Organic cofactors in the metabolism of Dehalococcoides mccartyi strains

A cDNA-microarray was designed and used to monitor the transcriptomic profile of Dehalococcoides mccartyi strain 195 (in a mixed community) respiring various chlorinated organics, including chloroethenes and 2,3-dichlorophenol. The cultures were continuously fed in order to establish steady-state respiration rates and substrate levels. The organization of array data into a clustered heat map revealed two major experimental partitions. This partitioning in the data set was further explored through principal component analysis. The first two principal components separated the experiments into those with slow (1.6 ؎ 0.6 M Cl ؊ /h)-and fast (22.9 ؎ 9.6 M Cl ؊ /h)-respiring cultures. Additionally, the transcripts with the highest loadings in these principal components were identified, suggesting that those transcripts were responsible for the partitioning of the experiments. By analyzing the transcriptomes (n ‫؍‬ 53) across experiments, relationships among transcripts were identified, and hypotheses about the relationships between electron transport chain members were proposed. One hypothesis, that the hydrogenases Hup and Hym and the formate dehydrogenase-like oxidoreductase (DET0186-DET0187) form a complex (as displayed by their tight clustering in the heat map analysis), was explored using a nondenaturing protein separation technique combined with proteomic sequencing. Although these proteins did not migrate as a single complex, DET0112 (an FdhB-like protein encoded in the Hup operon) was found to comigrate with DET0187 rather than with the catalytic Hup subunit DET0110. On closer inspection of the genome annotations of all Dehalococcoides strains, the DET0185-to-DET0187 operon was found to lack a key subunit, an FdhB-like protein. Therefore, on the basis of the transcriptomic, genomic, and proteomic evidence, the place of the missing subunit in the DET0185-to-DET0187 operon is likely filled by recruiting a subunit expressed from the Hup operon (DET0112). 5), which pass an electron to halogenated organics, and hydrogenases (H 2 ases), which oxidize the sole known electron donor of D. mccartyi, hydrogen (15, 16). Although expression has been confirmed at both the mRNA and protein levels for RDases (differing with the strain), H 2 ases (Hup, Hym-1, Hym-2, Vhu, Ech, and Hyc [5,17,18]), and other putative membrane-bound oxidoreductase proteins, the interactions among these proteins are not well described (19). Three of the other notable putative oxidoreductases are DET0187, a predicted (but not biochemically confirmed) molybdopterin-containing protein annotated as a formate dehydrogenase that maintains an atypical serine in its active site rather than the typical cysteine or selenocysteine (15); Nuo, a NADH-ubiquinone oxidoreductase that lacks the NADH-receiving subunits (NuoEFG) in its predicted operon; and Mod, an additional predicted (but not biochemically confirmed) molybdopterin-containing oxidoreductase.The main goal of this study was to observe broad transcriptional expression patterns during the steady-state...

Section: Genomic Differences Betweenmentioning

confidence: 99%

Meta-Analyses of Dehalococcoides mccartyi Strain 195 Transcriptomic Profiles Identify a Respiration Rate-Related Gene Expression Transition Point and Interoperon Recruitment of a Key Oxidoreductase Subunit

Mansfeldt

Rowe

Heavner

et al. 2014

“…However, the mechanisms that regulate the activity of D. mccartyi within natural ecosystems and shape its functional robustness in disturbed environments are poorly understood due to multiscale microbial community complexity and heterogeneity of biogeochemical processes involved in the sequential degradation (3,4). D. mccartyi exhibits specific restrictive metabolic requirements for a variety of exogenous compounds, such as hydrogen, acetate, corrinoids, biotin, and thiamine, which can be supplied by other microbial genera through a complex metabolic network (1,(5)(6)(7)(8). Therefore, the growth of D. mccartyi is more robust within functionally diverse microbial communities that are deterministically assembled than in pure cultures (5,8,9).…”

mentioning

confidence: 99%

“…D. mccartyi exhibits specific restrictive metabolic requirements for a variety of exogenous compounds, such as hydrogen, acetate, corrinoids, biotin, and thiamine, which can be supplied by other microbial genera through a complex metabolic network (1,(5)(6)(7)(8). Therefore, the growth of D. mccartyi is more robust within functionally diverse microbial communities that are deterministically assembled than in pure cultures (5,8,9). Previous studies have shown that D. mccartyi uses hydrogen as its sole electron donor for TCE dechlorination and outcompetes other terminal electron-accepting processes such as methanogenesis and acetogenesis at low hydrogen partial pressures (10,11).…”

mentioning

confidence: 99%

Efficient Metabolic Exchange and Electron Transfer within a Syntrophic Trichloroethene-Degrading Coculture of Dehalococcoides mccartyi 195 and Syntrophomonas wolfei

Mao

Stenuit

Polasko

et al. 2015

bDehalococcoides mccartyi 195 (strain 195) and Syntrophomonas wolfei were grown in a sustainable syntrophic coculture using butyrate as an electron donor and carbon source and trichloroethene (TCE) as an electron acceptor. The maximum dechlorination rate (9.9 ؎ 0.1 mol day ؊1 ) and cell yield [(1.1 ؎ 0.3) ؋ 10 8 cells mol ؊1 Cl ؊ ] of strain 195 maintained in coculture were, respectively, 2.6 and 1.6 times higher than those measured in the pure culture. The strain 195 cell concentration was about 16 times higher than that of S. wolfei in the coculture. Aqueous H 2 concentrations ranged from 24 to 180 nM during dechlorination and increased to 350 ؎ 20 nM when TCE was depleted, resulting in cessation of butyrate fermentation by S. wolfei with a theoretical Gibbs free energy of ؊13.7 ؎ 0.2 kJ mol ؊1 . Carbon monoxide in the coculture was around 0.06 mol per bottle, which was lower than that observed for strain 195 in isolation. The minimum H 2 threshold value for TCE dechlorination by strain 195 in the coculture was 0.6 ؎ 0.1 nM. Cell aggregates during syntrophic growth were observed by scanning electron microscopy. The interspecies distances to achieve H 2 fluxes required to support the measured dechlorination rates were predicted using Fick's law and demonstrated the need for aggregation. Filamentous appendages and extracellular polymeric substance (EPS)-like structures were present in the intercellular spaces. The transcriptome of strain 195 during exponential growth in the coculture indicated increased ATP-binding cassette transporter activities compared to the pure culture, while the membrane-bound energy metabolism related genes were expressed at stable levels. Groundwater contamination by trichloroethene (TCE), a potential human carcinogen, poses a serious threat to human health and can lead to the generation of vinyl chloride (VC), which is a known human carcinogen (1). Strains of Dehalococcoides mccartyi are the only known bacteria that can completely degrade TCE to the benign end product ethene. Biostimulation of indigenous Dehalococcoides spp. and bioaugmentation using Dehalococcoides-containing cultures are recognized as the most reliable in situ bioremediation technologies resulting in the complete dechlorination of TCE to ethene (2). However, the mechanisms that regulate the activity of D. mccartyi within natural ecosystems and shape its functional robustness in disturbed environments are poorly understood due to multiscale microbial community complexity and heterogeneity of biogeochemical processes involved in the sequential degradation (3, 4). D. mccartyi exhibits specific restrictive metabolic requirements for a variety of exogenous compounds, such as hydrogen, acetate, corrinoids, biotin, and thiamine, which can be supplied by other microbial genera through a complex metabolic network (1,(5)(6)(7)(8). Therefore, the growth of D. mccartyi is more robust within functionally diverse microbial communities that are deterministically assembled than in pure cultures (5,8,9). Previous studies have shown t...

“…Genomic analyses of sequenced D. mccartyi strains reveal a lack of complete corrinoid biosynthesis pathways, rendering D. mccartyi incapable of synthesizing corrinoids de novo (6,7). Exogenous cobalamin (a specific corrinoid also known as vitamin B 12 ) is generally added to grow D. mccartyi in isolation (8).…”

mentioning

confidence: 99%

Sustainable Growth of Dehalococcoides mccartyi 195 by Corrinoid Salvaging and Remodeling in Defined Lactate-Fermenting Consortia

Men

Seth

et al. 2014

dCorrinoids are essential cofactors of reductive dehalogenases in Dehalococcoides mccartyi, an important bacterium in bioremediation, yet sequenced D. mccartyi strains do not possess the complete pathway for de novo corrinoid biosynthesis. Pelosinus sp. and Desulfovibrio sp. have been detected in dechlorinating communities enriched from contaminated groundwater without exogenous cobalamin corrinoid. To investigate the corrinoid-related interactions among key members of these communities, we constructed consortia by growing D. mccartyi strain 195 (Dhc195) in cobalamin-free, trichloroethene (TCE)-and lactateamended medium in cocultures with Desulfovibrio vulgaris Hildenborough (DvH) or Pelosinus fermentans R7 (PfR7) and with both in tricultures. Only the triculture exhibited sustainable dechlorination and cell growth when a physiological level of 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, was provided. In the triculture, DvH provided hydrogen while PfR7 provided corrinoids to Dhc195, and the initiation of dechlorination and Dhc195 cell growth was highly dependent on the growth of PfR7. Corrinoid analysis indicated that Dhc195 imported and remodeled the phenolic corrinoids produced by PfR7 into cobalamin in the presence of DMB. Transcriptomic analyses of Dhc195 showed the induction of the CbiZ-dependent corrinoid-remodeling pathway and BtuFCD corrinoid ABC transporter genes during corrinoid salvaging and remodeling. In contrast, another operon annotated to encode a putative iron/cobalamin ABC transporter (DET1174-DET1176) was induced when cobalamin was exogenously provided. Interestingly, a global upregulation of phage-related genes was observed when PfR7 was present. These findings provide insights into both the gene regulation of corrinoid salvaging and remodeling in Dhc195 when it is grown without exogenous cobalamin and microbe-to-microbe interactions in dechlorinating microbial communities.C hlorinated solvents such as tetra-and trichloroethene (PCE/ TCE) have been among the most common subsurface contaminants in the United States for decades (1, 2). In situ bioremediation is an effective, economical, and environmentally friendly technology for treating chlorinated solvents (2, 3). Dehalococcoides mccartyi is the only known bacterium capable of carrying out respiratory reductive dechlorination of chloroethenes to nontoxic ethene and plays a key role in the bioremediation of contaminated groundwater (4, 5).As cofactors of reductive dehalogenases (RDases), the enzymes responsible for reductive dechlorination, corrinoids (e.g., cobalamin) are essential nutrients for supporting D. mccartyi growth and dechlorination. Genomic analyses of sequenced D. mccartyi strains reveal a lack of complete corrinoid biosynthesis pathways, rendering D. mccartyi incapable of synthesizing corrinoids de novo (6, 7). Exogenous cobalamin (a specific corrinoid also known as vitamin B 12 ) is generally added to grow D. mccartyi in isolation (8). Cobalamin, 5-methylbenzimidazolylcobamide ([5-MeBza]Cba), and 5-methoxyb...