Preferential mutagenesis of lacZ integrated at unique sites in the Escherichia coli chromosome

Abstract

To study the variation in spontaneous mutation frequencies in dierent chromosomal domains, a mini-Mu-kan-lacZ A transposable element was constructed to insert the lacZ A (Trp570 ® Opal) allele into many dierent loci in the Escherichia coli chromosome. Papillation on MacConkey lactose plates was used to screen for mini-Mu insertion mutants with elevated levels of spontaneous mutagenesis of lacZop ® LacZ + ; candidates were then screened for normal mutation frequencies in other genes. Two dierent insertion mutants with this enhanced mutagenesis phenotype were isolated from 14 000 colonies, and named plm-1 (preferential lacZ mutagenesis) and plm-2. The frequency of LacZ A ® LacZ + mutations in these plm mutants was over 400-fold higher than that in isogenic strains containing mini-Mu-kan-lacZop insertions at other loci. Six Lac + reversion (or suppression) mutations obtained from each of the two plm mutants were mapped by P1 transduction and all were found to be linked to the Kan r gene in the mini-Mu-kan-lacZop, suggesting that a localized mutagenic event is responsible for the preferential mutagenesis. Furthermore, both the LacZ + ® Lac-Z A and Kan r ® Kan s mutant frequencies of these Lac + revertants were in the range of 10 A3 to 10 A2 , indicating that this putative localized mutagenesis is neither allele nor gene speci®c. To identify the plm loci, the chromosomal regions¯anking the mini-Mu insertion sites were cloned and sequenced. A computer-assisted database search of homologous sequences revealed that the plm-1 locus is identical to the mutS gene; the mini-Mu insertion most probably results in the production of a truncated MutS protein. We suggest that the enhanced lacZ mutation frequency in plm-1 may be associated with an active process involving the putative truncated MutS protein. The DNA sequence of the plm-2 locus matched a putative malate oxidoreductase gene located at 55.5 min of the E. coli chromosome.