Characterization of succinate minus mutants of Escherichia Coli deficient in membrane-bound oxidoreductase activity



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N-methyl-N'-nitro-N-nitrosoguanidine treatment was employed to isolate Escherichia coli mutants having altered membrane-bound oxidoreductase activity. Mutants were selected for their ability to grow on glucose but not succinate as a sole carbon source. Five mutants were chosen for further study based on their differences in ability to grow on oxidizable substrates other than succinate. Mutant strain R23 failed to grow on glycerol, lactate, acetate and TCA intermediates and had negligible ability to oxidize succinate, formate and DL-lactate. Membrane analysis showed markedly reduced cytochrome content and low but significant oxidation of only NADH. Several classes of R23 transductants were isolated which had simultaneously regained the ability to grow on succinate, glycerol and lactate. Only one transductant class was able to oxidize formate. Mutant strain R25-1 exhibited leaky growth on lactate and demonstrated a conditional oxidation pattern. When grown on glucose, mutant R25-1 oxidized formate but was unable to oxidize this substrate when grown on fructose or glycerol. The same conditional pattern was observed for the membrane associated oxidation of formate and NADH. A drastic decrease also was noted in total cytochrome content of membranes from fructose grown R25-1 in comparison to membranes from the same R25-1 grown on glucose. R25-PC is a partial revertant of mutant R25-1 which was selected for study based on its ability to grow on lactate, while retaining the succinate minus characteristic. This revertant did not demonstrate a conditional oxidation of formate or NADH, and its cytochrome content did not vary with the growth substrate. Two classes of R25-1 transductants were isolated which were able to grow on succinate. Class II R25-1 transductants demonstrated the R25-1 conditional oxidation pattern, with the exception that now succinate oxidation was also conditional. When Class I transductants were grown on fructose, they exhibited a decrease in oxidase activities for formate and succinate, but this decrease was not as drastic as that noted for mutant R25-1 grown under identical conditions. Mutant strain R24 oxidized formate when grown on glucose, fructose, or glycerol, but failed to oxidize succinate regardless of the growth condition. Mutant SC43 grew on glycerol and lactate, but failed to grow on acetate and TCA intermediates. Whole cells of SC43 failed to oxidize formate, and oxidized succinate ineffectively. Isolated membranes from SC43 also failed to oxidize formate, but oxidized succinate at approximately one-half the rate of the parent strain, while exhibiting greatly reduced NADH oxidation.