MULTIPLE REPLICONS CONSTITUTING THE GENOME OF PSEUDOMONAS-CEPACIA-17616

Macrorestriction fragment analysis of DNA from Pseudomonas cepacia 176 16, in conjunction with Southern hybridization experiments using junct ion fragments containing rare restriction enzyme sites as probes, indi cated that this bacterium contains three large circular replicons of 3 .4, 2.5, and 0.9 megabases (Mb). Inclusion of the 170-kb cryptic plasm id present in this strain gave an overall estimate of genome size of 7 Mb. Other Southern hybridization experiments indicated that the three large replicons contained rRNA genes as well as insertion sequence el ements identified previously in this strain. The distribution of SwaI, PacI, and PmeI sites on the three replicons was determined. A derivat ive of Tn5-751 carrying a S,waI site was used to inactivate and map ge nes on the 2.5- and 3.4-Mb replicons. Mutants were isolated in which t he 2.5- and 0.9-Mb replicons had been reduced in size to 1.8 and 0.65 Mb, respectively. The loss of DNA from the 2.5-Mb replicon was associa ted with lysine auxotrophy, beta-lactamase deficiency, and failure to utilize ribitol and trehalose as carbon and energy sources. DNA fragme nts corresponding in size to randomly linearized forms of the differen t replicons were detected in unrestricted DNA by pulsed-field gel elec trophoresis. The results provide a framework for further genetic analy sis of strain 17616 and for evaluation of the genomic complexities of other P. cepacia isolates.