Plasmid-homologous sequences in the chromosome of plasmidless Coxiella burnetii Scurry Q217

Abstract

Chromosomal DNA from Coxiella burnetii Scurry Q217 was screened for the presence of plasmid-homologous sequences. Total DNA from Scurry Q217 was digested with NotI, and the resulting DNA fragments were separated by contour-clamped homogeneous electric field pulsed-field gel electrophoresis (CHEF-PFGE). Following hybridization with biotin-labeled QpH1 plasmid as a probe, two DNA fragments of 40 and 170 kb were identified as targets. These fragments were cloned, and subclones containing QpH1-homologous sequences were completely sequenced. The physical mapping of DNA fragments was achieved by PCR with primers derived from adjacent fragments, and a total of 18,360 bp was sequenced. ORFs were different in size, 6 ORFs were newly generated, and 25 ORFs were lost. It was found that plasmid-homologous sequences in Scurry Q217 were of chromosomal origin.Coxiella burnetii, the only member of the genus Coxiella, is the causative agent of Q fever in humans as well as of abortions in domestic animals, especially ruminants. So far, different criteria have been applied for the classification of C. burnetii isolates. Samuel et al. (7) distinguish between so-called acute and chronic isolates that cause acute or chronic Q fever in humans, apparently depending on the plasmid type. However, recent experimental findings (3) and PCR analysis of C. burnetii strains isolated from patients exhibiting chronic Q fever (11) revealed that isolates containing either QpH1 or QpRS can cause endocarditis. Yu and Raoult (15) confirmed these results by serotyping isolates with monoclonal antibodies and postulated that the development of acute or chronic Q fever depends on the patient's condition and immune status rather than on the plasmid type of the causative agent. Nevertheless, plasmids are still considered to be of major importance concerning virulence factors, mechanisms of pathogenicity, and proliferation in the phagolysosomal compartment.Assuming that the plasmid-coded sequences are of essential importance for C. burnetii, the existence of the plasmidless C. burnetii isolate Scurry Q217 (8) was rather unexpected. However, it was shown that Scurry Q217 contains chromosomally integrated plasmid-homologous DNA sequences (8). In an attempt to understand the significance of plasmids in C. burnetii, we examined plasmidless isolates with chromosomally integrated plasmid-homologous DNA fragments. MATERIALS AND METHODSDNA-modifying enzymes. Restriction endonucleases and other DNA-modifying enzymes were purchased from Stratagene (Heidelberg, Germany) and Amersham (Bad Homburg, Germany) and used under conditions recommended by the manufacturers.Plasmids and bacterial strains. C. burnetii DNA fragments were cloned in phagemid vector pBluescript II KS(ϩ), cosmid vector SuperCos 1, or lambda phage vector EMBL4 (Stratagene). Recombinant cosmid and lambda phage DNAs were packaged (Gigapack II packaging extract; Stratagene) and transfected into Escherichia coli XL1 Blue (Stratagene). C. burnetii reference isolates Scurry Q217, Priscilla Q177, Dugway 7E22-...

Genetic Diversity of the Q Fever Agent,Coxiella burnetii, Assessed by Microarray-Based Whole-Genome Comparisons

“…Plasmid polymorphisms specific to QpH1, QpRS, QpDV, and integrated plasmid-like sequences were confirmed via comparison with their deciphered sequences (38,63,79), while polymorphisms of QpDG are described here for the first time. Nine predicted functional plasmid ORFs are conserved among all plasmid types or integrated-plasmid sequences.…”

“…These plasmids range from 32 to 42 kb in size and share a common 25-kb "core" region along with unique regions (40,59,79). QpRS-like plasmid sequences are integrated into the chromosome of some isolates (40,60,79).…”

Is Q Fever an emerging or re-emerging zoonosis?

“…Among the strains, four plasmid types designated QpH1, QpRS, QpDV and one plasmid without designation derived from a chinese C. burnetii isolate have been described [60,70,88,112,163,172]. Plasmidless C. burnetii strains carry large plasmid-homologous sequences integrated into the chromosome [181]. Although the plasmids seem to be of major importance for virulence because their common sequences are conserved among all isolates of Coxiella, their biological significance is still unclear.…”

Plasmids and Rickettsial Evolution: Insight from Rickettsia felis

“…Given the phylogenetic position of R. felis , this implies multiple losses of the plasmid and raises the question of why, among all ten sequenced rickettsial genomes, a single maintenance of a plasmid system would remain in R. felis . Under this hypothesis, if some plasmid genes are essential for rickettsial fitness, then the lineages without plasmids may have had the plasmid genes incorporated into their chromosomes where they have become a permanent fixture, as is the case for the plasmidless C. burnetti isolate Scurry Q217 [37], [38] and some plasmidless Chlamydia spp. [35], [39], [40].…”