Activation of the lac genes of Tn951 by insertion sequences from Pseudomonas cepacia

Abstract

We have identified three transposable gene-activating elements from Pseudomonas cepacia on the basis of their abilities to increase expression of the lac genes of the broad-host-range plasmid pGC91.14 (pRPl::Tn951). When introduced into auxotrophic derivatives of P. cepacia 249 (ATCC 17616), this plasmid failed to confer the ability to utilize lactose. The lac genes of Tn951 were poorly expressed in P. cepacia and were not induced by isopropyl-I3-D-thiogalactopyranoside. Lac' variants of the pGC91.14-containing strains which formed I8-galactosidase at high constitutive levels as a consequence of transposition of insertion sequences from the P. cepacia genome to sites upstream of the lacZ gene of Tn951 were isolated. Certain of the elements also increased gene expression in other bacteria. For example, IS407 strongly activated the lacZ gene of Tn951 in Pseudomonas aeruginosa and Escherichia coli, and IS406 (but not IS407) did so in Zymomonas mobilis. The results indicate that IS elements from P. cepacia have potential for turning on the expression of foreign genes in a variety of gram-negative bacteria.Although Pseudomonas cepacia is notable for its extraordinary nutritional versatility (4,20,26,27,29), most soil isolates of this bacterium are unable to utilize lactose as a sole source of carbon and energy (4,20,21). Cornelis and co-workers identified a 17-kilobase (kb) transposon, Tn951 (Tnlac), in Yersinia enterolitica which carried lacI, lacZ, and lacY genes seemingly identical to those of the Escherichia coli lac operon and isolated several variants of the broadhost-range plasmid pRP1 (30) containing this element (12)(13)(14). We transferred one of these, pGC91.14 ( Fig. 1) into auxotrophic derivatives of P. cepacia 249 (ATCC 17616) by conjugation from E. coli JC3272. Although unable to utilize lactose themselves, the pGC91.14-containing transconjugants gave rise to Lac' variants (M. S. Wood, C. Lory, and T. G. Lessie, Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, H23, p. 143). In these strains, transposable gene-activating elements from the P. cepacia genome had inserted into Tn951 and increased expression of its lac genes. The results were similar to those we reported earlier for activation of the bla gene of Tnl in P. cepacia (20,28), which restored ability of 1-lactamase-deficient strains of this bacterium to utilize penicillin as a sole source of carbon and energy.We report here data describing the transposition of three P. cepacia insertion sequences, IS406, IS407, and IS415, to different sites within Tn951 and their effect on formation of lac-specific mRNA and of 3-galactosidase. We also describe the influence of P. cepacia insertion sequences on lac gene expression in other gram-negative bacteria. MATERIALS AND METHODSBacterial strains and plasmids. Table 1 lists strains and plasmids used in this study. Bacteria were grown in inorganic salts medium (33) supplemented with 1% (wt/vol) yeast extract or Casamino Acids or 0.5% of the specified carbon sources. Plasmid pGC91.14 was transferred from E. coli J...

High-Temperature-Induced Transposition of Insertion Elements in Burkholderia multivorans ATCC 17616

“…IS411 has been found to be an active IS element on pTGL1 (1). The remaining IS elements have been identified in a screening protocol, in which their insertion into the cI repressor gene resulted in the derepression of the cI-repressed genes (1,30,35,36). However, the available sequences of the IS elements in the public databases are limited to the complete sequences of IS401, IS402, IS406, and IS407 as well as the incomplete sequence of IS408.…”

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).…”

“…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.…”

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

“…In the insertion trap construct, pAH01 (Table 1), IS1413 activates transcription by creating a fusion promoter that is similar to that created by IS1490. Gene activation by creation of fusion promoters has been observed for other IS elements in clinical and environmental isolates (31)(32)(33)44).…”