Abstract
An Escherichia coli double mutant, sodAsodB, that is deficient in both bacterial superoxide dismutases (Mn superoxide dismutase and iron superoxide dismutase) is unable to grow on minimal medium in the presence of oxygen and exhibits increased sensitivity to paraquat and hydrogen peroxide. Expression of the evolutionarily unrelated eukaryotic CuZn superoxide dismutase in the sodAsodB E. coli mutant results in a wild-type phenotype with respect to aerobic growth on minimal medium and in resistance to paraquat and hydrogen peroxide. This supports the hypothesis that superoxide dismutation is the in vivo function of these proteins. Analysis of the growth of sodAsodB cells containing plasmids encoding partially active CuZn superoxide dismutases, produced by in vitro mutagenesis, shows a correlation between cell growth and enzyme activity. Thus, the sodAsodB strain provides a controlled selection for varying levels of superoxide dismutase activity.
Highlights
An Escherichia coli double mutant, sodAsodB, that is deficient in both bacterial superoxide dismutases (Mn superoxide dismutase and iron superoxide dismutase) is unable to grow on minimal medium in the presence of oxygen and exhibits increased sensitivity to paraquat and hydrogen peroxide
Our results indicate that human CuZn superoxide dismutase complements important phenotypic properties of the E. coli sodAsodB double mutant
T h e discovery that bovine erythrocuprein (CuZn superoxide dismutase) exhibits superoxide dismutase activity led to the E. coli Strains-E. coli K12 sodAsodB mutants were described hypothesis that this protein, assuperoxide scavenger, has a protective rolein the prevention of oxygen toxicity (1, 2)
Summary
Plasmids-pDT1-5 carriesthe sodA+ gene fromE. coli which that the superoxide dismutase activity of a cell is inversely encodes Mn superoxide dismutase (16). 2. Activity gel comparing superoxide dismutaselevels conferred by different plasmids and demonstrating the effect of charging CuZn superoxide dismutase with coEpapcher.lane received 5 pg of total cellular protein in disruption buffer with or without supplemental 2.0 mM CuSO,. Activity gel comparing superoxide dismutaselevels conferred by different plasmids and demonstrating the effect of charging CuZn superoxide dismutase with coEpapcher.lane received 5 pg of total cellular protein in disruption buffer with or without supplemental 2.0 mM CuSO, With this quantity of protein, extracts from pNco5AI137-containing cells do not produce a clearly visible band. Plating Efficiency on Rich and Minimal Media-Cells were grown overnight (37 "C, 200 rpm) to stationary phase in LB containing pg/ml ampicillinwith 1 mM CuS04, washedonce (centrifugation), serially diluted in phosphate-buffered saline, and plated(33pl) onto either LB or M9glucose agar medium.
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