FIG. 5.

Site-specific in vivo disulfide cross-linking between FecA (DALTV)/Cys and TonB/Cys as detected by anti-FecA and anti-TonB antisera. Pairwise combinations of strain CO1031 (tonB fecA) carrying each member of the series of pT7-7 FecA/Cys substitutions (D80C [DC], A81C [AC], L82C [LC], T83C [TC], and V84C [VC]) and TonBQ160C, TonBQ162C, and TonBY163C were grown in duplicate in NB. The cultures were adjusted to an A₅₇₈ of 1.0, whole-cell lysates were collected, and samples were electrophoresed on duplicate SDS-polyacrylamide gels followed by Western immunoblotting using either anti-TonB (A) or anti-FecA (B) antiserum. As negative controls, pairwise combinations of TonBC18A (containing no cysteine) together with FecA (DC) and TonB/Cys together with FecA (containing no cysteine) (WT) were included. The protein species detected are indicated on the right of each gel, and the molecular mass markers (in kilodaltons) are indicated on the left. An additional cross-linked species (X), which is likely to be a truncated TonB disulfide cross-linked to FecA (TonB′-FecA), was detected by both TonB and FecA antisera.