TraC of IncN Plasmid pKM101 Associates with Membranes and Extracellular High-Molecular-Weight Structures in Escherichia coli

Abstract

Conjugative transfer of IncN plasmid pKM101 is mediated by the TraI-TraII region-encoded transfer machinery components. Similar to the case for the related Agrobacterium tumefaciens T-complex transfer apparatus, this machinery is needed for assembly of pili to initiate cell-to-cell contact preceding DNA transfer. Biochemical and cell biological experiments presented here show extracellular localization of TraC, as suggested by extracellular complementation of TraC-deficient bacteria by helper cells expressing a functional plasmid transfer machinery (S. C. Winans, and G. C. Walker, J. Bacteriol. 161:402–410, 1985). Overexpression of TraC and its export in large amounts into the periplasm of Escherichia coliallowed purification by periplasmic extraction, ammonium sulfate precipitation, and column chromatography. Whereas TraC was soluble in overexpressing strains, it partly associated with the membranes in pKM101-carrying cells, possibly due to protein-protein interactions with other components of the TraI-TraII region-encoded transfer machinery. Membrane association of TraC was reduced in strains carrying pKM101 derivatives with transposon insertions in genes coding for other essential components of the transfer machinery,traM, traB, traD, andtraE but not eex, coding for an entry exclusion protein not required for DNA transfer. Cross-linking identified protein-protein interactions of TraC in E. coli carrying pKM101 but not derivatives with transposon insertions in essentialtra genes. Interactions with membrane-bound Tra proteins may incorporate TraC into a surface structure, suggested by its removal from the cell by shearing as part of a high-molecular-weight complex. Heterologous expression of TraC in A. tumefaciens partly compensated for the pilus assembly defect in strains deficient for its homolog VirB5, which further supported its role in assembly of conjugative pili. In addition to its association with high-molecular-weight structures, TraC was secreted into the extracellular milieu. Conjugation experiments showed that secreted TraC does not compensate transfer deficiency of TraC-deficient cells, suggesting that extracellular complementation may rely on cell-to-cell transfer of TraC only as part of a bona fide transfer apparatus.

Use of chimeric type IV secretion systems to define contributions of outer membrane subassemblies for contact‐dependent translocation

“…Accordingly, we propose that the pilus tip protein is recruited to and forms specific contacts with the OMCC as a prerequisite for pilus extension from the cell surface. Recruitment may occur at the extracellular surface, in view of evidence that VirB5-like subunits are exported across the OM independently of the T4SS (Schmidt-Eisenlohr et al, 1999). However, pilus tip proteins also have been reported to interact with IMC components (Yuan et al, 2005;Villamil Giraldo et al, 2012), raising the alternative possibility of their engagement with the T4SS in the periplasm.…”

The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights

“…In the presence of recipient cells, TraA is mobilized by an unknown mechanism to the surface of the donor cell where it polymerizes to form the pilus. In contrast, Vir-induced cultures of Agrobacterium assemble T-pili without host plant cells (Lai and Kado, 1998;Schmidt-Eisenlohr et al, 1999a). Strains of Agrobacterium with deletions of virB3, virB4, virB5, virB9 or virB10 produce VirB2, but the processed form accumulates inside rather than on the exterior of the cell, and pili are not observed (Lai and Kado, 1998).…”

“…In addition to VirB2, VirB5 is found as a minor component in T-pili preparations (Schmidt-Eisenlohr et al, 1999a). TraC, the IncN VirB5 homolog, also associates with an exocellular polymeric structure that may be the pilus of pKM101 (IncN) (Schmidt-Eisenlohr et al, 1999b) thus suggesting that VirB5 homologs may function as auxiliary structural proteins in pili of type IV secretion systems (Figure 2a). In contrast to VirB2, cellular levels of VirB5 were strongly correlated with the abundance of other Vir proteins, indicating its stabilization by protein±protein interactions (Schmidt-Eisenlohr et al, 1999a).…”

“…Thus, additional VirB components, as well as VirB2 and VirB5, are required for T-pilus formation. Isolated pili have terminal knobs, but it is unknown whether these knobs reside at the pilus tip or base anchoring the pilus in the bacterial envelope (Schmidt-Eisenlohr et al, 1999a). By analogy to pap±pili adhesins, the knob may be a distal feature of the T-pilus that mediates interaction with the plant cell.…”

A bacterial conjugation machinery recruited for pathogenesis

“…Functional interchangeability of homologous components between related T4SS has been reported in only a few instances (Schmidt-Eisenlohr et al, 1999). Based on the exceptional amino acid identities between Trw homologues of B. tribocorum and R388, we tested for heterologous complementation of a trwD mutant in R388 (Rivas et al, 1997) with the trwD gene of B. tribocorum.…”