Transfer of tra Proteins into the Recipient Cell during Bacterial ...

JOURNAL OF BACTERIOLOGY, June 1989, p. 3152-3157 0021-9193/89/063152-06$02.00/0 Copyright C) 1989, American Society for Microbiology

Vol. 171, No. 6

Transfer of tra Proteins into the Recipient Cell during Bacterial Conjugation Mediated by Plasmid ColIb-P9 CATHERINE E. D. REES AND BRIAN M. WILKINS*

Department of Genetics, University of Leicester, Leicester LE] 7RH, England Received 28 December 1988/Accepted 13 March 1989

Selective transfer of the two products of the ColIb primase gene, sog, from donor to recipient cell during was demonstrated by two independent methods. The transfer of these tra proteins was unidirectional and dependent on DNA transfer. The Sog polypeptides were localized to the cytoplasm of the donor cell, but they appeared to interact with other tra gene products located in the inner membrane. After cell mating, the transferred polypeptides were found to be in the cytoplasm of the recipient cell, and it is estimated that as many as 500 Sog polypeptides were transferred per round of conjugation. It is proposed that these proteins are transferred as a result of an interaction with the single-stranded DNA and that the transferred strand may be coated with Sog polypeptides.

conjugation

During bacterial conjugation mediated by Incll plasmids such as ColIb-P9, a specific plasmid strand is transferred to the recipient cell, where the double-stranded DNA molecule is regenerated (31). DNA synthesis on the transferred strand is initiated by a plasmid-encoded DNA primase that is the product of a gene called sog (11). This gene is a component of the I1 conjugation (tra) system, and it specifies two large, in-phase translation products of Mr 210,000 and 160,000, which are designated here Sog-210 and Sog-160 (7, 23, 27, 32). The primase generates short RNA primers, and this activity is dependent on the N-terminal third of Sog-210 (7, 20, 32). To initiate DNA synthesis on the transferred plasmid strand, sog DNA primase is supplied by the donor cell and transmitted to the recipient bacterium. The primase is remarkable in this respect because no general transfer of proteins occurs during bacterial conjugation (26, 28, 29). Primase transfer was first inferred from a functional test which showed that the enzyme originating from the donor strain could restore bacterial DNA synthesis in mated recipients defective in Escherichia coli primase (12). Subsequently, it was demonstrated that Sog-210 and Sog-160 from labeled donor cells were present in a set of proteins selectively retained by recipients after conjugation (23). The objective of this work was to characterize further the transfer of Sog polypeptides during bacterial conjugation. Identification of transferred proteins relies on separation of donor and recipient after mating. This was achieved either by destroying the labeled donor cells by lysis-from-without with bacteriophage T6 or by using minicells as the recipient cells and separating the cells according to size. This second method exploits the finding that minicells are competent recipients in matings mediated by Incll plasmids (15). We have investigated the location of the Sog polypeptides both in the donor cell and in the recipient cell after transfer. We have also determined their direction of transfer relative to that of the DNA and the number of Sog polypeptide molecules transmitted per conjugation cycle.

genotype, lac+ tsx+ [phage T6-sensitive]) or a T6-resistant

derivative, BW96T6r. Recipients were BW40 (lacYJ tsx-33 [T6']) and the homogenic lac+ tsx+ strain BW51. Further details of the strains are given elsewhere (12). Minicells were prepared from cultures of DS410 (14). Incll plasmids were pLG221 (ColIb-P9drd-J cib::TnS [11]) and pLG273 (ColIbP9drd-J cib::TnJO [17]). Recombinant plasmids used were pLG215 (pBR325fl [sog+ EcoRI, 8.4 kilobases] Apr Tcr [32]) and pLG2020 (pEMBLcos4fQ [ColIb-P9drd-1, 36.3 to 82 kilobases] Apr [27]). Media, enzymes, and radiochemicals. Media and general culture conditions were as described previously (11, 23). SGC is an M9 salts-glucose (0.4%)-Casamino Acids (0.2%) medium. For SGA, Casamino Acids were replaced by a mixture of all the common amino acids (each at 10 ,ug ml-') except methionine. Enzymes used in protein transfer were from Sigma Chemical Co. L-[35S]methionine and 14C-methylated-protein molecular weight markers were obtained from Amersham International. Protein transfer during conjugation. Donor cells were grown in SGA. At 20 min before mating, [35S]methionine (4.75 ng ml-', 45 TBq mmol-') was added, followed after 15 min by unlabeled methionine to 100 ,ug ml-' (i) Mating with minicells. Minicells were diluted in SGC to give -5.6 x 109 cells ml-'. A 1.5-ml portion of donor cells (-2 x 108 cells ml-1) was mixed with 1.5 ml of minicells, and the cells were incubated at 37°C for 1 h. Mating cells were disrupted by blending for 15 s, and sodium dodecyl sulfate (SDS) was then added to 0.01% to prevent further formation of mating pairs. Donor cells were then removed from minicells by sedimentation through a sucrose gradient. (ii) Removal of donor cells by T6 lysis. Recipient cells were grown in SGC. Mating mixtures contained 1.5 ml of each culture at -2 x 108 cells ml-1. After 30 min of mating at 37°C, chloramphenicol was added to 25 ,ug ml-', and the mixture was treated with UV-irradiated phage T6, DNase I, RNase A, protease K, and Brij 58 as described previously (23). T6W cells were harvested and washed by three rounds of centrifugation in a Sorvall SS34 rotor at 7,000 rpm for 5 min with resuspension in ice-cold phosphate buffer containing Brij 58 (0.5%). Two optical density (600 nm) units of BW40 cells were added to aid pelleting. The T6 treatment removed -98% of the labeled trichloroacetic acid-precipitable material present in the mating mixture. Harvested cells were

MATERIALS AND METHODS Bacterial strains and plasmids. Strains were derivatives of E. coli K-12. The donor host strain was BW96 (relevant *

Corresponding author. 3152

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either fractionated or analyzed by SDS-polyacrylamide gel electrophoresis for labeled polypeptides. Fractionation of cells. After sonication of cells in the presence of the protease inhibitor phenylmethylsulfonyl fluoride (0.1 mM), membranes were separated from soluble fractions by centrifugation in a Beckman 50Ti rotor at 30,000 rpm for 45 min as described by Baker et al. (4). Inner membrane proteins were solubilized from the washed membrane fraction by treatment with 0.5% Sarkosyl for 30 min (16). Periplasmic fractions were isolated following osmotic shock or the formation of spheroplasts. For osmotic shock (24), washed cells were suspended in 30% sucrose-30 mM Tris hydrochloride (pH 7.1)-0.6 mM EDTA and then transferred into ice-cold 5 mM MgCl2. After 10 min, shocked cells were separated by centrifugation from the supernatant, which contained periplasmic proteins. Spheroplasts were prepared by suspending washed cells in ice-cold 10% sucrose in 100 mM Tris hydrochloride (pH 7.6). Lysozyme was added to 100 ,ug ml-', and EDTA was added to 5 mM. After 15 min, cells were removed, resulting in a supernatant which contained periplasmic proteins. Formation of spheroplasts was monitored by phase-contrast microscopy. Proteins in the supernatant fractions were precipitated in 5% trichloroacetic acid before analysis by SDS-polyacrylamide gel electrophoresis. Enzyme assays. P-Galactosidase activity was determined by the method of Casaregola et al. (9) by using fractions derived from BW96 grown in the presence of 1 mM isopropyl-o-D-thiogalactopyranoside. P-Lactamase activity, specified by pBR328, was measured by the method of O'Callaghan et al. (25) by using the chromogenic cephalosporin Nitrocefin (Oxoid Ltd.) at 10 ,ug ml-' in 50 mM sodium phosphate buffer (pH 7). One unit is defined as the amount of enzyme giving a change in A482 of 1 min'. SDS-polyacrylamide gel electrophoresis and Western blotting (immunoblotting). The basic procedure was that of Laemmli (19). Gels consisted of a 7% stacking gel followed by a 10% separating gel (acrylamide monomer/dimer ratio of 44:0.8). Detection of radioactive bands was enhanced by fluorography (5). Whole-cell extracts were prepared from 10 optical density at 450 nm units of exponentially growing cells. Washed cells were suspended in 150 RI of sample buffer (62.5 mM Tris hydrochloride [pH 6.8]-3% SDS-10% glycerol-5% P-mercaptoethanol), heated at 60°C for 5 min, and then boiled for 7 min. Lysate was cleared by centrifugation in a Beckman 5OTi rotor at 30,000 rpm for 30 min, and 30 ,l was loaded per track. Western blotting was as described previously (23), with rabbit antiserum raised against a truncated form of sog primase (32). Molecular weight markers used were myosin (200 kilodaltons [kDa]), phosphorylase b (92.5 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), and carbonic anhydrase (30 kDa). Estimation of relative amounts of polypeptides. The relative intensity of individual bands in Coomassie blue-stained gels was determined from a positive-negative by using an LKB Ultroscan XL laser densitometer. The amount of 35S in an individual band was determined by the method of Ames (3). The band was cut out of the gel after fluorography and was wetted and solubilized in NCS tissue solubilizer (Amersham International) in scintillation fluid overnight at 37°C. RESULTS Location of Sog polypeptides in the donor cell. In a Coomassie blue-stained profile of whole-cell proteins prepared from strains carrying the sog gene, the larger Sog

PROTEIN TRANSFER DURING CONJUGATION

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TABLE 1. Enzyme activity per fraction derived from BW96(pLG221, pBR328) U of activity per fraction Fraction Fraction

~

Treatment cells of

per (600 optical of cellsunit nm)density

1-Galactosidase

3-Lactamase

1,172

99

Total

Sonication

Periplasm

Spheroplasting Osmotic shock

15 8

80 75

Cytoplasm + membranes

Spheroplasting Osmotic shock

478" 762"

18 1

Membranes

Osmotic shock

2

NTb

" Reduced activity was caused by loss of some material during the washing and sonication of the treated cells. b NT, Not tested.

polypeptide (Sog-210) is easily visualized as a band that runs above all other cellular proteins (see Fig. 4). However, the smaller polypeptide (Sog-160) comigrates with the RNA polymerase ,B and f' subunits, and its presence in whole-cell extracts is detected only by using antiserum raised against sog primase. To determine the locations of Sog-210 and Sog-160 in the donor cell, BW96 strains were lysed by sonication, and membrane fragments were separated from the soluble (cytoplasm plus periplasm) fraction. Inner membrane proteins were distinguished from outer membrane proteins on the basis of their solubility in Sarkosyl. Periplasmic samples were obtained by osmotic shock and by the preparation of spheroplasts, and the purity of the preparations was tested by assaying P-lactamase and P-galactosidase activities as periplasmic and cytoplasmic markers, respectively (Table 1). The various fractions obtained were analyzed by SDS-polyacrylamide gel electrophoresis, and the presence of Sog polypeptides was determined by Western blot (Fig. 1). In strains containing either the cloned sog gene on pLG215 (Fig. 1, lane 3) or the ColIbdrd-J plasmid pLG221 (lane 4), bands corresponding to the two Sog polypeptides were detected in the soluble fraction of the cell. Since no Sog polypeptides were detected in the periplasmic fractions, it was concluded that the majority of these polypeptides were localized in the cytoplasm of the cell, irrespective of whether the other tra genes were expressed in the cell. This localization of the Sog-210 to the cytoplasm agrees with that determined by Coomassie blue staining of cytoplasmic fractions of strains containing pLG221 and pLG215 (data not shown). In addition to the two expected Sog proteins, a new polypeptide of 77 kDa, which strongly cross-reacted with anti-Sog primase serum, was identified in the inner membrane fraction of cells carrying a complete I1 conjugation system (Fig. 1, lane 10). Transfer of polypeptides from ColIb donor cells. Transferred polypeptides were identified by labeling donor cell proteins with [35S]methionine and selectively harvesting recipients after mating. This was achieved in two ways. First, DS410 minicells were used as recipients, and after blending of mating cells, the donor cells were removed from the mating mixture by sedimentation through a sucrose gradient. Second, donor cells were lysed with phage T6, and cell debris was removed by extensive washing. Lanes 1 and 4 in Fig. 2 show the labeled proteins remaining associated

REES AND WILKINS

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J. BACTERIOL.

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FIG. 1. Location of Sog polypeptides in the donor cell. Cytoplasmic-plus-periplasmic (C+P), inner-membrane (IM), and outermembrane (OM) fractions were prepared from sonicated cells. Periplasmic proteins (P) were released after osmotic shock. Fractions were analyzed by Western blotting. Lanes: 1, whole-cell extract of BW96(pLG221); 2 to 4, cytoplasmic-plus-periplasmic fraction from BW96, BW96(pLG215), and BW96(pLG221), respectively; 5 to 7, periplasmic proteins from BW96, BW96(pLG215), and BW96(pLG221), respectively; 8, cytoplasmic-plus-periplasmic fraction from BW96(pLG221); 9 and 10, inner-membrane fraction from BW96 and BW96(pLG221), respectively; 11 and 12, outer-membrane fraction from BW96 and BW96(pLG221), respectively. Samples loaded contained material derived from an equivalent number of cells. Bands corresponding to Sog-210 and Sog-160 proteins are indicated by arrowheads, and horizontal lines mark the positions of molecular weight standards with myosin at the top.

with recipients harvested by each method after mating with BW96(pLG273) donors. Clearly, the major proteins retained by recipient bacteria were the 210- and 160-kDa polypeptides, previously identified as the products of the sog gene 2

3

5

4

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6

7

8

9

10

11

4

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-wi

FIG. Proteins

2.

AOs

Conjugative transfer

associated

with

of

Sog proteins. Lanes

minicells

after incubation

1

with

and 2, labeled

BW96(pLG273) and BW96(pLG215), respectively. For all other were removed by lysis with phage T6. Lane 3, Proteins retained by BW40 after mixing with T6-treated labeled BW96(pLG273); lane 4, proteins retained by BW40 after mating with BW96(pLG273); lanes 5, 7, and 9, proteins retained by BW40 lanes, the labeled cells

after incubation with labeled BW96,

BW96(pLG2020),

and BW96

(pLG273), respectively; lanes 6 and 8, labeled BW96 and BW96 (pLG2020), respectively, were treated with phage T6 before addition to BW40; lanes 10 and 11, proteins retained by BW96T6r(pLG273) donors after mating with labeled BW51 and BW51(pLG215) recipients, respectively. Bands corresponding to Sog-210 and Sog-160 proteins are indicated by closed arrowheads, and horizontal lines mark the positions of molecular weight standards with myosin at the top. The nonspecifically retained 38-kDa protein is indicated by open arrowheads.

(23). As a control, minicells were mixed with labeled BW96(pLG215) cells, and in this case, neither Sog polypeptide was retained (lane 2). Similarly, no Sog polypeptides were retained when donor cells were treated with T6 before they were added to the BW40 recipients (lane 3). The fainter bands present in all cases apparently correspond to a background of proteins retained by the recipients in a conjugation-independent process. While the level of this nonspecific background varied considerably between experiments, Sog210 and Sog-160 were always prevalent when mating had occurred and were never seen in the unmated controls. Figure 2 also shows the results of mixing BW40 recipients with BW96 cells containing either no plasmid (lane 5) or pLG2020 (lane 7). Plasmid pLG2020 carries most of the ColIb tra genes and specifies both Sog polypeptides (see Fig. 4), the two types of conjugative pilus, and entry exclusion (27) but lacks the oriT region of ColIb; hence, no DNA transfer can be initiated. No Sog polypeptide transfer was detected from BW96(pLG2020) cells, indicating that transfer of Sog polypeptides is dependent on DNA transfer. A discrete set of proteins originating from the donor cells was nonspecifically retained by the recipients even when the labeled cells contained no plasmid (Fig. 2, lanes 5 and 6). Predominant among these was a polypeptide of 38 kDa which precipitated with antiserum raised against OmpF protein (data not shown). This nonspecific retention is thought to be an artifact of the phage lysis procedure, which causes the release of a set of membrane proteins from the infected cells (22). Presumably these polypeptides become associated with the T6r cells. The extra low-molecularweight bands nonspecifically retained when the donor cells carried either pLG273 or pLG2020 (lanes 3, 7, and 8) were probably due to the presence of tra proteins in the membranes of these cells. To determine whether Sog polypeptides are transmissible from recipient to donor cell, T6-resistant donors of pLG273 were recovered after mating with T6-sensitive recipients that were labeled prior to mating. When the cloned sog gene was present in these recipient cells on plasmid pLG215, no labeled Sog proteins were recovered from the donor cells (Fig. 2, lane 11). Hence, Sog polypeptides are transferred unidirectionally and in the same direction as the plasmid DNA. Again a set of proteins from the T6-lysed cells was retained, and all were found to be membrane associated (data not shown). It is noted that when the recipients contained pLG215, retention of one of the low-molecularweight proteins apparent in the control (lane 10) did not occur, presumably because of some alteration conferred on the cell envelope of cells carrying this plasmid. Location of transferred Sog polypeptides in the recipient cell. Washed BW40 recipients, recovered from the T6 lysis procedure after mating with labeled donor cells, were fractionated as described above. When these fractions were analyzed by autoradiography, the transferred Sog proteins were associated solely with the soluble fraction of the cell, whereas the nonspecifically retained material fractionated primarily with the membrane proteins (Fig. 3, lanes 1 through 3). Isolation of periplasmic material was attempted both by osmotic shock (lanes 4 through 6) and by the formation of spheroplasts, and in both cases, small amounts of both Sog proteins were detected in the released material. However, Coomassie blue staining of the periplasmic-protein profile also revealed the presence of RNA polymerase 1 and 13' subunits, indicating that some lysis of recipient cells had occurred, allowing the release of cytoplasmic components. This may reflect fragility of the cells caused by their

PROTEIN TRANSFER DURING CONJUGATION

VOL. 6, 1989

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1

:

m

5

4

3

6

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q-

1_

*_

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011

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4

FIG. 3. Locations of transferred Sog polypeptides. After mating with labeled BW96(pLG273), BW40 recipient cells were fractionated to determine the locations of the transferred polypeptides. Lanes: 1 and 4, unfractionated recipients; 2 and 3, soluble (cytoplasmic plus periplasmic) and membrane fractions, respectively; 5, periplasmic proteins released by osmotic shock; 6, proteins remaining associated with the cells after osmotic shock. Bands corresponding to Sog-210 and Sog-160 proteins are indicated by arrowheads, and horizontal lines mark the positions of molecular weight standards with myosin at the top.

extensive treatment after mating. Consequently, it was not possible to determine how much, if any, of the Sog polypeptides was located in the periplasm of the recipient cells. However, it is clear that the majority of these proteins were transferred into the cytoplasm of the cell. Western blot analysis of fractions of mated recipients was used to search for small Sog proteins, as detected in fractionated donor cells. Although no 77-kDa polypeptide was detected, small amounts of a 50-kDa protein that crossreacted with anti-Sog primase serum were found in the inner membrane fraction of recipients after mating with donor cells carrying pLG273 (data not shown). Quantification of Sog polypeptide transfer. The number of molecules of Sog-210 present in donor cells was estimated by comparison with the and (' subunits of RNA polymerase. Protein extracts of whole cells were prepared from exponentially growing BW96(pLG273) cultures; they were separated on 10% acrylamide gels and stained with Coomassie blue 2

3

4

FIG. 4. Quantification of Sog-210 protein. Coomassie bluestained profiles of whole-cell extracts prepared from BW96 cells containing no plasmid (lane 1), pLG215 (lane 2), pLG2020 (lane 3), or pLG273 (lane 4). The bands containing Sog-210 (filled arrowhead) and RNA polymerase P and (' subunits and Sog-160 (open arrowhead) are indicated. Horizontal lines indicate molecular weight standards with myosin at the top.

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(Fig. 4). As determined by a scanning densitometer, the intensity of the Sog-210 band was 18% of that of the band containing the RNA polymerase ( and (' subunits and the Sog-160 protein. By taking a value of 9.6 x 103 ( plus 1' molecules cell-1 (T = 40 min [8]) and allowing for different molecular weights, it was estimated that there were approximately 1.25 x 103 molecules of Sog-210 per donor cell. The amount of Sog-210 protein transferred during conjugation was determined from the radioactivity in the Sog-210 band in the extract of mated recipients compared with the radioactivity in the corresponding band derived from an equivalent number of unmated donor cells. Values from four independent experiments were 15, 18, 24, and 26%. Therefore, approximately 20% of Sog-210 in the donors (-250 molecules) was transferred to the recipient. The amount of radioactivity in the Sog-160 band from mated recipients was at least 1.25 times greater than that in the Sog-210 band recovered from such cells. DISCUSSION It is shown here that both Sog polypeptides are located in the cytoplasm of the donor cell, which is consistent with the ability of sog DNA primase to substitute for E. coli primase in discontinuous bacterial DNA replication (32). The cellular locations of other ColIb tra products are unknown, but by analogy with the majority of F tra products (33), they are predicted to be predominantly membrane associated. One of the few F tra gene products located in the cytoplasm of the donor cell is traI DNA helicase (1). This is another tra protein known to be involved in conjugative DNA processing (18, 34), and because tral, like sog, specifies two translational products, it has been suggested that these genes may act analogously in conjugation (30). Besides the Sog-210 and Sog-160 proteins, immunoblotting also identified a 77-kDa polypeptide in extracts of pLG221-containing cells which was predominantly associated with the inner membrane. This protein was absent when the sog gene on ColIb was interrupted by a TnS insertion (see Fig. 3 in reference 23), indicating that it is determined by sog. It may be a third translational product of sog or, more likely, a breakdown product of either Sog-210 or Sog-160. Intriguingly, while this 77-kDa polypeptide was detectable in extracts of cells carrying the cloned sog gene on pLG215, it did not associate with the inner membrane (data not shown). This suggests that a part of the Sog protein(s) can interact with other products of tra genes which are located in the inner membrane. Conjugative transfer of Sog polypeptides to recipient cells was demonstrated by using two methods of separating donor and recipient after mating. These involved using either minicell recipients, which were separated according to size, or T6-resistant recipients, which were harvested after T6 lysis of donors. In both cases, the Sog-210 and Sog-160 proteins were predominant among a set of proteins selectively retained by the recipient cells. Fractionation of the mated recipients revealed that the retained Sog polypeptides were located in the cytoplasm of the cell. This confirmed the results of functional tests, which indicated that transmitted plasmid primase can interact with the chromosomal DNA of the recipient (12). The cytoplasmic location of Sog-210 and Sog-160 and their absence from the membrane fraction indicated that they were not retained by the recipients as remnants of a static conjugation "bridge" that spans the membranes but rather were actively transferred through the bacterial membranes.

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REES AND WILKINS

Our data point to the conclusion that the transfer of Sog polypeptides is dependent on DNA transfer. First, lysisfrom-without by phage T6 is known to block conjugative DNA transfer (13), and when donor cells were treated with T6 prior to addition to the recipients, no Sog proteins were transferred to the recipients. Second, no transfer was seen from cells harboring pLG2020, a nontransferable recombinant plasmid specifying the Sog polypeptides and both types of I1 conjugative pilus required to establish cell-to-cell contact. In addition, Sog polypeptides synthesized in recipients could not be transmitted to the donor cells during mating, indicating that protein transfer is unidirectional and in the same direction as DNA transfer. It is proposed that the Sog polypeptides are transferred to the recipient as a consequence of their interaction with the single-stranded DNA, perhaps mediated by the C-terminal region common to both polypeptides. This model is based on the observation that an intact C-terminal region is required for Sog protein transfer and for efficient ColIb transfer (23). The binding of Sog polypeptides to the DNA may, besides providing a mechanism of transfer for the primase, facilitate transfer of the DNA through the bacterial membranes. By comparison with the amounts of C and 1B' subunits of RNA polymerase, it was estimated that there are approximately 1,250 Sog-210 polypeptides per donor cell. Approximately 20% of the labeled Sog-210 protein was transferred from the donor to the recipient in 30-min matings. Since in conjugation mediated by IncIl plasmids DNA transfer is limited to one or two strands per recipient cell (2, 6), as many as 250 molecules of Sog-210 may be transferred per DNA strand. Hence, the primase protein is transferred in noncatalytic amounts, and in addition, at least an equal number of Sog-160 molecules is transferred. It is known that Incll plasmid primase protein purifies as a DNA-binding protein (21). Therefore a substantial fraction of the transferred single-stranded DNA may be complexed with Sog proteins. An appealing speculation is that the Sog polypeptides coat the transferred single strand, and besides protecting the DNA and generating RNA primers, they may act analogously to single-stranded-DNA-binding proteins to stimulate the activity of the DNA polymerase (10), thereby ensuring efficient regeneration of the intact plasmid molecule. It remains to be determined whether the transfer of specific tra proteins is a common feature of all conjugation systems.

J. BACTERIOL. J. Mol. Biol. 198:693-703. 5. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. 6. Boulnois, G. J., and B. M. Wilkins. 1978. A Coll-specified product, synthesized in newly infected recipients, limits the amount of DNA transferred during conjugation of Escherichia coli K-12. J. Bacteriol. 133:1-9. 7. Boulnois, G. J., B. M. Wilkins, and E. Lanka. 1982. Overlapping genes at the DNA primase locus of the large plasmid ColI. Nucleic Acids Res. 10:855-869. 8. Bremer, H., and P. P. Dennis. 1987. Modulation of chemical composition and other parameters of the cell by growth rate, p. 1527-1542. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Esche9.

10. 11.

12. 13.

14.

15.

16.

17. 18. 19.

ACKNOWLEDGMENTS We thank Karen Baker for her technical advice and Angela Thomas for her assistance with experimental work. This work was supported by Medical Research Council grant G8602116CB.

1.

2. 3. 4.

LITERATURE CITED Achtman, M., P. A. Manning, C. Edelbluth, and P. Herrlich. 1979. Export without proteolytic processing of inner and outer membrane proteins encoded by F sex factor tra cistrons in Escherichia coli minicells. Proc. NatI. Acad. Sci. USA 76: 4837-4841. Achtman, M., G. Morelli, and S. Schwuchow. 1978. Cell-cell interactions in conjugating Escherichia coli: role of F pili and fate of mating aggregates. J. Bacteriol. 135:1053-1061. Ames, G. F.-L. 1974. Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs. J. Biol. Chem. 249: 634-644. Baker, K., N. Mackman, M. Jackson, and I. B. Holland. 1987. Role of SecA and SecY in protein export as revealed by studies of TonA assembly into the outer membrane of Escherichia coli.

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