Dehalococcoides Mccartyi Strain Research Articles

Dehalococcoides mccartyi Strain GEO12 Has a Natural Tolerance to Chloroform Inhibition.

Cocontamination by chloroform and chloroethenes often confounds bioremediation efforts. Here, we describe Dehalococcoides mccartyi strain GEO12 that dechlorinates trichloroethene to ethene in 14 μM (1.6 mg·L-1) chloroform. The same chloroform concentration effectively inhibited dechlorination in Dehalococcoides strains ANAS2, 11a, and BAV1. Successive transfers of strain GEO12 in increasing concentrations of chloroform led to culture GEO12CF that tolerated 83 μM (10 mg·L-1) chloroform. The genome of strain GEO12 revealed seven reductive dehalogenase homologous (rdh) genes, including tceA and vcrA. Transcriptional analyses showed that chloroform (45 μM; 5.3 mg·L-1) in culture GEO12CF enhanced the transcription of tceA to a statistically significant degree (the median increase was 55.4 transcripts per 104 16S rRNA, CI95% = [12.9, 125]). The increase of vcrA transcripts in the presence of chloroform (45 μM; 5.3 mg·L-1) in culture GEO12CF was not statistically significant because the CI95% range spanned 0 (the median increase was 109 transcripts per 104 16S rRNA, CI95% = [-13.6, 246]). Inhibition of dehalogenation by chloroform is often seen in Dehalococcoides, but the mechanism remains unknown. Our results suggest that culture GEO12CF may overcome chloroform inhibition by rdh upregulation. The chloroform-adapted culture GEO12CF provides insights into the metabolic flexibility of Dehalococcoides and could be used to fight chloroethene contamination where chloroform is a cocontaminant.

Abundance of organohalide respiring bacteria and their role in dehalogenating antimicrobials in wastewater treatment plants

Anthropogenic organohalide contaminants present in wastewater treatment plants (WWTPs) often remain untreated and can be discharged into the environment. Although organohalide respiring bacteria (OHRB) contribute to the elimination of anthropogenic organohalides in natural anaerobic environments, reductive dehalogenation by OHRB in mainstream WWTPs remains poorly understood. In this study, we quantified OHRB during a long-term operation of a municipal WWTP with short hydraulic and sludge retention times (3 h and 1.5–5 days, respectively). The obligate OHRB were detected at high levels (averaging 2.56 ± 1.73 × 107 and 3.11 ± 1.16 × 107 16S rRNA gene copies/ml MLSS sludge in anoxic and aerobic zones, respectively) over the entire sampling period and throughout the wastewater treatment train. Microcosms derived from mainstream activated sludge contained an unidentified member of the Dehalococcoides genus that metabolically dechlorinated triclosan, used as a representative emerging organohalide antimicrobial, to diclosan, suggesting the potential of anaerobic degradation of emerging contaminants in WWTPs. To further understand the mechanisms for such antimicrobials’ removal, an investigation of dechlorination of triclosan by Dehalococcoides strains was conducted. Dechlorination of environmentally relevant concentrations of triclosan to diclosan was observed in Dehalococcoides mccartyi strain CG1, yielding 4.59 ± 0.34 × 108 cells/μmole Cl− removed at a rate of 0.062 μM/day and a minimal inhibitory concentration of 0.5 mg/L. Notably, both the tolerance of strain CG1 to triclosan and the rate of triclosan dechlorination increased when CG1 was cultured in the presence of both triclosan and tetrachloroethene. Taken together, our results suggest that anaerobic degradation of organohalide antimicrobials might be more prevalent in mainstream WWTPs than previously speculated, though the low growth yields that are supported by triclosan dechlorination seem to indicate that other organohalide substrates could be necessary to sustain OHRB populations in these systems.

Identification and characterization of a bacterial core methionine synthase

Methionine synthases are essential enzymes for amino acid and methyl group metabolism in all domains of life. Here, we describe a putatively anciently derived type of methionine synthase yet unknown in bacteria, here referred to as core-MetE. The enzyme appears to represent a minimal MetE form and transfers methyl groups from methylcobalamin instead of methyl-tetrahydrofolate to homocysteine. Accordingly, it does not possess the tetrahydrofolate binding domain described for canonical bacterial MetE proteins. In Dehalococcoides mccartyi strain CBDB1, an obligate anaerobic, mesophilic, slowly growing organohalide-respiring bacterium, it is encoded by the locus cbdbA481. In line with the observation to not accept methyl groups from methyl-tetrahydrofolate, all known genomes of bacteria of the class Dehalococcoidia lack metF encoding for methylene-tetrahydrofolate reductase synthesizing methyl-tetrahydrofolate, but all contain a core-metE gene. We heterologously expressed core-MetECBDB in E. coli and purified the 38 kDa protein. Core-MetECBDB exhibited Michaelis-Menten kinetics with respect to methylcob(III)alamin (KM ≈ 240 µM) and L-homocysteine (KM ≈ 50 µM). Only methylcob(III)alamin was found to be active as methyl donor with a kcat ≈ 60 s−1. Core-MetECBDB did not functionally complement metE-deficient E. coli strain DH5α (ΔmetE::kan) suggesting that core-MetECBDB and the canonical MetE enzyme from E. coli have different enzymatic specificities also in vivo. Core-MetE appears to be similar to a MetE-ancestor evolved before LUCA (last universal common ancestor) using methylated cobalamins as methyl donor whereas the canonical MetE consists of a tandem repeat and might have evolved by duplication of the core-MetE and diversification of the N-terminal part to a tetrahydrofolate-binding domain.

Effects of Arsenic on Trichloroethene-Dechlorination Activities of Dehalococcoides mccartyi 195.

Arsenic and trichloroethene (TCE) are among the most prevalent groundwater contaminants in the United States. Co-contamination of these two compounds has been detected at 63% of current TCE-contaminated National Priorities List sites. When in situ TCE reductive dechlorination is stimulated by the addition of fermentable substrates to generate a reducing environment, the presence of arsenic can be problematic because of the potential for increased mobilization and toxicity caused by the reduction of arsenate [As(V)] to arsenite [As(III)]. This study assesses the effects of arsenic exposure on the TCE-dechlorinating activities of Dehalococcoides mccartyi strain 195. Our results indicate that 9.1 μM As(III) caused a 50% decrease in D. mccartyi cell growth. While As(V) concentrations up to 200 μM did not initially impact TCE dechlorination, inhibition was observed in cultures amended with 200 μM As(V) and 100 μM As(V) in 12 and 17 days, respectively, corresponding with the accumulation of As(III). Transcriptomic and metabolomic analyses were performed to evaluate cellular responses to both As(V) and As(III) stress. Amendment of amino acids enhanced arsenic tolerance of D. mccartyi. Results from this study improve our understanding of potential inhibitions of D. mccartyi metabolism caused by arsenic and can inform the design of bioremediation strategies at co-contaminated sites.

Identification of a Chlorodibenzo-p-dioxin Dechlorinating Dehalococcoides mccartyi by Stable Isotope Probing.

Polychlorinated dibenzo-p-dioxins (PCDDs) are released into the environment from a variety of both anthropogenic and natural sources. While highly chlorinated dibenzo-p-dioxins are persistent under oxic conditions, in anoxic environments, these organohalogens can be reductively dechlorinated to less chlorinated compounds that are then more amenable to subsequent aerobic degradation. Identifying the microorganisms responsible for dechlorination is an important step in developing bioremediation approaches. In this study, we demonstrated the use of a DNA-stable isotope probing (SIP) approach to identify the bacteria active in dechlorination of PCDDs in river sediments, with 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD) as a model. In addition, pyrosequencing of reverse transcribed 16S rRNA of TeCDD dechlorinating enrichment cultures was used to reveal active members of the bacterial community. A set of operational taxonomic units (OTUs) responded positively to the addition of 1,2,3,4-TeCDD in SIP microcosms assimilating 13C-acetate as the carbon source. Analysis of bacterial community profiles of the 13C labeled heavy DNA fraction revealed that an OTU corresponding to Dehalococcoides mccartyi accounted for a significantly greater abundance in cultures amended with 1,2,3,4-TeCDD than in cultures without 1,2,3,4-TeCDD. This implies the involvement of this Dehalococcoides mccartyi strain in the reductive dechlorination of 1,2,3,4-TeCDD and suggests the applicability of SIP for a robust assessment of the bioremediation potential of organohalogen contaminated sites.

Deiodination in the presence of Dehalococcoides mccartyi strain CBDB1: comparison of the native enzyme and co-factor vitamin B12.

Triiodinated benzoic acid derivatives are widely used as contrast media for medical examinations and are found at high concentrations in urban aquatic environments. During bank filtration, deiodination of iodinated contrast media has been observed under anoxic/anaerobic conditions. While several bacterial strains capable of dechlorination and debromination have been isolated and characterized, deiodination has not yet been shown for an isolated strain. Here, we investigate dehalogenation of iodinated contrast media (ICM), triiodobenzoic acids (TIBA), and analogous chlorinated compounds by Dehalococcoides mccartyi strain CBDB1 and its corrinoid co-factor vitamin B12. No cell growth of CBDB1 was observed using iodinated compounds as electron acceptor. Only negligible deiodination occurred for ICM, whereas 2,3,5-TIBA was nearly completely deiodinated by CBDB1 without showing cell growth. Furthermore, TIBA inhibited growth with hexachlorobenzene which is usually a well-suited electron acceptor for strain CBDB1, indicating that TIBA is toxic for CBDB1. The involvement of CBDB1 enzymes in the deiodination of TIBA was verified by the absence of deiodination activity after heat inactivation. Adding iodopropane also inhibited the deiodination of TIBA by CBDB1 cells, indicating the involvement of a corrinoid-enzyme in the reductive TIBA deiodination. The results further suggest that the involved electron transport is decoupled from proton translocation and therefore growth. Graphical abstract.

Complete Genome Sequence of Dehalococcoides mccartyi Strain FL2, a Trichloroethene-Respiring Anaerobe Isolated from Pristine Freshwater Sediment.

Dehalococcoides mccartyi strain FL2 couples growth to hydrogen oxidation and reductive dechlorination of trichloroethene and cis- and trans-1,2-dichloroethenes. Strain FL2 has a 1.42-Mb genome with a G+C content of 47.0% and carries 1,465 protein-coding sequences, including 24 reductive dehalogenase genes.

Detection of Organohalide-Respiring Enzyme Biomarkers at a Bioaugmented TCE-Contaminated Field Site.

RNA-based biomarkers have been successfully detected at field sites undergoing in situ bioremediation, but the detection of expressed enzymes is a more direct way to prove activity for a particular biocatalytic process of interest since they provide evidence of potential in situ activity rather than simply confirming presence and abundance of genes in a given population by measurement of DNA copies using qPCR. Here we successfully applied shotgun proteomics to field samples from a trichloroethene (TCE)-contaminated industrial site in southern Ontario, Canada that had been bio-augmented with the commercially available KB-1TM microbial culture. The KB-1TM culture contains multiple strains of Dehalococcoides mccartyi (D. mccartyi) as well as an organohalide respiring Geobacter species. The relative abundances of specific enzymatic proteins were subsequently compared to corresponding qPCR-derived levels of DNA and RNA biomarkers in the same samples. Samples were obtained from two wells with high hydraulic connectivity to the KB-1TM-bioaugemented enhanced in situ bioremediation system, and two control wells that showed evidence of low levels of native organohalide respiring bacteria (OHRB), Dehalococcoides and Geobacter. Enzymes involved in organohalide respiration were detected in the metaproteomes of all four field samples, as were chaperonins of D. mccartyi, chemotaxis proteins, and ATPases. The most highly expressed RDase in the bioaugmentation culture (VcrA) was the most highly detected enzyme overall in the bioaugmented groundwater samples. In one background groundwater well, we found high expression of the Geobacter pceA RDase. The DNA and RNA biomarkers detected using qPCR-based assays were a set of orthologs of Dehalococcoides reductive dehalogenases (VcrA, TceA, BvcA, dehalogenase “DET1545”), and the Ni-Fe uptake hydrogenase, HupL. Within a sample, RNA levels for key enzymes correlated with relative protein abundance. These results indicate that laboratory observations of TCE-bioremediation biomarker protein expression are recapitulated in field environmental systems and that both RNA and protein biomarker monitoring hold promise for activity monitoring of in situ populations of OHRB.

Growth of Dehalococcoides spp. and increased abundance of reductive dehalogenase genes in anaerobic PCB-contaminated sediment microcosms

Polychlorinated biphenyls (PCBs) contaminate 19% of US Superfund sites and represent a serious risk to human and environmental health. One promising strategy to remediate PCB-contaminated sediments utilizes organohalide-respiring bacteria (OHRB) that dechlorinate PCBs.However, functional genes that act as biomarkers for PCB dechlorination processes (i.e., reductive dehalogenase genes) are poorly understood. Here, we developed anaerobic sediment microcosms that harbor an OHRB community dominated by the genus Dehalococcoides. During the 430-day microcosm incubation, Dehalococcoides 16S rRNA sequences increased two orders of magnitude to 107 copies/g of sediment, and at the same time, PCB118 decreased by as much as 70%. In addition, the OHRB community dechlorinated a range of penta- and tetra-chlorinated PCB congeners including PCBs 66, 70 + 74 + 76, 95, 90 + 101, and PCB110 without exogenous electron donor. We quantified candidate reductive dehalogenase (RDase) genes over a 430-day incubation period and found rd14, a reductive dehalogenase that belongs to Dehalococcoides mccartyi strain CG5, was enriched to 107 copies/g of sediment. At the same time, pcbA5 was enriched to only 105 copies/g of sediment. A survey for additional RDase genes revealed sequences similar to strain CG5's rd4 and rd8. In addition to demonstrating the PCB dechlorination potential of native microbial communities in contaminated freshwater sediments, our results suggest candidate functional genes with previously unexplored potential could serve as biomarkers of PCB dechlorination processes.

Structural dynamics and transcriptomic analysis of Dehalococcoides mccartyi within a TCE-Dechlorinating community in a completely mixed flow reactor

A trichloroethene (TCE)-dechlorinating community (CANAS) maintained in a completely mixed flow reactor was established from a semi-batch enrichment culture (ANAS) and was monitored for 400 days at a low solids retention time (SRT) under electron acceptor limitation. Around 85% of TCE supplied to CANAS (0.13 mmol d−1) was converted to ethene at a rate of 0.1 mmol d−1, with detection of low production rates of vinyl chloride (6.8 × 10−3 mmol d−1) and cis-dichloroethene (2.3 × 10−3 mmol d−1). Two distinct Dehalococcoides mccartyi strains (ANAS1 and ANAS2) were stably maintained at 6.2 ± 2.8 × 108 cells mL−1 and 5.8 ± 1.2 × 108 cells mL−1, respectively. Electron balance analysis showed 107% electron recovery, in which 6.1% were involved in dechlorination. 16 S rRNA amplicon sequencing revealed a structural regime shift between ANAS and CANAS while maintaining robust TCE dechlorination due to similar relative abundances of D. mccartyi and functional redundancy among each functional guild supporting D. mccartyi activity. D. mccartyi transcriptomic analysis identified the genes encoding for ribosomal RNA and the reductive dehalogenases tceA and vcrA as the most expressed genes in CANAS, while hup and vhu were the most critical hydrogenases utilized by D. mccartyi in the community.

The lower base of corrinoid small molecules regulates reductive dehalogenase enzyme function in Dehalococcoides species

Key interactions between proteins and small molecules regulate numerous environmental reactions. Such is the case with reductive dehalogenases that dechlorinate toxic chlorinated solvents only in the presence of corrinoid small molecules. Previous studies have demonstrated that the lower bases attached to these small molecule cofactors affect the activity of reductive dehalogenases differently in corrinoid auxotrophic Dehalococcoides mccartyi strains, which prefer the 5,6‐dimethylbenzimidazole (DMB) lower base. In contrast, Geobacter sulfurreducens strain PCA produces a small molecule corrinoid with 5‐hydroxybenzimidazole (5‐OHBza) as the lower base, which fails to support dechlorination catalyzed by reductive dehalogenases. Here, we cloned and heterologously expressed a unique Geobacter lovleyi O‐methyltransferase gene implicated in the methylation of 5‐OHBza to 5‐methoxybenzimidazole (5‐OMeBza). Unlike 5‐OHBza, 5‐OMeBza does support D. mccartyi reductive dechlorination activity. We demonstrated using HPLC and LC‐MS that the G. sulfurreducens mutant expressed the O‐methyltransferase, as evidenced by the native 5‐OHBza‐cobamide being converted into the 5‐OMeBza‐cobamide. Next, we co‐cultured D. mccartyi and the recombinant G. sulfurreducens in the absence of exogenous vitamin B12, which supported D. mccartyi growth and the complete dechlorination of cis‐1,2‐dichloroethene to ethene. These studies demonstrated that the O‐methyltransferase catalyzes a critical step in lower base biosynthesis and provides a genetic basis for the observation that D. mccartyi reductive dechlorination activity occurs in co‐cultures with G. lovleyi but not with G. sulfurreducens. Our results emphasize the critical role of small molecule interactions directing protein function in the case of dechlorinating toxic chlorinated solvents.Support or Funding InformationThe authors acknowledge funding support from the NIH and SERDP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proteogenomics Reveals Novel Reductive Dehalogenases and Methyltransferases Expressed during Anaerobic Dichloromethane Metabolism.

Dichloromethane (DCM) is susceptible to microbial degradation under anoxic conditions and is metabolized via the Wood-Ljungdahl pathway; however, mechanistic understanding of carbon-chlorine bond cleavage is lacking. The microbial consortium RM contains the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, which strictly requires DCM as a growth substrate. Proteomic workflows applied to DCM-grown consortium RM biomass revealed a total of 1,705 nonredundant proteins, 521 of which could be assigned to strain RM. In the presence of DCM, strain RM expressed a complete set of Wood-Ljungdahl pathway enzymes, as well as proteins implicated in chemotaxis, motility, sporulation, and vitamin/cofactor synthesis. Four corrinoid-dependent methyltransferases were among the most abundant proteins. Notably, two of three putative reductive dehalogenases (RDases) encoded within strain RM's genome were also detected in high abundance. Expressed RDase 1 and RDase 2 shared 30% amino acid identity, and RDase 1 was most similar to an RDase of Dehalococcoides mccartyi strain WBC-2 (AOV99960, 52% amino acid identity), while RDase 2 was most similar to an RDase of Dehalobacter sp. strain UNSWDHB (EQB22800, 72% amino acid identity). Although the involvement of RDases in anaerobic DCM metabolism has yet to be experimentally verified, the proteome characterization results implicated the possible participation of one or more reductive dechlorination steps and methyl group transfer reactions, leading to a revised proposal for an anaerobic DCM degradation pathway.IMPORTANCE Naturally produced and anthropogenically released DCM can reside in anoxic environments, yet little is known about the diversity of organisms, enzymes, and mechanisms involved in carbon-chlorine bond cleavage in the absence of oxygen. A proteogenomic approach identified two RDases and four corrinoid-dependent methyltransferases expressed by the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, suggesting that reductive dechlorination and methyl group transfer play roles in anaerobic DCM degradation. These findings suggest that the characterized DCM-degrading bacterium Dehalobacterium formicoaceticum and "Candidatus Dichloromethanomonas elyunquensis" strain RM utilize distinct strategies for carbon-chlorine bond cleavage, indicating that multiple pathways evolved for anaerobic DCM metabolism. The specific proteins (e.g., RDases and methyltransferases) identified in strain RM may have value as biomarkers for monitoring anaerobic DCM degradation in natural and contaminated environments.

Nitrous Oxide Is a Potent Inhibitor of Bacterial Reductive Dechlorination

Organohalide-respiring bacteria are key players for the turnover of organohalogens. At sites impacted with chlorinated ethenes, bioremediation promotes reductive dechlorination; however, stoichiometric conversion to environmentally benign ethene is not always achieved. We demonstrate that nitrous oxide (N2O), a compound commonly present in groundwater, inhibits organohalide respiration. N2O concentrations in the low micromolar range decreased dechlorination rates and resulted in incomplete dechlorination of tetrachloroethene (PCE) in Geobacter lovleyi strain SZ and of cis-1,2-dichloroethene ( cDCE) and vinyl chloride (VC) in Dehalococcoides mccartyi strain BAV1 axenic cultures. Presumably, N2O interferes with reductive dechlorination by reacting with super-reduced Co(I)-corrinoids of reductive dehalogenases, which is supported by the finding that N2O did not inhibit corrinoid-independent fumarate-to-succinate reduction in strain SZ. Kinetic analyses revealed a best fit to the noncompetitive Michaelis-Menten inhibition model and determined N2O inhibitory constants, KI, for PCE and cDCE dechlorination of 40.8 ± 3.8 and 21.2 ± 3.5 μM in strain SZ and strain BAV1, respectively. The lowest KI value of 9.6 ± 0.4 μM was determined for VC to ethene reductive dechlorination in strain BAV1, suggesting that this crucial dechlorination step for achieving detoxification is most susceptible to N2O inhibition. Groundwater N2O concentrations exceeding 100 μM are not uncommon, especially in watersheds impacted by nitrate runoff from agricultural sources. Thus, dissolved N2O measurements can inform about cDCE and VC stalls at sites impacted with chlorinated ethenes.

Strategy for the Rapid Dechlorination of Polychlorinated Biphenyls (PCBs) by Dehalococcoides mccartyi Strains.

Anaerobic bacteria play critical roles in the environmental bioremediation of polychlorinated biphenyls (PCBs). However, in situ applications of PCB dechlorinating anaerobes have been largely impeded by difficulties in growing PCB dechlorinators to a high cell abundance in short time periods. Here, we report the accelerated onset of PCB dechlorination by pre-cultivating Dehalococcoides mccartyi strains on chloroethenes as alternative electron acceptors. The extensive dechlorination of Aroclor 1260 was achieved within 1 week by D. mccartyi CG4 pregrown on trichloroethene (TCE) or tetrachloroethene (PCE). Compared to control cultures fed solely with Aroclor 1260, the PCB dechlorination rate was up to 30 times greater in cultures pre-cultivated with chloroethenes. However, when CG4 was simultaneously exposed to multiple potential substrates (PCE, TCE, and PCBs), PCB dechlorination was not observed until PCE was completely depleted. The expression of the bifunctional PCE and PCBs reductive dehalogenase (RDase) gene pcbA4 was inhibited by the presence of both substrates. Furthermore, in the presence of >0.3 mM TCE (produced as an intermediate from PCE dechlorination), the PCB dechlorination rate was an order of magnitude lower than in cultures amended with Aroclor 1260 after the complete depletion of TCE. This reduced PCB dechlorination rate corresponded with a sharp decrease in pcbA4 transcripts in the presence of both TCE and PCBs. The utilization of chloroethenes and PCBs as substrates by CG4 was found to be largely sequential rather than concurrent, suggesting that PCE and TCE are preferred substrates for the RDase responsible for PCE, TCE, and PCB dechlorination.

Reductive dechlorination of high concentrations of chloroethenes by a Dehalococcoides mccartyi strain 11G.

Chloroethenes are common groundwater and soil contaminants due to extensive historic utilization and inappropriate discharge. The tendency for chloroethenes to become sequestered as dense non-aqueous phase liquids (DNAPL)-a point source to groundwater contamination and causing high concentrations of chloroethenes in proximal aquifers poses a great challenge for remediation of chloroethene contaminated sites. In this study, we report isolation and characterization of a Dehalococcoides mccartyi strain 11G which couples growth with reductive dechlorination of trichloroethenes (TCE), dichloroethene (DCE) isomers and vinyl chloride (VC) to ethene at a growth yield ranging from 2.47±0.23 × 108 to 5.64±0.43 × 108 cells/µmoles Cl- released and co-metabolically dechlorinates tetrachloroethene (PCE) in the presence of TCE. Compared with previous D. mccartyi strains showing dechlorination of TCE at up to 2.0 mM, strain 11G is distinguished by its capacity to dechlorinate chloroethenes at initial concentrations of DCE isomers as high as 4 mM and TCE as high as 3.5 mM to ethene. Bioaugmentation of a contaminated microcosm with strain 11G resulted in complete detoxification of a mixture of 5 mM chloroethenes (2.5 mM of each TCE and cis-DCE) after 40 days. Strain 11G is a promising candidate for in situ bioremediation of high-concentration-chloroethene contaminated sites.

Draft Genome Sequences of the 1,2-Dichloropropane-Respiring Dehalococcoides mccartyi Strains RC and KS

Dehalococcoides mccartyi strains RC and KS respire toxic 1,2-dichloropropane to environmentally benign propene. Their genomes were sequenced with Ion Torrent technology, assembled, and annotated. The draft genomes of strains RC and KS were 1.50 and 1.49 Mb in size and carried 1,653 and 1,671 genes, respectively.

Syntrophic Partners Enhance Growth and Respiratory Dehalogenation of Hexachlorobenzene by Dehalococcoides mccartyi Strain CBDB1.

This study investigated syntrophic interactions between chlorinated benzene respiring Dehalococcoides mccartyi strain CBDB1 and fermenting partners (Desulfovibrio vulgaris, Syntrophobacter fumaroxidans, and Geobacter lovleyi) during hexachlorobenzene respiration. Dechlorination rates in syntrophic co-cultures were enhanced 2-3 fold compared to H2 fed CBDB1 pure cultures (0.23 ± 0.04 μmol Cl− day−1). Syntrophic partners were also able to supply cobalamins to CBDB1, albeit with 3–10 fold lower resultant dechlorination activity compared to cultures receiving exogenous cyanocobalamin. Strain CBDB1 pure cultures accumulated ~1 μmol of carbon monoxide per 87.5 μmol Cl− released during hexachlorobenzene respiration resulting in decreases in dechlorination activity. The syntrophic partners investigated were shown to consume carbon monoxide generated by CBDB1, thus relieving carbon monoxide autotoxicity. Accumulation of lesser chlorinated chlorobenzene congeners (1,3- and 1,4-dichlorobenzene and 1,3,5-trichlorobenzene) also inhibited dechlorination activity and their removal from the headspace through adsorption to granular activated carbon was shown to restore activity. Proteomic analysis revealed co-culturing strain CBDB1 with Geobacter lovleyi upregulated CBDB1 genes associated with reductive dehalogenases, hydrogenases, formate dehydrogenase, and ribosomal proteins. These data provide insight into CBDB1 ecology and inform strategies for application of CBDB1 in ex situ hexachlorobenzene destruction technologies.

Diastereoisomer-Specific Biotransformation of Hexabromocyclododecanes by a Mixed Culture Containing Dehalococcoides mccartyi Strain 195.

Hexabromocyclododecane (HBCD) stereoisomers may exhibit substantial differences in physicochemical, biological, and toxicological properties. However, there remains a lack of knowledge about stereoisomer-specific toxicity, metabolism, and environmental fate of HBCD. In this study, the biotransformation of (±)α-, (±)β-, and (±)γ-HBCD contained in technical HBCD by a mixed culture containing the organohalide-respiring bacterium Dehalococcoides mccartyi strain 195 was investigated. Results showed that the mixed culture was able to efficiently biotransform the technical HBCD mixture, with 75% of the initial HBCD (∼12 μM) in the growth medium being removed within 42 days. Based on the metabolites analysis, HBCD might be sequentially debrominated via dibromo elimination reaction to form tetrabromocyclododecene, dibromocyclododecadiene, and 1,5,9-cyclododecatriene. The biotransformation of the technical HBCD was likely diastereoisomer-specific. The transformation rates of α-, β-, and γ-HBCD were in the following order: α-HBCD > β-HBCD > γ-HBCD. The enantiomer fractions of (±)α-, (±)β-, and (±)γ-HBCD were maintained at about 0.5 during the 28 days of incubation, indicating a lack of enantioselective biotransformation of these diastereoisomers. Additionally, the amendment of another halogenated substrate tetrachloroethene (PCE), which supports the growth of strain 195, had a negligible impact on the transformation patterns of HBCD diastereoisomers and enantiomers. This study provided new insights into the stereoisomer-specific transformation patterns of HBCD by anaerobic microbes and has important implications for microbial remediation of anoxic environments contaminated by HBCD using the mixed culture containing Dehalococcoides.

Anaerobic biotransformation of hexachlorocyclohexane isomers by Dehalococcoides species and an enrichment culture.

The biotransformation of hexachlorocyclohexane isomers (HCH) by two Dehalococcoides mccartyi strains (195 and BTF08) and an enrichment culture was investigated and compared to conversion by the obligate anaerobic strain Clostridium pasteurianum strain DSMZ 525. The D. mccartyi strains preferentially transformed γ-HCH over α-HCH and δ-HCH isomers while β-HCH biotransformation was not significant. In case of the enrichment culture, γ-HCH was preferentially transformed over the δ-HCH, β-HCH and α-HCH isomers. Major observed metabolites in both cases were tetrachlorocyclohexene and as end products monochlorobenzene (MCB) and benzene. Dechlorination of the γ-HCH isomer was linked to an increase in cell numbers for strain 195. γ-HCH transformation was linked to considerable carbon stable isotope fractionation with the enrichment factor εc = - 5.5 ± 0.8‰ for D. mccartyi strain 195, εc = - 3.1 ± 0.4‰ for the enrichment culture and εc = - 4.1 ± 0.6‰ for co-metabolic transformation by C. pasteurianum.

The Complexome of Dehalococcoides mccartyi Reveals Its Organohalide Respiration-Complex Is Modular.

Dehalococcoides mccartyi strain CBDB1 is a slow growing strictly anaerobic microorganism dependent on halogenated compounds as terminal electron acceptor for anaerobic respiration. Indications have been described that the membrane-bound proteinaceous organohalide respiration complex of strain CBDB1 is functional without quinone-mediated electron transfer. We here study this multi-subunit protein complex in depth in regard to participating protein subunits and interactions between the subunits using blue native gel electrophoresis coupled to mass spectrometric label-free protein quantification. Applying three different solubilization modes to detach the respiration complex from the membrane we describe different solubilization snapshots of the organohalide respiration complex. The results demonstrate the existence of a two-subunit hydrogenase module loosely binding to the rest of the complex, tight binding of the subunit HupX to OmeA and OmeB, predicted to be the two subunits of a molybdopterin-binding redox subcomplex, to form a second module, and the presence of two distinct reductive dehalogenase module variants with different sizes. In our data we obtained biochemical evidence for the specificity between a reductive dehalogenase RdhA (CbdbA80) and its membrane anchor protein RdhB (CbdbB3). We also observed weak interactions between the reductive dehalogenase and the hydrogenase module suggesting a not yet recognized contact surface between these two modules. Especially an interaction between the two integral membrane subunits OmeB and RdhB seems to promote the integrity of the complex. With the different solubilization strengths we observe successive disintegration of the complex into its subunits. The observed architecture would allow the association of different reductive dehalogenase modules RdhA/RdhB with the other two protein complex modules when the strain is growing on different electron acceptors. In the search for other respiratory complexes in strain CBDB1 the remarkable result is not the detection of a standard ATPase but the absence of any other abundant membrane complex although an 11-subunit version of complex I (Nuo) is encoded in the genome.