Proteogenomics of the novel Dehalobacterium formicoaceticum strain EZ94 highlights a key role of methyltransferases during anaerobic dichloromethane degradation

Abstract

Dichloromethane (DCM, methylene chloride) is a toxic, high-volume industrial pollutant of long-standing. Anaerobic biodegradation is crucial for its removal from contaminated environments, yet prevailing mechanisms remain unresolved, especially concerning dehalogenation. In this study, we obtained an assembled genome of a novel DCM-degrading strain, Dehalobacterium formicoaceticum strain EZ94, from a stable DCM-degrading consortium, and we analyzed its proteome during degradation of DCM. A gene cluster recently predicted to play a major role in anaerobic DCM catabolism (the mec cassette) was found. Methyltransferases and other proteins encoded by the mec cassette were among the most abundant proteins produced, suggesting their involvement in DCM catabolism. Reductive dehalogenases were not detected. Genes and corresponding proteins for a complete Wood-Ljungdahl pathway, which could enable further metabolism of DCM carbon, were also found. Unlike for the anaerobic DCM degrader "Ca. F. warabiya," no genes for metabolism of the quaternary amines choline and glycine betaine were identified. This work provides independent and supporting evidence that mec-associated methyltransferases are key to anaerobic DCM metabolism.

Keywords: Anaerobic dichloromethane degradation; Dehalobacterium; Methyltransferases; Shotgun proteomics; Wood-Ljungdahl pathway.

Fig. 1

Phylogenomic tree based on proteins from 34 single copy marker gene genes derived from CheckM analysis (see the “Materials and methods” section). Circles on nodes indicate bootstrap values >90%. The scale bar represents 10% sequence divergence

Fig. 2

Gene synteny for the mec gene cluster among DCM-degrading bacteria. Blue shading represents tBLASTx identity values between corresponding proteins

Fig. 3

Proposed pathway for metabolism of DCM by D. formicoaceticum strain EZ94. Gene locus identifier numbers for corresponding enzymes are underlined, with mean protein abundance rank (“#” in orange boxes; “n.d.,” not detected, see Table S4). folD, methylene-H4F dehydrogenase/cyclohydrolase; fhs, formate-H4F ligase; metF, methylene-H4F reductase; acsE, methyltransferase; CoFeSP, methyl-Co(I) corrinoid iron sulfur protein; cooS, CO dehydrogenase; acsB, acetyl-CoA synthase; pta, phosphate acetyltransferase; ackA, acetate kinase