FIG. 1.

Cross-linking conditions influence the stability of VirB10 in Agrobacterium cells lacking wild-type VirB9. Cells of wild-type strain A348 (lanes 1 to 6) or strain Ax42 (virB9::Tn5virB) were induced overnight at 25°C in the presence of 200 μM AS. Cells were pelleted and incubated in 50 mM sodium phosphate buffer, pH 7.6, either at room temperature (RT) (lanes 1 to 4 and 7 to 10) or on ice (lanes 5 to 6 and 11 to 12) for the indicated lengths of time. For the samples in lanes 4, 6, 10, and 12, the cross-linking agent BS³ was included in the incubation at a final concentration of 500 μM (X). At the end of the incubation, cells were quenched with an equal volume of 50 mM Tris-Cl, pH 7.5, and processed for SDS-PAGE and immunoblotting analysis with anti-VirB10 antibodies, as described in Materials and Methods. The migration positions of proteins of known molecular mass (in kilodaltons) are shown at the left.