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

ABSTRACT: 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.

SUBMITTER: Kleindienst S

PROVIDER: S-EPMC6414379 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Project description:Dichloromethane (DCM, methylene chloride) is a toxic chemical with substantial ozone-depleting capacity that is released by anthropogenic activities and natural processes. Specialized anaerobic bacteria metabolize DCM; however, the genetic basis for this process has remained elusive. Comparative genomics of three known anaerobic DCM-degrading bacteria, including the bacterial isolate, Dehalobacterium formicoaceticum strain DMC (Defo), revealed a homologous gene cluster, designated the methylene chloride catabolism (mec) gene cassette, comprising eight to ten genes with predicted 79.6-99.7% amino acid identity. Functional annotation identified genes encoding a corrinoid-dependent methyltransferase system. To support these observations at the functional level, global proteome analysis of Defo cultures growing with DCM as the sole energy source was performed. Briefly, axenic cultures (n = 3 biological replicates) of Defo were grown in 100 mL of anoxic mineral basal salt medium with 0.2 mM sodium sulfide, 0.2 mM L-cysteine and 30 mM bicarbonate (pH 7.3) under a headspace of N2/CO2 (80:20, vol/vol) with 156 umol (10 uL) of DCM. Cultures were initiated with a 5% (vol/vol) inoculum, incubated at 30C in the dark without agitation, and provided one additional feeding of DCM once the initial amendment was consumed. Biomass for proteomic analyses was collected after 2 weeks of incubation when approximately 95% of the second DCM feedings were consumed. After protein extraction and digestion, global proteomics analyses of 2 ug peptide solutions were performed with an Orbitrap Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, US) equipped with a nano-electrospray source (ESI) interfaced with a Proxeon EASY-nLC 1200 system. Tandem mass spectrometry data (MS/MS) were searched against protein databases from the IMG Defo annotated genome (ID. 2757320304) to which common laboratory contaminant proteins were appended. For standard database searching, the peptide MS/MS data was searched using Proteome Discoverer v2.4. The MS/MS data were searched using the SEQUEST HT algorithm. Peptide spectrum match (PSM) confidence was evaluated with Percolator. PSM and peptides were considered identified at a q value of < 0.01. The shotgun proteomics analysis reported here revealed high expression of proteins encoded on the mec gene cluster during Defo anaerobic growth with DCM.

Project description: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.

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

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