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JOURNAL OF BACTERIOLOGY, May 1985, p. 584-590 Vol. 162, No.

Copyright X 1985, American Society for Microbiology

DNA Transfer Occurs During a Cell Surface Contact Stage of F Sex

Factor-Mediated Bacterial Conjugation
Department of Biological Sciences, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213
Received 17 September 1984/Accepted 4 February 1985

Donor bacteria containing JCFL39, a temperature-sensitive traD mutant of the F sex factor, were used at the
nonpermissive temperature to accumulate stable mating pairs with recipient cells. At this stage in conjugation,
extracellular F pili were removed by treatment with 0.01% sodium dodecyl sulfate. Upon then shifting to the
permissive temperature for JCFL39, transfer of the F plasmid was observed. The mating pairs that were
accumulated with JCFL39 at the nonpermissive temperature were readily observed by electron microscopy in
wall-to-wall contact with the recipient bacteria. These results demonstrate that the traD product, which is
known to be required in transferring DNA to a recipient bacterium, acts after the stage at which extracellular
F pili are required. In addition, we concluded that DNA transfer takes place while donor and recipient cells are
in surface contact and not necessarily through an extended F pilus as envisioned in some models of bacterial

Conjugation is the process whereby DNA is transferred Recipient bacteria which lack the outer membrane ompA

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from a donor to a recipient bacterium by a mechanism that protein are also unable to form stable mating pairs (21).
involves contact between the cells. Most conjugation studies Mutants in traM,D,I,Z, (and probably tra 1) are piliated and
have been performed with gram-negative bacteria and in are able to form stable mating pairs (26); the gene products
particular have centered on Escherichia coli and its sex have been implicated in donor conjugative DNA synthesis
factor, F. A central feature of F-mediated conjugation is the and transfer (15).
function of the F pilus, a hairlike extracellular filament that A shortcoming of these genetic studies is that they do not
is produced in one or few copies by an F plasmid-containing permit a definitive ordering of the inferred stages of conju-
donor bacterium (9). Although there is strong evidence that gation. As noted above, one area of particular concern is the
the F pilus is essential for the formation of the initial contact placement of the DNA transfer step at a time when the donor
between a donor and a recipient bacterium (4, 7), there is and recipient cell surfaces are in contact, and at which point
still a degree of uncertainity as to the role that this organelle extended F pili are presumably no longer required. Perhaps
plays in conjugative DNA transfer. The earliest observations the best attempt to demonstrate this point involved treating
(5, 16) indicated the possibility of direct cell surface contact mating mixtures with low concentrations of the detergent
between conjugating bacteria, but since these studies pre- sodium dodecyl sulfate (SDS), which depolymerizes F pilus
dated the discovery of F pili, no critical experiments were filaments (24). Achtman et al. (3) found that SDS treatment
performed at that time to distinguish between possible roles did not cause disaggregation of preformed mating pairs that
for the sex pilus. Brinton's studies on F pili led him to included an Hfr donor, and that the number of recombinants
propose a class of models in which the F pilus is directly continued to increase during further incubation in the pres-
involved in conjugative DNA transfer (6). However, no ence of SDS. This result is clearly consistent with the notion
direct evidence that demonstrates an association between F that extended F pili are not essential for DNA transfer, once
pili and DNA that is being transferred conjugatively has cell surface contact is established. However, stable mating
been reported in the literature. As an alternative, Curtiss pairs were not isolated as an intermediate, nor was subse-
(10) and Marvin and Hohn (18) have suggested that F pili quent DNA transfer demonstrated. The use of recombinant
might function by retracting and thereby drawing the donor formation as the assay for DNA transfer further introduced
and recipient cell surfaces together, at which point DNA a complication not present in an F plasmid mating.
transfer would occur. This idea is central to the currently From a genetic point of view, the most powerful method
favored model for conjugative transfer by F-like plasmids. of analyzing the order of events in a biological pathway is the
The central features of this model have most recently been one devised by Jarvik and Botstein (13). Their test employs
reviewed by Willetts and Skurray (26) and are presented in a pair of conditional mutants, one temperature sensitive and
Fig. 1. the other cold sensitive; by appropriate temperature shift
The model envisions conjugation as proceeding through a experiments it is possible to determine unambiguously the
series of ordered stages of cell surface and DNA metabolism relative times at which the corresponding gene functions are
events. Much of the evidence for this model is based upon required. To apply this approach to F-mediated bacterial
the phenotypes of F plasmid mutants that are deficient in conjugation, we have employed SDS and an F lac traD(Ts)
transfer (tra) (26). Mutants in traA,L,E,K,B,V,W,C,U, F,H, mutant as the requisite pair of conditional blocks. As in the
or the first part of traG do not synthesize F pili and are
defective in all stages of conjugation. Mutants in traN and experiments of Achtman et al. (3), SDS was used to elimi-
the second part of traG synthesize F pili and make unstable, nate extended F pilus filaments. Analysis of the phenotypes
but not stable (shear-resistant), cell surface contacts (17). of the various tra mutants that affect conjugative DNA
metabolism suggests that the traD product plays a direct role
in DNA transfer (15). We have used SDS and the traD(Ts)
Corresponding author. mutant to demonstrate that the traD product can act in

JC6140 donor bacteria was grown at 32C, diluted 25-fold

into 10 ml of LB broth in a 125-ml Erlenmeyer flask, and
Disaggregatio Recipient then shaken gently at 32C in a water bath. When the optical
and Donor SD
density at 550 nm (OD550) of the culture reached 0.4, a 0.4-ml
tra expression sensitivivt Pilus binding sample was mixed with 0.4 ml of a standing overnight culture
of XK1502 recipients that had been grown at 32C, and the
bacteria were allowed to mate for 30 min at 32C with gentle
agitation. Meanwhile, the remainder of the donor culture
was shifted to 42C, and a second 0.4-mi sample was
immediately added to 0.4 ml of an XK1502 culture that had
DNA troD function Pilus been grown as a standing culture overnight at 42C; these
transfer Nal sensitivity retraction were allowed to mate for 30 min at 42C. At various intervals
after the temperature shift, samples of JC6140 were taken,
and the mating procedure was repeated. Whenever the
OD550 of the donor culture reached 0.8, it was diluted
twofold with prewarmed LB broth to maintain exponential
Stable mating pair Unstable mating pair growth. Matings were stopped after 30 min by vortexing and
Stabilization chilling on ice. The mixtures were then diluted and plated
onto lactose-minimal agar plates containing nalidixic acid to
FIG. 1. Model for conjugative transfer by F-like plasmids. The assay for transconjugants and onto lactose-MacConkey agar
figure is modified from Willetts and Skurray (26) and highlights the plates containing streptomycin sulfate for donor counts.
involvement of F pili in the cell surface contact portion of the mating Reactivation of traD39 was followed in a similar manner,
cycle and the requirement of traD function for DNA transfer. F pili
are extremely sensitive to low concentrations of SDS (24), and using a culture of JC6140 grown overnight at 42C and then
extended F pili filaments quickly dissappear from the cell surface of shifted to 32C.
bacteria treated with the detergent, thereby effectively destroying Temperature shift experiments. JC6140 (donor) and

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donor activity (3). The details of conjugative DNA metabolism have XK1502 (recipient) bacteria were grown as 2.5-ml standing
been considerably simplified in the figure, and interested readers overnight cultures at 42C in 18- by 150-mm culture tubes. A
should consult the review by Willetts and Wilkins (27). For simplic- 0.1-ml sample of the JC6140 culture was subcultured into 2.5
ity, the donor bacterium is indicated as rod-shaped, whereas the ml of LB broth in a similar culture tube and incubated for 1
recipient is spherical. The F sex factor is indicated as a circle in the h at 42C. A 0.25-ml sample of this donor culture was then
donor bacterium, and chromosomal DNA is not shown for either added to 0.25 ml of the standing overnight culture of
XK1502, gently mixed, and incubated for 30 min at 42C
without shaking. At this time, the cultures were diluted
conjugation at a stage after that requiring the function of an 10-fold into LB broth containing SDS at 0.01%, and nalidixic
extended F pilus. The result further strengthens the sugges- acid at 20 ,ug/ml was added to some of the mating mixtures.
tion (3, 26) that DNA transfer normally occurs during a stage The cultures were then incubated for an additional 2 h at
in conjugation in which the donor and recipient cell surfaces either 42 or 32C. The numbers of transconjugants were
are in actual contact. obtained by plating various dilutions onto lactose-minimal
agar plates containing nalidixic acid at 20 ,ug/ml.
MATERIALS AND METHODS Electron microscopy. JC6140 and XK1502 strains were
Plasmids and strains. The plasmids used were JCFLO (F grown as 2.5-ml standing overnight cultures in LB broth in
lac tra+), JCFL8 [F lac traD8(Am)], and JCFL39 [F lac 18- by 150 mm tubes at 42C. Each of these was diluted
traD39(Ts)] from the collection of Achtman et al. (4). These 25-fold into LB broth and grown for 1 h at 42C. Samples of
were used in a JC3272 background (F- lac gal his trp lys Ar the donor, recipient, or an equal mixture of donor and
str) to give the donor strains JC3273, JC6129, and JC6140, recipient were incubated for 20 min at 42C, after which 9
respectively (4). The recipient strain used was XK1502 (F- volumes of LB broth containing 0.01% SDS and 1% glutar-
AlacUJ69 nalA) (19). aldehyde (freshly prepared) was added to each tube with
Mating efficiency determinations at 32 and 42C. JC6140, gentle swirling. The cultures were incubated at 42C for an
JC3273, and XK1502 were grown in duplicate at 32 and 42C additional 20 min and centrifuged for 10 min at 5,100 x g,
as standing overnight cultures in 10 ml of LB broth in 125-ml and the cell pellets were suspended in 0.5 ml of saline. Cells
Erlenmeyer flasks. Samples (0.1 ml) of the donor cultures were prepared for electron microscopy by spotting a sample
(JC6140 or JC3273) were subcultured into 2.4 ml of LB broth onto a 300-mesh, 0.2% Formvar carbon-coated grid. The
and allowed to stand for 2 h at either temperature. Then grids were stained with 1% aqueous uranyl acetate and
0.4-ml samples of these donor cultures were gently mixed examined in a Phillips 300 electron microscope at 60 kV.
with 0.4 ml of the standing overnight cultures of XK1502 Two grids per sample were used, and greater than 200 cells
(grown at 32 or 42C), and the mating mixture was allowed to were examined to determine the degree of aggregation.
stand for 2 hours at 32 or 42C. At the start of mating, Western blot analysis. Anti-traD protein (TraDp) immune
samples were diluted and plated onto lactose-MacConkey serum was raised in New Zealand White female rabbits by
agar plates containing 100 ,ug of streptomycin sulfate per ml using purified TraDp (manuscript in preparation). Individual
for donor cell counts. At the end of mating, samples of the strains for the immunoblot were grown as standing overnight
mating mixture were diluted and plated onto lactose-minimal cultures in LB broth and then diluted 50-fold into LB broth
agar plates containing 20 ,ug of nalidixic acid per ml to to obtain exponentially growing cells. Samples of 3 ml were
determine the number of transconjugants. taken when the OD550 reached 0.6 to 0.8. Cells were then
Kinetics of inactivation and reactivation of traD39. To collected by centrifugation and suspended in SDS-gel sam-
follow the time course of inactivation of the traD39 product ple buffer and incubated in boiling water for 3 min. Equiva-
in terms of ability to transfer, a standing overnight culture of lent amounts of material were loaded onto an 11- by 14-cm

9.5% SDS-polyacrylamide slab gel and electrophoresed as TABLE 1. Mating efficiencies of F lac( plasmids at 32 and 42C
described previously (19). Western blot analysis was per- No. of
formed by a modification of the procedure of Burnette (8). No. of
Nscon- Mating
The proteins were electrophoretically transferred onto an introduced
introduced obtained"
11.5- by 14.5-cm sheet of nitrocellulose at 50 mA overnight
in a Hoeffer Transphor apparatus with a transfer buffer of 25 JCFLO 32 1.5 x107 1.8 x 107 120
mM Tris-hydrochloride (pH 8.4)-192 mM glycine-20% meth- JCFLO 42 3.5 x107 1.6 x 107 46
anol. The nitrocellulose sheet was washed with distilled JCFL39 32 2.5 x107 4.8 x 106 19
water and then incubated for 1 h with 50 ml of 3% bovine JCFL39 42 4.2 107
x 2.5 x 102 5.2 x 10-4
serum albumin in PBSa (10 g of NaCl, 0.25 g of KCI, 2.71 g JCFLO is F lac tra'. JCFL39 is F lac traD39(Ts).
of Na2HPO4. 7H20, and 0.25 g of KH2PO4 per liter of The recipient was XK1502. Lac' Nal' colonies were scored as transconju-
distilled water with a final pH of 7.6), followed by another gants after mating for 2 h at the indicated temperature.
' Efficiency of mating was calculated as number of transconjugants ob-
1-h incubation in 1% bovine serum albumin-1% normal goat tained per 100 donors.
serum in PBSa. Immunoglobulin G (IgG)-enriched anti-
TraDp serum was used at a 1:50 dilution in blotting buffer
(see below), and 2 ml of serum per each cm of width of gel in 4 h (Fig. 2A). At this point it had not yet reached the value
lane was allowed to incubate for 2 h. This was followed by for cells grown continuously at 32C. The rate of initial
four washes of 15 min each with blotting buffer. Anti-rabbit increase corresponded to a doubling every 35 min, compared
Fc goat antibody conjugated to horseradish peroxidase was with the doubling time of 50 to 60 min for growth of the cells.
used at a dilution of 1:200 in blotting buffer, and 2 ml per gel Based on this result, we chose a 2-h mating period for the
lane was incubated with the paper for 2 h. The nitrocellulose temperature shift experiment described below (and the ex-
sheet was then washed three times with blotting buffer for 15 periment of Table 1).
min each followed by three 5-min washes with PBSa. As a corollary to this experiment, we determined the rate
Peroxidase buffer containing the chromogenic substrate 4- of inactivation of traD39 activity when a JCFL39 donor was
chloro-1-napthol (see below) was then added onto the nitro- shifted from 32 to 42C. The kinetics differed substantially

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cellulose. After the bands developed, the paper was rinsed in from those of activation (Fig. 2B). The initial rise in mating
distilled water and allowed to dry at room temperature. efficiency was reproducible and could be due to conjugative
Materials. Blotting buffer was 150 mM NaCl-5 mM transfer being inherently more efficient at 42C. This was
EDTA-50 mM Tris-hydrochloride (pH 7.4)-0.25% gel- followed by a rapid exponential decline in activity during the
atin-0.05% Tween 20. Peroxidase buffer was prepared by first hour (t412 of 8 to 10 min) and perhaps a slower decline
mixing 43.2 ml of distilled water, 10.8 ml of PBSa, and 0.2 ml thereafter. The value reached after 2 to 4 h was in agreement
of 30% hydrogen peroxide; just before use 6 ml of a 6% with the mating efficiency for a JCFL39 donor grown con-
solution of 4-chloro-1-napthol in methanol (freshly prepared) tinuously at 42C. Thus 95 to 99% of the traD39 product
was added. appears to be rapidly inactivated as a single species upon
Nitrocellulose (BA83) was from Schleicher & Schuell Co. temperature shift; there may be a residual active fraction
BSA, fraction V, was from Boehringer Mannheim Biochemi- that is diluted out more slowly.
cals, Indianapolis, Ind. Normal goal serum and horseradish Stage-specific involvement of traD in bacterial conjugation.
peroxidase-conjugated anti-rabbit Fc goat IgG were from We were now in a position to ask whether the stage in
Cappel Laboratories, Cochranville, Pa. Glutaraldehyde was conjugation at which extended F pili function could be
from Ladd Research Industries, Burlington, Vt. SDS was physically separated from the stage at which conjugative
from Pierce Chemical Co., Rockford, Ill. 4-Chloro-1-napthol DNA transfer occurs, by using SDS addition and the tem-
was from Sigma Chemical Co., St. Louis, Mo. perature-sensitive traD39 allele as the requisite pair of
conditional blocks. To do this, JCFL39-containing donors
RESULTS and an Lac-, nalidixic acid-resistant recipient, both grown
continuously at 42C, were mixed and incubated for 30 min
Further characterization of JCFL39. The plasmid JCFL39 at 42C. SDS was then added at 0.01% to remove extracel-
was isolated by Achtman et al. (4) as a nonsuppressible lular F pili (3) and prevent the further formation of stable
transfer-defective derivative of a wild-type F lac (JCFL0), mating pairs. One portion of the culture was shifted to 32C,
and the mutation defect was mapped in the F traD gene and another was maintained at 42C. After 2 h, these
(traD39) (25). In their standard 40-min mating, JCFL39 had cultures were assayed for Lac' Nalr transconjugants. The
a transfer efficiency of 10-2 at 42C and 100 at 32C, relative traD product can indeed be reactivated and can function
to JCFLO as 100 (4). As a first step in using their mutant, we after the addition of SDS; there was a greater than 100-fold
determined the transfer efficiency of JCFL39 under the increase in transconjugants at the permissive temperature
conditions that would be used in the temperature shift relative to continued incubation at the nonpermissive tem-
experiments. The data in Table 1 show that in transfer from perature (Table 2). In a control experiment, SDS was
a JC3272 host into an XK1502 recipient, JCFL39 was sixfold present throughout the initial 30-min period (Table 2). This
less efficient than JCFLO at 32C and 7.8 x 104-fold less demonstrated the importance of the preincubation step and
efficient than JCFL0 at 42C. Thus there is a 3.2 x 104-fold confirmed the SDS sensitivity of a stage in conjugation at
difference in transfer ability by JCFL39 at 32 and 42C. which extracellular F pili act in the formation of a stable
To carry out the temperature shift experiments, it was also mating pair. Nalidixic acid inhibits DNA gyrase (22) and is
necessary to determine the rate at which a JCFL39- known to stop DNA transfer immediately when added to a
containing donor regains activity when shifted from 42 to mating mixture (12), but will not affect the recipient that is
32C. To increase the sensitivity of this kinetics experiment, nalidixic acid resistant. Consistent with the role of traD in
a shortened mating time of 30 min was used. The mating DNA transfer, when nalidixic acid was added at the time of
efficiency of JCFL39 increased exponentially upon shifting the temperature shift, an increase in transconjugants was not
to the permissive temperature, rising approximately 100-fold observed in the presence of SDS at either 42 or 32C (Table

(A) 10-1 (B)


o c
c 0
0 0

0 0 0
0 0

0 0
0 c
C 0
0 CG
4- F0

6 6
z z

0 50 100
150 200 250 100 150 200 250
minutes minutes

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FIG. 2. (A) Kinetics of reactivation of traD39(Ts) activity in an exponentially growing culture of JC6140 shifted from 42 to 32C (B)
Kinetics of inactivation of traD39(Ts) activity in an exponentially growing culture of JC6140 donors shifted from 32 to 42C. Cultures were
grown and transconjugants were assayed as described in the text. The square in panel B is the value obtained for a 30-min mating between
JC6140 and XK1502 grown continuously and mated at 32C.

2). This indicates that DNA transfer had not yet taken place recipient bacteria at the nonpermissive temperature. After
during the preincubation at 42C and must have occurred at 30 min of incubation at 42C, SDS and glutaraldehyde were
32C when traD activity was recovered. added, and the culture was examined in the electron micro-
The transfer efficiency of JCFL39 in this experiment scope. Cells prepared in this manner were rarely seen in
(Table 2) was about 4. Although this value cannot be directly contact in the individual donor and recipient cultures,
related to either Table 1 or Fig. 2A, it does indicate that a whereas in the mixed mating population virtually all of the
high percentage of JCFL39 donors that have potentially bacteria were found to be in some sort of aggregate with the
regained traD activity at 32C are, in fact, able to transfer cells in surface contact (Fig. 3). In this experiment, it was
DNA to a recipient bacterium. This suggested efficient not possible to specifically identify the donor and recipient
formation of stable mating pairs during the 42"C preincuba- bacteria in the mating aggregates, since they are morpholog-
tion period and the resistance of these mating pairs to ically similar. Thus the correlation of aggregated cells to
dissociation by SDS (3). Since SDS might be expected to mating bacteria is based upon the observed statistical distri-
decrease the level of nonspecific aggregation of donor and bution. The mating aggregates observed in this manner were
recipient cells (3), we decided to look in the electron similar in appearance to those reported by Achtman et al.
microscope at the mating bacteria that were accumulated by (3). Although negative staining of whole cells readily dem-
the procedure in Table 2. onstrated the existence of such pairs, it was not able to
Electron microscopy of mating pairs. JCFL39-containing provide additional detail about the precise nature of the
donor bacteria were allowed to form mating aggregates with region in wall-to-wall contact. That analysis will require
carefully prepared thin sections of mating pairs, such as
these, to be viewed in the electron microscope. Such an
TABLE 2. Transfer of JCFL39 in preformed stable mating pairs analysis is in progress elsewhere (M. Durrenberger, M.S.
at 32 and 42C dissertation, Basel University, 1982; E. Kellenberger, per-
Temp of Temp of No. of sonal communication).
initial Additions at 30 min second Nonof Analysis of traD protein levels in JCFL39 donors. The
incubation incubation" transconjugants
per ml temperature-sensitive phenotype of the traD39 mutation
(OC) (OC)
could result from either temperature-sensitive synthesis or
42 SDS 42 1.5 x lo, temperature-sensitive function of the traD product. The
42 SDS 32 3.9 x 105 rapid decline in activity detected in the experiment of Fig.
42 SDS' 32 4.0 x 102 2B is consistent with temperature-sensitive function. How-
42 SDS, nalidixic acid 42 9.0 X 102 ever, the rate of increase in mating efficiency during traD39
42 SDS, nalidixic acid 32 9.0 x 102
activation parallelled cell growth (Fig. 2A) and suggests that
aAll second incubations were for 2 h. the functional traD39 product may have to be newly synthe-
All matings used JC6140 donors and XK1502 recipients and were per- sized. The level of traD product in F+ cells is very low, and
formed as described in the text. The number of donors introduced was 1.0 x
107 per ml. the protein has previously been observed only by using a
'SDS was added to the mating mixture during the initial incubation. high-copy-number plasmid or A transducing phage carrying

the gene in conjunction with a specific labeling protocol (14, (J D c d e f g h

20). Since we now have available rabbit antiserum against
purified traD protein, we were able to determine directly the
level of TraDp in the JCFL39 donor at both the permissive
and nonpermissive temperatures, using Western blotting as a ..,Tra Dp
highly sensitive assay. The results with JCFL39 containing
cells grown exponentially at 32C and 42C are shown in Fig.
4, along with F-, F lac tra+, and F lac traD8(Am) control
stains. The results indicated that the levels of TraDp in both
traD+ and traD39 cells were essentially identical at both
temperatures. In fact, there is an increase in the level of
TraDp in both types of cells grown at 42C. This indicates FIG. 4. Western blot analysis of traD protein levels in exponen-
that the defect associated with the traD39 allele results from tially growing cells at 32 and 42C. Anti-TraDp rabbit IgG and
a loss of function at the nonpermissive temperature, and not horseradish peroxidase-conjugated sesond antibody were used to
temperature-sensitive synthesis of the protein. visualize traD protein in an SDS-polyacrylamide slab gel of whole
cell protein (see the text). Lanes: a and b, JC3273 (F lac tra+) at 32
and 42C, respectively; c and d, JC3272 (F-) at 32 and 42C,
DISCUSSION respectively; e and f, JC6140 [F lac traD39(Ts)] at 32 and 42C,
These results provide direct evidence on the ordering of respectively; g and h, JC6190 [F lac traD8(Am)] at 32 and 42C,
respectively. Each lane contained the protein from approximately 3
two events in F sex factor-mediated bacterial conjugation. x 108cells.
With SDS addition and an F lac traD(Ts) mutant it was
possible to determine that the SDS-sensitive step in conjuga- clude, as also proposed by others, that the role of F pili in
tin (the function of extended F pili) precedes the step at conjugation is the generation of stable mating pairs that are
which traD function is required. Further, we found that the in cell surface contact, and that once these are formed there
timing of traD function is coincident with sensitivity to no longer exists a requirement for an extended F-pilus
nalidixic acid, providing additional evidence for the direct filament. DNA transfer then occurs from donor to recipient

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role of the traD product in DNA transfer. Combining these bacterium while the cell surfaces are in contact. Although it
results with electron microscopy of mating pairs, we con- is obvious that F pili play the essential role of first establish-
ing the cell surface contact between the mating bacteria, the
extended F pilus cannot be considered necessary in the
subsequent DNA transfer events.
75] Comparison of these results to earlier experiments high-
Donors alone lights the advantages inherent in the Jarvik and Botstein
50 approach to ordering events in a biological pathway (13). To
obtain a high percentage of mating aggregates (70% of all
25 cells), Achtman et al. (3) used a 20-min incubation period
before adding SDS. However, to obtain reasonably high
I. levels of recombinant formation, they employed an HfrC
donor and assayed for the lac genes, which are transferred at
7 min (23). The use of a late marker could have provided
X 75 more convincing evidence, but the Hfr's gradient of trans-
Recipient alone mission would have reduced the number of recombinants
2 50
significantly. Even then, the basis of their experiment is
kinetics, and thus there was not a conclusive argumefnt for a
discrete stage in conjugation where an extended F pilus is
' 25 not required. We place greater confidence in the use of the
traD(Ts) mutant, where an intermediate is accumulated
v- 1- which can be isolated and characterized and then shown to
proceed through the subsequent stages of conjugative trans-
Mating mixture Previous characterization of traD mutants placed the time
of traD action after the formation of a stable mating pair.
Si However, demonstration that a traD mutant can form a
stable mating pair does not rule out the possibility that traD
4 function is actually required at an earlier stage in conjugation
and that the observed stable mating pairs actually represent
r) 2_ 3 4 5 >6
an aborted or dead-end stage in conjugative transfer. In fact,
it has been reported that the F lac mutant JCFL60, which
carries a traD missense mutation, makes 2.5 times as many
No. of Cells in Aggregate
F pili as does the parent wild-type F lac strain (2). Also, cells
FIG. 3. Histograms of the state of aggregation of JC6140 donor carrying an F lac traD mutant are known to be resistant to
bacteria, XK1502 recipient bacteria, or a mixture of the two, after
incubation for 30 mim at 420C. Cells were prepared and observed in infection by the f2 class of bacteriophage. The phage parti-
the electron microscope as described in the text. At least 200 cells cles adsorb to the sides of the F pilus, but no RNA
were examined to obtain each histogram. The vertical bars repre- penetrates the cell (4). These additional traD phenotypes
sent the number of cells in each type of aggregate as a percentage of suggest the validity of questioning whether the timing of
the total number of cells examined. traD function may well be at an earlier stage in conjugation,

such as when an extended F pilus is present. However, as clumping induced by the action of the sex pheromone in
far as conjugation is concerned, our experiment explicitly gram-positive bacteria. It is possible that the subsequent
rules out this possibility. DNA transfer events that occur may be more directly related
The traD product is an inner membrane protein of molec- to each other.
ular weight about 78,000 (14, 20; our unpublished observa-
tions). Since it is a membrane protein, the temperature ACKNOWLEDGMENTS
response of the traD39(Ts) protein is potentially interesting. We thank Bonnie Chojnacki for her assistance with the electron
Western blot analysis showed that the protein is not subject microscopy experiments and John Bodner for introducing us to the
to temperature-sensitive synthesis. In temperature shift ex- Western blotting procedure.
periments, the traD39 mutant protein showed rapid inacti- This research was supported by Public Health Service grant
vation at the nonpermissive temperature, but extremely slow GM28925 from the National Institute of General Medical Sciences.
recovery of activity when shifted to the permissive temper-
ature. The recovery time of traD39 protein (more than 4 h to LITERATURE CITED
recover maximal activity) is especially remarkable when 1. Abdel-Monem, M., G. Taucher-Scholz, and M.-Q. Klinkert.
compared with the 30-min interval during which an entire 1983. Identification of Escherichia coli DNA helicase I as the
population of newly mated recipients becomes competent as tral gene product of the F sex factor. Proc. Natl. Acad. Sci.
donor bacteria (unpublished observations). One possibility
U.S.A. 80:4659-4663.
2. Achtman, M. 1973. Genetics of the F sex factor in enterobacte-
for this discrepancy is that the traD39 protein synthesized at riaceae. Curr. Top. Microbiol. Immunol. 60:79-123.
42C is irreversibly inactivated and that a tight regulation 3. Achtman, M., G. Morelli, and S. Schwuchow. 1978. Cell-cell
over the level of tra protein expression results in a very low interactions in conjugating Escherichia coli: role of F pili and
rate of synthesis of active traD39 product in an existing fate of mnating aggregates. J. Bacteriol. 135:1053-1061.
donor bacterium shifted to 32C. A second possibility is that 4. Achtman, M., N, Willetts, and A. J. Clark. 1971. Beginning a
inactive traD39 protein is incorporated into a cell structure genetic analysis of conjugational transfer determined by the F
(for example, the basal body of the F pilus), and it takes factor in Escherichia coli by isolation and characterization of
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