific cellulose synthase
activator is-bis - (3' * 5 ' ) - cyclic diguanylic acid depicted
in Figure 6 . They suggested that cellulose synthase activity is
controlled by a multicomponent regulatory system that includes
(1) diguanylic cyclase, the enzyme involved in the synthesis
of the cyclic diguanylic activator and (2) the activities of two
1991
0
\ /O'
/p\
0/p\ 0-
FIGURE 6. The structure of the diguanylic acid activator of cellulose
synthase.y
phosphodiesterases, which are involved in the degradation of
the activator. One phosphodiesterase is inhibited by Cat+ and
hydrolyzes the opening of the cyclic nucleotide and the second,
which is not affected by Ca2+, converts the dinucleotide into
5' GMF'. According to this model, the rate of polymerization
of UDP-glucose into cellulose is dependent on the intracellular
levels of diguanylic activator and the level of Ca2+. The in-
teractions of these components is summarized in the model
presented in Figure 7. Amikan and Ben~iman'~ have recently
shown the possible involvement of the same activator in the
modification/regulation of cellulose synthesis in Agrobacter-
ium tumefaciens.This observation suggests that both organisms
may utilize a similar regulatory mechanism, even though the
final cellulose product produced (bundles and simple flocs in
Agrobacterium vs. complex ribbons produced by Acetobacter)
and the rate of cellulose synthesis are markedly different.
VI. GENETICS OF CELLULOSE SYNTHESIS
Studies of the genes and gene products involved in cellulose
synthesis have been canied out in both Acetobacter xylinum
and Agrobacterium tumefaciens. The development of these
model systems should provide not only an understanding of
the genetics of cellulose biosynthesis in bacterial systems, but
also an insight into this process in higher plants.
The genetic analysis of cellulose biosynthesis in Acetobacter
qdinum has included the isolation of mutants that affect cel-
lulose production, characterization of indigenous plasmid spe-
cies, and the cloning of genes involved in this-process. A
number of researchers, beginning with Schramm and H e ~ t r i n , ~ ~
441
 Critical Reviews In

Cellulose fibril
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IMg+i
UDP-glucose

FIGURE 7. A proposed model for the regulation of cellulose synthesis by the cyclic diguanylic acid activator of cellulose synthase. Levels
of activator are determined by the synthetic activity of diguanylate cyclase, the degradative action of phosphodiesterase A and B, and the
inhibitory effect of Ca2+on phosphodiesterase activity."

have described the isolation of cellulose-negative (cel -) mu- Table 3

tants. Both spontaneous and mutagen-induced variants have Characteristics of Acetobacter xylinum Strains
For personal use only.

been isolated. Many reports have indicated that apparent spon-

taneous celluloseless mutants arise at a high rate when wild-
type cells are grown in aerated liquid The frequency I Characteristic

Colony morphology
Wild type

Small, rough
Cel-negative

Large, mucoid
Overproducer

Small, rough
with which these mutants occur also increases with the age of
Fluorescence with Bright Dull Bright
the culture. The spontaneous mutants have a mucoid appear- Tinopal
ance on solid agar like true cel- mutants, but the majority Pellicle production Pellicle No pellicle Thick pellicle
revert back to wild type when grown statically in broth culture. Relative cellulose 1 .o 0 . m . 1' 5.0-6.0
The most likely explanation for such behavior is that these are production (wet
weight)
not true genetic mutants, but instead represent alterations in
the phenotypic expression of genetic sequences involved in a
The trace amounts of cellulose produced in these pellicledeficient cultures
cellulose synthesis. These phenotypic mutants may be due to are thought to be cellulose II.
changes in gene expression, e.g., the repression of cellulose
synthesis genes during growth under aerated conditions, alter- will determine if these genetic changes represent mutations in
ations in the cell walkell membrane components induced by the structural or regulatory genes involved in cellulose bio-
the culture conditions, or other nongenetic events. A variety synthesis or pleiotropic mutations, e.g., those affecting the
of mutagens have been used in an effort to induce mutations lipopolysaccharide composition of the cell membrane.
in Acetobacter xylinuh. Nitrosoguanidine, ethyl methane sul- The existence of indigenous plasmids in A. xylinum was first
fonate, and nitrous acid are very effective mutagens for A . described by Valla et al?' They examined the plasmid content
xylinum, whereas hydroxylamine and ultraviolet light are rel- of a wild-type strain (ATCC 10245) and 13 cel- mutants de-
atively ineffective. 12.37*39 Cellulose-negative mutants can be rived from the wild type and showed that A. xylinum has a
identified by altered colony morphology (mucoid vs. rough), complex pattern of plasmids ranging in size from 16 to 300
fluorescence on agar plates containing tinopal or calcofluor, kb. A significant number of the cel- mutants (8/13) had iden-
and/or inability to produce a cellulose pellicle in liquid culture. tifiable changes in plasmid content. These changes included
In addition to cel- mutants, cellulose overproducer mutants both the loss of specific plasmids as well as apparent rear-
have also been isolated. These strains produce cellulose with rangements of plasmid sequences. A further analysis of cel-
the same characteristics as wild type, but at five to six times and transposon (Tn l)-induced mutants confirmed the complex
the wild-type level resulting in a thickened pellicle (Table 3). nature of the Acetobacter plasmid ~ o n t e n t . ~This
' series of
There has been little characterization of the overproducer strains mutants showed changes in plasmid content, changes in plas-
to date. Further study of these mutants as well as cel- mutants mid copy number, and often rearrangements between plasmid

442 Volume 17, Issue 6

 Microbiology
and chromosomal sequences. Valla et al. were unsuccessful in
attempts to cure A . xylinum of any of these indigenous plasmid
species. Their results were consistent with both plasmid genes
being involved in cellulose synthesis and the existence of es-
sential genes on Acerobucfer plasmids.
We have recently analyzed the plasmid content of another
wild-type strain of A . xylinum (ATCC 23769).42In contrast to
A . xylinum ATCC 10245, this wild-type strain has only a sin-
gle, low copy number plasmid of approximately 58 kb. Com-
parison of the plasmid content of this strain with a series of
nitrosoguanidine-induced,cellulose-negative mutants showed
no gross differences in plasmid content (Figure 8). Restriction
Critical Reviews in Microbiology Downloaded from informahealthcare.com by East Carolina University on 09/05/13
digestion analysis with a variety of enzymes revealed only a
few minor differences in these plasmid species. We were un-
able to cure this plasmid with a variety of known plasmid curing
agents, e.g., ethidium bromide, naladixic acid, mitomycin C,
and sodium dodecyl sulfate. Our results suggest cellulose syn-
thesis is not necessarily linked to the presence or absence of
plasmids in strain ATCC 23769.
Saxena and have also examined the plasmid content
of a variety ofA. xylinum wild-type strains. The various strains
had remarkably different plasmid content, with 0 to 10 plasmids
found per strain, and sizes ranging from 2 to 230 kb. Southern
blot analysis revealed little sequence homology between these
For personal use only.
WT cel-4 cel-10
plasmid
chr
FIGURE 8. The plasmid content of an Acerobacter qlinum wild-type strain (ATCC 23769) and cellulose-
negative (ce1-4, cel-10, cel-13, cel-15, and cel-20) rn~tants.~
1991
different plasmid species. Because some wild-type, cellulose-
producing strains contained no plasmid sequences, they also
concluded that plasmid genes probably have little involvement
in cellulose synthesis.
Acefobucter can act as both a recipient and donor for the
conjugative transfer of broad host range plasmids from incom-
patibility groups P and Q.4345 While plasmid sequences can
be transferred between Escherichiu coli and A. xylinum or
between two A . xylinurn strains, there is no evidence of the
mobilization of chromosomal genes during conjugation. The
conjugative transfer of plasmids has been useful in the intro-
duction of transposons such as Tn 1 and Tn 5,~ which can
then be used to tag and isolate specific genes. In addition,
conjugation has provided a means to introduce cloned DNA
sequences into Acerobucferand, by complementation of known
mutants, has led to the identification of the UDPG-pyrophos-
phorylase gene.* Transformation methodologies described for
other Gram-negative organisms have not proved successful for
A . ~ylinum.~.~However, a transformationprocedure and clon-
ing vectors for the related species A . uceri have been de-
scribed,49 and the possibility exists that a variation on this
method may be successful with A . xylinum. As an alternative
to the above methodologies, we have recently demonstrated
that high voltage electroporationS0can be used to transform A.
cel-13 cel-15 cel-20
443
 Critical Reviews In
xylinum with broad host range plasmids (pUCD2 and pRK
248). Transformation efficiencies of approximately lo7 colo-
nies per microgram of plasmid DNA can be achieved with
electroporation. Therefore, as genetic exchange mechanisms
are further developed and improved, our understanding of the
genetics of A. xylinum should increase.
Recently, two genes encoding enzymes involved in cellulose
biosynthesis, UDPG-pyrophosphorylaa and the catalytic
subunit of cellulose synthase, have been cloned. The UDPG-
pyrophosphorylase gene was isolated from a partial library of
Acerobacrer DNA by its ability to complement both cel- mu-
tants of A. xylinum and a galU mutant of E. coli. Cellulose-
Critical Reviews in Microbiology Downloaded from informahealthcare.com by East Carolina University on 09/05/13
negative mutants that received recombinant plasmid pVK
lOO(240) were able to synthesize cellulose at the wild-type rate
and had near wild-type levels of UDFG-pyrophosphorylase.
Assays for diguanylate cyclase, phosphodiesterase A, phos-
phoglucomutase, UDPG-pyrophosphorylase, and cellulose
synthase showed that the three cel- mutants that could be
complemented with pVK lOO(240) were deficient in only
UDPG-pyrophosphorylase activity. The 2.8 kb Hind III frag-
ment cloned in pVK lOO(240) also complemented an E. coli
galU mutant containing a structural gene mutation in UDPG-
pyrophosphorylase. Valla et al.& have suggested celA, as the
designation for this gene.
Saxena et aLS1used a different strategy to isolate the gene
For personal use only.
for the catalytic subunit of cellulose synthase. They used a
synthetic oligonucleotide probe homologous to the amino ter-
minus of this 83 kDa polypeptide to ident@ and clone a 9.5
kB Hind III fragment of Acerobacter DNA. Sequence analysis
has shown that this DNA fragment contains an open reading
frame sufficient to code for a 80 kDa polypeptide. In addition,
the amino terminal protein sequence deduced from the nucleic
acid sequence provides a match to the 83 kDa subunit, indi-
cating that in all likelihood it is the catalytic subunit for cel-
lulose synthase.
The genetic analysis of Acerobacter has provided a number
of interesting observations including (1) the existence of a
complex pattern of plasmids; (2) the utility of chemical and
transposon mutagenesis to induce cel- mutants; (3) the ap-
parent genetic instability in this organism, e.g., the high rate
of induction and reversion of spontaneous mutants, chromo-
somal and plasmid reakangements; and (4) the use of recom-
binant DNA technology to isolate genes involved in cellulose
synthesis. The ability to clone the genes involved in cellulose
biosynthesis will not only provide a better understanding of
this process but also lead to potential commercialization of
microbial cellulose production.
Because Agrobacterium tumefaciens can cause disease in a
wide variety of plants, it has been the object of study and
genetic analysis for quite some time.52Much attention has been
paid to the genetic interactions between bacterium and plant
cell in the development of crown gall tumors. One aspect of
this interaction is the specific attachment of bacterium to plant
444 Volume 17, Issue 6
host cell. Much of what is known concerning cellulose syn-
thesis in this organism centers around the role of cellulose in
this
A variety of mutants have been isolated that affect the ability
of Agrobacrerium to attach to plant cells. Included in this group
afe mutants deficient in cellulose produ~tion~.~as well as those
still capable of cellulose ~ynthesis.~ Further analysis of these
mutants has led to the proposal that certain low molecular
weight glucans are important in the initial attachment of bac-
terium to plant cell, whereas the synthesis of cellulose fibrils
promotes both the anchorage of the bacterium to the plant cell
surface and the formation of bacterial aggregates. Genetic anal-
ysis has indicated that many genedgene products are involved
in cellulose synthe~is~..~ and that the majority of genes map
to chromosomal sites. In fact, many genes are clustered, sug-
gesting that a specific region of the Agrobacrerium chromo-
some is concerned with the interaction of the bacterium with
the plant host cell. What remains to be determined is if the
clustering of genes is necessary for the coordinate expression
of these sequences and if other cellulose-synthesizing organ-
isms have a similar genetic organization.
In fact, recent work by Wong et al. suggests that at least
some of the genes involved in cellulose synthesis in Acero-
bacter xylinum are also organized in an operon (bcs, bacterial
cellulose synthesis). These genetic sequences were localized
to a segment of chromosomal DNA that could complement
. cellulose-negative strains deficient in cellulose synthase. The
operon contains at le& four protein-coding regions (bcsA,
bcsB, bcsC, and bcsD), with the catalytic subunit of cellulose
synthase encoded by the bcsB region. The functions of the
other genetic sequences are not known, but their expression
(along with bcsB) appears to be necessary for maximal cellulose
synthesis in vivo.
VII. ECOLOGICAL ASPECTS OF
CELLULOSE PRODUCING BACTERIA
Because A. xylinum was observed to produce a pellicle dur-
ing Static culture, and since it was considered to be a strict
aerobe, investigators proposed that the role of the pellicle was
to hold the organisms in an aerobic environment to facilitate
their ability to obtain ~ x y g e n . ~ ~
Could
. ~ there be other eco-
logical roles for cellulose secreted by A. xylinum?
Undried cellulosic pellicles retain water well. Thus, one
hypothetical role for cellulose could be moisture retention in
the microenvironment to allow for bacterial growth and prevent
dessication. Also, our experiments cultivating A . xylinum in
candle jars, 5% carbon dioxide gassed enclosures, and Biobags
show that A. xyxylinum can grow microaerophilically while con-
tinuing to synthesize cellulose,39 so the argument that the pel-
licle is vital for aerobic growth is no longer as compelling.
Laboratory experiments also indicated that pellicles could pro-
 Microbiology
tect bacteria from the killing effects of ultraviolet light. This
protective effect could be of considerable importance to the
organism as it grows on decaying fruits just as it could be
holding water for the biochemical degradation of substrates for
nutrition. Since A. xylinum is found primarily on decaying
fruits, could it be possible that cellulose pellicle synthesis plays
a role in successful colonization of a particular habitat? To
answer this question, a series of experiments were performed
in which pieces of apple were inoculated with various strains
of cellulose-producingand celluloselessA . xylinum, and assays
were done to determine density of various organisms growing
on the apples. Celluloseless and uninoculated controls tended
Critical Reviews in Microbiology Downloaded from informahealthcare.com by East Carolina University on 09/05/13
to become dry over the course of the experiment. Molds, fungi,
and other bacteria tended to predominate on the pieces of apples
that had been inoculated with the cellulose-negative strain.
When pellicles from a wild-type and a cellulose-over-producing
strain completely covered the pieces of apple, A. xylinum pre-
dominated over other microbes growing on the fruit. These
results indicate that the ability to synthesize a cellulosic pellicle
may allow A. xylinum to colonize food sources more effectively
than strains of A. xylinum that do not make cellulose. This
colonization ability may be important in the microenvironment
as A . xylinum seeks nutrients in competition with other de-
composing organisms such as other bacteria and fungi.
Cellulose fibrils have also been implicated in the infection
For personal use only.
process of plant tissue by Agrobacterium tumefaciens, which
is another example of the need of a microorganism to use an
exopolymer to hold to a particular substrate.8 Cellulose fibrils
anchor bacteria to plant surfaces. Cellulose-negative mutants
of Agrobacterium produced by transposon mutagenesis secrete
cellulose fibrils so no bacterial aggregates form on plants even
though the bacteria are still virulent. The ability of cellulose-
negative mutants to form tumors at the site of inoculation was
decreased if the site was rinsed with water. The absence of
cellulose fibrils apparently reduced the ability of the bacteria
to anchor and eventually infect plant tissue.
Cellulose fibril formation to enhance flocculation in acti-
vated sludge permits the aggregated growth of a variety of
Gram-negative bacteria. The ability to form a floc could allow
for enhanced microbial growth by giving cells a surface upon
which to cling while they are participating in the process of
wastewater decomposition.
To be successful in the environment, many microorganisms
have evolved efficient mechanisms to colonize surfaces whether
it be the bottom of a ship, the surface of teeth, or decaying
fruit. Colonization typically has involved production of exo-
polysaccharides that aid in attachment, act to prevent drying,
pH changes, and effects of toxic molecules upon the colonizer.
Cellulose is a highly organized exopolysaccharide, which may
Play a role-similar to exopolymers secreted by other bacteria.
VIII. SUMMARY AND CONCLUSIONS
Acetobacter, Agrobacterium, Pseudomonas, and Rhizobium
1991
are four genera of Gram-negative bacteria that synthesize cel-
lulose. Sarcina is the sole genus of Gram-positive bacteria to
do so. The quality or kind of cellulose synthesized varies among
the various microbes from the long microfibrils that form into
pellicles for Acetobacter to the short cellulosic fibrils of Agro-
bacterium and Rhizobium, which permit bacterial attachment
to plant tissue. Pseudomonas and Sarcina secrete cellulosethat,
while crystalline in form, is more amorphous in structure.
Cellulose producers are found among both prokaryotes and
eukaryotes; thus one assumes a possible evolutionary relation-
ship in the biosynthetic processes of bacterial and higher or-
ganisms. Brownlo has speculated that Sarcina was probably
the first cellulose producer. With the evolution of aerobiosis,
came the other bacterial cellulose producers that are Gram-
negative. The genetic information for cellulose synthesis by
eukaryotes probably came via an endosymbiotic mechanism
from prokaryotes. When nucleic acid andlor protein sequence
information becomes available for cellulose synthases of bac-
teria and higher organisms, the relationships among these var-
ious cellulose synthesizers will become clearer.
Cellulose fibrils of Acetobacter xylinum are the most highly
organized of those produced by all currently identified bacteria.
The fibrils are synthesized at or near the cytoplasmic membrane
probably from UDP-glucose via cellulose synthase that requires
a cyclic diguanylic acid activator. The molecules of glucose
that comprise the fibrils are polymerized as they pass through
the various membranes of the Gram-negative cell, possibly
with final extrusion through pores in the outer membrane. The
fibrils have the capacity to form into a highly ordered and
complex structure that under scanning electron microscopy re-
sembles tunnels. The function of this complex extracellular
matrix may be as simple as to hold the bacteria in the aerobic
environment or it may serve a diversity of functions such as
protection from ultraviolet light, retention of moisture, and
colonization of substrates.
This past decade has seen an explosion of information about
the biology of bacterially synthesized cellulose, but there is
still much that we do not know. In the future, we should know
more about evolutionary relationships between prokaryotic and
eukaryotic cellulose biogenesis. Further ecological analyses are
needed to understand the role(s) of cellulose for the bacteria
in their natural habitats. A better understanding of the actual
synthesis process as it occurs in the cell membrane may even-
tually permit the large scale in vitro synthesis of cellulose and
provide potentially an important alternative source of this nat-
ural polymer. Detailed genetic analyses will increase our
knowledge of the basic mechanisms of cellulose synthesis and
may eventually lead to many commercial applications of bac-
terial cellulose.
ACKNOWLEGMENTS
The authors wish to acknowledge the editorial assistance of
Barbara Randolph-Anderson and Janne Cannon and the sec-
445
 Critical Reviews In
retarial help of Pat McCarron. They also appreciate the will-
ingness of authors to provide permission for use of some of
the figures. Some of the authors research was supported by
the Research Council of the University of North Carolina at
Greensboro.
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