Vir proteins stabilize VirB5 and mediate its association with the T pilus of Agrobacterium tumefaciens

Three VirB proteins (VirB1*, VirB2, and VirB5) have been implicated as puta tive components of the T pilus from Agrobacterium tumefaciens, which likely mediates binding to plant cells followed by transfer of genetic material. Recently, VirB2 was indeed shown to be its major component (E.-M. Lai and C . I. Kado, J. Bacteriol. 180:2711-2717, 1998). Here, the influence of other Vir proteins on the stability and cellular localization of VirB1*, VirB2, and VirB5 was analyzed. Solubility of VirB1* and membrane association of Vi rB2 proved to be inherent features of these proteins, independent of virule nce gene induction. In contrast, cellular levels of VirB5 were strongly red uced in the absence of other Vir proteins, indicating its stabilization by protein-protein interactions. The assembly and composition of the T pilus w ere analyzed in nopaline strain C58(pTiC58), its flagellum-free derivative NT1REB(pJK270), and octopine strain A348(pTiA6) following optimized virulen ce gene induction on solid agar medium. In all strains VirB2 was the major pilus component and VirB5 cofractionated during several purification steps, such as ultracentrifugation, gel filtration, and sucrose gradient centrifu gation. VirB5 may therefore be directly involved in pilus assembly, possibl y as minor component. In contrast, secreted VirB1* showed no association wi th the T pilus. In-frame deletions in genes virB1, virB2, virB5, and virB6 blocked the formation of virulence gene-dependent extracellular high-molecu lar-weight structures. Thus, an intact VirB machinery as well as VirB2 and VirB5 are required for T-pilus formation.