Sequence diversity in haloalkane dehalogenases, as revealed by PCR using family-specific primers

Haloalkane dehalogenases (HLDs) are hydrolytic enzymes that cleave carbon-halogen bonds in various halogenated compounds. Interest initially grew in HLDs as biocatalysts for bioremediation and later for biotransformation applications; each specific HLD within the HLD family has its own substrate specificity, enantioselectivity and product inhibition characteristics. We developed degenerate oligonucleotide primers for HLD-encoding genes and used these to PCR-amplify large hld gene fragments using genomic DNA from the microbial community of a chlorinated-solvent-contaminated aquifer as a template. An analysis of small subunit ribosomal RNA genes revealed a high complexity in the eubacterial population, dominated by α-, β- and γ-Proteobacteria, and Acidobacteria. Using HLD-family-specific primers, we also retrieved transcribed hld homologues from the microbial consortium of this contaminated site. The DNA-derived hld sequences were phylogenetically broadly distributed over both HLD subclasses I and II. Most hld sequences of the environmental RNA data set clustered in three groups within both HLD subclasses, indicating that a considerable proportion of the microbial consortium carrying hld genes was actively involved in haloalkane dehalogenation. The small sequence variation in hld genes and transcripts within each HLD cluster inferred the presence of a substantial pool of highly related HLD genes. The sequence variability appeared to be unevenly distributed over the HLD genes, however, with no apparent preference for a particular protein segment or domain.