Increased Mutation Frequency in Redox-Impaired Escherichia coli Due to RelA- and RpoS-Mediated Repression of DNA Repair

Abstract

ABSTRACT Balancing of reducing equivalents is a fundamental issue in bacterial metabolism and metabolic engineering. Mutations in the key metabolic genes ldhA and pflB of Escherichia coli are known to stall anaerobic growth and fermentation due to a buildup of intracellular NADH. We observed that the rate of spontaneous mutation in E. coli BW25113 (Δ ldhA Δ pflB ) was an order of magnitude higher than that in wild-type (WT) E. coli BW25113. We hypothesized that the increased mutation frequency was due to an increased NADH/NAD + ratio in this strain. Using several redox-impaired strains of E. coli and different redox conditions, we confirmed a significant correlation ( P < 0.01) between intracellular-NADH/NAD + ratio and mutation frequency. To identify the genetic basis for this relationship, whole-genome transcriptional profiles were compared between BW25113 WT and BW25113 (Δ ldhA Δ pflB ). This analysis revealed that the genes involved in DNA repair were expressed at significantly lower levels in BW25113 (Δ ldhA Δ pflB ). Direct measurements of the extent of DNA repair in BW25113 (Δ ldhA Δ pflB ) subjected to UV exposure confirmed that DNA repair was inhibited. To identify a direct link between DNA repair and intracellular-redox ratio, the stringent-response-regulatory gene relA and the global-stress-response-regulatory gene rpoS were deleted. In both cases, the mutation frequencies were restored to BW25113 WT levels.