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Burkholderia cepacia has attracted attention because of its extraordinary degradative abilities and its potential as a pathogen for plants and for humans. This bacterium was formerly considered to belong to the genus Pseudomonas in the y-subclass of the Proteobacteria, but recently has been assigned to the P-subclass based on rrn gene sequence analyses and other key phenotypic characteristics. The B. cepacia genome is comprised of multiple chromosomes and is rich in insertion sequences. These two features may have played a key role in the evolution of novel degradative functions and the unusual adaptability of this bacterium.

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...

This study demonstrated that transposable elements in Pseudomonas cepacia could be inserted upstream of a poorly expressed gene and increase its expression more than 30-fold. Five elements, TnPcl, IS402, IS403, IS404, and IS405, were isolated by their ability to increase expression of the P-lactamase gene of the broad-host-range plasmid pRP1. Increased expression resulted only from insertion of these elements, suggesting that insertional activation is an important means of elevating gene expression in this organism. Four of the elements inserted between a Pstl site within the ,B-lactamase gene and a BamHI site located 375 base pairs upstream of its promoter. The element IS403 inserted distal to the BamHI site within the coding region for the gene tnpR, suggesting that insertional activation can act over greater than expected distances. In addition, the element IS402 activated the P-lactamase genes carried on plasmids pRP1 and pMR5 (temperature-sensitive pRP1) equally well in opposite orientations, demonstrating that insertional activation by this element occurs independent of its orientation.

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Extracts of Pseudomonas aeruginosa (ATCC 7700) cells grown on glucose, gluconate, or glycerol had enzyme activities related to the Entner-Doudoroff pathway. These activities were present in no more than trace amounts when the bacteria were grown on succinate. Fructose-1 ,6-diphosphate aldolase could not be detected in extracts of the bacteria grown on any of the above carbon sources. Therefore, it appears that P. aeruginosa degrades glucose via an inducible Entner-Doudoroff pathway. The apparent absence of fructose-1 , 6-diphosphate aldolase in cells growing on succinate suggests that the bacteria can form hexose and pentose phosphates from succinate by an alternate route. D-Glucose-6-phosphate dehydrogenase, a branch-point enzyme of the Entner-Doudoroff pathway, was purified 50-fold from glucose-grown cells. Its molecular weight, estimated by sucrose density gradient centrifugation, was found to be approximately 190,000. The enzyme was strongly inhibited by adenosine triphosphate, guanosine triphosphate, and deoxyguanosine triphosphate, which decreased the apparent binding of glucose-6-phosphate to the enzyme. It is suggested that adenine nucleotide-linked control of glucose-6-phosphate dehydrogenase may regulate the overall catabolism of hexose phosphates and prevent their wasteful degradation under certain conditions requiring gluconeogenesis.

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