TrbC propilin is the precursor of the pilin subunit TrbC of IncP conjugative pili in Escherichia coli. Likewise, its homologue, VirB2 propilin, is processed into T pilin of the Ti plasmid T pilus in Agrobacterium tumefaciens. TrbC and VirB2 propilin are truncated post-translationally at the N terminus by the removal of a 36/47-residue leader peptide, respectively. TrbC propilin undergoes a second processing step by the removal of 27 residues at the C terminus by host-encoded functions followed by the excision of four additional C-terminal residues by a plasmid-borne serine protease. The final product TrbC of 78 residues is cyclized via an intramolecular covalent head-to-tail peptide bond. The T pilin does not undergo additional truncation but is likewise cyclized. The circular structures of these pilins, as verified by mass spectrometry, represent novel primary configurations that conform and assemble into the conjugative apparatus.Horizontal gene transfer mediated by plasmid-borne conjugation from donor to recipient cells is initiated by a cell-to-cell bridging arrangement. These cellular interactions, whose complex is named mating pair formation (Mpf), 1 are initiated by conjugative pili encoded by broad-host range plasmids in Gram-negative bacteria. In the case of Agrobacterium-mediated T-DNA transfer to plant cells, the VirB complex is the Mpf structure (1, 2).Pili are usually long thin filaments extending from the surface of donor cells and upon close examination appear as tubelike structures with an outer diameter of about 8 -10 nm and a central, hydrophilic lumen of 2 nm (3, 4). With both the broadhost range plasmids of the IncP group and the Ti plasmids of the IncRh1 group, promiscuous pilus production requires genes of the Mpf system (5) and the virB operon (6, 7), respectively.Like the sex pili of F (8), pili of the above plasmids are essential for conjugative DNA transfer. Functional dissection of the Mpf apparatus and the virB genes has shown that DNA transfer is abolished by nonpolar inactivation of the pilin precursor genes or any gene of the pilus assembly machinery (5, 9). Besides DNA transfer, the transport of pilin subunits via the virB-encoded apparatus has been proposed to erect the T pilus (10). It remains unknown, however, if the transmembrane structures of the Mpf system and VirB complex are used as a channel to transfer DNA. Certainly, there exists a close evolutionary relationship between the IncP conjugative apparatus and the T-DNA transfer system (11,12). In this regard, the bacterial reliance of the pilus for DNA transfer raises a key question on whether or not the conjugative pilus is directly involved not only in bridging the donor to the recipient cell but also serving as a molecular conduit in DNA transmission. This resurrects an earlier key question raised on the possibility that the sex pilus, which appears to be tubular in structure, is used by the donor cell to transfer F-DNA rather than by direct cell-to-cell contact (3, 13). In this regard, the analyses of the structure as w...
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
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,...
The largest circular protein structures discovered define a class of transfer proteins acting in bacterial conjugation and type IV secretion. Proteins ranging from 73 to 78 residues with head-to-tail peptide bonds constitute the major subunit of conjugative pili of some type IV secretion systems. Their plasmid-encoded precursors are enzymatically processed and cyclized before being assembled into pili. These extra-cellular surface filaments mediate physical contact between donor and recipient cell or pathogen and host cell. Pili are essential prerequisites for DNA and protein transfer. A membrane-bound signal peptidase-like enzyme is responsible for the circularization reaction. Site-directed mutagenesis and mass spectrometry has been used extensively to unravel the mechanism of the enzyme-substrate interaction of the pilin maturation process.
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