FIG. 6.

Site-specific in vivo disulfide cross-linking between FecA and TonB is not enhanced by the presence of sodium citrate. Pairwise combinations of strain CO1031 (tonB fecA) carrying each member of the series of pBADFecA/Cys substitutions (D80C [DC], A81C [AC], L82C [LC], T83C [TC], and V84C [VC]) and TonBQ160C, TonBQ162C, and TonBY163C were grown in duplicate in NB (0.2% arabinose). Sodium citrate (1 mM) was added to one set (+) 15 min prior to harvesting. The cultures were adjusted to an A₅₇₈ of 1.0, and whole-cell lysates were collected and subjected to SDS-PAGE followed by Western immunoblotting using anti-TonB antisera. As negative controls, pairwise combinations of TonBC18A together with FecA (DC) and TonB/Cys together with FecA (wild type [WT]) were included. TonB cross-linked to FecA is indicated on the right.