A Defect in the Acetyl Coenzyme A↔Acetate Pathway Poisons Recombinational Repair-Deficient Mutants ofEscherichia coli

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Abstract
Recombinational repair-dependent mutants identify ways to avoid chromosomal lesions. Starting with arecBC(Ts) strain ofEscherichia coli, we looked for mutants unable to grow at 42°C in conditions that inactivate the RecBCD(Ts) enzyme. We isolated insertions inackAandpta, which comprise a two-gene operon responsible for the acetate↔acetyl coenzyme A interconversion. Using precise deletions of eitherackAorpta, we showed that either mutation makesE. colicells dependent on RecA or RecBCD enzymes at high temperature, suggesting dependence on recombinational repair rather than on the RecBCD-catalyzed linear DNA degradation. Complete inhibition of growth ofpta/ackA recmutants was observed only in the presence of nearby growing cells, indicating cross-inhibition.pta recmutants were sensitive to products of the mixed-acid fermentation of pyruvate, yet none of these substances inhibited growth of the double mutants in low-millimolar concentrations.pta, but notackA, mutants also depend on late recombinational repair functions RuvABC or RecG.pta/ackA recFmutants are viable, suggesting, together with the inviability ofpta/ackA recBCmutants, that chromosomal lesions due to thepta/ackAdefect are of the double-strand-break type. We have isolated three insertional suppressors that allow slow growth ofpta recBC(Ts) cells under nonpermissive conditions; all three are in or near genes with unknown functions. Although they do not form colonies,ackA recandpta recmutants are not killed under the nonpermissive conditions, exemplifying a case of synthetic inhibition rather than synthetic lethality.