title: Establishment of a replicating plasmid in Rickettsia prowazekii.
abstract: Rickettsia prowazekii, the causative agent of epidemic typhus, grows only within the cytosol of eukaryotic host cells. This obligate intracellular lifestyle has restricted the genetic analysis of this pathogen and critical tools, such as replicating plasmid vectors, have not been developed for this species. Although replicating plasmids have not been reported in R. prowazekii, the existence of well-characterized plasmids in several less pathogenic rickettsial species provides an opportunity to expand the genetic systems available for the study of this human pathogen. Competent R. prowazekii were transformed with pRAM18dRGA, a 10.3 kb vector derived from pRAM18 of R. amblyommii. A plasmid-containing population of R. prowazekii was obtained following growth under antibiotic selection, and the rickettsial plasmid was maintained extrachromosomally throughout multiple passages. The transformant population exhibited a generation time comparable to that of the wild type strain with a copy number of approximately 1 plasmid per rickettsia. These results demonstrate for the first time that a plasmid can be maintained in R. prowazekii, providing an important genetic tool for the study of this obligate intracellular pathogen.
authors.affiliation: Department of Microbiology and Immunology, Laboratory of Molecular Biology, University of South Alabama College of Medicine, Mobile, Alabama, United States of America. dowood@jaguar1.usouthal.edu
authors.forename: David O
authors.initials: DO
authors.lastname: Wood
authors.forename: Andria
authors.initials: A
authors.lastname: Hines
authors.forename: Aimee M
authors.initials: AM
authors.lastname: Tucker
authors.forename: Andrew
authors.initials: A
authors.lastname: Woodard
authors.forename: Lonnie O
authors.initials: LO
authors.lastname: Driskell
authors.forename: Nicole Y
authors.initials: NY
authors.lastname: Burkhardt
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Kurtti
authors.forename: Gerald D
authors.initials: GD
authors.lastname: Baldridge
authors.forename: Ulrike G
authors.initials: UG
authors.lastname: Munderloh
mesh_terms: D000818:Animals; D002460:Cell Line; D002642:Chick Embryo; D004261:DNA Replication; D018628:Gene Dosage; D051379:Mice; D010957:Plasmids; D012283:Rickettsia prowazekii / Q000235:genetics* / Q000254:growth & development