Multiple replicons constituting the genome of Pseudomonas cepacia 17616

Abstract

Macrorestriction fragment analysis of DNA from Pseudomonas cepacia 17616, in conjunction with Southern hybridization experiments using junction fragments containing rare restriction enzyme sites as probes, indicated that this bacterium contains three large circular replicons of 3.4, 2.5, and 0.9 megabases (Mb). Inclusion of the 170-kb cryptic plasmid 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 elements identified previously in this strain. The distribution of Swal, PacI, and Pmel sites on the three replicons was determined. A derivative of TnS-751 carrying a SwaI site was used to inactivate and map genes on the 2.5-and 3.4-Mb replicons. Mutants were isolated in which the 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 associated with lysine auxotrophy, P-lactamase deficiency, and failure to utilize ribitol and trehalose as carbon and energy sources. DNA fragments corresponding in size to randomly linearized forms of the different replicons were detected in unrestricted DNA by pulsed-field gel electrophoresis. The results provide a framework for further genetic analysis of strain 17616 and for evaluation of the genomic complexities of other P. cepacia isolates.Pseudomonas cepacia has attracted attention because of its extraordinary biodegradative abilities and its potential as an agent of bioremediation (2,13,22,25,35,44,45,51,56). The genome of this bacterium contains a large number of insertion sequences (ISs) (25, 27), which were identified on the basis of their abilities to promote genomic rearrangements (4, 12) and to activate the expression of neighboring genes (15,43,60). Such elements have been implicated in the recruitment of foreign genes for novel catabolic functions (15,43,54,60). Although there is considerable information about the biochemical activities of P. cepacia (2,3,11,25,26,35,40,45,51) and although certain genes and IS elements have been isolated and characterized (5,7,10,15,16,33,36,42,54,59,61), little is known about the overall organization of genes or the genomic distribution of IS elements. To gain such information we undertook the construction of a macrorestriction map of the genome of P. cepacia 17616 by using recently developed techniques for the manipulation of large DNA fragments (8,17,19,(46)(47)(48) To test the phenotypes of auxotrophic strains, 0.5% potassium phthalate or mannitol was substituted for the yeast extract and the medium was supplemented with 50 jig of each required amino acid per ml. For growth in liquid medium, 10-to 20-ml cultures were shaken in 125-ml flasks. For growth on plates, the medium was supplemented with 2% (wt/vol) agar. Preparation of genomic DNA for pulsed-field gel electrophoresis (PFGE). Agarose plugs containing intact chromosomal DNA were prepared essentially as described by Smith and Cantor (47). Approximatel...

Low-resolution sequencing of Rhodobacter sphaeroides 2.A.1T: chromosome II is a true chromosome

“…This discovery was rapidly followed by the finding that multiple chromosomes exist in at least four other bacterial genera, Brucella , Leptospira (Zuerner et al, 1993) , Agrobacterium (Allardet-Servent et al, 1993) and Pseudomonas (P. cepacia) (Cheng & Lessie, 1994; P. cepacia was recently renamed Burkholderia : Rodley et al, 1995). In the case of Agrobacterium one of these chromosomes is linear.…”

The marine bacterium Pseudoalteromonas haloplanktis has a complex genome structure composed of two separate genetic units.

“…Recently, however, multiple chromosomes have been described in five different bacterial species. The size of the "extra" chromosomes range from 350 kb in the case of Leptospira interrogans (Zuerner 1991;Zuerner et al 1993) to 2500 kb in the case of Pseudomonas cepacia 17616 (Cheng and Lessie 1994) (Table 3). A mathematical model has been proposed that explains the selective advantage of maintaining the genome in multiple, smaller replicons rather than a single, larger replicon (Stouthamer and Kooijman 1993).…”

“…However, with the discovery of two separate circular chromosomes in Rhodobacter sphaeroides 2.4.1 (Suwanto andKaplan 1989, 1992), the concept of the single circular chromosome that contains the entire genome of the prokaryote has been revised. A number of prokaryotes have complex genome organizations, including multiple chromosomes (Suwanto andKaplan 1989, 1992;Zuerner 1991;Allardet-Servent et al 1993;Michaux et al 1993;Zuerner et al 1993;Cheng and Lessie 1994) and large megaplasmids (Mergeay et al 1985;Friedrich et al 1986;Bancroft et al 1989;Sobral et al 1991;Lopez-Garcia et al 1994;Muro-Pastor et al 1994). The extent of such complexity has been largely unexplored, and, in particular, the genome structure of marine bacteria has, until now, been uncharacterized.…”

Pleiotropic roles of iron-responsive transcriptional regulator Fur in Burkholderia multivorans

“…B. multivorans ATCC 17616 is a soilderived strain whose extraordinary metabolic versatility has been well analysed. As is the case in other Burkholderia strains, the ATCC 17616 genome has multiple replicons consisting of three chromosomes of 3.4, 2.5 and 0.9 Mb (designated Chr I, Chr II and Chr III, respectively) and a 170 kb plasmid, pTGL1 (Cheng & Lessie, 1994). This strain shows extensive genomic plasticity, exhibiting, for example, frequent and large-scale insertions and deletions (Lessie et al, 1996).…”