Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life?
Mu transposons carrying the chloramphenicol resistance marker have been inserted into the cloned Escherichia coli genes sodA and sodB coding for manganese superoxide dismutase (MnSOD) and iron superoxide dismutase (FeSOD) respectively, creating mutations and gene fusions. The mutated sodA or sodB genes were introduced into the bacterial chromosome by allelic exchange. The resulting mutants were shown to lack the corresponding SOD by activity measurements and immunoblot analysis. Aerobically, in rich medium, the absence of FeSOD or MnSOD had no major effect on growth or sensitivity to the superoxide generator, paraquat. In minimal medium aerobic growth was not affected, but the sensitivity to paraquat was increased, especially in the sodA mutant. A sodA sodB double mutant completely devoid of SOD was also obtained. It was able to grow aerobically in rich medium, its catalase level was unaffected and it was highly sensitive to paraquat and hydrogen peroxide; the double mutant was unable to grow aerobically on minimal glucose medium. Growth could be restored by removing oxygen, by providing an SOD-overproducing plasmid or by supplementing the medium with the 20 amino acids. It is concluded that the total absence of SOD in E. coli creates a conditional sensitivity to oxygen.
D000332:Aerobiosis; D002701:Chloramphenicol / Q000494:pharmacology; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D007700:Kinetics; D009154:Mutation; D010269:Paraquat / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism
D004251:DNA Transposable Elements; D002701:Chloramphenicol; D013482:Superoxide Dismutase; D004262:DNA Restriction Enzymes; D010269:Paraquat