Abstract
Background Rickettsia spp. (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular, α-proteobacteria that have historically been associated with blood-feeding arthropods. Certain species cause typhus and spotted fevers in humans, but others are of uncertain pathogenicity or may be strict arthropod endosymbionts. Genetic manipulation of rickettsiae should facilitate a better understanding of their interactions with hosts.Methodology/Principal FindingsWe transformed a species never associated with human disease, Rickettsia montanensis, by electroporation with a TN5 transposon (pMOD700) containing green fluorescent protein (GFPuv) and chloramphenicol acetyltransferase (CAT) genes under regulation of promoters cloned from the Rickettsia rickettsii ompB gene, and isolated a Chloramphenicol-resistant GFP-fluorescent rickettsiae population (Rmontanensis700). The Rmontanensis700 rickettsiae contained a single transposon integrated near an acetyl-CoA acetyltransferase gene in the rickettsial chromosome. Northern blots showed that GFPuv and CAT mRNAs were both expressed as two transcripts of larger and smaller than predicted length. Western immunoblots showed that Rmontanensis700 and E. coli transformed with a plasmid containing the pMOD700 transposon both expressed GFPuv proteins of the predicted molecular weight.Conclusions/SignificanceLong-standing barriers to transformation of rickettsiae have been overcome by development of transposon-based rickettsial transformation vectors. The ompB promoter may be the most problematic of the four promoters so far employed in those vectors.
Highlights
The genus Rickettsia (Rickettsiales: Rickettsiaceae) consists of small, obligately intracellular Gram-negative bacteria that have been studied principally as established and newly emerging human pathogens transmitted by specific blood-feeding arthropods [1,2]
Similar to R. peacockii, R. montanensis may affect the epidemiology of Rocky Mountain spotted fever by interference with transovarial transmission of R. rickettsii infections in ticks [15,16,29], and by conference of protective immunity to infected mammalian hosts subsequently exposed to R. rickettsii [25,26]
The CHL-resistant rickettsiae were designated as R. montanensis pMODompBCAT\GFPuv line 700 (Rmontanensis700) and were serially passed by inoculation of
Summary
The genus Rickettsia (Rickettsiales: Rickettsiaceae) consists of small, obligately intracellular Gram-negative bacteria that have been studied principally as established and newly emerging human pathogens transmitted by specific blood-feeding arthropods [1,2]. Rickettsiae that are widely distributed in North American ticks that readily feed on humans but are not associated with human disease include the recently isolated (TJ Kurtti, unpublished) Ixodes scapularis endosymbiont [8,9,10], Rickettsia peacockii and R. montanensis. Recent studies have begun to explore the dynamics of the D. variabilis gene expression response to infection with R. montanensis [30,31,32], which is perhaps the best established laboratory model for study of the interactions of a Dermacentor tick host with a spotted fever group Rickettsia spp. that is not a human pathogen. Genetic manipulation of rickettsiae should facilitate a better understanding of their interactions with hosts
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