Methanooceanicella nereidis gen. nov., sp. nov., the first oceanic Methanocellaceae methanogen, isolated from potential methane hydrate bearing area offshore southwestern Taiwan.

A novel mesophilic, hydrogenotrophic methanogen, strain CWC-04T, was obtained from a sediment sample extracted from a gravity core retrieved at station 22 within the KP-9 area off the southwestern coast of Taiwan during the ORIII-1368 cruise in 2009. Cells of strain CWC-04T were rod-shaped, 1.4-2.9 µm long by 0.5-0.6 µm wide, and occurred singly. Strain CWC-04Tutilized formate, H2/CO2, 2-propanol/CO2 or 2-butanol/CO2 as catabolic substrates. The optimal growth conditions were 42 °C, 0.17 M NaCl and pH 5.35. The genomic DNA G+C content calculated from the genome sequence of strain CWC-04T was 46.19 mol%. Phylogenetic analysis of 16S rRNA gene revealed that strain CWC-04T is affiliated with the genus Methanocella. The 16S rRNA gene sequences similarities within strains Methanocella arvoryzae MRE50T, Methanocella paludicola SANAET and Methanocella conradii HZ254T were 93.7, 93.0 and 91.3 %, respectively. In addition, the optical density of CWC-04T culture dropped abruptly upon entering the late-log growth phase, with virus-like particles (150 nm in diameter) being observed on and around the cells. This observation suggests that strain CWC-04T harbours a lytic virus. Based on these phenotypic, phylogenetic and genomic results, we propose that strain CWC-04T represents a novel species of a novel genus in the family Methanocellaceae, for which the name Methanooceanicella nereidis gen. nov., sp. nov. is proposed. The type strain is CWC-04T (=BCRC AR10050T=NBRC 113165T).

Six novel species of the genus Methanoculleus isolated from various environments in Taiwan.

Taiwan is situated in the subtropical region and its geographical location and topographical features contribute to a rich ecological diversity and scenic landscapes. We investigated the diversity of methanogens in different environments of Taiwan using a culture-dependent method. This report presents the characterization and taxonomy of six hydrogenotrophic methanogens obtained from cold seep sediments (strain FWC-SCC1T and FWC-SCC3T), marine sediments (strain CWC-02T and YWC-01T), estuarine sediments (strain Afa-1T), and a hot spring well (strain Wushi-C6T) in Taiwan. The proposed names of the six novel species are Methanoculleus frigidifontis (type strain FWC-SCC1T=BCRC AR10056T=NBRC 113993T), Methanoculleus oceani (CWC-02T=BCRC AR10055T=NBRC 113992T), Methanoculleus methanifontis (FWC-SCC3T=BCRC AR10057T=NBRC 113994T), Methanoculleus nereidis (YWC-01T=BCRC AR10060T=NBRC 114597T), Methanoculleus formosensis (Afa-1T=BCRC AR10054T=NBRC 113995T), and Methanoculleus caldifontis (Wushi-06T=BCRC AR10059T= NBRC 114596T).

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

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

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

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

Methanolobus halotolerans sp. nov., isolated from the saline Lake Tus in Siberia.

A halotolerant, psychrotolerant and methylotrophic methanogen, strain SY-01T, was isolated from the saline Lake Tus in Siberia. Cells of strain SY-01T were non-motile, cocci and 0.8-1.0 µm in diameter. The only methanogenic substrate utilized by strain SY-01T was methanol. The temperature range of growth for strain SY-01T was from 4 to 40 °C and the optimal temperature for growth was 30 °C. The pH range of growth was from pH 7.2 to 9.0, with optimal growth at pH 8.0. The NaCl range of growth was 0-1.55 M with optimal growth at 0.51 M NaCl. The G+C content of the genome of strain SY-01T was 43.6 mol % as determined by genome sequencing. Phylogenetic analysis revealed that strain SY-01T was most closely related to Methanolobus zinderi SD1T (97.3 % 16S rRNA gene sequence similarity), and had 95.5-97.2 % similarities to other Methanolobus species with valid names. Genome relatedness between strain SY-01T and DSM 21339T was computed using average nucleotide identity and digital DNA-DNAhybridization, which yielded values of 79.7 and 21.7 %, respectively. Based on morphological, phenotypic, phylogenetic and genomic relatedness data presented here, it is evident that strain SY-01T represents a novel species of the genus Methanolobus, and the name Methanolobus halotolerans sp. nov. is proposed. The type strain is SY-01T (=BCRC AR10051T=NBRC 113166 T=DSM 107642T).

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).

Site-specific His/Asp phosphoproteomic analysis of prokaryotes reveals putative targets for drug resistance.

BACKGROUND: Phosphorylation of amino acid residues on proteins is an important and common post-translational modification in both eukaryotes and prokaryotes. Most research work has been focused on phosphorylation of serine, threonine or tyrosine residues, whereas phosphorylation of other amino acids are significantly less clear due to the controversy on their stability under standard bioanalytical conditions. RESULTS: Here we applied a shotgun strategy to analyze the histidine and aspartate phosphorylations in different microbes. Our results collectively indicate that histidine and aspartate phosphorylations frequently occur also in proteins that are not part of the two-component systems. Noticeably, a number of the modified proteins are pathogenesis-related or essential for survival in host. These include the zinc ion periplasmic transporter ZnuA in Acinetobacter baumannii SK17, the multidrug and toxic compound extrusion (MATE) channel YeeO in Klebsiella pneumoniae NTUH-K2044, branched amino acid transporter AzlC in Vibrio vulnificus and the RNA-modifying pseudouridine synthase in Helicobacter pylori. CONCLUSIONS: In summary, histidine and aspartate phosphorylation is likely to be ubiquitous and to take place in proteins of various functions. This work also sheds light into how these functionally important proteins and potential drug targets might be regulated at a post-translational level.

Haloterrigena mahii sp. nov., an extremely halophilic archaeon from a solar saltern.

A pleomorphic, gas-vesicle-containing, halophilic archaeon, designated strain H13T, was isolated from the solar saltern of the Western Salt Co., Chula Vista, California, USA. Cells of strain H13T were non-motile, rod-shaped and 3-10 µm in length. The optimum growth conditions were 3.5-5.0 M NaCl, 45-55 °C, and pH range of 6.5-8.2. The major polar lipids were C20C20 and C20C25 derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and disulfated diglycosyl diether-1. The G+C content of he genome of strain H13T was calculated as 65.10 mol%. Phylogenetic analysis of 16S rRNA and rpoB' genes revealed that strain H13 was most closely related to Haloterrigena saccharevitans AB14T (16S rRNA gene sequence similarity: 99.51 %; rpoB' sequence similarity: 96.19 %) and Haloterrigena thermotolerans PR5T (99.11 %; 95.50 %). Strain H13T showed low genome relatedness values with Htg. saccharevitans AB14T and Htg. thermotolerans PR5T based on estimated average nucleotide identity (ANI; 92.59 and 91.68 %, respectively) and genome-to-genome distance analysis (GGDA; 47.90 and 45.00 %, respectively). Based on the phenotypic, chemotaxonomic and phylogenetic properties and the genome relatedness, it is evident that strain H13T represents a novel species of the genus Haloterrigena, for which the name Haloterrigena mahiisp. nov. is proposed. The type strain is H13T (=BCRC 910151T=NBRC 111885T).

Methanoculleus sediminis sp. nov., a methanogen from sediments near a submarine mud volcano.

A mesophilic, hydrogenotrophic methanogen, strain S3Fa(T), was isolated from sediments collected by Ocean Researcher I cruise ORI-934 in 2010 near the submarine mud volcano MV4 located at the upper slope of south-west Taiwan. The methanogenic substrates utilized by strain S3Fa(T) were formate and H2/CO2 but not acetate, secondary alcohols, methylamines, methanol or ethanol. Cells of strain S3Fa(T) were non-motile, irregular cocci, 0.5-1.0 µm in diameter. The surface-layer protein showed an Mr of 128,000.The optimum growth conditions were 37 °C, pH 7.1 and 0.17 M NaCl. The DNA G+C content of the genome of strain S3Fa(T) was 62.3 mol%. Phylogenetic analysis revealed that strain S3Fa(T) was most closely related to Methanoculleus marisnigri JR1(T) (99.3% 16S rRNA gene sequence similarity). Genome relatedness between strain S3Fa(T) and Methanoculleus marisnigri JR1(T) was computed using both genome-to-genome distance analysis (GGDA) and average nucleotide identity (ANI) with values of 46.3-55.5% and 93.08%, respectively. Based on morphological, phenotypic, phylogenetic and genomic relatedness data, it is evident that strain S3Fa(T) represents a novel species of the genus Methanoculleus, for which the name Methanoculleus sediminis sp. nov. is proposed. The type strain is S3Fa(T) ( = BCRC AR10044(T) = DSM 29354(T)).

Methanoculleus taiwanensis sp. nov., a methanogen isolated from deep marine sediment at the deformation front area near Taiwan.

A mesophilic, hydrogenotrophic methanogen, strain CYW4(T), was isolated from deep-sea sediment obtained by the Ocean Researcher I cruiser, ORI-961, in 2011. The sediment was from the deformation front area offshore of south-western Taiwan. Here, seismic reflections indicated that methane hydrates were abundant. The methanogenic substrates utilized by strain CYW4(T) were formate and H2/CO2, but not acetate, secondary alcohols, methylamines, methanol and ethanol. Cells of strain CYW4(T) were non-motile, irregular cocci and 0.6-1.5 µm in diameter. The S-layer protein had an Mr of 112 000. The optimum growth conditions were at 37 °C, pH 8.1 and 0.08 M NaCl. Growth of the strain was stimulated by acetate. The G+C content of the chromosomal DNA of strain CYW4(T) was 61 mol%. Phylogenetic analysis revealed that strain CYW4(T) was most closely related to Methanoculleus marisnigri JR1(T) (96.82 % 16S rRNA gene sequence similarity). Based on the morphological, phenotypic and phylogenetic characteristics presented here, it is evident that strain CYW4(T) represents a novel species of the genus Methanoculleus, and the name Methanoculleus taiwanensis sp. nov. is proposed. The type strain is CYW4(T) ( = BCRC AR10043(T) = NBRC 110782(T)). The optical density of cultures of strain CYW4(T) dropped abruptly upon entering the stationary growth phase. During this time numerous particles of approximately 50 nm in diameter were observed on and around the cells. This suggests that strain CYW4(T) harbours a lytic virus that is induced in the stationary phase, which is of interest because only a few lytic viruses have been reported in methanogens.

Transgenic Arabidopsis expressing osmolyte glycine betaine synthesizing enzymes from halophilic methanogen promote tolerance to drought and salt stress.

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.

Methanogenic archaea isolated from Taiwan's Chelungpu fault.

Terrestrial rocks, petroleum reservoirs, faults, coal seams, and subseafloor gas hydrates contain an abundance of diverse methanoarchaea. However, reports on the isolation, purification, and characterization of methanoarchaea in the subsurface environment are rare. Currently, no studies investigating methanoarchaea within fault environments exist. In this report, we succeeded in obtaining two new methanogen isolates, St545Mb(T) of newly proposed species Methanolobus chelungpuianus and Methanobacterium palustre FG694aF, from the Chelungpu fault, which is the fault that caused a devastating earthquake in central Taiwan in 1999. Strain FG694aF was isolated from a fault gouge sample obtained at 694 m below land surface (mbls) and is an autotrophic, mesophilic, nonmotile, thin, filamentous-rod-shaped organism capable of using H(2)-CO(2) and formate as substrates for methanogenesis. The morphological, biochemical, and physiological characteristics and 16S rRNA gene sequence analysis revealed that this isolate belongs to Methanobacterium palustre. The mesophilic strain St545Mb(T), isolated from a sandstone sample at 545 mbls, is a nonmotile, irregular, coccoid organism that uses methanol and trimethylamine as substrates for methanogenesis. The 16S rRNA gene sequence of strain St545Mb(T) was 99.0% similar to that of Methanolobus psychrophilus strain R15 and was 96 to 97.5% similar to the those of other Methanolobus species. However, the optimal growth temperature and total cell protein profile of strain St545Mb(T) were different from those of M. psychrophilus strain R15, and whole-genome DNA-DNA hybridization revealed less than 20% relatedness between these two strains. On the basis of these observations, we propose that strain St545Mb(T) (DSM 19953(T); BCRC AR10030; JCM 15159) be named Methanolobus chelungpuianus sp. nov. Moreover, the environmental DNA database survey indicates that both Methanolobus chelungpuianus and Methanobacterium palustre are widespread in the subsurface environment.

Differentially expressed genes after hyper- and hypo-salt stress in the halophilic archaeon Methanohalophilus portucalensis.

Methanohalophilus portucalensis FDF1 can grow over a range of external NaCl concentrations, from 1.2 to 2.9 mol/L. Differential gene expression in response to long-term hyper-salt stress (3.1 mol/L of NaCl) and hypo-salt stress (0.9 mol/L of NaCl) were compared by differential display RT-PCR. Fourteen differentially expressed genes responding to long-term hyper- or hypo-salt stress were detected, cloned, and sequenced. Several of the differentially expressed genes were related to the unique energy-acquiring methanogenesis pathway in this organism, including the transmembrane protein MttP, cobalamin biosynthesis protein, methenyl-H4MPT cyclohydrolase and monomethylamine methyltransferase. One signal transduction histidine kinase was identified from the hyper-salt stress cultures. Moreover, 3 known stress-response gene homologues - the DNA mismatch repair protein, MutS, the universal stress protein, UspA, and a member of the protein-disaggregating multichaperone system, ClpB - were also detected. The transcriptional analysis of these long-term salt stress response and adaptation-related genes for cells immediately after salt stress indicated that the expression of the energy metabolism genes was arrested during hyper-salt shock, while the chaperone clpB gene was stimulated by both hypo- and hyper-salt shock.

The biotechnological potential of the extreme halophilic archaea Haloterrigena sp. H13 in xenobiotic metabolism using a comparative genomics approach.

Extreme halophilic archaea are thriving and dominant populations within hypersaline environments. Because of the extreme properties of the enzymes of halophilic archaea and similar metabolic abilities to their bacterial counterparts, our interests focus on their potential biotechnological applications. In this study, the partial genome of a newly isolated extreme halophilic archaeon, Haloterrigena sp. H13, was investigated. The genome size was estimated to be about 3.9 MB, and a genomic shotgun library was constructed. A total of 1479 clones from the library were sequenced once, and 1186 contigs were obtained. From these contigs, 580 open reading frames (ORFs) were identified, and 394 ORFs were annotated. From the partial genome of strain H13, we identified genes that may be involved in 1,2-dichloroethane degradation, naphthalene/anthracene degradation, gamma-hexachlorocyclohexane degradation, 1-/2-methylnaphthalene degradation and benzoate degradation via CoA ligation. Among the identified ORFs, gene homologs of (S)-2-haloacid dehalogenase (EC 3.8.1.2) and salicylate hydroxylase (EC 1.14.13.1), which might be involved in the degradation of dichloroethane, gamma-hexachlorocyclohexane and naphthalene, were found in the partial genome sequence of strain H13. According to the current genome annotation of peripheral metabolic pathways and the putative xenobiotic-degrading enzymes, the potential of extreme haloarchaea in bioremediation applications is proposed.

Characterization of osmolyte betaine synthesizing sarcosine dimethylglycine N-methyltransferase from Methanohalophilus portucalensis.

To overcome the extracellular salt stress, Methanohalophilus portucalensis FDF1(T) synthesizes the compatible solute betaine through the methylation of glycine, sarcosine, and N,N-dimethylglycine. S-adenosylmethionine (AdoMet) is the methyl donor. The enzyme sarcosine dimethylglycine N-methyltransferase (SDMT) of M. portucalensis, that catalyzes the formation of N,N-dimethylglycine and glycine betaine, has been purified and characterized. SDMT, a monomer of 33 kDa with a pI at 5.03, has a narrow substrate specificity limited to using only sarcosine and dimethylglycine as substrates for the methyl transferase reaction. The K(m) values for sarcosine and AdoMet were 2.29 and 0.21 mM, respectively, with a V(max) of 0.83 micromol/mg-min (k(cat) value of 0.44 s(-1)). The K(m) values for dimethylglycine and AdoMet were 3.76 and 0.59 mM, respectively, with a V(max) of 4.88 micromol/mg-min (k(cat) of 2.68 s(-1)). A high concentration of the end product betaine (2.0 M) did not affect the SMT activity, but it slightly inhibited the DMT activity. Both activities were also not affected by potassium or sodium ions in concentrations of 200-1,000 mM. We compared this novel archaeal SDMT enzyme to other similar bacterial transferases as well as to the glycine sarcosine dimethylglycine methyltransferase found also in M. portucalensis.

Comparative genomic analyses reveal trehalose synthase genes as the signature in genus Methanoculleus.

To date, the only methanoarchaea isolated directly from methane hydrate bearing sediments were Methanoculleus submarinus Nankai-1T and Methanoculleus sp. MH98A. Here, we provide the genome of Methanoculleus taiwanensis CYW4T isolated from the deep-sea subseafloor sediment at the Deformation Front offshore southwestern Taiwan, where methane hydrate deposits are likely located. Through comparative genomics analyses of nine Methanoculleus strains from various habitats, 2-3 coding genes for trehalose synthases were found in all nine Methanoculleus genomes, which were not detected in other methanogens and are therefore suggested as a signature of genus Methanoculleus among methane-producing archaea. In addition, the structural genes adjacent to trehalose synthase genes are comprised of the signaling module of Per-Arnt-Sim (PAS) domain-containing proteins, Hsp20 family proteins, arabinose efflux permeases and multiple surface proteins with fasciclin-like (FAS) repeat. This indicates that trehalose synthase gene clusters in Methanoculleus might play roles in the response to various stresses and regulate carbon storage and modification of surface proteins through accumulation of trehalose. The non-gas hydrate-associated Methanoculleus strains harbor carbon-monoxide dehydrogenase (cooS/acsA) genes, which are important for the conversion of acetate to methane at the step of CO oxidation/CO2 reduction in acetoclastic methanogens and further implies that these strains may be able to utilize CO for methanogenesis in their natural habitats. In addition, both genomes of M. bourgensis strains MS2T and MAB1 harbor highly abundant transposase genes, which may be disseminated from microbial communities in their habitats, sewage treatment plants and biogas reactors, which are breeding grounds for antibiotic resistance. Through comparative genomic analyses, we gained insight into understanding the life of strictly anaerobic methane-producing archaea in various habitats, especially in methane-based deep-sea ecosystems.

Comparative genomics of Vibrio campbellii strains and core species of the Vibrio Harveyi clade.

The core of the Vibrio Harveyi clade contains V. harveyi, V. campbellii, V. owensii, V. jasicida, and V. rotiferianus. They are well recognized aquatic animal pathogens, but misclassification has been common due to similarities in their rDNA sequences and phenotypes. To better understand their evolutionary relationships and functional features, we sequenced a shrimp pathogen strain V. harveyi 1114GL, reclassified it as V. campbellii and compared this and 47 other sequenced Vibrio genomes in the Harveryi clade. A phylogeny based on 1,775 genes revealed that both V. owensii and V. jasicida were closer to V. campbellii than to V. harveyi and that V. campbellii strains can be divided into two distinct groups. Species-specific genes such as intimin and iron acquisition genes were identified in V. campbellii. In particular, the 1114GL strain contains two bacterial immunoglobulin-like genes for cell adhesion with 22 Big_2 domains that have been extensively reshuffled and are by far the most expanded among all species surveyed in this study. The 1114GL strain differed from ATCC BAA-1116 by ~9% at the synonymous sites, indicating high diversity within V. campbellii. Our study revealed the characteristics of V. campbellii in the Harveyi clade and the genetic basis for their wide-spread pathogenicity.

Effects of substrate and potassium on the betaine-synthesizing enzyme glycine sarcosine dimethylglycine N-methyltransferase from a halophilic methanoarchaeon Methanohalophilus portucalensis.

Methanohalophilus portucalensis FDF1 can synthesize the compatible solute betaine de novo through the methylation of glycine, sarcosine and dimethylglycine with the methyl group from S-adenosylmethionine. After separation by DEAE-Sephacel ion chromatography using a KCl step gradient, glycine, sarcosine and dimethylglycine methytransfer (GSDMT) activities were detected in a single peak. The estimated molecular weight of GSDMT was 240 kDa and 2-D gel analysis indicated it was separated into four subunits (52 kDa) with different pI. The PBE94 chromatofocusing column also separated GSDMT into four protein peaks A, B, C, D. Both peak B and D proteins possessed GSDMT activity, while the peak A protein only exhibited SDMT activity. The multiple methyltransferase activities of the large complex appear to be unique compared to other methyltransferases used in betaine synthesis. Further methyltransferase assays in response to different concentrations of KCl indicated that the peak D protein exhibited low GSDMT activity only when K(+) < or = 0.4 M. The peak B protein exhibited a higher GSDMT activity at 0.4 M K(+), while the peak A protein exhibited SDMT activity only at higher K(+) (0.8 M). These results suggest that the internal K(+) concentration regulates GSDMT activities and affects the net betaine accumulation in the cells.

Comparison of Enzymatic Traits between Native and Recombinant Glycine Sarcosine N-Methyltransferase from Methanohalophilus portucalensis FDF1T.

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.

Draft Genome Sequence of Methanoculleus sediminis S3FaT, a Hydrogenotrophic Methanogen Isolated from a Submarine Mud Volcano in Taiwan.

Here, we announce the genome sequence of ITALIC! Methanoculleus sediminisS3Fa(T)(DSM 29354(T)), a strict anaerobic methanoarchaeon, which was isolated from sediments near the submarine mud volcano MV4 located offshore in southwestern Taiwan. The 2.49-Mb genome consists of 2,459 predicted genes, 3 rRNAs, 48 tRNAs, and 1 ncRNA. The sequence of this novel strain may provide more information for species delineation and the roles that this strain plays in the unique marine mud volcano habitat.