Metaproteomics reveals methyltransferases implicated in dichloromethane and glycine betaine fermentation by ' Candidatus Formimonas warabiya' strain DCMF

Abstract

Dichloromethane (DCM; CH₂Cl₂) is a widespread pollutant with anthropogenic and natural sources. Anaerobic DCM-dechlorinating bacteria use the Wood-Ljungdahl pathway, yet dechlorination reaction mechanisms remain unclear and the enzyme(s) responsible for carbon-chlorine bond cleavage have not been definitively identified. Of the three bacterial taxa known to carry out anaerobic dechlorination of DCM, 'Candidatus Formimonas warabiya' strain DCMF is the only organism that can also ferment non-chlorinated substrates, including quaternary amines (i.e., choline and glycine betaine) and methanol. Strain DCMF is present within enrichment culture DFE, which was derived from an organochlorine-contaminated aquifer. We utilized the metabolic versatility of strain DCMF to carry out comparative metaproteomics of cultures grown with DCM or glycine betaine. This revealed differential abundance of numerous proteins, including a methyltransferase gene cluster (the mec cassette) that was significantly more abundant during DCM degradation, as well as highly conserved amongst anaerobic DCM-degrading bacteria. This lends strong support to its involvement in DCM dechlorination. A putative glycine betaine methyltransferase was also discovered, adding to the limited knowledge about the fate of this widespread osmolyte in anoxic subsurface environments. Furthermore, the metagenome of enrichment culture DFE was assembled, resulting in five high quality and two low quality draft metagenome-assembled genomes. Metaproteogenomic analysis did not reveal any genes or proteins for utilization of DCM or glycine betaine in the cohabiting bacteria, supporting the previously held idea that they persist via necromass utilization.

Keywords: Wood–Ljungdahl pathway; anaerobic dechlorination; dichloromethane; glycine betaine; metaproteomics; methyltransferase; subsurface.

Figure 1

Volcano plot of strain DCMF protein expression with DCM and glycine betaine. Log₂-fold change (LFC) in abundance is shown as the difference between DCM and glycine betaine label-free quantitative intensity. A false discovery rate of 0.01 was the significance boundary, indicated by two lines on the graph. Proteins in the DCM-associated mec cassette are labeled as red triangles, those from the complete glycine betaine methyltransferase gene cluster by dark blue squares, those in the incomplete glycine betaine and/or dimethylglycine methyltransferase gene cluster by purple squares, and those from the sarcosine reductase gene cluster by light blue diamonds. IMG gene loci (prefaced by ‘Ga018035_’) are indicated for these proteins of interest. A full list of all significantly differentially abundant proteins is included in Supplementary Table S5.

Figure 2

Heatmap of relative protein abundance for genes/pathways of interest in DCM and glycine betaine-amended strain DCMF cells. Proteins for the Wood–Ljungdahl pathway and a F_OF₁-type ATPase were highly abundant under both growth conditions, while proteins in the DCM-associated mec cassette were significantly more abundant in DCM-grown cells, and proteins in a putative glycine betaine methyltransferase gene cluster and a sarcosine reductase gene cluster were significantly more abundant in glycine betaine-grown cells. Rows are labeled with gene symbols and the strain DCMF IMG gene loci (each prefaced with “Ga0180325_”).

Figure 3

Genetic organization of the mec cassette in strain DCMF and other anaerobic chlorinated methane-degrading bacteria. Loci are shown below the first and last gene in each cluster. Percentage sequence identity to strain DCMF is shown for each homologous protein; percentage identity for the DUF proteins (italicized) is in relation to D. formicoaceticum. Vertical lines represent contig boundaries. Reg, regulator; CoP, corrinoid protein; MTI, methyltransferase I; MTII, methyltransferase II; Trans, transporter; RACE, reductive activator of corrinoid-dependent enzymes.

Figure 4

Proposed DCM dechlorination schema in strain DCMF. (A) Reduced Co(I) corrinoid protein (CoP), acting in concert with a methyltransferase I protein (MTI), cleaves the first chlorine from DCM, producing a transient CH2Cl-CoP intermediate. Tetrahydrofolate (THF), bound to a methyltransferase II protein (MTII), then attacks the remaining carbon-chlorine bond, ultimately producing 5,10-methylene-tetrahydrofolate. The Co (III) in the corrinoid protein is reduced back to Co (I) by putative reductive activators of corrinoid-dependent enzymes (RACE) proteins, although this may not be required every reaction cycle. (B) The overall reaction showing DCM and THF producing 5,10-methylene-tetrahydrofolate. The carbon in DCM is highlighted in dark gray; the N5 and N10 nitrogen heteroatoms in tetrahydrofolate are highlighted in light gray.

Figure 5

Genetic organization of the glycine betaine methyltransferase gene cluster in strain DCMF and homologs in other glycine betaine-fermenting bacteria. Homologous clusters fell into two distinct groups, with similarity to the second (A) or first (B) half of the strain DCMF gene cluster. Homologous proteins are linked by dotted lines and percentage amino acid sequence identity to strain DCMF written within. Values in the unlinked BCCT and CoP proteins in Panel A are to the strain DCMF homolog with the highest percentage amino acid sequence identity: Ga0180325_114741 or Ga0180325_114736, respectively, in all cases). Loci shown are: Acetobacterium woodii DSM 1030 Awo_c07520 – Awo_07560, Acetobacterium dehalogenans DSM 11527 A3KSDRAFT_02713 – A3KSDRAFT_02709, D. hafniense Y51 DSY3157 – DSY3154, strain DCMF Ga0180325_114734 – Ga0180325_114742, ‘Ca. Frackibacter sp.’ T328-2 AWU54_1980 – AWU54_1975, Sporomusa ovata DSM 2662 SOV_3c09370—SOV_3c09310, S. ovata An4 SpAn4DRAFT_2140 – SpAn4DRAFT_2133. CoP, corrinoid protein; MTI, methyltransferase I; HydA, hydantoinase; MTII, methyltransferase II; BCCT, betaine/carnitine/choline family transporter; RACE, reductive activator of corrinoid-dependent enzymes; ProX, extracellular glycine betaine ligand binding protein. Asterisks indicate proteins with <30% query coverage of the strain DCMF homolog.