Physical mapping of the Coxiella burnetii genome

Abstract

Coxiella burnetii isolates from different genomic groups contain restriction fragment polymorphisms that were easily distinguishable using pulsed field gradient electrophoresis (PFGE). Conversely, isolates that belong to the same genomic group yield identical patterns indicating that PFGE can be used to identify the genomic grouping of new C. burnetii isolates. Intact C. burnetii cells were embedded in agarose and lysed in situ. The genomic DNA was digested with low-frequency cutting restriction endonucleases, and subjected to PFGE analysis. NotI and SfiI cut C. burnetii DNA least often and produced the largest fragments. ApaI, MluI, SalI, XbaI or XhoI produced only small DNA fragments (+/- 50 kbp). When PFGE was used to analyse C. burnetii genomes for the presence of plasmid-related sequences, all the plasmid sequences in Nine Mile and Priscilla were associated with their 36 kbp or 39 kbp plasmid bands, respectively. If these isolates contained plasmid sequences which had integrated into their chromosomes those sequences would have been visible as additional bands. These same studies also showed that plasmid sequences in the plasmidless-Ko isolate were completely contained within two NotI fragments, indicating that the integrated plasmid is localized to a concise region of the C. burnetii genome. Since it is difficult to conduct genetic analyses of obligate intracellular parasites using standard techniques, a physical map is being developed using PFGE. In addition to providing a means for determining gene loci, the physical maps provide a means for comparing genetic organization among the different strains of C. burnetii.