ISOLATION AND CHARACTERIZATION OF OXYGEN SENSITIVE MUTANTS OF ESCHERICHIA-COLI

Fifty-five oxygen sensitive mutants of Escherichia coli K-12 were isolated by chemical mutagenesis, two of which were manganese superoxide dismutase (Mn-SOD) defective mutants. These mutants (no. 34, no. 58) could grow anaerobically in minimal medium, but not under aerobic conditions. Mn-SOD of no. 58 was induced by adding paraquat or o-phenanthroline. However, the induction level was much lower than that of the wild-type strain without induction, inferring that strain no. 58 is a repressor-overproducing mutant. Strain no. 34 produced a mutant Mn-SOD enzyme (M') which migrated slowly in native PAGE. Protein M' was purified from strain no. 34 and the molecular weight was determined by gel filtration. Protein M' was found to be a dimer of identical subunits, as is the wild-type enzyme. The specific activity of M' is the same as that of the wild type. From these results, it was inferred that the mutant enzyme (M') may be more positively charged or less negatively charged than the wild type and that the productivity of M' was much lower than that of the wild-type enzyme. It was found that multicopies of the ilvD gene complemented the auxotrophic properties of mutant no. 34 under aerobic conditions. Since the gene product (alpha,beta-dihydroxyisovalerate dehydratase) is very susceptible to inactivation by superoxide radicals, a large amount of the enzyme must be needed for strain no. 34 to grow aerobically. In contrast, such a complementary gene was not cloned in strain no. 58.