PREFERENTIAL MUTAGENESIS OF LACZ INTEGRATED AT UNIQUE SITES IN THE ESCHERICHIA-COLI CHROMOSOME

To study the variation in spontaneous mutation frequencies in differen t chromosomal domains, a mini-Mu-kan-lacZ(-) transposable element was constructed to insert the lacZ(-)(Trp570 --> Opal) allele into many di fferent loci in the Escherichia coli chromosome. Papillation on MacCon key lactose plates was used to screen for mini-Mu insertion mutants wi th elevated levels of spontaneous mutagenesis of lacZop --> LacZ(+); c andidates were then screened for normal mutation frequencies in other genes. Two different insertion mutants with this enhanced mutagenesis phenotype were isolated from 14000 colonies, and named plm-1 (preferen tial lacZ mutagenesis) and plm-2. The frequency of LacZ(-) --> LacZ(+) mutations in these plm mutants was over 400-fold higher than that in isogenic strains containing mini-Mu-kan-lacZop insertions at other loc i. Six Lac(+) reversion (or suppression) mutations obtained from each of the two plm mutants were mapped by P1 transduction and all were fou nd to be linked to the Kan(r) gene in the mini-Mu-kan-lacZop suggestin g that a localized mutagenic event is responsible for the preferential mutagenesis. Furthermore, both the LacZ(+) --> LacZ(-) and Kan(r) --> Kan(s) mutant frequencies of these Lac(+) revertants were in the rang e of 10(-3) to 10(-2), indicating that this putative localized mutagen esis is neither allele nor gene specific. To identify the plm loci, th e chromosomal regions flanking the mini-Mu insertion sites were cloned and sequenced. A computer-assisted database search of homologous sequ ences revealed that the plm-1 locus is identical to the mutS gene; the mini-Mu insertion most probably results in the production of a trunca ted MutS protein. We suggest that the enhanced lacZ mutation frequency in plm-1 may be associated with an active process involving the putat ive truncated MutS protein. The DNA sequence of the plm-2 locus matche d a putative malate oxidoreductase gene located at 55.5 min of the E. coli chromosome.