The Escherichia coli galK2 papillation assay: its specificity and application to seven newly isolated mutator strains

• Published in: Mutation Research Vol. 292; no. 2; pp. 175 - 185
• Main Authors: Oller, Adriana R., Fijalkowska, Iwona J., Schaaper, Roel M.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.10.1993; Elsevier
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; DNA Mutational Analysis; DNA polymerase; DNA Polymerase III - genetics; dnaE gene; dnaQ gene; Drug Resistance - genetics; E. coli mutator strains; Escherichia coli; Escherichia coli - genetics; Exonucleolytic proofreading; Fundamental and applied biological sciences. Psychology; galK2 gene; Genes, Bacterial; Microbiology; Mutagenesis; Mutation; Papillation assay; Phenotype; Rifampin - pharmacology; Exonucleolytic proofreading; E. coli mutator strains; DNA polymerase; Papillation assay; dnaE gene; galK2 gene; dnaQ gene; Specific activity; Enzyme; Medium effect; Occurrence frequency; Escherichia coli; Bacteria; Mutator effect; DNA polymerase 3; Mutation; Thermal sensibility; Enterobacteriaceae
• ISSN: 0165-1161; 0027-5107
• DOI: 10.1016/0165-1161(93)90145-P

The Escherichia coli dnaE and dnaQ genes encode, respectively, the α (polymerase) and ε (proofreading) subunits of DNA polymerase III. Mutations in these genes resulting in mutator or antimutator phenotypes provide important tools to understand the mechanisms by which mutations occur. One way to isolate such strains is the use of papillation 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 pathways, and the characterization of 7 mutators isolated using this system. 1 mutator resided in dnaE and 6 in dnaQ. Sequencing of the galK2 allele revealed a G · C → T · A tranversion at base pair 571 that changed a glu codon (GAA) to a stop codon (TAA). The analysis of 319 revertants showed that a Gal + phenotype can be achieved by A · T → G · C transition, A · T → T · A transversion and A · T → C · G transversion. We characterized the mutator phenotypes of the newly isolated mutators by determining (i) their mutation frequencies to resistance to rifampicin and nalidixic acid in both wild-type and mutL backgrounds, (ii) 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 reversion pathways and magnitude of mutator effects, the mutators can be grouped into 3 classes. These classes may represent different mutational mechanisms that include defective base insertion, defective proofreading and interference with the postreplicative mismatch-repair system.