Conjugative Pili of IncP Plasmids, and the Ti Plasmid T Pilus Are Composed of Cyclic Subunits

Abstract

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 mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…Sex pili are composed of a small subunit, the pilin (VirB2), which assembles into a Wlamentous pilus structure at the cell surface (Eisenbrandt et al, 1999;Lai and Kado, 1998). Two additional proteins, VirB5 and VirB7, localize to the pilus (Sagulenko et al, 2001b;Schmidt-Eisenlohr et al, 1999a).…”

“…In case of the F plasmid pilin (TraA) signal peptide cleavage is followed by N-acetylation of the N-terminus (Moore et al, 1993). The pilins of A. tumefaciens (VirB2) and RP4 (TrbC) undergo an unusual kind of modiWcation: N-and C-termini of the precursors form a peptide bond resulting in cyclic proteins, composed of 74 and 78 aa, respectively (Eisenbrandt et al, 1999). In the A. tumefaciens T-pilus, VirB2 forms disulWde-cross-linked homodimers that are important for T-pilus stability (Sagulenko et al, 2001b).…”

Dimerization and interactions of Brucella suis VirB8 with VirB4 and VirB10 are required for its biological activity

“…The last group of T4SS components is that of surface-exposed proteins. VirB2 is the major pilus component (14,15) and VirB5 the minor component (16), and VirB7 and VirB3 also associate with the pilus or its components (17,18). Whereas most mechanistic studies have been performed with the model Agrobacterium tumefaciens, it is clear that there is some variation among the T4SSs from different organisms.…”

Spatial location and requirements for the assembly of the Agrobacterium tumefaciens type IV secretion apparatus

“…Ten VirB proteins (VirB2-VirB11) are essential for DNA transfer and the assembly of a T-pilus composed primarily of VirB2 (9)(10)(11). The VirB2 homolog of the Escherichia coli plasmid RP4, TrbC, assembles a RP4 pilus (12). VirB3, an F plasmid TraL homolog, and VirB5 are minor components of the T-pilus (13,14).…”