Establishment of a replicating plasmid in Rickettsia prowazekii

• Published in: PloS one Vol. 7; no. 4; p. e34715
• Main Authors: Wood, David O, Hines, Andria, Tucker, Aimee M, Woodard, Andrew, Driskell, Lonnie O, Burkhardt, Nicole Y, Kurtti, Timothy J, Baldridge, Gerald D, Munderloh, Ulrike G
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 17.04.2012; Public Library of Science (PLoS)
• Subjects: Animal behavior; Animals; Antibiotics; Bacillus subtilis; Biology; Cell Line; Chick Embryo; Cloning; Copy number; Cytosol; Deoxyribonucleic acid; DNA; DNA Replication; E coli; Epidemic typhus; Epidemics; Escherichia coli; Fibroblasts; Gene Dosage; Gene expression; Genetic analysis; Genetic engineering; Genetic vectors; Genomes; Identification; Immunology; Infections; Intracellular; Laboratories; Medicine; Mice; Molecular biology; Mutation; Pathogens; Physiology; Plasmids; Proteins; Replicating; Rickettsia; Rickettsia prowazekii; Rickettsia prowazekii - genetics; Rickettsia prowazekii - growth & development; Rickettsia rickettsii; Studies; Typhus; Vectors; Alabama; Minnesota
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0034715

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.