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

Abstract

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 genetic material and proteinaceous virulence factors. Here, we show that the absence of VirB6 leads to reduced cellular levels of VirB5 and VirB3, which were proposed to assist T pilus formation as minor component(s) or assembly factor(s), respectively. Overexpression of virB6 in trans restored levels of cell-bound and T pilus-associated VirB5 to wild type but did not restore 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 homodimer formation was abolished. This effect could not be restored by expression of VirB6 in trans. Expression of TraD, a component of the transfer machinery of the IncN plasmid pKM101, with significant sequence similarity to VirB6, restored neither protein levels nor bacterial virulence but partly permitted T pilus formation in a virB6 deletion strain. VirB6 may therefore regulate T pilus formation by direct interaction with VirB5, and wild-type levels of VirB3 and VirB7 homodimers are not required.

FIG. 1

Expression of VirB6 in wild-type strain C58 and virB6 deletion strain CB1006. (A) Analysis of subcellular fractions from virulence gene-induced (+AS) or uninduced (−AS) strain C58 after SDS-PAGE and Western blotting with VirB6-specific antiserum. T, total cell lysate; S, soluble fraction; M, membrane proteins. (B) Analysis of cell lysates from wild-type C58 (lanes 1) and virB6 deletion strain CB1006 carrying cloning vector pTrc200 (lane 2), pTrcB6 (lanes 3), or pTrcTraD (lanes 4) grown in the absence of induction, in the presence of AS for virulence gene induction, or in the presence of AS and IPTG for simultaneous induction of the trc promoter. Cell lysates were subjected to SDS-PAGE followed by Western blotting and detection with VirB6-specific antiserum. Arrowhead indicates aggregates of VirB6 detected in overexpressing cells. Numbers on the right are molecular masses of reference proteins in kilodaltons.

FIG. 2

Induction conditions affect levels of cell-bound virulence proteins. Wild-type strain C58 (wt) and virB6 deletion mutant CB1006 (Δ6) were grown on AB minimal medium plates at 20°C (lanes 1), in liquid culture at 20°C (lanes 2), or in liquid culture at 28°C (lanes 3). Cell lysates were subjected to SDS-PAGE followed by Western blotting and detection with specific antisera as indicated. Arrowheads indicate VirB proteins that accumulate to reduced levels in CB1006 compared to wild type only in liquid culture at 28°C. Numbers on the right are molecular masses of reference proteins.

FIG. 3

Effects of differential expression of VirB6 on other Vir proteins in cell lysates from wild-type strain C58 (lanes 1) and virB6 deletion strain CB1006 carrying cloning vector pTrc200 (lane 2), pTrcB6 (lanes 3), or pTrcTraD (lanes 4) grown on AB minimal medium plates at 20°C in the absence of induction, in the presence of AS for virulence gene induction, or in the presence of AS and IPTG for simultaneous induction of the trc promoter. Cell lysates were subjected to SDS-PAGE followed by Western blotting and detection with specific antisera as indicated. Arrowhead indicates putative degradation products of VirB5 detected in VirB6-overexpressing cells. Numbers on the right are molecular masses of reference proteins in kilodaltons.

FIG. 4

Complex formation of VirB7 and VirB9 in cell lysates wild-type strain C58 (lanes 1) and virB6 deletion strain CB1006 carrying cloning vector pTrc200 (lane 2), pTrcB6 (lanes 3), or pTrcTraD (lanes 4) grown on AB minimal medium plates at 20°C in the absence of induction, in the presence of AS for virulence gene induction, or in the presence of AS and IPTG for simultaneous induction of the trc promoter. Cell lysates were subjected to SDS-PAGE under nonreducing conditions followed by Western blotting and detection with VirB7- and VirB9-specific antisera. Numbers on the right are molecular masses of reference proteins in kilodaltons.

FIG. 5

Complementation of T pilus formation of strain CB1006. Extracellular T pili were isolated from wild-type strain C58 (lanes 1) and virB6 deletion strain CB1006 carrying cloning vector pTrc200 (lane 2), pTrcB6 (lanes 3), or pTrcTraD (lanes 4) grown on AB minimal medium plates at 20°C in the absence of induction, in the presence of AS for virulence gene induction, or in the presence of AS and IPTG for simultaneous induction of the trc promoter. T pilus fractions were subjected to SDS-PAGE, Western blotting, and detection with VirB2- and VirB5-specific antisera. Arrowhead indicates aberrantly migrating VirB2 in extracellular high-molecular mass structures isolated from TraD-expressing cells. Numbers on the right are molecular masses of reference proteins in kilodaltons.