Biochemistry of Catabolic Reductive Dehalogenation.

A wide range of phylogenetically diverse microorganisms couple the reductive dehalogenation of organohalides to energy conservation. Key enzymes of such anaerobic catabolic pathways are corrinoid and Fe-S cluster-containing, membrane-associated reductive dehalogenases. These enzymes catalyze the reductive elimination of a halide and constitute the terminal reductases of a short electron transfer chain. Enzymatic and physiological studies revealed the existence of quinone-dependent and quinone-independent reductive dehalogenases that are distinguishable at the amino acid sequence level, implying different modes of energy conservation in the respective microorganisms. In this review, we summarize current knowledge about catabolic reductive dehalogenases and the electron transfer chain they are part of. We review reaction mechanisms and the role of the corrinoid and Fe-S cluster cofactors and discuss physiological implications.

Structural basis of Qng1-mediated salvage of the micronutrient queuine from queuosine-5'-monophosphate as the biological substrate.

Eukaryotic life benefits from-and ofttimes critically relies upon-the de novo biosynthesis and supply of vitamins and micronutrients from bacteria. The micronutrient queuosine (Q), derived from diet and/or the gut microbiome, is used as a source of the nucleobase queuine, which once incorporated into the anticodon of tRNA contributes to translational efficiency and accuracy. Here, we report high-resolution, substrate-bound crystal structures of the Sphaerobacter thermophilus queuine salvage protein Qng1 (formerly DUF2419) and of its human ortholog QNG1 (C9orf64), which together with biochemical and genetic evidence demonstrate its function as the hydrolase releasing queuine from queuosine-5'-monophosphate as the biological substrate. We also show that QNG1 is highly expressed in the liver, with implications for Q salvage and recycling. The essential role of this family of hydrolases in supplying queuine in eukaryotes places it at the nexus of numerous (patho)physiological processes associated with queuine deficiency, including altered metabolism, proliferation, differentiation and cancer progression.

Recreating the natural evolutionary trend in key microdomains provides an effective strategy for engineering of a thermomicrobial N-demethylase.

N-demethylases have been reported to remove the methyl groups on primary or secondary amines, which could further affect the properties and functions of biomacromolecules or chemical compounds; however, the substrate scope and the robustness of N-demethylases have not been systematically investigated. Here we report the recreation of natural evolution in key microdomains of the Thermomicrobium roseum sarcosine oxidase (TrSOX), an N-demethylase with marked stability (melting temperature over 100 °C) and enantioselectivity, for enhanced substrate scope and catalytic efficiency on -C-N- bonds. We obtained the structure of TrSOX by crystallization and X-ray diffraction (XRD) for the initial framework. The natural evolution in the nonconserved residues of key microdomains-including the catalytic loop, coenzyme pocket, substrate pocket, and entrance site-was then identified using ancestral sequence reconstruction (ASR), and the substitutions that accrued during natural evolution were recreated by site-directed mutagenesis. The single and double substitution variants catalyzed the N-demethylation of N-methyl-L-amino acids up to 1800- and 6000-fold faster than the wild type, respectively. Additionally, these single substitution variants catalyzed the terminal N-demethylation of non-amino-acid compounds and the oxidation of the main chain -C-N- bond to a -C=N- bond in the nitrogen-containing heterocycle. Notably, these variants retained the enantioselectivity and stability of the initial framework. We conclude that the variants of TrSOX are of great potential use in N-methyl enantiomer resolution, main-chain Schiff base synthesis, and alkaloid modification or degradation.

P finder: genomic and metagenomic annotation of RNase P RNA gene (rnpB).

BACKGROUND: The rnpB gene encodes for an essential catalytic RNA (RNase P). Like other essential RNAs, RNase P's sequence is highly variable. However, unlike other essential RNAs (i.e. tRNA, 16 S, 6 S,...) its structure is also variable with at least 5 distinct structure types observed in prokaryotes. This structural variability makes it labor intensive and challenging to create and maintain covariance models for the detection of RNase P RNA in genomic and metagenomic sequences. The lack of a facile and rapid annotation algorithm has led to the rnpB gene being the most grossly under annotated essential gene in completed prokaryotic genomes with only a 24% annotation rate. Here we describe the coupling of the largest RNase P RNA database with the local alignment scoring algorithm to create the most sensitive and rapid prokaryote rnpB gene identification and annotation algorithm to date. RESULTS: Of the 2772 completed microbial genomes downloaded from GenBank only 665 genomes had an annotated rnpB gene. We applied P Finder to these genomes and were able to identify 2733 or nearly 99% of the 2772 microbial genomes examined. From these results four new rnpB genes that encode the minimal T-type P RNase P RNAs were identified computationally for the first time. In addition, only the second C-type RNase P RNA was identified in Sphaerobacter thermophilus. Of special note, no RNase P RNAs were detected in several obligate endosymbionts of sap sucking insects suggesting a novel evolutionary adaptation. CONCLUSIONS: The coupling of the largest RNase P RNA database and associated structure class identification with the P Finder algorithm is both sensitive and rapid, yielding high quality results to aid researchers annotating either genomic or metagenomic data. It is the only algorithm to date that can identify challenging RNAse P classes such as C-type and the minimal T-type RNase P RNAs. P Finder is written in C# and has a user-friendly GUI that can run on multiple 64-bit windows platforms (Windows Vista/7/8/10). P Finder is free available for download at https://github.com/JChristopherEllis/P-Finder as well as a small sample RNase P RNA file for testing.

Redox Coenzyme F420 Biosynthesis in Thermomicrobia Involves Reduction by Stand-Alone Nitroreductase Superfamily Enzymes.

Coenzyme F420 is a redox cofactor involved in hydride transfer reactions in archaea and bacteria. Since F420-dependent enzymes are attracting increasing interest as tools in biocatalysis, F420 biosynthesis is being revisited. While it was commonly accepted for a long time that the 2-phospho-l-lactate (2-PL) moiety of F420 is formed from free 2-PL, it was recently shown that phosphoenolpyruvate is incorporated in Actinobacteria and that the C-terminal domain of the FbiB protein, a member of the nitroreductase (NTR) superfamily, converts dehydro-F420 into saturated F420 Outside the Actinobacteria, however, the situation is still unclear because FbiB is missing in these organisms and enzymes of the NTR family are highly diversified. Here, we show by heterologous expression and in vitro assays that stand-alone NTR enzymes from Thermomicrobia exhibit dehydro-F420 reductase activity. Metabolome analysis and proteomics studies confirmed the proposed biosynthetic pathway in Thermomicrobium roseum These results clarify the biosynthetic route of coenzyme F420 in a class of Gram-negative bacteria, redefine functional subgroups of the NTR superfamily, and offer an alternative for large-scale production of F420 in Escherichia coli in the future.IMPORTANCE Coenzyme F420 is a redox cofactor of Archaea and Actinobacteria, as well as some Gram-negative bacteria. Its involvement in processes such as the biosynthesis of antibiotics, the degradation of xenobiotics, and asymmetric enzymatic reductions renders F420 of great relevance for biotechnology. Recently, a new biosynthetic step during the formation of F420 in Actinobacteria was discovered, involving an enzyme domain belonging to the versatile nitroreductase (NTR) superfamily, while this process remained blurred in Gram-negative bacteria. Here, we show that a similar biosynthetic route exists in Thermomicrobia, although key biosynthetic enzymes show different domain architectures and are only distantly related. Our results shed light on the biosynthesis of F420 in Gram-negative bacteria and refine the knowledge about sequence-function relationships within the NTR superfamily of enzymes. Appreciably, these results offer an alternative route to produce F420 in Gram-negative model organisms and unveil yet another biochemical facet of this pathway to be explored by synthetic microbiologists.

Enzymatic Synthesis of Methylated Terpene Analogues Using the Plasticity of Bacterial Terpene Synthases.

Methylated analogues of isopentenyl diphosphate were synthesised and enzymatically incorporated into methylated terpenes. A detailed stereochemical analysis of the obtained products is presented. The methylated terpene precursors were also used in conjunction with various isotopic labellings to gain insights into the mechanisms of their enzymatic formation.

Widening the bottleneck: Heterologous expression, purification, and characterization of the Ktedonobacter racemifer minimal type II polyketide synthase in Escherichia coli.

Enzyme assemblies such as type II polyketide synthases (PKSs) produce a wide array of bioactive secondary metabolites. While the molecules produced by type II PKSs have found remarkable clinical success, the biosynthetic prowess of these enzymes has been stymied by 1) the inability to reconstitute the bioactivity of the minimal PKS enzymes in vitro and 2) limited exploration of type II PKSs from diverse phyla. To begin filling this unmet need, we expressed, purified, and characterized the ketosynthase chain length factor (KS-CLF) and acyl carrier protein (ACP) from Ktedonobacter racemifer (Kr). Using E. coli as a heterologous host, we obtained soluble proteins in titers signifying improvements over previous KS-CLF heterologous expression efforts. Characterization of these enzymes reveals that KrACP has self-malonylating activity. Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of holo-KrACP and KrKS-CLF indicates that these enzymes do not interact in vitro, suggesting that the acylated state of these proteins might play an important role in facilitating biosynthetically relevant interactions. These results lay important groundwork for optimizing the interaction between KrKS-CLF and KrACP and exploring the biosynthetic potential of other non-actinomycete type II PKSs.

Structural insights into the enzyme specificity of a novel ω-transaminase from the thermophilic bacterium Sphaerobacter thermophilus.

Transaminases are pyridoxal 5'-phosphate-dependent enzymes that reversibly catalyze transamination reactions from an amino group donor substrate to an amino group acceptor substrate. ω-Transaminases (ωTAs) utilize compounds with an amino group not at α-carbon position as their amino group donor substrates. Recently, a novel ωTA with broad substrate specificity and high thermostability from the thermophilic bacterium Sphaerobacter thermophilus (St-ωTA) has been reported. Although St-ωTA has been biochemically characterized, little is known about its determinants of substrate specificity. In the present study, we determined the crystal structure of St-ωTA at 1.9 Šresolution to clarify in detail its mechanism of substrate recognition. The structure of St-ωTA revealed that it has a voluminous active site resulting from the unique spatial arrangement of residues comprising its active site. In addition, our molecular docking simulation results suggest that substrate compounds may bind to active site residues via electrostatic interactions or hydrophobic interactions that can be induced by subtle rearrangements of active site residues. On the basis of these structural analyses, we propose a plausible working model of the enzymatic mechanism of St-ωTA. Our results provide profound structural insights into the substrate specificity of St-ωTA and extend the boundaries of knowledge of TAs.

The C-terminal domain conformational switch revealed by the crystal structure of malyl-CoA lyase from Roseiflexus castenholzii.

Malyl-coenzyme A lyase (MCL) is a carbon-carbon bond lyase that catalyzes the reversible cleavage of coenzyme A (CoA) thioesters in multiple carbon metabolic pathways. This enzyme contains a CitE-like TIM barrel and an additional C-terminal domain that undergoes conformational changes upon substrate binding. However, the structural basis underlying these conformational changes is elusive. Here, we report the crystal structure of MCL from the thermophilic photosynthetic bacterium Roseiflexus castenholzii (RfxMCL) in the apo- and oxalate-bound forms at resolutions of 2.50 and 2.65 Å, respectively. Molecular dynamics simulations and structural comparisons with MCLs from other species reveal the deflection of the C-terminal domain to close the adjacent active site pocket in the trimer and contribute active site residues for CoA coordination. The deflection angles of the C-terminal domain are not only related to the occupation but also the type of bound substrates in the adjacent active site pocket. Our work illustrates that a conformational switch of the C-terminal domain accompanies the substrate-binding of MCLs. The results provide a framework for further investigating the reaction mechanism and multifunctionality of MCLs in different carbon metabolic pathways.

Enzymatic characterization of a thermostable phosphatase from Thermomicrobium roseum and its application for biosynthesis of fructose from maltodextrin.

Phosphatases, which catalyze the dephosphorylation of compounds containing phosphate groups, are important members of the haloacid dehalogenase (HAD)-like superfamily. Herein, a thermostable phosphatase encoded by an open reading frame of Trd_1070 from Thermomicrobium roseum was enzymologically characterized. This phosphatase showed promiscuous activity against more than ten sugar phosphates, with high specific activity toward ribose 5-phosphate, followed by ribulose 5-phosphate and fructose 6-phosphate. The half-life of Trd_1070 at 70 °C and pH 7.0 was about 14.2 h. Given that the catalytic efficiency of Trd_1070 on fructose 6-phosphate was 49-fold higher than that on glucose 6-phosphate, an in vitro synthetic biosystem containing alpha-glucan phosphorylase, phosphoglucomutase, phosphoglucose isomerase, and Trd_1070 was constructed for the production of fructose from maltodextrin by whole-cell catalysis, resulting in 21.6 g/L fructose with a ratio of fructose to glucose of approximately 2:1 from 50 g/L maltodextrin. This in vitro biosystem provides an alternative method to produce fructose with higher fructose content compared with the traditional production method using glucose isomerization. Further discovery and enzymologic characterization of phosphatases may promote further production of alternative monosaccharides through in vitro synthetic biosystems.

Characterisation of two self-sufficient CYP102 family monooxygenases from Ktedonobacter racemifer DSM44963 which have new fatty acid alcohol product profiles.

BACKGROUND: Two self-sufficient CYP102 family encoding genes (Krac_0936 and Krac_9955) from the bacterium Ktedonobacter racemifer DSM44963, which possesses one of the largest bacterial genomes, have been identified. METHODS: Phylogenetic analysis of both the encoded cytochrome P450 enzymes, Krac0936 and Krac9955. Both enzymes were produced and their turnovers with fatty acid substrates assessed in vitro and using a whole-cell oxidation system. RESULTS: Krac0936 hydroxylated straight chain, saturated fatty acids predominantly at the ω-1 and ω-2 positions using NADPH as the cofactor. Krac0936 was less active towards shorter unsaturated fatty acids but longer unsaturated acids were efficiently oxidised. cis,cis-9,12-Octadecadienoic and pentadecanoic acids were the most active substrates tested with Krac0936. Unusually Krac9955 showed very low levels of NAD(P)H oxidation activity though coupling of the reducing equivalents to product formation was high. The product distribution of tridecanoic, tetradecanoic and pentadecanoic acid oxidation by Krac9955 favoured oxidation at the ω-4, ω-5 and ω-6 positions, respectively. CONCLUSION: Krac0936 and Krac9955 are self-sufficient P450 monooxygenases. Krac0936 has a preference for pentadecanoic acid over other straight chain fatty acids and showed little or no activity with dodecanoic or octadecanoic acids. Krac9955 preferably oxidised shorter fatty acids compared to Krac0936 with tridecanoic having the highest levels of product formation. Unlike Krac0936 and P450Bm3, Krac9995 showed lower activities with unsaturated fatty acids. GENERAL SIGNIFICANCE: In this study of two of the CYP enzymes from K. racemifer we have shown that this bacterium from the Chloroflexi phylum contains genes which encode new proteins with novel activity.

The self-sufficient CYP102 family enzyme, Krac9955, from Ktedonobacter racemifer DSM44963 acts as an alkyl- and alkyloxy-benzoic acid hydroxylase.

A self-sufficient CYP102 family encoding gene (Krac_9955) has been identified from the bacterium Ktedonobacter racemifer DSM44963 which belongs to the Chloroflexi phylum. The characterisation of the substrate range of this enzyme was hampered by low levels of production using E. coli. The yield and purity of the Krac9555 enzyme was improved using a codon optimised gene, the introduction of a tag and modification of the purification protocol. The heme domain was isolated and in vitro analysis of substrate binding and turnover was performed. Krac9955 was found to preferentially bind alkyl- and alkyloxy-benzoic acids (≥95% high spin, Kd < 3 µM) over saturated and unsaturated fatty acids. Unusually for a self-sufficient CYP102 family member Krac9955 showed low levels of NAD(P)H oxidation activity for all the substrates tested though product formation was observed for many. For nearly all substrates the preferred site of hydroxylation of Krac9955 was eight carbons away from the carboxylate group with certain reactions proceeding at ≥ 90% selectivity. Krac9955 differs from CYP102A1 (P450Bm3), and is the first self-sufficient member of the CYP102 family of P450 enzymes which is not optimised for fast fatty acid hydroxylation close to the ω-terminus.

A manganese catalase from Thermomicrobium roseum with peroxidase and catecholase activity.

An enzyme with catechol oxidase activity was identified in Thermomicrobium roseum extracts via solution assays and activity-stained SDS-PAGE. Yet, the genome of T. roseum does not harbor a catecholase gene. The enzyme was purified with two anion exchange chromatography steps and ultimately identified to be a manganese catalase with additional peroxidase and catecholase activity. Catalase activity (6280 ± 430 IU/mg) clearly dominated over pyrogallol peroxidase (231 ± 53 IU/mg) and catecholase (3.07 ± 0.56 IU/mg) activity as determined at 70 °C. Most enzyme kinetic properties were comparable to previously characterized manganese catalase enzymes. Catalase activity was highest at alkaline pH values and showed inhibition by excess substrate and chloride. The apparent K m and k cat values were 20 mM and 2.02 × 104 s-1 subunit-1 at 25 °C and pH 7.0.

Development and application of a rapid, user-friendly, and inexpensive method to detect Dehalococcoides sp. reductive dehalogenase genes from groundwater.

TaqMan probe-based quantitative polymerase chain reaction (qPCR) specific to the biomarker reductive dehalogenase (RDase) genes is a widely accepted molecular biological tool (MBT) for determining the abundance of Dehalococcoides sp. in groundwater samples from chlorinated solvent-contaminated sites. However, there are significant costs associated with this MBT. In this study, we describe an approach that requires only low-cost laboratory equipment (a bench top centrifuge and a water bath) and requires less time and resources compared to qPCR. The method involves the concentration of biomass from groundwater, without DNA extraction, and loop-mediated isothermal amplification (LAMP) of the cell templates. The amplification products are detected by a simple visual color change (orange/green). The detection limits of the assay were determined using groundwater from a contaminated site. In addition, the assay was tested with groundwater from three additional contaminated sites. The final approach to detect RDase genes, without DNA extraction or a thermal cycler, was successful to 1.8 × 105 gene copies per L for vcrA and 1.3 × 105 gene copies per L for tceA. Both values are below the threshold recommended for effective in situ dechlorination.

Identification of a multi-protein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement.

Dehalococcoides mccartyi strain CBDB1 is an obligate organohalide-respiring bacterium using only hydrogen as electron donor and halogenated organics as electron acceptor. Here, we studied proteins involved in the respiratory chain under non-denaturing conditions. Using blue native gel electrophoresis (BN-PAGE), gel filtration and ultrafiltration an active dehalogenating protein complex with a molecular mass of 250-270 kDa was identified. The active subunit of reductive dehalogenase (RdhA) colocalised with a complex iron-sulfur molybdoenzyme (CISM) subunit (CbdbA195) and an iron-sulfur cluster containing subunit (CbdbA131) of the hydrogen uptake hydrogenase (Hup). No colocalisation between the catalytically active subunits of hydrogenase and reductive dehalogenase was found. By two-dimensional BN/SDS-PAGE the stability of the complex towards detergents was assessed, demonstrating stepwise disintegration with increasing detergent concentrations. Chemical cross-linking confirmed the presence of a higher molecular mass reductive dehalogenase protein complex composed of RdhA, CISM I and Hup hydrogenase and proved to be a potential tool for stabilising protein-protein interactions of the dehalogenating complex prior to membrane solubilisation. Taken together, the identification of the respiratory dehalogenase protein complex and the absence of indications for quinone participation in the respiration suggest a quinone-independent protein-based respiratory electron transfer chain in D. mccartyi.

Dehalogenimonas sp. Strain WBC-2 Genome and Identification of Its trans-Dichloroethene Reductive Dehalogenase, TdrA.

The Dehalogenimonas population in a dechlorinating enrichment culture referred to as WBC-2 was previously shown to be responsible for trans-dichloroethene (tDCE) hydrogenolysis to vinyl chloride (VC). In this study, blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzymatic assays and protein identification using liquid chromatography coupled with mass spectrometry (LC-MS/MS) led to the functional characterization of a novel dehalogenase, TdrA. This new reductive dehalogenase (RDase) catalyzes the dechlorination of tDCE to VC. A metagenome of the WBC-2 culture was sequenced, and a complete Dehalogenimonas genome, only the second Dehalogenimonas genome to become publicly available, was closed. The tdrA dehalogenase found within the Dehalogenimonas genome appears to be on a genomic island similar to genomic islands found in Dehalococcoides. TdrA itself is most similar to TceA from Dehalococcoides sp. strain FL2 with 76.4% amino acid pairwise identity. It is likely that the horizontal transfer of rdhA genes is not only a feature of Dehalococcoides but also a feature of other Dehalococcoidia, including Dehalogenimonas. A set of primers was developed to track tdrA in WBC-2 subcultures maintained on different electron acceptors. This newest dehalogenase is an addition to the short list of functionally defined RDases sharing the usual characteristic motifs (including an AB operon, a TAT export sequence, two iron-sulfur clusters, and a corrinoid binding domain), substrate flexibility, and evidence for horizontal gene transfer within the Dehalococcoidia.

Biochemical and EPR-spectroscopic investigation into heterologously expressed vinyl chloride reductive dehalogenase (VcrA) from Dehalococcoides mccartyi strain VS.

Reductive dehalogenases play a critical role in the microbial detoxification of aquifers contaminated with chloroethenes and chlorethanes by catalyzing the reductive elimination of a halogen. We report here the first heterologous production of vinyl chloride reductase VcrA from Dehalococcoides mccartyi strain VS. Heterologously expressed VcrA was reconstituted to its active form by addition of hydroxocobalamin/adenosylcobalamin, Fe(3+), and sulfide in the presence of mercaptoethanol. The kinetic properties of reconstituted VcrA catalyzing vinyl chloride reduction with Ti(III)-citrate as reductant and methyl viologen as mediator were similar to those obtained previously for VcrA as isolated from D. mccartyi strain VS. VcrA was also found to catalyze a novel reaction, the environmentally important dihaloelimination of 1,2-dichloroethane to ethene. Electron paramagnetic resonance (EPR) spectroscopic studies with reconstituted VcrA in the presence of mercaptoethanol revealed the presence of Cob(II)alamin. Addition of Ti(III)-citrate resulted in the appearance of a new signal characteristic of a reduced [4Fe-4S] cluster and the disappearance of the Cob(II)alamin signal. UV-vis absorption spectroscopy of Ti(III)citrate-treated samples revealed the formation of two new absorption maxima characteristic of Cob(I)alamin. No evidence for the presence of a [3Fe-4S] cluster was found. We postulate that during the reaction cycle of VcrA, a reduced [4Fe-4S] cluster reduces Co(II) to Co(I) of the enzyme-bound cobalamin. Vinyl chloride reduction to ethene would be initiated when Cob(I)alamin transfers an electron to the substrate, generating a vinyl radical as a potential reaction intermediate.

Heterologous complementation studies in Escherichia coli with the Hyp accessory protein machinery from Chloroflexi provide insight into [NiFe]-hydrogenase large subunit recognition by the HypC protein family.

Six Hyp maturation proteins (HypABCDEF) are conserved in micro-organisms that synthesize [NiFe]-hydrogenases (Hyd). Of these, the HypC chaperones interact directly with the apo-form of the catalytically active large subunit of Hyd enzymes and are believed to transfer the Fe(CN)2CO moiety of the bimetallic cofactor from the Hyp machinery to this large subunit. In E. coli, HypC is specifically required for maturation of Hyd-3 while its paralogue, HybG, is specifically required for Hyd-2 maturation; either HypC or HybG can mature Hyd-1. In this study, we demonstrate that the products of the hypABFCDE operon from the deeply branching hydrogen-dependent and obligate organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1 were capable of maturing and assembling active Hyd-1, Hyd-2 and Hyd-3 in an E. coli hyp mutant. Maturation of Hyd-1 was less efficient, presumably because HypB of E. coli was necessary to restore optimal enzyme activity. In a reciprocal maturation study, the highly O2-sensitive H2-uptake HupLS [NiFe]-hydrogenase from D. mccartyi CBDB1 was also synthesized in an active form in E. coli. Together, these findings indicated that HypC from D. mccartyi CBDB1 exhibits promiscuity in its large subunit interaction in E. coli. Based on these findings, we generated amino acid variants of E. coli HybG capable of partial recovery of Hyd-3-dependent H2 production in a hypC hybG double null mutant. Together, these findings identify amino acid regions in HypC accessory proteins that specify interaction with the large subunits of hydrogenase and demonstrate functional compatibility of Hyp accessory protein machineries.

Dehalococcoides mccartyi strain DCMB5 Respires a broad spectrum of chlorinated aromatic compounds.

Polyhalogenated aromatic compounds are harmful environmental contaminants and tend to persist in anoxic soils and sediments. Dehalococcoides mccartyi strain DCMB5, a strain originating from dioxin-polluted river sediment, was examined for its capacity to dehalogenate diverse chloroaromatic compounds. Strain DCMB5 used hexachlorobenzenes, pentachlorobenzenes, all three tetrachlorobenzenes, and 1,2,3-trichlorobenzene as well as 1,2,3,4-tetra- and 1,2,4-trichlorodibenzo-p-dioxin as electron acceptors for organohalide respiration. In addition, 1,2,3-trichlorodibenzo-p-dioxin and 1,3-, 1,2-, and 1,4-dichlorodibenzo-p-dioxin were dechlorinated, the latter to the nonchlorinated congener with a remarkably short lag phase of 1 to 4 days following transfer. Strain DCMB5 also dechlorinated pentachlorophenol and almost all tetra- and trichlorophenols. Tetrachloroethene was dechlorinated to trichloroethene and served as an electron acceptor for growth. To relate selected dechlorination activities to the expression of specific reductive dehalogenase genes, the proteomes of 1,2,3-trichlorobenzene-, pentachlorobenzene-, and tetrachloroethene-dechlorinating cultures were analyzed. Dcmb_86, an ortholog of the chlorobenzene reductive dehalogenase CbrA, was the most abundant reductive dehalogenase during growth with each electron acceptor, suggesting its pivotal role in organohalide respiration of strain DCMB5. Dcmb_1041 was specifically induced, however, by both chlorobenzenes, whereas 3 putative reductive dehalogenases, Dcmb_1434, Dcmb_1339, and Dcmb_1383, were detected only in tetrachloroethene-grown cells. The proteomes also harbored a type IV pilus protein and the components for its assembly, disassembly, and secretion. In addition, transmission electron microscopy of DCMB5 revealed an irregular mode of cell division as well as the presence of pili, indicating that pilus formation is a feature of D. mccartyi during organohalide respiration.

Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes.

Many reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediates cis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinating Geobacter and several Dehalococcoides strains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase from Geobacter was only detected transiently at the beginning of TCE dechlorination. The Dehalococcoides RDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. The Dehalococcoides RDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity. trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains of Dehalococcoides as a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities.