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

Abstract

Degenerate primers were used to amplify large fragments of reductive-dehalogenase-homologous (RDH) genes from genomic DNA of two Dehalococcoides populations, the chlorobenzene-and dioxin-dechlorinating strain CBDB1 and the trichloroethene-dechlorinating strain FL2. The amplicons (1,350 to 1,495 bp) corresponded to nearly complete open reading frames of known reductive dehalogenase genes and short fragments (approximately 90 bp) of genes encoding putative membrane-anchoring proteins. Cloning and restriction analysis revealed the presence of at least 14 different RDH genes in each strain. All amplified RDH genes showed sequence similarity with known reductive dehalogenase genes over the whole length of the sequence and shared all characteristics described for reductive dehalogenases. Deduced amino acid sequences of seven RDH genes from strain CBDB1 were 98.5 to 100% identical to seven different RDH genes from strain FL2, suggesting that both strains have an overlapping substrate range. All RDH genes identified in strains CBDB1 and FL2 were related to the RDH genes present in the genomes of Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain BAV1; however, sequence identity did not exceed 94.4 and 93.1%, respectively. The presence of RDH genes in strains CBDB1, FL2, and BAV1 that have no orthologs in strain 195 suggests that these strains possess dechlorination activities not present in strain 195. Comparative sequence analysis identified consensus sequences for cobalamin binding in deduced amino acid sequences of seven RDH genes. In conclusion, this study demonstrates that the presence of multiple nonidentical RDH genes is characteristic of Dehalococcoides strains.

Overview of organohalide-respiring bacteria and a proposal for a classification system for reductive dehalogenases

“…The preponderance of these organisms is partially due to the intense research efforts concentrated on these bacterial groups based on their environmental significance as dechlorinating organisms, but is also due to the fact that genomes from these organisms typically contain multiple rdhA genes [20,32,80,94].…”

“…Several molecular tools have been developed for examining a broad range of rdhA genes in a given sample (site, isolate or mixed culture), including both microarray-based methods and PCR-based protocols [60][61][62][63]. Identification of novel reductive dehalogenase genes has primarily come from PCR studies [32,33,[35][36][37]39,40,42] and subsequently from genome and metagenome sequencing efforts [6,13,14,41,63], with each new organism or environment sampled increasing the known diversity of putative rdhA genes.…”

“…The first gene sequences encoding reductive dehalogenases were identified using classical reverse genetics approaches based on partial amino acid sequences of purified enzymes [30,31]. PCR and genome studies have since identified new sequences with high sequence identity to the characterized genes [6,13,14,[32][33][34][35][36][37][38][39][40][41][42]. Reductive dehalogenase genes (or reductive dehalogenase homologous genes (rdh) if the encoded protein has not yet been biochemically characterized) typically comprise an operon containing rdhA, the gene for the catalytically active enzyme (RDase if characterized, otherwise RdhA), rdhB, a gene encoding a putative membrane-anchoring protein [34], and sometimes one or more members of rdhTKZECD, genes associated with reductive dehalogenase genes [43].…”

Role of “Dehalococcoides” spp. in the Anaerobic Transformation of Hexachlorobenzene in European Rivers

“…Even though the majority of known rdh genes from Dehalococcoides spp. were amplified using this primer set (21,39), it also failed to amplify some rdh genes (21,45). Additionally, in the full genome sequence of strain CBDB1, there are only four rdhA genes that have a perfect binding site to these primers.…”

“…Additionally, degenerate primers (forward primer, fdehal, and reverse primer, rdehal [see Table S2 in the supplemental material]) were used to amplify 550-to 660-bp fragments. These primers target conserved motifs in the upstream region of the iron-sulfur cluster binding motif of rdhA genes in strain CBDB1 (21). After cloning and sequencing of these PCR fragments, the sequences obtained were compared to those deposited in the National Center for Biotechnology Information (NCBI) database using the BLASTX tool (4).…”