articulo 3. conjugación articulo

1. Vol. 162, No. 2
DNA Transfer Occurs During a Cell Surface Contact Stage of F Sex
Factor-Mediated Bacterial Conjugation
MITRADAS M. PANICKER AND EDWIN G. MINKLEY, JR.*
Department ofBiological 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.
Conjugation is the process whereby DNA is transferred
from a donor to a recipient bacterium by a mechanism that
involves contact between the cells. Most conjugation studies
have been performed with gram-negative bacteria and in
particular have centered on Escherichia coli and its sex
factor, F. A central feature of F-mediated conjugation is the
function of the F pilus, a hairlike extracellular filament that
is produced in one or few copies by an F plasmid-containing
donor bacterium (9). Although there is strong evidence that
the F pilus is essential for the formation of the initial contact
between a donor and a recipient bacterium (4, 7), there is
still a degree of uncertainity as to the role that this organelle
plays in conjugative DNA transfer. The earliest observations
(5, 16) indicated the possibility of direct cell surface contact
between conjugating bacteria, but since these studies pre-
dated the discovery of F pili, no critical experiments were
performed at that time to distinguish between possible roles
for the sex pilus. Brinton's studies on F pili led him to
propose a class of models in which the F pilus is directly
involved in conjugative DNA transfer (6). However, no
direct evidence that demonstrates an association between F
pili and DNA that is being transferred conjugatively has
been reported in the literature. As an alternative, Curtiss
(10) and Marvin and Hohn (18) have suggested that F pili
might function by retracting and thereby drawing the donor
and recipient cell surfaces together, at which point DNA
transfer would occur. This idea is central to the currently
favored model for conjugative transfer by F-like plasmids.
The central features of this model have most recently been
reviewed by Willetts and Skurray (26) and are presented in
Fig. 1.
The model envisions conjugation as proceeding through a
series of ordered stages of cell surface and DNA metabolism
events. Much of the evidence for this model is based upon
the phenotypes of F plasmid mutants that are deficient in
transfer (tra) (26). Mutants in traA,L,E,K,B,V,W,C,U, F,H,
or the first part of traG do not synthesize F pili and are
defective in all stages of conjugation. Mutants in traN and
the second part of traG synthesize F pili and make unstable,
but not stable (shear-resistant), cell surface contacts (17).
*
Corresponding author.
Recipient bacteria which lack the outer membrane ompA
protein are also unable to form stable mating pairs (21).
Mutants in traM,D,I,Z, (and probably tra 1) are piliated and
are able to form stable mating pairs (26); the gene products
have been implicated in donor conjugative DNA synthesis
and transfer (15).
A shortcoming of these genetic studies is that they do not
permit a definitive ordering of the inferred stages of conju-
gation. As noted above, one area of particular concern is the
placement ofthe DNA transfer step at a time when the donor
and recipient cell surfaces are in contact, and at which point
extended F pili are presumably no longer required. Perhaps
the best attempt to demonstrate this point involved treating
mating mixtures with low concentrations of the detergent
sodium dodecyl sulfate (SDS), which depolymerizes F pilus
filaments (24). Achtman et al. (3) found that SDS treatment
did not cause disaggregation of preformed mating pairs that
included an Hfr donor, and that the number of recombinants
continued to increase during further incubation in the pres-
ence of SDS. This result is clearly consistent with the notion
that extended F pili are not essential for DNA transfer, once
cell surface contact is established. However, stable mating
pairs were not isolated as an intermediate, nor was subse-
quent DNA transfer demonstrated. The use of recombinant
formation as the assay for DNA transfer further introduced
a complication not present in an F plasmid mating.
From a genetic point of view, the most powerful method
of analyzing the order of events in a biological pathway is the
one devised by Jarvik and Botstein (13). Their test employs
a pair of conditional mutants, one temperature sensitive and
the other cold sensitive; by appropriate temperature shift
experiments it is possible to determine unambiguously the
relative times at which the corresponding gene functions are
required. To apply this approach to F-mediated bacterial
conjugation, we have employed SDS and an F lac traD(Ts)
mutant as the requisite pair of conditional blocks. As in the
experiments of Achtman et al. (3), SDS was used to elimi-
nate extended F pilus filaments. Analysis of the phenotypes
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)
mutant to demonstrate that the traD product can act in
584
JOURNAL OF BACTERIOLOGY, May 1985, p. 584-590
0021-9193/85/050584-07$02.00/0
Copyright X 1985, American Society for Microbiology

2. CELL SURFACE CONTACT DURING BACTERIAL CONJUGATION
Disaggregatio Recipient
and Donor SD
tra expression sensitivivt Pilus binding
DNA troD function Pilus
transfer Nal sensitivity retraction
Stable mating pair Unstable mating pair
Stabilization
FIG. 1. Model for conjugative transfer by F-like plasmids. The
figure is modified from Willetts and Skurray (26) and highlights the
involvement of F pili in the cell surface contact portion of the mating
cycle and the requirement of traD function for DNA transfer. F pili
are extremely sensitive to low concentrations of SDS (24), and
extended F pili filaments quickly dissappear from the cell surface of
bacteria treated with the detergent, thereby effectively destroying
donor activity (3). The details of conjugative DNA metabolism have
been considerably simplified in the figure, and interested readers
should consult the review by Willetts and Wilkins (27). For simplic-
ity, the donor bacterium is indicated as rod-shaped, whereas the
recipient is spherical. The F sex factor is indicated as a circle in the
donor bacterium, and chromosomal DNA is not shown for either
bacterium.
conjugation at a stage after that requiring the function of an
extended F pilus. The result further strengthens the sugges-
tion (3, 26) that DNA transfer normally occurs during a stage
in conjugation in which the donor and recipient cell surfaces
are in actual contact.
MATERIALS AND METHODS
Plasmids and strains. The plasmids used were JCFLO (F
lac tra+), JCFL8 [F lac traD8(Am)], and JCFL39 [F lac
traD39(Ts)] from the collection of Achtman et al. (4). These
were used in a JC3272 background (F- lac gal his trp lys Ar
str) to give the donor strains JC3273, JC6129, and JC6140,
respectively (4). The recipient strain used was XK1502 (F-
AlacUJ69 nalA) (19).
Mating efficiency determinations at 32 and 42°C. JC6140,
JC3273, and XK1502 were grown in duplicate at 32 and 42°C
as standing overnight cultures in 10 ml of LB broth in 125-ml
Erlenmeyer flasks. Samples (0.1 ml) of the donor cultures
(JC6140 or JC3273) were subcultured into 2.4 ml of LB broth
and allowed to stand for 2 h at either temperature. Then
0.4-ml samples of these donor cultures were gently mixed
with 0.4 ml of the standing overnight cultures of XK1502
(grown at 32 or 42°C), and the mating mixture was allowed to
stand for 2 hours at 32 or 42°C. At the start of mating,
samples were diluted and plated onto lactose-MacConkey
agar plates containing 100 ,ug of streptomycin sulfate per ml
for donor cell counts. At the end of mating, samples of the
mating mixture were diluted and plated onto lactose-minimal
agar plates containing 20 ,ug of nalidixic acid per ml to
determine the number of transconjugants.
Kinetics of inactivation and reactivation of traD39. To
follow the time course of inactivation of the traD39 product
in terms of ability to transfer, a standing overnight culture of
JC6140 donor bacteria was grown at 32°C, diluted 25-fold
into 10 ml of LB broth in a 125-ml Erlenmeyer flask, and
then shaken gently at 32°C in a water bath. When the optical
density at 550 nm (OD550) of the culture reached 0.4, a 0.4-ml
sample was mixed with 0.4 ml of a standing overnight culture
of XK1502 recipients that had been grown at 32°C, and the
bacteria were allowed to mate for 30 min at 32°C with gentle
agitation. Meanwhile, the remainder of the donor culture
was shifted to 42°C, and a second 0.4-mi sample was
immediately added to 0.4 ml of an XK1502 culture that had
been grown as a standing culture overnight at 42°C; these
were allowed to mate for 30 min at 42°C. 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
growth. Matings were stopped after 30 min by vortexing and
chilling on ice. The mixtures were then diluted and plated
onto lactose-minimal agar plates containing nalidixic acid to
assay for transconjugants and onto lactose-MacConkey agar
plates containing streptomycin sulfate for donor counts.
Reactivation of traD39 was followed in a similar manner,
using a culture of JC6140 grown overnight at 42°C and then
shifted to 32°C.
Temperature shift experiments. JC6140 (donor) and
XK1502 (recipient) bacteria were grown as 2.5-ml standing
overnight cultures at 42°C in 18- by 150-mm culture tubes. A
0.1-ml sample of the JC6140 culture was subcultured into 2.5
ml of LB broth in a similar culture tube and incubated for 1
h at 42°C. A 0.25-ml sample of this donor culture was then
added to 0.25 ml of the standing overnight culture of
XK1502, gently mixed, and incubated for 30 min at 42°C
without shaking. At this time, the cultures were diluted
10-fold into LB broth containing SDS at 0.01%, and nalidixic
acid at 20 ,ug/ml was added to some of the mating mixtures.
The cultures were then incubated for an additional 2 h at
either 42 or 32°C. The numbers of transconjugants were
obtained by plating various dilutions onto lactose-minimal
agar plates containing nalidixic acid at 20 ,ug/ml.
Electron microscopy. JC6140 and XK1502 strains were
grown as 2.5-ml standing overnight cultures in LB broth in
18- by 150 mm tubes at 42°C. Each of these was diluted
25-fold into LB broth and grown for 1 h at 42°C. Samples of
the donor, recipient, or an equal mixture of donor and
recipient were incubated for 20 min at 42°C, after which 9
volumes of LB broth containing 0.01% SDS and 1% glutar-
aldehyde (freshly prepared) was added to each tube with
gentle swirling. The cultures were incubated at 42°C for an
additional 20 min and centrifuged for 10 min at 5,100 x g,
and the cell pellets were suspended in 0.5 ml of saline. Cells
were prepared for electron microscopy by spotting a sample
onto a 300-mesh, 0.2% Formvar carbon-coated grid. The
grids were stained with 1% aqueous uranyl acetate and
examined in a Phillips 300 electron microscope at 60 kV.
Two grids per sample were used, and greater than 200 cells
were examined to determine the degree of aggregation.
Western blot analysis. Anti-traD protein (TraDp) immune
serum was raised in New Zealand White female rabbits by
using purified TraDp (manuscript in preparation). Individual
strains for the immunoblot were grown as standing overnight
cultures in LB broth and then diluted 50-fold into LB broth
to obtain exponentially growing cells. Samples of 3 ml were
taken when the OD550 reached 0.6 to 0.8. Cells were then
collected by centrifugation and suspended in SDS-gel sam-
ple buffer and incubated in boiling water for 3 min. Equiva-
lent amounts of material were loaded onto an 11- by 14-cm
585VOL. 162, 1985

3. 586 PANICKER AND MINKLEY
9.5% SDS-polyacrylamide slab gel and electrophoresed as
described previously (19). Western blot analysis was per-
formed by a modification of the procedure of Burnette (8).
The proteins were electrophoretically transferred onto an
11.5- by 14.5-cm sheet of nitrocellulose at 50 mA overnight
in a Hoeffer Transphor apparatus with a transfer buffer of 25
mM Tris-hydrochloride (pH 8.4)-192 mM glycine-20% meth-
anol. The nitrocellulose sheet was washed with distilled
water and then incubated for 1 h with 50 ml of 3% bovine
serum albumin in PBSa (10 g of NaCl, 0.25 g of KCI, 2.71 g
of Na2HPO4. 7H20, and 0.25 g of KH2PO4 per liter of
distilled water with a final pH of 7.6), followed by another
1-h incubation in 1% bovine serum albumin-1% normal goat
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
lane was allowed to incubate for 2 h. This was followed by
four washes of 15 min each with blotting buffer. Anti-rabbit
Fc goat antibody conjugated to horseradish peroxidase was
used at a dilution of 1:200 in blotting buffer, and 2 ml per gel
lane was incubated with the paper for 2 h. The nitrocellulose
sheet was then washed three times with blotting buffer for 15
min each followed by three 5-min washes with PBSa.
Peroxidase buffer containing the chromogenic substrate 4-
chloro-1-napthol (see below) was then added onto the nitro-
cellulose. After the bands developed, the paper was rinsed in
distilled water and allowed to dry at room temperature.
Materials. Blotting buffer was 150 mM NaCl-5 mM
EDTA-50 mM Tris-hydrochloride (pH 7.4)-0.25% gel-
atin-0.05% Tween 20. Peroxidase buffer was prepared by
mixing 43.2 ml of distilled water, 10.8 ml of PBSa, and 0.2 ml
of 30% hydrogen peroxide; just before use 6 ml of a 6%
solution of 4-chloro-1-napthol in methanol (freshly prepared)
was added.
Nitrocellulose (BA83) was from Schleicher & Schuell Co.
BSA, fraction V, was from Boehringer Mannheim Biochemi-
cals, Indianapolis, Ind. Normal goal serum and horseradish
peroxidase-conjugated anti-rabbit Fc goat IgG were from
Cappel Laboratories, Cochranville, Pa. Glutaraldehyde was
from Ladd Research Industries, Burlington, Vt. SDS was
from Pierce Chemical Co., Rockford, Ill. 4-Chloro-1-napthol
was from Sigma Chemical Co., St. Louis, Mo.
RESULTS
Further characterization of JCFL39. The plasmid JCFL39
was isolated by Achtman et al. (4) as a nonsuppressible
transfer-defective derivative of a wild-type F lac (JCFL0),
and the mutation defect was mapped in the F traD gene
(traD39) (25). In their standard 40-min mating, JCFL39 had
a transfer efficiency of 10-2 at 42°C and 100 at 32°C, relative
to JCFLO as 100 (4). As a first step in using their mutant, we
determined the transfer efficiency of JCFL39 under the
conditions that would be used in the temperature shift
experiments. The data in Table 1 show that in transfer from
a JC3272 host into an XK1502 recipient, JCFL39 was sixfold
less efficient than JCFLO at 32°C and 7.8 x 104-fold less
efficient than JCFL0 at 42°C. Thus there is a 3.2 x 104-fold
difference in transfer ability by JCFL39 at 32 and 42°C.
To carry out the temperature shift experiments, it was also
necessary to determine the rate at which a JCFL39-
containing donor regains activity when shifted from 42 to
32°C. To increase the sensitivity of this kinetics experiment,
a shortened mating time of 30 min was used. The mating
efficiency of JCFL39 increased exponentially upon shifting
to the permissive temperature, rising approximately 100-fold
TABLE 1. Mating efficiencies of F lac( plasmids at 32 and 42°C
No. of No. of
Donor Temp tcC)donors Nscon- Mating
plasmid introducedTjugants efficiency'introduced obtained"
JCFLO 32 1.5 x 107 1.8 x 107 120
JCFLO 42 3.5 x 107 1.6 x 107 46
JCFL39 32 2.5 x 107 4.8 x 106 19
JCFL39 42 4.2 x 107 2.5 x 102 5.2 x 10-4
JCFLO is F lac tra'. JCFL39 is F lac traD39(Ts).
The recipient was XK1502. Lac' Nal' colonies were scored as transconju-
gants after mating for 2 h at the indicated temperature.
' Efficiency of mating was calculated as number of transconjugants ob-
tained per 100 donors.
in 4 h (Fig. 2A). At this point it had not yet reached the value
for cells grown continuously at 32°C. The rate of initial
increase corresponded to a doubling every 35 min, compared
with the doubling time of 50 to 60 min for growth ofthe cells.
Based on this result, we chose a 2-h mating period for the
temperature shift experiment described below (and the ex-
periment of Table 1).
As a corollary to this experiment, we determined the rate
of inactivation of traD39 activity when a JCFL39 donor was
shifted from 32 to 42°C. The kinetics differed substantially
from those of activation (Fig. 2B). The initial rise in mating
efficiency was reproducible and could be due to conjugative
transfer being inherently more efficient at 42°C. This was
followed by a rapid exponential decline in activity during the
first hour (t412 of 8 to 10 min) and perhaps a slower decline
thereafter. The value reached after 2 to 4 h was in agreement
with the mating efficiency for a JCFL39 donor grown con-
tinuously at 42°C. Thus 95 to 99% of the traD39 product
appears to be rapidly inactivated as a single species upon
temperature shift; there may be a residual active fraction
that is diluted out more slowly.
Stage-specific involvement of traD in bacterial conjugation.
We were now in a position to ask whether the stage in
conjugation at which extended F pili function could be
physically separated from the stage at which conjugative
DNA transfer occurs, by using SDS addition and the tem-
perature-sensitive traD39 allele as the requisite pair of
conditional blocks. To do this, JCFL39-containing donors
and an Lac-, nalidixic acid-resistant recipient, both grown
continuously at 42°C, were mixed and incubated for 30 min
at 42°C. SDS was then added at 0.01% to remove extracel-
lular F pili (3) and prevent the further formation of stable
mating pairs. One portion of the culture was shifted to 32°C,
and another was maintained at 42°C. After 2 h, these
cultures were assayed for Lac' Nalr transconjugants. The
traD product can indeed be reactivated and can function
after the addition of SDS; there was a greater than 100-fold
increase in transconjugants at the permissive temperature
relative to continued incubation at the nonpermissive tem-
perature (Table 2). In a control experiment, SDS was
present throughout the initial 30-min period (Table 2). This
demonstrated the importance of the preincubation step and
confirmed the SDS sensitivity of a stage in conjugation at
which extracellular F pili act in the formation of a stable
mating pair. Nalidixic acid inhibits DNA gyrase (22) and is
known to stop DNA transfer immediately when added to a
mating mixture (12), but will not affect the recipient that is
nalidixic acid resistant. Consistent with the role of traD in
DNA transfer, when nalidixic acid was added at the time of
the temperature shift, an increase in transconjugants was not
observed in the presence of SDS at either 42 or 32°C (Table
J. BACTERIOL.

4. CELL SURFACE CONTACT DURING BACTERIAL CONJUGATION
(A)
0
0
0 50 100 150 200 250
10-1
0
c
0
0
0
0
CL
0
C)c
ot
c
CG
0
F04-
6
z
(B)
0
100 150 200 250
minutes minutes
FIG. 2. (A) Kinetics of reactivation of traD39(Ts) activity in an exponentially growing culture of JC6140 shifted from 42 to 32°C (B)
Kinetics of inactivation of traD39(Ts) activity in an exponentially growing culture of JC6140 donors shifted from 32 to 42°C. 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 32°C.
2). This indicates that DNA transfer had not yet taken place
during the preincubation at 42°C and must have occurred at
32°C when traD activity was recovered.
The transfer efficiency of JCFL39 in this experiment
(Table 2) was about 4. Although this value cannot be directly
related to either Table 1 or Fig. 2A, it does indicate that a
high percentage of JCFL39 donors that have potentially
regained traD activity at 32°C are, in fact, able to transfer
DNA to a recipient bacterium. This suggested efficient
formation of stable mating pairs during the 42"C preincuba-
tion period and the resistance of these mating pairs to
dissociation by SDS (3). Since SDS might be expected to
decrease the level of nonspecific aggregation of donor and
recipient cells (3), we decided to look in the electron
microscope at the mating bacteria that were accumulated by
the procedure in Table 2.
Electron microscopy of mating pairs. JCFL39-containing
donor bacteria were allowed to form mating aggregates with
TABLE 2. Transfer of JCFL39 in preformed stable mating pairs
at 32 and 42°C
Temp of Temp of No. of
initial Additions at 30 min second Nonof
incubation incubation" transconjugants
(OC) (OC) per ml
42 SDS 42 1.5 x lo,
42 SDS 32 3.9 x 105
42 SDS' 32 4.0 x 102
42 SDS, nalidixic acid 42 9.0 X 102
42 SDS, nalidixic acid 32 9.0 x 102
aAll second incubations were for 2 h.
b
All matings used JC6140 donors and XK1502 recipients and were per-
formed as described in the text. The number of donors introduced was 1.0 x
107 per ml.
'SDS was added to the mating mixture during the initial incubation.
recipient bacteria at the nonpermissive temperature. After
30 min of incubation at 42°C, SDS and glutaraldehyde were
added, and the culture was examined in the electron micro-
scope. Cells prepared in this manner were rarely seen in
contact in the individual donor and recipient cultures,
whereas in the mixed mating population virtually all of the
bacteria were found to be in some sort of aggregate with the
cells in surface contact (Fig. 3). In this experiment, it was
not possible to specifically identify the donor and recipient
bacteria in the mating aggregates, since they are morpholog-
ically similar. Thus the correlation of aggregated cells to
mating bacteria is based upon the observed statistical distri-
bution. The mating aggregates observed in this manner were
similar in appearance to those reported by Achtman et al.
(3). Although negative staining of whole cells readily dem-
onstrated the existence of such pairs, it was not able to
provide additional detail about the precise nature of the
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
analysis is in progress elsewhere (M. Durrenberger, M.S.
dissertation, Basel University, 1982; E. Kellenberger, per-
sonal communication).
Analysis of traD protein levels in JCFL39 donors. The
temperature-sensitive phenotype of the traD39 mutation
could result from either temperature-sensitive synthesis or
temperature-sensitive function of the traD product. The
rapid decline in activity detected in the experiment of Fig.
2B is consistent with temperature-sensitive function. How-
ever, the rate of increase in mating efficiency during traD39
activation parallelled cell growth (Fig. 2A) and suggests that
the functional traD39 product may have to be newly synthe-
sized. The level of traD product in F+ cells is very low, and
the protein has previously been observed only by using a
high-copy-number plasmid or A transducing phage carrying
0
o
c
0
0
0
0.
-
c
CS0
CP._.
0
C
cn
c
0
4-
6
z
587VOL. 162, 1985

5. 588 PANICKER AND MINKLEY
the gene in conjunction with a specific labeling protocol (14,
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
highly sensitive assay. The results with JCFL39 containing
cells grown exponentially at 32°C and 42°C 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 42°C. This indicates
that the defect associated with the traD39 allele results from
a loss of function at the nonpermissive temperature, and not
temperature-sensitive synthesis of the protein.
DISCUSSION
These results provide direct evidence on the ordering of
two events in F sex factor-mediated bacterial conjugation.
With SDS addition and an F lac traD(Ts) mutant it was
possible to determine that the SDS-sensitive step in conjuga-
tin (the function of extended F pili) precedes the step at
which traD function is required. Further, we found that the
timing of traD function is coincident with sensitivity to
nalidixic acid, providing additional evidence for the direct
role of the traD product in DNA transfer. Combining these
results with electron microscopy of mating pairs, we con-
75]
50
25
u)
X 75
O.-
2 50
0
' 25
CLA
7
I.
1-
Donors alone
Recipient alone
v-
75- Mating mixture
Si
r) _2 3 4 5 >6
No. of Cells in Aggregate
FIG. 3. Histograms of the state of aggregation of JC6140 donor
bacteria, XK1502 recipient bacteria, or a mixture of the two, after
incubation for 30 mim at 420C. Cells were prepared and observed in
the electron microscope as described in the text. At least 200 cells
were examined to obtain each histogram. The vertical bars repre-
sent the number of cells in each type of aggregate as a percentage of
the total number of cells examined.
(J D c d e f g h
..,Tra Dp
FIG. 4. Western blot analysis of traD protein levels in exponen-
tially growing cells at 32 and 42°C. Anti-TraDp rabbit IgG and
horseradish peroxidase-conjugated sesond antibody were used to
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 42°C, respectively; c and d, JC3272 (F-) at 32 and 42°C,
respectively; e and f, JC6140 [F lac traD39(Ts)] at 32 and 42°C,
respectively; g and h, JC6190 [F lac traD8(Am)] at 32 and 42°C,
respectively. Each lane contained the protein from approximately 3
x 108cells.
clude, as also proposed by others, that the role of F pili in
conjugation is the generation of stable mating pairs that are
in cell surface contact, and that once these are formed there
no longer exists a requirement for an extended F-pilus
filament. DNA transfer then occurs from donor to recipient
bacterium while the cell surfaces are in contact. Although it
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.
Comparison of these results to earlier experiments high-
lights the advantages inherent in the Jarvik and Botstein
approach to ordering events in a biological pathway (13). To
obtain a high percentage of mating aggregates (70% of all
cells), Achtman et al. (3) used a 20-min incubation period
before adding SDS. However, to obtain reasonably high
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
more convincing evidence, but the Hfr's gradient of trans-
mission would have reduced the number of recombinants
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
not required. We place greater confidence in the use of the
traD(Ts) mutant, where an intermediate is accumulated
which can be isolated and characterized and then shown to
proceed through the subsequent stages of conjugative trans-
fer.
Previous characterization of traD mutants placed the time
of traD action after the formation of a stable mating pair.
However, demonstration that a traD mutant can form a
stable mating pair does not rule out the possibility that traD
function is actually required at an earlier stage in conjugation
and that the observed stable mating pairs actually represent
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
F pili as does the parent wild-type F lac strain (2). Also, cells
carrying an F lac traD mutant are known to be resistant to
infection by the f2 class of bacteriophage. The phage parti-
cles adsorb to the sides of the F pilus, but no RNA
penetrates the cell (4). These additional traD phenotypes
suggest the validity of questioning whether the timing of
traD function may well be at an earlier stage in conjugation,
J. BACTERIOL.
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6. CELL SURFACE CONTACT DURING BACTERIAL CONJUGATION
such as when an extended F pilus is present. However, as
far as conjugation is concerned, our experiment explicitly
rules out this possibility.
The traD product is an inner membrane protein of molec-
ular weight about 78,000 (14, 20; our unpublished observa-
tions). Since it is a membrane protein, the temperature
response of the traD39(Ts) protein is potentially interesting.
Western blot analysis showed that the protein is not subject
to temperature-sensitive synthesis. In temperature shift ex-
periments, the traD39 mutant protein showed rapid inacti-
vation at the nonpermissive temperature, but extremely slow
recovery of activity when shifted to the permissive temper-
ature. The recovery time of traD39 protein (more than 4 h to
recover maximal activity) is especially remarkable when
compared with the 30-min interval during which an entire
population of newly mated recipients becomes competent as
donor bacteria (unpublished observations). One possibility
for this discrepancy is that the traD39 protein synthesized at
42°C is irreversibly inactivated and that a tight regulation
over the level of tra protein expression results in a very low
rate of synthesis of active traD39 product in an existing
donor bacterium shifted to 32°C. A second possibility is that
inactive traD39 protein is incorporated into a cell structure
(for example, the basal body of the F pilus), and it takes
several generations of growth at 32°C to dilute out the
inactive traD molecules already present at these sites. A
third possibility is that even at the permissive temperature
most of the traD39 product synthesized is inactive; it may
then take many generations to accumulate a level of the
active form of the protein sufficient for conjugative transfer
to occur. This latter suggestion is consistent with the rather
poor transfer efficiency of JCFL39-containing donors at the
permissive temperature.
Experiments currently in progress in this laboratory sug-
gest that a function of the traD protein in conjugation may be
to serve as a membrane anchor for DNA helicase I, the
product of the F traI gene (1). In this way, the energy of ATP
hydrolysis used to unwind the F-plasmid circle and to
translocate the unwinding enzyme with respect to the DNA
being unwound would be directly converted into the motive
force for transport of a strand of the plasmid's DNA into the
recipient bacterium. The fact that we have shown that traD
function is required when the mating bacteria are in cell
surface contact is consistent with the requirements of this
model.
Although these experiments help to clarify certain aspects
of the stages involved in bacterial conjugation, there still
remain a number of steps (outlined in Fig. 1) for which there
is less than definitive evidence. First, there is as yet no
convincing evidence that F pili function via retraction to
bring the mating cells into cell surface contact. Second, little
is known about the nature of the conversion of an unstable
mating pair into a stable mating pair. And third, as pointedly
noted in a recent review, much remains to be learned about
the biochemistry of the tra products involved in conjugative
DNA metabolism (27).
Finally, these experiments make possible ah interesting
comparison of F-mediated conjugation to the conjugative
transfer of plasmids that occurs in gram-positive bacteria,
such as Streptococcus faecalis, where sex pili have not been
found. In S.faecalis, there is evidence that a sex pheromone
induces cell clumping and that plasmid DNA is transferred
while bacteria are in cell surface contact (11). Since we no
longer believe that the F pilus plays a direct role in DNA
transfer, a comparison can be made between the function of
the sex pilus found in gram-negative organisms and the cell
clumping induced by the action of the sex pheromone in
gram-positive bacteria. It is possible that the subsequent
DNA transfer events that occur may be more directly related
to each other.
ACKNOWLEDGMENTS
We thank Bonnie Chojnacki for her assistance with the electron
microscopy experiments and John Bodner for introducing us to the
Western blotting procedure.
This research was supported by Public Health Service grant
GM28925 from the National Institute of General Medical Sciences.
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