Anaerobic Microbial Metabolism of Dichloroacetate

Abstract

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising "Candidatus Dichloromethanomonas elyunquensis" strain RM utilizes DCA as an energy source, and the transient formation of formate, H₂, and carbon monoxide (CO) was observed during growth. Only about half of the DCA was recovered as acetate, suggesting a fermentative catabolic route rather than a reductive dechlorination pathway. Sequencing of 16S rRNA gene amplicons and 16S rRNA gene-targeted quantitative real-time PCR (qPCR) implicated "Candidatus Dichloromethanomonas elyunquensis" strain RM in DCA degradation. An (S)-2-haloacid dehalogenase (HAD) encoded on the genome of strain RM was heterologously expressed, and the purified HAD demonstrated the cofactor-independent stoichiometric conversion of DCA to glyoxylate at a rate of 90 ± 4.6 nkat mg^-1 protein. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl coenzyme A (acetyl-CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway. Glyoxylate carboligase, which catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde, was highly abundant in DCA-grown cells. The physiological, biochemical, and proteogenomic data demonstrate the involvement of an HAD and the Wood-Ljungdahl pathway in the anaerobic fermentation of DCA, which has implications for DCA turnover in natural and engineered environments, as well as the metabolism of the cancer drug DCA by gut microbiota.IMPORTANCE Dichloroacetate (DCA) is ubiquitous in the environment due to natural formation via biological and abiotic chlorination processes and the turnover of chlorinated organic materials (e.g., humic substances). Additional sources include DCA usage as a chemical feedstock and cancer drug and its unintentional formation during drinking water disinfection by chlorination. Despite the ubiquitous presence of DCA, its fate under anoxic conditions has remained obscure. We discovered an anaerobic bacterium capable of metabolizing DCA, identified the enzyme responsible for DCA dehalogenation, and elucidated a novel DCA fermentation pathway. The findings have implications for the turnover of DCA and the carbon and electron flow in electron acceptor-depleted environments and the human gastrointestinal tract.

Keywords: anaerobic catabolic pathways; comparative proteomics; dichloroacetate; fermentation; haloacid dehalogenase.

FIG 1

DCA and DCM degradation by mixed culture RM. (A) Utilization of DCA in RM cultures that had consumed an initial feeding of 93 ± 15 μmol of DCM. DCA utilization commenced after a lag phase of about 3 weeks, and consumption was complete within 2 weeks. Replicate DCM-grown cultures rapidly consumed additional DCM feedings without a lag phase. (B) Formation of inorganic chloride, acetate, and formate during DCA catabolism by mixed culture RM. (C) Increase in 16S rRNA gene copies of “Ca. Dichloromethanomonas elyunquensis” during DCA catabolism by mixed culture RM under anoxic conditions. The data represent the average from triplicate incubations, and the error bars represent the standard deviations.

FIG 2

Transient formation of H₂ and CO during DCA catabolism by mixed culture RM. The data represent the average from triplicate incubations, and the error bars represent the standard deviations.

FIG 3

Microbial community structure responses to consecutive transfers of mixed culture RM with DCA as the sole energy source, as revealed by 16S rRNA gene amplicon sequencing. Taxa with relative abundances below 1% were categorized as “Others.” The operational taxonomic units (OTUs) representing bacteria and archaea are reported to the lowest taxonomic rank possible. “Ca. Dichloromethanomonas elyunquensis” was the dominant population in mixed culture RM, and continuous transfers with DCA resulted in further enrichment.

FIG 4

Amino acid sequence-based phylogenetic tree of select HADs. HAD1 (prokka_14346) and HAD2 (prokka_14344) of “Ca. Dichloromethanomonas elyunquensis” are shown in red font. Biochemically characterized HADs with demonstrated activity toward DCA are shown in blue font. The scale bar indicates the number of amino acid substitutions per site.

FIG 5

Enzymatic activity of the heterologously expressed HAD1 protein of “Ca. Dichloromethanomonas elyunquensis.” (A) SDS-PAGE illustrating HAD1 purification. Lane 1, protein size markers; lane 2, soluble crude extract of E. coli strain FEL153 carrying the had1 gene (prokka_14346); lane 3, purified His-tagged HAD1 protein. (B) Enzymatic activity of heterologously expressed and purified HAD1 of “Ca. Dichloromethanomonas elyunquensis” showing the stoichiometric conversion of DCA to glyoxylate. In abiotic control incubations without protein, DCA was stable. The data shown are from a single experiment, and independent experiments yielded similar results.

FIG 6

Proposed anaerobic catabolic pathway for DCA in “Ca. Dichloromethanomonas elyunquensis” strain RM. The shaded boxes indicate the log₂ fold change of normalized protein abundance values in DCA- versus DCM-grown cells at TP1 (dashed line boxes) and TP2 (solid line boxes). The protein abundance values represent average from three biological replicate cultures for each growth condition. Boxes marked with “+” signs indicate that the fold changes were statistically significant (P < 0.05) in the pairwise comparisons of DCA- versus DCM-grown cells at TP1 and/or TP2. Gene locus tags of each protein are depicted below protein names. Abbreviations: HAD1, haloacid dehalogenase 1; Gcl, glyoxylate carboligase; Hyi, hydroxypyruvate isomerase; Ghr, glyoxylate/hydroxypyruvate reductase; GlxR, 2-hydroxy-3-oxopropionate reductase; Gck, glycerate 2-kinase; Eno, enolase; Pyk, pyruvate kinase; POR, pyruvate-flavodoxin oxidoreductase; PTA, phosphate acetyltransferase; ACK, acetate kinase; ACS/CODH, acetyl coenzyme A synthase/carbon monoxide dehydrogenase; CFeSP, corrinoid iron-sulfur protein; MeTr, methyltransferase; MetF, methylene-tetrahydrofolate (H₄folate) reductase; MTHFD, methylene-H₄folate dehydrogenase; FolD, formyl-H₄folate cyclohydrolase; Fhs, formyl-H₄folate synthase; Fdh, formate dehydrogenase. WLP proteins are depicted in green font, and proteins involved in DCA reduction to acetate are shown in blue font. The fold change values are shown in Table S2.