Heterologous Expression of Active Dehalobacter Respiratory Reductive Dehalogenases in Escherichia coli.

Reductive dehalogenases (RDases) are a family of redox enzymes that are required for anaerobic organohalide respiration, a microbial process that is useful in bioremediation. Structural and mechanistic studies of these enzymes have been greatly impeded due to challenges in RDase heterologous expression, potentially because of their cobamide-dependence. There have been a few successful attempts at RDase production in unconventional heterologous hosts, but a robust method has yet to be developed. Here we outline a novel respiratory RDase expression system using Escherichia coli. The overexpression of E. coli's cobamide transport system, btu, and anaerobic expression conditions were found to be essential for production of active RDases from Dehalobacter-an obligate organohalide respiring bacterium. The expression system was validated on six enzymes with amino acid sequence identities as low as 28%. Dehalogenation activity was verified for each RDase by assaying cell extracts of small-scale expression cultures on various chlorinated substrates including chloroalkanes, chloroethenes, and hexachlorocyclohexanes. Two RDases, TmrA from Dehalobacter sp. UNSWDHB and HchA from Dehalobacter sp. HCH1, were purified by nickel affinity chromatography. Incorporation of the cobamide and iron-sulfur cluster cofactors was verified; however, the precise cobalamin incorporation could not be determined due to variance between methodologies, and the specific activity of TmrA was consistent with that of the native enzyme. The heterologous expression of respiratory RDases, particularly from obligate organohalide respiring bacteria, has been extremely challenging and unreliable. Here we present a relatively straightforward E. coli expression system that has performed well for a variety of Dehalobacter spp. RDases. IMPORTANCE Understanding microbial reductive dehalogenation is important to refine the global halogen cycle and to improve bioremediation of halogenated contaminants; however, studies of the family of enzymes responsible are limited. Characterization of reductive dehalogenase enzymes has largely eluded researchers due to the lack of a reliable and high-yielding production method. We are presenting an approach to express reductive dehalogenase enzymes from Dehalobacter, a key group of organisms used in bioremediation, in Escherichia coli. This expression system will propel the study of reductive dehalogenases by facilitating their production and isolation, allowing researchers to pursue more in-depth questions about the activity and structure of these enzymes. This platform will also provide a starting point to improve the expression of reductive dehalogenases from many other organisms.

Dehalococcoides-Containing Enrichment Cultures Transform Two Chlorinated Organophosphate Esters.

Although chlorinated organophosphate esters (Cl-OPEs) have been reported to be ubiquitously distributed in various anoxic environments, little information is available on their fate under anoxic conditions. In this study, we report two Dehalococcoides-containing enrichment cultures that transformed 3.88 ± 0.22 µmol tris(2-chloroethyl) phosphate (TCEP) and 2.61 ± 0.02 µmol tris(1-chloro-2-propyl) phosphate (TCPP) within 10 days. Based on the identification of the transformed products and deuteration experiments, we inferred that TCEP may be transformed to generate bis(2-chloroethyl) phosphate and ethene via one-electron transfer (radical mechanism), followed by C-O bond cleavage. Ethene was subsequently reduced to ethane. Similarly, TCPP was transformed to form bis(1-chloro-2-propyl) phosphate and propene. 16S rRNA gene amplicon sequencing and quantitative polymerase chain reaction analysis revealed that Dehalococcoides was the predominant contributor to the transformation of TCEP and TCPP. Two draft genomes of Dehalococcoides assembled from the metagenomes of the TCEP- and TCPP-transforming enrichment cultures contained 14 and 15 putative reductive dehalogenase (rdh) genes, respectively. Most of these rdh genes were actively transcribed, suggesting that they might contribute to the transformation of TCEP and TCPP. Taken together, this study provides insights into the role of Dehalococcoides during the transformation of representative Cl-OPEs.

Proteogenomics Reveals Novel Reductive Dehalogenases and Methyltransferases Expressed during Anaerobic Dichloromethane Metabolism.

Dichloromethane (DCM) is susceptible to microbial degradation under anoxic conditions and is metabolized via the Wood-Ljungdahl pathway; however, mechanistic understanding of carbon-chlorine bond cleavage is lacking. The microbial consortium RM contains the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, which strictly requires DCM as a growth substrate. Proteomic workflows applied to DCM-grown consortium RM biomass revealed a total of 1,705 nonredundant proteins, 521 of which could be assigned to strain RM. In the presence of DCM, strain RM expressed a complete set of Wood-Ljungdahl pathway enzymes, as well as proteins implicated in chemotaxis, motility, sporulation, and vitamin/cofactor synthesis. Four corrinoid-dependent methyltransferases were among the most abundant proteins. Notably, two of three putative reductive dehalogenases (RDases) encoded within strain RM's genome were also detected in high abundance. Expressed RDase 1 and RDase 2 shared 30% amino acid identity, and RDase 1 was most similar to an RDase of Dehalococcoides mccartyi strain WBC-2 (AOV99960, 52% amino acid identity), while RDase 2 was most similar to an RDase of Dehalobacter sp. strain UNSWDHB (EQB22800, 72% amino acid identity). Although the involvement of RDases in anaerobic DCM metabolism has yet to be experimentally verified, the proteome characterization results implicated the possible participation of one or more reductive dechlorination steps and methyl group transfer reactions, leading to a revised proposal for an anaerobic DCM degradation pathway.IMPORTANCE Naturally produced and anthropogenically released DCM can reside in anoxic environments, yet little is known about the diversity of organisms, enzymes, and mechanisms involved in carbon-chlorine bond cleavage in the absence of oxygen. A proteogenomic approach identified two RDases and four corrinoid-dependent methyltransferases expressed by the DCM degrader "Candidatus Dichloromethanomonas elyunquensis" strain RM, suggesting that reductive dechlorination and methyl group transfer play roles in anaerobic DCM degradation. These findings suggest that the characterized DCM-degrading bacterium Dehalobacterium formicoaceticum and "Candidatus Dichloromethanomonas elyunquensis" strain RM utilize distinct strategies for carbon-chlorine bond cleavage, indicating that multiple pathways evolved for anaerobic DCM metabolism. The specific proteins (e.g., RDases and methyltransferases) identified in strain RM may have value as biomarkers for monitoring anaerobic DCM degradation in natural and contaminated environments.

Uptake of Organohalide Pollutants, and Release of Partially Dehalogenated Products, by NpRdhA, a 'Base-Off' Cob(II)alamin-Dependent Reductive Dehalogenase from a Deep Sea Bacterium. A Molecular Dynamics Investigation.

This work shows that, during MD aided by external tiny random forces, 3-bromo-4-hydroxybenzoic acid (LHB), the product of reductive dehalogenation of 3,5-dibromo-4-hydroxybenzoic acid (LBB) by the corrin-based marine enzyme NpRdhA, is expelled along mainly the wide channel that connects the corrin to the external medium. In accordance, unbiased MD showed that LBB migrates relatively rapidly from the external medium to the inside of the channel, finally getting to the corrin active center of NpRdhA. The LBB pose, with bromide head and carboxylate tail nearly equidistant from the corrin Co ion, does not fit the results of previous automatic docking. Either the experimental structure of the NpRdhA-LBB complex, or a quantum-mechanical study of LBB at the corrin active site, are therefore urged.

Short-chain fatty acids facilitated long-term dechlorination of PCBs in Taihu Lake sediment microcosms: Evidence from PCB congener and microbial community analyses.

Microbial reductive dechlorination hosts great promise as an in situ bioremediation strategy for polychlorinated biphenyls (PCBs) contamination. However, the slow dechlorination in sediments limits natural attenuation. Short-chain fatty acids, as preferred carbon sources and electron donors for dechlorinating microorganisms, might stimulate PCB dechlorination. Herein, two sets of short-chain fatty acids, sole acetate and a fatty acid mixture (acetate, propionate, and butyrate), were amended periodically into Taihu Lake (China) sediment microcosms containing nine PCB congeners (PCB5, 12, 64, 71, 105, 114, 149, 153, and 170) after 24 weeks of incubation. Short-chain fatty acids facilitated the long-term PCB dechlorination and the promoting effect of the fatty acid mixture compared favorably with that of sole acetate. By the end of 108 weeks, the total PCB mass concentrations in acetate amended and fatty acid mixture amended microcosms significantly declined by 7.6% and 10.3% compared with non-amended microcosms (P < 0.05), respectively. Short-chain fatty acids selectively favored the removal of flanked meta and single-flanked para chlorines. Notably, a rare ortho dechlorination pathway, PCB25 (24-3-CB) to PCB13 (3-4-CB), was enhanced. Supplementary fatty acids significantly increased reductive dehalogenases (RDase) gene pcbA5 instead of improving the growth of Dehalococcoides. These findings highlight the merits of low cost short-chain fatty acids on in situ biostimulation in treating PCBs contamination.

Investigation of active site amino acid influence on carbon and chlorine isotope fractionation during reductive dechlorination.

Reductive dehalogenases (RDases) are corrinoid-dependent enzymes that reductively dehalogenate organohalides in respiratory processes. By comparing isotope effects in biotically catalyzed reactions to reference experiments with abiotic corrinoid catalysts, compound-specific isotope analysis (CSIA) has been shown to yield valuable insights into enzyme mechanisms and kinetics, including RDases. Here, we report isotopic fractionation (ε) during biotransformation of chloroform (CF) for carbon (εC = -1.52 ± 0.34‰) and chlorine (εCl = -1.84 ± 0.19‰), corresponding to a ΛC/Cl value of 1.13 ± 0.35. These results are highly suppressed compared to isotope effects observed both during CF biotransformation by another organism with a highly similar RDase (>95% sequence identity) at the amino acid level, and to those observed during abiotic dehalogenation of CF. Amino acid differences occur at four locations within the two different RDases' active sites, and this study examines whether these differences potentially affect the observed εC, εCl, and ΛC/Cl. Structural protein models approximating the locations of the residues elucidate possible controls on reaction mechanisms and/or substrate binding efficiency. These four locations are not conserved among other chloroalkane reducing RDases with high amino acid similarity (>90%), suggesting that these locations may be important in determining isotope fractionation within this homologous group of RDases.

Electronic structure studies of free and enzyme-bound B12 species by magnetic circular dichroism and complementary spectroscopic techniques.

Electronic absorption (Abs) and circular dichroism (CD) spectroscopic techniques have been used successfully for over half a century in studies of free and enzyme-bound B12 species. More recently, magnetic circular dichroism (MCD) spectroscopy and other complementary techniques have provided an increasingly detailed understanding of the electronic structure of cobalamins. While CD spectroscopy measures the difference in the absorption of left- and right-circularly polarized light, MCD spectroscopy adds the application of a magnetic field parallel to the direction of light propagation. Transitions that are formally forbidden according to the Abs and CD selection rules, such as ligand field (or d→d) transitions, can gain MCD intensity through spin-orbit coupling. As such, MCD spectroscopy provides a uniquely sensitive probe of the different binding modes, Co oxidation states, and axial ligand environments of B12 species in enzyme active sites, and thus the distinct reactivities displayed by these species. This chapter summarizes representative MCD studies of free and enzyme-bound B12 species, including those present in adenosyltransferases, isomerases, and reductive dehalogenases. Complementary spectroscopic and computational data are also presented and discussed where appropriate.

Flavin-dependent dehalogenases.

Flavin-dependent dehalogenases use flavin as a cofactor to catalyze carbon-halogen (C-X) bond cleavage from halogenated compounds which are mainly distributed as persistent environmental pollutants via anthropogenic activities. The accumulation of these compounds results in adaptation of bacteria to evolve metabolic pathways to metabolize the agents for four decades. Flavin-dependent enzymes have been evolved to catalyze dehalogenation in addition to its basal function. Apart from bacterial biodegradation, flavin-dependent dehalogenases also naturally appear in cellular metabolisms of higher organisms such as in human thyroid hormone. Although the removal of halogen is required in various applications, the usage of dehalogenases remains limited. In-depth understanding of their enzymatic mechanisms is useful for development of dehalogenases applications. Three main types of flavin-dependent dehalogenases are classified based on their reaction mechanisms reported to date: (1) flavin-dependent O2-utilizing dehalogenases; (2) flavin-dependent reductive dehalogenases; and (3) non-redox flavin-dependent dehalogenases. In this chapter, the catalytic properties, substrate scope, protein structures, enzymatic mechanisms, enzyme engineering, and also development of enzymes for novel applications are discussed.

Two distinct Dehalobacter strains sequentially dechlorinate 1,1,1-trichloroethane and 1,1-dichloroethane at a field site treated with granular zero valent iron and guar gum.

Chlorinated ethanes are environmental pollutants found frequently at many contaminated industrial sites. 1,1,1-Trichloroethane (1,1,1-TCA) can be dechlorinated and detoxified via abiotic transformation or biologically by the action of dechlorinating microorganisms such as Dehalobacter (Dhb). At a field site, it is challenging to distinguish abiotic vs. biotic mechanisms as both processes share common transformation products. In this study, we evaluated using the Dhb 16S rRNA gene and specific reductive dehalogenase genes as biomarkers for 1,1,1-TCA and 1,1-dichloroethane (1,1-DCA) dechlorination. We analyzed samples from laboratory groundwater microcosms and from an industrial site where a mixture of granular zero valent iron (ZVI) and guar gum was injected for 1,1,1-TCA remediation. Abiotic and biotic transformation products were monitored and the changes in dechlorinating organisms were tracked using quantitative PCR (qPCR) with primers targeting the Dhb 16S rRNA gene and two functional genes cfrA and dcrA encoding enzymes that dechlorinate 1,1,1-TCA to 1,1-DCA and 1,1-DCA to chloroethane (CA), respectively. The abundance of the cfrA- and dcrA-like genes confirmed that the two dechlorination steps were carried out by two distinct Dhb populations at the site. The biomarkers used in this study proved useful for monitoring different Dhb populations responsible for step-wise dechlorination and tracking biodegradation of 1,1,1-TCA and 1,1-DCA where both abiotic (e.g., with ZVI) and biotic processes co-occur.

Deiodination in the presence of Dehalococcoides mccartyi strain CBDB1: comparison of the native enzyme and co-factor vitamin B12.

Triiodinated benzoic acid derivatives are widely used as contrast media for medical examinations and are found at high concentrations in urban aquatic environments. During bank filtration, deiodination of iodinated contrast media has been observed under anoxic/anaerobic conditions. While several bacterial strains capable of dechlorination and debromination have been isolated and characterized, deiodination has not yet been shown for an isolated strain. Here, we investigate dehalogenation of iodinated contrast media (ICM), triiodobenzoic acids (TIBA), and analogous chlorinated compounds by Dehalococcoides mccartyi strain CBDB1 and its corrinoid co-factor vitamin B12. No cell growth of CBDB1 was observed using iodinated compounds as electron acceptor. Only negligible deiodination occurred for ICM, whereas 2,3,5-TIBA was nearly completely deiodinated by CBDB1 without showing cell growth. Furthermore, TIBA inhibited growth with hexachlorobenzene which is usually a well-suited electron acceptor for strain CBDB1, indicating that TIBA is toxic for CBDB1. The involvement of CBDB1 enzymes in the deiodination of TIBA was verified by the absence of deiodination activity after heat inactivation. Adding iodopropane also inhibited the deiodination of TIBA by CBDB1 cells, indicating the involvement of a corrinoid-enzyme in the reductive TIBA deiodination. The results further suggest that the involved electron transport is decoupled from proton translocation and therefore growth. Graphical abstract.

Inferring community dynamics of organohalide-respiring bacteria in chemostats by covariance of rdhA gene abundance.

We have developed a novel approach to identifying and quantifying closely related organohalide-respiring bacteria. Our approach made use of the unique genomic associations of specific reductive dehalogenase subunit A encoding genes (rdhA) that exist in known strains of Dehalococcoides mccartyi and Desulfitobacterium and the distinguishing covariance pattern of observed rdhA genes to assign genes to unknown strains. To test this approach, we operated five anaerobic reductively dechlorinating chemostats for 3-4 years with tetrachloroethene and trichloroethene as terminal electron acceptors and lactate/formate as electron donors. The presence and abundance of rdhA genes were determined comprehensively at the community level using a custom-developed Reductive Dehalogenase Chip (RDH Chip) DNA microarray and used to define putative strains of Dehalococcoides mccartyi and Desulfitobacterium sp. This monitoring revealed that stable chemical performance of chemostats was reflected by a stable community of reductively dechlorinating bacteria. However, perturbations introduced by, for example, electron donor limitation or addition of the competing electron acceptor sulfate led to overall changes in the chemostat performance, including incomplete reduction in the chloroethene substrates, and in the population composition of reductively dehalogenating bacteria. Interestingly, there was a high diversity of operationally defined D. mccartyi strains between the chemostats with almost all strains unique to their specific chemostats in spite of similar selective pressure and similar inocula shared between chemostats.

Complete genome sequence of Dehalobacter restrictus PER-K23(T.).

Dehalobacter restrictus strain PER-K23 (DSM 9455) is the type strain of the species Dehalobacter restrictus. D. restrictus strain PER-K23 grows by organohalide respiration, coupling the oxidation of H2 to the reductive dechlorination of tetra- or trichloroethene. Growth has not been observed with any other electron donor or acceptor, nor has fermentative growth been shown. Here we introduce the first full genome of a pure culture within the genus Dehalobacter. The 2,943,336 bp long genome contains 2,826 protein coding and 82 RNA genes, including 5 16S rRNA genes. Interestingly, the genome contains 25 predicted reductive dehalogenase genes, the majority of which appear to be full length. The reductive dehalogenase genes are mainly located in two clusters, suggesting a much larger potential for organohalide respiration than previously anticipated.