Organohalide respiration of a sulfate-reducer, Desulforhopalus singaporensis
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PROVIDER: PRJEB61466 | ENA |
REPOSITORIES: ENA
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Project description:The organohalide-respiring Sulfurospirillum multivorans uses chlorinated ethenes as electron acceptors for growth under anoxic conditions. However, little is known about the interaction of these substrates with proteins. Here, we apply thermal proteome profiling (TPP) to analyze enzyme-trichloroethene interactions. TPP is commonly used to investigate protein-ligand binding through protein melting curve shifts. Several modifications in the protocol, e.g. performing the incubation under anaerobic conditions and increasing the temperature range up to 97°C, improved the detection range and allowed the investigation of oxygen-sensitive proteins. Enzymatic reductive dehalogenation was prevented by omitting the electron donor during incubations. This enabled detecting the interaction of the tetrachloroethene reductive dehalogenase PceA with trichloroethene and confirms the enzyme’s specificity for this substrate. Another 19 proteins showed significant melting curve shifts with trichloroethene, pointing to other proteins directly or indirectly interacting with trichloroethene. Interestingly, a putative response regulator reacted similarly towards trichloroethene, which is potentially in line with its proposed role in regulating trichloroethene respiration. The TPP approach is here proven to facilitate the identification of substrate-enzyme interactions of strictly anaerobic reductive dehalogenases and probably their regulators. This strategy can be used to identify yet unknown substrate specificities and potential signal-sensing proteins in other difficult to study bacteria.
2018-06-06 | PXD009308 | Pride
Project description:Reductive Dehalogenation in Aarhus Bay
| PRJEB50583 | ENA
Project description:Organohalide respiration (OHR), catalysed by reductive dehalogenases (RDases), plays an important role in halogen cycling. Natural organohalides and putative RDase-encoding genes have been reported in Aarhus Bay sediments, however, OHR has not been experimentally verified. Here we show that sediments of Aarhus Bay can dehalogenate a range of organohalides, and different organohalides differentially affected microbial community compositions. PCE-dechlorinating cultures were further examined by 16S rRNA gene-targeted quantitative PCR and amplicon sequencing. Known organohalide-respiring bacteria (OHRB) including Dehalococcoides, Dehalobacter and Desulfitobacterium decreased in abundance during transfers and serial dilutions, suggesting the importance of yet uncharacterized OHRB in these cultures. Switching from PCE to 2,6-DBP led to its complete debromination to phenol in cultures with and without sulfate. 2,6-DBP debrominating cultures differed in microbial composition from PCE-dechlorinating cultures. Desulfobacterota genera recently verified to include OHRB, including Desulfovibrio and Desulfuromusa, were enriched in all microcosms, whereas Halodesulfovibrio was only enriched in cultures without sulfate. Hydrogen and methane were detected in cultures without sulfate. Hydrogen likely served as electron donor for OHR and methanogenesis. This study shows that OHR can occur in marine environments mediated by yet unknown OHRB, suggesting their role in natural halogen cycling.
| S-EPMC9303371 | biostudies-literature
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.
| S-EPMC6005880 | biostudies-literature
Project description: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 and (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.
2020-06-25 | PXD018595 | Pride
Project description: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 and (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.
2020-06-10 | PXD018558 | Pride
Project description:Desulfitobacterium hafniense strain TCE1 is capable of metabolically reducing tetra- and trichloroethenes by organohalide respiration. A previous study revealed that the pce gene cluster responsible for this process is located on an active composite transposon, Tn-Dha1. In the present work, we investigated the effects on the stability of the transposon during successive subcultivations of strain TCE1 in a medium depleted of tetrachloroethene. At the physiological level, an increased fitness of the population was observed after 9 successive transfers and was correlated with a decrease in the level of production of the PceA enzyme. The latter observation was a result of the gradual loss of the pce genes in the population of strain TCE1 and not of a regulation mechanism, as was postulated previously for a similar phenomenon described for Sulfurospirillum multivorans. A detailed molecular analysis of genetic rearrangements occurring around Tn-Dha1 showed two independent but concomitant events, namely, the transposition of the first insertion sequence, ISDha1-a, and homologous recombination across identical copies of ISDha1 flanking the transposon. A new model is proposed for the genetic heterogeneity around Tn-Dha1 in D. hafniense strain TCE1, along with some considerations for the cleavage mechanism mediated by the transposase TnpA1 encoded by ISDha1.
| S-EPMC3416639 | biostudies-literature
Project description:We compared the global transcriptomic analysis of Desulfoluna spongiiphila strain AA1, an organohalide-respiring Desulfobacterota isolated from a marine sponge, with 2,6-dibromophenol or with sulfate as electron acceptor. The most significant difference of the transcriptomic analysis was the expression of one reductive dehalogenase gene cluster (rdh16), which was significantly upregulated with 2,6-dibromophenol.
2020-01-10 | GSE141466 | GEO
Project description:<p>A variety of anthropogenic organohalide contaminants generated from industry are released into the environment, and thus cause serious pollution that endangers human health. In the present study, we investigated the microbial community composition of industrial saponification wastewater using 16S rRNA sequencing, providing genomic insights of potential organohalide dehalogenation bacteria (OHDBs) by whole-metagenome sequencing. We also explored yet-to-culture OHDBs involved in the microbial community. Microbial diversity analysis reveals that Proteobacteria and Patescibacteria phyla dominate microbiome abundance of the wastewater. In addition, a total of six bacterial groups (Rhizobiales, Rhodobacteraceae, Rhodospirillales, Flavobạcteriales, Micrococcales, and Saccharimonadales) were found as biomarkers in the key organohalide removal module. Ninety-four metagenome-assembled genomes (MAGs) were reconstructed from the microbial community, and 105 hydrolytic dehalogenase genes within 42 MAGs were identified, suggesting that the potential for hydrolytic organohalide dehalogenation is present in the microbial community. Subsequently, we characterized the organohalide dehalogenation of an isolated OHDB, Microbacterium sp. J1-1, which shows the dehalogenation activities of chloropropanol, dichloropropanol, and epichlorohydrin. This study provides a community-integrated multi-omics approach to gain functional OHDBs for industrial organohalide dehalogenation.</p>
2022-11-21 | MTBLS3916 | MetaboLights