The specific aims of this proposal are to sequence and annotate the genome of Rickettsia rickettsii, and to begin functional analysis of the genome. R. rickettsii is an obligately intracellular Gram negative bacterium that is widely distributed in the Americas. It is the etiologic agent of Rocky Mountain spotted fever, the most severe of the human rickettsioses. This microorganism has been considered as a potential biological weapon because of its high infectivity, since it causes disease with a short incubation period and relatively high mortality, especially in untreated cases. Currently, a serious discrepancy exists between the potential threat to human health posed by R. rickettsii and the number of laboratories studying this important pathogen. To add to existing methodologies presently available for study of this bacterium, this proposal will introduce genomic sequencing as a means to gain valuable insight into the intricacies of the pathogenesis of this microorganism. The derived database will lay the groundwork for further functional proteomic studies of R. rickettsii that may facilitate identification of new vaccine candidates and potential therapeutic targets. Additionally, this new information will refine our understanding of the biology of the evolution of this bacterium and perhaps result in recognition of novel pathogenic mechanisms used by R. rickettsii. We propose to complete the sequencing and primary annotation of the R. rickettsii genome within one year at a total cost of $0.36 per base pair. An initial computational approach will be used to identify putative open reading frames (ORF) encoding the proteins and genes expressed as RNAs. A newly developed Website will be constructed and maintained to display the R. rickettsii genome sequence and its annotation. Metabolic pathways and their components in R. rickettsii will be identified by computational analysis of operon structures and comparisons with proteins and pathways in R. prowazekii and other bacteria. To facilitate experimental functional genomic analyses of R. rickettsii, individual ORFs will be PCR amplified, cloned, expressed, catalogued, and made available to the scientific community. Biochemical functions of selected genes of R. rickettsii will be experimentally confirmed. Finally, selected transcriptional regulons of R. rickettsii which respond to external signals will be characterized.