β-galactosidase accumulation from a ctc-lacZ fusion in logarithmically growing cells, before and after 4% ethanol addition. (A) Wild type stressosome with all four co-antagonists and wild type RsbS and RsbRA (PB198, open circles); RsbS-S59A (PB470, closed circles); RsbRA-T171A (PB830, open squares); or RsbS-S59A together with RsbRA-T171A (PB1219, closed squares). (B) Minimal stressosome containing RsbRA as sole co-antagonist and wild type RsbS and RsbRA (PB1078, open circles); RsbS-S59A (PB1161, closed circles); RsbRA-T171A (PB1205, open squares); or RsbS-S59A with RsbRA-T171A (PB1190, closed squares). Representative results are shown; in independent experiments the T171A-S59A mutant manifested a response 0.2% that of wild type RsbRA and RsbS in the strain with all four co-antagonists (+/−0.03% SEM, n = 3); and 21% in the strain with RsbRA alone (+/−5.8%, n = 3).