All methane-producing Archaea (methanogens) are strict anaerobes, but the majority of species are tolerant to oxidants. Methanosarcina species are imp

The thioredoxin system, composed of thioredoxin reductase (TrxR) and thioredoxin (Trx), is widely distributed in nature, where it serves key roles in electron transfer and in the defense against oxidative stress. Although recent evidence reveals Trx homologues are almost universally present among the methane‐producing archaea (methanogens), a complete thioredoxin system has not been characterized from any methanogen. We examined the phylogeny of Trx homologues among methanogens and characterized the thioredoxin system from Methanosarcina acetivorans. Phylogenetic analysis of Trx homologues from methanogens revealed eight clades, with one clade containing Trxs broadly distributed among methanogens. The Methanococci and Methanobacteria each contain one additional Trx from another clade, respectively, whereas the Methanomicrobia contain an additional five distinct Trxs. Methanosarcina acetivorans, a member of the Methanomicrobia, contains a single TrxR (MaTrxR) and seven Trx homologues (MaTrx1–7), with representatives from five of the methanogen Trx clades. Purified recombinant MaTrxR had 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) reductase and oxidase activities. The apparent K_m value for NADPH was 115‐fold lower than that for NADH, consistent with NADPH as the physiological electron donor to MaTrxR. Purified recombinant MaTrx2, MaTrx6 and MaTrx7 exhibited dithiothreitol‐ and lipoamide‐dependent insulin disulfide reductase activities. However, only MaTrx7, which is encoded adjacent to MaTrxR, could serve as a redox partner to MaTrxR. These results reveal that M. acetivorans harbors at least three functional and distinct Trxs, and a complete thioredoxin system composed of NADPH, MaTrxR and at least MaTrx7. This is the first characterization of a complete thioredoxin system from a methanogen, which provides a foundation to understand the system in methanogens.

A plasmid-based expression system wherein mekB was fused to a constitutive Methanosarcina acetivorans promoter was used to express MekB, a broad-specificity esterase from Pseudomonas veronii , in M. acetivorans . The engineered strain had 80-fold greater esterase activity than wild-type M. acetivorans . Methyl acetate and methyl propionate esters served as the sole carbon and energy sources, resulting in robust growth and methane formation, with consumption of >97% of the substrates. Methanol was undetectable at the end of growth with methyl acetate, whereas acetate accumulated, a result consistent with methanol as the more favorable substrate. Acetate was consumed, and growth continued after a period of adaptation. Similar results were obtained with methyl propionate, except propionate was not metabolized. IMPORTANCE The fragile interactions of multispecies food chains converting complex biomass to methane are easily disrupted, a major impediment to efficient and reliable conversion of renewable biomass as an alternative to fossil fuels. The hybrid pathway, derived by combining catabolic pathways from a methanogen of the domain Archaea and a strictly aerobic species of the domain Bacteria , catalyzes the complete conversion of an industrial solvent that is also a naturally occurring compound to methane and carbon dioxide. The engineered pathway expands the exceptionally narrow range of substrates utilized by methanogens, exemplifying the simplification of food chains leading to the more-efficient conversion of complex biomass to methane.

CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N₂) fixation were targeted for dCas9-mediated repression. First, the nif operon (nifHI₁I₂DKEN) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNA), one targeting the promoter and the other nifD. Remarkably, growth of M. acetivorans with N₂ was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N₂. Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1 encoding a subunit of the repressor of the nif operon was targeted. The nrpR1 repression strain grew normally with N₂ but had increased nif operon transcript abundance consistent with a NrpR1 as repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivorans. IMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas and a biofuel. The CRISPRi-dCas9 system in the model methanogen M. acetivorans is the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens.

All nitrogen-fixing bacteria and archaea (diazotrophs) use molybdenum (Mo) nitrogenase to reduce dinitrogen (N ₂ ) to ammonia, with some also containing vanadium (V) and iron-only (Fe) nitrogenases that lack Mo. Among diazotrophs, the regulation and usage of the alternative V-nitrogenase and Fe-nitrogenase in methanogens are largely unknown. Methanosarcina acetivorans contains nif , vnf , and anf gene clusters encoding putative Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase, respectively. This study investigated nitrogenase expression and growth by M. acetivorans in response to fixed nitrogen, Mo/V availability, and CRISPRi repression of the nif , vnf , and/or anf gene clusters. The availability of Mo and V significantly affected growth of M. acetivorans with N ₂ but not with NH ₄ Cl. M. acetivorans exhibited the fastest growth rate and highest cell yield during growth with N ₂ in medium containing Mo, and the slowest growth in medium lacking Mo and V. qPCR analysis revealed the transcription of the nif operon is only moderately affected by depletion of fixed nitrogen and Mo, whereas vnf and anf transcription increased significantly when fixed nitrogen and Mo were depleted, with removal of Mo being key. Immunoblot analysis revealed Mo-nitrogenase is detected when fixed nitrogen is depleted regardless of Mo availability, while V-nitrogenase and Fe-nitrogenase are detected only in the absence of fixed nitrogen and Mo. CRISPRi repression studies revealed that V-nitrogenase and/or Fe-nitrogenase are required for Mo-independent diazotrophy, and unexpectedly that the expression of Mo-nitrogenase is also required. These results reveal that alternative nitrogenase production in M. acetivorans is tightly controlled and dependent on Mo-nitrogenase expression. IMPORTANCE: Methanogens and closely related methanotrophs are the only archaea known or predicted to possess nitrogenase. Methanogens play critical roles in both the global biological nitrogen and carbon cycles. Moreover, methanogens are an ancient microbial lineage and nitrogenase likely originated in methanogens. An understanding of the usage and properties of nitrogenases in methanogens can provide new insight into the evolution of nitrogen fixation and aid in the development nitrogenase-based biotechnology. This study provides the first evidence that a methanogen can produce all three forms of nitrogenases, including simultaneously. The results reveal components of Mo-nitrogenase regulate or are needed to produce V-nitrogenase and Fe-nitrogenase in methanogens, a result not seen in bacteria. Overall, this study provides a foundation to understand the assembly, regulation, and activity of the alternative nitrogenases in methanogens.

Haem-dependent catalase is an antioxidant enzyme that degrades H₂O₂, producing H₂O and O₂, and is common in aerobes. Catalase is present in some strictly anaerobic methane-producing archaea (methanogens), but the importance of catalase to the antioxidant system of methanogens is poorly understood. We report here that a survey of the sequenced genomes of methanogens revealed that the majority of species lack genes encoding catalase. Moreover, Methanosarcina acetivorans is a methanogen capable of synthesizing haem and encodes haem-dependent catalase in its genome; yet, Methanosarcina acetivorans cells lack detectable catalase activity. However, inducible expression of the haem-dependent catalase from Escherichia coli (EcKatG) in the chromosome of Methanosarcina acetivorans resulted in a 100-fold increase in the endogenous catalase activity compared with uninduced cells. The increased catalase activity conferred a 10-fold increase in the resistance of EcKatG-induced cells to H₂O₂ compared with uninduced cells. The EcKatG-induced cells were also able to grow when exposed to levels of H₂O₂ that inhibited or killed uninduced cells. However, despite the significant increase in catalase activity, growth studies revealed that EcKatG-induced cells did not exhibit increased tolerance to O₂ compared with uninduced cells. These results support the lack of catalase in the majority of methanogens, since methanogens are more likely to encounter O₂ rather than high concentrations of H₂O₂ in the natural environment. Catalase appears to be a minor component of the antioxidant system in methanogens, even those that are aerotolerant, including Methanosarcina acetivorans. Importantly, the experimental approach used here demonstrated the feasibility of engineering beneficial traits, such as H₂O₂ tolerance, in methanogens.