Methanovulcanius yangii gen. nov., sp. nov., a hydrogenotrophic methanogen, isolated from a submarine mud volcano in the offshore area of southwestern Taiwan.

A novel mesophilic, hydrogenotrophic methanogen, strain CYW5T, was isolated from a sediment sample of a piston core collected from submarine mud volcano MV5 located in the offshore area of southwestern Taiwan. Cells of strain CYW5T were irregular coccids, 0.5-1.0 µm in diameter and lysed easily by 0.01 % sodium dodecyl sulphate (SDS) treatment. Strain CYW5Tutilized formate or hydrogen plus carbon dioxide as catabolic substrates for methanogenesis. The optimal growth conditions were 37 °C, 0.043-0.085 M NaCl and pH 6.02-7.32. The genomic DNA G+C content calculated from the genome sequence of strain CYW5T was 56.2 mol%. The results of phylogenetic analysis of 16S rRNA gene sequences indicated that strain CYW5T represented a member of the family Methanomicrobiaceae in the order Methanomicrobiales, and was closely related to the members of the genus Methanogenium. The most closely related species was Methanogenium cariaci JR1T (94.9 % of 16S rRNA gene sequence identity). The average nucleotide identity and average amino acid identity values between strain CYW5T and members of the family Methanomicrobiaceae were 74.7-78.5 % and 49.1-64.9%, respectively. Although many of the morphological and physiological characteristics of strain CYW5T and the species of the genus Methanogenium were similar, they were distinguishable by the differences in genomic G+C content and temperature, NaCl and pH ranges for growth. Based on these phenotypic, phylogenetic and genomic results, we propose that strain CYW5T represents a novel species, of a novel genus, named Methanovulcanius yangii gen. nov., sp. nov. The type strain is CYW5T (=BCRC AR10048T=DSM 100756T=NBRC 111404T).

Lipid environment modulates processivity and kinetics of a presenilin homolog acting on multiple substrates in vitro.

Intramembrane proteases (IPs) hydrolyze peptides in the lipid membrane. IPs participate in a number of cellular pathways including immune response and surveillance, and cholesterol biosynthesis, and they are exploited by viruses for replication. Despite their broad importance across biology, how activity is regulated in the cell to control protein maturation and release of specific bioactive peptides at the right place and right time remains largely unanswered, particularly for the intramembrane aspartyl protease (IAP) subtype. At a molecular biochemical level, different IAP homologs can cleave non-biological substrates, and there is no sequence recognition motif among the nearly 150 substrates identified for just one IAP, presenilin-1, the catalytic component of γ-secretase known for its involvement in the production of amyloid-ß plaques associated with Alzheimer disease. Here we used gel-based assays combined with quantitative mass spectrometry and FRET-based kinetics assays to probe the cleavage profile of the presenilin homolog from the methanogen Methanoculleus marisnigri JR1 as a function of the surrounding lipid-mimicking environment, either detergent micelles or bicelles. We selected four biological IAP substrates that have not undergone extensive cleavage profiling previously, namely, the viral core protein of Hepatitis C virus, the viral core protein of Classical Swine Fever virus, the transmembrane segment of Notch-1, and the tyrosine receptor kinase ErbB4. Our study demonstrates a proclivity toward cleavage of substrates at positions of low average hydrophobicity and a consistent role for the lipid environment in modulating kinetic properties.

Methanofollis propanolicus sp. nov., a novel archaeal isolate from a Costa Rican oil well that uses propanol for methane production.

A novel methanogenic strain, CaP3V-MF-L2AT, was isolated from an exploratory oil well from Cahuita National Park, Costa Rica. The cells were irregular cocci, 0.8-1.8 µm in diameter, stained Gram-negative and were motile. The strain utilized H2/CO2, formate and the primary and secondary alcohols 1-propanol and 2-propanol for methanogenesis, but not acetate, methanol, ethanol, 1-butanol or 2-butanol. Acetate was required as carbon source. The novel isolate grew at 25-40 °C, pH 6.0-7.5 and 0-2.5% (w/v) NaCl. 16S rRNA gene sequence analysis revealed that the strain is affiliated to the genus Methanofollis. It shows 98.8% sequence similarity to its closest relative Methanofollis ethanolicus. The G + C content is 60.1 mol%. Based on the data presented here type strain CaP3V-MF-L2AT (= DSM 113321T = JCM 39176T) represents a novel species, Methanofollis propanolicus sp. nov.

High temperature shock threatens methane production via disturbing microbial interactions in anaerobic digestion.

Operational strategies shape microbial interactions determining anaerobic digesting process, but it is unclear whether and how the microbial network properties impact gas generation, especially in the transitional stage after operations. This research examined how the high temperature shock affected microbial diversity and network traits connected with the biogas production in a swine manure-fed anaerobic digester. Rising temperature (from 35 °C to 50 °C) significantly reduced biogas and methane production (p < 0.001) in the transitional stage due to the syntrophic loss of Methanomicrobiaceae and Firmicutes affiliated families. The high temperature shock reduced network modularity and thus caused the system functioning loss. Furthermore, the methanogenic stability was disrupted by high temperature shock (reduced the abundance of Methanosphaera but increased the abundance of Methanoculleus), which may result in the subsequent dysbiosis with other syntrophic communities. These findings suggest that the increased temperature-induced high network complexity and stability, but microbial communities need more time to restore the microenvironment via establishing the interactions of keystone species.

Development of real-time PCR methods for the quantification of Methanoculleus, Methanosarcina and Methanobacterium in anaerobic digestion.

Anaerobic digestion is a growing technology to manage organic waste and produce bioenergy. To promote this technology, it is essential to know, at the molecular level, the dynamics of microbial communities, specifically the methanogenic community. In the present study, three primer pairs were selected from seven primer pairs which were designed and tested with different concentrations and conditions to detect Methanosarcina, Methanoculleus and Methanobacterium by real-time PCR based on the SYBR Green System. The functionality of the developed methods was demonstrated by the high linear relationship of the standard curves, and the specificity of each primer was empirically verified by testing DNA isolated from methane-producing and non-producing strains. These assays also exhibited good repeatability and reproducibility, which indicates the robustness of the methods. The described primers were successfully used to investigate the methanogenic communities of 10 samples from an anaerobic co-digestion. The genus Methanosarcina was the dominant methanogenic group.

Characterizing the N- and O-linked glycans of the PGF-CTERM sorting domain-containing S-layer protein of Methanoculleus marisnigri.

The glycosylation of structural proteins is a widespread posttranslational modification in Archaea. Although only a handful of archaeal N-glycan structures have been determined to date, it is evident that the diversity of structures expressed is greater than in the other domains of life. Here, we report on our investigation of the N- and O-glycan modifications expressed by Methanoculleus marisnigri, a mesophilic methanogen from the Order Methanomicrobiales. Unusually, mass spectrometry (MS) analysis of purified archaella revealed no evidence for N- or O-glycosylation of the constituent archaellins, In contrast, the S-layer protein, identified as a PGF-CTERM sorting domain-containing protein encoded by MEMAR_RS02690, is both N- and O-glycosylated. Two N-glycans were identified by NMR and MS analysis: a trisaccharide α-GlcNAc-4-ß-GlcNAc3NGaAN-4-ß-Glc-Asn where the second residue is 2-N-acetyl, 3-N-glyceryl-glucosamide and a disaccharide ß-GlcNAc3NAcAN-4-ß-Glc-Asn, where the terminal residue is 2,3 di-N-acetyl-glucosamide. The same trisaccharide was also found N-linked to a type IV pilin. The S-layer protein is also extensively modified in the threonine-rich region near the C-terminus with O-glycans composed exclusively of hexoses. While the S-layer protein has a predicted PGF-CTERM processing site, no evidence of a truncated and lipidated C-terminus, the expected product of processing by an archaeosortase, was found. Finally, NMR also identified a polysaccharide expressed by M. marisnigri and composed of a repeating tetrasaccharide unit of [-2-ß-Ribf-3-α-Rha2OMe-3-α-Rha - 2-α-Rha-]. This is the first report of N- and O-glycosylation in an archaeon from the Order Methanomicrobiales.

Deconstructing Methanosarcina acetivorans into an acetogenic archaeon.

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

The glutamyl tail length of the cofactor F420 in the methanogenic Archaea Methanosarcina thermophila and Methanoculleus thermophilus.

The cofactor F420 is synthesized by many different organisms and as a redox cofactor, it plays a crucial role in the redox reactions of catabolic and biosynthetic metabolic pathways. It consists of a deazaflavin structure, which is linked via lactate to an oligoglutamate chain, that can vary in length. In the present study, the methanogenic Archaea Methanosarcina thermophila and Methanoculleus thermophilus were cultivated on different carbon sources and their coenzyme F420 composition has been assayed by reversed-phase ion-pair high-performance liquid chromatography with fluorometric detection regarding both, overall cofactor F420 production and distribution of F420 glutamyl tail length. In Methanosarcina thermophila cultivated on methanol, acetate, and a mixture of acetate and methanol, the most abundant cofactors were F420-5 and F420-4, whereby the last digit refers to the number of expressed glutamyl rests. By contrast, in the obligate CO2 reducing Methanoculleus thermophilus the most abundant cofactors were F420-3 and F420-4. In Methanosarcina thermophila, the relative proportions of the expressed F420 tail length changed during batch growth on all three carbon sources. Over time F420-3 and F420-4 decreased while F420-5 and F420-6 increased in their relative proportion in comparison to total F420 content. In contrast, in Methanoculleus thermophilus the relative abundance of the different F420 cofactors remained stable. It was also possible to differentiate the two methanogenic Archaea based on the glutamyl tail length of the cofactor F420. The cofactor F420-5 in concentrations >2% could only be assigned to Methanosarcina thermophila. In all four variants a trend for a positive correlation between the DNA concentration and the total concentration of the cofactor could be shown. Except for the variant Methanosarcinathermophila with acetate as sole carbon source the same could be shown between the concentration of the mcrA gene copy number and the total concentration of the cofactor.

Eubacterium coprostanoligenes and Methanoculleus identified as potential producers of metabolites that contribute to swine manure foaming.

AIM: Swine manure foaming is a major problem, causing damage to property, livestock, and people. Here, we identified the main chemicals and microbes that contribute to foaming. METHODS AND RESULTS: Foaming and non-foaming swine manure were sampled from farms in Iowa and Illinois. Targeted and untargeted metabolomics analyses identified chemical markers that differed between foaming and non-foaming manure and between manure layers. Microbial community analysis and metagenomics were performed on a subset of samples. Foam contained significantly higher levels of total bile acids and long chain fatty acids like palmitic, stearic and oleic acid than the other manure layers. Foam layers also had significantly higher levels of ubiquinone 9 and ubiquinone 10. The slurry layer of foaming samples contained more alanine, isoleucine/leucine, diacylglycerols (DG), phosphtatidylethanolamines, and vitamin K2, while ceramide was significantly increased in the slurry layer of non-foaming samples. Eubacterium coprostanoligenes and Methanoculleus were more abundant in foaming samples, and E. coprostanoligenes was significantly correlated with levels of DG. Genes involved in diacylglycerol biosynthesis and in the biosynthesis of branched-chain hydrophobic amino acids were overrepresented in foaming samples. CONCLUSIONS: A mechanism for manure foaming is hypothesized in which proliferation of Methanoculleus leads to excessive production of methane, while production of DG by E. coprostanoligenes and hydrophobic proteins by Methanosphaera stadtmanae facilitates bubble formation and stabilization. SIGNIFICANCE AND IMPACT OF STUDY: While some chemical and biological treatments have been developed to treat swine manure foaming, its causes remain unknown. We identified key microbes and metabolites that correlate with foaming and point to possible roles of other factors like animal feed.

Patterns of syntrophic interactions in methanogenic conversion of propionate.

Methanogenesis is central to anaerobic digestion processes. The conversion of propionate as a key intermediate for methanogenesis requires syntrophic interactions between bacterial and archaeal partners. In this study, a series of methanogenic enrichments with propionate as the sole substrate were developed to identify microbial populations specifically involved in syntrophic propionate conversion. These rigorously controlled propionate enrichments exhibited functional stability with consistent propionate conversion and methane production; yet, the methanogenic microbial communities experienced substantial temporal dynamics, which has important implications on the understanding of mechanisms involved in microbial community assembly in anaerobic digestion. Syntrophobacter was identified as the most abundant and consistent bacterial partner in syntrophic propionate conversion regardless of the origin of the source culture, the concentration of propionate, or the temporal dynamics of the culture. In contrast, the methanogen partners involved in syntrophic propionate conversion lacked consistency, as the dominant methanogens varied as a function of process condition and temporal dynamics. Methanoculleus populations were specifically enriched as the syntrophic partner at inhibitory levels of propionate, likely due to the ability to function under unfavorable environmental conditions. Syntrophic propionate conversion was carried out exclusively via transformation of propionate into acetate and hydrogen in enrichments established in this study. Microbial populations highly tolerant of elevated propionate, represented by Syntrophobacter and Methanoculleus, are of great significance in understanding methanogenic activities during process perturbations when propionate accumulation is frequently encountered. Key points • Syntrophobacter was the most consistent bacterial partner in propionate metabolism. • Diverse hydrogenotrophic methanogen populations could serve as syntrophic partners. • Methanoculleus emerged as a methanogen partner tolerant of elevated propionate.

Characterization of Blf4, an Archaeal Lytic Virus Targeting a Member of the Methanomicrobiales.

Today, the number of known viruses infecting methanogenic archaea is limited. Here, we report on a novel lytic virus, designated Blf4, and its host strain Methanoculleus bourgensis E02.3, a methanogenic archaeon belonging to the Methanomicrobiales, both isolated from a commercial biogas plant in Germany. The virus consists of an icosahedral head 60 nm in diameter and a long non-contractile tail of 125 nm in length, which is consistent with the new isolate belonging to the Siphoviridae family. Electron microscopy revealed that Blf4 attaches to the vegetative cells of M. bourgensis E02.3 as well as to cellular appendages. Apart from M. bourgensis E02.3, none of the tested Methanoculleus strains were lysed by Blf4, indicating a narrow host range. The complete 37 kb dsDNA genome of Blf4 contains 63 open reading frames (ORFs), all organized in the same transcriptional direction. For most of the ORFs, potential functions were predicted. In addition, the genome of the host M. bourgensis E02.3 was sequenced and assembled, resulting in a 2.6 Mbp draft genome consisting of nine contigs. All genes required for a hydrogenotrophic lifestyle were predicted. A CRISPR/Cas system (type I-U) was identified with six spacers directed against Blf4, indicating that this defense system might not be very efficient in fending off invading Blf4 virus.

Similar Methanogenic Shift but Divergent Syntrophic Partners in Anaerobic Digesters Exposed to Direct versus Successive Ammonium Additions.

During anaerobic digestion (AD) of protein-rich wastewater, ammonium (NH4+) is released by amino acid degradation. High NH4+ concentrations disturb the AD microbiome balance, leading to process impairments. The sensitivity of the AD microbiome to NH4+ and the inhibition threshold depend on multiple parameters, especially the previous microbial acclimation to ammonium stress. However, little is known about the effect of different NH4+ acclimation strategies on the differential expression of key active microbial taxa. Here, we applied NH4+ inputs of increasing intensity (from 1.7 to 15.2 g N-NH4+ liters-1) in batch assays fed with synthetic wastewater, according to two different strategies: (i) direct independent inputs at a unique target concentration and (ii) successive inputs in a stepwise manner. In both strategies, along the NH4+ gradient, the active methanogens shifted from acetoclastic Methanosaeta to Methanosarcina and eventually hydrogenotrophic Methanoculleus. Despite shorter latency times, the successive input modality led to lower methane production rate, lower soluble chemical oxygen demand (sCOD) removal efficiency, and lower half maximal inhibitory concentration, together with higher volatile fatty acid (VFA) accumulation, compared to the independent input modality. These differential performances were associated with a drastically distinct succession pattern of the active bacterial partners in both experiments. In particular, the direct exposure modality was characterized by a progressive enrichment of VFA producers (mainly Tepidimicrobium) and syntrophic VFA oxidizers (mainly Syntrophaceticus) with increasing NH4+ concentration, while the successive exposure modality was characterized by a more dynamic succession of VFA producers (mainly Clostridium, Sporanaerobacter, Terrisporobacter) and syntrophic VFA oxidizers (mainly Tepidanaerobacter, Syntrophomonas). These results bring relevant insights for improved process management through inoculum adaptation, bioaugmentation, or community-driven optimization. IMPORTANCE Anaerobic digestion (AD) is an attractive biotechnological process for wastewater bioremediation and bioenergy production in the form of methane-rich biogas. However, AD can be inhibited by ammonium generated by protein-rich effluent, commonly found in agro-industrial activities. Insights in the microbial community composition and identification of AD key players are crucial for anticipating process impairments in response to ammonium stress. They can also help in defining an optimal microbiome adapted to high ammonium levels. Here, we compared two strategies for acclimation of AD microbiome to increasing ammonium concentration to better understand the effect of this stress on the methanogens and their bacterial partners. Our results suggest that long-term cumulative exposure to ammonia disrupted the AD microbiome more strongly than direct (independent) ammonium additions. We identified bioindicators with different NH4+ tolerance capacity among VFA producers and syntrophic VFA oxidizers.

Early response of methanogenic archaea to H2 as evaluated by metagenomics and metatranscriptomics.

BACKGROUND: The molecular machinery of the complex microbiological cell factory of biomethane production is not fully understood. One of the process control elements is the regulatory role of hydrogen (H2). Reduction of carbon dioxide (CO2) by H2 is rate limiting factor in methanogenesis, but the community intends to keep H2 concentration low in order to maintain the redox balance of the overall system. H2 metabolism in methanogens becomes increasingly important in the Power-to-Gas renewable energy conversion and storage technologies. RESULTS: The early response of the mixed mesophilic microbial community to H2 gas injection was investigated with the goal of uncovering the first responses of the microbial community in the CH4 formation and CO2 mitigation Power-to-Gas process. The overall microbial composition changes, following a 10 min excessive bubbling of H2 through the reactor, was investigated via metagenome and metatranscriptome sequencing. The overall composition and taxonomic abundance of the biogas producing anaerobic community did not change appreciably 2 hours after the H2 treatment, indicating that this time period was too short to display differences in the proliferation of the members of the microbial community. There was, however, a substantial increase in the expression of genes related to hydrogenotrophic methanogenesis of certain groups of Archaea. As an early response to H2 exposure the activity of the hydrogenotrophic methanogenesis in the genus Methanoculleus was upregulated but the hydrogenotrophic pathway in genus Methanosarcina was downregulated. The RT-qPCR data corroborated the metatranscriptomic RESULTS: H2 injection also altered the metabolism of a number of microbes belonging in the kingdom Bacteria. Many Bacteria possess the enzyme sets for the Wood-Ljungdahl pathway. These and the homoacetogens are partners for syntrophic community interactions between the distinct kingdoms of Archaea and Bacteria. CONCLUSIONS: External H2 regulates the functional activity of certain Bacteria and Archaea. The syntrophic cross-kingdom interactions in H2 metabolism are important for the efficient operation of the Power-to-Gas process. Therefore, mixed communities are recommended for the large scale Power-to-Gas process rather than single hydrogenotrophic methanogen strains. Fast and reproducible response from the microbial community can be exploited in turn-off and turn-on of the Power-to-Gas microbial cell factories.

Crystal structure of an archaeal CorB magnesium transporter.

CNNM/CorB proteins are a broadly conserved family of integral membrane proteins with close to 90,000 protein sequences known. They are associated with Mg2+ transport but it is not known if they mediate transport themselves or regulate other transporters. Here, we determine the crystal structure of an archaeal CorB protein in two conformations (apo and Mg2+-ATP bound). The transmembrane DUF21 domain exists in an inward-facing conformation with a Mg2+ ion coordinated by a conserved π-helix. In the absence of Mg2+-ATP, the CBS-pair domain adopts an elongated dimeric configuration with previously unobserved domain-domain contacts. Hydrogen-deuterium exchange mass spectrometry, analytical ultracentrifugation, and molecular dynamics experiments support a role of the structural rearrangements in mediating Mg2+-ATP sensing. Lastly, we use an in vitro, liposome-based assay to demonstrate direct Mg2+ transport by CorB proteins. These structural and functional insights provide a framework for understanding function of CNNMs in Mg2+ transport and associated diseases.

Structural Insight into the Substrate Scope of Viperin and Viperin-like Enzymes from Three Domains of Life.

Viperin is a member of the radical S-adenosylmethionine superfamily and has been shown to restrict the replication of a wide range of RNA and DNA viruses. We recently demonstrated that human viperin (HsVip) catalyzes the conversion of CTP to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP or ddh-synthase), which acts as a chain terminator for virally encoded RNA-dependent RNA polymerases from several flaviviruses. Viperin homologues also exist in non-chordate eukaryotes (e.g., Cnidaria and Mollusca), numerous fungi, and members of the archaeal and eubacterial domains. Recently, it was reported that non-chordate and non-eukaryotic viperin-like homologues are also ddh-synthases and generate a diverse range of ddhNTPs, including the newly discovered ddhUTP and ddhGTP. Herein, we expand on the catalytic mechanism of mammalian, fungal, bacterial, and archaeal viperin-like enzymes with a combination of X-ray crystallography and enzymology. We demonstrate that, like mammalian viperins, these recently discovered viperin-like enzymes operate through the same mechanism and can be classified as ddh-synthases. Furthermore, we define the unique chemical and physical determinants supporting ddh-synthase activity and nucleotide selectivity, including the crystallographic characterization of a fungal viperin-like enzyme that utilizes UTP as a substrate and a cnidaria viperin-like enzyme that utilizes CTP as a substrate. Together, these results support the evolutionary conservation of the ddh-synthase activity and its broad phylogenetic role in innate antiviral immunity.

Green synthesis and biomedicinal applications of silver and gold nanoparticles functionalized with methanolic extract of Mentha longifolia.

This study deals with facile and rapid synthesis of silver nanoparticles (AgNPs) and Gold nanoparticles (AuNPs) using Mentha longifolia leaves extracts (MLE). The synthesized AgNPs and AuNPs were characterized by UV-visible spectroscopy (UV-Vis), Fourier transformed infra-red spectroscopy (FT-IR), atomic force microscopy (AFM) and transmission electron microscopy (TEM) techniques. The phytochemical analysis showed the presence of bioactive secondary metabolites, which are involved in the synthesis of nanoparticles (NPs). The surface plasmon resonance (SPR) observed at 435 and 550 nm, confirmed the green synthesis of AgNPs and AuNPs, respectively. The TEM images showed poly dispersed and round oval shapes of Ag and Au NPs with an average particles size of 10.23 ± 2 nm and 13.45 ± 2 nm, respectively. TEM results are in close agreements with that of AFM analysis. The FT-IR spectroscopy revealed the presence of OH, -NH2 and C = O groups, which involved in the synthesis of NPs. The MLE and their AgNPs and AuNP exhibited good in vitro antibacterial and anti-oxidant activities. Moreover, MLE and NPs also showed in vivo analgesic activities in mice, and excellent sedative properties in open field test paradigm.

Microbial community structure of the bovine rumen as affected by feeding cashew nut shell liquid, a methane-inhibiting and propionate-enhancing agent.

The effect of cashew nut shell liquid (CNSL) feeding on bacterial and archaeal community of the bovine rumen was investigated by analyzing clone libraries targeting 16S rRNA genes, methyl-coenzyme reductase A-encoding genes (mcrA), and their respective transcripts. Rumen samples were collected from three non-lactating cows fed on a hay and concentrate diet with or without CNSL supplementation. DNA and complementary DNA (cDNA) libraries were generated for investigating rumen microbial communities. MiSeq analysis also was performed to understand more comprehensively the changes in the microbial community structures. Following CNSL supplementation, the number of operational taxonomical unit (OTU) and diversity indices of bacterial and archaeal community were decreased. Bacterial OTUs belonging to Proteobacteria, including Succinivibrio, occurred at a higher frequency with CNSL feeding, especially in cDNA libraries. The methanogenic archaeal community became dominated by Methanomicrobium. A bacterial community shift also was observed in the MiSeq data, indicating that CNSL increased the proportion of Succinivibrio and other genera known to be involved in propionate production. Methanogenic archaeal community shifts to increase Methanoplanus and to decrease Methanobrevibacter also were observed. Together, these results imply the occurrence of significant changes in rumen communities, not only for bacteria but also for methanogens, following CNSL feeding.

Facile Stereoselective Reduction of Prochiral Ketones by using an F420 -dependent Alcohol Dehydrogenase.

Effective procedures for the synthesis of optically pure alcohols are highly valuable. A commonly employed method involves the biocatalytic reduction of prochiral ketones. This is typically achieved by using nicotinamide cofactor-dependent reductases. In this work, we demonstrate that a rather unexplored class of enzymes can also be used for this. We used an F420 -dependent alcohol dehydrogenase (ADF) from Methanoculleus thermophilicus that was found to reduce various ketones to enantiopure alcohols. The respective (S) alcohols were obtained in excellent enantiopurity (>99 % ee). Furthermore, we discovered that the deazaflavoenzyme can be used as a self-sufficient system by merely using a sacrificial cosubstrate (isopropanol) and a catalytic amount of cofactor F420 or the unnatural cofactor FOP to achieve full conversion. This study reveals that deazaflavoenzymes complement the biocatalytic toolbox for enantioselective ketone reductions.

Methanofollis fontis sp. nov., a methanogen isolated from marine sediment near a cold seep at Four-Way Closure Ridge offshore southwestern Taiwan.

A mesophilic, hydrogenotrophic methanogen, strain FWC-SCC2T, was isolated from deep-sea sediments collected by a real-time video multiple-corer at the C5-6 station near a cold seep at Four-Way Closure Ridge region during R/V Ocean Researcher III ORIII-1900 cruise in 2015. The cells were irregular cocci, non-motile and 0.8-1.2 µm in diameter. The methanogenic substrates utilized by strain FWC-SCC2T were formate or H2+CO2, but not acetate, methanol, ethanol or methylamines. Strain FWC-SCC2T was lysed in SDS (0.01 %, w/v). The M r of surface-layer protein was 116 400. The optimum growth conditions of strain FWC-SCC2T were 37 °C, 0.17 M NaCl and pH 6.7-7.0. The genomic DNA G+C content calculated from the genome sequence of strain FWC-SCC2T was 59.5 mol %. Phylogenetic analysis revealed that strain FWC-SCC2T was a member of the genus Methanofollis, and was most closely related to Methanofollis tationis Chile 9T (97.6 % similarity of 16S rRNA gene sequence) and shared 97.4, 95.9, 95.9 and 95.4 % with Methanofollis liminatans GKZPZT, Methanofollis formosanus ML15T, Methanofollis aquaemaris N2F9704T and Methanofollis ethanolicus HASUT, respectively. The genome relatedness values between strain FWC-SCC2T and M. tationis DSM 2702T were estimated by average nucleotide identity and digital DNA-DNA hybridization analyses and the results were 79.4 and 21.2 %, respectively. Based on the differences in physiological and biochemical properties, 16S rRNA gene phylogeny and genome relatedness presented here, it is suggested that strain FWC-SCC2T represents a novel species of the genus Methanofollis, and the name Methanofollis fontis sp. nov. is proposed. The type strain is FWC-SCC2T (=BCRC AR10052T=DSM 107935T= NBRC 113164T).

Type IV-Like Pili Facilitate Transformation in Naturally Competent Archaea.

Naturally competent organisms are capable of DNA uptake directly from the environment through the process of transformation. Despite the importance of transformation to microbial evolution, DNA uptake remains poorly characterized outside of the bacterial domain. Here, we identify the pilus as a necessary component of the transformation machinery in archaea. We describe two naturally competent organisms, Methanococcus maripaludis and Methanoculleus thermophilus In M. maripaludis, replicative vectors were transferred with an average efficiency of 2.4 × 103 transformants µg-1 DNA. In M. thermophilus, integrative vectors were transferred with an average efficiency of 2.7 × 103 transformants µg-1 DNA. Additionally, natural transformation of M. thermophilus could be used to introduce chromosomal mutations. To our knowledge, this is the first demonstration of a method to introduce targeted mutations in a member of the order Methanomicrobiales For both organisms, mutants lacking structural components of the type IV-like pilus filament were defective for DNA uptake, demonstrating the importance of pili for natural transformation. Interestingly, competence could be induced in a noncompetent strain of M. maripaludis by expressing pilin genes from a replicative vector. These results expand the known natural competence pili to include examples from the archaeal domain and highlight the importance of pili for DNA uptake in diverse microbial organisms.IMPORTANCE Microbial organisms adapt and evolve by acquiring new genetic material through horizontal gene transfer. One way that this occurs is natural transformation, the direct uptake and genomic incorporation of environmental DNA by competent organisms. Archaea represent up to a third of the biodiversity on Earth, yet little is known about transformation in these organisms. Here, we provide the first characterization of a component of the archaeal DNA uptake machinery. We show that the type IV-like pilus is essential for natural transformation in two archaeal species. This suggests that pili are important for transformation across the tree of life and further expands our understanding of gene flow in archaea.