Methanogenic Archaeon Research Articles

Phenotypic and genomic characterization of Methanothermobacter wolfeii strain BSEL, a CO2-capturing archaeon with minimal nutrient requirements.

A new variant of Methanothermobacter wolfeii was isolated from an anaerobic digester using enrichment cultivation in anaerobic conditions. The new isolate was taxonomically identified via 16S rRNA gene sequencing and tagged as M. wolfeii BSEL. The whole genome of the new variant was sequenced and de novo assembled. Genomic variations between the BSEL strain and the type strain were discovered, suggesting evolutionary adaptations of the BSEL strain that conferred advantages while growing under a low concentration of nutrients. M. wolfeii BSEL displayed the highest specific growth rate ever reported for the wolfeii species (0.27 ± 0.03 h-1) using carbon dioxide (CO2) as unique carbon source and hydrogen (H2) as electron donor. M. wolfeii BSEL grew at this rate in an environment with ammonium (NH4+) as sole nitrogen source. The minerals content required to cultivate the BSEL strain was relatively low and resembled the ionic background of tap water without mineral supplements. Optimum growth rate for the new isolate was observed at 64°C and pH 8.3. In this work, it was shown that wastewater from a wastewater treatment facility can be used as a low-cost alternative medium to cultivate M. wolfeii BSEL. Continuous gas fermentation fed with a synthetic biogas mimic along with H2 in a bubble column bioreactor using M. wolfeii BSEL as biocatalyst resulted in a CO2 conversion efficiency of 97% and a final methane (CH4) titer of 98.5%v, demonstrating the ability of the new strain for upgrading biogas to renewable natural gas.IMPORTANCEAs a methanogenic archaeon, Methanothermobacter wolfeii uses CO2 as electron acceptor, producing CH4 as final product. The metabolism of M. wolfeii can be harnessed to capture CO2 from industrial emissions, besides producing a drop-in renewable biofuel to substitute fossil natural gas. If used as biocatalyst in new-generation CO2 sequestration processes, M. wolfeii has the potential to accelerate the decarbonization of the energy generation sector, which is the biggest contributor of CO2 emissions worldwide. Nonetheless, the development of CO2 sequestration archaeal-based biotechnology is still limited by an uncertainty in the requirements to cultivate methanogenic archaea and the unknown longevity of archaeal cultures. In this study, we report the adaptation, isolation, and phenotypic characterization of a novel variant of M. wolfeii, which is capable of maximum growth with minimal nutrients input. Our findings demonstrate the potential of this variant for the production of renewable natural gas, paving the way for the development of more efficient and sustainable CO2 sequestration processes.

A hybrid photocatalytic system enables direct glucose utilization for methanogenesis

Integration of methanogenic archaea with photocatalysts presents a sustainable solution for solar-driven methanogenesis. However, maximizing CH4 conversion efficiency remains challenging due to the intrinsic energy conservation and strictly restricted substrates of methanogenic archaea. Here, we report a solar-driven biotic-abiotic hybrid (biohybrid) system by incorporating cadmium sulfide (CdS) nanoparticles with a rationally designed methanogenic archaeon Methanosarcina acetivorans C2A, in which the glucose synergist protein and glucose kinase, an energy-efficient route for glucose transport and phosphorylation from Zymomonas mobilis, were implemented to facilitate nonnative substrate glucose for methanogenesis. We demonstrate that the photo-excited electrons facilitate membrane-bound electron transport chain, thereby augmenting the Na+ and H+ ion gradients across membrane to enhance adenosine triphosphate (ATP) synthesis. Additionally, this biohybrid system promotes the metabolism of pyruvate to acetyl coenzyme A (AcCoA) and inhibits the flow of AcCoA to the tricarboxylic acid (TCA) cycle, resulting in a 1.26-fold augmentation in CH4 production from glucose-derived carbon. Our results provide a unique strategy for enhancing methanogenesis through rational biohybrid design and reprogramming, which gives a promising avenue for sustainably manufacturing value-added chemicals.

Development of a rapid and highly accurate method for 13C tracer-based metabolomics and its application on a hydrogenotrophic methanogen.

Microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a rapid and highly accurate method to determine isotopomer patterns in isotopically labeled compounds. Here, we developed a novel method for tracer-based metabolomics using CE-MS for underivatized proteinogenic amino acids. The method consisting of a ZipChip CE system and a high-resolution Orbitrap Fusion Tribrid mass spectrometer allows us to obtain highly accurate data from 1μl of 100nmol/l amino acids comparable to a mere 1 [Formula: see text] 104-105 prokaryotic cells. To validate the capability of the CE-MS method, we analyzed 16 protein-derived amino acids from a methanogenic archaeon Methanothermobacter thermautotrophicus as a model organism, and the mass spectra showed sharp peaks with low mass errors and background noise. Tracer-based metabolome analysis was then performed to identify the central carbon metabolism in M. thermautotrophicus using 13C-labeled substrates. The mass isotopomer distributions of serine, aspartate, and glutamate revealed the occurrence of both the Wood-Ljungdahl pathway and an incomplete reductive tricarboxylic acid cycle for carbon fixation. In addition, biosynthesis pathways of 15 amino acids were constructed based on the mass isotopomer distributions of the detected protein-derived amino acids, genomic information, and public databases. Among them, the presence of alternative enzymes of alanine dehydrogenase, ornithine cyclodeaminase, and homoserine kinase was suggested in the biosynthesis pathways of alanine, proline, and threonine, respectively. To our knowledge, the novel 13C tracer-based metabolomics using CE-MS can be considered the most efficient method to identify central carbon metabolism and amino acid biosynthesis pathways and is applicable to any kind of isolated microbe.

A shift between mineral and nonmineral sources of iron and sulfur causes proteome-wide changes in Methanosarcina barkeri.

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 (FeS2). Recently, paradigm-shifting studies indicate that Fe and S in FeS2 can be made bioavailable by methanogens through a reductive dissolution process. However, the impact of the utilization of FeS2, 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 FeS2, 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 FeS2, 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.

The minimal SUF system is not required for Fe–S cluster biogenesis in the methanogenic archaeon Methanosarcina acetivorans

Iron–sulfur (Fe–S) proteins are essential for the ability of methanogens to carry out methanogenesis and biological nitrogen fixation (diazotrophy). Nonetheless, the factors involved in Fe–S cluster biogenesis in methanogens remain largely unknown. The minimal SUF Fe–S cluster biogenesis system (i.e., SufBC) is postulated to serve as the primary system in methanogens. Here, the role of SufBC in Methanosarcina acetivorans, which contains two sufCB gene clusters, was investigated. The CRISPRi-dCas9 and CRISPR-Cas9 systems were utilized to repress or delete sufC1B1 and sufC2B2, respectively. Neither the dual repression of sufC1B1 and sufC2B2 nor the deletion of both sufC1B1 and sufC2B2 affected the growth of M. acetivorans under any conditions tested, including diazotrophy. Interestingly, deletion of only sufC1B1 led to a delayed-growth phenotype under all growth conditions, suggesting that the deletion of sufC2B2 acts as a suppressor mutation in the absence of sufC1B1. In addition, the deletion of sufC1B1 and/or sufC2B2 did not affect the total Fe–S cluster content in M. acetivorans cells. Overall, these results reveal that the minimal SUF system is not required for Fe–S cluster biogenesis in M. acetivorans and challenge the universal role of SufBC in Fe–S cluster biogenesis in methanogens.

Assimilatory sulfate reduction in the marine methanogen Methanothermococcus thermolithotrophicus

Methanothermococcus thermolithotrophicus is the only known methanogen that grows on sulfate as its sole sulfur source, uniquely uniting methanogenesis and sulfate reduction. Here we use physiological, biochemical and structural analyses to provide a snapshot of the complete sulfate reduction pathway of this methanogenic archaeon. We find that later steps in this pathway are catalysed by atypical enzymes. PAPS (3′-phosphoadenosine 5′-phosphosulfate) released by APS kinase is converted into sulfite and 3′-phosphoadenosine 5′-phosphate (PAP) by a PAPS reductase that is similar to the APS reductases of dissimilatory sulfate reduction. A non-canonical PAP phosphatase then hydrolyses PAP. Finally, the F420-dependent sulfite reductase converts sulfite to sulfide for cellular assimilation. While metagenomic and metatranscriptomic studies suggest that the sulfate reduction pathway is present in several methanogens, the sulfate assimilation pathway in M. thermolithotrophicus is distinct. We propose that this pathway was ‘mix-and-matched’ through the acquisition of assimilatory and dissimilatory enzymes from other microorganisms and then repurposed to fill a unique metabolic role.

In Situ Probing of the Intrinsic Adhesion Strength of Single Anaerobic Microbial Cells.

Probing the single-cell mechanobiology in situ is imperative for microbial processes in the medical, industrial, and agricultural realms, but it remains a challenge. Herein, we present a single-cell force microscopy method that can be used to measure microbial adhesion strength under anaerobic conditions in situ. This method integrates atomic force microscopy with an anaerobic liquid cell and inverted fluorescence microscopy. We obtained the nanomechanical measurements of the single anaerobic bacterium Ethanoligenens harbinense YUAN-3 and the methanogenic archaeon Methanosarcina acetivorans C2A and their nanoscale adhesion forces in the presence of sulfoxaflor, a successor of neonicotinoid pesticides. This study presents a new tool for in situ single-cell force measurements of various anoxic and anaerobic species and provides new perspectives for evaluating the potential environmental risk of neonicotinoid applications in ecosystems.

Structural and biochemical elucidation of class I hybrid cluster protein natively extracted from a marine methanogenic archaeon.

Whilst widespread in the microbial world, the hybrid cluster protein (HCP) has been paradoxically a long-time riddle for microbiologists. During three decades, numerous studies on a few model organisms unravelled its structure and dissected its metal-containing catalyst, but the physiological function of the enzyme remained elusive. Recent studies on bacteria point towards a nitric oxide reductase activity involved in resistance during nitrate and nitrite reduction as well as host infection. In this study, we isolated and characterised a naturally highly produced HCP class I from a marine methanogenic archaeon grown on ammonia. The crystal structures of the enzyme in a reduced and partially oxidised state, obtained at a resolution of 1.45 and 1.36-Å, respectively, offered a precise picture of the archaeal enzyme intimacy. There are striking similarities with the well-studied enzymes from Desulfovibrio species regarding sequence, kinetic parameters, structure, catalyst conformations, and internal channelling systems. The close phylogenetic relationship between the enzymes from Methanococcales and many Bacteria corroborates this similarity. Indeed, Methanococcales HCPs are closer to these bacterial homologues than to any other archaeal enzymes. The relatively high constitutive production of HCP in M. thermolithotrophicus, in the absence of a notable nitric oxide source, questions the physiological function of the enzyme in these ancient anaerobes.

A novel interdomain consortium from a Costa Rican oil well composed of Methanobacterium cahuitense sp. nov. and Desulfomicrobium aggregans sp. nov.

A novel interdomain consortium composed of a methanogenic Archaeon and a sulfate-reducing bacterium was isolated from a microbial biofilm in an oil well in Cahuita National Park, Costa Rica. Both organisms can be grown in pure culture or as stable co-culture. The methanogenic cells were non-motile rods producing CH4 exclusively from H2/CO2. Cells of the sulfate-reducing partner were motile rods forming cell aggregates. They utilized hydrogen, lactate, formate, and pyruvate as electron donors. Electron acceptors were sulfate, thiosulfate, and sulfite. 16S rRNA sequencing revealed 99% gene sequence similarity of strain CaP3V-M-L2AT to Methanobacterium subterraneum and 98.5% of strain CaP3V-S-L1AT to Desulfomicrobium baculatum. Both strains grew from 20 to 42 °C, pH 5.0–7.5, and 0–4% NaCl. Based on our data, type strains CaP3V-M-L2AT (= DSM 113354 T = JCM 39174 T) and CaP3V-S-L1AT (= DSM 113299 T = JCM 39179 T) represent novel species which we name Methanobacterium cahuitense sp. nov. and Desulfomicrobium aggregans sp. nov.

In situ Raman quantitative monitoring of methanogenesis: Culture experiments of a deep-sea cold seep methanogenic archaeon.

Gas production from several metabolic pathways is a necessary process that accompanies the growth and central metabolism of some microorganisms. However, accurate and rapid nondestructive detection of gas production is still challenging. To this end, gas chromatography (GC) is primarily used, which requires sampling and sample preparation. Furthermore, GC is expensive and difficult to operate. Several researchers working on microbial gases are looking forward to a new method to accurately capture the gas trends within a closed system in real-time. In this study, we developed a precise quantitative analysis for headspace gas in Hungate tubes using Raman spectroscopy. This method requires only a controlled focus on the gas portion inside Hungate tubes, enabling nondestructive, real-time, continuous monitoring without the need for sampling. The peak area ratio was selected to establish a calibration curve with nine different CH4-N2 gaseous mixtures and a linear relationship was observed between the peak area ratio of methane to nitrogen and their molar ratios (A(CH4)/A(N2) = 6.0739 × n(CH4)/n(N2)). The results of in situ quantitative analysis using Raman spectroscopy showed good agreement with those of GC in the continuous monitoring of culture experiments of a deep-sea cold seep methanogenic archaeon. This method significantly improves the detection efficiency and shows great potential for in situ quantitative gas detection in microbiology. It can be a powerful complementary tool to GC.

Complete genome assembly and methylome dissection of Methanococcus aeolicus PL15/H p.

Although restriction-modification systems are found in both Eubacterial and Archaeal kingdoms, comparatively less is known about patterns of DNA methylation and genome defense systems in archaea. Here we report the complete closed genome sequence and methylome analysis of Methanococcus aeolicus PL15/H p , a strain of the CO2-reducing methanogenic archaeon and a commercial source for MaeI, MaeII, and MaeIII restriction endonucleases. The M. aeolicus PL15/H p genome consists of a 1.68 megabase circular chromosome predicted to contain 1,615 protein coding genes and 38 tRNAs. A combination of methylome sequencing, homology-based genome annotation, and recombinant gene expression identified five restriction-modification systems encoded by this organism, including the methyltransferase and site-specific endonuclease of MaeIII. The MaeIII restriction endonuclease was recombinantly expressed, purified and shown to have site-specific DNA cleavage activity in vitro.

Nitrite reductase activity in F420-dependent sulphite reductase (Fsr) from Methanocaldococcus jannaschii.

Methanocaldococcus jannaschii (Mj), a hyperthermophilic and evolutionarily deeply rooted methanogenic archaeon from a deep-sea hydrothermal vent, produces F420-dependent sulphite reductase (Fsr) in response to exposure to sulphite. This enzyme allows Mj to detoxify sulphite, a potent inhibitor of methyl coenzyme-M reductase (Mcr), by reducing it to sulphide with reduced coenzyme F420 (F420H2) as an electron donor; Mcr is essential for energy production for a methanogen. Fsr allows Mj to utilize sulphite as a sulphur source. Nitrite is another potent inhibitor of Mcr and is toxic to methanogens. It is reduced by most sulphite reductases. In this study, we report that MjFsr reduced nitrite to ammonia with F420H2 with physiologically relevant K m values (nitrite, 8.9 µM; F420H2, 9.7 µM). The enzyme also reduced hydroxylamine with a K m value of 112.4 µM, indicating that it was an intermediate in the reduction of nitrite to ammonia. These results open the possibility that Mj could use nitrite as a nitrogen source if it is provided at a low concentration of the type that occurs in its habitat.

Isolation and physiological properties of methanogenic archaea that degrade tetramethylammonium hydroxide

Tetramethylammonium hydroxide (TMAH) is a known toxic chemical used in the photolithography process of semiconductor photoelectronic processes. Significant amounts of wastewater containing TMAH are discharged from electronic industries. It is therefore attractive to apply anaerobic treatment to industrial wastewater containing TMAH. In this study, a novel TMAH-degrading methanogenic archaeon was isolated from the granular sludge of a psychrophilic upflow anaerobic sludge blanket (UASB) reactor treating synthetic wastewater containing TMAH. Although the isolate (strain NY-STAYD) was phylogenetically related to Methanomethylovorans uponensis, it was the only isolated Methanomethylovorans strain capable of TMAH degradation. Strain NY-STAYD was capable of degrading methylamine compounds, similar to the previously isolated Methanomethylovorans spp. While the strain was able to grow at temperatures ranging from 15 to 37°C, the cell yield was higher at lower temperatures. The distribution of archaeal cells affiliated with the genus Methanomethylovorans in the original granular sludge was investigated by fluorescence in situ hybridization (FISH) using specific oligonucleotide probe targeting 16S rRNA. The results demonstrated that the TMAH-degrading cells associated with the genus Methanomethylovorans were not intermingled with other microorganisms but rather isolated on the granule's surface as a lone dominant archaeon. KEY POINTS: • A TMAH-degrading methanogenic Methanomethylovorans strain was isolated • This strain was the only known Methanomethylovorans isolate that can degrade TMAH • The highest cell yield of the isolate was obtained at psychrophilic conditions.

Crystal Structure of Pyrrolysyl-tRNA Synthetase from a Methanogenic Archaeon ISO4-G1 and Its Structure-Based Engineering for Highly-Productive Cell-Free Genetic Code Expansion with Non-Canonical Amino Acids.

Pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). Previously, we achieved full productivity of cell-free protein synthesis for bulky non-canonical amino acids, including Nε-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS with structure-based mutations in and around the amino acid binding pocket (first-layer and second-layer mutations, respectively). Recently, the PylRS·tRNAPyl pair from a methanogenic archaeon ISO4-G1 was used for genetic code expansion. In the present study, we determined the crystal structure of the methanogenic archaeon ISO4-G1 PylRS (ISO4-G1 PylRS) and compared it with those of structure-known PylRSs. Based on the ISO4-G1 PylRS structure, we attempted the site-specific incorporation of Nε-(p-ethynylbenzyloxycarbonyl)-L-lysine (pEtZLys) into proteins, but it was much less efficient than that of TCO*Lys with M. alvus PylRS mutants. Thus, the first-layer mutations (Y125A and M128L) of ISO4-G1 PylRS, with no additional second-layer mutations, increased the protein productivity with pEtZLys up to 57 ± 8% of that with TCO*Lys at high enzyme concentrations in the cell-free protein synthesis.

Effects of environmental factors on low temperature anaerobic digestion of pig manure

In this study, we investigated the inhibitory effect of low temperature on biogas fermentation system. Biogas fermentation inoculum was domesticated at 4 °C, and subsequently inoculated into pig manure feedstock in a batch biogas fermentation process. A low-temperature biogas fermentation system was maintained at 9 °C, and its abiotic factors, bacterial community, and archaeal community were determined. The results showed that (1) the biogas fermentation lasted for 160 days, with a total gas production of 19,150 ml, including N2 production of 11,370 ml, CO2 production of 3,534 ml, and CH4 production of 3,031 ml. (2) The average relative abundance of the primary dominant bacterium operational taxonomic unit (OTU) was 30%, with 100% similarity to Pseudomonas caeni , atypical denitrifying bacterium. The average relative abundance of the secondary dominant bacterium OTU was 20.36%, with 99% similarity to Clostridium cellulovorans, a typical cellulose- and hemicellulose-degrading bacterium. The average relative abundance of the primary dominant methanogenic archaeon OTU was 7.22%, with 99% similarity to Methanosphaera cuniculi, a typical methylotrophic methanogen, and the secondary dominant methanogenic archaeon OTU had an average relative abundance of 4.15%, with 98% similarity to Methanocorpusculum sinense, a typical hydrogenotrophic methanogen. (3) The primary dominant bacterial genus Pseudomonas exhibited highly significant negative correlations with the dominant biogas-producing microorganisms such as Clostridium, Terrisporobacter, Turicibacter, Methanosphaera, and Methanocorpusculum. We concluded that (1) high concentrations of abiotic factors such as ammonia nitrogen, acetic acid, and propionic acid were important indicators of relatively poor operation of low-temperature biogas fermentation systems, while (2) high numbers of MCG archaea and Pseudomonas were important biotic factors showing comparatively poor operation of the system.

The surface groups of polystyrene nanoparticles control their interaction with the methanogenic archaeon Methanosarcina acetivorans

A better understanding of the interaction between nanoplastics and archaea is crucial to fill the knowledge gaps regarding the ecological safety of nanoplastics. As a vital source for global methane emissions, methanogenic archaea have unique cell membranes that are distinctly different from those in all other forms of life, little is known about their interaction with nanoplastics. Here, we show that polystyrene nanoparticles functionalized with sulfonic acid (PS-SO3H) and amino (PS-NH2) interact with this methanogenic archaeon in distinct ways. Although both of them have no significant phenotype effects on Methanosarcina acetivorans C2A, these nanoparticles could affect DNA-mediated transposition of this methanogenic archaeon, and PS-SO3H also downregulated nitrogen fixation, nitrogen cycle metabolic process, oxidoreductase activity, etc. In addition, both nanoplastics decreased the protein contents in the extracellular polymer substances (EPS), with distinct binding sequences to the functional groups of the EPS. The single particle atomic force microscopy revealed that the force between the amino group and the M. acetivorans C2A was greater than that of sulfonic acid group. Our results exhibit that the surface groups of polystyrene nanoparticles control their risk on the methanogenic archaea, and these effects might influence their contribution on global methane emission.

Growth rate-dependent coordination of catabolism and anabolism in the archaeon Methanococcus maripaludis under phosphate limitation.

Catabolic and anabolic processes are finely coordinated in microorganisms to provide optimized fitness under varying environmental conditions. Understanding this coordination and the resulting physiological traits reveals fundamental strategies of microbial acclimation. Here, we characterized the system-level physiology of Methanococcus maripaludis, a niche-specialized methanogenic archaeon, at different dilution rates ranging from 0.09 to 0.003 h-1 in chemostat experiments under phosphate (i.e., anabolic) limitation. Phosphate was supplied as the limiting nutrient, while formate was supplied in excess as the catabolic substrate and carbon source. We observed a decoupling of catabolism and anabolism resulting in lower biomass yield relative to catabolically limited cells at the same dilution rates. In addition, the mass abundance of several coarse-grained proteome sectors (i.e., combined abundance of proteins grouped based on their function) exhibited a linear relationship with growth rate, mostly ribosomes and their biogenesis. Accordingly, cellular RNA content also correlated with growth rate. Although the methanogenesis proteome sector was invariant, the metabolic capacity for methanogenesis, measured as methane production rates immediately after transfer to batch culture, correlated with growth rate suggesting translationally independent regulation that allows cells to only increase catabolic activity under growth-permissible conditions. These observations are in stark contrast to the physiology of M. maripaludis under formate (i.e., catabolic) limitation, where cells keep an invariant proteome including ribosomal content and a high methanogenesis capacity across a wide range of growth rates. Our findings reveal that M. maripaludis employs fundamentally different strategies to coordinate global physiology during anabolic phosphate and catabolic formate limitation.

CRISPR-Cas9 Toolkit for Genome Editing in an Autotrophic CO2-Fixing Methanogenic Archaeon.

ABSTRACTThe CRISPR-Cas9 system is a robust genome editing tool that is widely applied in eukaryotes and bacteria. However, use of this technique has only been developed for one species of Archaea, a domain of life ranking in parallel with Eukarya and Bacteria. In this study, we applied the CRISPR-Cas9 genome editing technique to Methanococcus maripaludis, an autotrophic and hydrogenotrophic methanogenic archaeon with a remarkably polyploid genome comprising up to ~55 chromosomal copies per cell. An editing plasmid was designed that encodes small guide RNA (sgRNA), Cas9 protein and an ~1-kb repair template (donor). Highly efficient (75% to 100%) and precise genome editing was achieved following one-step transformation. Significantly, the Cas9-based system efficiently deleted one or two genes and a large DNA fragment (~9 kb) and even synchronously deleted 13 genes located at three loci in all chromosomal copies of M. maripaludis. Moreover, precise in situ genome modifications, such as gene tagging and multiple- and even single-nucleotide mutagenesis, were also introduced with high efficiency. Further, as a proof of concept, precise mutagenesis at the nucleotide level allowed the engineering of both transcriptional and translational activities. Mutations were introduced into an archaeal promoter BRE (transcription factor B [TFB] recognition element), a terminator U-tract region, and a gene coding region. Stop codon introduction into a gene through single-nucleotide substitution shut down its expression, providing an alternative strategy for gene inactivation. In conclusion, the robust CRISPR-Cas9 genetic toolkit developed in this investigation greatly facilitates the application of M. maripaludis as a model system in the study of archaeal biology and biotechnology development, particularly CO2-based biotechnologies.IMPORTANCE Archaea are prokaryotes with intriguing biological characteristics. They possess bacterial cell structures but eukaryotic homologous information processing machinery and eukaryotic featured proteins. Archaea also display excellent adaptability to extreme environments and play pivotal roles in ecological processes, thus exhibiting valuable biotechnological potential. However, the in-depth understanding and practical application of archaea are much lagging, because only a minority of pure cultures are available, and even worse, very few can be genetically manipulated. This work developed CRISPR-Cas9-based genome editing technology in Methanococcus maripaludis, a CO2-fixing methanogenic archaeon. The CRISPR-Cas9 approach developed in this study provides an elegant and efficient genome editing toolkit that can be applied in the knockout of single or multiple genes, in situ gene tagging, multiple- or single-nucleotide mutagenesis, and inactivation of gene expression by introduction of stop codons. The successful development of the CRISPR-Cas9 toolkit will facilitate the application of M. maripaludis in archaeal biology research and biotechnology development, particularly CO2-derived biotechnologies.

Metabolic Synergy between Human Symbionts Bacteroides and Methanobrevibacter.

ABSTRACTTrophic interactions between microbes are postulated to determine whether a host microbiome is healthy or causes predisposition to disease. Two abundant taxa, the Gram-negative heterotrophic bacterium Bacteroides thetaiotaomicron and the methanogenic archaeon Methanobrevibacter smithii, are proposed to have a synergistic metabolic relationship. Both organisms play vital roles in human gut health; B. thetaiotaomicron assists the host by fermenting dietary polysaccharides, whereas M. smithii consumes end-stage fermentation products and is hypothesized to relieve feedback inhibition of upstream microbes such as B. thetaiotaomicron. To study their metabolic interactions, we defined and optimized a coculture system and used software testing techniques to analyze growth under a range of conditions representing the nutrient environment of the host. We verify that B. thetaiotaomicron fermentation products are sufficient for M. smithii growth and that accumulation of fermentation products alters secretion of metabolites by B. thetaiotaomicron to benefit M. smithii. Studies suggest that B. thetaiotaomicron metabolic efficiency is greater in the absence of fermentation products or in the presence of M. smithii. Under certain conditions, B. thetaiotaomicron and M. smithii form interspecies granules consistent with behavior observed for syntrophic partnerships between microbes in soil or sediment enrichments and anaerobic digesters. Furthermore, when vitamin B12, hematin, and hydrogen gas are abundant, coculture growth is greater than the sum of growth observed for monocultures, suggesting that both organisms benefit from a synergistic mutual metabolic relationship.IMPORTANCE The human gut functions through a complex system of interactions between the host human tissue and the microbes which inhabit it. These diverse interactions are difficult to model or examine under controlled laboratory conditions. We studied the interactions between two dominant human gut microbes, B. thetaiotaomicron and M. smithii, using a seven-component culturing approach that allows the systematic examination of the metabolic complexity of this binary microbial system. By combining high-throughput methods with machine learning techniques, we were able to investigate the interactions between two dominant genera of the gut microbiome in a wide variety of environmental conditions. Our approach can be broadly applied to studying microbial interactions and may be extended to evaluate and curate computational metabolic models. The software tools developed for this study are available as user-friendly tutorials in the Department of Energy KBase.

An Archaea-specific c-type cytochrome maturation machinery is crucial for methanogenesis in Methanosarcina acetivorans

c-Type cytochromes (cyt c) are proteins that undergo post-translational modification to covalently bind heme, which allows them to facilitate redox reactions in electron transport chains across all domains of life. Genomic evidence suggests that cyt c are involved in electron transfer processes among the Archaea, especially in members that produce or consume the potent greenhouse gas methane. However, neither the maturation machinery for cyt c in Archaea nor their role in methane metabolism has ever been functionally characterized. Here, we have used CRISPR-Cas9 genome editing tools to map a distinct pathway for cyt c biogenesis in the model methanogenic archaeon Methanosarcina acetivorans, and have also identified substrate-specific functional roles for cyt c during methanogenesis. Although the cyt c maturation machinery from M. acetivorans is universally conserved in the Archaea, our evolutionary analyses indicate that different clades of Archaea acquired this machinery through multiple independent horizontal gene transfer events from different groups of Bacteria. Overall, we demonstrate the convergent evolution of a novel Archaea-specific cyt c maturation machinery and its physiological role during methanogenesis, a process which contributes substantially to global methane emissions.