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Methanogens often inhabit sulfidic environments that favor the precipitation of transition metals such as iron (Fe) as metal sulfides, including mackinawite (FeS) and pyrite (FeS 2 ). These metal sulfides have historically been considered biologically unavailable. Nonetheless, methanogens are commonly cultivated with sulfide (HS - ) as a sulfur source, a condition that would be expected to favor metal precipitation and thus limit metal availability. Recent studies have shown that methanogens can access Fe and sulfur (S) from FeS and FeS 2 to sustain growth. As such, medium supplied with FeS 2 should lead to higher availability of transition metals when compared to medium supplied with HS - . Here, we examined how transition metal availability under sulfidic (i.e., cells provided with HS - as sole S source) versus non-sulfidic (cells provided with FeS 2 as sole S source) conditions impact the metalloproteome of Methanosarcina barkeri Fusaro. To achieve this, we employed size exclusion chromatography coupled with inductively coupled plasma mass spectrometry and shotgun proteomics. Significant changes were observed in the composition and abundance of iron, cobalt, nickel, zinc, and molybdenum proteins. Among the differences were alterations in the stoichiometry and abundance of multisubunit protein complexes involved in methanogenesis and electron transport chains. Our data suggest that M. barkeri utilizes the minimal iron-sulfur cluster complex and canonical cysteine biosynthesis proteins when grown on FeS 2 but uses the canonical Suf pathway in conjunction with the tRNA-Sep cysteine pathway for iron-sulfur cluster and cysteine biosynthesis under sulfidic growth conditions.IMPORTANCEProteins that catalyze biochemical reactions often require transition metals that can have a high affinity for sulfur, another required element for life. Thus, the availability of metals and sulfur are intertwined and can have large impacts on an organismismal biochemistry. Methanogens often occupy anoxic, sulfide-rich (euxinic) environments that favor the precipitation of transition metals as metal sulfides, thereby creating presumed metal limitation. Recently, several methanogens have been shown to acquire iron and sulfur from pyrite, an abundant iron-sulfide mineral that was traditionally considered to be unavailable to biology. The work presented here provides new insights into the distribution of metalloproteins, and metal uptake of Methanosarcina barkeri Fusaro grown under euxinic or pyritic growth conditions. Thorough characterizations of this methanogen under different metal and sulfur conditions increase our understanding of the influence of metal availability on methanogens, and presumably other anaerobes, that inhabit euxinic environments., Competing Interests: The authors declare no conflict of interest.

Accumulating evidences are challenging the paradigm that methane in surface water primarily stems from the anaerobic transformation of organic matters. Yet, the contribution of oxygenic photosynthetic bacteria, a dominant species in surface water, to methane production remains unclear. Here we show methanogenesis triggered by the interaction between oxygenic photosynthetic bacteria and anaerobic methanogenic archaea. By introducing cyanobacterium Synechocystis PCC6803 and methanogenic archaea Methanosarcina barkeri with the redox cycling of iron, CH 4 production was induced in coculture biofilms through both syntrophic methanogenesis (under anoxic conditions in darkness) and abiotic methanogenesis (under oxic conditions in illumination) during the periodic dark-light cycles. We have further demonstrated CH 4 production by other model oxygenic photosynthetic bacteria from various phyla, in conjunction with different anaerobic methanogenic archaea exhibiting diverse energy conservation modes, as well as various common Fe-species. These findings have revealed an unexpected link between oxygenic photosynthesis and methanogenesis and would advance our understanding of photosynthetic bacteria's ecological role in the global CH 4 cycle. Such light-driven methanogenesis may be widely present in nature., (© 2024. The Author(s).)

Methanogenic archaea play a key role in the global carbon cycle because these microorganisms remineralize organic compounds in various anaerobic environments. The microorganism Methanosarcina barkeri is a metabolically versatile methanogen, which can utilize acetate, methanol, and H 2 /CO 2 to synthesize methane. However, the regulatory mechanisms underlying methanogenesis for different substrates remain unknown. In this study, RNA-seq analysis was used to investigate M. barkeri growth and gene transcription under different substrate regimes. According to the results, M. barkeri showed the best growth under methanol, followed by H 2 /CO 2 and acetate, and these findings corresponded well with the observed variations in genes transcription abundance for different substrates. In addition, we identified a novel regulator, MSBRM_RS03855 (designated as HdrR), which specifically activates the transcription of the heterodisulfide reductase hdrBCA operon in M. barkeri . HdrR was able to bind to the hdrBCA operon promoter to regulate transcription. Furthermore, the structural model analyses revealed a helix-turn-helix domain, which is likely involved in DNA binding. Taken together, HdrR serves as a model to reveal how certain regulatory factors control the expression of key enzymes in the methanogenic pathway.IMPORTANCEThe microorganism Methanosarcina barkeri has a pivotal role in the global carbon cycle and contributes to global temperature homeostasis. The consequences of biological methanogenesis are far-reaching, including impacts on atmospheric methane and CO 2 concentrations, agriculture, energy production, waste treatment, and human health. As such, reducing methane emissions is crucial to meeting set climate goals. The methanogenic activity of certain microorganisms can be drastically reduced by inhibiting the transcription of the hdrBCA operon, which encodes heterodisulfide reductases. Here, we provide novel insight into the mechanisms regulating hdrBCA operon transcription in the model methanogen M. barkeri . The results clarified that HdrR serves as a regulator of heterodisulfide reductase hdrBCA operon transcription during methanogenesis, which expands our understanding of the unique regulatory mechanisms that govern methanogenesis. The findings presented in this study can further our understanding of how genetic regulation can effectively reduce the methane emissions caused by methanogens., Competing Interests: The authors declare no conflict of interest.

Iron (Fe) and sulfur (S) are required elements for life, and changes in their availability can limit the ecological distribution and function of microorganisms. In anoxic environments, soluble Fe typically exists as ferrous iron [Fe(II)] and S as sulfide (HS - ). These species exhibit a strong affinity that ultimately drives the formation of sedimentary pyrite (FeS 2 ). Recently, paradigm-shifting studies indicate that Fe and S in FeS 2 can be made bioavailable by methanogens through a reductive dissolution process. However, the impact of the utilization of FeS 2 , as opposed to canonical Fe and S sources, on the phenotype of cells is not fully understood. Here, shotgun proteomics was utilized to measure changes in the phenotype of Methanosarcina barkeri MS grown with FeS 2 , Fe(II)/HS - , or Fe(II)/cysteine. Shotgun proteomics tracked 1,019 proteins overall, with 307 observed to change between growth conditions. Functional characterization and pathway analyses revealed these changes to be systemic and largely tangential to Fe/S metabolism. As a final step, the proteomics data were viewed with respect to previously collected transcriptomics data to deepen the analysis. Presented here is evidence that M. barkeri adopts distinct phenotypes to exploit specific sources of Fe and S in its environment. This is supported by observed protein abundance changes across broad categories of cellular biology. DNA adjacent metabolism, central carbon metabolism methanogenesis, metal trafficking, quorum sensing, and porphyrin biosynthesis pathways are all features in the phenotypic differentiation. Differences in trace metal availability attributed to complexation with HS - , either as a component of the growth medium [Fe(II)/HS - ] or generated through reduction of FeS 2 , were likely a major factor underpinning these phenotypic differences.IMPORTANCEThe methanogenic archaeon Methanosarcina barkeri holds great potential for industrial bio-mining and energy generation technologies. Much of the biochemistry of this microbe is poorly understood, and its characterization will provide a glimpse into biological processes that evolved close to life's origin. The discovery of its ability to extract iron and sulfur from bulk, solid-phase minerals shifted a longstanding paradigm that these elements were inaccessible to biological systems. The full elucidation of this process has the potential to help scientists and engineers extract valuable metals from low-grade ore and mine waste generating energy in the form of methane while doing so., Competing Interests: The authors declare no conflict of interest.

To significantly advance the bio-electrochemical CO 2 -conversion rate and unfold the correlation between the abiotic electrode and the attached microorganisms, an atomic-nanoparticle bridge of Co-N 4 @Co-NP crafted in metal-organic frameworks-derived nanosheets is integrated with a model methanogen of Methanosarcina barkeri (M. barkeri). The direct bonding of N in Co-N 4 and Fe in member protein of Cytochrome b (Cytb) activates a fast direct electron transfer path while the Co nanoparticles further strengthen this bonding via decreasing the energy gap between the p-band center of N and the d-band center of Fe. This multiorbital tuning operation of Co nanoparticles also enhances the coenzyme F420-mediated electron transfer by enabling the electron flow direct to the hydrogenation sites. Particularly, the increased surface electric field of the Co-N 4 @Co-NP bridge-based nanosheet electrode facilitates the interfacial Na + accumulation to expedite ATPase transport for powering intracellular CO 2 conversion. Remarkably, the self-assembled M.barkeri-Co-N 4 @Co-NP biohybrid achieves a high methane production rate of 3860 mmol m -2 day -1 , which greatly outperforms other reported biohybrid systems. This work demonstrates a comprehensive scrutinization of biotic-abiotic energy transfer, which may serve as a guiding principle for efficient bio-electrochemical system design., (© 2023 Wiley-VCH GmbH.)

Nickel (Ni) is a key component of the active site metallocofactors of numerous enzymes required for methanogenesis, including [NiFe]-hydrogenase, carbon monoxide dehydrogenase, and methyl CoM reductase, leading to a high demand for Ni among methanogens. However, methanogens often inhabit euxinic environments that favor the sequestration of nickel as metal-sulfide minerals, such as nickelian pyrite [(Ni,Fe)S 2 ], that have low solubilities and that are not considered bioavailable. Recently, however, several different model methanogens ( Methanosarcina barkeri , Methanococcus voltae , Methanococcus maripaludis ) were shown to reductively dissolve pyrite (FeS 2 ) and to utilize dissolution products to meet iron and sulfur biosynthetic demands. Here, using M. barkeri Fusaro, and laboratory-synthesized (Ni,Fe)S 2 that was physically isolated from cells using dialysis membranes, we show that trace nickel (<20 nM) abiotically solubilized from the mineral can support methanogenesis and limited growth, roughly fivefold less than the minimum concentration known to support methanogenesis. Furthermore, when provided direct contact with (Ni,Fe)S 2 , M. barkeri promoted the reductive dissolution of (Ni,Fe)S 2 and assimilated solubilized nickel, iron, and sulfur as its sole source of these elements. Cells that reductively dissolved (Ni,Fe)S 2 bioaccumulated approximately fourfold more nickel than those grown with soluble nickel and sulfide but had similar metabolic coupling efficiencies. While the mechanism for Ni uptake in archaeal methanogens is not known, homologs of the bacterial Nik uptake system were shown to be ubiquitous across methanogen genomes. Collectively, these observations indicate that (Ni,Fe)S 2 is bioavailable in anoxic environments and that methanogens can convert this mineral into nickel-, iron-, and sulfur-containing metalloenzymes to support methanogenesis and growth. IMPORTANCE Nickel is an essential metal, and its availability has changed dramatically over Earth history due to shifts in the predominant type of volcanism in the late Archean that limited its availability and an increase in euxinic conditions in the early Proterozoic that favored its precipitation as nickel sulfide minerals. Observations presented herein indicate that the methanogen, Methanosarcina barkeri , can acquire nickel at low concentration (<20 nM) from soluble and mineral sources. Furthermore, M. barkeri was shown to actively reduce nickelian pyrite; use dissolution products to meet their iron, sulfur, and nickel demands; and bioaccumulate nickel. These data help to explain how M. barkeri (and possibly other methanogens and anaerobes) can acquire nickel in contemporary and past anoxic or euxinic environments., Competing Interests: The authors declare no conflict of interest.

Ammonia is a ubiquitous potential inhibitor of anaerobic digestion processes, mainly exhibiting inhibition towards methanogenic activity. However, knowledge as to how ammonia affects the methanogens is still limited. In this study, we cultured a multitrophic methanogen, Methanosarcina barkeri DSM 800, with acetate, H 2 /CO 2 , and methanol to evaluate the influence of ammonia on different methanogenic pathways. Aceticlastic methanogenesis was more sensitive to increased ammonia concentrations than hydrogenotrophic and methylotrophic methanogenesis. Theoretical maximum NH 3 tolerances of M. barkeri fed with acetate, H 2 /CO 2 , and methanol were calculated to be 39.1 ± 9.0, 104.3 ± 7.4, and 85.7 ± 1.0 mg/L, respectively. The order of the ΔG range of M. barkeri under three methanogenic pathways reflected the order of ammonia tolerance of M. barkeri. Our results provide insights into the role of the thermodynamic potential of methanogenesis on the tolerance of ammonia stress; and shed light on the mechanism of ammonia inhibition on anaerobic digestion., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Acetotrophic methanogens' dysfunction in anaerobic digestion under ammonia pressure has been widely concerned. Lipids, the main cytomembrane structural biomolecules, normally play indispensable roles in guaranteeing cell functionality. However, no studies explored the effects of high ammonia on acetotrophic methanogens' lipids. Here, a high-throughput lipidomic interrogation deciphered lipid reprogramming in representative acetoclastic methanogen ( Methanosarcina barkeri ) upon high ammonia exposure. The results showed that high ammonia conspicuously reduced polyunsaturated lipids and longer-chain lipids, while accumulating lipids with shorter chains and/or more saturation. Also, the correlation network analysis visualized some sphingolipids as the most active participant in lipid-lipid communications, implying that the ammonia-induced enrichment in these sphingolipids triggered other lipid changes. In addition, we discovered the decreased integrity, elevated permeability, depolarization, and diminished fluidity of lipid-supported membranes under ammonia restraint, verifying the noxious ramifications of lipid abnormalities. Additional analysis revealed that high ammonia destabilized the structure of extracellular polymeric substances (EPSs) capable of protecting lipids, e.g., declining α-helix/(β-sheet + random coil) and 3-turn helix ratios. Furthermore, the abiotic impairment of critical EPS bonds, including C-OH, C═O-NH-, and S-S, and the biotic downregulation of functional proteins involved in transcription, translation, and EPS building blocks' supply were unraveled under ammonia stress and implied as the crucial mechanisms for EPS reshaping.

Integration of methanogens with semiconductors is an effective approach to sustainable solar-driven methanogenesis. However, the H 2 production rate by semiconductors largely exceeds that of methanogen metabolism, resulting in abundant H 2 as side product. Here, we report that binary metallic active sites (namely, NiCu alloys) are incorporated into the interface between CdS semiconductors and Methanosarcina barkeri. The self-assembled Methanosarcina barkeri-NiCu@CdS exhibits nearly 100% CH 4 selectivity with a quantum yield of 12.41 ± 0.16% under light illumination, which not only exceeds the reported biotic-abiotic hybrid systems but also is superior to most photocatalytic systems. Further investigation reveal that the Ni-Cu-Cu hollow sites in NiCu alloys can directly supply hydrogen atoms and electrons through photocatalysis to the Methanosarcina barkeri for methanogenesis via both extracellular and intracellular hydrogen cycles, effectively turning down the H 2 production. This work provides important insights into the biotic-abiotic hybrid interface, and offers an avenue for engineering the methanogenesis process., (© 2022. The Author(s).)

Fe(III) has been recognized as a potential electron sink for the anaerobic oxidation of methane (Fe-AOM) in diverse environments. However, most of previous Fe-AOM processes are limited to ANME archaea and the Fe-AOM mechanism remains unclear. Here we investigate, for the first time, the Fe-AOM performance and mechanisms by a single methanogen Methanosarcina barkeri. The results showed that M. barkeri was capable of oxidizing methane to CO 2 and reducing ferrihydrite to siderite simultaneously. The presence of methane enhanced both the abundances of redox-active species (such as cytochromes) and electrochemical activity of M. barkeri. The proteomic analyses revealed that M. barkeri up-regulated the expressions of a number of methanogenic enzymes during Fe-AOM, and significantly enriched metabolic pathways of amino acid synthesis and nitrogen fixation. Metabolic inhibition experiments indicated that membrane-bound redox-active components (cytochromes, methanophenazine and F 420 H 2 :quinone oxidoreductase) were probably involved in extracellular electron transfer (EET) from cells to ferrihydrite. Overall, these results provide a deep insight into the single‑carbon metabolism and survival strategy for methanogens and suggest that methanogens may play an important role in linking methane and iron cycling in the substrate-limited environments., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Undesirable ammonium concentrations can lead to unstable anaerobic digestion processes, and Methanosarcina spp. are the representative methanogens under inhibition. However, no known work seems to exist for directly exploring the detailed metabolic regulation of pure cultured representative Methanosarcina spp. to ammonium inhibition. We used transcriptomics and proteomics to profile the metabolic regulation of Methanosarcina barkeri to 1, 4, and 7 g N/L of total ammoniacal nitrogen (TAN), where free ammonia concentrations were between 1.5 and 36.1 mg N/L. At the initial stages of ammonium inhibition, the genes participating in the acquisition and assimilation of reduced nitrogen sources showed significant upregulation where the minimal fold change of gene transcription was about 2. Apart from nitrogen metabolism, the transcription of some genes in methanogenesis also significantly increased at the initial stages. For example, the genes encoding alternative heterodisulfide reductase subunits (HdrAB), energy-converting hydrogenase subunit (EchC), and methanophenazine-dependent hydrogenase subunits (VhtAC) were significantly upregulated by at least 2.05 times. For the element translocation at the initial stages, the genes participating in the uptake of ferrous iron, potassium ion, and molybdate were significantly upregulated with a minimal fold change of 2.10. As the cultivation proceeded, the gene encoding the cell division protein subunit (FtsH) was significantly upregulated by 13.0 times at 7 g N/L of TAN; meanwhile, an increment in OD 600 was observed at the terminal sampling point of 7 g N/L of TAN. The present study explored the metabolic regulation of M. barkeri in stress response, protein synthesis, signal transduction, nitrogen metabolism, methanogenesis, and element translocation. The results would contribute to the understanding of the metabolic effects of ammonium inhibition on methanogens and have significant practical implication in inhibited anaerobic digestion.

The direct conversion of CO 2 to value-added chemical commodities, thereby storing solar energy, offers a promising option for alleviating both the current energy crisis and global warming. Semiconductor-biological hybrid systems are novel approaches. However, the inherent defects of photocorrosion, photodegradation, and the toxicity of the semiconductor limit the application of these biohybrid systems. We report here that Rhodopseudomonas palustris was able to directly act as a living photosensitizer to drive CO 2 to CH 4 conversion by Methanosarcina barkeri under illumination after coculturing. Specifically, R. palustris formed a direct electric syntrophic coculture with M. barkeri. Here, R. palustris harvested solar energy, performed anoxygenic photosynthesis using sodium thiosulfate as an electron donor, and transferred electrons extracellularly to M. barkeri to drive methane generation. The methanogenesis of M. barkeri in coculture was a light-dependent process with a production rate of 4.73 ± 0.23 μM/h under light, which is slightly higher than that of typical semiconductor-biohybrid systems (approximately 4.36 μM/h). Mechanistic and transcriptomic analyses showed that electrons were transferred either directly or indirectly (via electron shuttles), subsequently driving CH 4 production. Our study suggests that R. palustris acts as a natural photosensitizer that, in coculture with M. barkeri, results in a new way to harvest solar energy that could potentially replace semiconductors in biohybrid systems., (© 2021. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Dual stable isotope probing has been used to infer rates of microbial biomass production and modes of carbon fixation. In order to validate this approach for assessing archaeal production, the methanogenic archaeon Methanosarcina barkeri was grown either with H 2 , acetate or methanol with D 2 O and 13 C-dissolved inorganic carbon (DIC). Our results revealed unexpectedly low D incorporation into lipids, with the net fraction of water-derived hydrogen amounting to 0.357 ± 0.042, 0.226 ± 0.003 and 0.393 ± 0.029 for growth on H 2 /CO 2 , acetate and methanol respectively. The variability in net water H assimilation into lipids during the growth of M. barkeri on different substrates is possibly attributed to different Gibbs free energy yields, such that higher energy yield promoted the exchange of hydrogen between medium water and lipids. Because NADPH likely serves as the portal for H transfer, increased NADPH production and/or turnover associated with high energy yield may explain the apparent differences in net water H assimilation into lipids. The variable DIC and water H incorporation into M. barkeri lipids imply systematic, metabolic patterns of isotope incorporation and suggest that the ratio of 13 C-DIC versus D 2 O assimilation in environmental samples may serve as a proxy for microbial energetics in addition to microbial production and carbon assimilation pathways., (© 2020 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Methanogens are responsible for the last step in anaerobic digestion (AD), in which methane (a biofuel) is produced. Some methanogens can cometabolize chlorinated pollutants, contributing for their removal during AD. Methanogenic cofactors involved in cometabolic reductive dechlorination, such as F 430 and cobalamin, contain metal ions (nickel, cobalt, iron) in their structure. We hypothesized that the supplementation of trace metals could improve methane production and the cometabolic dechlorination of 1,2-dichloroethene (DCE) by pure cultures of Methanosarcina barkeri. Nickel, cobalt, and iron were added to cultures of M. barkeri growing on methanol and methanol plus DCE. Metal amendment improved DCE dechlorination to vinyl chloride (VC): assays with 20 µM of Fe 3+ showed the highest final concentration of VC (5× higher than in controls without Fe 3+ ), but also in assays with 5.5 µM of Co 2+ and 5 µM of Ni 2+ VC formation was improved (3.5-4× higher than in controls without the respective metals). Dosing of metals could be useful to improve anaerobic removal of chlorinated compounds, and more importantly decrease the detrimental effect of DCE on methane production in anaerobic digesters., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

Methane-producing microbial communities are of ecological and biotechnological interest. Syntrophic interactions among sulfate reducers and aceto/hydrogenotrophic and obligate hydrogenotrophic methanogens form a key component of these communities, yet, the impact of these different syntrophic routes on methane production and their stability against sulfate availability are not well understood. Here, we construct model synthetic communities using a sulfate reducer and two types of methanogens representing different methanogenesis routes. We find that tri-cultures with both routes increase methane production by almost twofold compared to co-cultures and are stable in the absence of sulfate. With increasing sulfate, system stability and productivity decreases and does so faster in communities with aceto/hydrogenotrophic methanogens despite the continued presence of acetate. We show that this is due to a shift in the metabolism of these methanogens towards co-utilization of hydrogen with acetate. These findings indicate the important role of hydrogen dynamics in the stability and productivity of syntrophic communities.

The Methanosarcinales , a lineage of cytochrome-containing methanogens, have recently been proposed to participate in direct extracellular electron transfer interactions within syntrophic communities. To shed light on this phenomenon, we applied electrochemical techniques to measure electron uptake from cathodes by Methanosarcina barkeri , which is an important model organism that is genetically tractable and utilizes a wide range of substrates for methanogenesis. Here, we confirm the ability of M. barkeri to perform electron uptake from cathodes and show that this cathodic current is linked to quantitative increases in methane production. The underlying mechanisms we identified include, but are not limited to, a recently proposed association between cathodes and methanogen-derived extracellular enzymes (e.g., hydrogenases) that can facilitate current generation through the formation of reduced and diffusible methanogenic substrates (e.g., hydrogen). However, after minimizing the contributions of such extracellular enzymes and using a mutant lacking hydrogenases, we observe a lower-potential hydrogen-independent pathway that facilitates cathodic activity coupled to methane production in M. barkeri Our electrochemical measurements of wild-type and mutant strains point to a novel and hydrogenase-free mode of electron uptake with a potential near -484 mV versus standard hydrogen electrode (SHE) (over 100 mV more reduced than the observed hydrogenase midpoint potential under these conditions). These results suggest that M. barkeri can perform multiple modes (hydrogenase-mediated and free extracellular enzyme-independent modes) of electrode interactions on cathodes, including a mechanism pointing to a direct interaction, which has significant applied and ecological implications. IMPORTANCE Methanogenic archaea are of fundamental applied and environmental relevance. This is largely due to their activities in a wide range of anaerobic environments, generating gaseous reduced carbon that can be utilized as a fuel source. While the bioenergetics of a wide variety of methanogens have been well studied with respect to soluble substrates, a mechanistic understanding of their interaction with solid-phase redox-active compounds is limited. This work provides insight into solid-phase redox interactions in Methanosarcina spp. using electrochemical methods. We highlight a previously undescribed mode of electron uptake from cathodes that is potentially informative of direct interspecies electron transfer interactions in the Methanosarcinales ., (Copyright © 2019 Rowe et al.)

Energy conservation via hydrogen cycling, which generates proton motive force by intracellular H 2 production coupled to extracellular consumption, has been controversial since it was first proposed in 1981. It was hypothesized that the methanogenic archaeon Methanosarcina barkeri is capable of energy conservation via H 2 cycling, based on genetic data that suggest that H 2 is a preferred, but nonessential, intermediate in the electron transport chain of this organism. Here, we characterize a series of hydrogenase mutants to provide direct evidence of H 2 cycling. M. barkeri produces H 2 during growth on methanol, a phenotype that is lost upon mutation of the cytoplasmic hydrogenase encoded by frhADGB , although low levels of H 2 , attributable to the Ech hydrogenase, accumulate during stationary phase. In contrast, mutations that conditionally inactivate the extracellular Vht hydrogenase are lethal when expression of the vhtGACD operon is repressed. Under these conditions, H 2 accumulates, with concomitant cessation of methane production and subsequent cell lysis, suggesting that the inability to recapture extracellular H 2 is responsible for the lethal phenotype. Consistent with this interpretation, double mutants that lack both Vht and Frh are viable. Thus, when intracellular hydrogen production is abrogated, loss of extracellular H 2 consumption is no longer lethal. The common occurrence of both intracellular and extracellular hydrogenases in anaerobic microorganisms suggests that this unusual mechanism of energy conservation may be widespread in nature. IMPORTANCE ATP is required by all living organisms to facilitate essential endergonic reactions required for growth and maintenance. Although synthesis of ATP by substrate-level phosphorylation is widespread and significant, most ATP is made via the enzyme ATP synthase, which is energized by transmembrane chemiosmotic gradients. Therefore, establishing this gradient across the membrane is of central importance to sustaining life. Experimental validation of H 2 cycling adds to a short list of mechanisms for generating a transmembrane electrochemical gradient that is likely to be widespread, especially among anaerobic microorganisms., (Copyright © 2018 Kulkarni et al.)

Wetlands contribute to 30% of global methane emissions due to an imbalance between microbial methane production and consumption. Methanogenesis and methanotrophy have mainly been studied separately, and little is known about their potential interactions in aquatic environments. To mimic the interaction between methane producers and oxidizers in the environment, we co-cultivated the methanogenic archaeon Methanosarcina barkeri with aerobic Methylocystaceae methanotrophs in an oxygen-limited bioreactor using acetate as methanogenic substrate. Methane, acetate, dissolved oxygen, available nitrogen, pH, temperature, and cell density were monitored to follow system stability and activity. Stable reactor operation was achieved for two consecutive periods of 2 months. Fluorescence in situ hybridization micrographs indicated close association between both groups of microorganisms. This association suggests that the methanotrophs profit from direct access to the methane that is produced from acetate, while methanogens are protected by the concomitant oxygen consumption of the methanotrophs. This proof of principle study can be used to set up systems to study their responses to environmental changes.

A metabolic network model provides a computational framework for studying the metabolism of a cell at the system level. The organization of metabolic networks has been investigated in different studies. One of the organization aspects considered in these studies is the decomposition of a metabolic network. The decompositions produced by different methods are very different and there is no comprehensive evaluation framework to compare the results with each other. In this study, these methods are reviewed and compared in the first place. Then they are applied to six different metabolic network models and the results are evaluated and compared based on two existing and two newly proposed criteria. Results show that no single method can beat others in all criteria but it seems that the methods introduced by Guimera and Amaral and Verwoerd do better on among metabolite-based methods and the method introduced by Sridharan et al. does better among reaction-based ones. Also, the methods are applied to several artificial networks, each constructed from merging a few KEGG pathways. Then, their capability to recover those pathways are compared. Results show that among metabolite-based methods, the method of Guimera and Amaral does better again, however, no notable difference between the performances of reaction-based methods was detected.

Methanosarcina barkeri (DSM 800) is a metabolically versatile methanogen and shows distinct metabolic status under different substrate regimes. However, the mechanisms underlying distinct transcriptional profiles under different substrate regimes remain elusive. In this study, based on transcriptional analysis, the growth performances and gene expressions of M. barkeri fed on acetate, H 2  + CO 2 , and methanol, respectively, were investigated. M. barkeri showed higher growth performances under methanol, followed by H 2  + CO 2 and acetate, which corresponded well with the variations of gene expressions. The α diversity (evenness) of gene expressions was highest under the acetate regime, followed by H 2  + CO 2 and methanol, and significantly and negatively correlated with growth performances. The gene co-expression analysis showed that "Energy production and conversion," "Coenzyme transport and metabolism," and "Translation, ribosomal structure, and biogenesis" showed deterministic cooperation patterns of intra- and inter-functional classes. However, "Posttranslational modification, protein turnover, chaperones" showed exclusion with other functional classes. The gene expressions and especially the relationships among them potentially drove the shifts of metabolic status under different substrate regimes. Consequently, this study revealed the diversity-related ecological strategies that a high α diversity probably provided more fitness and tolerance under natural environments and oppositely a low α diversity strengthened some specific physiological functions, as well as the co-responses of gene expressions to different substrate regimes.

The incorporation of noncanonical amino acids (ncAAs) with different side chains into a peptide is a promising technique for changing the functional properties of that peptide. Of particular interest is the incorporation of ncAAs into peptide-derived natural products to optimize their biophysical properties for medical and industrial applications. Here, we present the first instance of ncAA incorporation into the natural product cinnamycin in streptomycetes using the orthogonal pyrrolysyl-tRNA synthetase/tRNA Pyl pair from Methanosarcina barkeri. This approach allows site-specific incorporation of ncAAs via the read-through of a stop codon by the suppressor tRNA Pyl , which can carry different pyrrolysine analogues. Five new deoxycinnamycin derivatives were obtained with three distinct pyrrolysine analogues incorporated into diverse positions of the antibiotic. The combination of partial hydrolysis and MS/MS fragmentation analysis was used to verify the exact position of the incorporation events. The introduction of ncAAs into different positions of the peptide had opposite effects on the peptide's biological activity.

Methane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilization of methane through anaerobic methane oxidation. The enzyme uses an ancillary factor called coenzyme F 430 , a nickel-containing modified tetrapyrrole that promotes catalysis through a methyl radical/Ni(ii)-thiolate intermediate. However, it is unclear how coenzyme F 430 is synthesized from the common primogenitor uroporphyrinogen iii, incorporating 11 steric centres into the macrocycle, although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamization and carbocyclic ring formation. Here we identify the proteins that catalyse the biosynthesis of coenzyme F 430 from sirohydrochlorin, termed CfbA-CfbE, and demonstrate their activity. The research completes our understanding of how the repertoire of tetrapyrrole-based pigments are constructed, permitting the development of recombinant systems to use these metalloprosthetic groups more widely.

Hydrogen (H2) consumption and methane (CH4) production in pure cultures of three different methanogens were investigated during cultivation with 0, 0.2 and 4.21 μM added nickel (Ni). The results showed that the level of dissolved Ni in the anaerobic growth medium did not notably affect CH4 production in the cytochrome-free methanogenic species Methanobacterium bryantii and Methanoculleus bourgensis MAB1, but affected CH4 formation rate in the cytochrome-containing Methanosarcina barkeri grown on H2 and CO2. Methanosarcina barkeri also had the highest amounts of Ni in its cells, indicating that more Ni is needed by cytochrome-containing than by cytochrome-free methanogenic species. The concentration of Ni affected threshold values of H2 partial pressure (pH2) for all three methanogen species studied, with M. bourgensis MAB1 reaching pH2 values as low as 0.1 Pa when Ni was available in amounts used in normal anaerobic growth medium. To our knowledge, this is the lowest pH2 threshold recorded to date in pure methanogen culture, which suggests that M.bourgensis MAB1 have a competitive advantage over other species through its ability to grow at low H2 concentrations. Our study has implications for research on the H2-driven deep subsurface biosphere and biogas reactor performance., Competing Interests: The authors have declared that no competing interests exist.

Unlabelled: The transformation of ferrihydrite to stable iron oxides over time has important consequences for biogeochemical cycling of many metals and nutrients. The response of methanogenic activity to the presence of iron oxides depends on the type of iron mineral, but the effects of changes in iron mineralogy on methanogenesis have not been characterized. To address these issues, we constructed methanogenic cocultures of Geobacter and Methanosarcina strains with different ferrihydrite mineralization pathways. In this system, secondary mineralization products from ferrihydrite are regulated by the presence or absence of phosphate. In cultures producing magnetite as the secondary mineralization product, the rates of methanogenesis from acetate and ethanol increased by 30.2% and 135.3%, respectively, compared with a control lacking ferrihydrite. Biogenic magnetite was proposed to promote direct interspecies electron transfer between Geobacter and Methanosarcina in a manner similar to that of c-type cytochrome and thus facilitate methanogenesis. Vivianite biomineralization from ferrihydrite in the presence of phosphate did not significantly influence the methanogenesis processes. The correlation between magnetite occurrence and facilitated methanogenesis was supported by increased rates of methane production from acetate and ethanol with magnetite supplementation in the defined cocultures. Our data provide a new perspective on the important role of iron biomineralization in biogeochemical cycling of carbon in diverse anaerobic environments., Importance: It has been found that microbial methanogenesis is affected by the presence of iron minerals, and their influences on methanogenesis are associated with the mineralogical properties of the iron minerals. However, how changes in iron mineralogy affect microbial methanogenesis has not been characterized. To address this issue, we constructed methanogenic cocultures of Geobacter and Methanosarcina strains with different ferrihydrite mineralization pathways. The experimental results led to two contributions, i.e., (i) the transformation of iron minerals might exert an important influence on methanogenesis under anaerobic conditions and (ii) both biogenic and chemical magnetite can accelerate syntrophic ethanol oxidization between Geobacter metallireducens and Methanosarcina barkeri This study sheds new light on the important role of iron biomineralization in the biogeochemical cycling of carbon in diverse anaerobic environments, particularly in iron-rich natural and agricultural wetland soils., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

All methanogenic and methanotrophic archaea known to date contain methyl-coenzyme M reductase (MCR) that catalyzes the reversible reduction of methyl-coenzyme M to methane. This enzyme contains the nickel porphinoid F430 as a prosthetic group and, highly conserved, a thioglycine and four methylated amino acid residues near the active site. We describe herein the presence of a novel post-translationally modified amino acid, didehydroaspartate, adjacent to the thioglycine as revealed by mass spectrometry and high-resolution X-ray crystallography. Upon chemical reduction, the didehydroaspartate residue was converted into aspartate. Didehydroaspartate was found in MCR I and II from Methanothermobacter marburgensis and in MCR of phylogenetically distantly related Methanosarcina barkeri but not in MCR I and II of Methanothermobacter wolfeii, which indicates that didehydroaspartate is dispensable but might have a role in fine-tuning the active site to increase the catalytic efficiency., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We describe the genetically directed incorporation of aminooxy functionality into recombinant proteins by using a mutant Methanosarcina barkeri pyrrolysyl-tRNA synthetase/tRNACUA pair. This allows the general production of nonhydrolysable ubiquitin conjugates of recombinant origin by bioorthogonal oxime ligation. This was exemplified by the preparation of nonhydrolysable versions of diubiquitin, polymeric ubiquitin chains and ubiquitylated SUMO. The conjugates exhibited unrivalled isostery with the native isopeptide bond, as inferred from structural and biophysical characterisation. Furthermore, the conjugates functioned as nanomolar inhibitors of deubiquitylating enzymes and were recognised by linkage-specific antibodies. This technology should provide a versatile platform for the development of powerful tools for studying deubiquitylating enzymes and for elucidating the cellular roles of diverse polyubiquitin linkages., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Methanogenesis, the microbial methane (CH4 ) production, is traditionally thought to anchor the mineralization of organic matter as the ultimate respiratory process in deep sediments, despite the presence of oxidized mineral phases, such as iron oxides. This process is carried out by archaea that have also been shown to be capable of reducing iron in high levels of electron donors such as hydrogen. The current pure culture study demonstrates that methanogenic archaea (Methanosarcina barkeri) rapidly switch from methanogenesis to iron-oxide reduction close to natural conditions, with nitrogen atmosphere, even when faced with substrate limitations. Intensive, biotic iron reduction was observed following the addition of poorly crystalline ferrihydrite and complex organic matter and was accompanied by inhibition of methane production. The reaction rate of this process was of the first order and was dependent only on the initial iron concentrations. Ferrous iron production did not accelerate significantly with the addition of 9,10-anthraquinone-2,6-disulfonate (AQDS) but increased by 11-28% with the addition of phenazine-1-carboxylate (PCA), suggesting the possible role of methanophenazines in the electron transport. The coupling between ferrous iron and methane production has important global implications. The rapid transition from methanogenesis to reduction of iron-oxides close to the natural conditions in sediments may help to explain the globally-distributed phenomena of increasing ferrous concentrations below the traditional iron reduction zone in the deep 'methanogenic' sediment horizon, with implications for metabolic networking in these subsurface ecosystems and in past geological settings., (© 2016 John Wiley & Sons Ltd.)

Two subtypes of class III anaerobic ribonucleotide reductases (RNRs) studied so far couple the reduction of ribonucleotides to the oxidation of formate, or the oxidation of NADPH via thioredoxin and thioredoxin reductase. Certain methanogenic archaea contain a phylogenetically distinct third subtype of class III RNR, with distinct active-site residues. Here we report the cloning and recombinant expression of the Methanosarcina barkeri class III RNR and show that the electrons required for ribonucleotide reduction can be delivered by a [4Fe-4S] protein ferredoxin disulfide reductase, and a conserved thioredoxin-like protein NrdH present in the RNR operon. The diversity of class III RNRs reflects the diversity of electron carriers used in anaerobic metabolism.

Unlabelled: Pyruvate, a central intermediate in the carbon fixation pathway of methanogenic archaea, is rarely used as an energy source by these organisms. The sole exception to this rule is a genetically uncharacterized Methanosarcina barkeri mutant capable of using pyruvate as a sole energy and carbon source (the Pyr(+) phenotype). Here, we provide evidence that suggests that the Pyr(+) mutant is able to metabolize pyruvate by overexpressing pyruvate ferredoxin oxidoreductase (por) and mutating genes involved in central carbon metabolism. Genomic analysis showed that the Pyr(+) strain has two mutations localized to Mbar&#95;A1588, the biotin protein ligase subunit of the pyruvate carboxylase (pyc) operon, and Mbar&#95;A2165, a putative transcriptional regulator. Mutants expressing the Mbar&#95;A1588 mutation showed no growth defect compared to the wild type (WT), yet the strains lacked pyc activity. Recreation of the Mbar&#95;A2165 mutation resulted in a 2-fold increase of Por activity and gene expression, suggesting a role in por transcriptional regulation. Further transcriptomic analysis revealed that Pyr(+) strains also overexpress the gene encoding phosphoenolpyruvate carboxylase, indicating the presence of a previously uncharacterized route for synthesizing oxaloacetate in M. barkeri and explaining the unimpaired growth in the absence of Pyc. Surprisingly, stringent repression of the por operon was lethal, even when the media were supplemented with pyruvate and/or Casamino Acids, suggesting that por plays an unidentified essential function in M. barkeri., Importance: The work presented here reveals a complex interaction between anabolic and catabolic pathways involving pyruvate metabolism in Methanosarcina barkeri Fusaro. Among the unexpected findings were an essential role for the enzyme pyruvate-ferredoxin oxidoreductase and an alternate pathway for synthesis of oxaloacetate. These results clarify the mechanism of methanogenic catabolism of pyruvate and expand our understanding of carbon assimilation in methanogens., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Natural photosynthesis harnesses solar energy to convert CO2 and water to value-added chemical products for sustaining life. We present a hybrid bioinorganic approach to solar-to-chemical conversion in which sustainable electrical and/or solar input drives production of hydrogen from water splitting using biocompatible inorganic catalysts. The hydrogen is then used by living cells as a source of reducing equivalents for conversion of CO2 to the value-added chemical product methane. Using platinum or an earth-abundant substitute, α-NiS, as biocompatible hydrogen evolution reaction (HER) electrocatalysts and Methanosarcina barkeri as a biocatalyst for CO2 fixation, we demonstrate robust and efficient electrochemical CO2 to CH4 conversion at up to 86% overall Faradaic efficiency for ≥ 7 d. Introduction of indium phosphide photocathodes and titanium dioxide photoanodes affords a fully solar-driven system for methane generation from water and CO2, establishing that compatible inorganic and biological components can synergistically couple light-harvesting and catalytic functions for solar-to-chemical conversion.

Microbial life inhabits deeply buried marine sediments, but the extent of this vast ecosystem remains poorly constrained. Here we provide evidence for the existence of microbial communities in ~40° to 60°C sediment associated with lignite coal beds at ~1.5 to 2.5 km below the seafloor in the Pacific Ocean off Japan. Microbial methanogenesis was indicated by the isotopic compositions of methane and carbon dioxide, biomarkers, cultivation data, and gas compositions. Concentrations of indigenous microbial cells below 1.5 km ranged from <10 to ~10(4) cells cm(-3). Peak concentrations occurred in lignite layers, where communities differed markedly from shallower subseafloor communities and instead resembled organotrophic communities in forest soils. This suggests that terrigenous sediments retain indigenous community members tens of millions of years after burial in the seabed., (Copyright © 2015, American Association for the Advancement of Science.)

Microbial syntrophic metabolism has been well accepted as the heart of how methanogenic and other anaerobic microbial communities function. In this work, we applied a single-cell RT-qPCR approach to reveal gene-expression heterogeneity in a model syntrophic system of Desulfovibrio vulgaris and Methanosarcina barkeri, as compared with the D. vulgaris monoculture. Using the optimized primers and single-cell analytical protocol, we quantitatively determine gene-expression levels of 6 selected target genes in each of the 120 single cells of D. vulgaris isolated from its monoculture and dual-culture with M. barkeri. The results demonstrated very significant cell-to-cell gene-expression heterogeneity for the selected D. vulgaris genes in both the monoculture and the syntrophic dual-culture. Interestingly, no obvious increase in gene-expression heterogeneity for the selected genes was observed for the syntrophic dual-culture when compared with its monoculture, although the community structure and cell-cell interactions have become more complicated in the syntrophic dual-culture. In addition, the single-cell RT-qPCR analysis also provided further evidence that the gene cluster (DVU0148-DVU0150) may be involved syntrophic metabolism between D. vulgaris and M. barkeri. Finally, the study validated that single-cell RT-qPCR analysis could be a valuable tool in deciphering gene functions and metabolism in mixed-cultured microbial communities.

Site-specific incorporation of bioorthogonal unnatural amino acids into proteins provides a useful tool for the installation of specific functionalities that will allow for the labeling of proteins with virtually any probe. We demonstrate the genetic encoding of a set of alkene lysines using the orthogonal PylRS/PylTCUA pair in Escherichia coli. The installed double bond functionality was then applied in a photoinitiated thiol-ene reaction of the protein with a fluorescent thiol-bearing probe, as well as a cysteine residue of a second protein, showing the applicability of this approach in the formation of heterogeneous non-linear fused proteins.

Incorporation of multiple different unnatural amino acids into the same polypeptide remains a significant challenge. Orthogonal ribosomes, which are evolvable as they direct the translation of a single dedicated orthogonal mRNA, can provide an avenue to produce such polypeptides routinely. Recent advances in engineering orthogonal ribosomes have created a prototype system to enable genetically encoded introduction of two different functional groups, albeit with limited efficiency. Here, we systematically investigated the limiting factors of this system by using assays to measure the levels and activities of individual components; we identified Methanosarcina barkeri PylRS as a limiting factor for protein yield. Balancing the expression levels of individual components significantly improved growth rate and protein yield. This optimization of the system is likely to increase the scope of evolved orthogonal ribosome-mediated incorporation of multiple different unnatural amino acids., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Direct interspecies electron transfer (DIET) is potentially an effective form of syntrophy in methanogenic communities, but little is known about the diversity of methanogens capable of DIET. The ability of Methanosarcina barkeri to participate in DIET was evaluated in coculture with Geobacter metallireducens. Cocultures formed aggregates that shared electrons via DIET during the stoichiometric conversion of ethanol to methane. Cocultures could not be initiated with a pilin-deficient G. metallireducens strain, suggesting that long-range electron transfer along pili was important for DIET. Amendments of granular activated carbon permitted the pilin-deficient G. metallireducens isolates to share electrons with M. barkeri, demonstrating that this conductive material could substitute for pili in promoting DIET. When M. barkeri was grown in coculture with the H2-producing Pelobacter carbinolicus, incapable of DIET, M. barkeri utilized H2 as an electron donor but metabolized little of the acetate that P.carbinolicus produced. This suggested that H2, but not electrons derived from DIET, inhibited acetate metabolism. P. carbinolicus-M. barkeri cocultures did not aggregate, demonstrating that, unlike DIET, close physical contact was not necessary for interspecies H2 transfer. M. barkeri is the second methanogen found to accept electrons via DIET and the first methanogen known to be capable of using either H2 or electrons derived from DIET for CO2 reduction. Furthermore, M. barkeri is genetically tractable,making it a model organism for elucidating mechanisms by which methanogens make biological electrical connections with other cells.

Biochar, a charcoal-like product of the incomplete combustion of organic materials, is an increasingly popular soil amendment designed to improve soil fertility. We investigated the possibility that biochar could promote direct interspecies electron transfer (DIET) in a manner similar to that previously reported for granular activated carbon (GAC). Although the biochars investigated were 1000 times less conductive than GAC, they stimulated DIET in co-cultures of Geobacter metallireducens with Geobacter sulfurreducens or Methanosarcina barkeri in which ethanol was the electron donor. Cells were attached to the biochar, yet not in close contact, suggesting that electrons were likely conducted through the biochar, rather than biological electrical connections. The finding that biochar can stimulate DIET may be an important consideration when amending soils with biochar and can help explain why biochar may enhance methane production from organic wastes under anaerobic conditions.

In living organisms heme is formed from the common precursor uroporphyrinogen III by either one of two substantially different pathways. In contrast to eukaryotes and most bacteria which employ the so-called "classical" heme biosynthesis pathway, the archaea use an alternative route. In this pathway, heme is formed from uroporphyrinogen III via the intermediates precorrin-2, sirohydrochlorin, siroheme, 12,18-didecarboxysiroheme, and iron-coproporphyrin III. In this study the heme biosynthesis proteins AhbAB, AhbC, and AhbD from Methanosarcina barkeri were functionally characterized. Using an in vivo enzyme activity assay it was shown that AhbA and AhbB (Mbar&#95;A1459 and Mbar&#95;A1460) together catalyze the conversion of siroheme into 12,18-didecarboxysiroheme. The two proteins form a heterodimeric complex which might be subject to feedback regulation by the pathway end-product heme. Further, AhbC (Mbar&#95;A1793) was shown to catalyze the formation of iron-coproporphyrin III in vivo. Finally, recombinant AhbD (Mbar&#95;A1458) was produced in E. coli and purified indicating that this protein most likely contains two [4Fe-4S] clusters. Using an in vitro enzyme activity assay it was demonstrated that AhbD catalyzes the conversion of iron-coproporphyrin III into heme.

Methanosarcina barkeri is an Archaeon that produces methane anaerobically as the primary byproduct of its metabolism. M. barkeri can utilize several substrates for ATP and biomass production including methanol, acetate, methyl amines, and a combination of hydrogen and carbon dioxide. In 2006, a metabolic reconstruction of M. barkeri, iAF692, was generated based on a draft genome annotation. The iAF692 reconstruction enabled the first genome-Scale simulations for Archaea. Since the publication of the first metabolic reconstruction of M. barkeri, additional genomic, biochemical, and phenotypic data have clarified several metabolic pathways. We have used this newly available data to improve the M. barkeri metabolic reconstruction. Modeling simulations using the updated model, iMG746, have led to increased accuracy in predicting gene knockout phenotypes and simulations of batch growth behavior. We used the model to examine knockout lethality data and make predictions about metabolic regulation under different growth conditions. Thus, the updated metabolic reconstruction of M. barkeri metabolism is a useful tool for predicting cellular behavior, studying the methanogenic lifestyle, guiding experimental studies, and making predictions relevant to metabolic engineering applications., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In natural environments methane is usually produced by aceticlastic and hydrogenotrophic methanogenic archaea. However, some methanogens can use C(1) compounds such as methanol as the substrate. To determine the contributions of individual substrates to methane production, the stable-isotope values of the substrates and the released methane are often used. Additional information can be obtained by using selective inhibitors (e.g., methyl fluoride, a selective inhibitor of acetoclastic methanogenesis). We studied stable carbon isotope fractionation during the conversion of methanol to methane in Methanosarcina acetivorans, Methanosarcina barkeri, and Methanolobus zinderi and generally found large fractionation factors (-83‰ to -72‰). We further tested whether methyl fluoride impairs methylotrophic methanogenesis. Our experiments showed that even though a slight inhibition occurred, the carbon isotope fractionation was not affected. Therefore, the production of isotopically light methane observed in the presence of methyl fluoride may be due to the strong fractionation by methylotrophic methanogens and not only by hydrogenotrophic methanogens as previously assumed.

The discovery of pyrrolysine not only expanded the set of the known proteinogenic amino acids but also revealed unusual features of its encoding mechanism. The engagement of a canonical stop codon and a unique aminoacyl-tRNA synthetase-tRNA pair that can be used to accommodate a broad range of unnatural amino acids while maintaining strict orthogonality in a variety of prokaryotic and eukaryotic expression systems has proven an invaluable combination. Within a few years since its properties were elucidated, the pyrrolysine translational machinery has become a popular choice for the synthesis of recombinant proteins bearing a wide variety of otherwise hard-to-introduce functional groups. It is also central to the development of new synthetic strategies that rely on stop-codon suppression., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Lys120 in the DNA-binding domain (DBD) of p53 becomes acetylated in response to DNA damage. But, the role and effects of acetylation are obscure. We prepared p53 specifically acetylated at Lys120, AcK120p53, by in vivo incorporation of acetylated lysine to study biophysical and structural consequences of acetylation that may shed light on its biological role. Acetylation had no affect on the overall crystal structure of the DBD at 1.9-Å resolution, but significantly altered the effects of salt concentration on specificity of DNA binding. p53 binds DNA randomly in vitro at effective physiological salt concentration and does not bind specifically to DNA or distinguish among its different response elements until higher salt concentrations. But, on acetylation, AcK120p53 exhibited specific DNA binding and discriminated among response elements at effective physiological salt concentration. AcK120p53 and p53 had the highest affinity to the same DNA sequence, although acetylation reduced the importance of the consensus C and G at positions 4 and 7, respectively. Mass spectrometry of p53 and AcK120p53 DBDs bound to DNA showed they preferentially segregated into complexes that were either DNA(p53DBD)(4) or DNA(AcK120DBD)(4), indicating that the different DBDs prefer different quaternary structures. These results are consistent with electron microscopy observations that p53 binds to nonspecific DNA in different, relaxed, quaternary states from those bound to specific sequences. Evidence is accumulating that p53 can be sequestered by random DNA, and target search requires acetylation of Lys120 and/or interaction with other factors to impose specificity of binding via modulating changes in quaternary structure.

Methanogens are the only significant biological producers of methane. A limited number of C(1) substrates, such as methanol, methylamines, methyl sulfate, formate, H(2)+CO(2) or CO, and acetate, serve as carbon and energy source. During degradation of these compounds, a primary proton as well as a primary sodium ion gradient is established, which is a unique feature of methanogens. This raises the question about the coupling ion for ATP synthesis by the unique A(1)A(o) ATP synthase. Here, we describe how to analyze and determine the Na(+) dependence of two model methanogens, the hydrogenotrophic Methanothermobacter thermautotrophicus and the methylotrophic Methanosarcina barkeri. Furthermore, the determination of important bioenergetic parameters like the ΔpH, ΔΨ, or the intracellular volume in M. barkeri is described. For the analyses of the A(1)A(O) ATP synthase, methods for measurement of ATP synthesis as well as ATP hydrolysis in Methanosarcina mazei Gö1 are described., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The family Methanosarcinaceae has an expanded repertoire of growth substrates relative to most other methanogenic archaea. Various methylamines, methylated thiols, and methanol can serve as precursors to both methane and carbon dioxide. These compounds are mobilized into metabolism by methyltransferases that use the growth substrate to methylate a cognate corrinoid protein, which in turn is used as a substrate by a second methyltransferase to methylate Coenzyme M (CoM), forming methyl-SCoM, the precursor to both methane and carbon dioxide. Orthologs of the methyltransferases, as well as the small corrinoid proteins, are found in many archaeal and bacterial genomes. Some of these are homologs of the methylamine methyltransferases predicted to require pyrrolysine, an atypical genetically encoded amino acid, for synthesis. As a resource for the study of these sizable families of proteins, we describe here techniques our laboratories have used for the study of methanogen corrinoid-dependent methyltransferases, focusing especially on isolation and assay techniques useful for various activities of components of the methylamine- and methylthiol-dependent CoM methyltransferase systems., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Desulfovibrio vulgaris is a metabolically flexible micro-organism. It can use sulfate as an electron acceptor to catabolize a variety of substrates, or in the absence of sulfate can utilize organic acids and alcohols by forming a syntrophic association with a hydrogen-scavenging partner to relieve inhibition by hydrogen. These alternative metabolic types increase the chance of survival for D. vulgaris in environments where one of the potential external electron acceptors becomes depleted. In this work, whole-genome D. vulgaris microarrays were used to determine relative transcript levels as D. vulgaris shifted its metabolism from syntrophic in a lactate-oxidizing dual-culture with Methanosarcina barkeri to a sulfidogenic metabolism. Syntrophic dual-cultures were grown in two independent chemostats and perturbation was introduced after six volume changes with the addition of sulfate. The results showed that 132 genes were differentially expressed in D. vulgaris 2 h after addition of sulfate. Functional analyses suggested that genes involved in cell envelope and energy metabolism were the most regulated when comparing syntrophic and sulfidogenic metabolism. Upregulation was observed for genes encoding ATPase and the membrane-integrated energy-conserving hydrogenase (Ech) when cells shifted to a sulfidogenic metabolism. A five-gene cluster encoding several lipoproteins and membrane-bound proteins was downregulated when cells were shifted to a sulfidogenic metabolism. Interestingly, this gene cluster has orthologues found only in another syntrophic bacterium, Syntrophobacter fumaroxidans, and four recently sequenced Desulfovibrio strains. This study also identified several novel c-type cytochrome-encoding genes, which may be involved in the sulfidogenic metabolism.

The recently discovered antibacterial compound alaremycin, produced by Streptomyces sp. A012304, structurally closely resembles 5-aminolevulinic acid, the substrate of porphobilinogen synthase. During the initial steps of heme biosynthesis, two molecules of 5-aminolevulinic acid are asymmetrically condensed to porphobilinogen. Alaremycin was found to efficiently inhibit the growth of both Gram-negative and Gram-positive bacteria. Using the newly created heme-permeable strain Escherichia coli CSA1, we are able to uncouple heme biosynthesis from bacterial growth and demonstrate that alaremycin targets the heme biosynthetic pathway. Further studies focused on the activity of alaremycin against the opportunistic pathogenic bacterium Pseudomonas aeruginosa. The MIC of alaremycin was determined to be 12 mM. Alaremycin was identified as a direct inhibitor of recombinant purified P. aeruginosa porphobilinogen synthase and had a K(i) of 1.33 mM. To understand the molecular basis of alaremycin's antibiotic activity at the atomic level, the P. aeruginosa porphobilinogen synthase was cocrystallized with the alaremycin. At 1.75-A resolution, the crystal structure reveals that the antibiotic efficiently blocks the active site of porphobilinogen synthase. The antibiotic binds as a reduced derivative of 5-acetamido-4-oxo-5-hexenoic acid. The corresponding methyl group is, however, not coordinated by any amino acid residues of the active site, excluding its functional relevance for alaremycin inhibition. Alaremycin is covalently bound by the catalytically important active-site lysine residue 260 and is tightly coordinated by several active-site amino acids. Our data provide a solid structural basis to further improve the activity of alaremycin for rational drug design. Potential approaches are discussed.

Methanogens use an unusual energy-conserving electron transport chain that involves reduction of a limited number of electron acceptors to methane gas. Previous biochemical studies suggested that the proton-pumping F(420)H(2) dehydrogenase (Fpo) plays a crucial role in this process during growth on methanol. However, Methanosarcina barkeri Delta fpo mutants constructed in this study display no measurable phenotype on this substrate, indicating that Fpo plays a minor role, if any. In contrast, Delta frh mutants lacking the cytoplasmic F(420)-reducing hydrogenase (Frh) are severely affected in their ability to grow and make methane from methanol, and double Delta fpo/Delta frh mutants are completely unable to use this substrate. These data suggest that the preferred electron transport chain involves production of hydrogen gas in the cytoplasm, which then diffuses out of the cell, where it is reoxidized with transfer of electrons into the energy-conserving electron transport chain. This hydrogen-cycling metabolism leads directly to production of a proton motive force that can be used by the cell for ATP synthesis. Nevertheless, M. barkeri does have the flexibility to use the Fpo-dependent electron transport chain when needed, as shown by the poor growth of the Delta frh mutant. Our data suggest that the rapid enzymatic turnover of hydrogenases may allow a competitive advantage via faster growth rates in this freshwater organism. The mutant analysis also confirms the proposed role of Frh in growth on hydrogen/carbon dioxide and suggests that either Frh or Fpo is needed for aceticlastic growth of M. barkeri.

Archaeal methane formation from methylamines is initiated by distinct methyltransferases with specificity for monomethylamine, dimethylamine, or trimethylamine. Each methylamine methyltransferase methylates a cognate corrinoid protein, which is subsequently demethylated by a second methyltransferase to form methyl-coenzyme M, the direct methane precursor. Methylation of the corrinoid protein requires reduction of the central cobalt to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric iron-sulfur protein, was isolated from Methanosarcina barkeri and is required for in vitro ATP-dependent reductive activation of methylamine:CoM methyl transfer from all three methylamines. In the absence of the methyltransferases, highly purified RamA was shown to mediate the ATP-dependent reductive activation of Co(II) corrinoid to the Co(I) state for the monomethylamine corrinoid protein, MtmC. The ramA gene is located near a cluster of genes required for monomethylamine methyltransferase activity, including MtbA, the methylamine-specific CoM methylase and the pyl operon required for co-translational insertion of pyrrolysine into the active site of methylamine methyltransferases. RamA possesses a C-terminal ferredoxin-like domain capable of binding two tetranuclear iron-sulfur proteins. Mutliple ramA homologs were identified in genomes of methanogenic Archaea, often encoded near methyltrophic methyltransferase genes. RamA homologs are also encoded in a diverse selection of bacterial genomes, often located near genes for corrinoid-dependent methyltransferases. These results suggest that RamA mediates reductive activation of corrinoid proteins and that it is the first functional archetype of COG3894, a family of redox proteins of unknown function.