Investigation of Two Evolutionarily Unrelated Halocarboxylic Acid Dehalogenase Gene Families

Abstract

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 α-halocarboxylic acid (αHA) dehalogenase genes, called group I and group II deh genes. The two families are evolutionarily unrelated and together represent almost all of the αHAdeh 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 dehgenes. Amino acid sequences derived from 10 of 11 group Ideh 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 exceptdehII°P11 showed conservation of previously identified essential residues. Molecular analysis of the twodeh families highlighted four subdivisions in each, which were supported by high bootstrap values in phylogenetic trees and by enzyme structure-function considerations. Group Ideh genes included two putative cryptic or silent genes, dehI°PP3 anddehI°17a, produced by different organisms. Group II deh genes included two cryptic genes and an active gene, dehII PP3, that can be switched off and on. All αHA-degrading bacteria so far described were Proteobacteria, a result that may be explained by limitations either in the host range fordeh genes or in isolation methods.

Detection and enumeration of haloacetic acid-degrading bacteria in drinking water distribution systems using dehalogenase genes

“…PCR protocols using degenerate primers to amplify deh I and deh II genes in the isolates, consortium, and drinking water distribution system samples were adapted from Hill et al. (1999).…”

“…Amplification program: 94°C for 10 min; with 36 cycles of 94°C for 45 s; 55°C for 1 min; 75°C for 45 s; finally 75°C with 7 min extension. Primers for both deh I and deh II were synthesized by Integrated DNA Technologies (Coralville, IA, USA) as published by Hill et al. (1999).…”

“…To design an effective molecular screen, it is necessary to have a functional target gene that is relevant to HAA degradation in drinking water. In bacteria that have the ability to utilize and dehalogenate HAAs, the haloacid dehalogenase enzyme is critical (Hill et al. 1999; Janssen et al.…”

“…2004). Hill et al. (1999) used a systematic molecular approach to group dehalogenase genes as either deh I or deh II according to gene similarity.…”

“…(1999) used a systematic molecular approach to group dehalogenase genes as either deh I or deh II according to gene similarity. Either the deh I or deh II genes could provide a good functional target for non‐cultivation based detection of HAA degraders (Fetzner and Lingens 1994; Hill et al. 1999; Janssen et al.…”