Mutation of rqlH rescues the rqlI mutant defect in vitro.

(A) A schematic of the screen used to search for suppressor mutations of the ΔrqlI mutant. Bacteria carrying a PsulA-lacZ reporter plasmid (pCWR2) were transposon-mutagenized and screened on tetrazolium lactose agar for suppressor mutants with decreased sulA promoter activity, (distinguishable by darker colony color). (B) A tetrazolium lactose plate demonstrating the colony colors for WT, F11ΔrqlI, and four independent F11ΔrqlIΔrqlH::tn transposon mutants that were isolated from the screen. (C) Bacteria carrying pJLJ3 (PsulA-GFP) were grown microaerobically and GFP levels and culture densities were measured to quantify sulA expression. (D) Microaerobic growth of WT, ΔrqlH, ΔrqlI, and ΔrqlHI strains, as measured by dilution plating of 24 h cultures. (E) Bacteria were grown aerobically for 4 h, at which point mitomycin C was added to a final concentration of 0.25 μg/mL for 1 h. The cultures were titered immediately before and 1 h after addition of mitomycin C, and survival was calculated. (F) WT and ΔrqlI cells carrying either an empty vector control (pRR48), or the RqlI expression plasmid pCWR16 were grown and tested for mitomycin C survival as in (E). (G) Bacteria were grown aerobically for 4 h, then diluted and spread onto plates to get ~100 colonies per plate. The plates were then exposed to various levels of UV radiation and incubated overnight, at which time percent survival was calculated. In C-G, bars indicate mean values ± SEM from three or more independent experiments performed in triplicate. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001 in comparison with WT; as determined by Student’s t tests. (H) Bacteria were grown either for 4 h aerobically or 24 h microaerobically. Aliquots from each culture were used to titer viable bacteria, and the remaining cultures were plated onto agar containing nalidixic acid. The numbers of nalidixic acid resistant mutants recovered were used as input for FALCOR to calculate mutation rates (number of mutations per 1010 cells per generation). Bars indicate mean values ± 95% confidence intervals.