FIG. 3.

The KefB and KefC systems, RpoS, and Dps can provide protection against NEM independently. Cells were grown in K₁₀ medium, and cell viability and Western blot assays were conducted with stationary-phase cells exactly as described in Materials and Methods. (a) NEM was added at 0.1 mM to cells of MJF274 (•), MJF276 (kefB kefC::Tn10; ▪), MJF358 (rpoS::kan; ⧫), MJF371 (dps::kan; ▴), MJF359 (kefB kefC::Tn10 rpoS::kan; ○), and MJF376 (kefB kefC::Tn10 dps::kan; □) at time zero, and cell viability was determined. (b) Western blot analysis using an RpoS monoclonal antibody. Lane 1, MJF274, no addition; lane 2, MJF274, 0.2 mM NEM (10 min); lane 3, MJF274, 0.2 mM NEM (30 min); lane 4, MJF276, no addition; lane 5, MJF276, 0.2 mM NEM (10 min); lane 6, MJF276, 0.2 mM NEM (30 min). Lanes 7 to 12 were the same except that sodium acetate was added to a final concentration of 50 mM instead of NEM. (c) NEM was added at 0.1 mM to cells of MJF359 (kefB kefC::Tn10 rpoS::kan; ○), MJF413 (kefB kefC::Tn10 dps::cam; □), and MJF411 (kefB kefC::Tn10 rpoS::kan dps::cam; ▵) at time zero, and cell viability was determined. The arrows represent no viable cells by the next time point; error bars represent the standard deviation from the mean for one experiment.