Project description:Dehalococcoides mccartyi strains conserve energy from reductive dechlorination reactions catalyzed by corrinoid-dependent reductive dehalogenase enzyme systems. Dehalococcoides lacks the ability for de novo corrinoid synthesis, and pure cultures require the addition of cyanocobalamin (vitamin B(12)) for growth. In contrast, Geobacter lovleyi, which dechlorinates tetrachloroethene to cis-1,2-dichloroethene (cis-DCE), and the nondechlorinating species Geobacter sulfurreducens have complete sets of cobamide biosynthesis genes and produced 12.9 ± 2.4 and 24.2 ± 5.8 ng of extracellular cobamide per liter of culture suspension, respectively, during growth with acetate and fumarate in a completely synthetic medium. G. lovleyi-D. mccartyi strain BAV1 or strain FL2 cocultures provided evidence for interspecies corrinoid transfer, and cis-DCE was dechlorinated to vinyl chloride and ethene concomitant with Dehalococcoides growth. In contrast, negligible increase in Dehalococcoides 16S rRNA gene copies and insignificant dechlorination occurred in G. sulfurreducens-D. mccartyi strain BAV1 or strain FL2 cocultures. Apparently, G. lovleyi produces a cobamide that complements Dehalococcoides' nutritional requirements, whereas G. sulfurreducens does not. Interestingly, Dehalococcoides dechlorination activity and growth could be restored in G. sulfurreducens-Dehalococcoides cocultures by adding 10 μM 5',6'-dimethylbenzimidazole. Observations made with the G. sulfurreducens-Dehalococcoides cocultures suggest that the exchange of the lower ligand generated a cobalamin, which supported Dehalococcoides activity. These findings have implications for in situ bioremediation and suggest that the corrinoid metabolism of Dehalococcoides must be understood to faithfully predict, and possibly enhance, reductive dechlorination activities.
Project description:Organohalide respiration, mediated by Dehalococcoides mccartyi, is a useful bioremediation process that transforms ground water pollutants and known human carcinogens such as trichloroethene and vinyl chloride into benign ethenes. Successful application of this process depends on the fundamental understanding of the respiration and metabolism of D. mccartyi. Reductive dehalogenases, encoded by rdhA genes of these anaerobic bacteria, exclusively catalyze organohalide respiration and drive metabolism. To better elucidate D. mccartyi metabolism and physiology, we analyzed available transcriptomic data for a pure isolate (Dehalococcoides mccartyi strain 195) and a mixed microbial consortium (KB-1) using the previously developed pan-genome-scale reconstructed metabolic network of D. mccartyi. The transcriptomic data, together with available proteomic data helped confirm transcription and expression of the majority genes in D. mccartyi genomes. A composite genome of two highly similar D. mccartyi strains (KB-1 Dhc) from the KB-1 metagenome sequence was constructed, and operon prediction was conducted for this composite genome and other single genomes. This operon analysis, together with the quality threshold clustering analysis of transcriptomic data helped generate experimentally testable hypotheses regarding the function of a number of hypothetical proteins and the poorly understood mechanism of energy conservation in D. mccartyi. We also identified functionally enriched important clusters (13 for strain 195 and 11 for KB-1 Dhc) of co-expressed metabolic genes using information from the reconstructed metabolic network. This analysis highlighted some metabolic genes and processes, including lipid metabolism, energy metabolism, and transport that potentially play important roles in organohalide respiration. Overall, this study shows the importance of an organism's metabolic reconstruction in analyzing various "omics" data to obtain improved understanding of the metabolism and physiology of the organism.
Project description:Dehalococcoides mccartyi strain WBC-2 dechlorinates carcinogen vinyl chloride to ethene in the West Branch Canal Creek (WBC-2) microbial consortium used for bioaugmentation. We assembled and closed the complete genome sequence of this prokaryote using metagenomic sequencing from an enrichment culture.
Project description:Transcriptional profiling of the Donna II mixed community containing Dehalococcoides mccartyi strain 195 comparing a batch starved control to the mixed community being fed 1,2,3,4-tetrachlorobenzene as an electron acceptor. The goal was to determine which transcripts are regulated in response to a shift in a different electron acceptor rather than the consistent tetrachloroethene (PCE) that the parent reactor was maintained on. Overall design: Three-condition experiment, triplicate Donna II batch fed 1,2,3,4-tetrachlorobenzene sampled at 2 days vs. pooled Donna II batch fed PCE sampled at 3 days; triplicate Donna II batch fed 1,2,3,4-tetrachlorobenzene sampled at 3 days vs. pooled Donna II batch fed PCE sampled at 3 days . The additional two microarrays are the technical dye-swap of the 3 day starved control.
Project description:We present a statistical model designed to identify the effect of experimental perturbations on the aggregate behavior of the transcriptome expressed by the bacterium Dehalococcoides mccartyi strain 195. Strains of Dehalococcoides are used in sub-surface bioremediation applications because they organohalorespire tetrachloroethene and trichloroethene (common chlorinated solvents that contaminate the environment) to non-toxic ethene. However, the biochemical mechanism of this process remains incompletely described. Additionally, the response of Dehalococcoides to stress-inducing conditions that may be encountered at field-sites is not well understood. The constructed statistical model captured the aggregate behavior of gene expression phenotypes by modeling the distinct eigengenes of 100 transcript clusters, determining stable relationships among these clusters of gene transcripts with a sparse network-inference algorithm, and directly modeling the effect of changes in experimental conditions by constructing networks conditioned on the experimental state. Based on the model predictions, we discovered new response mechanisms for DMC, notably when the bacterium is exposed to solvent toxicity. The network identified a cluster containing thirteen gene transcripts directly connected to the solvent toxicity condition. Transcripts in this cluster include an iron-dependent regulator (DET0096-97) and a methylglyoxal synthase (DET0137). To validate these predictions, additional experiments were performed. Continuously fed cultures were exposed to saturating levels of tetrachloethene, thereby causing solvent toxicity, and transcripts that were predicted to be linked to solvent toxicity were monitored by quantitative reverse-transcription polymerase chain reaction. Twelve hours after being shocked with saturating levels of tetrachloroethene, the control transcripts (encoding for a key hydrogenase and the 16S rRNA) did not significantly change. By contrast, transcripts for DET0137 and DET0097 displayed a 46.8±11.5 and 14.6±9.3 fold up-regulation, respectively, supporting the model. This is the first study to identify transcripts in Dehalococcoides that potentially respond to tetrachloroethene solvent-toxicity conditions that may be encountered near contamination source zones in sub-surface environments.
Project description:We have developed and characterized a bacterial consortium that reductively dechlorinates trichloroethene to ethene. Quantitative PCR analysis for the 16S rRNA and reductive dehalogenase genes showed that the consortium is highly enriched with Dehalococcoides spp. that have two vinyl chloride reductive dehalogenase genes, bvcA and vcrA, and a trichloroethene reductive dehalogenase gene, tceA. The metagenome analysis of the consortium by the next generation sequencer SOLiD 3 Plus suggests that a Dehalococcoides sp. that is highly homologous to D. mccartyi 195 and equipped with vcrA and tceA exists in the consortium. We isolated this Dehalococcoides sp. and designated it as D. mccartyi UCH-ATV1. As the growth of D. mccartyi UCH-ATV1 is too slow under isolated conditions, we constructed a consortium by mixing D. mccartyi UCH-ATV1 with several other bacteria and performed metagenomic sequencing using the single molecule DNA sequencer PacBio RS II. We successfully determined the complete genome sequence of D. mccartyi UCH-ATV1. The strain is equipped with vcrA and tceA, but lacks bvcA. Comparison with tag sequences of SOLiD 3 Plus from the original consortium shows a few differences between the sequences. This suggests that a genome rearrangement of Dehalococcoides sp. occurred during culture.
Project description:Dehalococcoides mccartyi strains are corrinoid-auxotrophic Bacteria and axenic cultures that require vitamin B12 (CN-Cbl) to conserve energy via organohalide respiration. Cultures of D. mccartyi strains BAV1, GT and FL2 grown with limiting amounts of 1 µg l(-1) CN-Cbl quickly depleted CN-Cbl, and reductive dechlorination of polychlorinated ethenes was incomplete leading to vinyl chloride (VC) accumulation. In contrast, the same cultures amended with 25 µg l(-1) CN-Cbl exhibited up to 2.3-fold higher dechlorination rates, 2.8-9.1-fold increased growth yields, and completely consumed growth-supporting chlorinated ethenes. To explore whether known cobamide-producing microbes supply Dehalococcoides with the required corrinoid cofactor, co-culture experiments were performed with the methanogen Methanosarcina barkeri strain Fusaro and two acetogens, Sporomusa ovata and Sporomusa sp. strain KB-1, as Dehalococcoides partner populations. During growth with H2/CO2, M. barkeri axenic cultures produced 4.2 ± 0.1 µg l(-1) extracellular cobamide (factor III), whereas the Sporomusa cultures produced phenolyl- and p-cresolyl-cobamides. Neither factor III nor the phenolic cobamides supported Dehalococcoides reductive dechlorination activity suggesting that M. barkeri and the Sporomusa sp. cannot fulfil Dehalococcoides' nutritional requirements. Dehalococcoides dechlorination activity and growth occurred in M. barkeri and Sporomusa sp. co-cultures amended with 10 µM 5',6'-dimethylbenzimidazole (DMB), indicating that a cobalamin is a preferred corrinoid cofactor of strains BAV1, GT and FL2 when grown with chlorinated ethenes as electron acceptors. Even though the methanogen and acetogen populations tested did not produce cobalamin, the addition of DMB enabled guided biosynthesis and generated a cobalamin that supported Dehalococcoides' activity and growth. Guided cobalamin biosynthesis may offer opportunities to sustain and enhance Dehalococcoides activity in contaminated subsurface environments.
Project description:Corrinoids are cobalt-containing molecules that function as enzyme cofactors in a wide variety of organisms but are produced solely by a subset of prokaryotes. Specific corrinoids are identified by the structure of their axial ligands. The lower axial ligand of a corrinoid can be a benzimidazole, purine, or phenolic compound. Though it is known that many organisms obtain corrinoids from the environment, the variety of corrinoids that can serve as cofactors for any one organism is largely unstudied. Here, we examine the range of corrinoids that function as cofactors for corrinoid-dependent metabolism in Dehalococcoides mccartyi strain 195. Dehalococcoides bacteria play an important role in the bioremediation of chlorinated solvents in the environment because of their unique ability to convert the common groundwater contaminants perchloroethene and trichloroethene to the innocuous end product ethene. All isolated D. mccartyi strains require exogenous corrinoids such as vitamin B(12) for growth. However, like many other corrinoid-dependent bacteria, none of the well-characterized D. mccartyi strains has been shown to be capable of synthesizing corrinoids de novo. In this study, we investigate the ability of D. mccartyi strain 195 to use specific corrinoids, as well as its ability to modify imported corrinoids to a functional form. We show that strain 195 can use only specific corrinoids containing benzimidazole lower ligands but is capable of remodeling other corrinoids by lower ligand replacement when provided a functional benzimidazole base. This study of corrinoid utilization and modification by D. mccartyi provides insight into the array of strategies that microorganisms employ in acquiring essential nutrients from the environment.
Project description: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.