Bacterial conjugation mediated by plasmid RP4: RSF1010 mobilization, donor-specific phage propagation, and pilus production require the same Tra2 core components of a proposed DNA transport complex.

DNA transfer by bacterial conjugation requires a mating pair formation (Mpf) system that specifies functions for establishing the physical contact between the donor and the recipient cell and for DNA transport across membranes. Plasmid RP4 (IncP alpha) contains two transfer regions designated Tra1 and Tra2, both of which contribute to Mpf. Twelve components are essential for Mpf, TraF of Tra1 and 11 Tra2 proteins, TrbB, -C, -D, -E, -F, -G, -H, -I, -J, -K, and -L. The phenotype of defined mutants in each of the Tra2 genes was determined. Each of the genes, except trbK, was found to be essential for RP4-specific plasmid transfer and for mobilization of the IncQ plasmid RSF1010. The latter process did not absolutely require trbF, but a severe reduction of the mobilization frequency occurred in its absence. Transfer proficiency of the mutants was restored by complementation with defined Tra2 segments containing single trb genes. Donor-specific phage propagation showed that traF and each of the genes encoded by Tra2 are involved. Phage PRD1, however, still adsorbed to the trbK mutant strain but not to any of the other mutant strains, suggesting the existence of a plasmid-encoded receptor complex. Strains containing the Tra2 plasmid in concert with traF were found to overexpress trb products as well as extracellular filaments visualized by electron microscopy. Each trb gene and traF are needed for the formation of the pilus-like structures. The trbK gene, which is required for PRD1 propagation and for pilus production but not for DNA transfer on solid media, encodes the RP4 entry-exclusion function. The components of the RP4 Mpf system are discussed in the context of related macromolecule export systems.     

TraG from RP4 and TraG and VirD4 from Ti plasmids confer relaxosome specificity to the conjugal transfer system of pTiC58

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG(RP4)) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG(RP4). A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG(RP4) was expressed in the donors. Mutations in traG(RP4) with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG(RP4) nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG(RP4) mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG(RP4)-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG(RP4) and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.     

Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer.

Transfer genes of the IncP plasmid RP4 are grouped in two separate regions, designated Tra1 and Tra2. Tra2 gene products are proposed to be mainly responsible for the formation of mating pairs in conjugating cells. To provide information relevant to understanding the function of Tra2 gene products, the nucleotide sequence of the entire RP4 Tra2 region is presented here. Twelve open reading frames were identified in the Tra2 core region, being essential for intraspecific Escherichia coli matings. Predicted sizes of 11 of the 12 Tra2 polypeptides could be verified by expression in E. coli. Based on hydropathy plot analysis, most of the Tra2 open reading frames encode proteins that may interact with membranes. Interestingly, six of the predicted Tra2 gene products exhibited significant sequence similarities to gene products encoded by the VirB operon of the Agrobacterium Ti plasmid. VirB proteins are thought to function in the formation of a transmembrane structure that mediates the passage of T-DNA molecules from bacteria into plant cells. Because of this analogy and the hydropathy of Tra2 gene products, we assume that the DNA transfer machineries acting in bacterial conjugation and T-DNA transfer are structurally and functionally similar. Therefore, the data presented here, support the hypothesis that Ti vir and IncP tra genes evolved from a common ancestor. This suggestion is favored by previous findings of sequence similarities between the IncP and Ti DNA transfer system.     

Conjugative junctions in RP4-mediated mating of Escherichia coli

The physical association of bacteria during conjugation mediated by the IncPalpha plasmid RP4 was investigated. Escherichia coli mating aggregates prepared on semisolid medium were ultrarapidly frozen using copper block freezing, followed by freeze substitution, thin sectioning, and transmission electron microscopy. In matings where the donor bacteria contained conjugative plasmids, distinctive junctions were observed between the outer membranes of the aggregates of mating cells. An electron-dense layer linked the stiffly parallel outer membranes in the junction zone, but there were no cytoplasmic bridges nor apparent breaks in the cell walls or membranes. In control experiments where the donors lacked conjugative plasmids, junctions were not observed. Previous studies have shown that plasmid RP4 carries operons for both plasmid DNA processing (Tra1) and mating pair formation (Tra2). In matings where donor strains carried Tra2 only or Tra2 plus the pilin-processing protease TraF, junctions were found but they were shorter and more interrupted than the wild type. If the donor strain had the pilin gene knocked out (trbC), junctions were still found. Thus, it appears that the electron-dense layer between the outer membranes of the conjugating cells is not composed of pilin.     

Isolation of fla-lacZ fusions in Escherichia coli K-12: most fusions result in soluble beta-galactosidase

A series of fusions of flagellar genes to the lacZ gene was generated by insertion of Mu dII301 (Apr lac) bacteriophage into the genome of Escherichia coli. The beta-galactosidase activity in each resulting mutant was measured, and the location of the activity in the membrane, periplasmic, or cytoplasmic fraction of the cell was determined. There were three classes of mutants: those which had beta-galactosidase activity mainly in the membrane fraction, those which had it distributed in the soluble and membrane fractions, and those which had it in the cytoplasmic fraction only. The last, soluble-fraction-only, class was predominant in fla-lac gene fusions. In particular, the following mutants were shown to have beta-galactosidase activity in the membrane fractions: on the inner membrane, mutants with flaB fusions, and on the inner and outer membranes, mutants with flaA4850, flaM, and flaU4849 fusions. These results suggest that fla-lacZ gene fusions produce proteins which are able to detect the signals of the leader sequence and the membrane-anchoring region of the flagellar system.     

Cytoplasmic membrane vesicles of Escherichia coli. A simple method for preparing the cytoplasmic and outer membranes.

A simple preparative method is described for isolation of the cytoplasmic and outer membranes from E. coli. The characteristics of both membrane fractions were studied chemically, biologically, and morphologically. Spheroplasts of E. coli K-12 strain W3092, prepared by treating cells with EDTA-lysozyme [EC 3.2.1.17], were disrupted in a French press. The crude membrane fraction was washed with 3 mM EDTA-10% (w/v) sucrose, pH 7.2, and the cytoplasmic membranes and outer membranes were separated by sucrose isopycnic density gradient centrifugation. The crude membrane fraction contained approximately 10% of the protein of the whole cells, 0.3% of the DNA, 0.7% of the RNA, 0.3% of the peptidoglycan, and about 30% of the lipopolysaccharide. The cytoplasmic membrane fraction was rich in phospholipid, while the outer membrane fraction contained much lipopolysaccharide and carbohydrate; the relative contents of lipopolysaccharide and carbohydrate per mg protein in the cytoplasmic membrane fraction were 12 and 40%, respectively, of the contents in the outer membrane fraction. Cytochrome b1, NADH oxidase, D-lactate dehydrogenase [EC 1.1.1.28], succinate dehydrogenase [EC 1.3.99.1], ATPase [EC 3.5.1.3], and activity for concentrative uptake of proline were found to be localized mainly in the cytoplasmic membranes; their specific activities in the outer membrane fraction were 1.5 to 3% of those in the cytoplasmic membrane fraction. In contrast, a phospholipase A appeared to be localized mainly in the outer membranes and its specific activity in the cytoplasmic membrane fraction was only 5% of that in the outer membrane fraction. The cytoplasmic and outer membrane fractions both appeared homogeneous in size and shape and show vesicular structures by electron microscopy. The advantages of this method for large scale preparation of the cytoplasmic and outer membrane fractions are discussed.     

Assembly of a functional phage PRD1 receptor depends on 11 genes of the IncP plasmid mating pair formation complex.

PRD1, a lipid-containing double-stranded DNA bacteriophage, uses the mating pair formation (Mpf) complex encoded by conjugative IncP plasmids as a receptor. Functions responsible for conjugative transfer of IncP plasmids are encoded by two distinct regions, Tra1 and Tra2. Ten Tra2 region gene products (TrbB to TrbL) and one from the Tra1 region (TraF) form the Mpf complex. We carried out a mutational analysis of the PRD1 receptor complex proteins by isolating spontaneous PRD1-resistant mutants. The mutations were distributed among the trb genes in the Tra2 region and accumulated predominantly in three genes, trbC, trbE, and trbL. Three of 307 phage-resistant mutants were weakly transfer proficient. Mutations causing a phage adsorption-deficient, transfer-positive phenotype were analyzed by sequencing.     

Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation.

In order to characterize the protein composition of the outer membrane of Borrelia burgdorferi, we have isolated inner and outer membranes by using discontinuous sucrose density step gradients. Outer and inner membrane fractions isolated by this method contained less than 1 and 2%, respectively, of the total lactate dehydrogenase activity (soluble marker) in cell lysate. More importantly, the purified outer membranes contained less than 4% contamination by the C subunit of F1/F0 ATPase (inner membrane marker). Very little flagellin protein was present in the outer membrane sample. This indicated that the outer membranes were relatively free of contamination by cytoplasmic, inner membrane or flagellar components. The outer membrane fractions (rho = 1.19 g/cm3) contained 0.15 mg (dry weight) of protein per mg. Inner membrane samples (rho = 1.12 g/cm3) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer membrane vesicles contained about 1,700 intramembranous particles per micron 2 while inner membrane densities for inner and outer membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel electrophoresis-SDS-PAGE analyses of inner and outer membrane samples revealed several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly shows that the inner and outer membranes isolated by this technique are unique structures.     

Interaction of Bacteroides fragilis pLV22a relaxase and transfer DNA with Escherichia coli RP4-TraG coupling protein

Many Bacteroides transfer factors are mobilizable in Escherichia coli when coresident with the IncP conjugative plasmid RP4, but not F. To begin characterization and potential interaction between Bacteroides mobilizable transfer factors and the RP4 mating channel, both mutants and deletions of the DNA processing (dtr), mating pair formation (mpf) and traG coupling genes of RP4 were tested for mobilization of Bacteroides plasmid pLV22a. All 10 mpf but none of the four dtr genes were required for mobilization of pLV22a. The RP4 TraG coupling protein (CP) was also required for mobilization of pLV22a, but could be substituted by a C-terminal deletion mutant of the F TraD CP. Potential interactions of the TraG CP with relaxase protein(s) and transfer DNA of both RP4 and pLV22a were assessed. Overlay assays identified productive interactions between TraG and the relaxase proteins of both MbpB and TraI from pLV22a and RP4 respectively. The Agrobacterium Transfer-ImmunoPrecipitation (TrIP) assay also identified an interaction between TraG and both RP4 and pLV22a transfer DNA. Thus, mobilization of the Bacteroides pLV22a in E. coli utilizes both RP4 Mpf and CP functions including an interaction between the relaxosome and the RP4 CP similar to that of cognate RP4 plasmid.     

TrbK, a small cytoplasmic membrane lipoprotein, functions in entry exclusion of the IncP alpha plasmid RP4.

TrbK is the only plasmid-encoded gene product involved in entry exclusion of the broad-host-range plasmid RP4. The corresponding gene, trbK, coding for a protein of 69 amino acid residues maps in the Tra2 region within the mating pair formation genes. TrbK carries a lipid moiety at the N-terminal cysteine of the mature 47-residue polypeptide. The mutant protein TrbKC23G cannot be modified or proteolytically processed but still acts in entry exclusion with reduced efficiency. An 8-amino-acid truncation at the C terminus of TrbK results in a complete loss of the entry exclusion activity but still allows the protein to be processed. TrbK localizes predominately to the cytoplasmic membrane. Its function depends on presence in the recipient cell but not in the donor cell. TrbK excludes plasmids of homologous systems of the P complex; it is inert towards the IncI system. The likely target for TrbK action is the mating pair formation system, because DNA or any of the components of the relaxosome were excluded as possible targets.     

Interaction between the IncHI1 plasmid R27 coupling protein and type IV secretion system: TraG associates with the coiled-coil mating pair formation protein TrhB

Assemblies of plasmid-encoded proteins direct the conjugative transfer of plasmid DNA molecules between bacteria. These include the membrane-associated mating pair formation (Mpf) complex necessary for pilus production and the cytoplasmic relaxosome required for DNA processing. The proposed link between these distinct protein complexes is the coupling protein (the TraG family of proteins). Interactions between the coupling protein and relaxosome components have been previously characterized and we document here, for the first time, a direct interaction between the coupling protein and an Mpf protein. Using the adenylate cyclase bacterial two-hybrid (BTH) system, we present in vivo evidence that the IncHI1 plasmid R27-encoded proteins TraG and TrhB interact. This interaction was verified through a co-immunoprecipitation reaction. We have also been able to delineate the interaction domain of TrhB to TraG by showing a positive interaction using the first 220 amino acids of TrhB (452 aa). TrhB has a proline-rich domain from amino acids 135-173 which may serve to facilitate protein interactions and/or periplasmic extension. TrhB self association was detected using far-Western, co-immunoprecipitation, and also BTH analysis, which was used to define the homotypic interaction domain, comprising a predicted coiled-coil region at residues 77-124 of TrhB. These data support a model in which the coupling protein interacts with an Mpf component to target the transferring DNA strand held by the relaxosome to the transmembrane Mpf complex.     

Functional and mutational analysis of conjugative transfer region 2 (Tra2) from the IncHI1 plasmid R27

The transfer 2 region (Tra2) of the conjugative plasmid drR27 (derepressed R27) was analyzed by PSI-BLAST, insertional mutagenesis, genetic complementation, and an H-pilus assay. Tra2 contains 11 mating-pair formation (Mpf) genes that are essential for conjugative transfer, 9 of which are essential for H-pilus production (trhA, -L, -E, -K, -B, -V, -C, -P, and -W). TrhK has similarity to secretin proteins, suggesting a mechanism by which DNA could traverse the outer membrane of donors. The remaining two Mpf genes, trhU and trhN, play an auxiliary role in H-pilus synthesis and are proposed to be involved in DNA transfer and mating-pair stabilization, respectively. Conjugative transfer abilities were restored for each mutant when complemented with the corresponding transfer gene. In addition to the essential Mpf genes, three genes, trhO, trhZ, and htdA, modulate R27 transfer frequency. Disruption of trhO and trhZ severely reduced the transfer frequencies of drR27, whereas disruption of htdA greatly increased the transfer frequency of wild-type R27 to drR27 levels. A comparison of the essential transfer genes encoded by the Tra2 and Tra1 (T. D. Lawley, M. W. Gilmour, J. E. Gunton, L. J. Standeven, and D. E. Taylor, J. Bacteriol. 184:2173-2183, 2002) of R27 to other transfer systems illustrates that the R27 conjugative transfer system is a chimera composed of IncF-like and IncP-like transfer systems. Furthermore, the Mpf/type IV secretion systems encoded by IncH and IncF transfer systems are distinct from that of the IncP transfer system. The phenotypic and ecological significance of these observations is discussed.     

Characterization of membrane-bound spermidine dehydrogenase of Citrobacter freundii.

Spermidine dehydrogenase found in the membrane fraction of Citrobacter freundii IFO 12681 was solubilized with Triton X-100 and further purified to homogeneity. The properties of the membrane enzyme were almost identical to those obtained from the soluble fraction of the organism with respect to molecular and catalytic properties. Thus, binding properties of the enzyme to the bacterial membrane were checked. The ratio of enzyme activity found in the soluble fraction to the membrane fraction was dependent on salt concentration during cell disruption. A hydrophobic interaction was largely involved in anchoring the enzyme to the membrane fraction. Purified spermidine dehydrogenase from the soluble fraction was readily adsorbed into the membrane fraction in the presence of salt. Spermidine dehydrogenase appeared to be a membrane-bound enzyme localized in the cytoplasmic membranes in a manner that makes a partial release of the enzyme possible during mechanical cell disruption. When spermidine oxidation was done with the resting cells of C. freundii, a stoichiometric formation of two reaction products, 1,3-diaminopropane and gamma-aminobutyraldeyde, was observed without any lag time. These facts indicate that the enzyme is localized on the outer surface of the cytoplasmic membranes or in the periplasmic space of the organism.     

Comparison of methods used to separate the inner and outer membranes of cell envelopes of Campylobacter spp

The outer membrane of Campylobacter coli, C. jejuni and C. fetus cell envelopes appeared as three fractions after sucrose gradient centrifugation. Each outer membrane fraction was contaminated with succinate dehydrogenase activity from the cytoplasmic membrane fraction. Similarly the inner membrane fraction was contaminated with 2-ketodeoxyoctonate and outer membrane proteins including the porin(s). The separation of these two membranes was not facilitated by variations in lysozyme treatment, cell age, presence or absence of flagella, or longer lipopolysaccharide chain length. Sodium lauroyl sarcosinate extraction resulted in an outer membrane fraction which contained some inner membrane contamination and produced multiple bands upon sucrose gradient centrifugation. Triton X-100 extraction removed the inner membrane from the outer membrane and Triton X-100/EDTA treatment extracted lipopolysaccharide-rich regions of the outer membrane which contained almost exclusively the Campylobacter porin(s). These data indicated that the inner and outer membranes of the Campylobacter cell envelope were very difficult to separate, possibly because of extensive fusions between these two membranes.     

Localization of proteins controlling motility and chemotaxis in Escherichia coli.

Flagellar proteins controlling motility and chemotaxis in Escherichia coli were selectively labeled in vivo with [35S]methionine. This distribution of these proteins in subcellular fractions was examined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography. The motA, motB, cheM, and cheD gene products were found to be confined exclusively to the inner cytoplasmic membrane fraction, whereas the cheY, cheW, and cheA (66,000 daltons) polypeptides appeared only in the soluble cytoplasmic fraction. The cheB, cheX, cheZ, and cheA (76,000 daltons) proteins, however, were distributed in both the cytoplasm and the inner membrane fractions. The hag gene product (flagellin) was the only flagellar protein examined that copurified with the outer lipopolysaccharide membrane. Differences in the intracellular locations of the che and mot gene prodcuts presumably reflect the functional attributes of these components.     

TraG-like proteins of DNA transfer systems and of the Helicobacter pylori type IV secretion system: inner membrane gate for exported substrates?

TraG-like proteins are potential NTP hydrolases (NTPases) that are essential for DNA transfer in bacterial conjugation. They are thought to mediate interactions between the DNA-processing (Dtr) and the mating pair formation (Mpf) systems. TraG-like proteins also function as essential components of type IV secretion systems of several bacterial pathogens such as Helicobacter pylori. Here we present the biochemical characterization of three members of the family of TraG-like proteins, TraG (RP4), TraD (F), and HP0524 (H. pylori). These proteins were found to have a pronounced tendency to form oligomers and were shown to bind DNA without sequence specificity. Standard NTPase assays indicated that these TraG-like proteins do not possess postulated NTP-hydrolyzing activity. Surface plasmon resonance was used to demonstrate an interaction between TraG and relaxase TraI of RP4. Topology analysis of TraG revealed that TraG is a transmembrane protein with cytosolic N and C termini and a short periplasmic domain close to the N terminus. We predict that multimeric inner membrane protein TraG forms a pore. A model suggesting that the relaxosome binds to the TraG pore via TraG-DNA and TraG-TraI interactions is presented.     

Subcellular localization of seven VirB proteins of Agrobacterium tumefaciens: implications for the formation of a T-DNA transport structure.

Plant cell transformation by Agrobacterium tumefaciens involves the transfer of a single-stranded DNA-protein complex (T-complex) from the bacterium to the plant cell. One of the least understood and important aspects of this process is how the T-complex exits the bacterium. The eleven virB gene products have been proposed to specify the DNA export channel on the basis of their predicted hydrophobicity. To determine the cellular localization of the VirB proteins, two different cell fractionation methods were employed to separate inner and outer membranes. Seven VirB-specific antibodies were used on Western blots (immunoblots) to detect the proteins in the inner and outer membranes and soluble (containing cytoplasm and periplasm) fractions. VirB5 was in both the inner membrane and cytoplasm. Six of the VirB proteins were detected in the membrane fractions only. Three of these, VirB8, VirB9, and VirB10, were present in both inner and outer membrane fractions regardless of the fractionation method used. Three additional VirB proteins, VirB1, VirB4, and VirB11, were found mainly in the inner membrane fraction by one method and were found in both inner and outer membrane fractions by a second method. These results confirm the membrane localization of seven VirB proteins and strengthen the hypothesis that VirB proteins are involved in the formation of a T-DNA export channel or gate. That most of the VirB proteins analyzed are found in both inner and outer membrane fractions suggest that they form a complex pore structure that spans both membranes, and their relative amounts in the two membrane fractions reflect their differential sensitivity to the experimental conditions.     

IncP Plasmids Are Unusually Effective in Mediating Conjugation of Escherichia coli and Saccharomyces cerevisiae: Involvement of the Tra2 Mating System

Mobilizable shuttle plasmids containing the origin-of-transfer (oriT) region of plasmids F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPα) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Saccharomyces cerevisiae. Only the Pα system caused detectable mobilization to yeast, giving peak values of 5 × 10⁻⁵ transconjugants per recipient cell in 30 min. Transfer of the shuttle plasmid required carriage of oriT in cis and the provision in trans of the Pα Tra1 core and Tra2 core regions. Genes outside the Tra1 core did not increase the mobilization efficiency. All 10 Tra2 core genes (trbB, -C, -D, -E, -F, -G, -H, -I, -J, and -L) required for plasmid transfer to E. coli K-12 were needed for transfer to yeast. To assess whether the mating-pair formation (Mpf) system or DNA-processing apparatus of the Pα conjugation system is critical in transkingdom transfer, an assay using an IncQ-based shuttle plasmid specifying its own DNA-processing system was devised. RP1 but not ColIb mobilized the construct to yeast, indicating that the Mpf complex determined by the Tra2 core genes plus traF is primarily responsible for the remarkable fertility of the Pα system in mediating gene transfer from bacteria to eukaryotes.     

A specific protease encoded by the conjugative DNA transfer systems of IncP and Ti plasmids is essential for pilus synthesis.

TraF, an essential component of the conjugative transfer apparatus of the broad-host-range plasmid RP4 (IncP), which is located at the periplasmic side of the cytoplasmic membrane, encodes a specific protease. The traF gene products of IncP and Ti plasmids show extensive similarities to prokaryotic and eukaryotic signal peptidases. Mutational analysis of RP4 TraF revealed that the mechanism of the proteolytic cleavage reaction resembles that of signal and LexA-like peptidases. Among the RP4 transfer functions, the product of the Tra2 gene, trbC, was identified as a target for the TraF protease activity. TrbC is homologous to VirB2 of Ti plasmids and thought to encode the RP4 prepilin. The maturation of TrbC involves three processing reactions: (i) the removal of the N-terminal signal peptide by Escherichia coli signal peptidase I (Lep), (ii) a proteolytic cleavage at the C terminus by an as yet unidentified host cell enzyme, and (iii) C-terminal processing by TraF. The third reaction of the maturation process is critical for conjugative transfer, pilus synthesis, and the propagation of the donor-specific bacteriophage PRD1. Thus, cleavage of TrbC by TraF appears to be one of the initial steps in a cascade of processes involved in export of the RP4 pilus subunit and pilus assembly mediated by the RP4 mating pair formation function.