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Characterization of multiple large replicons comprising the genome of Pseudomonas cepacia 17616
Abstract
Preparations of unrestricted DNA from Pseudomonas cepacia obtained by lysis of bacteria in agarose plugs contained small amounts of DNA with three different electrophoretic mobilities. The behavior the different species in CHEF gels corresponded to DNA fragments of 3.4, 2.5, and 0.9 Mb. Further analyses were consistent with the notion that the different species represented populations of linear DNAs derived by random double strand breakage of three distinct circular replicons. Treatment of DNA from strain 17616 with SwaI, PacI, and PmeI, restriction enzymes which recognize 8-bp sequences, generated four, six, and three fragments, respectively. Double digestion of the DNA with SwaI and PacI or with SwaI and PmeI generated fragments whose combined molecular weights was 6.8 Mb. Treatment of the DNA with AflII AseI, DraI, SpeI, and XbaI, restriction enzymes which recognize 6-bp sequences, generated about 50 fragments whose combined molecular weight also was about 6.8 Mb. The results indicate that the size of the genome of strain 17616 was 6.8 Mb. The combined molecular weights of the sets of SwaI, PacI, and PmeI fragments was 4.3, 5.9, and 5.9 Mb, respectively, reflecting the fact that the 2.5 Mb replicon contained no SwaI site and that the 0.9 Mb replicon lacked PacI and PmeI sites. The organization of SwaI, PacI, and PmeI fragments comprising each of the replicons was determined by Southern hybridization experiments using various junction fragments as probes and by further restriction analyses. Southern hybridization experiments using r-RNAs and various IS elements from P. cepacia as probes indicated that each of the replicons contained r-RNA genes and IS elements. A SwaI site was introduced into Tn5-751 and the resulting transposon was used to generate and map mutations related to amino acid biosynthesis and the degradation of various carbon sources. Derivatives of strain 17616 were identified in which large segments of the 2.5 and 0.9 Mb replicons had been deleted. The results provide a framework for further genetic analysis of P. cepacia.
Subject Area
Microbiology|Molecular biology|Genetics
Recommended Citation
Cheng, Hai-ping, "Characterization of multiple large replicons comprising the genome of Pseudomonas cepacia 17616" (1993). Doctoral Dissertations Available from Proquest. AAI9408263.
https://scholarworks.umass.edu/dissertations/AAI9408263