Multidrug resistance (MDR) is an increasingly more common presentation of antibiotic resistance in hospitals and communities worldwide. Resistance may occur through acquisition of resistance determinants from other organisms, or through chromosomal mutations in the drug target genes or in transcriptional regulators such as MarA and its homologs, or by mutations in, or duplications of, genes for multidrug efflux pumps. MarA of the marRAB operon in E. coli controls the expression of 80 or more genes in the mar regulon. Initially discovered in E. coli, MarA homologs are found in all bacterial species examined. MarA has its own functional homologs (SoxS and Rob) in E. coli. Recent studies in this laboratory and others have demonstrated that MarA and its homologs can affect not only MDR but also influence the infectivity of the organism. Thus this single regulatory protein contributes to both MDR and virulence. This proposal asks for funding to continue work, ongoing for two decades since the discovery of the mar locus in this laboratory, on the molecular basis for the functions of MarA and its marRAB operon. The new project period will examine: (1) the control of expression of the marRAB operon, both by its repressor, MarR, which has recently been shown to interact directly with other cell proteins which modify its activity, as well as by other proteins that regulate marRAB; the crystal structure of MarR with DNA and other ligands;(2) the molecular requirements for MarA-mediated repression of gene expression, the interaction of MarA with RNA polymerase/DNA, the interplay of MarA with other regulators of the hdeAB operon involved in pH stress response; (3) a gene newly-found to affect MDR by controlling the amount of OmpF porin; (4) MarA-regulated products which are associated with the infectious process in an E. coli pyelonephritis mouse model; (5) MarA homologs in Serratia marcescens, an opportunistic pathogen of increasing threat to immunocompromised patients. With the E. coli marRAB operon as the focus, the proposed studies seek to define the role of the proteins regulated by MarA, including the effect of interactions of other cellular proteins with MarR and MarA on drug resistance and virulence. Work in this area should serve as a paradigm for similar regulatory proteins in other bacteria and suggest novel approaches towards preventing and curing bacterial infectious diseases. This project focuses on a family of important regulatory proteins found in all bacteria examined, but not in mammalian cells, which controls the expression of more than 80 other genes in the bacterial cell. Among its many effects, the protein produces antibiotic resistance and increased virulence, two important challenges facing the treatment of infectious diseases. Therefore, understanding how this protein both regulates others and is regulated itself provides a number of novel possible targets for drugs in the control of infectious diseases.