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Background: In anoxic coastal and marine sediments, degradation of methylated compounds is the major route to the production of methane, a powerful greenhouse gas. Dimethylsulphide (DMS) is the most abundant biogenic organic sulphur compound in the environment and an abundant methylated compound leading to methane production in anoxic sediments. However, understanding of the microbial diversity driving DMS-dependent methanogenesis is limited, and the metabolic pathways underlying this process in the environment remain unexplored. To address this, we used anoxic incubations, amplicon sequencing, genome-centric metagenomics and metatranscriptomics of brackish sediments collected along the depth profile of the Baltic Sea with varying sulphate concentrations., Results: We identified Methanolobus as the dominant methylotrophic methanogens in all our DMS-amended sediment incubations (61-99%) regardless of their sulphate concentrations. We also showed that the mtt and mta genes (trimethylamine- and methanol-methyltransferases) from Methanolobus were highly expressed when the sediment samples were incubated with DMS. Furthermore, we did not find mtsA and mtsB (methylsulphide-methyltransferases) in metatranscriptomes, metagenomes or in the Methanolobus MAGs, whilst mtsD and mtsF were found 2-3 orders of magnitude lower in selected samples., Conclusions: Our study demonstrated that the Methanolobus genus is likely the key player in anaerobic DMS degradation in brackish Baltic Sea sediments. This is also the first study analysing the metabolic pathways of anaerobic DMS degradation in the environment and showing that methylotrophic methane production from DMS may not require a substrate-specific methyltransferase as was previously accepted. This highlights the versatility of the key enzymes in methane production in anoxic sediments, which would have significant implications for the global greenhouse gas budget and the methane cycle. Video Abstract., (© 2023. The Author(s).)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Methanonatronarchaeia represents a deep-branching phylogenetic lineage of extremely halo(alkali)philic and moderately thermophilic methyl-reducing methanogens belonging to the phylum Halobacteriota. It includes two genera, the alkaliphilic Methanonatronarchaeum and the neutrophilic Ca. Methanohalarchaeum. The former is represented by multiple closely related pure culture isolates from hypersaline soda lakes, while the knowledge about the latter is limited to a few mixed cultures with anaerobic haloarchaea. To get more insight into the distribution and ecophysiology of this enigmatic group of extremophilic methanogens, potential activity tests and enrichment cultivation with different substrates and at different conditions were performed with anaerobic sediment slurries from various hypersaline lakes in Russia. Methanonatronarchaeum proliferated exclusively in hypersaline soda lake samples mostly at elevated temperature, while at mesophilic conditions it coexisted with the extremely salt-tolerant methylotroph Methanosalsum natronophilum. Methanonatronarchaeum was also able to serve as a methylotrophic or hydrogenotrophic partner in several thermophilic enrichment cultures with fermentative bacteria. Ca. Methanohalarchaeum did not proliferate at mesophilic conditions and at thermophilic conditions it competed with extremely halophilic and moderately thermophilic methylotroph Methanohalobium, which it outcompeted at a combination of elevated temperature and methyl-reducing conditions. Overall, the results demonstrated that Methanonatronarchaeia are specialized extremophiles specifically proliferating in conditions of elevated temperature coupled with extreme salinity and simultaneous availability of a wide range of C 1 -methylated compounds and H 2 /formate., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The methanol-derived methanogenetic pathway contributes to bulk methane production in cold regions, but the cold adaptation mechanisms are obscure. This work investigated the mechanisms using a psychrophilic methylotrophic methanogen Methanolobus psychrophilus R15. R15 possesses two mtaCB operon paralogues-encoding methanol:corrinoid methyltransferase that is key to methanol-based methanogenesis. Molecular combined methanogenic assays determined that MtaC1 is important in methanogenesis at the optimal temperature of 18°C, but MtaC2 can be a cold-adaptive paralogue by highly upregulated at 8°C. The 5'P-seq and 5'RACE all assayed that processing occurred at the 5' untranslated region (5'-UTR) of mtaC2; reporter genes detected higher protein expression, and RNA half-life experiments assayed prolonged lifespan of the processed transcript. Therefore, mtaC2 5'-UTR processing to move the bulged structure elevated both the translation efficiency and transcript stability. 5'P-seq, quantitative RT-PCR and northern blot all identified enhanced mtaC2 5'-UTR processing at 8°C, which could contribute to the upregulation of mtaC2 at cold. The R15 cell extract contains an endoribonuclease cleaving an identified 10 nt-processing motif and the native mtaC2 5'-UTR particularly folded at 8°C. Therefore, this study revealed a 5'-UTR processing mediated post-transcriptional regulation mechanism controlling the cold-adaptive methanol-supported methanogenetic pathway, which may be used by other methylotrophic methanogens., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Methane is a potent greenhouse gas; methane production and consumption within seafloor sediments has generated intense interest. Anaerobic oxidation of methane (AOM) and methanogenesis (MOG) primarily occur at the depth of the sulfate-methane transition zone or underlying sediment respectively. Methanogenesis can also occur in the sulfate-reducing sediments through the utilization of non-competitive methylated compounds; however, the occurrence and importance of this process are not fully understood. Here, we combined a variety of data, including geochemical measurements, rate measurements and molecular analyses to demonstrate the presence of a cryptic methane cycle in sulfate-reducing sediments from the continental shelf of the northern South China Sea. The abundance of methanogenic substrates as well as the high MOG rates from methylated compounds indicated that methylotrophic methanogenesis was the dominant methanogenic pathway; this conclusion was further supported by the presence of the methylotrophic genus Methanococcoides. High potential rates of AOM were observed in the sediments, indicating that methane produced in situ could be oxidized simultaneously by AOM, presumably by ANME-2a/b as indicated by 16S rRNA gene analysis. A significant correlation between the relative abundance of methanogens and methanotrophs was observed over sediment depth, indicating that methylotrophic methanogenesis could potentially fuel AOM in this environment. In addition, higher potential rates of AOM than sulfate reduction rates at in situ methane conditions were observed, making alternative electron acceptors important to support AOM in sulfate-reducing sediment. AOM rates were stimulated by the addition of Fe/Mn oxides, suggesting AOM could be partially coupled to metal oxide reduction. These results suggest that methyl-compounds driven methane production drives a cryptic methane cycling and fuels AOM coupled to the reduction of sulfate and other electron acceptors., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Anaerobic bacteria are known to produce neurotoxic methylmercury [MeHg] when elemental mercury [Hg(0)] is provided as the sole mercury source. In this study, we examined the formation of MeHg in anaerobic incubations of sediment collected from the San Jacinto River estuary (Texas, USA) amended with aqueous Hg(0) to investigate the microbial communities involved in the conversion of Hg(0) to MeHg. The results show that the addition of the methanogen inhibitor 2-bromoethanesulfonate (BES) significantly decreased MeHg production. The mercury methylation gene, hgcA, was detected in these sediments using archaeal specific primers, and 16S rRNA sequencing showed that a member of the Methanosarcinaceae family of methanogens was active. These results suggest that methanogenic archaea play an underappreciated role in the production of MeHg in estuarine sediments contaminated with Hg(0)., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)

Anaerobic biological techniques are widely used in the reductive decolorization of textile wastewater. However, the decolorization efficiency of textile wastewater by conventional anaerobic biological techniques is generally limited due to the low biomass retention capacity and short hydraulic retention time (HRT). In this study, a methane-based hollow fiber membrane bioreactor (HfMBR) was initially inoculated with an enriched anaerobic methane oxidation (AOM) culture to rapidly form an anaerobic biofilm. Then, synthetic azo dye wastewater containing methyl orange (MO) was fed into the HfMBR. MO decolorization efficiency of ∼ 100 % (HRT = 2 to 1.5 days) and maximum decolorization rate of 883 mg/L/day (HRT = 0.5 day) were obtained by the stepwise increase of the MO loading rate into the methane-based HfMBR. Scanning electron microscopy (SEM) and fluorescence in situ hybridization (FISH) analysis visually revealed that archaea clusters formed synergistic consortia with adjacent bacteria. Quantitative PCR (qPCR), phylogenetic and high-throughput sequencing analysis results further confirmed the biological consortia formation of methane-related archaea and partner bacteria, which played a synergistic role in MO decolorization. The high removal efficiency and stable microbial structure in HfMBR suggest it is a potentially effective technique for high-toxic azo dyes removal from textile wastewater., 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 © 2019 Elsevier B.V. All rights reserved.)

Monomeric isocitrate dehydrogenase (IDH) stands for a separated subgroup among IDH protein family. Up to now, all reported monomeric IDHs are from prokaryotes. Here, a monomeric IDH from a marine methanogenic archaeon Methanococcoides methylutens (MmIDH) was reported for the first time. BLAST search demonstrated that only a few marine archaea encode the monomeric IDH and all these organisms are methylotrophic. MmIDH shows the highest homology (~ 70%) to the monomeric IDHs from some marine bacteria, suggesting a lateral gene transfer event between marine bacteria and archaea. The monomeric state of MmIDH was determined by size exclusion chromatography. MmIDH is divalent cation-dependent and Mn 2+ is the most favored. Kinetic analysis showed that MmIDH is highly specific to NADP + and cannot utilize the NAD + . The optimal temperature for MmIDH activity is 50 °C and the optimal pH is 8.2. Heat inactivation assay revealed that MmIDH is a mesophilic enzyme. It sustained 50% activity after incubation at 39 °C for 20 min. Moreover, the putative coenzyme binding residues (His590, Arg601, and Arg650) of MmIDH were explored by mutagenesis. The triple mutant H590L/R601D/R650S displayed a 5.93-fold preference for NAD + over NADP + , indicating that the coenzyme specificity of MmIDH was significantly switched from NADP + to NAD + by three key mutations.

MazF is a sequence-specific endoribonuclease or mRNA interferase, which cleaves RNA at a specific sequence. Since the expression of a specific gene or a group of specific genes can be regulated by MazF, expanding the repertoire of recognition sequences by MazF mRNA interferases is highly desirable for biotechnological and medical applications. Here, we identified a gene for a MazF homologue (MazFme) from Methanohalobium evestigatum, an extremely halophilic archaeon. In order to suppress the toxicity of MazFme to the E. coli cells, the C-terminal half of the cognate antitoxin MazEme was fused to the N-terminal end of MazFme. Since the fusion of the C-terminal half of MazEme to MazFme was able to neutralize MazFme toxicity, the MazEme-MazFme fusion protein was expressed in a large amount without any toxic effects. After purification of the MazEme, the free MazFme RNA cleavage specificity was determined by primer extension and synthetic ribonucleotides, revealing that MazFme is a CUGGU/UUGGU-specific endoribonuclease., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Despite the significance of biogenic methane generation in coal beds, there has never been a systematic long-term evaluation of the ecological response to biostimulation for enhanced methanogenesis in situ. Biostimulation tests in a gas-free coal seam were analysed over 1.5 years encompassing methane production, cell abundance, planktonic and surface associated community composition and chemical parameters of the coal formation water. Evidence is presented that sulfate reducing bacteria are energy limited whilst methanogenic archaea are nutrient limited. Methane production was highest in a nutrient amended well after an oxic preincubation phase to enhance coal biofragmentation (calcium peroxide amendment). Compound-specific isotope analyses indicated the predominance of acetoclastic methanogenesis. Acetoclastic methanogenic archaea of the Methanosaeta and Methanosarcina genera increased with methane concentration. Acetate was the main precursor for methanogenesis, however more acetate was consumed than methane produced in an acetate amended well. DNA stable isotope probing showed incorporation of 13 C-labelled acetate into methanogenic archaea, Geobacter species and sulfate reducing bacteria. Community characterisation of coal surfaces confirmed that methanogenic archaea make up a substantial proportion of coal associated biofilm communities. Ultimately, methane production from a gas-free subbituminous coal seam was stimulated despite high concentrations of sulfate and sulfate-reducing bacteria in the coal formation water. These findings provide a new conceptual framework for understanding the coal reservoir biosphere.

Coastal saltmarsh sediments represent an important source of natural methane emissions, much of which originates from quaternary and methylated amines, such as choline and trimethylamine. In this study, we combine DNA stable isotope probing with high throughput sequencing of 16S rRNA genes and 13 C 2 -choline enriched metagenomes, followed by metagenome data assembly, to identify the key microbes responsible for methanogenesis from choline. Microcosm incubation with 13 C 2 -choline leads to the formation of trimethylamine and subsequent methane production, suggesting that choline-dependent methanogenesis is a two-step process involving trimethylamine as the key intermediate. Amplicon sequencing analysis identifies Deltaproteobacteria of the genera Pelobacter as the major choline utilizers. Methanogenic Archaea of the genera Methanococcoides become enriched in choline-amended microcosms, indicating their role in methane formation from trimethylamine. The binning of metagenomic DNA results in the identification of bins classified as Pelobacter and Methanococcoides. Analyses of these bins reveal that Pelobacter have the genetic potential to degrade choline to trimethylamine using the choline-trimethylamine lyase pathway, whereas Methanococcoides are capable of methanogenesis using the pyrrolysine-containing trimethylamine methyltransferase pathway. Together, our data provide a new insight on the diversity of choline utilizing organisms in coastal sediments and support a syntrophic relationship between Bacteria and Archaea as the dominant route for methanogenesis from choline in this environment.

About 60% of natural gas production in the United States comes from hydraulic fracturing of unconventional reservoirs, such as shales or organic-rich micrites. This process inoculates and enriches for halotolerant microorganisms in these reservoirs over time, resulting in a saline ecosystem that includes methane producing archaea. Here, we survey the biogeography of methanogens across unconventional reservoirs, and report that members of genus Methanohalophilus are recovered from every hydraulically fractured unconventional reservoir sampled by metagenomics. We provide the first genomic sequencing of three isolate genomes, as well as two metagenome assembled genomes (MAGs). Utilizing six other previously sequenced isolate genomes and MAGs, we perform comparative analysis of the 11 genomes representing this genus. This genomic investigation revealed distinctions between surface and subsurface derived genomes that are consistent with constraints encountered in each environment. Genotypic differences were also uncovered between isolate genomes recovered from the same well, suggesting niche partitioning among closely related strains. These genomic substrate utilization predictions were then confirmed by physiological investigation. Fine-scale microdiversity was observed in CRISPR-Cas systems of Methanohalophilus, with genomes from geographically distinct unconventional reservoirs sharing spacers targeting the same viral population. These findings have implications for augmentation strategies resulting in enhanced biogenic methane production in hydraulically fractured unconventional reservoirs., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

This study evaluated the methanogens responsible for methanogenic degradation of tetramethylammonium hydroxide (TMAH) in a continuous flow bioreactor. The enriched methanogens attained an estimated maximum specific TMAH degradation rate and half-saturation constant of 39.5 mg TMAH/gVSS/h and 820 mg/L, following the Monod-type kinetic expression for methanogenic TMAH degradation. Presence of sulfide more than 20 mg/L significantly extended lag period and slowed down specific TMAH degradation rates. The results of terminal restriction fragment length polymorphism (T-RFLP), cloning/sequencing, and quantitative real-time PCR analyses targeting on the methyl coenzyme M reductase alpha subunit (mcrA) genes retrieved from the bioreactor and batch experiments indicated that Methanomethylovorans species were the dominant methanogens responsible for methanogenic degradation of TMAH. The isolated TMAH-degrading methanogen from the bioreactor, however, was identified closely related to Methanosarcina mazei. It is likely that a very low TMAH environment in the bioreactor favored the growth of Methanomethylovorans hollandica, while the much higher TMAH in the isolation growth medium proliferated Methanosarcina mazei., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A psychrotolerant, methylotrophic methanogen, strain YSF-03 T , was isolated from the saline meromictic Lake Shira in Siberia. Cells of strain YSF-03 T were non-motile, irregular cocci and 0.8-1.2 µm in diameter. The methanogenic substrates utilized by strain YSF-03 T were methanol and trimethylamine. The temperature range of growth for strain YSF-03 T was from 0 to 37 °C. The optimum growth conditions were 30-37 °C, pH 7.0-7.4 and 0.17 M NaCl. The G+C content of the genome of strain YSF-03 T was 41.3 mol%. Phylogenetic analysis revealed that strain YSF-03 T was most closely related to Methanolobus profundi MobM T (98.15 % similarity in 16S rRNA gene sequence). Genome relatedness between strain YSF-03 T and MobM T was computed using the Genome-to-Genome Distance Calculator and average nucleotide identity, which gave values of 23.5 and 79.3 %, respectively. Based on the morphological, phenotypic, phylogenetic and genomic relatedness data presented here, it is evident that strain YSF-03 T represents a novel species of the genus Methanolobus, for which the name Methanolobus psychrotolerans sp. nov. is proposed. The type strain is YSF-03 T (=BCRC AR10049 T =DSM 104044 T =NBRC 112514 T ).

We examined the effect of ammonium and temperature on methane production in high rate upflow anaerobic sludge bed reactors treating pig manure supernatant. We operated four reactors at two ammonium concentrations ('low' at 1.9, 'high' at 3.7 g L -1 , termed LA and HA reactors, respectively) and at variable temperatures over 358 days. Archaeal and bacterial communities were characterized by Illumina sequencing of 16S rRNA amplicons. Ammonium was a major selective factor for bacterial and archaeal community structure. After ~200 days of adaptation to high ammonium levels, acetate and propionate removal and methane production improved substantially in HA reactors. Aceticlastic Methanosaeta was abundant and positively correlated to methane yield in the HA reactors, whereas Methanosarcina was more abundant in LA reactors. Furthermore, a group of monophyletic OTUs that was related to Thaumarchaeota in phylogenetic analysis was highly abundant in the archaeal communities, particularly in the HA reactors. The most abundant bacterial OTU in LA reactors, representing Syntrophomonadaceae, was also positively correlated to methane yield in the HA reactors, indicating its importance in methane production under ammonia stress. In conclusion, efficient methane production, involving aceticlastic methanogenesis by Methanosaeta took place in the reactors at free ammonia concentrations as high as 1 g L -1 .

Archaea are cosmopolitan in aerated soils around the world. While the dominance of Thaumarchaeota has been reported in most soils, the methanogens are recently found to be ubiquitous but with low abundances in the aerated soil globally. However, the seasonal changes of Archaea community in the aerated soils are still in the mist. In this study, we investigated the change of Archaea in the context of environmental variables over a period of 12 months in a subtropical soil on the Chongming Island, China. The results showed that Nitrososphaera spp. were the dominant archaeal population while the methanogens were in low proportions but highly diverse (including five genera: Methanobacterium, Methanocella, Methanosaeta, Methanosarcina, and Methanomassiliicoccus) in the aerated soil samples determined by high throughput sequencing. A total of 126 LSA correlations were found in the dataset including all the 72 archaeal OTUs and 8 environmental factors. A significance index defined as the pagerank score of each OTU divided by its relative abundance was used to evaluate the significance of each OTU. The results showed that five out of 17 methanogen OTUs were significantly positively correlated with temperature, suggesting those methanogens might increase with temperature rather than being dormant in the aerated soils. Given the metabolic response of methanogens to temperature under aerated soil conditions, their contribution to the global methane cycle warrants evaluation.

Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an 'all-or-none' pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We used paddy soil slurries amended with rice straw to identify the microbial populations involved in the methanogenic breakdown of plant polymers. Rice straw greatly stimulated microbial activity over the 28-day incubation period. On day 7, the transient peak concentration of acetate (24 mM) coincided with the onset of increased methane production. Microbial 16S rRNA transcript numbers increased by one to two orders of magnitude, but not the 16S rRNA gene copy numbers. Using metatranscriptomic rRNA, Clostridiaceae, Lachnospiraceae, Ruminococcaceae, Veillonellaceae and Pseudomonadaceae were identified to be the most abundant and the most dynamic bacterial groups. Changes in methanogen rRNA and mRNA abundances corresponded well with methanogenic activity. Acetate determined the abundance ratio between Methanosarcinaceae and Methanosaetaceae. Methanocellaceae dominated hydrogenotrophic methanogenesis. Transcript levels of mRNA families involved in plant polymer breakdown increased slightly with time. Glycosyl hydrolase (GH) transcripts involved in cellulose and chitin breakdown were predominantly expressed by the Firmicutes, whereas those involved in hemicellulose breakdown exhibited more diverse taxonomic sources, including Acidobacteria, Bacteriodetes and Chloroflexi. Taken together, we observed strong population dynamics and the expression of taxonomically diverse GH families, suggesting that not only Firmicutes, but also less abundant groups play a major functional role in the decomposition of rice straw., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

TRAM domain proteins present in Archaea and Bacteria have a β-barrel shape with anti-parallel β-sheets that form a nucleic acid binding surface; a structure also present in cold shock proteins (Csps). Aside from protein structures, experimental data defining the function of TRAM domains is lacking. Here, we explore the possible functional properties of a single TRAM domain protein, Ctr3 (cold-responsive TRAM domain protein 3) from the Antarctic archaeon Methanococcoides burtonii that has increased abundance during low temperature growth. Ribonucleic acid (RNA) bound by Ctr3 in vitro was determined using RNA-seq. Ctr3-bound M. burtonii RNA with a preference for transfer (t)RNA and 5S ribosomal RNA, and a potential binding motif was identified. In tRNA, the motif represented the C loop; a region that is conserved in tRNA from all domains of life and appears to be solvent exposed, potentially providing access for Ctr3 to bind. Ctr3 and Csps are structurally similar and are both inferred to function in low temperature translation. The broad representation of single TRAM domain proteins within Archaea compared with their apparent absence in Bacteria, and scarcity of Csps in Archaea but prevalence in Bacteria, suggests they represent distinct evolutionary lineages of functionally equivalent RNA-binding proteins., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1(T), a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change.

Cold environments dominate the Earth's biosphere and the resident microorganisms play critical roles in fulfilling global biogeochemical cycles. However, only few studies have examined the molecular basis of thermosensing; an ability that microorganisms must possess in order to respond to environmental temperature and regulate cellular processes. Two component regulatory systems have been inferred to function in thermal regulation of gene expression, but biochemical studies assessing these systems in Bacteria are rare, and none have been performed in Archaea or psychrophiles. Here we examined the LtrK/LtrR two component regulatory system from the Antarctic archaeon, Methanococcoides burtonii, assessing kinase and phosphatase activities of wild-type and mutant proteins. LtrK was thermally unstable and had optimal phosphorylation activity at 10 °C (the lowest optimum activity for any psychrophilic enzyme), high activity at 0 °C and was rapidly thermally inactivated at 30 °C. These biochemical properties match well with normal environmental temperatures of M. burtonii (0-4 °C) and the temperature this psychrophile is capable of growing at in the laboratory (-2 to 28 °C). Our findings are consistent with a role for LtrK in performing phosphotransfer reactions with LtrR that could lead to temperature-dependent gene regulation.

iso-Alkanes are major components of petroleum and have been considered recalcitrant to biodegradation under methanogenic conditions. However, indigenous microbes in oil sands tailings ponds exposed to solvents rich in 2-methylbutane, 2-methylpentane, 3-methylpentane, n-pentane, and n-hexane produce methane in situ. We incubated defined mixtures of iso- or n-alkanes with mature fine tailings from two tailings ponds of different ages historically exposed to different solvents: one, ~10 years old, receiving C5-C6 paraffins and the other, ~35 years old, receiving naphtha. A lengthy incubation (>6 years) revealed iso-alkane biodegradation after lag phases of 900-1800 and ~280 days, respectively, before the onset of methanogenesis, although lag phases were shorter with n-alkanes (~650-1675 and ~170 days, respectively). 2-Methylpentane and both n-alkanes were completely depleted during ~2400 days of incubation, whereas 2-methylbutane and 3-methylpentane were partially depleted only during active degradation of 2-methylpentane, suggesting co-metabolism. In both cases, pyrotag sequencing of 16S rRNA genes showed codominance of Peptococcaceae with acetoclastic (Methanosaeta) and hydrogenotrophic (Methanoregula and Methanolinea) methanogens. These observations are important for predicting long-term greenhouse-gas emissions from oil sands tailings ponds and extend the known range of hydrocarbons susceptible to methanogenic biodegradation in petroleum-impacted anaerobic environments.

Two groups of haloalkaliphilic methanogenic archaea were dominating in enrichments from hypersaline soda lake sediments at pH 10. At moderate salt concentrations with formate or H2 as electron donor, methanogens belonging to the genus Methanocalculus were enriched, while at high salt concentrations with methylated substrates, a group related to Methanosalsum zhilinae was dominating. For both groups, several pure cultures were obtained including the type strains AMF2T for the Methanocalculus group and AME2T for the Methanosalsum group. The Methanocalculus group is characterized by lithoheterotrophic growth with either formate (preferable substrate) or H2 at moderate salinity up to 1.5-2 M total Na+ and obligate alkaliphilic growth with an optimum at pH 9.5. According to phylogenetic analysis, the group also includes closely related strains isolated previously from the low-salt alkaline Lonar Lake. The novel Methanosalsum group is characterized by high salt tolerance (up to 3.5 M total Na+) and obligate alkaliphilic growth with an optimum at pH 9.5. It has a typical methylotrophic substrate profile, utilizing methanol, methylamines and dimethyl sulfide (at low concentrations) as methanogenic substrates. On the basis of physiological and phylogenetic data, it is proposed that the two groups of soda lake methanogenic isolates are assigned into two novel species, Methanocalculus alkaliphilus sp. nov. (type strain AMF2T = DSM 24457T = UNIQEM U859T) and Methanosalsum natronophilum sp. nov. (type strain AME2T = DSM 24634T = NBRC 110091T).

In this study, a control anaerobic membrane bioreactor (C-AnMBR) and a bioaugmented anaerobic membrane bioreactor (B-AnMBR) were operated for 210 d to treat pharmaceutical wastewater. Both the bioreactors were fed with the pharmaceutical wastewater containing TCOD of 16,249 ± 714 mg/L and total dissolved solids (TDS) of 29,450 ± 2209 mg/L with an organic loading rate (OLR) of 13.0 ± 0.6 kgCOD/m(3)d. Under steady-state condition, an average total chemical oxygen demand (TCOD) removal efficiency of 46.1 ± 2.9% and 60.3 ± 2.8% was achieved by the C-AnMBR and the B-AnMBR, respectively. The conventional anaerobes in the C-AnMBR cannot tolerate the hypersaline conditions well, resulting in lower TCOD removal efficiency, biogas production and methane yield than the B-AnMBR seeded from the coastal shore. Pyrosequencing analysis indicated that marine bacterial species (Oliephilus sp.) and halophilic bacterial species (Thermohalobacter sp.) were only present in the B-AnMBR; these species could possibly degrade complex and recalcitrant organic matter and withstand hypersaline environments. Two different dominant archaeal communities, genus Methanosaeta (43.4%) and Methanolobus (61.7%), were identified as the dominant methanogens in the C-AnMBR and the B-AnMBR, respectively. The species of genus Methanolobus was reported resistant to penicillin and required sodium and magnesium for growth, which could enable it to thrive in the hypersaline environment., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The influence of solution chemistry on microbial transport was examined using the strictly anaerobic trichloroethene (TCE) bioaugmentation culture KB-1(®). A column was employed to determine transport behaviors and deposition kinetics of three distinct functional species in KB-1(®), Dehalococcoides, Geobacter, and Methanomethylovorans, over a range of ionic strengths under a well-controlled anaerobic condition. A quantitative polymerase chain reaction (qPCR) was utilized to enumerate cell concentration and complementary techniques were implemented to evaluate cell surface electrokinetic potentials. Solution chemistry was found to positively affect the deposition rates, which was consistent with calculated Derjaguin-Landau-Verwey-Overbeek (DLVO) interaction energies. Retained microbial profiles showed spatially constant colloid deposition rate coefficients, in agreement with classical colloid filtration theory (CFT). It was interesting to note that the three KB-1(®) species displayed similar transport and retention behaviors under the defined experimental conditions despite their different cell electrokinetic properties. A deeper analysis of cell characteristics showed that factors, such as cell size and shape, concentration, and motility were involved in determining adhesion behavior., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Microbial methanogenesis at extreme conditions of saline alkaline soda lakes has, so far, been poorly investigated. Despite the obvious domination of sulfidogenesis as the therminal anaerobic process in the hypersaline soda lakes of Kulunda Steppe (Altai, southwestern Siberia), high concentrations of methane were detected in the anaerobic sediments. Potential activity measurements with different substrates gave results significantly deviating from what is commonly found in hypersaline habitats with neutral pH. In particular, not only a non-competitive methylotrophic pathway was active, but also lithotrophic and, in some cases, even acetate-dependent methanogenesis was found to be present in hypersaline soda lake sediments. All three pathways were functioning exclusively within the alkaline pH range between 8 and 10.5, while the salt concentration was the key factor influencing the activity. Methylotrophic and, to a lesser extent, lithotrophic methanogenesis were active up to soda-saturating conditions (4 M total Na(+)). Acetate-dependent methanogenesis was observed at salinities below 3 M total Na(+). Detection of methanogens in sediments using the mcrA gene as a functional marker demonstrated domination of methylotrophic genera Methanolobus and Methanosalsum and lithotrophic Methanocalculus. In a few cases, acetoclastic Methanosaeta was detected, as well as two deep lineage methanogens. Cultivation results corresponded well to the mcrA-based observations. Enrichments for natronophilic methylotrophic methanogens resulted in isolation of Methanolobus strains at moderate salinity, while at salt concentrations above 2 M Na(+) a novel member of the genus Methanosalsum was dominating. Enrichments with H2 or formate invariably resulted in domination of close relatives of Methanocalculus natronophilus. Enrichments with acetate at low salt concentration yielded two acetoclastic alkaliphilic Methanosaeta cultures, while at salinity above 1 M Na(+) syntrophic associations were apparently responsible for the observed acetate conversion to methane. Overall, the results indicated the presence of functionally structured and active methanogenic populations in Siberian hypersaline soda lakes., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Psychrophilic methanogenic Archaea contribute significantly to global methane emissions, but archaeal cold adaptation mechanisms remain poorly understood. Hinted by that mRNA architecture determined secondary structure respond to cold more promptly than proteins, differential RNA-seq was used in this work to examine the genome-wide transcription start sites (TSSs) of the psychrophilic methanogen Methanolobus psychrophilus R15 and its response to cold. Unlike most prokaryotic mRNAs with short 5' untranslated regions (5' UTR, median lengths of 20-40 nt), 51% mRNAs of this methanogen have large 5' UTR (>50 nt). For 24% of the mRNAs, the 5' UTR is >150 nt. This implies that post-transcriptional regulation may be significance in the psychrophile. Remarkably, 219 (14%) genes possessed multiple gene TSSs (gTSSs), and 84 genes exhibited temperature-regulated gTSS selection to express alternative 5' UTR. Primer extension studies confirmed the temperature-dependent TSS selection and a stem-loop masking of ribosome binding sites was predicted from the longer 5' UTRs, suggesting alternative 5' UTRs-mediated translation regulation in the cold adaptation as well. In addition, 195 small RNAs (sRNAs) were detected, and Northern blots confirmed that many sRNAs were induced by cold. Thus, this study revealed an integrated transcriptional and post-transcriptional regulation for cold adaptation in a psychrophilic methanogen.

Cold-adaptive methanogens contribute significantly to methane emission from the cold area, while the cold-adaptive mechanisms used by Archaea remain elusive. Methanolobus psychrophilus R15, a cold-adaptive methanogen isolated from a Tibetan plateau wetland, grows at 0-25 °C and optimally at 18 °C when isolated; however, it grows optimally at 30 °C after culturing at 18 °C for several years. Aiming to gain insights into the protein profiles that are involved in optimal growth and cold adaptation of this methanogen, here, we performed a comparative proteomic study using 2D DIGE on the cultures grown at 30, 18 and 4 °C. 1439 protein spots (3167 ORFs annotated in the R15 genome) were detected, and 202 of 322 differentially expressed protein spots were identified by MALDI-TOF/TOF. The protein abundance of most enzymes involved in methanogenesis, energy conservation and central metabolism were increased at 30 °C, while most ribosome proteins were decreased at 30 °C. Proteasome and ROS scavengers increased expressions at 4 °C, suggesting more aberrant proteins and ROS formed at lower temperatures. Different from the cold-adaptive Methanococcoides burtonii, some chaperones were increased at 4 °C, implying that protein folding was impaired at cold in this psychrophilic archaeon. This study indicates that diverse cold-adaptive mechanisms can be used by different methanogenic Archaea.

The impact of temperature on the largely unresolved intermediary ecosystem metabolism and associated unknown microbiota that link cellulose degradation and methane production in soils of a moderately acidic (pH 4.5) fen was investigated. Supplemental [(13) C]cellulose stimulated the accumulation of propionate, acetate and carbon dioxide as well as initial methane production in anoxic peat soil slurries at 15°C and 5°C. Accumulation of organic acids at 15°C was twice as fast as that at 5°C. 16S rRNA [(13) C]cellulose stable isotope probing identified novel unclassified Bacteria (79% identity to the next cultured relative Fibrobacter succinogenes), unclassified Bacteroidetes (89% identity to Prolixibacter bellariivorans), Porphyromonadaceae, Acidobacteriaceae and Ruminococcaceae as main anaerobic degraders of cellulose-derived carbon at both 15°C and 5°C. Holophagaceae and Spirochaetaceae were more abundant at 15°C. Clostridiaceae dominated the degradation of cellulose-derived carbon only at 5°C. Methanosarcina was the dominant methanogenic taxa at both 15°C and 5°C. Relative abundance of Methanocella increased at 15°C whereas that of Methanoregula and Methanosaeta increased at 5°C. Thaumarchaeota closely related to Nitrosotalea (presently not known to grow anaerobically) were abundant at 5°C but absent at 15°C indicating that Nitrosotalea sp. might be capable of anaerobic growth at low temperatures in peat., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Two novel strains of methanogens were isolated from an estuarine sediment with the capability to utilize quaternary amines. Based on the 16S rRNA analysis, strain B1d shared 99 % sequence identity with Methanolobus vulcani PL-12/M(T) and strain Q3c shared 99 % identity with Methanococcoides sp. PM1 and PM2, but our current isolates display clearly different capabilities of growth on quaternary amines and were isolated based on these capabilities. Strain Q3c was capable of growth on tetramethylammonium and choline, while strain B1d was capable of growth on glycine betaine. Ml. vulcani PL-12/M(T) was incapable of growth on glycine betaine, indicating an obvious distinction between strains B1d and PL-12/M(T). Strain Q3c now represents the only known tetramethylammonium-utilizing methanogen in isolation. Strain B1d is the first quaternary amine-utilizing methanogen from the genus Methanolobus. This study suggests that quaternary amines may serve as ready precursors of biological methane production in marine environments.

Glycine betaine (betaine) has the highest cellular osmoprotective efficiency which does not accumulate in most glycophytes. The biosynthetic pathway for betaine in higher plants is derived from the oxidation of low-accumulating metabolite choline that limiting the ability of most plants to produce betaine. Halophilic methanoarchaeon Methanohalophilus portucalensis FDF1(T) is a model anaerobic methanogen to study the acclimation of water-deficit stresses which de novo synthesize betaine by the stepwise methylation of glycine, catalyzed by glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase. In this report, genes encoding these betaine biosynthesizing enzymes, Mpgsmt and Mpsdmt, were introduced into Arabidopsis. The homozygous Mpgsmt (G), Mpsdmt (S), and their cross, Mpgsmt and Mpsdmt (G × S) plants showed increased accumulation of betaine. Water loss from detached leaves was slower in G, S, and G × S lines than wild-type (WT). Pot-grown transgenic plants showed better growth than WT after 9 days of withholding water or irrigating with 300 mM NaCl. G, S, G × S lines also maintained higher relative water content and photosystem II activity than WT under salt stress. This suggests heterologously expressed Mpgsmt and Mpsdmt could enhance tolerance to drought and salt stress in Arabidopsis. We also found a twofold increase in quaternary ammonium compounds in salt-stressed leaves of G lines, presumably due to the activation of GSMT activity by high salinity. This study demonstrates that introducing stress-activated enzymes is a way of avoiding the divergence of primary metabolites under normal growing conditions, while also providing protection in stressful environments.

Sulfur dioxide from flue gas was converted into sulfate after the absorption of alkaline solutions. Haloalkaliphilic microorganisms have been used in reducing sulfate to decrease expenses and avoid sulfide inhibition. The effects of different COD/SO4(2-) ratios and hydraulic retention times (HRTs) on the sulfate removal efficiency and bacterial community were investigated in model experiments. Ethanol showed better performance as an electron donor than lactate. The optimum COD/SO4(2-) ratio and HRT were 4.0 and 18 h, respectively, with respective sulfate removal efficiency and rate of 97.8 ± 1.11% and 6.26 ± 0.0710 g/Ld. Sulfide concentrations reached 1,603 ± 3.38 mg/L. Based on denaturing gradient gel electrophoresis analysis of 16S rDNA, the major sulfate-reducing bacterium (SRB) was Desulfonatronovibrio sp., which was only detected at a COD/SO4(2-) ratio of 4.0 using ethanol as an electron donor. Different HRTs had no significant effect on the band corresponding to this species. PCR results show that methane-producing archaea (MPA) were from the acetoclastic methanogenic family Methanosarcinaceae. Quantitative real-time PCR did not demonstrate any significant competition between SRB and MPA. The findings of this study indicate that sulfate reduction, nitrate reduction, and sulfide oxidization may occur in the same bioreactor., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A mesophilic, slightly halophilic, obligately methylotrophic, methanogenic archaeon, designated strain GTA13(T), was isolated from natural gas-bearing confined aquifers in the Minami-Kanto gas field, Japan. The cells were non-motile, slightly irregular cocci, 0.7-1.0 µm in diameter and occurred singly, in pairs or as small aggregates. The cells grew with tri- or dimethylamine but not with H2/CO2, formate, acetate, methanol or dimethyl sulphide. Vitamins, sodium and magnesium were required for growth. Optimal growth occurred at pH 7.0-7.5, 35 °C, 0.35-0.40 M NaCl and 15-50 mM MgCl2. The NaCl range for growth was 0.2-1.3 M. The DNA G+C content was 43.7 mol%. Strain GTA13(T) showed highest levels of 16S rRNA gene sequence similarity with Methanohalophilus portucalensis FDF-1(T) (96.4% sequence similarity) and Methanohalophilus halophilus DSM 3094(T) (96.0%). On the basis of physiological and phylogenetic features, strain GTA13(T) is considered to represent a novel species of the genus Methanohalophilus, for which the name Methanohalophilus levihalophilus sp. nov. is proposed. The type strain is GTA13(T) ( = NBRC 110099(T) = DSM 28452(T)). An emended description of the genus Methanohalophilus is also proposed., (© 2014 IUMS.)

A novel, strictly anaerobic, methylotrophic marine methanogen, strain SLH33(T), was isolated from deep sediment samples covered by an orange microbial mat collected from the Napoli Mud Volcano. Cells of strain SLH33(T) were Gram-stain-negative, motile, irregular cocci that occurred singly. Cells utilized trimethylamine, dimethylamine, monomethylamine, methanol, betaine, N,N-dimethylethanolamine and choline (N,N,N-trimethylethanolamine) as substrates for growth and methanogenesis. The optimal growth temperature was 30 °C; maximum growth rate was obtained at pH 7.0 in the presence of 0.5 M Na(+). The DNA G+C content of strain SLH33(T) was 43.4 mol%. Phylogenetic analyses based on 16S rRNA gene sequences placed strain SLH33(T) within the genus Methanococcoides. The novel isolate was related most closely to Methanococcoides methylutens TMA-10(T) (98.8% 16S rRNA gene sequence similarity) but distantly related to Methanococcoides burtonii DSM 6242(T) (97.6%) and Methanococcoides alaskense AK-5(T) (97.6%). DNA-DNA hybridization studies indicated that strain SLH33(T) represents a novel species, given that it shared less than 16% DNA-DNA relatedness with Methanococcoides methylutens TMA-10(T). The name Methanococcoides vulcani sp. nov. is proposed for this novel species, with strain SLH33(T) ( = DSM 26966(T) = JCM 19278(T)) as the type strain. An emended description of the genus Methanococcoides is also proposed., (© 2014 IUMS.)

Two-component signal transduction systems (TCSs) are a major mechanism used by bacteria in response to environmental changes. Although many sequenced archaeal genomes encode TCSs, they remain poorly understood. Previously, we reported that a methanogenic archaeon, Methanosaeta harundinacea, encodes FilI, which synthesizes carboxyl-acyl homoserine lactones, to regulate transitions of cellular morphology and carbon metabolic fluxes. Here, we report that filI, the cotranscribed filR2, and the adjacent filR1 constitute an archaeal TCS. FilI possesses a cytoplasmic kinase domain (histidine kinase A and histidine kinase-like ATPase) and its cognate response regulator. FilR1 carries a receiver (REC) domain coupled with an ArsR-related domain with potential DNA-binding ability, while FilR2 carries only a REC domain. In a phosphorelay assay, FilI was autophosphorylated and specifically transferred the phosphoryl group to FilR1 and FilR2, confirming that the three formed a cognate TCS. Through chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) using an anti-FilR1 antibody, FilR1 was shown to form in vivo associations with its own promoter and the promoter of the filI-filR2 operon, demonstrating a regulatory pattern common among TCSs. ChIP-qPCR also detected FilR1 associations with key genes involved in acetoclastic methanogenesis, acs4 and acs1. Electrophoretic mobility shift assays confirmed the in vitro tight binding of FilR1 to its own promoter and those of filI-filR2, acs4, and mtrABC. This also proves the DNA-binding ability of the ArsR-related domain, which is found primarily in Archaea. The archaeal promoters of acs4, filI, acs1, and mtrABC also initiated FilR1-modulated expression in an Escherichia coli lux reporter system, suggesting that FilR1 can up-regulate both archaeal and bacterial transcription. In conclusion, this work identifies an archaeal FilI/FilRs TCS that regulates the methanogenesis of M. harundinacea.

A novel mesophilic, methylotrophic, methanogenic archaeon, designated strain EK1(T), was enriched and isolated from wetland sediment. Phylogenetic analysis showed that strain EK1(T) was affiliated with the genus Methanomethylovorans within the family Methanosarcinaceae, and shared the highest 16S rRNA and methyl-coenzyme M reductase alpha-subunit gene sequence similarity with the type strain of Methanomethylovorans hollandica (98.8 and 92.6 %, respectively). The cells of strain EK1(T) were observed to be Gram-negative, non-motile and irregular cocci that did not lyse in 0.1 % (w/v) sodium dodecyl sulfate. Methanol, mono-, di- and trimethylamine, dimethyl sulfide and methanethiol were found to be used as catabolic and methanogenic substrates, whereas H2/CO2, formate, 2-propanol and acetate were not. Growth was observed at 25-40 °C (optimum, 37 °C), at pH 5.5-7.5 (optimum, pH 6.0-6.5) and in the presence of 0-0.1 M NaCl (optimum, 0 M). Growth and methane production rates were stimulated in the presence of H2/CO2 although methane production and growth yields were not significantly affected; acetate, formate, 2-propanol and CO/CO2/N2 did not affect methane production. CoCl2 (0.6-2.0 μM) and FeCl2 (25 mg/l) stimulated growth, while yeast extract and peptone did not. The DNA-DNA hybridization experiment revealed a relatedness of <20 % between EK1(T) and the type strains of the genus Methanomethylovorans. The DNA G+C content of strain EK1(T) was determined to be 39.2 mol%. Based on the polyphasic taxonomic study, strain EK1(T) represents a novel species belonging to the genus Methanomethylovorans, for which the name Methanomethylovorans uponensis sp. nov. is proposed. The type strain is strain EK1(T)(=NBRC 109636(T) = KCTC 4119(T) = JCM 19217(T)).

A methanogenic hexadecane-degrading consortium designated SK originating from the Shengli oil field was cultured at 55 °C. The structure and dynamics of the microbial community during successive transfers were examined using terminal restriction fragment length polymorphism (T-RFLP) fingerprinting and sequencing of 16S rRNA gene fragments. The archaeal community was mainly composed of hydrogenotrophic methanogens affiliated with Methanothermobacter crinale and acetoclastic methanogens related to Methanosaeta thermophila. Over four-fifths of the bacterial clones in the hexadecane-degrading subcultures exhibited < 90% similarity to sequences of known type strains, and clones were mainly grouped into unclassified bacteria (66.3-66.7%), Firmicutes (9.6-10.6%), Thermotogae (7.0-7.7%), and Nitrospira (5.3-5.8%). The dominant operating taxonomic unit (OTU) (41.3-43.0% of all clones) representing terminal restriction fragment (T-RF) 125 bp exhibited only 82.6% sequence similarity to Thermotoga maritime and clustered in a monophyletic, deep-branching lineage (designated Shengli cluster). Two other OTUs (T-RFs 66 and 67 bp) were assigned to uncultured members of the candidate phylum OP8 and Firmicutes, respectively. These novel bacterial assemblages are likely to be involved in the process of hexadecane degradation because of their high abundance in the enrichments. These result substantially expand the knowledge of the extent of bacterial diversity associated with the anaerobic degradation of alkanes under thermophilic conditions., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Hyperthermophile proteins commonly have higher numbers of surface ionic interactions than homologous proteins from other domains of life. PfuTIM, a triosephosphate isomerase (TIM) from the hyperthermophile archaeon, Pyrococcus furiosus, contains an intricate network of 4 ion pairs in its 4th beta/alpha unit, (β/α)4, whereas MbuTIM, a triosephosphate isomerase from a psychrophile archaeon, Methanococcoides burtonii, lacks this network. Notably, (β/α)4 is the first element of the structure formed during folding of certain TIM-type (beta/alpha)8 barrel proteins. Previously, we have shown that elimination of PfuTIM's ion pair network in PfuTIM significantly decreases its kinetic structural stability. Here, we describe the reciprocal experiment in which this ion pair network is introduced into MbuTIM, to produce MutMbuTIM. Recombinant MbuTIM displays multi-state unfolding with apparent Tm values of autonomous structural elements approaching, or above, 70°C, when a temperature scanning rate of 90°C/h is used. The protein displays significant intrinsic kinetic stability, i.e., there is a marked temperature scan rate-dependence of the Tm values associated with unfolding transitions. The Tm values drop by as much as ~10°C when the temperature scanning rate is lowered to 5°C/h. MutMbuTIM, incorporating PfuTIM's ion pair network, shows significantly higher apparent Tm values (raised by 4-6°C over those displayed by MbuTIM). MutMbuTIM also displays significantly higher kinetic thermal stability. Thus, it appears that the thermal stability of triosephosphate isomerase can be increased, or decreased, by either enhancing, or reducing, the strength of ion pair interactions stabilizing (β/α)4, presumably through reduced cooperativity (and increased autonomy) in unfolding transitions., (Copyright © 2013. Published by Elsevier B.V.)

Choline (N,N,N-trimethylethanolamine), which is widely distributed in membrane lipids and is a component of sediment biota, has been shown to be utilized anaerobically by mixed prokaryote cultures to produce methane but not by pure cultures of methanogens. Here, we show that five recently isolated Methanococcoides strains from a range of sediments (Aarhus Bay, Denmark; Severn Estuary mudflats at Portishead, United Kingdom; Darwin Mud Volcano, Gulf of Cadiz; Napoli mud volcano, eastern Mediterranean) can directly utilize choline for methanogenesis producing ethanolamine, which is not further metabolized. Di- and monomethylethanolamine are metabolic intermediates that temporarily accumulate. Consistent with this, dimethylethanolamine was shown to be another new growth substrate, but monomethylethanolamine was not. The specific methanogen inhibitor 2-bromoethanesulfonate (BES) inhibited methane production from choline. When choline and trimethylamine are provided together, diauxic growth occurs, with trimethylamine being utilized first, and then after a lag (∼7 days) choline is metabolized. Three type strains of Methanococcoides (M. methylutens, M. burtonii, and M. alaskense), in contrast, did not utilize choline. However, two of them (M. methylutens and M. burtonii) did metabolize dimethylethanolamine. These results extend the known substrates that can be directly utilized by some methanogens, giving them the advantage that they would not be reliant on bacterial syntrophs for their substrate supply.

In Arctic wet tundra, microbial controls on organic matter decomposition are likely to be altered as a result of climatic disruption. Here, we present a study on the activity, diversity and vertical distribution of methane-cycling microbial communities in the active layer of wet polygonal tundra on Herschel Island. We recorded potential methane production rates from 5 to 40 nmol h(-1) g(-1) wet soil at 10 °C and significantly higher methane oxidation rates reaching values of 6-10 μmol h(-1) g(-1) wet soil. Terminal restriction fragment length polymorphism (T-RFLP) and cloning analyses of mcrA and pmoA genes demonstrated that both communities were stratified along the active layer vertical profile. Similar to other wet Arctic tundra, the methanogenic community hosted hydrogenotrophic (Methanobacterium) as well as acetoclastic (Methanosarcina and Methanosaeta) members. A pronounced shift toward a dominance of acetoclastic methanogens was observed in deeper soil layers. In contrast to related circum-Arctic studies, the methane-oxidizing (methanotrophic) community on Herschel Island was dominated by members of the type II group (Methylocystis, Methylosinus, and a cluster related to Methylocapsa). The present study represents the first on methane-cycling communities in the Canadian Western Arctic, thus advancing our understanding of these communities in a changing Arctic., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The diversity and density of methanogenic archaea and methane production were investigated ex situ at different growth stages of rice plant cultivated in compost-treated tropical rice fields. The qPCR analysis revealed variation in methanogens population from 3.40 × 10(6) to 1.11 × 10(7)  copies g(-1)  dws, in the year 2009 and 4.37 × 10(6) to 1.36 × 10(7)  copies g(-1)  dws in the year 2010. Apart from methanogens, a large number of bacterial (9.60 × 10(9) -1.44 × 10(10)  copies g(-1)  dws) and archaeal (7.13 × 10(7) -3.02 × 10(8)  copies g(-1)  dws) communities were also associated with methanogenesis. Methanogen population size varied in the order: flowering > ripening > tillering > postharvest > preplantation stage. The RFLP-based 16S rRNA gene-targeted phylogenetic analysis showed that clones were closely related to diverse group of methanogens comprising members of Methanomicrobiaceae, Methanosarcinaceae, Methanosaetaceae and RC I. Laboratory incubation studies revealed higher amount of cumulative CH(4) at the flowering stage. The integration of methanogenic community structure and CH(4) production potential of soil resulted in a better understanding of the dynamics of CH(4) production in organically treated rice-field soil. The hypothesis that the stages of plant development influence the methanogenic community structure leading to temporal variation in the CH(4) production has been successfully tested., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Propionate is one of the major intermediary products in the anaerobic decomposition of organic matter in wetlands and paddy fields. Under methanogenic conditions, propionate is decomposed through syntrophic interaction between proton-reducing and propionate-oxidizing bacteria and H(2)-consuming methanogens. Temperature is an important environmental regulator; yet its effect on syntrophic propionate oxidation has been poorly understood. In the present study, we investigated the syntrophic oxidation of propionate in a rice field soil at 15°C and 30°C. [U-(13)C]propionate (99 atom%) was applied to anoxic soil slurries, and the bacteria and archaea assimilating (13)C were traced by DNA-based stable isotope probing. Syntrophobacter spp., Pelotomaculum spp., and Smithella spp. were found significantly incorporating (13)C into their nucleic acids after [(13)C]propionate incubation at 30°C. The activity of Smithella spp. increased in the later stage, and concurrently that of Syntrophomonas spp. increased. Aceticlastic Methanosaetaceae and hydrogenotrophic Methanomicrobiales and Methanocellales acted as methanogenic partners at 30°C. Syntrophic oxidation of propionate also occurred actively at 15°C. Syntrophobacter spp. were significantly labeled with (13)C, whereas Pelotomaculum spp. were less active at this temperature. In addition, Methanomicrobiales, Methanocellales, and Methanosarcinaceae dominated the methanogenic community, while Methanosaetaceae decreased. Collectively, temperature markedly influenced the activity and community structure of syntrophic guilds degrading propionate in the rice field soil. Interestingly, Geobacter spp. and some other anaerobic organisms like Rhodocyclaceae, Acidobacteria, Actinobacteria, and Thermomicrobia probably also assimilated propionate-derived (13)C. The mechanisms for the involvement of these organisms remain unclear.

Soda lakes constitute extreme aquatic ecosystems with remarkably high primary productivity rates, but information on the diversity and activity of methanogens in such environments is sparse. Using 16S rRNA and functional genes, we investigated the diversity of methanogens in the sediments of Lonar Lake, a unique saline and alkaline ecosystem formed by meteorite impact in the Deccan basalts. Although domain and phylum level 16S rRNA gene libraries were dominated by phylotypes related to Halobacteriales, sequences related to potentially novel Archaea within the orders Methanosarcinales and Methanomicrobiales were obtained together with a significant fraction of sequences representing uncultivated Euryarchaeota [Correction added after online publication 16 April 2012: orders 'Methanosarcina and Methanomicrobiaceae' changed to 'Methanosarcinales and Methanomicrobiales']. To identify the active methylotrophic Archaea involved in methanogenesis, mRNA transcripts of mcrA were retrieved from methanol consuming and methane emitting sediment microcosms at two different time points. Reverse-transcription PCR, qPCR, DGGE fingerprint, and clone library analysis showed that the active Archaea were closely related to Methanolobus oregonensis. To our knowledge, this is the first study identifying active methylotrophic methanogens in such an environment., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Marine mud volcanoes are geological structures emitting large amounts of methane from their active centres. The Amsterdam mud volcano (AMV), located in the Anaximander Mountains south of Turkey, is characterized by intense active methane seepage produced in part by methanogens. To date, information about the diversity or the metabolic pathways used by the methanogens in active centres of marine mud volcanoes is limited. (14)C-radiotracer measurements showed that methylamines/methanol, H(2)/CO(2) and acetate were used for methanogenesis in the AMV. Methylotrophic methanogenesis was measured all along the sediment core, Methanosarcinales affiliated sequences were detected using archaeal 16S PCR-DGGE and mcrA gene libraries, and enrichments of methanogens showed the presence of Methanococcoides in the shallow sediment layers. Overall acetoclastic methanogenesis was higher than hydrogenotrophic methanogenesis, which is unusual for cold seep sediments. Interestingly, acetate porewater concentrations were extremely high in the AMV sediments. This might be the result of organic matter cracking in deeper hotter sediment layers. Methane was also produced from hexadecanes. For the most part, the methanogenic community diversity was in accordance with the depth distribution of the H(2)/CO(2) and acetate methanogenesis. These results demonstrate the importance of methanogenic communities in the centres of marine mud volcanoes., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Earthworms emit denitrification-derived nitrous oxide and fermentation-derived molecular hydrogen. The present study demonstrated that the earthworm Eudrilus eugeniae, obtained in Brazil, emitted methane. Other worms displayed a lesser or no capacity to emit methane. Gene and transcript analyses of mcrA (encoding the alpha subunit of methyl-CoM reductase) in gut contents of E. eugeniae suggested that Methanosarcinaceae, Methanobacteriaceae, and Methanomicrobiaceae might be associated with this emission.

Methanogens have been reported in complex microbial communities from hypersaline environments, but little is known about their phylogenetic diversity. In this work, methane concentrations in environmental gas samples were determined while methane production rates were measured in microcosm experiments with competitive and non-competitive substrates. In addition, the phylogenetic diversity of methanogens in microbial mats from two geographical locations was analyzed: the well studied Guerrero Negro hypersaline ecosystem, and a site not previously investigated, namely Laguna San Ignacio, Baja California Sur, Mexico. Methanogenesis in these microbial mats was suspected based on the detection of methane (in the range of 0.00086 to 3.204 %) in environmental gas samples. Microcosm experiments confirmed methane production by the mats and demonstrated that it was promoted only by non-competitive substrates (trimethylamine and methanol), suggesting that methylotrophy is the main characteristic process by which these hypersaline microbial mats produce methane. Phylogenetic analysis of amino acid sequences of the methyl coenzyme-M reductase (mcrA) gene from natural and manipulated samples revealed various methylotrophic methanogens belonging exclusively to the family Methanosarcinaceae. Moderately halophilic microorganisms of the genus Methanohalophilus were predominant (>60 % of mcrA sequences retrieved). Slightly halophilic and marine microorganisms of the genera Methanococcoides and Methanolobus, respectively, were also identified, but in lower abundances.

RNA polymerase in Archaea is composed of 11 or 12 subunits - 9 or 10 that form the core, and a heterodimer formed from subunits E and F that associates with the core and can interact with general transcription factors and facilitate transcription. While the ability of the heterodimer to bind RNA has been demonstrated, it has not been determined whether it can recognize specific RNA targets. In this study we used a recombinant archaeal MbRpoE/F to capture cellular mRNA in vitro and a microarray to determine which transcripts it specifically binds. Only transcripts for 117 genes (4% of the total) representing 48 regions of the genome were bound by MbRpoE/F. The transcripts represented important genes in a number of functional classes: methanogenesis, cofactor biosynthesis, nucleotide metabolism, transcription, translation, import/export. The arrangement and characteristics (e.g. codon and amino acid usage) of genes relative to the putative origin of replication indicate that MbRpoE/F preferentially binds to mRNA of genes whose expression may be important for cellular fitness. We also compared the biophysical properties of RpoE/F from M. burtonii and Methanocaldococcus jannaschii, demonstrating a 50°C difference in their apparent melting temperatures. By using MbRpoE/F to capture and characterize cellular RNA we have identified a previously unknown functional property of the MbRpoE/F heterodimer., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The growth of all microorganisms is limited to a specific temperature range. However, it has not previously been determined to what extent global protein profiles change in response to temperatures that incrementally span the complete growth temperature range of a microorganism. As a result it has remained unclear to what extent cellular processes (inferred from protein abundance profiles) are affected by growth temperature and which, in particular, constrain growth at upper and lower temperature limits. To evaluate this, 8-plex iTRAQ proteomics was performed on the Antarctic microorganism, Methanococcoides burtonii. Methanococcoides burtonii was chosen due to its importance as a model psychrophilic (cold-adapted) member of the Archaea, and the fact that proteomic methods, including subcellular fractionation procedures, have been well developed. Differential abundance patterns were obtained for cells grown at seven different growth temperatures (-2°C, 1°C, 4°C, 10°C, 16°C, 23°C, 28°C) and a principal component analysis (PCA) was performed to identify trends in protein abundances. The multiplex analysis enabled three largely distinct physiological states to be described: cold stress (-2°C), cold adaptation (1°C, 4°C, 10°C and 16°C), and heat stress (23°C and 28°C). A particular feature of the thermal extremes was the synthesis of heat- and cold-specific stress proteins, reflecting the important, yet distinct ways in which temperature-induced stress manifests in the cell. This is the first quantitative proteomic investigation to simultaneously assess the response of a microorganism to numerous growth temperatures, including the upper and lower growth temperatures limits, and has revealed a new level of understanding about cellular adaptive responses., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Methanococcoides burtonii is a member of the Archaea that was isolated from Ace Lake in Antarctica and is a valuable model for studying cold adaptation. Low temperature transcriptional regulation of global gene expression, and the arrangement of transcriptional units in cold-adapted archaea has not been studied. We developed a microarray for determining which genes are expressed in operons, and which are differentially expressed at low (4°C) or high (23°C) temperature. Approximately 55% of genes were found to be arranged in operons that range in length from 2 to 23 genes, and mRNA abundance tended to increase with operon length. Analysing microarray data previously obtained by others for Halobacterium salinarum revealed a similar correlation between operon length and mRNA abundance, suggesting that operons may play a similar role more broadly in the Archaea. More than 500 genes were differentially expressed at levels up to ≈ 24-fold. A notable feature was the upregulation of genes involved in maintaining RNA in a state suitable for translation in the cold. Comparison between microarray experiments and results previously obtained using proteomics indicates that transcriptional regulation (rather than translation) is primarily responsible for controlling gene expression in M. burtonii. In addition, certain genes (e.g. involved in ribosome structure and methanogenesis) appear to be regulated post-transcriptionally. This is one of few experimental studies describing the genome-wide distribution and regulation of operons in archaea., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Archaeal Group II chaperonins (Cpns) are strongly conserved, considering that their growth temperatures range from 23 to 122°C. The C-terminal 15-25 residues are hypervariable, and highly charged in thermophilic species. Our hypothesis is that the C-terminal is a key determinant of stabilization of the Cpn complex. The C-terminus of the Cpn from the hyperthermophile Pyrococcus furiosus was mutated to test this hypothesis. C-terminal deletions and replacement of charged residues resulted in destabilization. The stability of ATPase activity declined in proportion to the reduction in charged residues with Ala or Gly. An EK-rich motif ((528)EKEKEKEGEK5(37)) proved to be a key domain for stabilization at or near 100°C. Mutations "tuned" the Cpn for optimal protein folding at lower optimal temperatures, and Glu substitution was more potent than Lys replacement. Pf Cpn stability was enhanced by Ca(2+), especially in the mutant Cpn lacking C-terminal Lys residues. This suggests that Glu-Glu interactions between C termini might be mediated by Ca(2+). The C-terminal of a Cpn from the psychrophilic archaeon Methanococcoides burtonii was replaced by a domain from the hyperthermophile, resulting in increased thermostability and thermoactivity. We conclude that localized evolutionary variation in the C-terminus modulates the temperature range of archaeal Cpns., (Copyright © 2011 Elsevier Inc. All rights reserved.)