Rickettsiae prowazekii, the etiologic agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of its eucaryotic host cell, unbound by a vacuolar membrane. The overall goal of this research is to increase our understanding of the biochemical and genetic mechanisms involved in the successful intracellular parasitism by this organism. Previous work, employing recombinant DNA techniques, has led to the isolation of a number of R. prowazekii genes. A major goal of the work described in this application is the characterization of these genes and gene products using DNA sequence analysis and subsequent comparison studies with corresponding genes and gene products found in Escherichia coli and/or eucaryotic systems. Additional genes, coding for enzymes of the tricarboxylic acid cycle, will be isolated in order to investigate coordinate expression of rickettsial genes. The cloning of genes coding for proteins of the rickettsial cell surface will also be vigorously pursued. Isolation and characterization of these genes and gene products will form the foundation for subsequent studies on rickettsial recognition and penetration of its host cell. A major objective of this grant will be to use the knowledge gained from the isolation and characterization of rickettsial genes to probe the regulatory control mechanisms in viable rickettsiae. Cloned genes or appropriate fragments will be used as probes to detect specific messenger RNA. Finally, this work will begin to establish a system for the direct genetic analysis of R. prowazekii. This will involve screening low passaged R. prowazekii strains for naturally-occurring plasmids potentially useful as rickettsial vectors. In addition, studies designed to assess the ability or R. prowazekii to take-up and express exogenous DNA will also be performed. This work should provide an understanding of rickettsial gene structure and function and define the limits of direct genetic manipulation in this obligate intracellular parasite.