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Anaerobic microbial metabolism of dichloroacetate

ABSTRACT: Anaerobic degradation of dichloroacetate (DCA) in the environment is not well understood. Recently, a microbial mixed culture (RM) was derived from pristine freshwater sediment enriched with dichloromethane (CH2Cl2, DCM) as the sole energy source under anoxic conditions. RM comprises of Candidatus Dichloromethanomonas elyunquensis strain RM which cannot only utilize DCM as an energy source, but also DCA, with the transient formation of formate, H2, and carbon monoxide (CO). To understand the metabolic capabilities of Ca. Dichloromethanomonas elyunquensi, a comparative global proteomic analysis between DCA-grown (MS datasets 1,2,3 - T8,T15) and DCM-grown (MS datasets 4,5,6 - T12,T15) cells was performed. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl-coenzyme A (CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway in DCA fermentation. Combined with physiological, biochemical, and genomics information, these results demonstrate the involvement of a haloacid dehalogenase and the Wood-Ljungdahl pathway in the anaerobic metabolism of DCA, which has broader implications in our understanding of DCA turnover in natural and engineered environments.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Candidatus Dichloromethanomonas Elyunquensis Strain Rm

SUBMITTER: Frank E. Loffler

PROVIDER: MSV000086520 | MassIVE | Wed Nov 25 11:31:00 GMT 2020

SECONDARY ACCESSION(S): PXD022742

REPOSITORIES: MassIVE

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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.

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Anaerobic microbial metabolism of dichloroacetate

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