Fig. 4. V. cholerae EF-Tu is exquisitely sensitive to oxidative thiol modifications.

To determine the redox status of EF-Tu in vivo, E. coli MC4100 and V. cholerae O395 were grown in LB medium at 37°C under aerobic conditions until mid-log phase was reached. Cells were either left untreated or were treated with 3 mM HOCl for 20 min. Samples were taken and cysteines were labeled with NEM, followed by the reduction of all oxidized thiols and the labeling of all newly reduced cysteines with the 500 Da thiol-alkylating molecule AMS. Addition of AMS molecules is visualized as migration difference on SDS-PAGE, whose extent directly reflects the number of in vivo oxidized cysteines. To define the migration behavior of fully NEM-labeled EF-Tu (equivalent to reduced species) and fully AMS labeled EF-Tu (equivalent to completely oxidized species), aliquots of non-stressed MC4100 or O395 cells (indicated by E.c. and V.c., respectively) were reduced with DTT and labeled exclusively with either NEM (lanes 1 and 2) or AMS (lanes 3 and 4). Proteins were separated on SDS-PAGE and EF-Tu was visualized with Western blot analysis using antibodies against E. coli EF-Tu.