Figure 1.
Development of an in vivo disulfide cross-linking protocol. Cells of strain MC4100ΔBC (ΔtatBC) harbouring plasmid p101C*BC producing TatBL9C alongside TatCM205C were incubated with either LB medium (control, C), or LB supplemented with 10 mM DTT (reduced; R) or (a) the indicated concentrations of CuP for 15 min or (b) with 1.8 mM CuP for 1–15 min. The reaction was quenched by addition of 8 mM NEM/12 mM EDTA, membranes were prepared and proteins were separated by SDS-PAGE (10% polyacrylamide). Cross-linked products were visualized by immunoblotting using anti-TatBFL or anti-TatC antibodies, as indicated. (c) Aliquots of cells from the oxidized and control samples in (b) were spread on LB plates containing chloramphenicol and the number of colonies enumerated following growth at 37°C for 24 h. The y-axis shows the ratio of the number of colony forming units (cfu) obtained after incubation with 1.8 mM CuP compared to the number after incubation in LB medium only; n = 3 biological replicates, error bars are ±s.d.