Occurrence of several genes encoding putative reductive dehalogenases inDesulfitobacterium hafniense/frappieriandDehalococcoides ethenogenes

Abstract

Desulfitobacterium frappieri PCP-1 has the capacity to dehalogenate several halogenated aromatic compounds by reductive dehalogenation, however, the genes encoding the enzymes involved in such processes have not yet been identified. Using a degenerate oligonucleotide corresponding to a conserved sequence of CprA/PceA reductive dehalogenases, a cprA-like gene fragment was amplified by PCR from this bacterial strain. A Desulfitobacterium frappieri PCP-1 cosmid library was screened with the PCR product, allowing the cloning and sequencing of a 1.9-kb fragment. This fragment contains a nucleic acid sequence identical to one genomic contig of Desulfitobacterium hafniense, a bacterium closely related to Desulfitobacterium frappieri that is also involved in reductive dehalogenation. Other genes related to the Desulfitobacterium dehalogenans cpr locus were identified in this contig. Interestingly, the gene arrangement shows the presence of two copies of cprA-, cprB-, cprC-, cprD-, cprK-, and cprT-related genes, suggesting that gene duplication occurred within this chromosomic region. The screening of Delfitobacterium hafniense genomic contigs with a CprA-deduced amino acid sequence revealed two other cprA-like genes. Microbial genomes available in gene databases were also analyzed for sequences related to CprA/PceA. Two open reading frames encoding other putative reductive dehalogenases in Desulfitobacterium hafniense contigs were detected, along with 17 in the Dehalococcoides ethenogenes genome, a bacterium involved in the reductive dehalogenation of tetrachloroethene to ethene. The fact that several gene encoding putative reductive dehalogenases exist in Delfitobacterium hafniense, probably in other members of the genus Desulfitobacterium, and in Dehalococcoides ethenogenes suggests that these bacteria use distinct but related enzymes to achieve the dehalogenation of several chlorinated compounds [corrected].

Genetic Identification of a Putative Vinyl Chloride Reductase in Dehalococcoides sp. Strain BAV1

“…and to identify RDase genes implicated in VC reductive dechlorination. Multiple alignments of full-length protein and DNA sequences of TceA (GenBank accession numbers AAN85590, AAN85588, and AAF73916A) and putative RDase genes identified in the genome of D. ethenogenes strain 195 (35) were constructed by using ClustalW and ClustalX (32). Conserved amino acid sequences were identified and used to design degenerate PCR primers (Fig.…”

“…A comparison of the translated amino acid sequence BvcA with putative RDases found on other Dehalococcoides sp. genomes (13,35) showed that sequence identity did not exceed 39% at the protein level. These findings indicate that bvcA is a unique gene not present in other described Dehalococcoides isolates.…”

Multiple Nonidentical Reductive-Dehalogenase-Homologous Genes Are Common in Dehalococcoides

“…Access to whole-genome sequence data for D. ethenogenes strain 195 (http://www.tigr.org/tdb/mdb/mdbinprogress.html) allowed for a systematic screening for dehalogenase sequences (48). Besides the TCE dehalogenase-encoding tceA gene, 17 different reductive-dehalogenase-homologous (RDH) genes were found in strain 195, and all share the above-described features characteristic of dehalogenases (44,48).…”

“…Besides the TCE dehalogenase-encoding tceA gene, 17 different reductive-dehalogenase-homologous (RDH) genes were found in strain 195, and all share the above-described features characteristic of dehalogenases (44,48). Hence, it was hypothesized that the genomes of Dehalococcoides populations contain multiple RDH genes, corresponding to the individual range of chlorinated electron acceptors used by individual strains.…”

Regulation of Anaerobic Dehalorespiration by the Transcriptional Activator CprK

“…The low G ϩ C, Gram-positive bacterium D. dehalogenans, from which the protein studied here originates, can utilize hydroxylated polychlorinated biphenyls (10), and the Desulfitobacterium chlororespirans dehalogenase can use hydroxy-polychlorinated biphenyls as substrates (11). Multiple gene clusters encoding reductive dehalogenases have been detected in the genomes of dehalorespiring microbes, including 17 potential dehalogenases in Dehalococcoides ethenogenes (12). Some of the proteins involved in dehalorespiration are encoded in the cpr (chlorophenol reduction) gene cluster, which includes the reductive dehalogenase (cprA), its membrane anchor (cprB), several proposed chaperonins (cprT, -Z, -E, and -D), and two putative regulatory proteins (cprC and cprK) that are organized into four transcriptional units encoding cprT, cprBA, cprZE, and cprBACD (13).…”