Methanolobus use unspecific methyltransferases to produce methane from dimethylsulphide in Baltic Sea sediments.

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.

Isolation and physiological properties of methanogenic archaea that degrade tetramethylammonium hydroxide.

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

Ecology of Methanonatronarchaeia.

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 C1 -methylated compounds and H2 /formate.

Post-transcriptional regulation is involved in the cold-active methanol-based methanogenic pathway of a psychrophilic methanogen.

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.

Methyl-compounds driven benthic carbon cycling in the sulfate-reducing sediments of South China Sea.

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.

Production of methylmercury by methanogens in mercury contaminated estuarine sediments.

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).

Biochemical and phylogenetic characterization of a monomeric isocitrate dehydrogenase from a marine methanogenic archaeon Methanococcoides methylutens.

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 Mn2+ 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.

High-rate anaerobic decolorization of methyl orange from synthetic azo dye wastewater in a methane-based hollow fiber membrane bioreactor.

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.

A CUGGU/UUGGU-specific MazF homologue from Methanohalobium evestigatum.

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.

Long-term succession in a coal seam microbiome during in situ biostimulation of coalbed-methane generation.

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 13C-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.

Deltaproteobacteria (Pelobacter) and Methanococcoides are responsible for choline-dependent methanogenesis in a coastal saltmarsh sediment.

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 13C2-choline enriched metagenomes, followed by metagenome data assembly, to identify the key microbes responsible for methanogenesis from choline. Microcosm incubation with 13C2-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.

Comparative genomics and physiology of the genus Methanohalophilus, a prevalent methanogen in hydraulically fractured shale.

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.

Methanogenic degradation of tetramethylammonium hydroxide by Methanomethylovorans and Methanosarcina.

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.

Methanolobus psychrotolerans sp. nov., a psychrotolerant methanoarchaeon isolated from a saline meromictic lake in Siberia.

A psychrotolerant, methylotrophic methanogen, strain YSF-03T, was isolated from the saline meromictic Lake Shira in Siberia. Cells of strain YSF-03T were non-motile, irregular cocci and 0.8-1.2 µm in diameter. The methanogenic substrates utilized by strain YSF-03T were methanol and trimethylamine. The temperature range of growth for strain YSF-03T 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-03T was 41.3 mol%. Phylogenetic analysis revealed that strain YSF-03T was most closely related to Methanolobus profundi MobMT (98.15 % similarity in 16S rRNA gene sequence). Genome relatedness between strain YSF-03T and MobMT 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-03T represents a novel species of the genus Methanolobus, for which the name Methanolobus psychrotolerans sp. nov. is proposed. The type strain is YSF-03T (=BCRC AR10049T=DSM 104044T=NBRC 112514T).

Anaerobic digestion of pig manure supernatant at high ammonia concentrations characterized by high abundances of Methanosaeta and non-euryarchaeotal archaea.

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.

Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis.

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 response of archaeal species to seasonal variables in a subtropical aerated soil: insight into the low abundant methanogens.

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.

Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1(T) identified the role of protein phosphorylation in methanogenesis and osmoregulation.

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.

Characterization of a temperature-responsive two component regulatory system from the Antarctic archaeon, Methanococcoides burtonii.

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.

Single TRAM domain RNA-binding proteins in Archaea: functional insight from Ctr3 from the Antarctic methanogen Methanococcoides burtonii.

TRAM domain proteins present in Archaea and Bacteria have a ß-barrel shape with anti-parallel ß-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.