FIG. 7.

Nonspecific in vivo cross-linking between TonB and FecA is enhanced in the presence of sodium citrate. Cross-linking experiments were performed in E. coli strain CO1031 carrying TonBC18ANP and either pBAD18 or pBADfecA (wild type [WT]). The cells were grown in NB (0.2% arabinose) for 4 h, adjusted to an A₅₇₈ of 1.0, and resuspended in 10 mM sodium phosphate buffer (pH 6.8). The cells were incubated for a further 15 min in the presence (+) or absence (−) of 1 or 10 mM sodium citrate premixed with 100 μm FeCl₃. One percent FA was added to some of the cultures (+), and the cultures were incubated for a further 25 min. Whole-cell lysates were collected and subjected to SDS-PAGE followed by Western immunoblotting using anti-TonB antisera. The molecular mass markers (in kilodaltons) are indicated on the left. However, as FA cross-links are heat labile, samples were not boiled, so it is not possible to designate the exact molecular masses of protein species based on mobility in SDS-PAGE. TonB cross-linked with FecA is indicated.