Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life?

Abstract

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.

Implication of (Mn)superoxide dismutase of Enterococcus faecalis in oxidative stress responses and survival inside macrophages

“…H 2 O 2 can diffuse rapidly through cell membranes. A higher sensitivity of SOD mutants to exogenous H 2 O 2 has been reported (Carlioz & Touati, 1986;Poyart et al, 2001). Since only insignificant differences in growth characteristics were observed between the wild-type and the DsodA mutant, it may be concluded that the quantity of ROS produced by Ent.…”

“…O N{ 2 is a powerful oxidant that reacts with biomolecules, including DNA, at virtually diffusion-limited rates. Thus, the higher free iron levels in SOD mutants also explain why they are more sensitive to killing by H 2 O 2 than their wild-type counterparts (Carlioz & Touati, 1986). This effect of H 2 O 2 can be blocked by cellpermeable iron chelators, confirming that the killing is mediated by Fenton chemistry (Imlay & Linn, 1988).…”

Iron is required to relieve inhibitory effects on NifL on transcriptional activation by NifA in Klebsiella pneumoniae

“…Samples of the culture were harvested at different times during growth, and the cells were suspended in potassium phosphate buffer (100 mM; pH 7.0). They were disrupted by five cycles of freezing and thawing (dry ice-42ЊC) (10). Protein concentrations were determined by the method of Bradford (7) with the Bio-Rad protein assay (6), using bovine serum albumin (BSA) as standard.…”

Cellular Defenses against Superoxide and Hydrogen Peroxide

“…This deficiency may arise from the oxidation of the dihydroxyethyl thiamine intermediate of transketolase (26), but firm structural evidence is lacking. SOD mutants of both E. coli and S. cerevisiae are also unable to perform normal sulfur metabolism (12,27); in this case the causal lesion remains completely unknown. It is likely that additional injuries remain to be identified.…”