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. Within the QpH1-homologous region spanning 16,624 bp, homology was as high as 99%. Deletions were identified within EcoRI fragments A(H)-C(H)-K(H)-B(H) (13,490 bp) and J(H)-G(H)-E(H)-L+-D(H) (6,509 bp) and in fragment A(H) alone (619 bp). An insertion of 744 bp was identified within the JDc region of Scurry Q217. A search for putative coding regions identified a total of 17 open reading frames (ORFs). Compared to plasmid QpH1, 6 ORFs were identical, 5 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.