Dehalococcoides Mccartyi Strain Research Articles

Reductive dehalogenase of Dehalococcoides mccartyi strain CBDB1 reduces cobalt- containing metal complexes enabling anodic respiration

Microorganisms capable of direct or mediated extracellular electron transfer (EET) have garnered significant attention for their various biotechnological applications, such as bioremediation, metal recovery, wastewater treatment, energy generation in microbial fuel cells, and microbial or enzymatic electrosynthesis. One microorganism of particular interest is the organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1, known for its ability to reductively dehalogenate toxic and persistent halogenated organic compounds through organohalide respiration (OHR), using halogenated organics as terminal electron acceptors. A membrane-bound OHR protein complex couples electron transfer to proton translocation across the membrane, generating a proton motive force, which enables metabolism and proliferation. In this study we show that the halogenated compounds can be replaced with redox mediators that can putatively shuttle electrons between the OHR complex and the anode, coupling D. mccartyi cells to an electrode via mediated EET. We identified cobalt-containing metal complexes, referred to as cobalt chelates, as promising mediators using a photometric high throughput methyl viologen-based enzyme activity assay. Through various biochemical approaches, we show that cobalt chelates are specifically reduced by CBDB1 cells, putatively by the reductive dehalogenase subunit (RdhA) of the OHR complex. Using cyclic voltammetry, we also demonstrate that cobalt chelates exchange electrons with a gold electrode, making them promising candidates for bioelectrochemical cultivation. Furthermore, using the AlphaFold 2-calculated RdhA structure and molecular docking, we found that one of the identified cobalt chelates exhibits favorable binding to RdhA, with a binding energy of approximately −28 kJ mol−1. Taken together, our results indicate that bioelectrochemical cultivation of D. mccartyi with cobalt chelates as anode mediators, instead of toxic halogenated compounds, is feasible, which opens new perspectives for bioremediation and other biotechnological applications of strain CBDB1.

Combatting multiple aromatic organohalide pollutants in sediments by bioaugmentation with a single Dehalococcoides

Dehalococcoides are capable of dehalogenating various organohalide pollutants under anaerobic conditions, and they have been applied in bioremediation. However, the presence of multiple aromatic organohalides, including polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and tetrabromobisphenol A (TBBPA), at contaminated sites may pose challenges to Dehalococcoides-mediated bioremediation due to the lack of knowledge about the influence of co-contamination on bioremediation. In this study, we investigated the bioremediation of aromatic organohalides present as individual and co-contaminants in sediments by bioaugmentation with a single population of Dehalococcoides. Bioaugmentation with Dehalococcoides significantly increased the dehalogenation rate of PCBs, PBDEs, and TBBPA in sediments contaminated with individual pollutants, being up to 19.7, 27.4 and 2.1 times as that in the controls not receiving bioinoculants. For sediments containing all the three classes of pollutants, bioaugmentation with Dehalococcoides also effectively enhanced dehalogenation, and the extent of enhancement depended on the bioinoculants and types of pollutants. Interestingly, in many cases co-contaminated sediments bioaugmented with Dehalococcoides mccartyi strain CG1 displayed a greater enhancement in dehalogenation rates compared to the sediments polluted with individual pollutant. For instance, when augmented with a low quantity of strain CG1, the dehalogenation rates of Aroclor1260 and PBDEs in co-contaminated sediments were approximately two times as that in sediments containing individual pollutants (0.428 and 9.03 vs. 0.195 and 4.20 × 10−3d−1). Additionally, D. mccartyi CG1 grew to higher abundances in co-contaminated sediments. These findings demonstrate that a single Dehalococcoides population can sustain dehalogenation of multiple aromatic organohalides in contaminated sediments, suggesting that co-contamination does not necessarily impede the use of Dehalococcoides for bioremediation. The study also underscores the significance of anaerobic organohalide respiration for effective bioremediation.

Dehalococcoides mccartyi strain CBDB1 takes up protons from the cytoplasm to reductively dehalogenate organohalides indicating a new modus of proton motive force generation.

Proton translocation across the cytoplasmic membrane is a vital process for all organisms. Dehalococcoides strains are strictly anaerobic organohalide respiring bacteria that lack quinones and cytochromes but express a large membrane-bound protein complex (OHR complex) proposed to generate a proton gradient. However, its functioning is unclear. By using a dehalogenase-based enzyme activity assay with deuterium-labelled water in various experimental designs, we obtained evidence that the halogen atom of the halogenated electron acceptor is substituted with a proton from the cytoplasm. This suggests that the protein complex couples exergonic electron flux through the periplasmic subunits of the OHR complex to the endergonic transport of protons from the cytoplasm across the cytoplasmic membrane against the proton gradient to the halogenated electron acceptor. Using computational tools, we located two proton-conducting half-channels in the AlphaFold2-predicted structure of the OmeB subunit of the OHR complex, converging in a highly conserved arginine residue that could play a proton gatekeeper role. The cytoplasmic proton half-channel in OmeB is connected to a putative proton-conducting path within the reductive dehalogenase subunit. Our results indicate that the reductive dehalogenase and its halogenated substrate serve as both electron and proton acceptors, providing insights into the proton translocation mechanism within the OHR complex and contributing to a better understanding of energy conservation in D. mccartyi strains. Our results reveal a very simple mode of energy conservation in anaerobic bacteria, showing that proton translocation coupled to periplasmic electron flow might have importance also in other microbial processes and biotechnological applications.

Syntrophic Interactions Ameliorate Arsenic Inhibition of Solvent-Dechlorinating Dehalococcoides mccartyi.

Interactions and nutrient exchanges among members of microbial communities are important for understanding functional relationships in environmental microbiology. We can begin to elucidate the nature of these complex systems by taking a bottom-up approach utilizing simplified, but representative, community members. Here, we assess the effects of a toxic stress event, the addition of arsenite (As(III)), on a syntrophic co-culture containing lactate-fermenting Desulfovibrio vulgaris Hildenborough and solvent-dechlorinating Dehalococcoides mccartyi strain 195. Arsenic and trichloroethene (TCE) are two highly prevalent groundwater contaminants in the United States, and the presence of bioavailable arsenic is of particular concern at remediation sites in which reductive dechlorination has been employed. While we previously showed that low concentrations of arsenite (As(III)) inhibit the keystone TCE-reducing microorganism, D. mccartyi, this study reports the utilization of physiological analysis, transcriptomics, and metabolomics to assess the effects of arsenic on the metabolisms, gene expression, and nutrient exchanges in the described co-culture. It was found that the presence of D. vulgaris ameliorated arsenic stress on D. mccartyi, improving TCE dechlorination under arsenic-contaminated conditions. Nutrient and amino acid export by D. vulgaris may be a stress-ameliorating exchange in this syntrophic co-culture under arsenic stress, based on upregulation of transporters and increased extracellular nutrients like sarcosine and ornithine. These results broaden our knowledge of microbial community interactions and will support the further development and implementation of robust bioremediation strategies at multi-contaminant sites.

Dehalococcoides mccartyi strain NIT01 grows more stably in vessels made of pure titanium rather than the stainless alloy SUS304.

Advances in many isolation studies have revealed that pure Dehalococcoides grow stably, although the large-scale pure cultivation of Dehalococcoides has yet to be established. In this study, 7 L-culturing of Dehalococcoides mccartyi strain NIT01 was first performed using vessels made of glass and stainless alloy SUS304. All batches cultured in the glass vessel successfully dechlorinated >95% of 1 mM trichloroethene (TCE) to ethene (ETH), whereas only 5 out of 13 batches cultured in the SUS304 vessel did the same. The difference in dechlorination efficiency suggested the possible inhibition of dechlorination by SUS304. Also, the strain NIT01 showed long delays in dechlorination with pieces of SUS316, steel, and a repeatedly used SUS304, but not with titanium. The repeatedly used SUS304 cracked and increased the Fe2+ concentration to ≥76 μM. Dechlorination by this strain was also inhibited with ≥1000 μM Fe2+ and ≥23 μM Cr3+ but not with ≤100 μM Ni2+ , suggesting that Cr3+ eluted from solid stainless alloys inhibited the dechlorination. Culturing in a titanium vessel instead of a stainless alloy showed the complete dechlorination of 1 mM TCE within 12-28 days with a growth yield of 2.7 × 107 cells/μmol-released Cl- , even after repeating use of the vessels six times.

Insight into the Mechanism Underlying Dehalococcoides mccartyi Strain CBDB1-Mediated B12-Dependent Aromatic Reductive Dehalogenation.

Anaerobic bacteria transform aromatic halides through reductive dehalogenation. This dehalorespiration is catalyzed by the supernucleophilic coenzyme vitamin B12, cob(I)alamin, in reductive dehalogenases. So far, the underlying inner-sphere electron transfer (ET) mechanism has been discussed controversially. In the present study, all 36 chloro-, bromo-, and fluorobenzenes and full-size cobalamin are analyzed at the quantum chemical density functional theory level with respect to a wide range of theoretically possible inner-sphere ET mechanisms. The calculated reaction free energies within the framework of CoI···X (X = F, Cl, and Br) attack rule out most of the inner-sphere pathways. The only route with feasible energetics is a proton-coupled two-ET mechanism that involves a B12 side-chain tyrosine (modeled by phenol) as a proton donor. For 12 chlorobenzenes and 9 bromobenzenes with experimental data from Dehalococcoides mccartyi strain CBDB1, the newly proposed PC-TET mechanism successfully discriminates 16 of 17 active from 4 inactive substrates and correctly predicts the observed regiospecificity to 100%. Moreover, fluorobenzenes are predicted to be recalcitrant in agreement with experimental findings. Conceptually, based on the Bell-Evans-Polanyi principle, the computational approach provides novel mechanistic insights and may serve as a tool for predicting the energetic feasibility of reductive aromatic dehalogenation.

Mechanistic insight into the Dehalococcoides-mediated reductive dechlorination of polychlorinated biphenyls.

Microbial reductive dechlorination provides a green and highly desirable approach to address the pollution raised by the substantial legacies of polychlorinated biphenyls (PCBs) in soil, sediment, and underground water. It has been shown that the reaction event is catalyzed by supernucleophilic cob(I)alamin housed in reductive dehalogenases (RDases). However, the mechanism still remains elusive. Herein, we unravel the mechanism via quantum chemical calculations, considering a general model of RDase and the dechlorination regioselectivity of two representative PCB congeners, 234-236-CB and 2345-236-CB. The B12-catalzyed reductive dechlorination of PCBs starts with the formation of a reactant complex, followed by a proton-coupled two-electron transfer (PC-TET) and a subsequent single-electron transfer (SET). The PC-TET yields a cob(III)alamin-featured intermediate, which is quickly reduced by the latter SET fueled by significant energetic benefits (∼100 kcal mol-1). It rationalizes the exclusive detection and characterization of cob(I/II)alamins in RDase-mediated dehalogenation experiments. The determined mechanism successfully reproduces the experimental dechlorination regioselectivity and reactivity, as observed with Dehalococcoides mccartyi strain CG1.

Continuous cultivation of Dehalococcoides mccartyi with brominated tyrosine avoids toxic byproducts and gives tight reactor control

Dehalococcoides mccartyi strain CBDB1 is a strictly anaerobic organohalide-respiring bacterium with strong application potential to remediate aquifers and soils contaminated with halogenated aromatics. To date, cultivation of strain CBDB1 has mostly been done in bottles or fed-batch reactors. Challenges with such systems include low biomass yield and difficulties in controlling the growth conditions. Here, we report the cultivation of planktonic D. mccartyi strain CBDB1 in a continuous stirring tank reactor (CSTR) that led to high cell densities (∼8 × 108 cells mL-1) and dominance of strain CBDB1. The reactor culture received acetate, hydrogen, and the brominated amino acid D- or L-3,5-dibromotyrosine as substrates. Both D- and L-3,5-dibromotyrosine were utilized as respiratory electron acceptors and are promising for biomass production due to their decent solubility in water and the formation of a non-toxic debromination product, tyrosine. By monitoring headspace pressure decrease which is indicative of hydrogen consumption, the organohalide respiration rate was followed in real time. Proteomics analyses revealed that the reductive dehalogenase CbdbA238 was highly expressed with both D- and L-3,5-dibromotyrosine, while other reductive dehalogenases including those that were previously suggested to be constitutively expressed, were repressed. Denaturing gradient gel electrophoresis (DGGE) of amplified 16S rRNA genes indicated that the majority of cells in the community belonged to the Dehalococcoides although the CSTR was operated under non-sterile conditions. Hence, tightly controlled CSTR cultivation of Dehalococcoides opens novel options to improve biomass production for bioaugmentation and for advanced biochemical studies.

Long-term survival of Dehalococcoides mccartyi strains in mixed cultures under electron acceptor and ammonium limitation.

Few strains of Dehalococcoides mccartyi harbour and express the vinyl chloride reductase (VcrA) that catalyzes the dechlorination of vinyl chloride (VC), a carcinogenic soil and groundwater contaminant. The vcrA operon is found on a Genomic Island (GI) and, therefore, believed to participate in horizontal gene transfer (HGT). To try to induce HGT of the vcrA-GI, we blended two enrichment cultures in medium without ammonium while providing VC. We hypothesized that these conditions would select for a mutant strain of D. mccartyi that could both fix nitrogen and respire VC. However, after more than 4 years of incubation, we found no evidence for HGT of the vcrA-GI. Rather, we observed VC-dechlorinating activity attributed to the trichloroethene reductase TceA. Sequencing and protein modelling revealed a mutation in the predicted active site of TceA, which may have influenced substrate specificity. We also identified two nitrogen-fixing D. mccartyi strains in the KB-1 culture. The presence of multiple strains of D. mccartyi with distinct phenotypes is a feature of natural environments and certain enrichment cultures (such as KB-1), and may enhance bioaugmentation success. The fact that multiple distinct strains persist in the culture for decades and that we could not induce HGT of the vcrA-GI suggests that it is not as mobile as predicted, or that mobility is restricted in ways yet to be discovered to specific subclades of Dehalococcoides.

Dehalogenation of Chlorinated Ethenes to Ethene by a Novel Isolate, "Candidatus Dehalogenimonas etheniformans".

Dehalococcoides mccartyi strains harboring vinyl chloride (VC) reductive dehalogenase (RDase) genes are keystone bacteria for VC detoxification in groundwater aquifers, and bioremediation monitoring regimens focus on D. mccartyi biomarkers. We isolated a novel anaerobic bacterium, "Candidatus Dehalogenimonas etheniformans" strain GP, capable of respiratory dechlorination of VC to ethene. This bacterium couples formate and hydrogen (H2) oxidation to the reduction of trichloro-ethene (TCE), all dichloroethene (DCE) isomers, and VC with acetate as the carbon source. Cultures that received formate and H2 consumed the two electron donors concomitantly at similar rates. A 16S rRNA gene-targeted quantitative PCR (qPCR) assay measured growth yields of (1.2 ± 0.2) × 108 and (1.9 ± 0.2) × 108 cells per μmol of VC dechlorinated in cultures with H2 or formate as electron donor, respectively. About 1.5-fold higher cell numbers were measured with qPCR targeting cerA, a single-copy gene encoding a putative VC RDase. A VC dechlorination rate of 215 ± 40 μmol L-1 day-1 was measured at 30°C, with about 25% of this activity occurring at 15°C. Increasing NaCl concentrations progressively impacted VC dechlorination rates, and dechlorination ceased at 15 g NaCl L-1. During growth with TCE, all DCE isomers were intermediates. Tetrachloroethene was not dechlorinated and inhibited dechlorination of other chlorinated ethenes. Carbon monoxide formed and accumulated as a metabolic by-product in dechlorinating cultures and impacted reductive dechlorination activity. The isolation of a new Dehalogenimonas species able to effectively dechlorinate toxic chlorinated ethenes to benign ethene expands our understanding of the reductive dechlorination process, with implications for bioremediation and environmental monitoring. IMPORTANCE Chlorinated ethenes are risk drivers at many contaminated sites, and current bioremediation efforts focus on organohalide-respiring Dehalococcoides mccartyi strains to achieve detoxification. We isolated and characterized the first non-Dehalococcoides bacterium, "Candidatus Dehalogenimonas etheniformans" strain GP, capable of metabolic reductive dechlorination of TCE, all DCE isomers, and VC to environmentally benign ethene. In addition to hydrogen, the new isolate utilizes formate as electron donor for reductive dechlorination, providing opportunities for more effective electron donor delivery to the contaminated subsurface. The discovery that a broader microbial diversity can achieve detoxification of toxic chlorinated ethenes in anoxic aquifers illustrates the potential of naturally occurring microbes for biotechnological applications.

Diversity of organohalide respiring bacteria and reductive dehalogenases that detoxify polybrominated diphenyl ethers in E-waste recycling sites.

Widespread polybrominated diphenyl ethers (PBDEs) contamination poses risks to human health and ecosystems. Bioremediation is widely considered to be a less ecologically disruptive strategy for remediation of organohalide contamination, but bioremediation of PBDE-contaminated sites is limited by a lack of knowledge about PBDE-dehalogenating microbial populations. Here we report anaerobic PBDE debromination in microcosms established from geographically distinct e-waste recycling sites. Complete debromination of a penta-BDE mixture to diphenyl ether was detected in 16 of 24 investigated microcosms; further enrichment of these 16 microcosms implicated microbial populations belonging to the bacterial genera Dehalococcoides, Dehalogenimonas, and Dehalobacter in PBDE debromination. Debrominating microcosms tended to contain either both Dehalogenimonas and Dehalobacter or Dehalococcoides alone. Separately, complete debromination of a penta-BDE mixture was also observed by axenic cultures of Dehalococcoides mccartyi strains CG1, CG4, and 11a5, suggesting that this phenotype may be fairly common amongst Dehalococcoides. PBDE debromination in these isolates was mediated by four reductive dehalogenases not previously known to debrominate PBDEs. Debromination of an octa-BDE mixture was less prevalent and less complete in microcosms. The PBDE reductive dehalogenase homologous genes in Dehalococcoides genomes represent plausible molecular markers to predict PBDE debromination in microbial communities via their prevalence and transcriptions analysis.

Efficient and Complete Detoxification of Polybrominated Diphenyl Ethers in Sediments Achieved by Bioaugmentation with Dehalococcoides and Microbial Ecological Insights.

Polybrominated diphenyl ethers (PBDEs) are prevalent environmental pollutants, but bioremediation of PBDEs remains to be reported. Here we report accelerated remediation of a penta-BDE mixture in sediments by bioaugmentation with Dehalococcoides mccartyi strains CG1 and TZ50. Bioaugmentation with different amounts of each Dehalococcoides strain enhanced debromination of penta-BDEs compared with the controls. The sediment microcosm spiked with 6.8 × 106 cells/mL strain CG1 showed the highest penta-BDEs removal (89.9 ± 7.3%) to diphenyl ether within 60 days. Interestingly, co-contaminant tetrachloroethene (PCE) improved bioaugmentation performance, resulting in faster and more extensive penta-BDEs debromination using less bioinoculants, which was also completely dechlorinated to ethene by introducing D. mccartyi strain 11a. The better bioaugmentation performance in sediments with PCE could be attributed to the boosted growth of the augmented Dehalococcoides and capability of the PCE-induced reductive dehalogenases to debrominate penta-BDEs. Finally, ecological analyses showed that bioaugmentation resulted in more deterministic microbial communities, where the augmented Dehalococcoides established linkages with indigenous microorganisms but without causing obvious alterations of the overall community diversity and structure. Collectively, this study demonstrates that bioaugmentation with Dehalococcoides is a feasible strategy to completely remove PBDEs in sediments.

Enhanced perchloroethene dechlorination by humic acids via increasing the dehalogenase activity of Dehalococcoides strains.

Perchloroethene (PCE) is a widely used chlorinated solvent. PCE is toxic to humans and has been identified as an environmental contaminant at thousands of sites worldwide. Several Dehalococcoides mccartyi strains can transform PCE to ethene, and thus contribute to bioremediation of contaminated sites. Humic acids (HA) are ubiquitous redox-active compounds of natural aquatic and soil systems and have been intensively studied because of their effect in electron transfer. In this study, we observed the dechlorination of PCE was accelerated by HA in mixed cultures containing Dehalococcoides strains. Anthraquinone-2,6-disulfonic acid (AQDS), a humic acid analogue, inhibited PCE dechlorination in our cultures and thus induced an opposite effect on PCE dehalogenation than HA. We observed the same effect on PCE dechlorination with the pure culture of Dehalococcoides mccartyi strain CBDB1. Not only in mixed cultures but also in pure cultures, growth of Dehalococcoides was not influenced by HA but inhibited by AQDS. Enzymatic activity tests confirmed the dehalogenating activity of strain CBDB1 was increased by HA, especially when using hydrogen as electron donor. We conclude that HA enhanced PCE dechlorination by increasing the reaction speed between hydrogen and the dehalogenase enzyme rather than acting as electron shuttle through its quinone moieties.

Dehalogenation of Polybrominated Diphenyl Ethers and Polychlorinated Biphenyls Catalyzed by a Reductive Dehalogenase in Dehalococcoides mccartyi Strain MB.

Polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) are notorious persistent organic pollutants. However, few organohalide-respiring bacteria that harbor reductive dehalogenases (RDases) capable of dehalogenating these pollutants have been identified. Here, we report reductive dehalogenation of penta-BDEs and PCBs byDehalococcoides mccartyi strain MB. The PCE-pregrown cultures of strain MB debrominated 86.6 ± 7.4% penta-BDEs to di- to tetra-BDEs within 5 days. Similarly, extensive dechlorination of Aroclor1260 and Aroclor1254 was observed in the PCE-pregrown cultures of strain MB, with the average chlorine per PCB decreasing from 6.40 ± 0.02 and 5.40 ± 0.03 to 5.98 ± 0.11 and 5.19 ± 0.07 within 14 days, respectively; para-substituents were preferentially dechlorinated from PCBs. Moreover, strain MB showed distinct enantioselective dechlorination of different chiral PCB congeners. Dehalogenation activity and cell growth were maintained during the successive transfer of cultures when amended with penta-BDEs as the sole electron acceptors but not when amended with only PCBs, suggesting metabolic and co-metabolic dehalogenation of these compounds, respectively. Transcriptional analysis, proteomic profiling, and in vitro activity assays indicated that MbrA was involved in dehalogenating PCE, PCBs, and PBDEs. Interestingly, resequencing of mbrA in strain MB identified three nonsynonymous mutations within the nucleotide sequence, although the consequences of which remain unknown. The substrate versatility of MbrA enabled strain MB to dechlorinate PCBs in the presence of either penta-BDEs or PCE, suggesting that co-metabolic dehalogenation initiated by multifunctional RDases may contribute to PCB attenuation at sites contaminated with multiple organohalide pollutants.

Debromination of TetraBromoBisphenol-A (TBBPA) depicting the metabolic versatility of Dehalococcoides

TetraBromoBisphenol-A (TBBPA) is a widely used brominated flame retardant and an emerging contaminant that has amassed significant environmental impacts. Though there are a few studies that report the bioremediation of TBBPA, there is no direct evidence to suggest a metabolic use of TBBPA as the sole electron acceptor, which offers an advantage in the complete and energy-efficient process of debromination under anaerobic conditions. In this study, Dehalococcoides mccartyi strain CG1 was identified to be capable of utilizing TBBPA as the sole electron acceptor at its maximum soluble concentrations (7.3 μM) coupled with cell growth. A previously characterized reductive dehalogenase (RDase), PcbA1, and six other RDases of strain CG1 were detected during TBBPA debromination via transcriptional and proteomic analyses. Furthermore, as a commonly co-contaminated brominated flame retardant of TBBPA, penta-BDEs were debrominated synchronously with TBBPA by strain CG1. This study provides deeper insights into the versatile dehalogenation capabilities of D. mccartyi strain CG1 and its role in in situ remediations of persistent organic pollutants in the environment.

Iron Sulfide Enhanced the Dechlorination of Trichloroethene by Dehalococcoides mccartyi Strain 195.

Iron sulfide (FeS) nanoparticles have great potential in environmental remediation. Using the representative species Dehalococcoides mccartyi strain 195 (Dhc 195), the effect of FeS on trichloroethene (TCE) dechlorination was studied with hydrogen and acetate as the electron donor and carbon source, respectively. With the addition of 0.2 mM Fe2+ and S2–, the dechlorination rate of TCE was enhanced from 25.46 ± 1.15 to 37.84 ± 1.89 μmol⋅L–1⋅day–1 by the in situ formed FeS nanoparticles, as revealed through X-ray diffraction. Comparing the tceA gene copy numbers between with FeS and without FeS, real-time polymerase chain reaction (PCR) indicated that the abundance of the tceA gene increased from (2.83 ± 0.13) × 107 to (4.27 ± 0.21) × 108 copies/ml on day 12. The transcriptional activity of key genes involved in the electron transport chain was upregulated after the addition of FeS, including those responsible for the iron–sulfur cluster assembly protein gene (DET1632) and transmembrane transport of iron (DET1503, DET0685), cobalamin (DET0685, DET1139), and molybdenum (DET1161) genes. Meanwhile, the reverse transcription of tceA was increased approximately five times on the 12th day. These upregulations together suggested that the electron transport of D. mccartyi strain 195 was enhanced by FeS for apparent TCE dechlorination. Overall, the present study provided an eco-friendly and effective method to achieve high remediation efficiency for organohalide-polluted groundwater and soil.

Respiratory Vinyl Chloride Reductive Dechlorination to Ethene in TceA-Expressing Dehalococcoides mccartyi.

Bioremediation of chlorinated ethenes in anoxic aquifers hinges on organohalide-respiring Dehalococcoidia expressing vinyl chloride (VC) reductive dehalogenase (RDase). The tceA gene encoding the trichloroethene-dechlorinating RDase TceA is frequently detected in contaminated groundwater but not recognized as a biomarker for VC detoxification. We demonstrate that tceA-carrying Dehalococcoides mccartyi (Dhc) strains FL2 and 195 grow with VC as an electron acceptor when sufficient vitamin B12 (B12) is provided. Strain FL2 cultures that received 50 μg L-1 B12 completely dechlorinated VC to ethene at rates of 14.80 ± 1.30 μM day-1 and attained 1.64 ± 0.11 × 108 cells per μmol of VC consumed. Strain 195 attained similar growth yields of 1.80 ± 1.00 × 108 cells per μmol of VC consumed, and both strains could be consecutively transferred with VC as the electron acceptor. Proteomic analysis demonstrated TceA expression in VC-grown strain FL2 cultures. Resequencing of the strain FL2 and strain 195 tceA genes identified non-synonymous substitutions, although their consequences for TceA function are currently unknown. The finding that Dhc strains expressing TceA respire VC can explain ethene formation at chlorinated solvent sites, where quantitative polymerase chain reaction analysis indicates that tceA dominates the RDase gene pool.

Changes of the Proteome and Acetylome during Transition into the Stationary Phase in the Organohalide-Respiring Dehalococcoides mccartyi Strain CBDB1.

The strictly anaerobic bactGIerium Dehalococcoides mccartyi obligatorily depends on organohalide respiration for energy conservation and growth. The bacterium also plays an important role in bioremediation. Since there is no guarantee of a continuous supply of halogenated substrates in its natural environment, the question arises of how D. mccartyi maintains the synthesis and activity of dehalogenating enzymes under these conditions. Acetylation is a means by which energy-restricted microorganisms can modulate and maintain protein levels and their functionality. Here, we analyzed the proteome and Nε-lysine acetylome of D. mccartyi strain CBDB1 during growth with 1,2,3-trichlorobenzene as an electron acceptor. The high abundance of the membrane-localized organohalide respiration complex, consisting of the reductive dehalogenases CbrA and CbdbA80, the uptake hydrogenase HupLS, and the organohalide respiration-associated molybdoenzyme OmeA, was shown throughout growth. In addition, the number of acetylated proteins increased from 5% to 11% during the transition from the exponential to the stationary phase. Acetylation of the key proteins of central acetate metabolism and of CbrA, CbdbA80, and TatA, a component of the twin-arginine translocation machinery, suggests that acetylation might contribute to maintenance of the organohalide-respiring capacity of the bacterium during the stationary phase, thus providing a means of ensuring membrane protein integrity and a proton gradient.

Interspecies metabolite transfer and aggregate formation in a co-culture of Dehalococcoides and Sulfurospirillum dehalogenating tetrachloroethene to ethene

Microbial communities involving dehalogenating bacteria assist in bioremediation of areas contaminated with halocarbons. To understand molecular interactions between dehalogenating bacteria, we co-cultured Sulfurospirillum multivorans, dechlorinating tetrachloroethene (PCE) to cis−1,2-dichloroethene (cDCE), and Dehalococcoides mccartyi strains BTF08 or 195, dehalogenating PCE to ethene. The co-cultures were cultivated with lactate as electron donor. In co-cultures, the bacterial cells formed aggregates and D. mccartyi established an unusual, barrel-like morphology. An extracellular matrix surrounding bacterial cells in the aggregates enhanced cell-to-cell contact. PCE was dehalogenated to ethene at least three times faster in the co-culture. The dehalogenation was carried out via PceA of S. multivorans, and PteA (a recently described PCE dehalogenase) and VcrA of D. mccartyi BTF08, as supported by protein abundance. The co-culture was not dependent on exogenous hydrogen and acetate, suggesting a syntrophic relationship in which the obligate hydrogen consumer D. mccartyi consumes hydrogen and acetate produced by S. multivorans. The cobamide cofactor of the reductive dehalogenase—mandatory for D. mccartyi—was also produced by S. multivorans. D. mccartyi strain 195 dechlorinated cDCE in the presence of norpseudo-B12 produced by S. multivorans, but D. mccartyi strain BTF08 depended on an exogenous lower cobamide ligand. This observation is important for bioremediation, since cofactor supply in the environment might be a limiting factor for PCE dehalogenation to ethene, described for D. mccartyi exclusively. The findings from this co-culture give new insights into aggregate formation and the physiology of D. mccartyi within a bacterial community.

Dual Element (C/Cl) Isotope Analysis Indicates Distinct Mechanisms of Reductive Dehalogenation of Chlorinated Ethenes and Dichloroethane in Dehalococcoides mccartyi Strain BTF08 With Defined Reductive Dehalogenase Inventories.

Dehalococcoides mccartyi strain BTF08 has the unique property to couple complete dechlorination of tetrachloroethene and 1,2-dichloroethane to ethene with growth by using the halogenated compounds as terminal electron acceptor. The genome of strain BTF08 encodes 20 genes for reductive dehalogenase homologous proteins (RdhA) including those described for dehalogenation of tetrachloroethene (PceA, PteA), trichloroethene (TceA) and vinyl chloride (VcrA). Thus far it is unknown under which conditions the different RdhAs are expressed, what their substrate specificity is and if different reaction mechanisms are employed. Here we found by proteomic analysis from differentially activated batches that PteA and VcrA were expressed during dechlorination of tetrachloroethene to ethene, while TceA was expressed during 1,2-dichloroethane dehalogenation. Carbon and chlorine compound-specific stable isotope analysis suggested distinct reaction mechanisms for the dechlorination of (i) cis-dichloroethene and vinyl chloride versus (ii) tetrachloroethene. This differentiation was observed independent of the expressed RdhA proteins. Differently, two stable isotope fractionation patterns were observed for 1,2-dichloroethane transformation, for cells with distinct RdhA inventories. Conclusively, we could link specific RdhA expression with functions and provide an insight into the apparently substrate-specific reaction mechanisms in the pathway of reductive dehalogenation in D. mccartyi strain BTF08. Data are available via ProteomeXchange with identifiers PXD018558 and PXD018595.