Effect of repeat copy number on variable-number tandem repeat mutations in Escherichia coli O157:H7

Abstract

Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 x 10(-4) mutations/generation and a combined 28-locus rate of 6.4 x 10(-4) mutations/generation. We observed single- and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 (r2= 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included (r2= 0.833, P < 0.0001) or excluded (r2= 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single- and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data.

FIG. 1.

Frequency distributions of mutation products. Shown are frequencies of insertion (A), deletion (B), and total (C) mutations involving <21 repeat units plotted as a percentage of total mutations.

FIG. 2.

Diversity as a function of mutation rate. Two diversity measures, diversity (A and B) and total number of alleles (C and D), are plotted against mutation rate. Diversity was calculated from a collection of 344 diverse E. coli O157/O55 isolates. Regression plots are presented both including (A and C) and excluding (B and D) data from locus O157-10 (indicated by an arrow in panels A and C). Correlations from data excluding locus O157-10 are presented for both diversity measure plots.

FIG. 3.

Locus O157-10 mutation rate as a function of repeat copy number. (A) Allele frequency distribution for 344 diverse E. coli O157/O55 isolates is presented with the PSPE strain repeat copy numbers labeled 1 to 9 and colored black. Strains 1 to 9 are H6436, ATCC 700927, Spain 401, F6750, EC536 and EC1212, 01A6820, DEC4C, 01A7146, and Spain 41, respectively. (B) A correlation between repeat copy number and mutation rate for locus O157-10 is presented along with the regression line equation.

FIG. 4.

Mutation rate across loci as a function of repeat copy number. Correlations between repeat copy number and mutation rate are presented for all mutating loci in the PSPEs, both including (A) and excluding (B) data from locus O157-10. Equations for the two regression lines are also presented.

FIG. 5.

Large repeat copy mutations occur in alleles with higher repeat copy numbers. The number of mutations involving >4 repeats at locus O157-10 is presented for the nine PSPE strains according to their repeat copy numbers.