The majority of urinary tract infections (UTIs) are caused by uropathogenic strains of Escherichia coli. These strains are known to harbor many virulence factors such as hemolysin, aerobactin and several types of fimbriae including P, S and type 1. The genes encoding bacterial virulence factors are often clustered together on the bacterial chromosome in areas termed "pathogenicity islands" (PAIs). The PAIs vary in size, location and genetic content between strains. However, at present they share the characteristic of being largely undefined. The overall goal of this research is to further elucidate the unique attributes of uropathogenic E. coli (UPEC) that distinguish them from other commensal and pathogenic strains. A problem with identifying novel virulence factors is that many or most of these genes are not expressed under in vitro conditions. However, new techniques have been developed to overcome this barrier. One of these techniques, differential fluorescence induction (DFI), has been particularly successful in the identification of genes differentially expressed under various environmental conditions (including inside of eukaryotic cells) and will be utilized in these studies. Additionally, putative virulence factors will be identified on the basis of sequence homologies to other known virulence genes. All candidate virulence factors will be further investigated by making allelic knockouts with subsequent testing in the murine model of ascending urinary tract infection to determine their significance in vivo. An epidemiological survey of candidate virulence genes will then be carried out. The discovery of new virulence factors may lead to a better understanding of how urinary tract isolates differ from benign, commensal strains of E. coli. In addition, these studies may provide insight into how bacterial gene expression is modulated in response to the host cell environment and lead to new targets for therapeutics and vaccine development.