Figure 3.

ToxR–TonB makes the pmf-dependent crosslink with ExbD. Crosslinking was performed as described in Section “Materials and Methods” with the following strains: cTonB (W3110 expressing chromosomally encoded TonB and ExbD); ΔTonB (KP1344); pTonB (KP1344/pKP325); pToxR–TonB (KP1344/pKP1529); and pToxR–TonB CCCP (KP1344/pKP1529 treated with 50 μM CCCP). l-Arabinose was added to induce expression of plasmid-encoded TonB as follows: 0.00075% for pTonB and 0.00046% for pToxR–TonB. Complexes were detected on immunoblots of 13% SDS-polyacrylamide gels using ExbD polyclonal antibodies (upper two panels). Uncrosslinked monomeric TonB and ToxR-TonB levels were visualized with the monoclonal anti-TonB antibody 4F1 (bottom panel). The molecular mass standards (kDa) are shown on the left.