Deconstructing Methanosarcina acetivorans into an acetogenic archaeon

The reductive acetyl-coenzyme A (acetyl-CoA) pathway, whereby carbon dioxide is sequentially reduced to acetyl-CoA via coenzyme-bound C1 intermediates, is the only autotrophic pathway that can at the same time be the means for energy conservation. A conceptually similar metabolism and a key process in the global carbon cycle is methanogenesis, the biogenic formation of methane. All known methanogenic archaea depend on methanogenesis to sustain growth and use the reductive acetyl-CoA pathway for autotrophic carbon fixation. Here, we converted a methanogen into an acetogen and show that Methanosarcina acetivorans can dispense with methanogenesis for energy conservation completely. By targeted disruption of the methanogenic pathway, followed by adaptive evolution, a strain was created that sustained growth via carbon monoxide–dependent acetogenesis. A minute flux (less than 0.2% of the carbon monoxide consumed) through the methane-liberating reaction remained essential, indicating that currently living methanogens utilize metabolites of this reaction also for anabolic purposes. These results suggest that the metabolic flexibility of methanogenic archaea might be much greater than currently known. Also, our ability to deconstruct a methanogen into an acetogen by merely removing cellular functions provides experimental support for the notion that methanogenesis could have evolved from the reductive acetyl-coenzyme A pathway.

Vht hydrogenase is required for hydrogen cycling during nitrogen fixation by the non-hydrogenotrophic methanogen Methanosarcina acetivorans.

Methanosarcina acetivorans is the primary model to understand the physiology of methanogens that do not use hydrogenase to consume or produce hydrogen (H2) during methanogenesis. The genome of M. acetivorans encodes putative methanophenazine-reducing hydrogenases (Vht and Vhx), F420-reducing hydrogenase (Frh), and hydrogenase maturation machinery (Hyp), yet cells lack significant hydrogenase activity under all growth conditions tested to date. Thus, the importance of hydrogenase to the physiology of M. acetivorans has remained a mystery. M. acetivorans can fix dinitrogen (N2) using nitrogenase that is documented in bacteria to produce H2 during the reduction of N2 to ammonia. Therefore, we hypothesized that M. acetivorans uses hydrogenase to recycle H2 produced by nitrogenase during N2 fixation. Results demonstrate that hydrogenase expression and activity is higher in N2-grown cells compared to cells grown with fixed nitrogen (NH4Cl). To test the importance of each hydrogenase and the maturation machinery, the CRISPRi-dCas9 system was used to generate separate M. acetivorans strains where transcription of the vht, frh, vhx, or hyp operons is repressed. Repression of vhx and frh does not alter growth with either NH4Cl or N2 and has no effect on H2 metabolism. However, repression of vht or hyp results in impaired growth with N2 but not NH4Cl. Importantly, H2 produced endogenously by nitrogenase is detected in the headspace of culture tubes containing the vht or hyp repression strains. Overall, the results reveal that Vht hydrogenase recycles H2 produced by nitrogenase that is required for optimal growth of M. acetivorans during N2 fixation.

Mechanisms for Electron Uptake by Methanosarcina acetivorans during Direct Interspecies Electron Transfer

The conversion of organic matter to methane plays an important role in the global carbon cycle and is an effective strategy for converting wastes to a useful biofuel. The reduction of carbon dioxide to methane accounts for approximately a third of the methane produced in anaerobic soils and sediments as well as waste digesters.

Mechanisms for Electron Uptake by Methanosarcina acetivorans During Direct Interspecies Electron Transfer

Direct interspecies electron transfer (DIET) between bacteria and methanogenic archaea appears to be an important syntrophy in both natural and engineered methanogenic environments. However, the electrical connections on the outer surface of methanogens and the subsequent processing of electrons for carbon dioxide reduction to methane are poorly understood. Here we report that the genetically tractable methanogen Methanosarcina acetivorans can grow via DIET in co-culture with Geobacter metallireducens serving as the electron-donating partner. Comparison of gene expression patterns in M. acetivorans grown in co-culture versus pure culture growth on acetate revealed that transcripts for the outer-surface, multi-heme, c-type cytochrome MmcA were higher during DIET-based growth. Deletion of mmcA inhibited DIET. The high aromatic amino acid content of M. acetivorans archaellins suggests that they might assemble into electrically conductive archaella. A mutant that could not express archaella was deficient in DIET. However, this mutant grew in DIET-based co-culture as well as the archaella-expressing parental strain in the presence of granular activated carbon, which was previously shown to serve as a substitute for electrically conductive pili as a conduit for long-range interspecies electron transfer in other DIET-based co-cultures. Transcriptomic data suggesting that the membrane-bound Rnf, Fpo, and HdrED complexes also play a role in DIET were incorporated into a charge-balanced model illustrating how electrons entering the cell through MmcA can yield energy to support growth from carbon dioxide reduction. The results are the first genetics-based functional demonstration of likely outer-surface electrical contacts for DIET in a methanogen.

Methanosarcina acetivorans simultaneously produces molybdenum, vanadium, and iron-only nitrogenases in response to fixed nitrogen and molybdenum depletion

All nitrogen-fixing bacteria and archaea (diazotrophs) use molybdenum (Mo) nitrogenase to reduce dinitrogen (N2) to ammonia. Some diazotrophs also contain alternative nitrogenases that lack Mo: vanadium (V) and iron-only (Fe) nitrogenases. Among diazotrophs, the regulation and usage of the alternative nitrogenases in methanogens is largely unknown. Methanosarcina acetivorans contains nif, vnf, and anf gene clusters encoding putative Mo-, V-, and Fe-nitrogenases, respectively. This study investigated the effect of fixed nitrogen and Mo/V availability on nitrogenase expression and growth by M. acetivorans. The availability of Mo and V did not affect growth of M. acetivorans with fixed nitrogen but significantly affected growth with N2. M. acetivorans exhibited the fastest growth rate and highest cell yield during growth with N2 in medium containing Mo. Depletion of Mo (Fe-only condition) resulted in a significant decrease in growth rate and cell yield. The addition of V to Mo-depleted medium stimulated diazotrophic growth but was still less than growth in Mo-replete medium. qPCR analysis revealed transcription of the nif operon is only moderately affected by depletion of fixed nitrogen and Mo. However, vnf and anf transcription increased significantly when fixed nitrogen and Mo were depleted, with removal of Mo being the key factor. Immunoblot analysis revealed Mo-nitrogenase is produced when fixed nitrogen is depleted regardless of Mo availability, while V- and Fe-nitrogenases are produced only in the absence of fixed nitrogen and Mo. These results reveal that alternative nitrogenase production in M. acetivorans is tightly controlled and that all three nitrogenases can be simultaneously produced.

Isoprene Production from Municipal Wastewater Biosolids by Engineered Archaeon Methanosarcina acetivorans

Wastewater biosolids are a promising feedstock for production of value-added renewable chemicals. Methane-producing archaea (methanogens) are already used to produce renewable biogas via the anaerobic treatment of wastewater. The ability of methanogens to efficiently convert dissolved organic carbon into methane makes them an appealing potential platform for biorefining using metabolic engineering. We have engineered a strain of the methanogen Methanosarcina acetivorans to produce the volatile hemiterpene isoprene in addition to methane. The engineered strain was adapted to grow in municipal wastewater through cultivation in a synthetic wastewater medium. When introduced to municipal wastewater the engineered methanogens were able to compete with the indigenous microorganisms and produce 0.97 mM of isoprene (65.9 ± 21.3 g per m3 of effluent). The production of isoprene in wastewater appears to be dependent on the quantity of available methanogenic substrate produced during upstream digestion by heterotrophic fermenters. This shows that with minimal adaptation it is possible to drop-in engineered methanogens to existing wastewater environments and attain value-added products in addition to the processing of wastewater. This shows the potential for utilizing methanogens as a platform for low-cost production of renewable materials without expensive feedstocks or the need to build or adapt existing facilities.

Putative Extracellular Electron Transfer in Methanogenic Archaea

It has been suggested that a few methanogens are capable of extracellular electron transfers. For instance, Methanosarcina barkeri can directly capture electrons from the coexisting microbial cells of other species. Methanothrix harundinacea and Methanosarcina horonobensis retrieve electrons from Geobacter metallireducens via direct interspecies electron transfer (DIET). Recently, Methanobacterium, designated strain YSL, has been found to grow via DIET in the co-culture with Geobacter metallireducens. Methanosarcina acetivorans can perform anaerobic methane oxidation and respiratory growth relying on Fe(III) reduction through the extracellular electron transfer. Methanosarcina mazei is capable of electromethanogenesis under the conditions where electron-transfer mediators like H2 or formate are limited. The membrane-bound multiheme c-type cytochromes (MHC) and electrically-conductive cellular appendages have been assumed to mediate the extracellular electron transfer in bacteria like Geobacter and Shewanella species. These molecules or structures are rare but have been recently identified in a few methanogens. Here, we review the current state of knowledge for the putative extracellular electron transfers in methanogens and highlight the opportunities and challenges for future research.

A bacterial signal transduction phosphorelay in the methanogenic archaeon Methanosarcina acetivorans

Signal transduction via two-component systems is a powerful tool for microorganisms to respond to environmental changes. Histidine kinases originating from Bacteria are the most common signaling enzymes and are also present in Archaea, but not in all phyla. A total of 124 bacterial-type histidine kinases and/or regulators were identified in a screen of 149 Euryarchaeota genomes, but little is known about the signal transfer and molecular regulation of these systems. In this work, the hybrid kinase MA4377 from the methanogenic archaeon Methanosarcina acetivorans was investigated. MA4377 is a multidomain protein resembling a bacterial-type histidine kinase with two additional receiver domains at the C-terminus. Recombinant protein was employed to investigate the intra- and intermolecular phosphorelay in vitro. The kinase displays autophosphorylation activity of histidine residue 497. While no intramolecular phosphorelay was observed, the CheY-like receiver protein MA4376 was identified as part of the multi-component system that also seems to include the Msr-type transcription factor MA4375. This study reveals the presence and in vitro function of a bacterial-type hybrid histidine kinase integrated into an archaeal phosphorelay system.

Methanosarcina acetivorans contains a functional ISC system for iron-sulfur cluster biogenesis

Background The production of methane by methanogens is dependent on numerous iron-sulfur (Fe-S) cluster proteins; yet, the machinery involved in Fe-S cluster biogenesis in methanogens remains largely unknown. Methanogen genomes encode uncharacterized homologs of the core components of the ISC (IscS and IscU) and SUF (SufBC) Fe-S cluster biogenesis systems found in bacteria and eukaryotes. Methanosarcina acetivorans contains three iscSU and two sufCB gene clusters. Here, we report genetic and biochemical characterization of M. acetivorans iscSU2. Results Purified IscS2 exhibited pyridoxal 5′- phosphate-dependent release of sulfur from L-cysteine. Incubation of purified IscU2 with IscS2, cysteine, and iron (Fe2+) resulted in the formation of [4Fe-4S] clusters in IscU2. IscU2 transferred a [4Fe-4S] cluster to purified M. acetivorans apo-aconitase. IscU2 also restored the aconitase activity in air-exposed M. acetivorans cell lysate. These biochemical results demonstrate that IscS2 is a cysteine desulfurase and that IscU2 is a Fe-S cluster scaffold. M. acetivorans strain DJL60 deleted of iscSU2 was generated to ascertain the in vivo importance of IscSU2. Strain DJL60 had Fe-S cluster content and growth similar to the parent strain but lower cysteine desulfurase activity. Strain DJL60 also had lower intracellular persulfide content compared to the parent strain when cysteine was an exogenous sulfur source, linking IscSU2 to sulfur metabolism. Conclusions This study establishes that M. acetivorans contains functional IscS and IscU, the core components of the ISC Fe-S cluster biogenesis system and provides the first evidence that ISC operates in methanogens.