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Journal of Bacteriology, April 1999, p. 2535-2547, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Investigation of Two Evolutionarily Unrelated Halocarboxylic Acid Dehalogenase Gene Families

Katja E. Hill, Julian R. Marchesi,* and Andrew J. Weightman

Cardiff School of Biosciences, Cardiff University, Cardiff, CF1 3TL, Wales, United Kingdom

Received 1 September 1998/Accepted 29 January 1999

Dehalogenases are key enzymes in the metabolism of halo-organic compounds. This paper describes a systematic approach to the isolation and molecular analysis of two families of bacterial alpha -halocarboxylic acid (alpha HA) dehalogenase genes, called group I and group II deh genes. The two families are evolutionarily unrelated and together represent almost all of the alpha HA deh genes described to date. We report the design and evaluation of degenerate PCR primer pairs for the separate amplification and isolation of group I and II deh genes. Amino acid sequences derived from 10 of 11 group I deh partial gene products of new and previously reported bacterial isolates showed conservation of five residues previously identified as essential for activity. The exception, DehD from a Rhizobium sp., had only two of these five residues. Group II deh gene sequences were amplified from 54 newly isolated strains, and seven of these sequences were cloned and fully characterized. Group II dehalogenases were stereoselective, dechlorinating L- but not D-2-chloropropionic acid, and derived amino acid sequences for all of the genes except dehII°P11 showed conservation of previously identified essential residues. Molecular analysis of the two deh families highlighted four subdivisions in each, which were supported by high bootstrap values in phylogenetic trees and by enzyme structure-function considerations. Group I deh genes included two putative cryptic or silent genes, dehI°PP3 and dehI°17a, produced by different organisms. Group II deh genes included two cryptic genes and an active gene, dehIIPP3, that can be switched off and on. All alpha HA-degrading bacteria so far described were Proteobacteria, a result that may be explained by limitations either in the host range for deh genes or in isolation methods.

* Corresponding author. Mailing address: Cardiff School of Biosciences, Cardiff University, P.O. Box 915, Cardiff, CF1 3TL, Wales, United Kingdom. Phone: 44 (0)1222 874309. Fax: 44 (0)1222 874305.E-mailmarchesi{at}cardiff.ac.uk.

Journal of Bacteriology, April 1999, p. 2535-2547, Vol. 181, No. 8
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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