Regulation of mutY and nature of mutator mutations in Escherichia coli populations under nutrient limitation

Abstract

Previous analysis of aerobic, glucose-limited continuous cultures of Escherichia coli revealed that G:C-to-T:A (G:C-->T:A) transversions were the most commonly occurring type of spontaneous mutation. One possible explanation for the preponderance of these mutations was that nutrient limitation repressed MutY-dependent DNA repair, resulting in increased proportions of G:C-->T:A transversions. The regulation of the mutY-dependent DNA repair system was therefore studied with a transcriptional mutY-lacZ fusion recombined into the chromosome. Expression from the mutY promoter was fourfold higher under aerobic conditions than under anaerobic conditions. But mutY expression was higher in glucose- or ammonia-limited chemostats than in nutrient-excess batch culture, so mutY was not downregulated by nutrient limitation. An alternative explanation for the frequency of G:C-->T:A transversions was the common appearance of mutY mutator mutations in the chemostat populations. Of 11 chemostat populations screened in detail, six contained mutators, and the mutator mutation in four cultures was located in the region of mutY at 66 min on the chromosome. The spectrum of mutations and rate of mutation in these isolates were fully consistent with a mutY-deficiency in each strain. Based on PCR analysis of the region within and around mutY, isolates from three individual populations contained deletions extending at least 2 kb upstream of mutY and more than 5 kb downstream. In the fourth population, the deletion was even longer, extending at least 5 kb upstream and 5 kb downstream of mutY. The isolation of mutY mutator strains from four independent populations with extensive chromosomal rearrangements suggests that mutY inactivation by deletion is a means of increasing mutation rates under nutrient limitation and explains the observed frequency of G:C-->T:A mutations in glucose-limited chemostats.

FIG. 1.

Expression of a mutY-lacZ fusion in strain BW3500 was monitored by measuring β-galactosidase activity during growth in batch culture in minimal medium containing 0.02% (wt/vol) glucose. Growth was monitored by measuring the absorbance at 580 nm.

FIG. 2.

(A) Locations of primers used in PCR, as probes in dot blots, and for construction of mutY-lacZ transcriptional fusion. (B) Primer pairs (specified below gels) were used to amplify the mutY gene and surrounding region in several chemostat mutator mutants. Agarose concentrations in the gels were 1 and 0.8%, respectively. The gel lane numbered 1 corresponded to strain BW3526 carrying the mutY::Tn10 insertion. Lanes 2, WT strain BW2952; lanes 3, isolate 21Qa1; lanes 4, isolate L3Oa1; lanes 5, isolate 10U6; lanes 6, isolate C7F1; lanes 7, DNA markers with sizes of 23,130, 9,416, 6,557, 4,361, 2,322, and 2,027 bp. (C) Results of slot blotting of DNA extracted from mutator strains. Hybridization was at 50°C with DIG-dUTP-tailed probes mutYF2, mutYF3, and mutYF9 as well as the positive control probe for an unlinked housekeeping gene, eno.