Expression of Reductive Dehalogenase Genes in Dehalococcoides ethenogenes Strain 195 Growing on Tetrachloroethene, Trichloroethene, or 2,3-Dichlorophenol

Abstract

Reductive dehalogenase (RD) gene transcript levels in Dehalococcoides ethenogenes strain 195 were investigated using reverse transcriptase quantitative PCR during growth and reductive dechlorination of tetrachloroethene (PCE), trichloroethene (TCE), or 2,3-dichlorophenol (2,3-DCP). Cells grown with PCE or TCE had high transcript levels (greater than that for rpoB) for tceA, which encodes the TCE RD, pceA, which encodes the PCE RD, and DET0162, which contains a predicted stop codon and is considered nonfunctional. In cells grown with 2,3-DCP, tceA mRNA was less than 1% of that for rpoB, indicating that its transcription was regulated. pceA and DET0162 were the only RD genes with high transcript levels in cells grown with 2,3-DCP. Proteomic analysis of PCE-grown cells detected both PceA and TceA with high peptide coverage but not DET0162, and analysis of 2,3-DCP-grown cells detected PceA with high coverage but not TceA, DET0162, or any other potential RD. Cells grown with PCE or 2,3-DCP were tested for the ability to dechlorinate PCE, TCE, or 2,3-DCP with H 2 as the electron donor. 2,3-DCP-grown cells were unable to dechlorinate TCE but dechlorinated PCE to TCE without a lag, and PCE-grown cells dechlorinated 2,3-DCP without a lag. These results show that 2,3-DCP-grown cells do not produce TceA and that DET0162 is transcribed but its translation product is not detectable in cells and are consistent with PceA's being bifunctional, also serving as the 2,3-DCP RD. Chlorophenols naturally occur in soils and are good candidates for the original substrates for PceA.Dehalococcoides ethenogenes strain 195 reductively dechlorinates tetrachloroethene (PCE) and trichloroethene (TCE) to vinyl chloride (VC) and ethene (21,32). In addition to chlorinated ethenes, strain 195 has been found to reductively dechlorinate chlorobenzenes and other chloroaromatics (7) and more recently 2,3-dichlorophenol (2,3-DCP) and 2,3,4-trichlorophenol in the ortho position to 3-monochlorophenol or 3,4-dichlorophenol, respectively (1). The reduction of halogenated compounds by Dehalococcoides is carried out by membranebound respiratory reductive dehalogenases (RDs) (12,19,26,27), and although more than 90 RD-homologous genes have been identified in this genus (11,15,26,30), little is known about their specific functions. PCE-RD (PceA) and TCE-RD (TceA) were first characterized in mixed dechlorinating enrichment cultures containing strain 195 and were found to reductively dehalogenate PCE to TCE and TCE to VC and ethene, respectively (19). The gene encoding TCE-RD was subsequently cloned, sequenced, and designated tceA (18).The genome sequence of strain 195 (30) revealed 17 RDhomologous genes in addition to tceA (designated DET0079) and pceA (designated DET0318; J. Magnuson, personal communication). Common features of RDs include the presence of a putative twin-arginine transport signal sequence used for transport into the periplasm of folded proteins that can contain prosthetic groups, iron-sulfur cluster-binding motifs, and an adjacent "B" RD gene...

Regulation of reductive dehalogenase gene transcription in Dehalococcoides mccartyi

“…In addition, albeit at lower levels, transcripts of further rdhA genes were formed, suggesting that multiple rdhA genes are induced by a single chlorinated ethene [24]. Studies with trichloroethene (TCE)-and tetrachloroethene (PCE)-grown cells of strain 195 indicated that the genes encoding the PCE and TCE reductive dehalogenases (PceA, TceA) were transcribed to the highest levels among all 17 rdhA genes [25], and the corresponding proteins were identified with a high coverage in the proteome of the respective cells [26]. Furthermore, rdhA gene expression levels depended on the growth phase and the PCE respiration rate [27][28][29].…”

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.…”

Quantifying Enhanced Microbial Dehalogenation Impacting the Fate and Transport of Organohalide Mixtures in Contaminated Sediments

“…Cbdb_A88 (rdh 05) was also one of the three reductive dehalogenase protein fragments detected in a culture of Dehalococcoides sp. strain CBDB1 growing on 2,3-dichlorophenol (Morris et al, 2007). However, the transcription of all 32 putative rdh genes, including cbdb_A1495 (rdh 04) and cbdb_A88 (rdh 05), was observed in a culture of Dehalococcoides sp.…”

“…strain CBDB1 spiked with 1,2,3-and 1,2,4-trichlorobenzene (Adrian et al, 2007). Cbdb_A88 (rdh 05) was also one of the three reductive dehalogenase protein fragments detected in a culture of Dehalococcoides sp.…”

“…The presence and amount of genes in environmental samples have been used as molecular markers to assess their related activity, such as dechlorinating genes for monitoring dehalogenation of chlorinated ethenes (Fung et al, 2007;Futamata et al, 2007;Behrens et al, 2008;Rahm and Richardson, 2008;Carreon-Diazconti et al, 2009). Since no known rdh gene involved in PCB dechlorination has been identified to date, further study on these two rdh genes (rdh 04 and rdh 05) may help in developing methods to monitor PCB dechlorinating bacteria.…”