The MarR-Type Regulator Rdh2R Regulates rdh Gene Transcription in Dehalococcoides mccartyi Strain CBDB1.

Reductive dehalogenases are essential enzymes in organohalide respiration and consist of a catalytic subunit A and a membrane protein B, encoded by rdhAB genes. Thirty-two rdhAB genes exist in the genome of Dehalococcoides mccartyi strain CBDB1. To gain a first insight into the regulation of rdh operons, the control of gene expression of two rdhAB genes (cbdbA1453/cbdbA1452 and cbdbA1455/cbdbA1454) by the MarR-type regulator Rdh2R (cbdbA1456) encoded directly upstream was studied using heterologous expression and in vitro studies. Promoter-lacZ reporter fusions were generated and integrated into the genome of the Escherichia coli host. The lacZ reporter activities of both rdhA promoters decreased upon transformation of the cells with a plasmid carrying the rdh2R gene, suggesting that Rdh2R acts as repressor, whereas the lacZ reporter activity of the rdh2R promoter was not affected. The transcriptional start sites of both rdhA genes in strain CBDB1 and/or the heterologous host mapped to a conserved direct repeat with 11- to 13-bp half-sites. DNase I footprinting revealed binding of Rdh2R to a ∼30-bp sequence covering the complete direct repeat in both promoters, including the transcriptional start sites. Equilibrium sedimentation ultracentrifugation revealed that Rdh2R binds as tetramer to the direct-repeat motif of the rdhA (cbdbA1455) promoter. Using electrophoretic mobility shift assays, a similar binding affinity was found for both rdhA promoters. In the presence of only one half-site of the direct repeat, the interaction was strongly reduced, suggesting a positive cooperativity of binding, for which unusual short palindromes within the direct-repeat half-sites might play an important role. IMPORTANCE: Dehalococcoides mccartyi strains are obligate anaerobes that grow by organohalide respiration. They have an important bioremediation potential because they are capable of reducing a multitude of halogenated compounds to less toxic products. We are now beginning to understand how these organisms make use of this large catabolic potential, whereby D. mccartyi expresses dehalogenases in a compound-specific fashion. MarR-type regulators are often encoded in the vicinity of reductive dehalogenase genes. In this study, we made use of heterologous expression and in vitro studies to demonstrate that the MarR-type transcription factor Rdh2R acts as a negative regulator. We identify its binding site on the DNA, which suggests a mechanism by which it controls the expression of two adjacent reductive dehalogenase operons.

Identification of a multi-protein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement.

Dehalococcoides mccartyi strain CBDB1 is an obligate organohalide-respiring bacterium using only hydrogen as electron donor and halogenated organics as electron acceptor. Here, we studied proteins involved in the respiratory chain under non-denaturing conditions. Using blue native gel electrophoresis (BN-PAGE), gel filtration and ultrafiltration an active dehalogenating protein complex with a molecular mass of 250-270 kDa was identified. The active subunit of reductive dehalogenase (RdhA) colocalised with a complex iron-sulfur molybdoenzyme (CISM) subunit (CbdbA195) and an iron-sulfur cluster containing subunit (CbdbA131) of the hydrogen uptake hydrogenase (Hup). No colocalisation between the catalytically active subunits of hydrogenase and reductive dehalogenase was found. By two-dimensional BN/SDS-PAGE the stability of the complex towards detergents was assessed, demonstrating stepwise disintegration with increasing detergent concentrations. Chemical cross-linking confirmed the presence of a higher molecular mass reductive dehalogenase protein complex composed of RdhA, CISM I and Hup hydrogenase and proved to be a potential tool for stabilising protein-protein interactions of the dehalogenating complex prior to membrane solubilisation. Taken together, the identification of the respiratory dehalogenase protein complex and the absence of indications for quinone participation in the respiration suggest a quinone-independent protein-based respiratory electron transfer chain in D. mccartyi.

Reductive Dehalogenation of Oligocyclic Phenolic Bromoaromatics by Dehalococcoides mccartyi Strain CBDB1.

Dehalococcoides mccartyi strains transform many halogenated compounds and are used for bioremediation. Such anaerobic transformations were intensively studied with chlorinated and simply structured compounds such as chlorinated benzenes, ethenes, and ethanes. However, many halogenated oligocyclic aromatic compounds occur in nature as either naturally produced materials or as part of commercial products such as pharmaceuticals, pesticides, or flame retardants. Here, we demonstrate that the D. mccartyi strain CBDB1 reductively debrominated two oligocyclic aromatic phenolic compounds, tetrabromobisphenol A (TBBPA) and bromophenol blue (BPB). The strain CBDB1 completely converted TBBPA to bisphenol A and BPB to phenol red with a stepwise removal of all bromide substituents. Debromination (but no cell growth) was detected in the cultures cultivated with TBBPA. In contrast, strain CBDB1 grew when interacting with BPB, demonstrating that this substrate was used as an electron acceptor for organobromine respiration. High doses of BPB delayed debromination and inhibited growth in the early cultivation phase. A higher toxicity of TBBPA compared with that of BPB might be due to the higher lipophilicity of TBBPA. Mass spectrometric analyses of whole-cell extracts demonstrated that two proteins encoded by the reductive dehalogenase homologous genes CbdbA1092 and CbdbA1503 were specifically induced by the used oligocyclic compounds, whereas others (e.g., CbdbA84 (CbrA)) were downregulated.

Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM).

Targeted absolute protein quantification yields valuable information about physiological adaptation of organisms and is thereby of high interest. Especially for this purpose, two proteomic mass spectrometry-based techniques namely selective reaction monitoring (SRM) and precursor reaction monitoring (PRM) are commonly applied. The objective of this study was to establish an optimal quantification assay for proteins with the focus on those involved in housekeeping functions and putative reductive dehalogenase proteins from the strictly anaerobic bacterium Dehalococcoides mccartyi strain CBDB1. This microbe is small and slow-growing; hence, it provides little biomass for comprehensive proteomic analysis. We therefore compared SRM and PRM techniques. Eleven peptides were successfully quantified by both methods. In addition, six peptides were solely quantified by SRM and four by PRM, respectively. Peptides were spiked into a background of Escherichia coli lysate and the majority of peptides were quantifiable down to 500 amol absolute on column by both methods. Peptide quantification in CBDB1 lysate resulted in the detection of 15 peptides using SRM and 14 peptides with the PRM assay. Resulting quantification of five dehalogenases revealed copy numbers of <10 to 115 protein molecules per cell indicating clear differences in abundance of RdhA proteins during growth on hexachlorobenzene. Our results indicated that both methods show comparable sensitivity and that the combination of the mass spectrometry assays resulted in higher peptide coverage and thus more reliable protein quantification.

A H2 -oxidizing, 1,2,3-trichlorobenzene-reducing multienzyme complex isolated from the obligately organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1.

Dehalococcoides mccartyi is a small, slow-growing bacterium of the phylum Chloroflexi that conserves energy using aliphatic and aromatic organohalides as electron acceptors, and hydrogen as sole electron donor. A recent study identified a protein complex in the membrane of strain CBDB1 comprising a Hup hydrogenase, a complex iron-sulphur molybdoprotein and a reductive dehalogenase (RdhA) that catalyses reduction of 1,2,3,4-tetrachlorobenzene. Using a combination of size-exclusion chromatography, in-gel hydrogenase activity-staining, immunological analysis and mass spectrometry, we identified here a large molecular mass protein complex solubilized from the cytoplasmic membrane of D. mccartyi strain CBDB1 that catalysed H2 -dependent reduction of 1,2,3-trichlorobenzene (1,2,3-TCB) to 1,3-DCB. In-gel zymographic staining revealed H2 :benzyl viologen oxidoreductase activity associated with the complex and immunological analysis identified co-elution of CdbdA195, the predicted catalytic subunit of the iron-sulphur molybdoenzyme, the chlorobenzene-specific RdhA, CbrA, and traces of HupL, the catalytic subunit of the Hup hydrogenase. Quantitative reverse transcriptase PCR analyses indicated that the expression of the hupL and cbdbA195 genes was induced by 1,2,3-TCB but not by hydrogen. Together, these data identify and describe a protein-based electron-transfer complex catalysing H2 oxidation coupled to chlorobenzene reduction.

Organic cofactors in the metabolism of Dehalococcoides mccartyi strains.

Dehalococcoides mccartyi strains are strictly anaerobic organisms specialized to grow with halogenated compounds as electron acceptor via a respiratory process. Their genomes are among the smallest known for free-living organisms, and the embedded gene set reflects their strong specialization. Here, we briefly review main characteristics of published Dehalococcoides genomes and show how genome information together with cultivation and biochemical experiments have contributed to our understanding of Dehalococcoides physiology and biochemistry. We extend this approach by the detailed analysis of cofactor metabolism in Dehalococcoides strain CBDB1. Dehalococcoides genomes were screened for encoded proteins annotated to contain or interact with organic cofactors, and the expression of these proteins was analysed by shotgun proteomics to shed light on cofactor requirements. In parallel, cultivation experiments testing for vitamin requirements showed that cyanocobalamin (vitamin B12), thiamine and biotin were essential supplements and that cyanocobalamin could be substituted by dicyanocobinamide and dimethylbenzimidazole. Dehalococcoides genome analysis, detection of single enzymes by shotgun proteomics and inhibition studies confirmed the expression of the biosynthetic pathways for pyridoxal-5-phosphate, flavin nucleotides, folate, S-adenosylmethionine, pantothenate and nicotinic acids in strain CBDB1. Haem/cytochromes, quinones and lipoic acids were not necessary for cultivation or dechlorination activity and no biosynthetic pathways were identified in the genomes.