Multiple Reductive-Dehalogenase-Homologous Genes Are Simultaneously Transcribed during Dechlorination by Dehalococcoides- Containing Cultures

Abstract

Degenerate primers were used to amplify 14 distinct reductive-dehalogenase-homologous (RDH) genes from the Dehalococcoides-containing mixed culture KB1. Most of the corresponding predicted proteins were highly similar (97 to >99% amino acid identity) to previously reported Dehalococcoides reductive dehalogenases. To examine the differential transcription of these RDH genes, KB1 was split into five subcultures amended with either trichloroethene, cis-1,2-dichloroethene, vinyl chloride, 1,2-dichlorethane, or no chlorinated electron acceptor. Total RNA was extracted following the onset of reductive dechlorination, and RDH transcripts were reverse transcribed and amplified using degenerate primers. The results indicate that the transcription of RDH genes requires the presence of a chlorinated electron acceptor, and for all treatments, multiple RDH genes were simultaneously transcribed, with transcripts of two of the genes being present under all four electron-accepting conditions. Two of the transcribed sequences were highly similar to reported vinyl chloride reductase genes, namely, vcrA from Dehalococcoides sp. strain VS and bvcA from Dehalococcoides sp. strain BAV1. These findings suggest that multiple RDH genes are induced by a single chlorinated substrate and that multiple reductive dehalogenases contribute to chloroethene degradation in KB1.

Identification and transcriptional analysis of trans-DCE-producing reductive dehalogenases in Dehalococcoides species

“…None of the seven identified RDase genes of MB are similar to the TCE RDase genes in Dehalococcoides (for example, strains 195 and FL2). Phylogenetic analysis of the RDase genes in strain MB and other Dehalococcoides using MAFFT software generated phylogenetic trees that are similar in topology to previous studies Waller et al, 2005) (Figure 1).…”

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].…”

“…While these proteins often cannot be assigned substrate specificity with certainty, these experiments provide evidence for function and can be used to better illustrate the sequence similarity/substrate affinity relationship within this group of enzymes. These methods have been somewhat confounded in the case of Dehalococcoides, where multiple reductive dehalogenase genes are typically expressed in the presence of a single halogenated substrate [79,80].…”

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

“…Therefore, it can be speculated that at mesophilic temperatures, besides cbrA and cbdbA1624, some other rdh genes contribute significantly to the metabolic activity. Complex transcriptional responses to various chlorinated compounds were previously reported for Dehalococcoides isolates and cocultures (16,44,45) and were predicted to be tightly regulated by two-component and/or MarR-type regulators (29,35). The majority of rdh genes detected with T-RFLP and sequencing analysis were similar to those of strain CBDB1, which are located close to MarR-type regulators.…”

“…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.…”