Atpase activity and multimer formation of Pilq protein are required for thin pilus biogenesis in plasmid R64

Plasmid R64 pilQ gene is essential for the formation of thin pilus, a type IV pilus. The pilQ product contains NTP binding motifs and belongs to the PulE-VirB11 family of NTPases. The pilQ gene was overexpressed with an N-terminal His tag, and PilQ protein was purified. Purified His tag PilQ protein displayed ATPase activity with a V(max) of 0.71 nmol/min/mg of protein and a K(m) of 0.26 mm at pH 6.5. By gel filtration chromatography, PilQ protein was eluted at the position corresponding to 460 kDa, suggesting that PilQ protein forms a homooctamer. To analyze the relationship between structure and function of PilQ protein, amino acid substitutions were introduced within several conserved motifs. Among 11 missense mutants, 7 mutants exhibited various levels of reduced DNA transfer frequencies in liquid matings. Four mutant genes (T234I, K238Q, D263N, and H328A) were overexpressed with a His tag. The purified mutant PilQ proteins contained various levels of reduced ATPase activity. Three mutant PilQ proteins formed stable multimers similar to wild-type PilQ, whereas the PilQ D263N multimer was unstable. PilQ D263N monomer exhibited low ATPase activity, while PilQ D263N multimer did not. These results indicate that ATPase activity of the PilQ multimer is essential for R64 thin pilus biogenesis.     

An inner membrane platform in the type II secretion machinery of Gram-negative bacteria

The type II secretion machinery allows most Gram-negative bacteria to deliver virulence factors into their surroundings. We report that in Erwinia chrysanthemi, GspE (the putative NTPase), GspF, GspL and GspM constitute a complex in the inner membrane that is presumably used as a platform for assembling other parts of the secretion machinery. The GspE–GspF–GspL–GspM complex was demonstrated by two methods: (i) co-immunoprecipitation of GspE–GspF–GspL with antibodies raised against either GspE or GspF; (ii) interactions in the yeast two-hybrid system between GspF and GspE, GspF and GspL, GspL and GspM. GspL was found to have an essential role in complex formation. We propose a model in which the GspE–GspF–GspL–GspM proteins constitute a building block within the secretion machinery on top of which another building block, referred to as a pseudopilus, assembles. By analogy, we predict that a similar platform is required for the biogenesis of the type IV pilus.     

Crystal structure of PilF: functional implication in the type 4 pilus biogenesis in Pseudomonas aeruginosa

PilF is a requisite protein involved in the type 4 pilus biogenesis system from the Gram-negative human pathogenic bacteria, Pseudomonas aeruginosa. We determined the PilF structure at a 2.2A resolution; this includes six tandem tetratrico peptide repeat (TPR) units forming right-handed superhelix. PilF structure was similar to the heat shock protein organizing protein, which interacts with the C-terminal peptide of Hsp90 and Hsp70 via a concave Asn ladder in the inner groove of TPR superhelix. After simulated screening, the C-terminal pentapeptides of PilG, PilU, PilY, and PilZ proved to be a likely candidate binding to PilF, which are ones of 25 necessary components involved in the type 4 pilus biogenesis system. We proposed that PilF would be critical as a bridgehead in protein-protein interaction and thereby, PilF may bind a necessary molecule in type 4 pilus biogenesis system such as PilG, PilU, PilY, and PilZ.     

Novel Topology of BfpE, a Cytoplasmic Membrane Protein Required for Type IV Fimbrial Biogenesis in Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) produces the bundle-forming pilus (BFP), a type IV fimbria that has been implicated in virulence, autoaggregation, and localized adherence to epithelial cells. The bfpE gene is one of a cluster of bfp genes previously shown to encode functions that direct BFP biosynthesis. Here, we show that an EPEC strain carrying a nonpolar mutation in bfpE fails to autoaggregate, adhere to HEp-2 cells, or form BFP, thereby demonstrating that BfpE is required for BFP biogenesis. BfpE is a cytoplasmic membrane protein of the GspF family. To determine the membrane topology of BfpE, we fused bfpE derivatives containing 3' truncations and/or internal deletions to alkaline phosphatase and/or beta-galactosidase reporter genes, whose products are active only when localized to the periplasm or cytoplasm, respectively. In addition, we constructed BfpE sandwich fusions using a dual alkaline phosphatase/beta-galactosidase reporter cassette and analyzed BfpE deletion derivatives by sucrose density flotation gradient fractionation. The data from these analyses support a topology in which BfpE contains four hydrophobic transmembrane (TM) segments, a large cytoplasmic segment at its N terminus, and a large periplasmic segment near its C terminus. This topology is dramatically different from that of OutF, another member of the GspF family, which has three TM segments and is predominantly cytoplasmic. These findings provide a structural basis for predicting protein-protein interactions required for assembly of the BFP biogenesis machinery.     

In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae

The type II secretion system is a multi-protein complex that spans the cell envelope of Gram-negative bacteria and promotes the secretion of proteins, including several virulence factors. This system is homologous to the type IV pilus biogenesis machinery and contains five proteins, EpsG-K, termed the pseudopilins that are structurally homologous to the type IV pilins. The major pseudopilin EpsG has been proposed to form a pilus-like structure in an energy-dependent process that requires the ATPase, EpsE. A key remaining question is how the membrane-bound EpsG interacts with the cytoplasmic ATPase, and if this is a direct or indirect interaction. Previous studies have established an interaction between the bitopic inner membrane protein EpsL and EpsE; therefore, in this study we used in vivo cross-linking to test the hypothesis that EpsG interacts with EpsL. Our findings suggest that EpsL may function as a scaffold to link EpsG and EpsE and thereby transduce the energy generated by ATP hydrolysis to support secretion. The recent discovery of structural homology between EpsL and a protein in the type IV pilus system implies that this interaction may be conserved and represent an important functional interaction for both the type II secretion and type IV pilus systems.     

Secretion of TcpF by the Vibrio cholerae Toxin-Coregulated Pilus Biogenesis Apparatus Requires an N-Terminal Determinant

Type IV pili are important for microcolony formation, biofilm formation, twitching motility, and attachment. We and others have shown that type IV pili are important for protein secretion across the outer membrane, similar to type II secretion systems. This study explored the relationship between protein secretion and pilus formation in Vibrio cholerae. The toxin-coregulated pilus (TCP), a type IV pilus required for V. cholerae pathogenesis, is necessary for the secretion of the colonization factor TcpF (T. J. Kirn, N. Bose, and R. K. Taylor, Mol. Microbiol. 49:81–92, 2003). This phenomenon is not unique to V. cholerae; secreted virulence factors that are dependent on the presence of components of the type IV pilus biogenesis apparatus for secretion have been reported with Dichelobacter nodosus (R. M. Kennan, O. P. Dhungyel, R. J. Whittington, J. R. Egerton, and J. I. Rood, J. Bacteriol. 183:4451–4458, 2001) and Francisella tularensis (A. J. Hager et al., Mol. Microbiol. 62:227–237, 2006). Using site-directed mutagenesis, we demonstrated that the secretion of TcpF is dependent on the presence of selected amino acid R groups at position five. We were unable to find other secretion determinants, suggesting that Y5 is the major secretion determinant within TcpF. We also report that proteins secreted in a type IV pilus biogenesis apparatus-dependent manner have a YXS motif within the first 15 amino acids following the Sec cleavage site. The YXS motif is not present in proteins secreted by type II secretion systems, indicating that this is unique to type IV pilus-mediated secretion. Moreover, we show that TcpF interacts with the pilin TcpA, suggesting that these proteins are secreted by the type IV pilus biogenesis system. These data provide a starting point for understanding how type IV pili can mediate secretion of virulence factors important for bacterial pathogenesis.     

Biology of type II secretion

The type II secretion pathway or the main terminal branch of the general secretion pathway, as it has also been referred to, is widely distributed among Proteobacteria, in which it is responsible for the extracellular secretion of toxins and hydrolytic enzymes, many of which contribute to pathogenesis in both plants and animals. Secretion through this pathway differs from most other membrane transport systems, in that its substrates consist of folded proteins. The type II secretion apparatus is composed of at least 12 different gene products that are thought to form a multiprotein complex, which spans the periplasmic compartment and is specifically required for translocation of the secreted proteins across the outer membrane. This pathway shares many features with the type IV pilus biogenesis system, including the ability to assemble a pilus-like structure. This review discusses recent findings on the organization of the secretion apparatus and the role of its various components in secretion. Different models for pilus-mediated secretion through the gated pore in the outer membrane are also presented, as are the possible properties that determine whether a protein is recognized and secreted by the type II pathway.     

The plasmid R64 thin pilus identified as a type IV pilus.

The entire nucleotide sequence of the pil region of the IncI1 plasmid R64 was determined. Analysis of the sequence indicated that 14 genes, designated pilI through pilV, are involved in the formation of the R64 thin pilus. Protein products of eight pil genes were identified by the maxicell procedure. The pilN product was shown to be a lipoprotein by an experiment using globomycin. A computer search revealed that several R64 pil genes have amino acid sequence homology with proteins involved in type IV pilus biogenesis, protein secretion, and transformation competence. The pilS and pilV products were suggested to be prepilins for the R64 thin pilus, and the pilU product appears to be a prepilin peptidase. These results suggest that the R64 thin pilus belongs to the type IV family, specifically group IVB, of pili. The requirement of the pilR and pilU genes for R64 liquid mating was demonstrated by constructing their frameshift mutations. Comparison of three type IVB pilus biogenesis systems, the pil system of R64, the toxin-coregulated pilus (tcp) system of Vibrio cholerae, and the bundle-forming pilus (bfp) system of enteropathogenic Escherichia coli, suggests that they have evolved from a common ancestral gene system.     

An Aeromonas salmonicida type IV pilin is required for virulence in rainbow trout Oncorhynchus mykiss

Aeromonas salmonicida expresses a large number of proven and suspected virulence factors including bacterial surface proteins, extracellular degradative enzymes, and toxins. We report the isolation and characterization of a 4-gene cluster, tapABCD, from virulent A. salmonicida A450 that encodes proteins homologous to components required for type IV pilus biogenesis. One gene, tapA, encodes a protein with high homology to type IV pilus subunit proteins from many gram-negative bacterial pathogens, including Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio vulnificus. A survey of A. salmonicida isolates from a variety of sources shows that the tapA gene is as ubiquitous in this species as it is in other members of the Aeromonads. Immunoblotting experiments demonstrate that it is expressed in vitro and is antigenically conserved among the A. salmonicida strains tested. A mutant A. salmonicida strain defective in expression of TapA was constructed by allelic exchange and found to be slightly less pathogenic for juvenile Oncorhynchus mykiss (rainbow trout) than wild type when delivered by intraperitoneal injection. In addition, fish initially challenged with a high dose of wild type were slightly more resistant to rechallenge with wild type than those initially challenged with the tapA mutant strain, suggesting that presence of TapA contributes to immunity. Two of the other three genes identified, tapB and tapC, encode proteins with homology to factors known to be required for type IV pilus assembly in P. aeruginosa, but in an as yet unidentified manner. TapB is a member of the ABC-transporter family of proteins that contain characteristic nucleotide-binding regions, and which may provide energy for type IV pilus assembly through the hydrolysis of ATP. TapC homologs are integral cytoplasmic membrane proteins that may play a role in pilus anchoring or initiation of assembly. The fourth gene, tapD, encodes a product that shares homology with a family of proteins with a known biochemical function, namely, the type IV prepilin leader peptidases. These bifunctional enzymes proteolytically cleave the leader peptide from the pilin precursor (prepilin) and then N-methylate the newly exposed N-terminal amino acid prior to assembly of the subunits into the pilus structure. We demonstrate that A. salmonicida TapD is able to restore type IV pilus assembly and type II secretion in a P. aeruginosa strain carrying a mutation in its type IV peptidase gene, suggesting that it plays the same role in A. salmonicida.     

XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL

GspE belongs to a secretion NTPase superfamily, members of which are involved in type II/IV secretion, type IV pilus biogenesis and DNA transport in conjugation or natural transformation. Predicted to be a cytoplasmic protein, GspE has nonetheless been shown to be membrane-associated by interacting with the N-terminal cytoplasmic domain of GspL. By taking biochemical and genetic approaches, we observed that ATP binding triggers oligomerization of Xanthomonas campestris XpsE (a GspE homolog) as well as its association with the N-terminal domain of XpsL (a GspL homolog). While isolated XpsE exhibits very low intrinsic ATPase activity, association with XpsL appears to stimulate ATP hydrolysis. Mutation at a conserved lysine residue in the XpsE Walker A motif causes reduction in its ATPase activity without significantly influencing its interaction with XpsL, congruent with the notion that XpsE-XpsL association precedes ATP hydrolysis. For the first time, functional significance of ATP binding to GspE in type II secretion system is clearly demonstrated. The implications may also be applicable to type IV pilus biogenesis.     

Structure-function analysis of BfpB, a secretin-like protein encoded by the bundle-forming-pilus operon of enteropathogenic Escherichia coli

Production of type IV bundle-forming pili by enteropathogenic Escherichia coli (EPEC) requires BfpB, an outer-membrane lipoprotein and member of the secretin protein superfamily. BfpB was found to compose a ring-shaped, high-molecular-weight outer-membrane complex that is stable in 4% sodium dodecyl sulfate at temperatures of < or = 65 degrees C. Chemical cross-linking and immunoprecipitation experiments disclosed that the BfpB multimeric complex interacts with BfpG, and mutational studies showed that BfpG is required for the formation and/or stability of the multimer but not for the outer-membrane localization of BfpB. Formation of the BfpB multimer also does not require BfpA, the repeating subunit of the pilus filament. Functional studies of the BfpB-BfpG complex revealed that its presence confers vancomycin sensitivity, indicating that it may form an incompletely gated channel through the outer membrane. BfpB expression is also associated with accumulation of EPEC proteins in growth medium, suggesting that it may support both pilus biogenesis and protein secretion.     

PapD, a periplasmic transport protein in P-pilus biogenesis.

The product of the papD gene of uropathogenic Escherichia coli is required for the biogenesis of digalactoside-binding P pili. Mutations within papD result in complete degradation of the major pilus subunit, PapA, and of the pilinlike proteins PapE and PapF and also cause partial breakdown of the PapG adhesin. The papD gene was sequenced, and the gene product was purified from the periplasm. The deduced amino acid sequence and the N-terminal sequence obtained from the purified protein revealed that PapD is a basic and hydrophilic peripheral protein. A periplasmic complex between PapD and PapE was purified from cells that overproduced and accumulated these proteins in the periplasm. Antibodies raised against this complex reacted with purified wild-type P pili but not with pili purified from a papE mutant. In contrast, anti-PapD serum did not react with purified pili or with the culture fluid of piliated cells. However, this serum was able to specifically precipitate the PapE protein from periplasmic extracts, confirming that PapD and PapE were associated as a complex. It is suggested that PapD functions in P-pilus biogenesis as a periplasmic transport protein. Probably PapD forms complexes with pilus subunits at the outer surface of the inner membrane and transports them in a stable configuration across the periplasmic space before delivering them to the site(s) of pilus polymerization.     

The usher N terminus is the initial targeting site for chaperone-subunit complexes and participates in subsequent pilus biogenesis events

Pilus biogenesis on the surface of uropathogenic Escherichia coli requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway, periplasmic chaperone-subunit complexes target an outer membrane (OM) usher for subunit assembly into pili and secretion to the cell surface. The molecular mechanisms of protein secretion across the OM are not well understood. Mutagenesis of the P pilus usher PapC and the type 1 pilus usher FimD was undertaken to elucidate the initial stages of pilus biogenesis at the OM. Deletion of residues 2 to 11 of the mature PapC N terminus abolished the targeting of the usher by chaperone-subunit complexes and rendered PapC nonfunctional for pilus biogenesis. Similarly, an intact FimD N terminus was required for chaperone-subunit binding and pilus biogenesis. Analysis of PapC-FimD chimeras and N-terminal fragments of PapC localized the chaperone-subunit targeting domain to the first 124 residues of PapC. Single alanine substitution mutations were made in this domain that blocked pilus biogenesis but did not affect targeting of chaperone-subunit complexes. Thus, the usher N terminus does not function simply as a static binding site for chaperone-subunit complexes but also participates in subsequent pilus assembly events.     

Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus

Type IV pili are expressed from a wide variety of Gram‐negative bacteria and play a major role in host cell adhesion and bacterial motility. PilC is one of at least a dozen different proteins that are implicated in Type IV pilus assembly in Thermus thermophilus and a member of a conserved family of integral inner membrane proteins which are components of the Type II secretion system (GspF) and the archeal flagellum. PilC/GspF family members contain repeats of a conserved helix‐rich domain of around 100 residues in length. Here, we describe the crystal structure of one of these domains, derived from the N‐terminal domain of Thermus thermophilus PilC. The N‐domain forms a dimer, adopting a six helix bundle structure with an up‐down‐up‐down‐up‐down topology. The monomers are related by a rotation of 170°, followed by a translation along the axis of the final α‐helix of approximately one helical turn. This means that the regions of contact on helices 5 and 6 in each monomer are overlapping, but different. Contact between the two monomers is mediated by a network of hydrophobic residues which are highly conserved in PilC homologs from other Gram‐negative bacteria. Site‐directed mutagenesis of residues at the dimer interface resulted in a change in oligomeric state of PilC from tetramers to dimers, providing evidence that this interface is also found in the intact membrane protein and suggesting that it is important to its function. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

PilP, a pilus biogenesis lipoprotein in Neisseria gonorrhoeae, affects expression of PilQ as a high-molecular-mass multimer

Studies of gonococcal pilus biogenesis are fundamental to understanding organelle structure/function relationships and identifying new approaches to controlling disease. This area of research is also relevant to elucidating the basic mechanisms of outer membrane translocation of macromolecules, which requires components highly related to those involved in type IV pilus expression. Previous studies have shown that products of several ancillary pil genes are required for organelle biogenesis but of these only PilQ, a member of the GspD protein family, is a component of the outer membrane. DNA sequencing of the region upstream of pilQ revealed the presence of two open reading frames (ORFs) whose deduced polypeptides shared significant identities with proteins required for pilus expression in Pseudomonas aeruginosa and Pseudomonas syringae, the genes for which are arrayed upstream of a gene encoding a PilQ homologue. Gonococcal mutants bearing transposon insertions in these ORFs were non-piliated and failed to express pilus-associated phenotypes, and the corresponding genes were designated pilO and pilP. The piliation defects in the mutants could not be ascribed to polarity on distal pilQ expression as shown by direct measurement of PilQ antigen in those backgrounds and the use of a novel technique to create tandem duplications in the gonococcus (Gc) genome. As predicted by the presence of a consensus lipoprotein signal sequence, PilP expressed in both Escherichia coli and Gc could be labelled with [³H]-palmitic acid. PilP^? as well as PilQ^? mutants shed PilC, a protein which facilitates pilus assembly and is implicated in epithelial cell adherence, in a soluble form. Combined with the finding that levels of multimerized PilQ were greatly reduced in PilP^? mutants, the results suggest that PilP is required for PilQ function and that PilQ and PilC may interact during the terminal stages of pilus biogenesis. The findings also support the hypothesis that the Gc PilQ multimer corresponds to a physiologically relevant form of the protein required for pilus biogenesis.     

Bacterial outer membrane ushers contain distinct targeting and assembly domains for pilus biogenesis

Biogenesis of a superfamily of surface structures by gram-negative bacteria requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway a periplasmic chaperone works together with an outer membrane usher to direct substrate folding, assembly, and secretion to the cell surface. We analyzed the structure and function of the PapC usher required for P pilus biogenesis by uropathogenic Escherichia coli. Structural analysis indicated PapC folds as a beta-barrel with short extracellular loops and extensive periplasmic domains. Several periplasmic regions were localized, including two domains containing conserved cysteine pairs. Functional analysis of deletion mutants revealed that the PapC C terminus was not required for insertion of the usher into the outer membrane or for proper folding. The usher C terminus was not necessary for interaction with chaperone-subunit complexes in vitro but was required for pilus biogenesis in vivo. Interestingly, coexpression of PapC C-terminal truncation mutants with the chromosomal fim gene cluster coding for type 1 pili allowed P pilus biogenesis in vivo. These studies suggest that chaperone-subunit complexes target an N-terminal domain of the usher and that subunit assembly into pili depends on a subsequent function provided by the usher C terminus.     

Structure of the PilM-PilN inner membrane type IV pilus biogenesis complex from Thermus thermophilus

Type IV pili are surface-exposed filaments, which extend from a variety of bacterial pathogens and play a major role in pathogenesis, motility, and DNA uptake. Here, we present the crystal structure of a complex between a cytoplasmic component of the type IV pilus biogenesis system from Thermus thermophilus, PilM, in complex with a peptide derived from the cytoplasmic portion of the inner membrane protein PilN. PilM also binds ATP, and its structure is most similar to the actin-like protein FtsA. PilN binds in a narrow channel between the 1A and 1C subdomains in PilM; the binding site is well conserved in other gram-negative bacteria, notably Neisseria meningitidis, Pseudomonas aeruginosa, and Vibrio cholerae. We find no evidence for the catalysis of ATP hydrolysis by PilM; fluorescence data indicate that the protein is likely to be saturated by ATP at physiological concentrations. In addition, binding of the PilN peptide appears to influence the environment of the ATP binding site. This is the first reported structure of a complex between two type IV pilus biogenesis proteins. We propose a model in which PilM binds ATP and then PilN as one of the first steps in the formation of the inner membrane platform of the type IV pilus biogenesis complex.     

The Inner Membrane Protein PilG Interacts with DNA and the Secretin PilQ in Transformation

Expression of type IV pili (Tfp), filamentous appendages emanating from the bacterial surface, is indispensable for efficient neisserial transformation. Tfp pass through the secretin pore consisting of the membrane protein PilQ. PilG is a polytopic membrane protein, conserved in Gram-positive and Gram-negative bacteria, that is required for the biogenesis of neisserial Tfp. PilG null mutants are devoid of pili and non-competent for transformation. Here, recombinant full-length, truncated and mutated variants of meningococcal PilG were overexpressed, purified and characterized. We report that meningococcal PilG directly binds DNA in vitro, detected by both an electromobility shift analysis and a solid phase overlay assay. PilG DNA binding activity was independent of the presence of the consensus DNA uptake sequence. PilG-mediated DNA binding affinity was mapped to the N-terminus and was inactivated by mutation of residues 43 to 45. Notably, reduced meningococcal transformation of DNA in vivo was observed when PilG residues 43 to 45 were substituted by alanine in situ, defining a biologically significant DNA binding domain. N-terminal PilG also interacted with the N-terminal region of PilQ, which previously was shown to bind DNA. Collectively, these data suggest that PilG and PilQ in concert bind DNA during Tfp-mediated transformation.     

Interaction with type IV pili induces structural changes in the bacterial outer membrane secretin PilQ

Type IV pili are cell surface organelles found on many Gram-negative bacteria. They mediate a variety of functions, including adhesion, twitching motility, and competence for DNA uptake. The type IV pilus is a helical polymer of pilin protein subunits and is capable of rapid polymerization or depolymerization, generating large motor forces in the process. Here we show that a specific interaction between the outer membrane secretin PilQ and the type IV pilus fiber can be detected by far-Western analysis and sucrose density gradient centrifugation. Transmission electron microscopy of preparations of purified pili, to which the purified PilQ oligomer had been added, showed that PilQ was uniquely located at one end of the pilus fiber, effectively forming a "mallet-type" structure. Determination of the three-dimensional structure of the PilQ-type IV pilus complex at 26-angstroms resolution showed that the cavity within the protein complex was filled. Comparison with a previously determined structure of PilQ at 12-angstroms resolution indicated that binding of the pilus fiber induced a dissociation of the "cap" feature and lateral movement of the "arms" of the PilQ oligomer. The results demonstrate that the PilQ structure exhibits a dynamic response to the binding of its transported substrate and suggest that the secretin could play an active role in type IV pilus assembly as well as secretion.     

Interaction between protein subunits of the type IV secretion system of Bartonella henselae

In this study we used the yeast two-hybrid system to identify interactions between protein subunits of the virB type IV secretion system of Bartonella henselae. We report interactions between inner membrane and periplasmic proteins, the pilus polypeptide, and the core complex and a novel interaction between VirB3 and VirB5.