The overall goal of these studies is to extend our knowledge of mutation in stationary-phase E. coli cells. In one system, reversion of an F'- borne lac frameshift mutation occurs in stationary-phase cells by a novel mutational mechanism requiring RecA, RecBCD, and RuvABC, functions involved in recombination and double-strand break-repair. The proposed experiments will determine whether chromosomal mutations occur by this mechanism, and reveal other routes to mutation in stationary-phase cells as well. The experimental design follows: (1) Independent chromosomal insertions of the lac frameshift mutation target into isogenic F- and F strains will be screened for those that revert to Lac+ during stationary-phase. (2) The dependence of the reversion phenotype of such insertions on homologous recombination functions and the SOS response will be determined. Lac+ mutations themselves will be characterized by mapping and sequence analysis. (3) Chromosomal insertions that are permissive for RecBCD-dependent stationary-phase mutation and some that are not will be exactly located on the E. coli chromosome, creating a map of hot and cold sites. (4) Permissive insertions will be used in a genetic screen to identify additional mechanistic and regulatory components of this mutational mechanism. This work will elucidate universal and F'-specific features of stationary-phase mutation and provide new links between recombination, repair, and mutation. Understanding the mechanisms and regulation of recombination-dependent mutation in stationary-phase or stressed cells has important implications for mutations that lead to bacterial resistance to antibiotics and to the origins of cancer tumor progression and resistance to chemotherapeutic drugs.
