The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein

Abstract

The process of T-DNA transfer from Agrobacterium tumefaciens to plant cells is thought to involve passage of a DNA-protein complex through a specialized structure in the bacterial membrane. The virB operon of A. tumefaciens encodes 11 proteins, of which 9 are known to be located in the membranes and 10 have been shown to be essential for virulence. Sequence comparisons between proteins encoded by the virB operon and those encoded by operons from conjugative plasmids indicated that VirB proteins may form a structure similar to a conjugative pilus. Here, we examine the effects of mutations in virB4 on the accumulation and localization of other VirB proteins. VirB4 shares amino acid sequence similarity with the TraC protein of plasmid F, which is essential for pilus formation in Escherichia coli, and with the PtlC protein of Bordetella pertussis, which is required for toxin secretion. Polar and nonpolar virB4 mutants were examined, and all were shown to be unable to accumulate VirB3 protein to wild-type levels. A low level of VirB3 protein which was present in induced NT1RE cells harboring virB4 nonpolar mutant pBM1130 was found to associate with the inner membrane fraction only, whereas in wild-type cells VirB3 associated with both inner and outer membranes. The results indicate that for VirB3 to accumulate in the outer membrane, VirB4 must also be present, and it is possible that one role of VirB4 is in the correct assembly of a VirB protein membrane structure.

Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens

“…Separation by DDM suggests that their interaction with the T4SS complex can easily be disrupted, and it will be interesting to identify their binding partners. VirB3 is located in the outer membrane in a VirB4-dependent manner (45). Inefficient extraction with DDM suggests that VirB3 may associate with VirB4 more closely than anticipated, and it may play a role in VirB4-mediated transmission of energy for pilus assembly.…”

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

“…Stable expression of VirB3 requires the presence of the Mpf components VirB4 and VirB6 (Hapfelmeier et al, 2000;Jones et al, 1994). Since VirB4 is also required for targeting of VirB3 to the outer membrane (Jones et al, 1994), it is likely that VirB3 and VirB4 retain a close interaction in the Mpf complex, a presumption supported by the fact that VirB3 and VirB4 are encoded by a single gene (TriC) in case of the Cos100 conjugation system (Strauch et al, 2003). A second interaction partner of VirB3 may be the pilus-associated protein VirB5, which has been detected to interact with VirB3 in a yeast two-hybrid screen, a result which was conWrmed by in vitro data (Shamaei-Tousi et al, 2004).…”

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

“…While a connection to T-pilus assembly has not been established for either VirB4 or VirB3, they may physically interact. In a virB4 deletion strain, cellular levels of VirB3 are reduced, Transfer of DNA from A. tumefaciens 21 and VirB3 is localized to the inner membrane rather than both inner and outer membranes as in wild-type strains (Jones et al, 1994). VirB3 speci®c localization, mediated by VirB4, may promote mobilization of VirB2 to the exterior of the cell.…”

“…The homology between VirB4 and VirB3 and the IncF proteins TraL and TraC, respectively, suggests that these Agrobacterium proteins have a role in pilus assembly (Jones et al, 1994). The IncF proteins are essential for pilus formation but are not pilus structural components.…”