Biogenesis of T Pili in Agrobacterium tumefaciens Requires Precise VirB2 Propilin Cleavage and Cyclization

VirB2 propilin is processed by the removal of a 47-amino-acid signal peptide to generate a 74-amino-acid peptide product in both Escherichia coli and Agrobacterium tumefaciens. The cleaved VirB2 protein is further cyclized to form the T pilin in A. tumefaciens but not in E. coli. Mutations in the signal peptidase cleavage sequence of VirB2 propilin cause the formation of aberrant T pilin and also severely attenuate virulence. No T pilus was observed in these mutants. The potential role of the exact VirB2 propilin cleavage and cyclization in T pilus biogenesis and virulence is discussed.     

Membrane location of the Ti plasmid VirB proteins involved in the biosynthesis of a pilin-like conjugative structure on Agrobacterium tumefaciens

Abstract The virB operon of the Agrobacterium tumefaciens Ti plasmid encodes 11 proteins. Specific antisera to VirB2, VirB3 and VirB9 were used to locate these virulence proteins in the A. tumefaciens cell. Immunoblot analysis located VirB2 protein to the inner and outer membranes; VirB3 and VirB9 were likewise associated with both membranes, but mainly in the outer membrane. VirB2 is processed from a 12.3-kDa protein into a 7.2-kDa polypeptide. Such sized protein results from cleavage at residue Ala⁴⁷, upstream of which two additional alanine residues Ala⁴⁵-Ala⁴⁶ are contained and bearing resemblance to a signal peptide peptidase-I cleavage sequence. VirB2 and VirB3 sequences are strikingly similar to the pilin biosynthetic proteins TraA and TraL encoded by the tra operon of F and R1-19 plasmids. Since traA encodes a propilin that is cleaved into a 7.2-kDa conjugative pilin product and since this cleavage site is present in both TraA and VirB2, we propose that virB2 encodes a pilin-like protein which together with VirB3 and VirB9 as well as other VirB proteins may be used for interkingdom T-DNA transfer between bacteria and plants.     

Maturation of IncP pilin precursors resembles the catalytic Dyad-like mechanism of leader peptidases

The pilus subunit, the pilin, of conjugative IncP pili is encoded by the trbC gene. IncP pilin is composed of 78 amino acids forming a ring structure (R. Eisenbrandt, M. Kalkum, E.-M. Lai, C. I. Kado, and E. Lanka, J. Biol. Chem. 274:22548-22555, 1999). Three enzymes are involved in maturation of the pilin: LepB of Escherichia coli for signal peptide removal and a yet-unidentified protease for removal of 27 C-terminal residues. Both enzymes are chromosome encoded. Finally, the inner membrane-associated IncP TraF replaces a four-amino-acid C-terminal peptide with the truncated N terminus, yielding the cyclic polypeptide. We refer to the latter process as "prepilin cyclization." We have used site-directed mutagenesis of trbC and traF to unravel the pilin maturation process. Each of the mutants was analyzed for its phenotypes of prepilin cyclization, pilus formation, donor-specific phage adsorption, and conjugative DNA transfer abilities. Effective prepilin cyclization was determined by matrix-assisted laser desorption-ionization-mass spectrometry using an optimized sample preparation technique of whole cells and trans-3-indolyl acrylic acid as a matrix. We found that several amino acid exchanges in the TrbC core sequence allow prepilin cyclization but disable the succeeding pilus assembly. We propose a mechanism explaining how the signal peptidase homologue TraF attacks a C-terminal section of the TrbC core sequence via an activated serine residue. Rather than cleaving and releasing hydrolyzed peptides, TraF presumably reacts as a peptidyl transferase, involving the N terminus of TrbC in the aminolysis of a postulated TraF-acetyl-TrbC intermediate. Under formal loss of a C-terminal tetrapeptide, a new peptide bond is formed in a concerted action, connecting serine 37 with glycine 114 of TrbC.     

Membrane insertion of the F-pilin subunit is Sec independent but requires leader peptidase B and the proton motive force.

F pilin is the subunit required for the assembly of conjugative pili on the cell surface of Escherichia coli carrying the F plasmid. Maturation of the F-pilin precursor, propilin, involves three F plasmid transfer products: TraA, the propilin precursor; TraQ, which promotes efficient propilin processing; and TraX, which is required for acetylation of the amino terminus of the 7-kDa pilin polypeptide. The mature pilin begins at amino acid 52 of the TraA propilin sequence. We performed experiments to determine the involvement of host cell factors in propilin maturation. At the nonpermissive temperature in a LepBts (leader peptidase B) host, propilin processing was inhibited. Furthermore, under these conditions, only full-length precursor was observed, suggesting that LepB is responsible for the removal of the entire propilin leader peptide. Using propilin processing as a measure of propilin insertion into the plasma membrane, we found that inhibition or depletion of SecA and SecY does not affect propilin maturation. Addition of a general membrane perturbant such as ethanol also had no effect. However, dissipation of the proton motive force did cause a marked inhibition of propilin processing, indicating that membrane insertion requires this energy source. We propose that propilin insertion in the plasma membrane proceeds independently of the SecA-SecY secretion machinery but requires the proton motive force. These results present a model whereby propilin insertion leads to processing by leader peptidase B to generate the 7-kDa peptide, which is then acetylated in the presence of TraX.     

Processed VirB2 Is the Major Subunit of the Promiscuous Pilus of Agrobacterium tumefaciens

Previous studies have implicated the obligatory requirement for the vir regulon (or “virulon”) of the Ti plasmid for the transfer of oncogenes from Agrobacterium tumefaciens to plant cells. The machinery used in this horizontal gene transfer has been long thought to be a transformation or conjugative delivery system. Based on recent protein sequence comparisons, the proteins encoded by the virB operon are strikingly similar to proteins involved in the synthesis and assembly of conjugative pili such as the conjugative pilus of F plasmid in Escherichia coli. The F pilus is composed of TraA pilin subunits derived from TraA propilin. In the present study, evidence is provided showing that the counterpart of TraA is VirB2, which like TraA propilin is processed into a 7.2-kDa product that comprises the pilus subunit as demonstrated by biochemical and electron microscopic analyses. The processed VirB2 protein is present exocellularly on medium on which induced A. tumefaciens had grown and appears as thin filaments of 10 nm that react specifically to VirB2 antibody. Exocellular VirB2 is produced abundantly at 19°C as compared with 28°C, an observation that parallels the effect of low temperature on the production of vir gene-specific pili observed previously (K. J. Fullner, L. C. Lara, and E. W. Nester, Science 273:1107–1109, 1996). Export of the processed VirB2 requires other virB genes since mutations in these genes cause the loss of VirB2 pilus formation and result in processed VirB2 accumulation in the cell. The presence of exocellular processed VirB2 is directly correlated with the formation of pili, and it appears as the major protein in the purified pilus preparation. The evidence provides a compelling argument for VirB2 as the propilin whose 7.2-kDa processed product is the pilin subunit of the promiscuous conjugative pilus, hereafter called the “T pilus” of A. tumefaciens.     

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.     

Promiscuous DNA transfer system of Agrobacterium tumefaciens: role of the virB operon in sex pilus assembly and synthesis

Conjugative transfer of DNA that occurs between bacteria also operates between bacteria and higher organisms. The transfer of DNA between Gram-negative bacteria requires initial contact by a sex pilus followed by DNA traversing four membranes (donor plus recipient) using a transmembrane pore. Accumulating evidence suggests that transfer of the T-DNA from Agrobacterium tumefaciens to plants may also occur via a conjugative mechanism. The virB operon of the Ti plasmid exhibits close homologies to genes that are known to encode the pilin subunits and pilin assembly proteins. The proteins encoded by the PilW operon of IncW plasmid R388 share strong similarities (average similarity=50.8%) with VirB proteins. Similarly, the TraA, TraL and TraC proteins of IncF plasmid F have similarities to VirB2, VirB3 and VirB4 respectively (average similarity = 45.3%). VirB2 protein (12.3 kDa) contains a signal peptidase-I cleavage sequence that generates a polypeptide of 7.2 kDa. Likewise, the 12.8 kDa propilin protein TraA of plasmid F also possesses a peptidase-I cleavage site that generates the 7.2 kDa pilin structural protein. Similar amino acid sequences of the conjugative transfer genes of F, R388 as well as plasmid RP4 and the genes of the ptl operon of Bortedella pertussis suggest the existence of a superfamily of transmembrane proteins adapted to the promiscuous transfer of DNA-protein complexes.     

Evidence for VirB4-Mediated Dislocation of Membrane-Integrated VirB2 Pilin during Biogenesis of the Agrobacterium VirB/VirD4 Type IV Secretion System

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.The Agrobacterium tumefaciens VirB/VirD4 type IV secretion system (T4SS) delivers effector proteins and DNA to plant cells during infection (1, 14). The 11 VirB proteins and VirD4 substrate receptor mediate assembly of the envelope-spanning translocation channel, whereas the VirB proteins independently of VirD4 are required for polymerization of the extracellular T pilus (6, 32, 46). These T4SS subunits include the three ATPases VirD4, VirB4, and VirB11; a trans-envelope core complex comprised of VirB7, VirB9, and VirB10; subunits involved in assembly or spatial positioning of the core complex (VirB1, VirB6, and VirB8); and other structural components (VirB2 pilin, VirB3, and pilus-associated VirB5) (1, 14, 43, 48, 55, 70). The VirB/VirD4 subunits are conserved among many Gram-negative bacterial T4SSs, and recent structures of homologs of VirD4, VirB5, VirB8, VirB10, and VirB11 and a VirB7/VirB9/VirB10 machine subassembly are supplying exciting new information about T4SS machine architectures (11, 28, 29).The pilin subunit VirB2 is a component of both the secretion channel and T pilus (39, 47, 48). Its role in substrate transfer was established with a modified chromatin immunoprecipitation (ChIP) assay termed transfer DNA (T-DNA) immunoprecipitation (TrIP), wherein the pilin (but not the T pilus) was shown to form formaldehyde-cross-linkable contacts with the translocating T-DNA substrate (10). TrIP studies with virB mutant strains also supplied evidence that VirB2 occupies a distal portion of the translocation channel near or at the outer membrane (OM) (10). Complementary genetic studies identified mutations in several VirB subunits, including VirB6, VirB9, VirB10, and VirB11, that selectively block T pilus production without affecting substrate transfer (39, 40, 41, 62). These Tra⁺ Pil⁻ “uncoupling” mutations do not bypass the requirement for VirB2 production for substrate transfer, as the further deletion of virB2 from the Tra⁺ Pil⁻ mutant strains renders these strains transfer defective (39, 41, 62). Therefore, VirB2 pilin, but not an intact T pilus, is required for passage of substrates to target cells.The pathways culminating in the integration of VirB2 into the two terminal organelles, the secretion channel and T pilus, are fundamentally poorly understood. The early VirB protein-independent reactions involve insertion of the 12.3-kDa propilin into the inner membrane (IM); cleavage of a long, 47-residue signal sequence, presumably by LepB signal peptidase; and covalent joining of the N-terminal Gln48 and C-terminal Ser121 to form the mature, cyclic pilin (24). This unusual head-to-tail cyclization reaction was also shown for the VirB2 homolog, TrbC (24/51% sequence identity/similarity) of plasmid RP4 (24, 34, 44). Other VirB2 homologs, such as F plasmid TraA (19/47% identity/similarity) (67), remain linear although their N termini are modified by N acetylation (54).Prevailing models suggest that mature forms of conjugative pilins accumulate in the IM as pools for use in assembly of the channel/pilus upon receipt of an unknown morphogenetic signal(s). The IM-integrated VirB2, TraA_F, and TrbC_RP4 pilins likely adopt similar topologies, as deduced from similar predicted secondary structures and results of reporter fusion studies with periplasmically active alkaline phosphatase (PhoA) (5, 22, 56). Two hydrophobic domains are thought to orient across the IM so that a small, intervening hydrophilic loop is cytoplasmic and the hydrophilic N and C termini are periplasmic. Detailed studies confirming this overall topology are lacking, and limited information exists regarding the nature of pilin interactions with other T4SS subunits (36, 51). Furthermore, little is known about the mechanism or energetic requirements for dislocation of membrane-integrated forms of conjugative pilins during machine morphogenesis.In A. tumefaciens, mutations in the Walker A nucleoside triphosphate (NTP) binding site motifs of the VirB4 and VirB11 ATPases render cells defective for substrate transfer and pilus production, indicating that NTP energy consumption by both ATPases is essential for assembly of the two terminal organelles (6, 7, 58, 62, 68). VirB4-like subunits are signatures of all T4SSs described to date, whereas VirB11-like proteins are common but not ubiquitous among the T4SSs (1). Some T4SSs, such as the conjugation machines encoded by Escherichia coli F-like plasmids, lack VirB11 homologs, and yet their conjugative pili extend and retract dynamically by a mechanism(s) dependent on VirB4 homologs (18, 65). On the basis of these observations, it is reasonable to propose that the VirB4-like subunits catalyze early reactions associated with assembly of conjugative pili.Here, we used the scanning cysteine accessibility method (SCAM) (9) to define the IM topology of cyclized VirB2. We then assayed for contributions of VirB subunits to the pilin structural organization. We present biochemical evidence for VirB4-mediated dislocation of VirB2 pilin from the membrane and also for a contribution by VirB11 in modulating pilin tertiary or quaternary structure. We discuss our findings in the context of recent advances in our understanding of T4SS machine assembly and architecture.     

VirB2 is a processed pilin-like protein encoded by the Agrobacterium tumefaciens Ti plasmid.

The mechanism of DNA transmission between distinct organisms has remained a subject of long-standing interest. Agrobacterium tumefaciens mediates the transfer of plant oncogenes in the form of a 25-kb T-DNA sector of a resident Ti plasmid. A growing body of evidence leading to the elucidation of the mechanism involved in T-DNA transfer comes from studies on the vir genes contained in six major operons that are required for the T-DNA transfer process. Recent comparative amino acid sequence studies of the products of these vir genes have revealed interesting similarities between Tra proteins of Escherichia coli F factor, which are involved in the biosynthesis and assembly of a conjugative pilus, and VirB proteins encoded by genes of the virB operon of A. tumefaciens pTiC58. We have previously identified VirB2 as a pilin-like protein with processing features similar to those of TraA of the F plasmid and have shown that VirB2 is required for the biosynthesis of pilin on a flagella-free Agrobacterium strain. In the present work, VirB2 is found to be processed and localized primarily to the cytoplasmic membrane in E. coli. Cleavage of VirB2 was predicted previously to occur between alanine and glutamine in the sequence -Pro-Ala-Ala-Ala-Glu-Ser-. This peptidase cleavage sequence was mutated by an amino acid substitution for one of the alanine residues (D for A at position 45 [A45D]), by deletion of the three adjacent alanines, and by a frameshift mutation 22 bp upstream of the predicted Ala-Glu cleavage site. With the exception of the frameshift mutation, the alanine mutations do not prevent VirB2 processing in E. coli, while in A. tumefaciens they result in VirB2 instability, since no holo- or processed protein is detectable. All of the above mutations abolish virulence. The frameshift mutation abolishes processing in both organisms. These results indicate that VirB2 is processed into a 7.2-kDa structural protein. The cleavage site in E. coli appears to differ from that predicted in A. tumefaciens. Yet, the cleavage sites are relatively close to each other since the final cleavage products are similar in size and are produced irrespective of the length of the amino-terminal portion of the holoprotein. As we observed previously, the similarity between the processing of VirB2 in A. tumefaciens and the processing of the propilin TraA of the F plasmid now extends to E. coli.     

Nucleotide sequence of the tra YALE region from IncFV plasmid pED208

The pED208 plasmid is a 90-kilobase conjugative plasmid which is the derepressed form of Fo lac plasmid (IncFV). A 3.3-kilobase HindIII-PstI fragment from the pED208 plasmid was cloned and sequenced and was found to contain four open reading frames which were highly homologous to the traA, traL, traE, and traY gene products of the F plasmid. The pED208 traA propilin protein was 119 amino acids in length, consisting of a leader sequence of 55 amino acids and a mature pilin subunit of 64 residues. The leader sequence contained a hydrophobic region followed by a classic signal peptidase cleavage site (Ala-Ser-Ala-55). F and pED208 pilin proteins shared 27 conserved residues and had similar predicted secondary structures. The pED208 traA and traL genes were separated by a single base pair, and no ribosome binding site preceded the traL gene. The pED208 traY gene contained an IS2 insertion element in orientation II 180 nucleotides (60 residues) upstream of the traY stop codon. This insertion of IS2 resulted in a predicted fusion peptide of 69 residues for traY which may provide the observed traY activity. Since IS2 is absent in the wild-type plasmid, Fo lac, derepression and concomitant multipiliation may be due to the insertion of IS2 providing constitutive expression of the pED208 tra operon.     

Role of the propilin leader peptide in the maturation of F pilin.

F-pilin maturation and translocation result in the cleavage of a 51-amino-acid leader sequence from propilin and require LepB and TraQ but not the SecA-SecY secretion pathway. The unusual propilin leader peptide and the dependence of its cleavage on TraQ suggested that TraQ recognition may be specific for the leader peptide. An in vitro propilin cleavage assay yielded propilin (13 kDa), the pilin polypeptide (7 kDa), and a 5.5-kDa protein as the traA products. The 5.5-kDa protein comigrates with the full-length 51-amino-acid leader peptide, and [14C]proline labeling confirmed its identity since the only proline residues of propilin are found within the leader peptide. The in vitro and in vivo propilin-processing reactions proceed similarly in a single polypeptide cleavage step. Furthermore, TraQ dependence is a property of F-pilin maturation specifically rather than a property of the leader peptide. A propilin derivative with an amino-terminal signal sequence generated by deleting codons 2 to 28 required TraQ for processing in vivo. On the other hand, a chimeric protein with the propilin wild-type leader peptide fused to the mature portion of beta-lactamase was processed in a TraQ-independent manner. Thus, despite its unusual length, the propilin leader peptide seems to perform a function similar to that of the typical amino-terminal signal sequence. This work suggests that TraQ is not necessary for the proteolysis of propilin and therefore is likely to act as a chaperone-like protein that promotes the translocation of propilin.     

Topology of the VirB4 C terminus in the Agrobacterium tumefaciens VirB/D4 type IV secretion system

Gram-negative type IV secretion systems (T4SSs) transfer proteins and DNA to eukaryotic and/or prokaryotic recipients resulting in pathogenesis or conjugative DNA transfer. VirB4, one of the most conserved proteins in these systems, has both energetic and structural roles in substrate translocation. We previously predicted a structural model for the large C-terminal domain (residues 425-789) of VirB4 of Agrobacterium tumefaciens. Here we have defined a homology-based structural model for Agrobacterium VirB11. Both VirB4 and VirB11 models predict hexameric oligomers. Yeast two-hybrid interactions define peptides in the C terminus of VirB4 and the N terminus of VirB11 that interact with each other. These interactions were mapped onto the homology models to predict direct interactions between the hexameric interfaces of VirB4 and VirB11 such that the VirB4 C terminus stacks above VirB11 in the periplasm. In support of this, fractionation and Western blotting show that the VirB4 C terminus is localized to the membrane and periplasm rather than the cytoplasm of cells. Additional high resolution yeast two-hybrid results demonstrate interactions between the C terminus of VirB4 and the periplasmic portions of VirB1, VirB8, and VirB10. Genetic studies reveal dominant negative interactions and thus function of the VirB4 C terminus in vivo. The above data are integrated with the existing body of literature to propose a structural, periplasmic role for the C-terminal half of the Agrobacterium VirB4 protein.     

Processing and maturation of the pilin of the type IV secretion system encoded within the gonococcal genetic island

The type IV secretion system (T4SS) encoded within the gonococcal genetic island (GGI) of Neisseria gonorrhoeae has homology to the T4SS encoded on the F plasmid. The GGI encodes the putative pilin protein TraA and a serine protease TrbI, which is homologous to the TraF protein of the RP4 plasmid involved in circularization of pilin subunits of P-type pili. TraA was processed to a 68-amino acid long circular peptide by leader peptidase and TrbI. Processing occurred after co-translational membrane insertion and was independent of other proteins. Circularization occurred after removal of three C-terminal amino acids. Mutational analysis of TraA revealed limited flexibility at the cleavage and joining sites. Mutagenesis of TrbI showed that the conserved Lys-93 and Asp-155 are essential, whereas mutagenesis of Ser-52, the putative catalytic serine did not influence circularization. Further mutagenesis of other serine residues did not identify a catalytic serine, indicating that TrbI either contains redundant catalytic serine residues or does not function via a serine-lysine dyad mechanism. In vitro studies revealed that circularization occurs via a covalent intermediate between the C terminus of TraA and TrbI. The intermediate is processed to the circular form after cleavage of the N-terminal signal sequence. This is the first demonstration of a covalent intermediate in the circularization mechanism of conjugative pili.     

Major antigenic determinants of F and ColB2 pili.

F-like conjugative pili are expressed by plasmids with closely related transfer systems. They are tubular filaments that are composed of repeating pilin subunits arranged in a helical array. Both F and ColB2 pilin have nearly identical protein sequences, and both contain an acetylated amino-terminal alanine residue. However, they differ by a few amino acid residues at their amino termini. Rabbit antisera raised against purified F and ColB2 pili are immunologically cross-reactive by only 25%, as measured by a competition enzyme-linked immunosorbent assay (ELISA). A tryptic peptide corresponding to the first 15 amino acid residues of ColB2 pilin was isolated and found to remove nearly 80% of ColB2 pilus-directed rabbit antibodies. The corresponding tryptic peptide from F pilin, which reacted with anti-F pilus antibodies to remove 80%, was less than 20% reactive with anti-ColB2 pilus antiserum. Cleavage of these peptides with cyanogen bromide (at a methionine residue approximately in the middle of the peptide) did not affect the antigenicity of these peptides. Synthetic N alpha-acetylated peptides corresponding to the first eight amino acids of F pilin (Ac-Ala-Gly-Ser-Ser-Gly-Gln-Asp-Leu-COOH) and the first six amino acids of ColB2 pilin (Ac-Ala-Gln-Gly-Gln-Asp-Leu-COOH) were prepared and tested by competition ELISA with homologous and heterologous anti-pilus antisera. The F peptide F(1-8) inhibited the interaction of F pili and anti-F pilus antiserum to 80%, while the ColB2 peptide ColB2(1-6) inhibited anti-ColB2 pilus antiserum reacting with ColB2 pili by greater than 60%. The two peptides F(1-8) and ColB2(1-6) were inactive by competition ELISAs with heterologous antisera. These results suggest that the major antigenic determinant of both F and ColB2 pili is at the amino terminus of the pilin subunit and that 80% of antibodies raised against these pili are specific for this region of the pilin molecule.     

VirB1* promotes T-pilus formation in the vir-Type IV secretion system of Agrobacterium tumefaciens

The vir-type IV secretion system of Agrobacterium is assembled from 12 proteins encoded by the virB operon and virD4. VirB1 is one of the least-studied proteins encoded by the virB operon. Its N terminus is a lytic transglycosylase. The C-terminal third of the protein, VirB1*, is cleaved from VirB1 and secreted to the outside of the bacterial cell, suggesting an additional function. We show that both nopaline and octopine strains produce abundant amounts of VirB1* and perform detailed studies on nopaline VirB1*. Both domains are required for wild-type virulence. We show here that the nopaline type VirB1* is essential for the formation of the T pilus, a subassembly of the vir-T4SS composed of processed and cyclized VirB2 (major subunit) and VirB5 (minor subunit). A nopaline virB1 deletion strain does not produce T pili. Complementation with full-length VirB1 or C-terminal VirB1*, but not the N-terminal lytic transglycosylase domain, restores T pili containing VirB2 and VirB5. T-pilus preparations also contain extracellular VirB1*. Protein-protein interactions between VirB1* and VirB2 and VirB5 were detected in the yeast two-hybrid assay. We propose that VirB1 is a bifunctional protein required for virT4SS assembly. The N-terminal lytic transglycosylase domain provides localized lysis of the peptidoglycan cell wall to allow insertion of the T4SS. The C-terminal VirB1* promotes T-pilus assembly through protein-protein interactions with T-pilus subunits.     

Agrobacterium tumefaciens VirB6 domains direct the ordered export of a DNA substrate through a type IV secretion System

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) translocates DNA and protein substrates across the bacterial cell envelope. Six presumptive channel subunits of this T4SS (VirD4, VirBll, VirB6, VirB8, VirB2, and VirB9) form close contacts with the VirD2-T-strand transfer intermediate during export, as shown recently by a novel transfer DNA immunoprecipitation (TrIP) assay. Here, we characterize the contribution of the hydrophobic channel component VirB6 to substrate translocation. Results of reporter protein fusion and cysteine accessibility studies support a model for VirB6 as a polytopic membrane protein with a periplasmic N terminus, five transmembrane segments, and a cytoplasmic C terminus. TrIP studies aimed at characterizing the effects of VirB6 insertion and deletion mutations on substrate translocation identified several VirB6 functional domains: (i) a central region composed of a large periplasmic loop (P2) (residues 84 to 165) mediates the interaction of VirB6 with the exiting T-strand; (ii) a multi-membrane-spanning region carboxyl-terminal to loop P2 (residues 165 to 245) is required for substrate transfer from VirB6 to the bitopic membrane subunit VirB8; and (iii) the two terminal regions (residues 1 to 64 and 245 to 290) are required for substrate transfer to the periplasmic and outer membrane-associated VirB2 and VirB9 subunits. Our findings support a model whereby the periplasmic loop P2 comprises a portion of the secretion channel and distinct domains of VirB6 participate in channel subunit interactions required for substrate passage to the cell exterior.     

Localization, cloning, and sequence determination of the conjugative plasmid ColB2 pilin gene.

ColB2 is a colicin-producing, 96-kilobase plasmid which encodes a conjugative system that is similar, but not identical, to F. A restriction map of this plasmid was generated, and DNA homology studies between F and ColB2 plasmids revealed homology only between their transfer operons. The locations of the ColB2 transfer operon and ColB2 pilin gene were localized on this restriction map. The gene encoding ColB2 pilin, traA, was cloned and sequenced. The pilin protein of ColB2 is identical to F, except at the amino terminus, where ala-gln of ColB2 pilin corresponds to Ala-Gly-Ser-Ser of F pilin. This is due to a 6-base-pair deletion in the ColB2 pilin gene. Biochemical studies on tryptic peptides derived from ColB2 pilin demonstrate the location of this gene to be correct. There is a putative signal peptidase cleavage site after the sequence Ala-Met-Ala, giving a signal peptide of 51 amino acids and a mature pilin protein of 68 amino acids (7,000 daltons). The amino terminus is blocked, probably with an acetyl group. A chimera containing the ColB2 pilin gene was able to complement an F traA mutant, demonstrating that the pilus assembly proteins of F can utilize the ColB2 pilin protein to form a pilus.     

VirB7 lipoprotein is exocellular and associates with the Agrobacterium tumefaciens T pilus

Agrobacterium tumefaciens transfers oncogenic T-DNA and effector proteins to plant cells via a type IV secretion pathway. This transfer system, assembled from the products of the virB operon, is thought to consist of a transenvelope mating channel and the T pilus. When screened for the presence of VirB and VirE proteins, material sheared from the cell surface of octopine strain A348 was seen to possess detectable levels of VirB2 pilin, VirB5, and the VirB7 outer membrane lipoprotein. Material sheared from the cell surface of most virB gene deletion mutants also possessed VirB7, but not VirB2 or VirB5. During purification of the T pilus from wild-type cells, VirB2, VirB5, and VirB7 cofractionated through successive steps of gel filtration chromatography and sucrose density gradient centrifugation. A complex containing VirB2 and VirB7 was precipitated from a gel filtration fraction enriched for T pilus with both anti-VirB2 and anti-VirB7 antiserum. Both the exocellular and cellular forms of VirB7 migrated as disulfide-cross-linked dimers and monomers when samples were electrophoresed under nonreducing conditions. A mutant synthesizing VirB7 with a Ser substitution of the lipid-modified Cys15 residue failed to elaborate the T pilus, whereas a mutant synthesizing VirB7 with a Ser substitution for the disulfide-reactive Cys24 residue produced very low levels of T pilus. Together, these findings establish that the VirB7 lipoprotein localizes exocellularly, it associates with the T pilus, and both VirB7 lipid modification and disulfide cross-linking are important for T-pilus assembly. T-pilus-associated VirB2 migrated in nonreducing gels as a monomer and a disulfide-cross-linked homodimer, whereas cellular VirB2 migrated as a monomer. A strain synthesizing a VirB2 mutant with a Ser substitution for the reactive Cys64 residue elaborated T pilus but exhibited an attenuated virulence phenotype. Dithiothreitol-treated T pilus composed of native VirB2 pilin and untreated T pilus composed of the VirB2C64S mutant pilin distributed in sucrose gradients more predominantly in regions of lower sucrose density than untreated, native T pili. These findings indicate that intermolecular cross-linking of pilin monomers is not required for T-pilus production, but cross-linking does contribute to T-pilus stabilization.     

DNA sequence of the F traALE region that includes the gene for F pilin

The complete sequence of a 1.4-kilobase PstI fragment containing the F transfer genes traA, -L, and -E is presented. The traA reading frame has been located both genetically and by comparing the primary structure of F pilin (the traA product) predicted by the DNA sequence to the amino acid composition and sequence of N- and C-terminal peptides isolated from purified F pilin. Taken together, these data show that there is a leader peptide of 51 amino acids and that F pilin contains 70 amino acids, giving molecular weights of 13,200 for F propilin and 7,200 for mature F pilin. Secondary structure predictions for F pilin revealed a reverse turn that precedes the sequence Ala-Met-Ala51, a classic signal peptidase cleavage site. The N-terminal alanine residue is blocked by an acetyl group as determined by 1H-nuclear magnetic resonance spectroscopy. The traL and traE genes encode proteins of molecular weights 10,350 and 21,200, respectively. According to DNA sequence predictions, these proteins do not contain signal peptide leader sequences. Secondary structure predictions for these proteins are in accord with traLp and traEp being membrane proteins in which hydrophobic regions capable of spanning the membrane are linked by sequences that form turns and carry positively charged residues capable of interacting with the membrane surface.     

Assembly of mutant pilins in Pseudomonas aeruginosa: formation of pili composed of heterologous subunits.

Recently, we reported the degree of N-terminal processing within the cytoplasmic membranes of three mutant pilins from Pseudomonas aeruginosa PAK with respect to leader peptide removal and the methylation of the N-terminal phenylalanine (B. L. Pasloske and W. Paranchych, Mol. Microbiol. 2:489-495, 1988). The results of those experiments showed that the deletion of 4 or 8 amino acids within the highly conserved N terminus greatly inhibited leader peptide removal. On the other hand, the mutation of the glutamate at position 5 to a lysine permitted leader peptide cleavage but inhibited transmethylase activity. In this report, we have examined the effects of these mutant pilins upon pilus assembly in a P. aeruginosa PAO host with or without the chromosomally encoded pilin gene present. Pilins with deletions of 4 or 8 amino acids in the N-terminal region were not incorporated into pili. Interestingly, pilin subunits containing the glutamate-to-lysine mutation were incorporated into compound pili together with PAO wild-type subunits. However, the mutant pilins were unable to polymerize as a homopolymer. When wild-type PAK and PAO pilin subunits were expressed in the same bacterial strain, the pilin subunits assembled into homopolymeric pili containing one or the other type of subunit.