Direct selection for mutators in Escherichia coli

We have constructed strains that allow a direct selection for mutators of E scherichia coli on a single plate medium. The plate selection is based on u sing two different markers whose reversion is enhanced by a given mutator, Plates containing limiting amounts of each respective nutrient allow the gr owth of ghost colonies or microcolonies that give rise to full-size colonie s only if a reversion event occurs. Because two successive mutational event s are required, mutator cells are favored to generate full-size colonies. R eversion of a third marker allows direct visualization of the mutator pheno type by the large number of blue papillae in the full-size colonies. We als o describe plate selections involving three successive nutrient markers fol lowed by a fourth papillation step. Different frameshift or base substituti on mutations are used to select for mismatch-repair-defective strains (mutH LS and uvrD). We can detect and monitor mutator cells arising spontaneously , at frequencies lower than 10(-5) in the population. Also, we can measure a mutator cascade, in which one type of mutator (mutT) generates a second m utator (mutHLS) that then allows stepwise frameshift mutations. We discuss the relevance of mutators arising on a single medium as a result of cells o vercoming successive growth barriers to the development and progression of cancerous tumors, some of which are mutator cell lines.