Insertion-sequence-dependent rearrangements of Pseudomonas cepacia plasmid pTGL1

Abstract

Pseudomonas cepacia 249 (ATCC 17616) harbors a 170-kilobase (kb) plasmid designated pTGL1. We identified three insertion sequences, IS405, IS408, and IS411, on this plasmid. Various prototrophic and auxotrophic derivatives in our collection contained variants of pTGL1 formed by accretion and deletion of other elements. Plasmid pTGL6, the variant in one prototroph, evolved from pTGL1 by the addition of three copies of IS401 (1.3 kb) and one of IS402 (1 kb), to generate pTGL5, and recombination between two of the copies of IS401 on pTGL5 to form pTGL6. The latter event entailed loss of one copy of IS401 and an additional 5.4 kb of plasmid DNA. Derivatives of the broad-host-range plasmid pRPl carrying the above insertion sequences and recombinant plasmids carrying fragments of plasmids pTGL6 and pTGL5 were used as probes to ascertain the extent of reiteration of the various elements in the P. cepacia genome. The data indicate a high frequency of genomic rearrangements which presumably contributes to the extraordinary adaptability of this bacterium.Pseudomonas cepacia is notable for the extraordinary number of compounds it can utilize as sole carbon and energy sources (1,4,17,21,26,27,36). It is both an opportunistic pathogen for humans and a phytopathogen (1,14,26 (18) was used for the isolation of recombinant derivatives of plasmids pACYC184 (8) and pBR325 (7) carrying fragments of pTGL5 and pTGL6. These and other plasmids described in this paper are listed in Tables 1 and 2. Bacteria were grown at 37°C with shaking in flasks filled to one-fifth of their nominal capacity. For P. cepacia the medium consisted of inorganic salts solution (20) supplemented with 1% (wt/vol) Casamino Acids (Difco Laboratories, Detroit, Mich.). Strain SK1592 was grown in LB medium supplemented with either ampicillin (25 pig/ml) or chloramphenicol (20 jig/ml) depending on the vector used and the insert location.Isolation of DNA. Plasmid DNA was isolated by the procedure of Currier and Nester (12). When used as probes in Southern hybridization experiments the samples were subjected to two rounds of cesium chloride centrifugation. In some experiments plasmid DNA from E. coli Was isolated by the procedure of Bimboim and Doly (6

Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors

“…As far as gram-negative bacteria are concerned, most IS elements have been isolated from enteric bacteria (mainly E. coli; for reviews and lists of isolated elements, see references 14 and 20). However, there have been a substantial number of reports on IS elements identified in other gram-negative bacterial species, such as Agrobacterium tumefaciens (3,15,24,42), Xanthomonas campestris (22, 23), and various Rhizobium (9,28,32) and Pseudomonas (6,13,35,41,45) strains.In general, these elements have been found more or less by chance because of the capacity to inactivate or activate particular genes. However, there have been successful attempts to screen for transposable elements by means of direct selection procedures (8,15,30,35).…”

Multiple replicons constituting the genome of Pseudomonas cepacia 17616

“…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).…”

A fusion promoter created by a new insertion sequence, IS1490, activates transcription of 2,4,5-trichlorophenoxyacetic acid catabolic genes in Burkholderia cepacia AC1100

“…Southern blotting performed with chromosomal digests of B. cepacia AC1100, 17616 (249) and 25416 and E. coli JM109 showed that the new IS elements and IS931 were observed only in B. cepacia AC1100 (data not shown). Additionally, no homology to the genomic DNA of AC1100 was detected by using several IS elements isolated from B. cepacia 17616 (IS401, IS402, and IS408 [6,17,18,36]) as DNA probes. These results suggest the presence of a wide diversity of IS elements among isolates of B. cepacia.…”

“…A large number of IS elements in B. cepacia strains have been identified on the basis of their capability to insertionally activate downstream genes (6,17,18,30,36). Two IS elements that increase the expression of neighboring genes, IS931 and IS932, have been isolated previously from strain AC1100.…”