Vol. 162, No. 2

DNA Transfer Occurs During a Cell Surface Contact Stage of F SexFactor-Mediated Bacterial ConjugationMITRADAS M. PANICKER AND EDWIN G. MINKLEY, JR.*

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 thenonpermissive 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 thepermissive temperature for JCFL39, transfer of the F plasmid was observed. The mating pairs that wereaccumulated with JCFL39 at the nonpermissive temperature were readily observed by electron microscopy inwall-to-wall contact with the recipient bacteria. These results demonstrate that the traD product, which isknown to be required in transferring DNA to a recipient bacterium, acts after the stage at which extracellularF pili are required. In addition, we concluded that DNA transfer takes place while donor and recipient cells arein surface contact and not necessarily through an extended F pilus as envisioned in some models of bacterialconjugation.

Conjugation is the process whereby DNA is transferredfrom a donor to a recipient bacterium by a mechanism thatinvolves contact between the cells. Most conjugation studieshave been performed with gram-negative bacteria and inparticular have centered on Escherichia coli and its sexfactor, F. A central feature of F-mediated conjugation is thefunction of the F pilus, a hairlike extracellular filament thatis produced in one or few copies by an F plasmid-containingdonor bacterium (9). Although there is strong evidence thatthe F pilus is essential for the formation of the initial contactbetween a donor and a recipient bacterium (4, 7), there isstill a degree of uncertainity as to the role that this organelleplays in conjugative DNA transfer. The earliest observations(5, 16) indicated the possibility of direct cell surface contactbetween 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 rolesfor the sex pilus. Brinton's studies on F pili led him topropose a class of models in which the F pilus is directlyinvolved in conjugative DNA transfer (6). However, nodirect evidence that demonstrates an association between Fpili and DNA that is being transferred conjugatively hasbeen reported in the literature. As an alternative, Curtiss(10) and Marvin and Hohn (18) have suggested that F pilimight function by retracting and thereby drawing the donorand recipient cell surfaces together, at which point DNAtransfer would occur. This idea is central to the currentlyfavored model for conjugative transfer by F-like plasmids.The central features of this model have most recently beenreviewed by Willetts and Skurray (26) and are presented inFig. 1.The model envisions conjugation as proceeding through a

series of ordered stages of cell surface and DNA metabolismevents. Much of the evidence for this model is based uponthe phenotypes of F plasmid mutants that are deficient intransfer (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 andthe 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 ompAprotein are also unable to form stable mating pairs (21).Mutants in traM,D,I,Z, (and probably tra 1) are piliated andare able to form stable mating pairs (26); the gene productshave been implicated in donor conjugative DNA synthesisand 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 theplacement of the DNA transfer step at a time when the donorand recipient cell surfaces are in contact, and at which pointextended F pili are presumably no longer required. Perhapsthe best attempt to demonstrate this point involved treatingmating mixtures with low concentrations of the detergentsodium dodecyl sulfate (SDS), which depolymerizes F pilusfilaments (24). Achtman et al. (3) found that SDS treatmentdid not cause disaggregation of preformed mating pairs thatincluded an Hfr donor, and that the number of recombinantscontinued to increase during further incubation in the pres-ence of SDS. This result is clearly consistent with the notionthat extended F pili are not essential for DNA transfer, oncecell surface contact is established. However, stable matingpairs were not isolated as an intermediate, nor was subse-quent DNA transfer demonstrated. The use of recombinantformation as the assay for DNA transfer further introduceda 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 theone devised by Jarvik and Botstein (13). Their test employsa pair of conditional mutants, one temperature sensitive andthe other cold sensitive; by appropriate temperature shiftexperiments it is possible to determine unambiguously therelative times at which the corresponding gene functions are

required. To apply this approach to F-mediated bacterialconjugation, we have employed SDS and an F lac traD(Ts)mutant as the requisite pair of conditional blocks. As in theexperiments of Achtman et al. (3), SDS was used to elimi-nate extended F pilus filaments. Analysis of the phenotypesof the various tra mutants that affect conjugative DNAmetabolism suggests that the traD product plays a direct rolein 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. 584-5900021-9193/85/050584-07$02.00/0Copyright X 1985, American Society for Microbiology

CELL SURFACE CONTACT DURING BACTERIAL CONJUGATION

Disaggregatio Recipientand Donor SD

tra expression sensitivivt Pilus binding

DNA troD function Pilustransfer Nal sensitivity retraction

Stable mating pair Unstable mating pair

Stabilization

FIG. 1. Model for conjugative transfer by F-like plasmids. Thefigure is modified from Willetts and Skurray (26) and highlights theinvolvement of F pili in the cell surface contact portion of the matingcycle and the requirement of traD function for DNA transfer. F piliare extremely sensitive to low concentrations of SDS (24), andextended F pili filaments quickly dissappear from the cell surface ofbacteria treated with the detergent, thereby effectively destroyingdonor activity (3). The details of conjugative DNA metabolism havebeen considerably simplified in the figure, and interested readersshould consult the review by Willetts and Wilkins (27). For simplic-ity, the donor bacterium is indicated as rod-shaped, whereas therecipient is spherical. The F sex factor is indicated as a circle in thedonor bacterium, and chromosomal DNA is not shown for eitherbacterium.

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 surfacesare in actual contact.

MATERIALS AND METHODSPlasmids and strains. The plasmids used were JCFLO (F

lac tra+), JCFL8 [F lac traD8(Am)], and JCFL39 [F lactraD39(Ts)] from the collection of Achtman et al. (4). Thesewere used in a JC3272 background (F- lac gal his trp lys Arstr) 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°Cas standing overnight cultures in 10 ml of LB broth in 125-mlErlenmeyer flasks. Samples (0.1 ml) of the donor cultures(JC6140 or JC3273) were subcultured into 2.4 ml of LB brothand allowed to stand for 2 h at either temperature. Then0.4-ml samples of these donor cultures were gently mixedwith 0.4 ml of the standing overnight cultures of XK1502(grown at 32 or 42°C), and the mating mixture was allowed tostand for 2 hours at 32 or 42°C. At the start of mating,samples were diluted and plated onto lactose-MacConkeyagar plates containing 100 ,ug of streptomycin sulfate per mlfor donor cell counts. At the end of mating, samples of themating mixture were diluted and plated onto lactose-minimalagar plates containing 20 ,ug of nalidixic acid per ml todetermine the number of transconjugants.

Kinetics of inactivation and reactivation of traD39. Tofollow the time course of inactivation of the traD39 productin terms of ability to transfer, a standing overnight culture of

JC6140 donor bacteria was grown at 32°C, diluted 25-foldinto 10 ml of LB broth in a 125-ml Erlenmeyer flask, andthen shaken gently at 32°C in a water bath. When the opticaldensity at 550 nm (OD550) of the culture reached 0.4, a 0.4-mlsample was mixed with 0.4 ml of a standing overnight cultureof XK1502 recipients that had been grown at 32°C, and thebacteria were allowed to mate for 30 min at 32°C with gentleagitation. Meanwhile, the remainder of the donor culturewas shifted to 42°C, and a second 0.4-mi sample wasimmediately added to 0.4 ml of an XK1502 culture that hadbeen grown as a standing culture overnight at 42°C; thesewere allowed to mate for 30 min at 42°C. At various intervalsafter the temperature shift, samples of JC6140 were taken,and the mating procedure was repeated. Whenever theOD550 of the donor culture reached 0.8, it was dilutedtwofold with prewarmed LB broth to maintain exponentialgrowth. Matings were stopped after 30 min by vortexing andchilling on ice. The mixtures were then diluted and platedonto lactose-minimal agar plates containing nalidixic acid toassay for transconjugants and onto lactose-MacConkey agarplates 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 thenshifted to 32°C.Temperature shift experiments. JC6140 (donor) and

XK1502 (recipient) bacteria were grown as 2.5-ml standingovernight cultures at 42°C in 18- by 150-mm culture tubes. A0.1-ml sample of the JC6140 culture was subcultured into 2.5ml of LB broth in a similar culture tube and incubated for 1h at 42°C. A 0.25-ml sample of this donor culture was thenadded to 0.25 ml of the standing overnight culture ofXK1502, gently mixed, and incubated for 30 min at 42°Cwithout shaking. At this time, the cultures were diluted10-fold into LB broth containing SDS at 0.01%, and nalidixicacid at 20 ,ug/ml was added to some of the mating mixtures.The cultures were then incubated for an additional 2 h ateither 42 or 32°C. The numbers of transconjugants wereobtained by plating various dilutions onto lactose-minimalagar plates containing nalidixic acid at 20 ,ug/ml.

Electron microscopy. JC6140 and XK1502 strains weregrown as 2.5-ml standing overnight cultures in LB broth in18- by 150 mm tubes at 42°C. Each of these was diluted25-fold into LB broth and grown for 1 h at 42°C. Samples ofthe donor, recipient, or an equal mixture of donor andrecipient were incubated for 20 min at 42°C, after which 9volumes of LB broth containing 0.01% SDS and 1% glutar-aldehyde (freshly prepared) was added to each tube withgentle swirling. The cultures were incubated at 42°C for anadditional 20 min and centrifuged for 10 min at 5,100 x g,and the cell pellets were suspended in 0.5 ml of saline. Cellswere prepared for electron microscopy by spotting a sampleonto a 300-mesh, 0.2% Formvar carbon-coated grid. Thegrids were stained with 1% aqueous uranyl acetate andexamined in a Phillips 300 electron microscope at 60 kV.Two grids per sample were used, and greater than 200 cellswere examined to determine the degree of aggregation.

Western blot analysis. Anti-traD protein (TraDp) immuneserum was raised in New Zealand White female rabbits byusing purified TraDp (manuscript in preparation). Individualstrains for the immunoblot were grown as standing overnightcultures in LB broth and then diluted 50-fold into LB brothto obtain exponentially growing cells. Samples of 3 ml weretaken when the OD550 reached 0.6 to 0.8. Cells were thencollected 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

586 PANICKER AND MINKLEY

9.5% SDS-polyacrylamide slab gel and electrophoresed asdescribed previously (19). Western blot analysis was per-formed by a modification of the procedure of Burnette (8).The proteins were electrophoretically transferred onto an11.5- by 14.5-cm sheet of nitrocellulose at 50 mA overnightin a Hoeffer Transphor apparatus with a transfer buffer of 25mM Tris-hydrochloride (pH 8.4)-192 mM glycine-20% meth-anol. The nitrocellulose sheet was washed with distilledwater and then incubated for 1 h with 50 ml of 3% bovineserum albumin in PBSa (10 g of NaCl, 0.25 g of KCI, 2.71 gof Na2HPO4. 7H20, and 0.25 g of KH2PO4 per liter ofdistilled water with a final pH of 7.6), followed by another1-h incubation in 1% bovine serum albumin-1% normal goatserum 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 gellane was allowed to incubate for 2 h. This was followed byfour washes of 15 min each with blotting buffer. Anti-rabbitFc goat antibody conjugated to horseradish peroxidase was

used at a dilution of 1:200 in blotting buffer, and 2 ml per gellane was incubated with the paper for 2 h. The nitrocellulosesheet was then washed three times with blotting buffer for 15min 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 indistilled water and allowed to dry at room temperature.

Materials. Blotting buffer was 150 mM NaCl-5 mMEDTA-50 mM Tris-hydrochloride (pH 7.4)-0.25% gel-atin-0.05% Tween 20. Peroxidase buffer was prepared bymixing 43.2 ml of distilled water, 10.8 ml of PBSa, and 0.2 mlof 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 horseradishperoxidase-conjugated anti-rabbit Fc goat IgG were fromCappel Laboratories, Cochranville, Pa. Glutaraldehyde was

from Ladd Research Industries, Burlington, Vt. SDS was

from Pierce Chemical Co., Rockford, Ill. 4-Chloro-1-naptholwas from Sigma Chemical Co., St. Louis, Mo.

RESULTS

Further characterization of JCFL39. The plasmid JCFL39was isolated by Achtman et al. (4) as a nonsuppressibletransfer-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 hada transfer efficiency of 10-2 at 42°C and 100 at 32°C, relativeto JCFLO as 100 (4). As a first step in using their mutant, wedetermined the transfer efficiency of JCFL39 under theconditions that would be used in the temperature shiftexperiments. The data in Table 1 show that in transfer froma JC3272 host into an XK1502 recipient, JCFL39 was sixfoldless efficient than JCFLO at 32°C and 7.8 x 104-fold lessefficient than JCFL0 at 42°C. Thus there is a 3.2 x 104-folddifference 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 to32°C. To increase the sensitivity of this kinetics experiment,a shortened mating time of 30 min was used. The matingefficiency of JCFL39 increased exponentially upon shiftingto the permissive temperature, rising approximately 100-fold

TABLE 1. Mating efficiencies of F lac( plasmids at 32 and 42°C

No. of No. ofDonor Temp tcC)donors Nscon- Mating

plasmid introducedTjugants efficiency'introduced obtained"JCFLO 32 1.5 x 107 1.8 x 107 120JCFLO 42 3.5 x 107 1.6 x 107 46JCFL39 32 2.5 x 107 4.8 x 106 19JCFL39 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 valuefor cells grown continuously at 32°C. The rate of initialincrease corresponded to a doubling every 35 min, comparedwith the doubling time of 50 to 60 min for growth of the cells.Based on this result, we chose a 2-h mating period for thetemperature 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 wasshifted from 32 to 42°C. The kinetics differed substantiallyfrom those of activation (Fig. 2B). The initial rise in matingefficiency was reproducible and could be due to conjugativetransfer being inherently more efficient at 42°C. This wasfollowed by a rapid exponential decline in activity during thefirst hour (t412 of 8 to 10 min) and perhaps a slower declinethereafter. The value reached after 2 to 4 h was in agreementwith the mating efficiency for a JCFL39 donor grown con-tinuously at 42°C. Thus 95 to 99% of the traD39 productappears to be rapidly inactivated as a single species upontemperature shift; there may be a residual active fractionthat is diluted out more slowly.

Stage-specific involvement of traD in bacterial conjugation.We were now in a position to ask whether the stage inconjugation at which extended F pili function could bephysically separated from the stage at which conjugativeDNA transfer occurs, by using SDS addition and the tem-perature-sensitive traD39 allele as the requisite pair ofconditional blocks. To do this, JCFL39-containing donorsand an Lac-, nalidixic acid-resistant recipient, both growncontinuously at 42°C, were mixed and incubated for 30 minat 42°C. SDS was then added at 0.01% to remove extracel-lular F pili (3) and prevent the further formation of stablemating pairs. One portion of the culture was shifted to 32°C,and another was maintained at 42°C. After 2 h, thesecultures were assayed for Lac' Nalr transconjugants. ThetraD product can indeed be reactivated and can functionafter the addition of SDS; there was a greater than 100-foldincrease in transconjugants at the permissive temperaturerelative to continued incubation at the nonpermissive tem-perature (Table 2). In a control experiment, SDS waspresent throughout the initial 30-min period (Table 2). Thisdemonstrated the importance of the preincubation step andconfirmed the SDS sensitivity of a stage in conjugation atwhich extracellular F pili act in the formation of a stablemating pair. Nalidixic acid inhibits DNA gyrase (22) and isknown to stop DNA transfer immediately when added to amating mixture (12), but will not affect the recipient that isnalidixic acid resistant. Consistent with the role of traD inDNA transfer, when nalidixic acid was added at the time ofthe temperature shift, an increase in transconjugants was notobserved in the presence of SDS at either 42 or 32°C (Table

J. BACTERIOL.

CELL SURFACE CONTACT DURING BACTERIAL CONJUGATION

(A)

0

0

0 50 100 150 200 250

10-1

0

c0

00

0

CL

0

C)c

ot

c

CG0

F04-

6z

(B)0

100 150 200 250minutes 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 betweenJC6140 and XK1502 grown continuously and mated at 32°C.

2). This indicates that DNA transfer had not yet taken placeduring the preincubation at 42°C and must have occurred at32°C when traD activity was recovered.The transfer efficiency of JCFL39 in this experiment

(Table 2) was about 4. Although this value cannot be directlyrelated to either Table 1 or Fig. 2A, it does indicate that ahigh percentage of JCFL39 donors that have potentiallyregained traD activity at 32°C are, in fact, able to transferDNA to a recipient bacterium. This suggested efficientformation of stable mating pairs during the 42"C preincuba-tion period and the resistance of these mating pairs todissociation by SDS (3). Since SDS might be expected todecrease the level of nonspecific aggregation of donor andrecipient cells (3), we decided to look in the electronmicroscope at the mating bacteria that were accumulated bythe procedure in Table 2.

Electron microscopy of mating pairs. JCFL39-containingdonor bacteria were allowed to form mating aggregates with

TABLE 2. Transfer of JCFL39 in preformed stable mating pairsat 32 and 42°C

Temp of Temp of No. ofinitial 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 10542 SDS' 32 4.0 x 10242 SDS, nalidixic acid 42 9.0 X 10242 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. After30 min of incubation at 42°C, SDS and glutaraldehyde wereadded, and the culture was examined in the electron micro-scope. Cells prepared in this manner were rarely seen incontact in the individual donor and recipient cultures,whereas in the mixed mating population virtually all of thebacteria were found to be in some sort of aggregate with thecells in surface contact (Fig. 3). In this experiment, it wasnot possible to specifically identify the donor and recipientbacteria in the mating aggregates, since they are morpholog-ically similar. Thus the correlation of aggregated cells tomating bacteria is based upon the observed statistical distri-bution. The mating aggregates observed in this manner weresimilar 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 toprovide additional detail about the precise nature of theregion in wall-to-wall contact. That analysis will requirecarefully prepared thin sections of mating pairs, such as

these, to be viewed in the electron microscope. Such ananalysis 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. Thetemperature-sensitive phenotype of the traD39 mutationcould result from either temperature-sensitive synthesis or

temperature-sensitive function of the traD product. Therapid 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 traD39activation parallelled cell growth (Fig. 2A) and suggests thatthe functional traD39 product may have to be newly synthe-sized. The level of traD product in F+ cells is very low, andthe protein has previously been observed only by using a

high-copy-number plasmid or A transducing phage carrying

0

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0Ccnc0

4-

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587VOL. 162, 1985

588 PANICKER AND MINKLEY

the gene in conjunction with a specific labeling protocol (14,20). Since we now have available rabbit antiserum againstpurified traD protein, we were able to determine directly thelevel of TraDp in the JCFL39 donor at both the permissiveand nonpermissive temperatures, using Western blotting as ahighly sensitive assay. The results with JCFL39 containingcells grown exponentially at 32°C and 42°C are shown in Fig.4, along with F-, F lac tra+, and F lac traD8(Am) controlstains. The results indicated that the levels of TraDp in bothtraD+ and traD39 cells were essentially identical at bothtemperatures. In fact, there is an increase in the level ofTraDp in both types of cells grown at 42°C. This indicatesthat the defect associated with the traD39 allele results froma loss of function at the nonpermissive temperature, and nottemperature-sensitive synthesis of the protein.

DISCUSSIONThese 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 waspossible to determine that the SDS-sensitive step in conjuga-tin (the function of extended F pili) precedes the step atwhich traD function is required. Further, we found that thetiming of traD function is coincident with sensitivity tonalidixic acid, providing additional evidence for the directrole of the traD product in DNA transfer. Combining theseresults with electron microscopy of mating pairs, we con-

75]

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7

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Donors alone

Recipient alone

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FIG. 3. Histograms of the state of aggregation of JC6140 donorbacteria, XK1502 recipient bacteria, or a mixture of the two, afterincubation for 30 mim at 420C. Cells were prepared and observed inthe electron microscope as described in the text. At least 200 cellswere examined to obtain each histogram. The vertical bars repre-

sent the number of cells in each type of aggregate as a percentage ofthe 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 andhorseradish peroxidase-conjugated sesond antibody were used tovisualize traD protein in an SDS-polyacrylamide slab gel of wholecell protein (see the text). Lanes: a and b, JC3273 (F lac tra+) at 32and 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 3x 108cells.

clude, as also proposed by others, that the role of F pili inconjugation is the generation of stable mating pairs that arein cell surface contact, and that once these are formed thereno longer exists a requirement for an extended F-pilusfilament. DNA transfer then occurs from donor to recipientbacterium while the cell surfaces are in contact. Although itis obvious that F pili play the essential role of first establish-ing the cell surface contact between the mating bacteria, theextended F pilus cannot be considered necessary in thesubsequent DNA transfer events.Comparison of these results to earlier experiments high-

lights the advantages inherent in the Jarvik and Botsteinapproach to ordering events in a biological pathway (13). Toobtain a high percentage of mating aggregates (70% of allcells), Achtman et al. (3) used a 20-min incubation periodbefore adding SDS. However, to obtain reasonably highlevels of recombinant formation, they employed an HfrCdonor and assayed for the lac genes, which are transferred at7 min (23). The use of a late marker could have providedmore convincing evidence, but the Hfr's gradient of trans-mission would have reduced the number of recombinantssignificantly. Even then, the basis of their experiment iskinetics, and thus there was not a conclusive argumefnt for adiscrete stage in conjugation where an extended F pilus isnot required. We place greater confidence in the use of thetraD(Ts) mutant, where an intermediate is accumulatedwhich can be isolated and characterized and then shown toproceed through the subsequent stages of conjugative trans-fer.

Previous characterization of traD mutants placed the timeof traD action after the formation of a stable mating pair.However, demonstration that a traD mutant can form astable mating pair does not rule out the possibility that traDfunction is actually required at an earlier stage in conjugationand that the observed stable mating pairs actually representan aborted or dead-end stage in conjugative transfer. In fact,it has been reported that the F lac mutant JCFL60, whichcarries a traD missense mutation, makes 2.5 times as manyF pili as does the parent wild-type F lac strain (2). Also, cellscarrying an F lac traD mutant are known to be resistant toinfection by the f2 class of bacteriophage. The phage parti-cles adsorb to the sides of the F pilus, but no RNApenetrates the cell (4). These additional traD phenotypessuggest the validity of questioning whether the timing oftraD function may well be at an earlier stage in conjugation,

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CELL SURFACE CONTACT DURING BACTERIAL CONJUGATION

such as when an extended F pilus is present. However, asfar as conjugation is concerned, our experiment explicitlyrules 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 temperatureresponse of the traD39(Ts) protein is potentially interesting.Western blot analysis showed that the protein is not subjectto temperature-sensitive synthesis. In temperature shift ex-

periments, the traD39 mutant protein showed rapid inacti-vation at the nonpermissive temperature, but extremely slowrecovery of activity when shifted to the permissive temper-ature. The recovery time of traD39 protein (more than 4 h torecover maximal activity) is especially remarkable whencompared with the 30-min interval during which an entirepopulation of newly mated recipients becomes competent asdonor bacteria (unpublished observations). One possibilityfor this discrepancy is that the traD39 protein synthesized at42°C is irreversibly inactivated and that a tight regulationover the level of tra protein expression results in a very lowrate of synthesis of active traD39 product in an existingdonor bacterium shifted to 32°C. A second possibility is thatinactive traD39 protein is incorporated into a cell structure(for example, the basal body of the F pilus), and it takesseveral generations of growth at 32°C to dilute out theinactive traD molecules already present at these sites. Athird possibility is that even at the permissive temperaturemost of the traD39 product synthesized is inactive; it maythen take many generations to accumulate a level of theactive form of the protein sufficient for conjugative transferto occur. This latter suggestion is consistent with the ratherpoor transfer efficiency of JCFL39-containing donors at thepermissive temperature.

Experiments currently in progress in this laboratory sug-

gest that a function of the traD protein in conjugation may beto serve as a membrane anchor for DNA helicase I, theproduct of the F traI gene (1). In this way, the energy of ATPhydrolysis used to unwind the F-plasmid circle and totranslocate the unwinding enzyme with respect to the DNAbeing unwound would be directly converted into the motiveforce for transport of a strand of the plasmid's DNA into therecipient bacterium. The fact that we have shown that traDfunction is required when the mating bacteria are in cellsurface contact is consistent with the requirements of thismodel.Although these experiments help to clarify certain aspects

of the stages involved in bacterial conjugation, there stillremain a number of steps (outlined in Fig. 1) for which thereis less than definitive evidence. First, there is as yet no

convincing evidence that F pili function via retraction tobring the mating cells into cell surface contact. Second, littleis known about the nature of the conversion of an unstablemating pair into a stable mating pair. And third, as pointedlynoted in a recent review, much remains to be learned aboutthe biochemistry of the tra products involved in conjugativeDNA metabolism (27).

Finally, these experiments make possible ah interestingcomparison of F-mediated conjugation to the conjugativetransfer of plasmids that occurs in gram-positive bacteria,such as Streptococcus faecalis, where sex pili have not beenfound. In S.faecalis, there is evidence that a sex pheromoneinduces cell clumping and that plasmid DNA is transferredwhile bacteria are in cell surface contact (11). Since we no

longer believe that the F pilus plays a direct role in DNAtransfer, a comparison can be made between the function ofthe sex pilus found in gram-negative organisms and the cell

clumping induced by the action of the sex pheromone ingram-positive bacteria. It is possible that the subsequentDNA transfer events that occur may be more directly relatedto each other.

ACKNOWLEDGMENTS

We thank Bonnie Chojnacki for her assistance with the electronmicroscopy experiments and John Bodner for introducing us to theWestern blotting procedure.

This research was supported by Public Health Service grantGM28925 from the National Institute of General Medical Sciences.

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