Molecular analysis of mutS expression and mutation in natural isolates of pathogenic Escherichia coli

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Abstract
Deficiencies in the MutS protein disrupt methyl-directed mismatch repair (MMR), generating a mutator phenotype typified by high mutation rates and promiscuous recombination. How such deficiencies might arise in the natural environment was determined by analysing pathogenic strains ofEscherichia coli. Quantitative Western immunoblotting showed that the amount of MutS in a wild-type strain of the enterohaemorrhagic pathogenE. coliO157 : H7 decreased about 26-fold in stationary-phase cells as compared with the amount present during exponential-phase growth. The depletion of MutS in O157 : H7 is significantly greater than that observed for a laboratory-attenuatedE. coliK-12 strain. In the case of stable mutators,mutSdefects in strains identified among natural isolates were analysed, including twoE. coliO157 : H7 strains, a diarrhoeagenicE. coliO55 : H7 strain, and a uropathogenic strain from theE. colireference (ECOR) collection. No MutS could be detected in the four strains by Western immunoblot analyses. RNase T2 protection assays showed that the strains were either deficient inmutStranscripts or produced transcripts truncated at the 3′ end. Nucleotide sequence analysis revealed extensive deletions in themutSregion of three strains, ranging from 7·5 to 17·3 kb relative toE. coliK-12 sequence, while the ECOR mutator contained a premature stop codon in addition to other nucleotide changes in themutScoding sequence. These results provide insights into the status of themutSgene and its product in pathogenic strains ofE. coli.