THE ESCHERICHIA-COLI GALK2 PAPILLATION ASSAY - ITS SPECIFICITY AND APPLICATION TO 7 NEWLY ISOLATED MUTATOR STRAINS

The Escherichia coli dnaE and dnaQ genes encode, respectively, the alp ha (polymerase) and epsilon (proofreading) subunits of DNA polymerase III. Mutations in these genes resulting in mutator or antimutator phen otypes provide important tools to understand the mechanisms by which m utations occur. One way to isolate such strains is the use of papillat ion assays. We used one such assay based on the reversion of the galK2 allele in cells grown on MacConkey-Gal plates. Here, we describe the identification of the galK2 mutation and its possible reversion pathwa ys, and the characterization of 7 mutators isolated using this system. 1 mutator resided in dnaE and 6 in dnaQ. Sequencing of the galK2 alle le revealed a G . C --> T . A transversion at base pair 571 that chang ed a glu codon (GAA) to a stop codon (TAA). The analysis of 319 revert ants showed that a Gal+ phenotype can be achieved by A . T --> G . C t ransition, A . T --> T . A transversion and A . T --> C . G transversi on. We characterized the mutator phenotypes of the newly isolated muta tors by determining (i) their mutation frequencies to resistance to ri fampicin and nalidixic acid in both wild-type and mutL backgrounds, (i i) their temperature sensitivity and medium dependence and (iii) their mutational specificity (by analyzing the nature of galK revertants). Based on the genomic locations of their mutations, specificity of reve rsion pathways and magnitude of mutator effects, the mutators can be g rouped into 3 classes. These classes may represent different mutationa l mechanisms that include defective base insertion, defective proofrea ding and interference with the postreplicative mismatch-repair system.