Roles of respiratory oxidases in protecting Escherichia coli K12 from oxidative stress

Isogenic strains of Escherichia coli that were defective in either of the t wo major aerobic terminal respiratory oxidases (cytochromes bo' and bd) or in the putative third oxidase (cytochrome bd-II) were studied to elucidate role(s) for oxidases in protecting cells from oxidative stress in the form of H2O2 and paraquat. Exponential phase cultures of all three oxidase mutan ts exhibited a greater decline in cell viability when exposed to H2O2 stres s compared to the isogenic parent wild-type strain. Cytochrome bo' mutants showed the greatest sensitivity to H2O2 under all conditions studied indica ting that this oxidase was crucial for protection from H2O2 in E. coli. Cel l killing of all oxidase mutants by H2O2 was by an uncharacterized mechanis m (mode 2 killing) with cell growth rate affected. The expression of Phi(ka tG-lacZ), an indicator of intracellular H2O2, was 2-fold higher in a cydAB: :kan mutant compared to the wild-type strain at low H2O2 concentrations (< 100 mu M) suggesting that cytochrome bd mutants were experiencing higher in tracellular levels of H2O2. Protein fusions to the three oxidase genes demo nstrated that expression of genes encoding cytochrome bd, but not cytochrom e bo' or cytochrome bd-II was increased in the presence of external H2O2. T his increase in expression of Phi(cydA-lacZ) by H2O2 was further enhanced i n a cyo::kan mutant. The level of cytochrome bd determined spectrally and P hi(cydA-lacZ) expression was 5-fold and 2-fold higher respectively in an rp oS mutant compared to isogenic wild-type cells suggesting that RpoS was a n egative regulator of cytochrome bd. Whether the effect of RpoS is direct or indirect remains to be determined.