Understanding Homology in Genetic Recombination

Antibiotic resistance is an escalating problem for modern chemotherapy of bacterial infectious diseases, and, in combination with the deteriorating pipeline of new antibacterials, is creating a clear and urgent danger to public health and national biodefense. Although the mechanisms that facilitate the de novo development, clonal spread, and horizontal transfer of resistance factors are not fully understood, the rapid rate at which antibiotic-resistant bacteria arise is likely due to a combination of mutations introduced during SOS mutagenesis and gene transfer between organisms. Recently, the Escherichia coli RecA protein[unreadable]s activities in SOS induction and homologous recombination have revealed RecA as a crucial player in these phenomena. A combination of primary literature, patent data, and unpublished results demonstrate that RecA mediates a range of phenomena related to bacterial pathogenecity, particularly the development and transmission of antibiotic resistance genes. Although the high conservation of RecA among bacterial species compellingly suggests the possibility that RecA may play similar roles in species other than E. coli, many questions remain as to how the properties of individual variants are related to their specific biological functions. To delimit possible models for the RecA-mediated activities that occur in pathogenic bacteria, we propose three Specific Aims to exploit our recently developed methods for rapid, parallel purification and rigorous characterization of RecA proteins to elucidate the relationships between RecA structure, in vitro activities, and physiologic functions. Briefly, we will (1) systematically define and evaluate structure-function relationships among RecA proteins from 31 pathogenic bacteria using biochemical and cellular activity assays; (2) provide insight into the species-specific molecular mechanisms of RecADNA filament activation using directed mutagenesis and substrate analogs; and (3) demonstrate that RecA effectors can be delivered into living bacteria to produce physiological consequences. The successful realization of the Aims will provide (1) substantial and novel insights into the molecular mechanisms by which different RecA proteins from select bacterial pathogens carry out their biological functions; (2) a novel microbiological toolbox that will be central to teasing apart the various roles of RecA in pathogenicity; and (3) novel methods for the delivery of small-molecule RecA effectors into bacterial pathogens.