Rickettsia prowazekii is the etiologic agent of epidemic typhus, a louseborne disease of man. Understanding the biology of this obligate intracellular bacterial parasite, and the factors which contribute to its virulence has been hampered by the difficulties associated with growing rickettsiae in eggs and cell cultures, the impossibility of controlling growth conditions within a eukaryotic cell, and the absence of a genetic system. Rickettsial mutants are practically nonexistent and gene transfer mechanisms have not been identified. Accordingly, in order to investigate some of the fundamental attributes of R. prowazekii, we have elected to apply recombinant DNA techniques and clone R. prowazekii DNA into Escherichia coli hosts. In this way rickettsial genes, gene products, and regulatory mechanisms can be examined and manipulated in a highly manageable system. Clone banks will be established using a variety of plasmid and cosmid vectors and cloning methods designed to vary the size and copy number of the cloned DNA. In this way, the probability of cloning any specific gene is enhanced. Clone banks will be screened by immunological methods to detect the presence of rickettsial antigens. In addition, direct selection techniques will be used to isolate rickettsial genes complementing known E. coli auxotrophic mutations. The rickettsial genes to be screened for expression in E. coli include cytoplasmic enzymes, membrane-bound activities and R. prowazekii surface antigens. Clones expressing rickettsial activities will provide gene products for biochemical studies and precisely defined R. prowazekii DNA segments for studies on rickettsial gene structure and expression.
Showing the most recent 10 out of 31 publications
