Depletion of the cellular amounts of the MutS and MutH methyl-directed mismatch repair proteins in stationary-phase Escherichia coli K-12 cells

Abstract

The MutL, MutS, and MutH proteins mediate methyl-directed mismatch (MDM) repair and help to maintain chromosome stability in Escherichia coli. We determined the amounts of the MDM repair proteins in exponentially growing, stationary-phase, and nutrient-starved bacteria by quantitative Western immunoblotting. Extracts of null mutants containing various amounts of purified MDM repair proteins were used as quantitation standards. In bacteria growing exponentially in enriched minimal salts-glucose medium, about 113 MutL dimers, 186 MutS dimers, and 135 MutH monomers were present per cell. Calculations with the in vitro dissociation constants of MutS binding to different mismatches suggested that MutS is not present in excess, and may be nearly limiting in some cases, for MDM repair in exponentially growing cells. Remarkably, when bacteria entered late stationary phase or were deprived of a utilizable carbon source for several days, the cellular amount of MutS dropped at least 10-fold and became barely detectable by the methods used. In contrast, the amount of MutH dropped only about threefold and the amount of MutL remained essentially constant in late-stationary-phase and carbon-starved cells compared with those in exponentially growing bacteria. RNase T2 protection assays showed that the amounts of mutS, mutH, and mutL, but not miaA, transcripts decreased to undetectable levels in late-stationary-phase cells. These results suggested that depletion of MutS in nutritionally stressed cells was possibly caused by the relative instability of MutS compared with MutL and MutH. Our findings suggest that the MDM repair capacity is repressed in nutritionally stressed bacteria and correlate with conclusions from recent studies of adaptive mutagenesis. On the other hand, we did not detect induction of MutS or MutL in cells containing stable mismatches in multicopy single-stranded DNA encoded by bacterial retrons.