Project description:We report here the complete genome sequence (2.08 Mb) of Methanohalophilus portucalensis strain FDF-1T, a halophilic methylotrophic methanogen isolated from the sediment of a saltern in Figeria da Foz, Portugal. The average nucleotide identity and DNA-DNA hybridization analyses show that Methanohalophilus mahii, M. halophilus, and M. portucalensis are three different species within the Methanosarcinaceae family.
Project description:The complete genome sequence of Methanohalophilus halophilus DSM 3094T, a member of the Methanosarcinaceae family and the Methanosarcianales order, consists of 2,022,959 bp in one contig and contains 2,137 predicted genes. The genome is consistent with a halophilic methylotrophic anaerobic lifestyle, including the methylotrophic and CO2-H2 methanogensis pathways.
Project description:The halophilic methanoarchaeon Methanohalophilus portucalensis can synthesize the osmolyte betaine de novo in response to extracellular salt stress. Betaine is generated by the stepwise methylation of glycine to form sarcosine, N, N-dimethylglycine and betaine by using S-adenosyl-L-methionine (AdoMet) as the methyl donor. The complete gene cluster of Mpgsmt-sdmt was cloned from Southern hybridization and heterologous expressed in E. coli respectively. The recombinant MpGSMT and MpSDMT both retained their in vivo functional activities in E. coli BL21(DE3)RIL to synthesize and accumulate betaine and conferred elevated survival ability in betaine transport deficient mutant E. coli MKH13 under high salt stress. The dramatic activating effects of sodium and potassium ions on the in vitro methyltransferase activities of MpGSMT, but not MpSDMT or bacterial GSMT and SDMT, revealed that GSMT from halophilic methanoarchaeon possesses novel regulate mechanism in betaine biosynthesis pathway. The circular dichroism spectra showed the fluctuated peaks at 206 nm were detected in the MpGSMT under various concentrations of potassium or sodium ions. This fluctuated difference may cause by a change in the ?-turn structure located at the conserved glycine- and sarcosine-binding residue Arg167 of MpGSMT. The analytical ultracentrifugation analysis indicated that the monomer MpGSMT switched to dimeric form increased from 7.6% to 70% with KCl concentration increased from 0 to 2.0 M. The level of potassium and sodium ions may modulate the substrate binding activity of MpGSMT through the conformational change. Additionally, MpGSMT showed a strong end product, betaine, inhibitory effect and was more sensitive to the inhibitor AdoHcy. The above results indicated that the first enzymatic step involved in synthesizing the osmolyte betaine in halophilic archaea, namely, GSMT, may also play a major role in coupling the salt-in and compatible solute (osmolyte) osmoadaptative strategies in halophilic methanogens for adapting to high salt environments.
Project description:Methane is a well-known factor in green house effect and also could be an alternative energy, which produced via methanogenesis by methanogenic archaea. Methanohalophilus portucalensis FDF1T is a halophilic methylotrophic methanogen and has been used as a model to study osmoadaptation in fluctuated abiotic environmental conditions. Here we report a genome-wide phosphoproteomic analysis of M. portucalensis FDF1T to uncover the possible phosphorylation-mediated regulations in methanogenesis and osmoadaptation. According to the biological function analysis revealed 20.6% of total 149 phosphoproteins were involved in the regulation of the single carbon metabolism for methanogenesis and osmoadaptation. Together with genome sequencing and phosphoproteomic analysis, we provided the first evidence to indicate protein phosphorylation networks in regulating the pyrrolysine (Pyl) mediated translation of methylotrophic methyltransferase. Furthermore, the functional assays of glycine sarcosine N-methyltransferase revealed that the enzymatic activity was modulated by threonine phosphorylation. Collectively, our results not only provide new insight into phosphorylation-mediated regulations in single carbon energy metabolism but also clearly demonstrate that phosphorylation plays a critical role in osmolyte betaine synthesis through a phospho-switch mechanism.
Project description:The halophilic methanoarchaeon Methanohalophilus portucalensis FDF1T possesses the ability to synthesize the osmolyte betaine from its precursor, glycine, in response to extracellular salt stress through a three-step transmethylation process. Analysis of recombinant glycine sarcosine N-methyltransferase (rGSMT) and recombinant sarcosine dimethylglycine N-methyltransferase (rSDMT) from Escherichia coli indicated that betaine synthesis is rate-limited by rGSMT and is constitutively activated by rSDMT. Therefore, it is of interest to purify native GSMT from Methanohalophilus portucalensis to further compare its enzymatic characteristics and kinetics with rGSMT. In this study, native GSMT was purified through DEAE ion exchange and gel filtration chromatography with 95% purity. The enzymatic characteristics of GSMT and rGSMT showed similar trends of activities that were activated by high concentrations of monovalent cations. Both were feedback-inhibited by the end product, betaine, and competitively inhibited by S-adenosylhomocysteine (SAH). Native GSMT was 2-fold more sensitive to SAH than rGSMT. Notably, comparison of the kinetic parameters illustrated that the turnover rate of glycine methylation of GSMT was promoted by potassium ions, whereas rGSMT was activated by increasing protein-glycine binding affinity. These results suggest that GSMT and rGSMT may have different levels of post-translational modifications. Our preliminary mass spectrometry evidence indicated that there was no detectable phosphosite on GSMT after the complicated purification processes, whereas purified rGSMT still possessed 23.1% of its initial phosphorylation level. We believe that a phosphorylation-mediated modification may be involved in the regulation of this energy consuming betaine synthesis pathway during the stress response in halophilic methanoarchaea.
Project description:Methanohalophilus mahii is the type species of the genus Methanohalophilus, which currently comprises three distinct species with validly published names. Mhp. mahii represents moderately halophilic methanogenic archaea with a strictly methylotrophic metabolism. The type strain SLP(T) was isolated from hypersaline sediments collected from the southern arm of Great Salt Lake, Utah. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,012,424 bp genome is a single replicon with 2032 protein-coding and 63 RNA genes and part of the Genomic Encyclopedia of Bacteria and Archaea project. A comparison of the reconstructed energy metabolism in the halophilic species Mhp. mahii with other representatives of the Methanosarcinaceae reveals some interesting differences to freshwater species.
Project description:<h4>Background</h4>Chromohalobacter salexigens (formerly Halomonas elongata DSM 3043) is a halophilic extremophile with a very broad salinity range and is used as a model organism to elucidate prokaryotic osmoadaptation due to its strong euryhaline phenotype.<h4>Results</h4>C. salexigens DSM 3043's metabolism was reconstructed based on genomic, biochemical and physiological information via a non-automated but iterative process. This manually-curated reconstruction accounts for 584 genes, 1386 reactions, and 1411 metabolites. By using flux balance analysis, the model was extensively validated against literature data on the C. salexigens phenotypic features, the transport and use of different substrates for growth as well as against experimental observations on the uptake and accumulation of industrially important organic osmolytes, ectoine, betaine, and its precursor choline, which play important roles in the adaptive response to osmotic stress.<h4>Conclusions</h4>This work presents the first comprehensive genome-scale metabolic model of a halophilic bacterium. Being a useful guide for identification and filling of knowledge gaps, the reconstructed metabolic network iOA584 will accelerate the research on halophilic bacteria towards application of systems biology approaches and design of metabolic engineering strategies.
Project description:Intorduction:Dead Sea is a hypersaline lake with 34% salinity, gains its name due to the absence of any living macroscopic creatures. Despite the extreme hypersaline environment, it is a unique ecosystem for various halophilic microorganisms adapted to this environment. Aims & Objectives:Halophilic microorganisms are known for various potential biotechnological applications, the purpose of the current research is isolation and screening of halophilic bacteria from Dead Sea mud for potential antimicrobial applications. Methods & Materials:Screening for antagonistic bacteria was conducted by bacterial isolation from Dead Sea mud samples and agar plate antagonistic assay. The potential antagonistic isolates were subjected to biochemical characterization and identification by 16S-rRNA sequencing. Among the collected isolates, four isolates showed potential antagonistic activity against Bacillus subtilis 6633 and Escherichia coli 8739. The most active isolate (24-DSM) was subjected for antagonistic activity and minimal inhibitory concentration against different gram positive and negative bacterial strains after cultivation in different salt concentration media. Results: The results of 16S-rRNA analysis revealed that 24-DSM is very closely related to Bacillus persicus strain B48, which was isolated from hypersaline lake in Iran. Conclusion:Therefore, the isolate 24-DSM is assigned as a new strain of B. persicusi isolated from the Dead Sea mud. B. persicusi 24-DSM showed higher antimicrobial activity, when it was cultivated with saline medium, against all tested bacterial strains, where the most sensitive bacterial strain was Corynebacterium diphtheria 51696.
Project description:In deep ocean hypersaline basins, the combination of high salinity, unusual ionic composition and anoxic conditions represents significant challenges for microbial life. We used geochemical porewater characterization and DNA sequencing based taxonomic surveys to enable environmental and microbial characterization of anoxic hypersaline sediments and brines in the Orca Basin, the largest brine basin in the Gulf of Mexico. Full-length bacterial 16S rRNA gene clone libraries from hypersaline sediments and the overlying brine were dominated by the uncultured halophilic KB1 lineage, Deltaproteobacteria related to cultured sulfate-reducing halophilic genera, and specific lineages of heterotrophic Bacteroidetes. Archaeal clones were dominated by members of the halophilic methanogen genus Methanohalophilus, and the ammonia-oxidizing Marine Group I (MG-I) within the Thaumarchaeota. Illumina sequencing revealed higher phylum- and subphylum-level complexity, especially in lower-salinity sediments from the Orca Basin slope. Illumina and clone library surveys consistently detected MG-I Thaumarchaeota and halotolerant Deltaproteobacteria in the hypersaline anoxic sediments, but relative abundances of the KB1 lineage differed between the two sequencing methods. The stable isotopic composition of dissolved inorganic carbon and methane in porewater, and sulfate concentrations decreasing downcore indicated methanogenesis and sulfate reduction in the anoxic sediments. While anaerobic microbial processes likely occur at low rates near their maximal salinity thresholds in Orca Basin, long-term accumulation of reaction products leads to high methane concentrations and reducing conditions within the Orca Basin brine and sediments.