GENETIC ANALYSIS OF RICKETTSIA PROWAZEKII

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PROVIDED. Members of the genus Rickettsia are the etiologic agents of rocky mountainand other spotted fevers and endemic, scrub, and epidemic typhus, diseases that pose a pernicious health threat worldwide. Rickettsiaprowazekii, thz etiologic agent of epidemic typhus is an obligate intracellular parasitic bacterium that can grow only within the cytoplasm of a eucaryotic host cell. The ability of rickettsiae to exploit this intracellularniche in animalsas diverse as arthropods and humans and to subsequently cause serious human disease provides the impetus for this study. This proposal focuses on the development and application of genetic techniques to address questions about the pathogenic bacterium R. prowazekii and its obligate intracytoplasmic existence. It exploits the availability of the R. prowazekii genome sequence and the development of rickettsial genetic technologies to test hypotheses related torickettsial gene function, DNA replication, and pathogenic mechanisms. In Specific Aim 1 our goal is to capitalize on a rickettsial transformation system and the identification of a selectable antibiotic resistance gene that can beexpressed inR.prowazekii to discriminate, via knockouts, essential function at the level of single genes. Specifically targeted genes include those that encode products withhomology to known virulencegenes of other bacteria, genes hypothesized to be expressed only in the arthropod vector, genes hypothesized to be non-functional and part of the process of rickettsial reductiveevolution,and finally, genes with homologs within the R.prowazekii genome. In addition, a transposon-based approach will be used to generate random insertion mutants. In Specific Aim 2 our goal is to isolate the functional origin of replication. One approach will attempt to generate a rickettsial minichromosome by linkingputative origin fragments with the selectable erythromycin-resistant gene, ereB. An alternate method will identify the originby the bindingof rickettsial DnaA. Specific Aim 3 will continue our characterization of transcription termination and our identificationof rickettsialtranscriptional changes that occur just prior to lysis of the host cell. Using ribonuclease protection studies we will determine whether these changes reflect a general property of the rickettsiae by examining additional non-intrinsic termination sites and the effect of cell number on terminationat these sites. Modulation of Rho and its correlation to these changes will be: assessed.