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Journal of Bacteriology, December 2002, p. 6581-6591, Vol. 184, No. 23
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.23.6581-6591.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Transposition of DEH, a Broad-Host-Range Transposon Flanked by ISPpu12, in Pseudomonas putida Is Associated with Genomic Rearrangements and Dehalogenase Gene Silencing

Andrew J. Weightman,* Andrew W. Topping,{dagger} Katja E. Hill,{ddagger} Li Ling Lee, Kenji Sakai,§ J. Howard Slater, and Andrew W. Thomas||

Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3TL, Wales, United Kingdom

Received 29 April 2002/ Accepted 27 August 2002

Pseudomonas putida strain PP3 produces two hydrolytic dehalogenases encoded by dehI and dehII, which are members of different deh gene families. The 9.74-kb DEH transposon containing dehI and its cognate regulatory gene, dehRI, was isolated from strain PP3 by using the TOL plasmid pWW0. DEH was fully sequenced and shown to have a composite transposon structure, within which dehI and dehRI were divergently transcribed and were flanked on either side by 3.73-kb identical direct repeats. The flanking repeat unit, designated ISPpu12, had the structure of an insertion sequence in that it was bordered by 24-bp near-perfect inverted repeats and contained four open reading frames (ORFs), one of which was identified as tnpA, putatively encoding an ISL3 family transposase. A putative lipoprotein signal peptidase was encoded by an adjacent ORF, lspA, and the others, ISPpu12 orf1 and orf2, were tentatively identified as a truncated cation efflux transporter gene and a PbrR family regulator gene, respectively. The orf1-orf2 intergenic region contained an exact match with a previously described active, outward-orientated promoter, Pout. Transposition of DEH-ISPpu12 was investigated by cloning the whole transposon into a suicide plasmid donor, pAWT34, and transferring the construct to various recipients. In this way DEH-ISPpu12 was shown to transpose in a broad range of Proteobacteria. Transposition of ISPpu12 independently from DEH, and inverse transposition, whereby the vector DNA and ISPpu12 inserted into the target genome without the deh genes, were also observed to occur at high frequencies in P. putida PaW340. Transposition of a second DEH-ISPpu12 derivative introduced exogenously into P. putida PP3 via the suicide donor pAWT50 resulted in silencing of resident dehI and dehII genes in about 10% of transposition transconjugants and provided a genetic link between transposition of ISPpu12 and dehalogenase gene silencing. Database searches identified ISPpu12-related sequences in several bacterial species, predominantly associated with plasmids and xenobiotic degradative genes. The potential role of ISPpu12 in gene silencing and activation, as well as the adaptation of bacteria to degrade xenobiotic compounds, is discussed.

* Corresponding author. Mailing address: Cardiff School of Biosciences, Main Building, Cardiff University, P.O. Box 915, Cardiff CF10 3TL, Wales, United Kingdom. Phone: 44 (0)29 2087 5877. Fax: 44 (0)29 2087 4305. E-mail: weightman{at}cardiff.ac.uk.

{dagger} Present address: Avecia, Billingham, Cleveland TS23 1YN, United Kingdom.

{ddagger} Present address: Oral Surgery, Medicine and Pathology, University of Wales, College of Medicine, Cardiff CF4 4XY, Wales, United Kingdom.

§ Present address: Faculty of Engineering, Oita University, Oita 870-11, Japan.

|| Present address: School of Biomedical Sciences, University of Wales Institute Cardiff, Cardiff CF5 2YB, Wales, United Kingdom.

Journal of Bacteriology, December 2002, p. 6581-6591, Vol. 184, No. 23
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.23.6581-6591.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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