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

Abstract

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.T pili are generated when Agrobacterium tumefaciens cells are induced by plant phenolic compounds such as acetosyringone. Induction leads to the expression of virulence (vir) genes located on a resident Ti plasmid (10). Of these vir genes, the 11 genes of the virB operon are involved in the synthesis and assembly of the T pilus (5). The T pilus is composed mainly of processed VirB2 protein (6), with VirB5 (12) and VirB7 (11) as associated components. Jones et al. (4) first characterized the processing steps of VirB2 that is cleaved into a 7.2-kDa product in both Escherichia coli and A. tumefaciens. Further studies by mass spectrometry (MS) demonstrated that the processed VirB2 product is composed of 74 amino acid residues linked by a peptide bond between the amino-and carboxylterminal residues to generate a cyclic peptide (the T pilin) (1). T pilin subunits are transported across the bacterial membranes and assembled into an exocellular, flexuous T pilus filament 10 nm in diameter protruding from the bacterial cell wall (5-7). Transport of T pilin subunits to the cell surface requires each of the 11 genes of the virB operon (5), while propilin cleavage can occur independently of any other gene of the Ti plasmid (1). The 47-residue signal peptide is cleaved off amino terminally from VirB2 propilin, most likely by a general signal peptidase I, LepB (1, 4, 13). Whether peptidyl cyclization of the resulting 74-residue peptide takes place simultaneously or is an individual reaction remains unclear.In the present study, we examined the propilin cleavage and cyclization reactions that lead to T pilin formation in E. coli and A. tumefaciens and addressed the roles of these reactions in T pilus biogenesis and tumorigenesis.T pilin is cyclized in A. tumefaciens but not in E. coli. Based on immunoblotting and pulse-chase analyses, it was demonstrated that VirB2 propilin is cleaved in A. tumefaciens, as well as in E. coli (4). To determine whether the processed VirB2 protein is cyclized in E. coli like that in A. tumefaciens, we expressed virB2 under tac promoter control in both E. coli and A. tumefaciens as previously described (1). VirB2 was induced by isopropyl-␤-D-thiogalactopyranoside (IPTG) in E. coli and A. tumefaciens (in the absence of the Ti plasmid), and cells were analyzed by matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) MS and by immunoblotting with anti-VirB2 serum as previously described (1, 6). Briefly,...

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

“…As might be expected, some substitutions near the processing/cyclization junction were destabilizing (Cys51 and Cys54) or blocked protein function without discernible effects on stability (Cys117). Similar effects were described earlier for mutations located near the cleavage site but within the signal sequence (47). However, some substitutions near the cyclization junction (Cys54, Cys58, and Cys119) selectively blocked substrate transfer without affecting T pilus biogenesis.…”

“…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).…”

Structures of Naturally Occurring Circular Proteins from Bacteria

“…However no TraF homolog is present on the Ti plasmid. Interestingly, cyclization of T pilin was found to occur in Agrobacterium tumefaciens but not in E. coli, indicating that the enzyme or mechanism responsible for cyclization is of chromosomal origin and species specific (43).…”

“…A characteristic of IncP pili is their tendency to aggregate in bundles (21), which suggests that the surface of the pili is hydrophobic, and therefore it can be surmised that at least some extended hydrophobic areas exist on the surface of the TrbC subunits comprising the pili. On polyacrylamide gels, the linear form of T pilin has lower mobility than the mature circular protein, indicating that the three-dimensional structures of the two proteins most likely differ significantly (43). Finally, secondary-structure prediction programs have identified two putative transmembrane helices in both the circular pilins (21).…”

“…Despite being very similar in function and size (78 versus 74 amino acids), TrbC and T pilin do not show a high degree of sequence similarity. However, there seem to be several conserved residues across various pilin homologues within the core region, including six absolutely conserved glycine residues (21,43).…”