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Summary: Anaerobic oxidation of methane (AOM) is a microbial process degrading ample methane in anoxic environments, and Ca. Methanoperedens mediated nitrate‐ or metal‐reduction linked AOM is believed important in freshwater systems. This work, via 16S rRNA gene diversity survey and 16S rRNA quantification, found abundant Ca. Methanoperedens along with iron in the cold Zoige wetland at Tibetan Plateau. The wetland soil microcosm performed Fe(III) reduction, rather than nitrate‐ nor sulphate‐reduction, coupled methane oxidation (3.87 μmol d−1) with 32.33 μmol Fe(II) accumulation per day at 18°C, but not at 30°C. A metagenome‐assembled genome (MAG) recovered from the microcosm exhibits ~74% average nucleotide identity with the reported Ca. Methanoperedens spp. that perform Fe(III) reduction linked AOM, thus a novel species Ca. Methanoperedens psychrophilus was proposed. Ca. M. psychrophilus contains the whole suite of CO2 reductive methanogenic genes presumably involving in AOM via a reverse direction, and comparative genome analysis revealed its unique gene categories: the multi‐heme clusters (MHCs) cytochromes, the S‐layer proteins highly homologous to those recovered from lower temperature environments and type IV pili, those could confer Ca. M. psychrophilus of cold adaptability. Therefore, this work reports the first methanotroph implementing AOM in an alpine wetland. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

Adaptation to higher temperatures would increase the environmental competitiveness of psychrophiles, organisms that thrive in low‐temperature environments. Methanolobus psychrophilus, a cold wetland methanogen, 'evolved' as a mesophile, growing optimally at 30 °C after subculturings, and cells grown with ample substrates exhibited higher integrity. Here, we investigated N‐glycosylation of S‐layer proteins, the major archaeal envelope component, with respect to mesophilic adaptation. Lectin affinity enriched a glycoprotein in cells grown at 30 °C under ample substrate availability, which was identified as the S‐layer protein Mpsy_1486. Four N‐glycosylation sites were identified on Mpsy_1486, which exhibited different glycosylation profiles, with N94 only found in cells cultured at 30 °C. An N‐linked glycosylation inhibitor, tunicamycin, reduced glycosylation levels of Mpsy_1486 and growth at 30 °C, thus establishing a link between S‐layer protein glycosylation and higher temperature adaptation of the psychrophilic archaeon M. psychrophilus. [ABSTRACT FROM AUTHOR]

Summary: It has been recently shown that magnetite nanoparticles (nanoFe3O4) can facilitate methanogenic syntrophy but the effect of magnetite on methanogenesis alone remains elusive. Here we show that aceticlastic methanogenesis by Methanosarcina mazei is accelerated by magnetite and is correlated with the redox cycling of structural Fe(II) and Fe(III) in the mineral. An enrichment and its closest pure culture relative, Ms. mazei zm‐15, both obtained from a natural wetland of the Tibetan plateau were tested in this experiment. The Fe(II) to Fe(III) ratios in magnetite, as measured by multiple approaches, show an initial increase in both the methanogenic cultures and the blank preparations containing no microbes. The Fe(II)/Fe(III) ratio then displays a distinct decline followed by an increase towards the end of incubation only in the enrichment and pure culture cultivations. This redox cycling of magnetite is in accordance with the stimulation of aceticlastic methanogenesis. Microscopic observation reveals the precipitation of nanoFe3O4 on methanogen cell surface. The genomic analysis predicts that in addition to electron transfer components essential for aceticlastic methanogenesis, Ms. mazei zm‐15 contains an outer‐surface multiheme c‐type cytochrome (MHC) and a few function‐unknown surface proteins that harbour monoheme motif. We hypothesize that the redox cycling of nanoFe3O4 delivers a positive influence via the MHC to the membrane electron transfer chain and hence promote the aceticlastic methanogenesis. [ABSTRACT FROM AUTHOR]

Summary: Natural transformation increases the genetic diversity of bacteria, but is costly and must be strictly controlled. We previously found that deletion of ccpA, a key regulator of carbon catabolite repression (CCR), reduced transformation efficiency of Streptococcus oligofermentans, the current work further investigated the regulatory mechanisms of CcpA. The competence operon comCDE is subjected to basal and autoregulatory transcription. A luciferase reporter detected a transcriptional readthrough (TRT) from the upstream tRNAArg into the comCDE operon, which was induced by L‐arginine. Insertion of the Escherichia coli T1T2 terminator downstream of tRNAArg abolished TRT, and reduced the basal comCDE transcription by 77% and also the transformation efficiency. Deletion of ccpA increased tRNAArg TRT and tRNAArg‐comCDE polycistronic transcript by twofold. An in vitro transcription assay determined that CcpA promoted the transcription termination of tRNAArg TRT, and RNA EMSA and SPR assays detected equal binding affinity of CcpA to both the RNA and DNA of tRNAArg. These results indicate that CcpA controls the basal comCDE transcription by post‐transcriptional actions. Overexpression of comDE or its phospho‐mimicking mutant comDED58E reduced transformation efficiency, indicating that excessive ComE impairs competence development. CCR‐regulated competence was further confirmed by higher tRNAArg TRT but lower transformation efficiency in galactose than in glucose. [ABSTRACT FROM AUTHOR]

RNase J, a prokaryotic 5′-3′ exo/endoribonuclease, contributes to mRNA decay, rRNA maturation and post-transcriptional regulation. Yet the processive-exoribonucleolysis mechanism remains obscure. Here, we solved the first RNA-free and RNA-bound structures of an archaeal RNase J, and through intensive biochemical studies provided detailed mechanistic insights into the catalysis and processivity. Distinct dimerization/tetramerization patterns were observed for archaeal and bacterial RNase Js, and unique archaeal Loops I and II were found involved in RNA interaction. A hydrogen-bond-network was identified for the first time that assists catalysis by facilitating efficient proton transfer in the catalytic center. A conserved 5′-monophosphate-binding pocket that coordinates the RNA 5′-end ensures the 5′-monophosphate preferential exoribonucleolysis. To achieve exoribonucleolytic processivity, the 5′-monophosphate-binding pocket and nucleotide +4 binding site anchor RNA within the catalytic track; the 5′-capping residue Leu37 of the sandwich pocket coupled with the 5′-monophosphate-binding pocket are dedicated to translocating and controlling the RNA orientation for each exoribonucleolytic cycle. The processive-exoribonucleolysis mechanism was verified as conserved in bacterial RNase J and also exposes striking parallels with the non-homologous eukaryotic 5′-3′ exoribonuclease, Xrn1. The findings in this work shed light on not only the molecular mechanism of the RNase J family, but also the evolutionary convergence of divergent exoribonucleases. [ABSTRACT FROM AUTHOR]

RATIONALE Methanol, ethanol, and acetic acid are not easily extracted from aqueous samples and are susceptible to isotope fractionation in gas chromatography/isotope ratio mass spectrometry (GC/IRMS) analysis. Developing a direct dilution GC/IRMS method for aqueous samples, by adjusting the sample concentrations in common solvents to be similar to each other and using a fixed GC split ratio, is very convenient and important because any linearity effects caused by amount-dependent isotope fractionation can be avoided. METHODS The suitability of acetonitrile and acetone solvents for the GC/IRMS analysis of pure methanol, ethanol and acetic acid, and commercial liquor and vinegar samples was evaluated using n-hexane and water as control solvents. All the solvents including water were separated from the analyte on a HP-INNOWAX column and were diverted away from the combustion interface. The influence of liquor matrix on the ethanol GC/IRMS analyses was evaluated by adding pure ethanol to liquor samples. RESULTS Acetonitrile and acetone gave similar δ13C values for pure ethanol and pure acetic acid to those obtained in water and n-hexane, and also gave similar δ13C values of ethanol in liquor and acetic acid in white vinegar to that obtained in water. For methanol analysis, acetonitrile and refined acetone gave similar δ13C values to that obtained in water, but n-hexane was not a suitable solvent. In addition, isotopic fractionation caused by solvent and solute interactions was observed. CONCLUSIONS We recommend using acetonitrile for the GC/IRMS analysis of aqueous alcoholic samples, and acetone for the analysis of aqueous acetic acid samples. This direct dilution method can provide high accurate and precise GC/IRMS analysis of the relative changes in δ13C values of methanol, ethanol, and acetic acid. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

RATIONALE The stable carbon isotope ratios of methanogen-produced CH4 and CO2 are useful information for identifying and quantifying methanogenic pathways. Isotope ratio mass spectrometry combined with gas chromatography (GC/IRMS) is a very attractive tool for performing high-precision compound-specific isotope analysis. However, no GC/IRMS techniques have yet been available or been validated that give baseline separation of methanogen-produced CH4 and CO2 from N2/N-oxides and H2O vapor at ambient temperature and compatibility with analysis by mass spectrometry. METHODS Microbe-produced CH4 and CO2 in headspace gases were separated from N2/N-oxides and H2O vapor in a single run on a GS-CarbonPLOT column at 35°C and with a maximum operating temperature of 120-140°C. The simultaneous characterization of CH4 and CO2 was then performed by GC/IRMS using an optimized backflush time to eliminate the interference from N2/N-oxides and H2O vapor, and by GC/MS due to there being no interference from O2 gas in the culture. RESULTS GC/MS and GC/IRMS were used to calculate the ionization efficiency of CO2 as 8.22-8.84 times that of CH4 in GC/MS analysis, and it was deduced that the N-oxides, which can potentially interfere with δ13C analysis, were probably produced mainly in the source of the isotope ratio mass spectrometer. We also determined the aceticlastic methanogenic pathway. CONCLUSIONS The established GC/MS and GC/IRMS techniques are suitable for characterizing the gaseous carbon-containing compounds produced by microbial cultures. Through high-precision carbon isotope analysis by GC/IRMS combined with low concentrations of 13C-labelled substrates, the technique has great potential for identifying and quantifying methanogen-mediated carbon metabolic processes and pathways. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

We analysed the cold-responsive gene repertoire for a psychrophilic methanogen, Methanolobus psychrophilus R15 through genomic and RNA-seq assayed transcriptomic comparisons for cultures at 18° C (optimal temperature) versus 4° C. The differences found by RNA-seq analysis were verified using quantitative real time- PCR assay. The results showed that as in the Antarctic methanogen, Methanococcoides burtonii, genes for methanogenesis, biosynthesis and protein synthesis were all downregulated by the cold in R15. However, the RNA polymerase complex was upregulated at cold, as well as a gene cluster for a putative exosome complex, suggesting that exosome-mediated RNA decay may be cold-accelerated. Unexpectedly, the chaperonin genes for both thermosome and GroES/ EL were all upregulated at 4° C. Strain R15 possessed eight protein families for oxygen detoxification, including both anaerobe-specific superoxide reductase ( SOR) and the aerobe-typical superoxide dismutase ( SOD)-catalase oxidant-removing system, implying the higher oxidative tolerance. Compared with a mesophilic methanogen, R15 survived in higher paraquat, a redox-cycling drug. Moreover, 71 one-component systems and 50 two-component systems for signal transduction ranked strain R15, together with M. burtonii, as being highly adaptive among archaea. Most of them exhibited cold-enhanced expression, indicating their involvement in cold adaptation. This study has added new perspectives on the cold adaptation of methanogenic archaea. [ABSTRACT FROM AUTHOR]