Proteomic dataset of the organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1 grown on hexachlorobenzene as electron acceptor.
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ABSTRACT:
SUBMITTER: Schiffmann CL
PROVIDER: S-EPMC4773567 | biostudies-literature | 2016 Jun
REPOSITORIES: biostudies-literature
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Proteomic dataset of the organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1 grown on hexachlorobenzene as electron acceptor.
Schiffmann Christian L CL Otto Wolfgang W Hansen Rasmus R Nielsen Per Halkjær PH Adrian Lorenz L Seifert Jana J von Bergen Martin M Jehmlich Nico N
Data in brief 20160222
The proteome of the anaerobic organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1 was analyzed by nano liquid chromatography coupled to mass spectrometry (LC-MS/MS). Two different preparation methods, (i) in-solution and (ii) in-gel proteolytic digestion were assessed to elucidate the core and the functional proteome of bacterial cultures grown in synthetic anaerobic medium with hexachlorobenzene as sole electron acceptor. A detailed analysis of the data presented is available ...[more]
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Project description:The proteome of the anaerobic bacterium Dehalococcoides mccartyi strain CBDB1 from the phylum Chloroflexi was investigated. D. mccartyi strain CBDB1 is a model organism for organohalide respiration where halogenated organic compounds serve as terminal electron acceptors. A wide range of halogenated organic compounds have been shown to be dehalogenated by the strain CBDB1. Therefore, D. mccartyi strain CBDB1 is a promising candidate for bioremediation application. Proteomic analysis of cultures grown with hexachlorobenzene as only electron acceptor resulted in identification of 8,491 distinct peptides which represents 1,023 proteins. A coverage of 70% of the 1,458 annotated proteins for strain CBDB1 was achieved. In addition, a spectral library was created from the annotated spectra. By using proteogenomics, 18 previously not annotated peptides were identified which contribute to four proteins previously not annotated and corrections in length of eight protein coding sequences.
2016-03-11 | PXD003081 | Pride
Project description: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.
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Project description:The acetyl-CoA "Wood-Ljungdahl" pathway couples the folate-mediated one-carbon (C1) metabolism to either CO2 reduction or acetate oxidation via acetyl-CoA. This pathway is distributed in diverse anaerobes and is used for both energy conservation and assimilation of C1 compounds. Genome annotations for all sequenced strains of Dehalococcoides mccartyi, an important bacterium involved in the bioremediation of chlorinated solvents, reveal homologous genes encoding an incomplete Wood-Ljungdahl pathway. Because this pathway lacks key enzymes for both C1 metabolism and CO2 reduction, its cellular functions remain elusive. Here we used D. mccartyi strain 195 as a model organism to investigate the metabolic function of this pathway and its impacts on the growth of strain 195. Surprisingly, this pathway cleaves acetyl-CoA to donate a methyl group for production of methyl-tetrahydrofolate (CH3-THF) for methionine biosynthesis, representing an unconventional strategy for generating CH3-THF in organisms without methylene-tetrahydrofolate reductase. Carbon monoxide (CO) was found to accumulate as an obligate by-product from the acetyl-CoA cleavage because of the lack of a CO dehydrogenase in strain 195. CO accumulation inhibits the sustainable growth and dechlorination of strain 195 maintained in pure cultures, but can be prevented by CO-metabolizing anaerobes that coexist with D. mccartyi, resulting in an unusual syntrophic association. We also found that this pathway incorporates exogenous formate to support serine biosynthesis. This study of the incomplete Wood-Ljungdahl pathway in D. mccartyi indicates a unique bacterial C1 metabolism that is critical for D. mccartyi growth and interactions in dechlorinating communities and may play a role in other anaerobic communities.
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Project description: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.
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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 ?M 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.
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Project description:Corrinoid auxotrophic organohalide-respiring Dehalococcoides mccartyi (Dhc) strains are keystone bacteria for reductive dechlorination of toxic and carcinogenic chloroorganic contaminants. We demonstrate that the lower base attached to the essential corrinoid cofactor of reductive dehalogenase (RDase) enzyme systems modulates dechlorination activity and affects the vinyl chloride (VC) RDases BvcA and VcrA differently. Amendment of 5,6-dimethylbenzimidazolyl-cobamide (DMB-Cba) to Dhc strain BAV1 and strain GT cultures supported cis-1,2-dichloroethene-to-ethene reductive dechlorination at rates of 107.0 (±12.0)??M and 67.4 (±1.4)??M Cl(-) released per day, respectively. Strain BAV1, expressing the BvcA RDase, reductively dechlorinated VC to ethene, although at up to fivefold lower rates in cultures amended with cobamides carrying 5-methylbenzimidazole (5-MeBza), 5-methoxybenzimidazole (5-OMeBza) or benzimidazole (Bza) as the lower base. In contrast, strain GT harboring the VcrA RDase failed to grow and dechlorinate VC to ethene in medium amended with 5-OMeBza-Cba or Bza-Cba. The amendment with DMB to inactive strain GT cultures restored the VC-to-ethene-dechlorinating phenotype and intracellular DMB-Cba was produced, demonstrating cobamide uptake and remodeling. The distinct responses of Dhc strains with BvcA versus VcrA RDases to different cobamides implicate that the lower base exerts control over Dhc reductive dechlorination rates and extents (that is, detoxification), and therefore the dynamics of Dhc strains with discrete reductive dechlorination capabilities. These findings emphasize that the role of the corrinoid/lower base synthesizing community must be understood to predict strain-specific Dhc activity and achieve efficacious contaminated site cleanup.
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Project description:Anaerobic organism capable of dechlorinating a wide variety of environmental pollutants
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Project description:The strictly anaerobic bacterium Dehalococcoides mccartyi is obligatory dependent on organohalide respiration for energy conservation and growth. Due to its capability to reductively dehalogenate a multitude of toxic halogenated electron acceptors, it plays an important role in the attenuation of these compounds at respective contaminated sites. Here, D. mccartyi strain CBDB1, specialized on the dehalogenation of chloroaromatic compounds, was grown in a two-liquid phase system with 1,2,3-trichlorobenzene as electron acceptor, acetate plus CO2 as carbon source and hydrogen as electron donor. The proteome and Nε-lysine acetylome were analyzed in the lag, exponential and stationary phases. The high and almost invariable abundance of the membrane-localized organohalide respiration complex consisting of the reductive dehalogenases CbrA and CbdbA80, the uptake hydrogenase HupLS and the organohalide respiration molybdoenzyme OmeAB was shown throughout growth and also after a prolonged stationary phase. Quantification of transcripts of reductive dehalogenase genes revealed their major synthesis starting in the lag phase, which might be a prerequisite for balanced growth in the exponential phase. The analyses of the coverage of functional pathways as well as indicator analysis revealed the growth-phase specificity of the proteome, with regulatory proteins identified as important indicators for the stationary phase. The number of acetylated proteins increased from the lag to the stationary phase. We detected pronounced acetylation of key proteins of the acetate metabolism, i.e. the synthesis of acetyl-CoA and its processing via gluconeogenesis and the incomplete Wood-Ljungdahl pathway, as well as of proteins central for the biosynthesis of amino acids, co-factors and terpenoids. In addition, the partial acetylation of the reductive dehalogenases as well as of TatA, a component of the twin-arginine translocation machinery, suggests that acetylation might be directly involved in the maintenance of the organohalide respiration capacity of D. mccartyi over periods without access to halogenated electron acceptors.
2021-02-15 | PXD018912 | Pride
Project description: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.
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Project description:Reductive dehalogenases are essential enzymes in organohalide respiration and consist of a catalytic subunit A and a membrane protein B, encoded by rdhAB genes. Thirty-two rdhAB genes exist in the genome of Dehalococcoides mccartyi strain CBDB1. To gain a first insight into the regulation of rdh operons, the control of gene expression of two rdhAB genes (cbdbA1453/cbdbA1452 and cbdbA1455/cbdbA1454) by the MarR-type regulator Rdh2R (cbdbA1456) encoded directly upstream was studied using heterologous expression and in vitro studies. Promoter-lacZ reporter fusions were generated and integrated into the genome of the Escherichia coli host. The lacZ reporter activities of both rdhA promoters decreased upon transformation of the cells with a plasmid carrying the rdh2R gene, suggesting that Rdh2R acts as repressor, whereas the lacZ reporter activity of the rdh2R promoter was not affected. The transcriptional start sites of both rdhA genes in strain CBDB1 and/or the heterologous host mapped to a conserved direct repeat with 11- to 13-bp half-sites. DNase I footprinting revealed binding of Rdh2R to a ∼30-bp sequence covering the complete direct repeat in both promoters, including the transcriptional start sites. Equilibrium sedimentation ultracentrifugation revealed that Rdh2R binds as tetramer to the direct-repeat motif of the rdhA (cbdbA1455) promoter. Using electrophoretic mobility shift assays, a similar binding affinity was found for both rdhA promoters. In the presence of only one half-site of the direct repeat, the interaction was strongly reduced, suggesting a positive cooperativity of binding, for which unusual short palindromes within the direct-repeat half-sites might play an important role.ImportanceDehalococcoides mccartyi strains are obligate anaerobes that grow by organohalide respiration. They have an important bioremediation potential because they are capable of reducing a multitude of halogenated compounds to less toxic products. We are now beginning to understand how these organisms make use of this large catabolic potential, whereby D. mccartyi expresses dehalogenases in a compound-specific fashion. MarR-type regulators are often encoded in the vicinity of reductive dehalogenase genes. In this study, we made use of heterologous expression and in vitro studies to demonstrate that the MarR-type transcription factor Rdh2R acts as a negative regulator. We identify its binding site on the DNA, which suggests a mechanism by which it controls the expression of two adjacent reductive dehalogenase operons.
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