VirB6 is required for stabilization of VirB5 and VirB3 and formation of VirB7 homodimers in Agrobacterium tumefaciens

VirB6 from Agrobacterium tumefaciens is an essential component of the type IV secretion machinery for T pilus formation and genetic transformation of plants. Due to its predicted topology as a polytopic inner membrane protein , it was proposed to form the transport pore for cell-to-cell transfer of g enetic material and proteinaceous virulence factors. Here, we show that the absence of VirB6 leads to reduced cellular levels of VirB5 and VirB3, whic h were proposed to assist T pilus formation as minor component(s) or assemb ly factor(s), respectively. Overexpression of virB6 in trans restored level s of cell-bound and T pilus-associated VirB5 to wild type but did not resto re VirB3 levels. Thus, VirB6 has a stabilizing effect on VirB5 accumulation , thereby regulating T pilus assembly. In the absence of VirB6, cell-bound VirB7 monomers and VirB7-VirB9 heterodimers were reduced and VirB7 homodime r formation was abolished. This effect could not be restored by expression of VirB6 in trans. Expression of TraD, a component of the transfer machiner y of the IncN plasmid pKM101, with significant sequence similarity to VirB6 , restored neither protein levels nor bacterial virulence but partly permit ted T pilus formation in a virB6 deletion strain. VirB6 may therefore regul ate T pilus formation by direct interaction with VirB5, and wild-type level s of VirB3 and VirB7 homodimers are not required.