INTRODUCTION

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The F plasmid is a naturally derepressed conjugative plasmid of 100 kb that was used in the early 1970s to investigate the genetics of bacterial conjugation. With the complete sequence of the F transfer region becoming available in 1994 (28), the wealth of data on the five discernible functions in the F transfer region: DNA transport, F-pilus assembly and retraction, mating pair stabilization, surface and entry exclusion, and regulation could be reconciled with information gleaned from computer analyses and data bank searches for homologues of known function. While homologies between different transfer systems were often noted, the functions of only a few gene products were discerned by using this approach.

Studies on the F pilus revealed that it is composed of a major subunit, pilin, of 70 amino acids. Pilin is processed from a 121-amino-acid precursor, propilin, encoded by the traA gene. Propilin is inserted in the inner membrane through the action of TraQ, an F-pilin-specific chaperone in a process which requires ATP and an active proton motive force (43). Propilin is cleaved to pilin by the host leader peptidase and is acetylated at its N terminus by TraX (54). Mature pilin is stored as a large pool in the inner membrane, where it is assembled into a functional pilus filament by the assembly proteins TraL, -E, -K, -B, -V, -C, -F, -W, -U, and -H, TrbC, and the N-terminal portion of TraG. The F pilus is also capable of retraction or disassembly presumably into the host cell; however, it has been difficult to discern which proteins are responsible for this process, with only TrbI being named as a candidate (49). TraN and the C-terminal portion of TraG are important for mating pair stabilization (Mps), a process that allows F⁺ cells to mate more efficiently in liquid media and to resist disaggregation by shear forces or the addition of chaotropic agents such as sodium dodecyl sulfate (50). Two proteins are involved in preventing redundant plasmid exchange between F⁺ cells: TraT, an outer membrane protein, blocks mating pair stabilization (surface exclusion) between donor cells, while TraS, an inner membrane protein, blocks the signal for DNA transfer between donor cells (entry exclusion) (2).

While substantial progress has been made in understanding the protein-DNA interactions at the oriT (origin of transfer) region prior to transfer, as well as the regulation of this process, little is known about the functions of the proteins involved in pilus assembly, mating pair stabilization, or exclusion. In most cases, mutations in the genes for these functions resulted in a loss of pili and/or DNA transfer leading to the conclusion that the F pilus is intimately involved in DNA transfer. Computer analysis of the F transfer region sequence has revealed that orf169 encodes a transglycolase related to the lysozyme family (8); TraB is a homologue and/or analogue of VirB10, suggesting that it might be part of a periplasmic complex similar to that for the VirB proteins (13). However, little else is known about the function of the majority of mating pair formation (Mpf) gene products.

The R100 (100-kb) plasmid, also known as NR1, R222 (84), which carries multiple drug resistance, was able to repress F transfer through the process of fertility inhibition (58). The pili of the derepressed mutant, R100-1 (73), are serologically distinct from F pili (27). Cells carrying R100-1 show reduced sensitivity to F-specific phages. Unlike F, R100-1 pili do not attach the isometric RNA phages such as R17 laterally and, although the filamentous phages such as f1 or M13 bind to the pilus tip, they do not infect the cells efficiently (83). Unlike F⁺ cells, R100-1⁺ cells do not require the outer membrane protein, OmpA, or the pyrophosphorylethanolamine (PPEA) group in the inner core of the lipopolysaccharide (LPS) of the recipient cell for efficient conjugation (5). Both plasmids specify different surface (TraT) and entry (TraS) exclusion systems (83). The genes for DNA transport (TraI, -M, and -Y but not TraD) and regulation of conjugation (TraJ, -M, and -Y and the antisense RNA, FinP, but not FinO) are specific for their cognate plasmid (reviewed in reference 39).

In a study on the allelic specificity of related F-like plasmids, Willetts and Maule (83) concluded that, unlike the genes that conferred plasmid specificity, the genes for Mpf were interchangeable. However, that analysis used the complete R100-1 plasmid to complement mutations in F, where subtle differences between the two plasmids could have been overlooked. Studies on the effect of complementing a traA (pilin) mutation in F with cloned pilin genes from R100-1 and ColB2, another F-like plasmid, revealed that the sequence differences in pilin do not determine the requirement for OmpA and PPEA in the recipient cell, surface exclusion specificity or sensitivity to f1 phage infection (5). These results suggested that there was another protein either at the pilus tip or in the outer membrane of the donor cell responsible for identifying these moieties on the recipient cell surface. Consequently, we undertook to complete the sequence of the R100-1 transfer region, which included the remaining genes for Mpf. The sequences for traA (27), traL to traP (4), and orfJ to traF (39) were combined with the sequence of the 6.4-kb region (trbA to traS) reported here to generate a complete view of the transfer region of R100-1. Complementation data revealed that the 15 genes known to be involved in pilus synthesis and assembly are interchangeable between the two plasmids and that none of these proteins imparts specificity to the mating system. TraN was found to be involved in the requirement for OmpA and PPEA in the recipient cell (40). TraG is apparently involved in recognizing TraS during the process of entry exclusion, a phenomenon which provides interesting clues to the mechanism of signal transmission during the initiation of the transfer process.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The Escherichia coli strains used in this study include ED2149 [F lacU124 (nadA aroG gal att  bio)], ED24 (F Lac Spc^r), XK1200 (ED2149 gyrA), JE2571 (leu thr str fla pil), MC4100 (F araD139 (argF-lac)U169 rpsL150 relA1 flb3501 deoC1 ptsF25 rbsR), and DH5 (hsdR17 lacU169 80 lacZM15 recA1 supE44). A list of F and R100-1 plasmids and chimeric plasmids derived from F and R100-1 are given in Tables 1 and 2, respectively. Bacterial cultures were grown in Luria-Bertani (LB) medium with appropriate antibiotics at the following concentrations: ampicillin (50 µg/ml), kanamycin (30 µg/ml), streptomycin (200 µg/ml), and naladixic acid (40 µg/ml).

DNA preparation. The 100-kb R100-1 plasmid was purified by the method of Humphries et al. (34). A 2-ml overnight culture of E. coli JE2571 harboring the R100-1 plasmid was inoculated into 1 liter of LB broth containing streptomycin (20 µg/ml), and the culture was grown overnight. Cells were harvested by centrifugation at 2,000 × g for 15 min at 4°C. The cell pellet was resuspended in 20 ml of 25% sucrose-50 mM Tris-HCl (pH 8.0) and incubated on ice for 5 min. Three milliliters of lysozyme solution (20 mg of lysozyme per ml in 0.25 M EDTA [pH 8.0]) was added and mixed by inversion, and the mixture was further incubated on ice for 5 min. Then, 20 ml of 0.25 M EDTA (pH 8.0) was added, mixed by inversion, and incubated on ice for 30 min. After the addition of 30 ml of Triton lysis solution (4 ml of Triton X-100; 50 ml of 0.25 EDTA, pH 8.0; 10 ml of 1 M Tris-HCl, pH 8.0; and 2 ml of a 5-mg/ml mixture of RNase A per 200 ml of solution), the mixture was further incubated on ice for 60 min with periodic mixing by gentle inversion. The supernatant was collected after centrifugation at 12,000 × g for 2 h at 4°C. After the addition of 12 ml of 50% polyethylene glycol 8000 and 4 ml of 5 M NaCl, the DNA was precipitated at 4°C overnight. The precipitate was collected following centrifugation at 3,000 × g for 15 min at 4°C. The precipitate was dissolved in 23 ml of CsCl in TE buffer (10 mM Tris-HCl-1 mM EDTA, pH 7.5; density, 1.397 g/cm³) and was recentrifuged at 12,000 × g for 15 min at 4°C. The liquid portion was transferred to a solid-wall ultracentrifuge tube, to which 200 µl of ethidium bromide (10 mg/ml) was added. The gradient was formed in a 50.2 Ti fixed angle rotor in a Beckman ultracentrifuge at 38,000 rpm for 48 h at 18°C. The appropriate band was drawn off with a syringe and was dialyzed extensively in TES buffer (40 ml of 1 M Tris-HCl [pH 8.0]-16 ml of 0.25 M EDTA [pH 8.0]-8 ml of 0.5 M NaCl in 4 liters of water). After phenol extraction, the DNA was recovered by ethanol precipitation and stored in TE buffer at 20°C. Small preparations of DNA were prepared by the method of Birnboim and Doly (9) or by using a Qiagen kit (40).

Recombinant DNA techniques, DNA sequencing, and PCR. Techniques for cloning restriction fragments or PCR fragments were as described previously (68). Restriction endonuclease digestion, generation of blunt ends, and ligation of DNA molecules were performed as described by Ausubel et al. (7). All enzymes were supplied by Boehringer Mannheim except Vent polymerase (New England Biolabs). The method for generating 3' deletions in pKAR-5, as well as sequencing, either by the dideoxy sequencing method or automated sequencing, has been described previously (39). All sequences that diverged from the F sequence were sequenced in both directions, while sequences that were nearly completely homologous to F were sequenced more than once in the same direction. All restriction sites used in cloning were sequenced over by using other, overlapping clones or PCR products to ensure that the sequences were complete. The sequences of primers used in this project are available upon request.

Immunogold electron microscopy. The procedure for immunogold electron microscopy was as described in Rondot et al. (66). The monoclonal antibody (MAb) JEL92 (29) was used at a 1:500 dilution and gold-conjugated anti-mouse goat antibodies (EY Labs) were used at a 1:100 dilution.

Phage sensitivity, mating, and surface exclusion assays. Mating assays, as well as surface exclusion assays, have been described by Anthony et al. (5) and Klimke and Frost (40). The surface exclusion index is the ratio of transconjugants formed in the absence or the presence of an exclusion system. Flac donors were selected on MacConkey lactose medium (68) containing either ampicillin or kanamycin, while transconjugants were selected on M9 minimal medium (7) supplemented with 0.2% lactose and appropriate antibiotics.

	RESULTS

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Updating the sequences of the R100-1 and F transfer regions. Portions of the sequence of the R100 or R100-1 transfer regions have been presented previously. These include geneX (22), the oriT region (51), traM (21), traJ and traY (23, 35), traA (27), traL-traP (4), orfJ-traF (39), traT (60), traT-traI (86), and finO-orfA (52, 86). The entire sequence including trbA-traS which is determined in this study is included under the accession number AF112468. A comparison of the entire R100-1 and F transfer regions is presented in Fig. 1. Four sequencing errors were identified in the F traD sequence first presented by Jalajakumari and Manning (36) and reprinted in Frost et al. (28). These errors result in frameshift mutations over short regions of the gene. When corrected (sequence correction incorporated into U01159) the homology between TraD of R100-1 and F increases, with the two proteins being virtually identical over the N-terminal region. Two important mutations in traD were also sequenced. The traD8 mutation, which is associated with loss of DNA transfer and increased piliation (6), is due to a G-to-A transition which changes a tryptophan codon to an amber at position 103. The temperature-sensitive mutation traD39 characterized by decreased DNA transfer at the nonpermissive temperature (63) is due to a C-to-T transition which results in a proline to leucine substitution at position 396. 

View larger version (36K): [in this window] [in a new window]   FIG. 1.   Comparison of the F and R100-1 transfer operons with the chimeric plasmids used in this study depicted below. Detailed information on the clones is given in Table 2.

Differences between F and R100-1 transfer regions. A compilation of the gene products encoded by F and R100-1 plasmids, the percent similarity and identity between each protein, and a brief description of their function or important characteristics is given in Table 3. In the new sequence presented here (trbA-traS), major differences in the sequences of the inessential genes trbA and trbF were identified between F and R100-1. In addition, artA, a gene which maps between trbA and traQ in F and is oriented opposite to the direction of transcription of the transfer region, is missing in R100-1. We have continued to use the orf designations initiated by Yoshioka et al. (86), where the gene order is orfC, finO, orfB, and orfA at the distal end of the operon. orfC is designated orf286 in R6-5 and is missing in F (76). The fertility inhibition gene finO is interrupted by an IS3 element in F (12) and contains a frameshift mutation in R100-1 (87) which is responsible for the derepressed phenotype of both plasmids. The orfD gene in R100-1 is the homologue of F traX (54), whose gene product is required for N-terminal acetylation of pilin. The orfE gene lies between traT and traD in R100-1 and has no known function. orfF, orfG1, orfG2, orfH, orfI, and orfJ have been discussed in Klimke et al. (39) and are summarized in Table 3.

Characterization of F tra mutants for sensitivity to the phagemid M13K07. A transducing phage assay was originally used to demonstrate that f1 phage could infect F cells at low efficiency (67). F-specific filamentous phages do not absolutely require the pilus for penetration into the cell but instead use the pilus to access a secondary receptor, TolA, located in the periplasm, more efficiently (80). Two domains in the attachment protein, pIII, are responsible for F-pilus tip recognition and TolA binding, as suggested by this two-step process (14, 65). A temperature-sensitive mutation in traC (traC1044) does not form F-pilus filaments but is fully sensitive to transducing phage such as M13K07 at the nonpermissive temperature (70). This mutation appears to permit pilus tip formation but prevents pilus extension beyond the cell surface. Thus, only the pilus tip exposed at the cell surface is theoretically required for successful f1 phage infection. The M13K07 assay provides a simple means to assess formation of the pilus tip prior to protrusion of the pilus filament and is more sensitive than assays based on plaque formation.

Complementation of F tra mutants with R100-1 variants. In this study we concentrated on the genes for Mpf, which includes the genes for pilus synthesis, mating pair stabilization, and entry and/or surface exclusion. The plasmid specificities of genes involved in DNA metabolism and regulation of the transfer region have been discussed previously (83).

Characteristics of pili synthesized by an F traX mutant. Pili expressed by pOX38-traX482 contain unacetylated pilin subunits, but these pili are proficient in conjugation and attach both f1 and R17 phages well (54). However, overexpression of pili in the absence of traX results in a subtle change in the conformation of the pilin subunits within the filament such that R17 no longer attaches and the N-terminal domain is exposed on the sides of the pilus, as monitored by the use of JEL92 MAbs (31). Since F and R100-1 TraX proteins diverge in sequence near their N termini, their ability to acetylate F and R100-1 pilin might vary, leading to slight conformational changes of pilin within the pilus filament. Acetylation of F pilin expressed by pOX38-traX482 was restored by supplying either F or R100-1 traX in trans, suggesting that the F and R100-1 enzymes are equally active (data not shown). Electron micrographs of pili expressed by pOX38-traX482 revealed that they were more kinked than wild-type pili and contained more vesicle-like structures (Fig. 2). Like wild-type F pili, pOX38-traX482 pili did not bind the MAb JEL92 on their sides (Fig. 2), a finding which is in contrast to the data presented by Grossman et al. (31). Thus, overexpression of pili in the absence of traX alters the configuration of the pilin subunits within the pilus.

View larger version (63K): [in this window] [in a new window]   FIG. 2.   The effect of pilin acetylation on structural characteristics of pili. Immunogold electron microscopy was carried out as described in Materials and Methods. The binding of MAb JEL92 to pili elaborated by MC4100 cells harboring either pOX38-Km (A) or pOX38-traX482 (B) is shown. Arrows indicate the terminal knobs or vesicular materials coated with gold particles. Magnification is ×14,500, and the size bar represents 1 µm.

	DISCUSSION

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The analysis of the sequence of the R100-1 transfer region has revealed a number of instances where genes have been inserted or deleted when compared to F. In addition, there are instances of gene fusions (orfJ), serial repeats of genes (R100-1 orfG1 and orfG2) and duplications within genes (F traY). Other sequences showed mosaicism with clearly defined stretches of sequence being nonhomologous (traB, traP, traN, trbA, and traG). Several proteins known to have a role in conjugation (TraJ, -Y, -M, and -S) vary considerably in sequence, which defines the specificity of those proteins for their cognate plasmids. Others vary in sequence with little to no effect on the conjugative process (TraB and -P). The differences in TraN between F and R100-1 are striking and have been related to the requirement for OmpA and LPS in the recipient cell. The differences in TraG, the only other protein besides TraN involved in mating pair stabilization, are localized to the C-terminal half of the protein which corresponds to TraG* and is responsible for this step in the conjugation process.

A major goal of this study was to identify a transfer protein that could confer resistance to f1 phage without affecting functional pilus production, as seen with the R100-1 plasmid. Complementation studies revealed that neither pilin nor any other pilus synthesis genes from R100-1 gave the desired phenotype. Thus, the precise nature of the pilus tip remains unknown. Proposed models for the nature of the pilus tip include a unique configuration of pilin molecules which is not dependent on pilin sequence or a host-encoded moiety at the tip. Alternatively, more than one transfer protein may be required to generate the precise tip structure which gives each transfer system its characteristic pattern of filamentous phage sensitivity.

One interesting aspect of these results is the characterization of the F tra mutants with M13K07. Our results suggest that mutations in traB, traF, traH, traW and possibly traV, as well as traC1044 (70), allow pilus tip formation but block pilus outgrowth beyond the cell surface. Similarly, mutations in traL, traE, traK, traC, and traG106 appear to affect formation of the tip structure. None of these mutations affect the level of pilin subunits in the inner membrane pools (56). TraL is an interesting protein which has never been detected in in vivo expression experiments. An mRNA processing site lies within the coding sequence for TraL, and its initiating methionine abuts a complex secondary structure thought to stabilize the highly translated traA message (41). One function for such a rarely expressed protein could be to limit the number of sites for pilus assembly, since there are usually fewer than three pili/cell under derepressed conditions. Mutations in trbI, an inessential transfer gene, give longer-than-usual pili, suggesting a defect in pilus retraction or length determination (49). Mutations in traP yield pili which function almost normally but have a tendency to be easily dislodged from the cell during preparation of specimens for electron microscopy (reference 4 and unpublished observation).

While much remains to be discovered about the precise function of the genes involved in Mpf, the various genes are beginning to fall into functional subgroups within the five major classes of transfer genes. For the Mpf genes, there are subgroups involved in pilin maturation (TraQ and -X), pilus tip formation (TraL, -E, -K, -C, and -G) pilus outgrowth (TraB, -F, -H, and -W and possibly TraV), retraction (TrbI), pilus stabilization (TraP), and possibly DNA transfer (TraU). There are also surface exclusion (TraT) and entry exclusion (TraS) proteins that block transfer between donor cells at different stages.

It appears to be important to differentiate between the initiation of DNA synthesis and DNA transport, which are separate events controlled by different mechanisms (3, 38, 69). These reports suggested that the pilus was required for generating a signal that initiated DNA synthesis (62). Mutations in the mating pair stabilization genes (traN548 and traG101) allowed this signal to occur after loose cell-to-cell contacts had formed but blocked DNA transport to the recipient cell (38). The surface exclusion and entry exclusion proteins prevented stabilization and signalling from occurring, respectively. TraT in the recipient cell blocks mating pair stabilization but not the signal for DNA synthesis in the donor cell (2, 3); TraS in the recipient cell blocks a plasmid-specific signal for DNA synthesis and subsequent transport (3, 61). Nonpiliated traG mutants such as traG106, when complemented by R100-1 traG, are unaffected by F surface exclusion genes (most probably TraS) in the recipient cell. Thus, the inner membrane protein, TraG, and/or the periplasmic derivative TraG* (25), in the donor cell can differentiate between F and R100-1 TraS, an inner membrane protein, in the recipient cell. This suggests that TraG might be translocated into the recipient cell or that TraG and TraS communicate with each other perhaps via the pilus.

The reason for the acetylation of F pilin has been puzzling since traX mutants have no obvious phenotype (46). Our results suggest that acetylation by TraX might ensure correct assembly of the pilus. The deformities in pilus structure appear to be proportional to the level of tra gene expression in the absence of traX with the traX mutation in single-copy pOX38 leading to kinked pili and overexpression of traA-traG in pTG801 (31), giving F pili with a very different structure. Thus, TraX might act as a governor on the speed and accuracy of pilus assembly. In agreement with this, the pili expressed by pOX38-tra715, where the entire transfer region including traX is overexpressed from a T7 promoter (45), appear to be normal (unpublished observations), suggesting that the levels of pilin and TraX are balanced in this system and are capable of correctly assembling the F pilus.

The nature of the pilus tip has eluded identification for many years. One clue has been a decrease in mating pair formation by the addition of 1 mM Zn²⁺, which is thought to bind to the pilus tip (62). Cross-linking experiments with f1 or M13 pIII protein have been inconclusive (unpublished observations), and the complementation analyses presented here have not yielded any clues. It is interesting that f1 phage attach equally well to F⁺ and R100-1-containing cells but that R100-1 is resistant (1%) to these phages. Perhaps f1 attached to the F pilus tip mimics a recipient cell and triggers pilus retraction, while the R100-1 pilus remains immune. These results suggest that a signal is transmitted down the pilus, perhaps by a conformational change in the pilin subunits triggered at the tip and passing through the length of the pilus. Since pilin can assume more than one configuration in a functional pilus, as seen in the experiments of Grossman et al. (31), this remains an attractive possibility. Another question that has continually eluded researchers is the nature of the conjugative pore. While there is probably a role for the pilus in this process, an analysis of previous results plus the complementation data presented here suggests that the mating pair stabilization genes might form this elusive channel. If the pilus is involved in signalling that DNA synthesis should begin in the donor cell, the mechanism by which it transmits this signal to TraI, bound at oriT, to begin unwinding the DNA as a prelude to conjugative DNA replication and/or transfer remains the central question in this process. Our results suggest that TraG might have an important role in this signalling mechanism.