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

Wasmund, Kenneth; Trueba-Santiso, Alba; Vicent, Teresa; Adrian, Lorenz; Vuilleumier, Stéphane; Marco‐Urrea, Ernest

doi:10.1007/s11356-023-28144-1

Cited by 3 publications

(2 citation statements)

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“…Additionally, MecA was not detected in D. formicoaceticum strain EZ94, and MecJ has never been detected in studies of either D. formicoaceticum strain or Ca . Dichloromethanomonas elyunquensis (23, 28). Overall, high expression of the key methyltransferases and corrinoid proteins show the possibility of DCM assimilation to methylenetetrahydrofolate in SC05, after which methylenetetrahydrofolate could then enter the WLP for further metabolism (Figure 3).…”

Section: Resultsmentioning

confidence: 99%

“…The DCM fermentation pathway-first discovered in Dehalobacterium formicoaceticum strain DMC-uses the WLP to release chloride ions from DCM, ultimately producing formate and acetate (24,25). Candidatus Formimonas warabiya and D. formicoaceticum strain EZ94 are also predicted to use this pathway (26)(27)(28). The second pathway, DCM mineralization, also employs the WLP to metabolize DCM, but instead reduces carbon dioxide and hydrogen as end products (29).…”

Section: Introductionmentioning

confidence: 99%

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Frommeccassette tordhA: a keyDehalobactergenomic neighborhood in a chloroform and dichloromethane–transforming microbial consortium

Bulka,

Picott,

Mahadevan

et al. 2023

Preprint

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Chloroform (CF) and dichloromethane (DCM) are groundwater contaminants of concern due to their high toxicity and inhibition of important biogeochemical processes. Biotransformation of CF and DCM have been well documented but always independent of one another. CF is dechlorinated to DCM by organohalide-respiring bacteria using reductive dehalogenases (RDases), while known DCM-degraders either ferment or mineralize DCM, both of which use themeccassette to facilitate the entry of DCM to the Wood-Ljungdahl pathway. The SC05 culture, used commercially for bioaugmentation, is the first and only known stable enrichment culture to transform CF and DCM simultaneously. Here we use metagenomic and metaproteomic analysis to identify the functional genes involved in each of these transformations. A singleDehalobactermetagenome assembled genome contains the genes for an RDase, namedacdA, and a completemeccassette encoded on a single contig. AcdA and several of the Mec proteins were also highly expressed. Furthermore, AcdA displayed high dechlorination activity on CF and other chloroalkanes but did not show significant dechlorination of DCM. Overall, the high expression of Mec proteins and the activity of AcdA suggest aDehalobactercapable of dechlorination of CF to DCM, and subsequent mineralization of DCM using themeccassette.ImportanceChloroform (CF) and dichloromethane (DCM) are harmful groundwater contaminants. SC05 is the first anaerobic microbial culture that can effectively and simultaneously remove both CF and DCM. Here we identify the key CF and DCM-degrading pathways in the culture, which are both found in aDehalobacterstrain. ThisDehalobactercontains and expresses all the genes necessary for both CF dechlorination and DCM mineralization, which has never been observed. Identifying these functional genes will improve our knowledge of how CF and DCM bioremediation progress and provide means to monitor the process when SC05 is deployed in the field.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Frommeccassette tordhA: a keyDehalobactergenomic neighborhood in a chloroform and dichloromethane–transforming microbial consortium

Bulka,

Picott,

Mahadevan

et al. 2023

Preprint

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From mec cassette to rdhA : a key Dehalobacter genomic neighborhood in a chloroform and dichloromethane–transforming microbial consortium

Bulka,

Picott,

Mahadevan

et al. 2024

Appl Environ Microbiol

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Chloroform (CF) and dichloromethane (DCM) are groundwater contaminants of concern due to their high toxicity and inhibition of important biogeochemical processes such as methanogenesis. Anaerobic biotransformation of CF and DCM has been well documented but typically independently of one another. CF is the electron acceptor for certain organohalide-respiring bacteria that use reductive dehalogenases (RDases) to dechlorinate CF to DCM. In contrast, known DCM degraders use DCM as their electron donor, which is oxidized using a series of methyltransferases and associated proteins encoded by the mec cassette to facilitate the entry of DCM to the Wood–Ljungdahl pathway. The SC05 culture is an enrichment culture sold commercially for bioaugmentation, which transforms CF via DCM to CO 2 . This culture has the unique ability to dechlorinate CF to DCM using electron equivalents provided by the oxidation of DCM to CO 2 . Here, we use metagenomic and metaproteomic analyses to identify the functional genes involved in each of these transformations. Though 91 metagenome-assembled genomes were assembled, the genes for an RDase—named acdA —and a complete mec cassette were found to be encoded on a single contig belonging to Dehalobacter . AcdA and critical Mec proteins were also highly expressed by the culture. Heterologously expressed AcdA dechlorinated CF and other chloroalkanes but had 100-fold lower activity on DCM. Overall, the high expression of Mec proteins and the activity of AcdA suggest a Dehalobacter capable of dechlorination of CF to DCM and subsequent mineralization of DCM using the mec cassette. IMPORTANCE Chloroform (CF) and dichloromethane (DCM) are regulated groundwater contaminants. A cost-effective approach to remove these pollutants from contaminated groundwater is to employ microbes that transform CF and DCM as part of their metabolism, thus depleting the contamination as the microbes continue to grow. In this work, we investigate bioaugmentation culture SC05, a mixed microbial consortium that effectively and simultaneously degrades both CF and DCM coupled to the growth of Dehalobacter . We identified the functional genes responsible for the transformation of CF and DCM in SC05. These genetic biomarkers provide a means to monitor the remediation process in the field.

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MecE, MecB, and MecC proteins orchestrate methyl group transfer during dichloromethane fermentation

Soder-Walz,

Deobald,

Vicent

et al. 2024

Appl Environ Microbiol

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Dichloromethane (DCM), a common hazardous industrial chemical, is anaerobically metabolized by four bacterial genera: Dehalobacter , Dehalobacterium , Ca . Dichloromethanomonas, and Ca . Formimonas. However, the pivotal methyltransferases responsible for DCM transformation have remained elusive. In this study, we investigated the DCM catabolism of Dehalobacterium formicoaceticum strain EZ94, contained in an enriched culture, using a combination of biochemical approaches. Initially, enzymatic assays were conducted with cell-free protein extracts, after protein separation by blue native polyacrylamide gel electrophoresis. In the slices with the highest DCM transformation activity, a high absolute abundance of the methyltransferase MecC was revealed by mass spectrometry. Enzymatic activity assays with heterologously expressed MecB, MecC, and MecE from strain EZ94 showed complete DCM transformation only when all three enzymes were present. Our experimental results, coupled with the computational analysis of MecB, MecC, and MecE sequences, enabled us to assign specific roles in DCM transformation to each of the proteins. Our findings reveal that both MecE and MecC are zinc-dependent methyltransferases responsible for DCM demethylation and re-methylation of a product, respectively. MecB functions as a cobalamin-dependent shuttle protein transferring the methyl group between MecE and MecC. This study provides the first biochemical evidence of the enzymes involved in the anaerobic metabolism of DCM. IMPORTANCE Dichloromethane (DCM) is a priority regulated pollutant frequently detected in groundwater. In this work, we identify the proteins responsible for the transformation of DCM fermentation in Dehalobacterium formicoaceticum strain EZ94 using a combination of biochemical approaches, heterologous expression of proteins, and computational analysis. These findings provide the basis to apply these proteins as biological markers to monitor bioremediation processes in the field.

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

Cited by 3 publications

References 43 publications

Frommeccassette tordhA: a keyDehalobactergenomic neighborhood in a chloroform and dichloromethane–transforming microbial consortium

Frommeccassette tordhA: a keyDehalobactergenomic neighborhood in a chloroform and dichloromethane–transforming microbial consortium

From mec cassette to rdhA : a key Dehalobacter genomic neighborhood in a chloroform and dichloromethane–transforming microbial consortium

MecE, MecB, and MecC proteins orchestrate methyl group transfer during dichloromethane fermentation

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