Survey of RpoS activity in evolved populations and phenotype study of a Δ rpoS mutant. RpoS activity was assayed in evolved populations and selected single mutants using an I2 staining assay and Western blot analysis. (A) I2 staining assay. Overnight cultures were streaked on glucose minimal medium agar spiked with 0.35% (w/v) isobutanol, incubated at 30°C for 48 hours, and then stained with USP tincture of iodine. Samples are as follows (from top left, to bottom right): E. coli EcNR1 ΔacrA::kan (ΔacrA), E. coli EcHW24 miaA-hfq 4407505 -7:AGGAAAA (hfq*), E. coli EcNR1 (WT), Xylose #1 end population (X1), Xylose #2 end population (X2), Xylose #3 end population (X3), Glucose #1 end population (G1), Glucose #2 end population (G2), Glucose #3 end population (G3). (B) Western blot analysis of RpoS in total cellular protein extracted from cultures of E. coli EcNR1 (WT), G3.2, and E. coli EcHW24 miaA-hfq 4407505 -7:AGGAAAA (hfq*) grown to early exponential phase either with (+) or without (-) 0.5% (w/v) isobutanol in NG50 medium. Experiment was repeated several times to verify results; representative Western blot shown. Purified E. coli RpoS (NeoClone) was used as a positive control (ctrl). (C) Phenotype study of a ΔrpoS mutant. E. coli BW25113 ΔrpoS::kan (obtained from the Keio collection ; strain # JW5437-1) and parent strain E. coli BW25113 were grown in 0%, 0.5%, and 1% (w/v) isobutanol glucose media. To facilitate comparison, we report normalized relative fitness (RF/RF0%), defined as relative fitness divided by relative fitness at 0% (w/v) isobutanol; relative fitness (RF) was calculated as μΔrpoS/μWT where μΔrpoS is the maximum specific growth rate (h-1) of E. coli BW25113 ΔrpoS::kan and μWT is the maximum specific growth rate (h-1) of E. coli BW25113.