Putative novel hydrogen- and iron-oxidizing sheath-producing Zetaproteobacteria thrive at the Fåvne deep-sea hydrothermal vent field.

IMPORTANCE: Knowledge on microbial iron oxidation is important for understanding the cycling of iron, carbon, nitrogen, nutrients, and metals. The current study yields important insights into the niche sharing, diversification, and Fe(III) oxyhydroxide morphology of Ghiorsea, an iron- and hydrogen-oxidizing Zetaproteobacteria representative belonging to Zetaproteobacteria operational taxonomic unit 9. The study proposes that Ghiorsea exhibits a more extensive morphology of Fe(III) oxyhydroxide than previously observed. Overall, the results increase our knowledge on potential drivers of Zetaproteobacteria diversity in iron microbial mats and can eventually be used to develop strategies for the cultivation of sheath-forming Zetaproteobacteria.

Activation mechanism and activity of globupain, a thermostable C11 protease from the Arctic Mid-Ocean Ridge hydrothermal system.

Deep-sea hydrothermal vents offer unique habitats for heat tolerant enzymes with potential new enzymatic properties. Here, we present the novel C11 protease globupain, which was prospected from a metagenome-assembled genome of uncultivated Archaeoglobales sampled from the Soria Moria hydrothermal vent system located on the Arctic Mid-Ocean Ridge. Sequence comparisons against the MEROPS-MPRO database showed that globupain has the highest sequence identity to C11-like proteases present in human gut and intestinal bacteria. Successful recombinant expression in Escherichia coli of the wild-type zymogen and 13 mutant substitution variants allowed assessment of residues involved in maturation and activity of the enzyme. For activation, globupain required the addition of DTT and Ca2+. When activated, the 52kDa proenzyme was processed at K137 and K144 into a 12kDa light- and 32kDa heavy chain heterodimer. A structurally conserved H132/C185 catalytic dyad was responsible for the proteolytic activity, and the enzyme demonstrated the ability to activate in-trans. Globupain exhibited caseinolytic activity and showed a strong preference for arginine in the P1 position, with Boc-QAR-aminomethylcoumarin (AMC) as the best substrate out of a total of 17 fluorogenic AMC substrates tested. Globupain was thermostable (Tm activated enzyme = 94.51°C ± 0.09°C) with optimal activity at 75°C and pH 7.1. Characterization of globupain has expanded our knowledge of the catalytic properties and activation mechanisms of temperature tolerant marine C11 proteases. The unique combination of features such as elevated thermostability, activity at relatively low pH values, and ability to operate under high reducing conditions makes globupain a potential intriguing candidate for use in diverse industrial and biotechnology sectors.

Exploring Codon Adjustment Strategies towards Escherichia coli-Based Production of Viral Proteins Encoded by HTH1, a Novel Prophage of the Marine Bacterium Hypnocyclicus thermotrophus.

Marine viral sequence space is immense and presents a promising resource for the discovery of new enzymes interesting for research and biotechnology. However, bottlenecks in the functional annotation of viral genes and soluble heterologous production of proteins hinder access to downstream characterization, subsequently impeding the discovery process. While commonly utilized for the heterologous expression of prokaryotic genes, codon adjustment approaches have not been fully explored for viral genes. Herein, the sequence-based identification of a putative prophage is reported from within the genome of Hypnocyclicus thermotrophus, a Gram-negative, moderately thermophilic bacterium isolated from the Seven Sisters hydrothermal vent field. A prophage-associated gene cluster, consisting of 46 protein coding genes, was identified and given the proposed name Hypnocyclicus thermotrophus phage H1 (HTH1). HTH1 was taxonomically assigned to the viral family Siphoviridae, by lowest common ancestor analysis of its genome and phylogeny analyses based on proteins predicted as holin and DNA polymerase. The gene neighbourhood around the HTH1 lytic cassette was found most similar to viruses infecting Gram-positive bacteria. In the HTH1 lytic cassette, an N-acetylmuramoyl-L-alanine amidase (Amidase_2) with a peptidoglycan binding motif (LysM) was identified. A total of nine genes coding for enzymes putatively related to lysis, nucleic acid modification and of unknown function were subjected to heterologous expression in Escherichia coli. Codon optimization and codon harmonization approaches were applied in parallel to compare their effects on produced proteins. Comparison of protein yields and thermostability demonstrated that codon optimization yielded higher levels of soluble protein, but codon harmonization led to proteins with higher thermostability, implying a higher folding quality. Altogether, our study suggests that both codon optimization and codon harmonization are valuable approaches for successful heterologous expression of viral genes in E. coli, but codon harmonization may be preferable in obtaining recombinant viral proteins of higher folding quality.

"Candidatus Ethanoperedens," a Thermophilic Genus of Archaea Mediating the Anaerobic Oxidation of Ethane.

Cold seeps and hydrothermal vents deliver large amounts of methane and other gaseous alkanes into marine surface sediments. Consortia of archaea and partner bacteria thrive on the oxidation of these alkanes and its coupling to sulfate reduction. The inherently slow growth of the involved organisms and the lack of pure cultures have impeded the understanding of the molecular mechanisms of archaeal alkane degradation. Here, using hydrothermal sediments of the Guaymas Basin (Gulf of California) and ethane as the substrate, we cultured microbial consortia of a novel anaerobic ethane oxidizer, "Candidatus Ethanoperedens thermophilum" (GoM-Arc1 clade), and its partner bacterium "Candidatus Desulfofervidus auxilii," previously known from methane-oxidizing consortia. The sulfate reduction activity of the culture doubled within one week, indicating a much faster growth than in any other alkane-oxidizing archaea described before. The dominance of a single archaeal phylotype in this culture allowed retrieval of a closed genome of "Ca. Ethanoperedens," a sister genus of the recently reported ethane oxidizer "Candidatus Argoarchaeum." The metagenome-assembled genome of "Ca. Ethanoperedens" encoded a complete methanogenesis pathway including a methyl-coenzyme M reductase (MCR) that is highly divergent from those of methanogens and methanotrophs. Combined substrate and metabolite analysis showed ethane as the sole growth substrate and production of ethyl-coenzyme M as the activation product. Stable isotope probing demonstrated that the enzymatic mechanism of ethane oxidation in "Ca. Ethanoperedens" is fully reversible; thus, its enzymatic machinery has potential for the biotechnological development of microbial ethane production from carbon dioxide.IMPORTANCE In the seabed, gaseous alkanes are oxidized by syntrophic microbial consortia that thereby reduce fluxes of these compounds into the water column. Because of the immense quantities of seabed alkane fluxes, these consortia are key catalysts of the global carbon cycle. Due to their obligate syntrophic lifestyle, the physiology of alkane-degrading archaea remains poorly understood. We have now cultivated a thermophilic, relatively fast-growing ethane oxidizer in partnership with a sulfate-reducing bacterium known to aid in methane oxidation and have retrieved the first complete genome of a short-chain alkane-degrading archaeon. This will greatly enhance the understanding of nonmethane alkane activation by noncanonical methyl-coenzyme M reductase enzymes and provide insights into additional metabolic steps and the mechanisms underlying syntrophic partnerships. Ultimately, this knowledge could lead to the biotechnological development of alkanogenic microorganisms to support the carbon neutrality of industrial processes.

Profundibacter amoris gen. nov., sp. nov., a new member of the Roseobacter clade isolated from Loki's Castle Vent Field on the Arctic Mid-Ocean Ridge.

A bacterial strain, designated BAR1T, was isolated from a microbial mat growing on the surface of a barite chimney at the Loki's Castle Vent Field, at a depth of 2216 m. Cells of strain BAR1T were rod-shaped, Gram-reaction-negative and grew on marine broth 2216 at 10-37 °C (optimum 27-35 °C), pH 5.5-8.0 (optimum pH 6.5-7.5) and 0.5-5.0 % NaCl (optimum 2 %). The DNA G+C content was 57.38 mol%. The membrane-associated major ubiquinone was Q-10, the fatty acid profile was dominated by C18 : 1ω7c (91 %), and the polar lipids detected were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminolipid, one unidentified lipid and one unidentified phospholipid. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain BAR1T clustered together with Rhodobacterales bacterium PRT1, as well as the genera Halocynthiibacter and Pseudohalocynthiibacter in a polyphyletic clade within the Roseobacter clade. Several characteristics differentiate strain BAR1T from the aforementioned genera, including its motility, its piezophilic behaviour and its ability to grow at 35 °C and under anaerobic conditions. Accordingly, strain BAR1T is considered to represent a novel genus and species within the Roseobacter clade, for which the name Profundibacter amoris gen. nov., sp. nov. is proposed. The type strain is Profundibacter amoris BAR1T (=JCM 31874T=DSM 104147T).

Distribution Patterns of Iron-Oxidizing Zeta- and Beta-Proteobacteria From Different Environmental Settings at the Jan Mayen Vent Fields.

Iron oxidizers are widespread in marine environments and play an important role in marine iron cycling. However, little is known about the overall distribution of iron oxidizers within hydrothermal systems, including settings with little hydrothermal activity. Moreover, the extent to which different phylogenetic groups of iron oxidizers exhibit niche specialization toward different environmental settings, remains largely unknown. Obtaining such knowledge is critical to unraveling the impact of the activity of iron oxidizers and how they are adapted. Here, we used 16S rRNA sequencing to characterize the distribution of iron oxidizers in different environmental settings within the Jan Mayen hydrothermal vent fields (JMVFs). Putative iron oxidizers affiliated to Zetaproteobacteria and Betaproteobacteria were detected within iron mounds, bottom seawater, basalt surfaces, and surface layers of sediments. The detected iron oxidizers were compared to sequence types previously observed in patchily distributed iron mats associated with diffuse venting at the JMVFs. Most OTUs of iron oxidizers reoccurred under different environmental settings, suggesting a limited degree of niche specialization. Consequently, most of the detected iron oxidizers seem to be generalists with a large habitat range. Our study highlights the importance of gathering information about the overall distribution of iron oxidizers in hydrothermal systems to fully understand the role of this metabolic group regarding cycling of iron. Furthermore, our results provide further evidence of the presence of iron-oxidizing members of Betaproteobacteria in marine environments.

Genome Analysis of Vallitalea guaymasensis Strain L81 Isolated from a Deep-Sea Hydrothermal Vent System.

Abyssivirga alkaniphila strain L81T, recently isolated from a black smoker biofilm at the Loki's Castle hydrothermal vent field, was previously described as a mesophilic, obligately anaerobic heterotroph able to ferment carbohydrates, peptides, and aliphatic hydrocarbons. The strain was classified as a new genus within the family Lachnospiraceae. Herein, its genome is analyzed and A. alkaniphila is reassigned to the genus Vallitalea as a new strain of V. guaymasensis, designated V. guaymasensis strain L81. The 6.4 Mbp genome contained 5651 protein encoding genes, whereof 4043 were given a functional prediction. Pathways for fermentation of mono-saccharides, di-saccharides, peptides, and amino acids were identified whereas a complete pathway for the fermentation of n-alkanes was not found. Growth on carbohydrates and proteinous compounds supported methane production in co-cultures with Methanoplanus limicola. Multiple confurcating hydrogen-producing hydrogenases, a putative bifurcating electron-transferring flavoprotein­butyryl-CoA dehydrogenase complex, and a Rnf-complex form a basis for the observed hydrogen-production and a putative reverse electron-transport in V. guaymasensis strain L81. Combined with the observation that n-alkanes did not support growth in co-cultures with M. limicola, it seemed more plausible that the previously observed degradation patterns of crude-oil in strain L81 are explained by unspecific activation and may represent a detoxification mechanism, representing an interesting ecological function. Genes encoding a capacity for polyketide synthesis, prophages, and resistance to antibiotics shows interactions with the co-occurring microorganisms. This study enlightens the function of the fermentative microorganisms from hydrothermal vents systems and adds valuable information on the bioprospecting potential emerging in deep-sea hydrothermal systems.

Complete genome sequence of Lutibacter profundi LP1T isolated from an Arctic deep-sea hydrothermal vent system.

Lutibacter profundi LP1T within the family Flavobacteriaceae was isolated from a biofilm growing on the surface of a black smoker chimney at the Loki's Castle vent field, located on the Arctic Mid-Ocean Ridge. The complete genome of L. profundi LP1T is the first genome to be published within the genus Lutibacter. L. profundi LP1T consists of a single 2,966,978 bp circular chromosome with a GC content of 29.8%. The genome comprises 2,537 protein-coding genes, 40 tRNA species and 2 rRNA operons. The microaerophilic, organotrophic isolate contains genes for all central carbohydrate metabolic pathways. However, genes for the oxidative branch of the pentose-phosphate-pathway, the glyoxylate shunt of the tricarboxylic acid cycle and the ATP citrate lyase for reverse TCA are not present. L. profundi LP1T utilizes starch, sucrose and diverse proteinous carbon sources. In accordance, the genome harbours 130 proteases and 104 carbohydrate-active enzymes, indicating a specialization in degrading organic matter. Among a small arsenal of 24 glycosyl hydrolases, which offer the possibility to hydrolyse diverse poly- and oligosaccharides, a starch utilization cluster was identified. Furthermore, a variety of enzymes may be secreted via T9SS and contribute to the hydrolytic variety of the microorganism. Genes for gliding motility are present, which may enable the bacteria to move within the biofilm. A substantial number of genes encoding for extracellular polysaccharide synthesis pathways, curli fibres and attachment to surfaces could mediate adhesion in the biofilm and may contribute to the biofilm formation. In addition to aerobic respiration, the complete denitrification pathway and genes for sulphide oxidation e.g. sulphide:quinone reductase are present in the genome. sulphide:quinone reductase and denitrification may serve as detoxification systems allowing L. profundi LP1T to thrive in a sulphide and nitrate enriched environment. The information gained from the genome gives a greater insight in the functional role of L. profundi LP1T in the biofilm and its adaption strategy in an extreme environment.

Lutibacter profundi sp. nov., isolated from a deep-sea hydrothermal system on the Arctic Mid-Ocean Ridge and emended description of the genus Lutibacter.

A bacterial strain designated LP1T was isolated from a microbial mat growing on the surface of a black smoker chimney at the Loki's Castle hydrothermal system, which is located on the Arctic Mid-Ocean Ridge. Phylogenetic analyses based on 16S rRNA gene sequences positioned strain LP1T within the family Flavobacteriaceae with Lutibacterholmesii as the closest relative (97.5 % 16S rRNA gene sequence similarity). Strain LP1T was rod-shaped, Gram-reaction-negative and non-motile. It grew in a modified artificial seawater medium supplemented with tryptone and vitamins at pH 5.5-7.5 (optimum pH 6.0-6.5), within a temperature range of 13-34 °C (optimum 23 °C), and under microaerobic conditions. The most abundant fatty acids (>10 %) were iso-C15 : 0 (25.2 %) and iso-C15 : 0 3-OH (14.5 %). The genome of strain LP1T has a DNA G+C content of 29.8 mol%. Based on the results of the polyphasic characterization presented here, strain LP1T is considered to represent a novel species of the genus Lutibacter, for which the name Lutibacter profundi sp. nov. is proposed. The type strain is LP1T (=DSM 100437T =JCM 30585T). An emended description of the genus Lutibacter is also provided to fit the description of strain LP1T.

Isolation and complete genome sequence of the thermophilic Geobacillus sp. 12AMOR1 from an Arctic deep-sea hydrothermal vent site.

Members of the genus Geobacillus have been isolated from a wide variety of habitats worldwide and are the subject for targeted enzyme utilization in various industrial applications. Here we report the isolation and complete genome sequence of the thermophilic starch-degrading Geobacillus sp. 12AMOR1. The strain 12AMOR1 was isolated from deep-sea hot sediment at the Jan Mayen hydrothermal Vent Site. Geobacillus sp. 12AMOR1 consists of a 3,410,035 bp circular chromosome and a 32,689 bp plasmid with a G + C content of 52 % and 47 %, respectively. The genome comprises 3323 protein-coding genes, 88 tRNA species and 10 rRNA operons. The isolate grows on a suite of sugars, complex polysaccharides and proteinous carbon sources. Accordingly, a versatility of genes encoding carbohydrate-active enzymes (CAZy) and peptidases were identified in the genome. Expression, purification and characterization of an enzyme of the glycoside hydrolase family 13 revealed a starch-degrading capacity and high thermal stability with a melting temperature of 76.4 °C. Altogether, the data obtained point to a new isolate from a marine hydrothermal vent with a large bioprospecting potential.

Abyssivirga alkaniphila gen. nov., sp. nov., an alkane-degrading, anaerobic bacterium from a deep-sea hydrothermal vent system, and emended descriptions of Natranaerovirga pectinivora and Natranaerovirga hydrolytica.

A strictly anaerobic, mesophilic, syntrophic, alkane-degrading strain, L81T, was isolated from a biofilm sampled from a black smoker chimney at the Loki's Castle vent field. Cells were straight, rod-shaped, Gram-positive-staining and motile. Growth was observed at pH 6.2-9.5, 14-42 °C and 0.5-6 % (w/w) NaCl, with optima at pH 7.0-8.2, 37 °C and 3% (w/w) NaCl. Proteinaceous substrates, sugars, organic acids and hydrocarbons were utilized for growth. Thiosulfate was used as an external electron acceptor during growth on crude oil. Strain L81T was capable of syntrophic hydrocarbon degradation when co-cultured with a methanogenic archaeon, designated strain LG6, isolated from the same enrichment. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain L81T is affiliated with the family Lachnospiraceae, and is most closely related to the type strains of Natranaerovirga pectinivora (92 % sequence similarity) and Natranaerovirga hydrolytica (90%). The major cellular fatty acids of strain L81T were C15 : 0, anteiso-C15 : 0 and C16 : 0, and the profile was distinct from those of the species of the genus Natranaerovirga. The polar lipids were phosphatidylglycerol, diphosphatidylglycerol, three unidentified phospholipids, four unidentified glycolipids and two unidentified phosphoglycolipids. The G+C content of genomic DNA was determined to be 31.7 mol%. Based on our phenotypic, phylogenetic and chemotaxonomic results, strain L81T is considered to represent a novel species of a new genus of the family Lachnospiraceae, for which we propose the name Abyssivirga alkaniphila gen. nov., sp. nov. The type strain of Abyssivirga alkaniphila is L81T (=DSM 29592T=JCM 30920T). We also provide emended descriptions of Natranaerovirga pectinivora and Natranaerovirga hydrolytica.

Hypnocyclicus thermotrophus gen. nov., sp. nov. isolated from a microbial mat in a hydrothermal vent field.

The bacterial strain, IR-2T, was isolated from a microbial mat sampled near a hydrothermal vent in the Greenland Sea. Phylogenetic analysis, based on the 16S rRNA gene, showed that the closest relatives of IR-2T were Ilyobacter tartaricus, Ilyobacter insuetus, Propionigenium modestum and Fusobacterium varium (91 % 16S rRNA gene sequence similarity). The cells of the novel strain were Gram-stain-negative and pleomorphic; changing from long motile rods to non-motile ring structures during the growth cycle. Growth occurred at 20-55 °C (optimally at 48 °C), with 1-6 % (w/v) NaCl (optimally with 2 %), and at pH 5.3-8.0 (optimally at pH 6.0-8.0). The strain had obligate fermentative growth on various sugars and yeast extract. The DNA G+C content of strain IR-2T was 25.7 mol%. The cell sugars comprised mainly ribose, mannose and glucose, while the main polar lipids were glycolipids, phospholipids, phosphatidylglycerol and diphosphatidylglycerol. The fatty acid content of strain IR-2 was dominated by saturated and unsaturated iso-branched or anteiso-branched forms. Strain IR-2 represents a novel genus and species, for which the name Hypnocyclicus thermotrophus gen. nov., sp. nov. is proposed. The type strain is IR-2T ( = DSM 100055 = JCM 30901).

Functional interactions among filamentous Epsilonproteobacteria and Bacteroidetes in a deep-sea hydrothermal vent biofilm.

Little is known about how lithoautotrophic primary production is connected to microbial organotrophic consumption in hydrothermal systems. Using a multifaceted approach, we analysed the structure and metabolic capabilities within a biofilm growing on the surface of a black smoker chimney in the Loki's Castle vent field. Imaging revealed the presence of rod-shaped Bacteroidetes growing as ectobionts on long, sheathed microbial filaments (> 100 µm) affiliated with the Sulfurovum genus within Epsilonproteobacteria. The filaments were composed of a thick (> 200 nm) stable polysaccharide, representing a substantial fraction of organic carbon produced by primary production. An integrated -omics approach enabled us to assess the metabolic potential and in situ metabolism of individual taxonomic and morphological groups identified by imaging. Specifically, we provide evidence that organotrophic Bacteroidetes attach to and glide along the surface of Sulfurovum filaments utilizing organic polymers produced by the lithoautotrophic Sulfurovum. Furthermore, in situ expression of acetyl-CoA synthetase by Sulfurovum suggested the ability to assimilate acetate, indicating recycling of organic matter in the biofilm. This study expands our understanding of the lifestyles of Epsilonproteobacteria in hydrothermal vents, their metabolic properties and co-operative interactions in deep-sea hydrothermal vent food webs.

Complete Genome Sequence of the Thermophilic and Facultatively Chemolithoautotrophic Sulfate Reducer Archaeoglobus sulfaticallidus Strain PM70-1T.

Dissimilatory sulfate-reducing archaea of the genus Archaeoglobus display divergent preferences in the use of energy sources and electron acceptors. Here we present the complete genome sequence of the thermophilic Archaeoglobus sulfaticallidus strain PM70-1(T), which distinctly couples chemolithoautotrophic growth on H2/CO2 to sulfate reduction in addition to heterotrophic growth.

Fine-Scale Community Structure Analysis of ANME in Nyegga Sediments with High and Low Methane Flux.

To obtain knowledge on how regional variations in methane seepage rates influence the stratification, abundance, and diversity of anaerobic methanotrophs (ANME), we analyzed the vertical microbial stratification in a gravity core from a methane micro-seeping area at Nyegga by using 454-pyrosequencing of 16S rRNA gene tagged amplicons and quantitative PCR. These data were compared with previously obtained data from the more active G11 pockmark, characterized by higher methane flux. A down core stratification and high relative abundance of ANME were observed in both cores, with transition from an ANME-2a/b dominated community in low-sulfide and low methane horizons to ANME-1 dominance in horizons near the sulfate-methane transition zone. The stratification was over a wider spatial region and at greater depth in the core with lower methane flux, and the total 16S rRNA copy numbers were two orders of magnitude lower than in the sediments at G11 pockmark. A fine-scale view into the ANME communities at each location was achieved through operational taxonomical units (OTU) clustering of ANME-affiliated sequences. The majority of ANME-1 sequences from both sampling sites clustered within one OTU, while ANME-2a/b sequences were represented in unique OTUs. We suggest that free-living ANME-1 is the most abundant taxon in Nyegga cold seeps, and also the main consumer of methane. The observation of specific ANME-2a/b OTUs at each location could reflect that organisms within this clade are adapted to different geochemical settings, perhaps due to differences in methane affinity. Given that the ANME-2a/b population could be sustained in less active seepage areas, this subgroup could be potential seed populations in newly developed methane-enriched environments.

Finding a needle in the virus metagenome haystack--micro-metagenome analysis captures a snapshot of the diversity of a bacteriophage armoire.

Viruses are ubiquitous in the oceans and critical components of marine microbial communities, regulating nutrient transfer to higher trophic levels or to the dissolved organic pool through lysis of host cells. Hydrothermal vent systems are oases of biological activity in the deep oceans, for which knowledge of biodiversity and its impact on global ocean biogeochemical cycling is still in its infancy. In order to gain biological insight into viral communities present in hydrothermal vent systems, we developed a method based on deep-sequencing of pulsed field gel electrophoretic bands representing key viral fractions present in seawater within and surrounding a hydrothermal plume derived from Loki's Castle vent field at the Arctic Mid-Ocean Ridge. The reduction in virus community complexity afforded by this novel approach enabled the near-complete reconstruction of a lambda-like phage genome from the virus fraction of the plume. Phylogenetic examination of distinct gene regions in this lambdoid phage genome unveiled diversity at loci encoding superinfection exclusion- and integrase-like proteins. This suggests the importance of fine-tuning lyosgenic conversion as a viral survival strategy, and provides insights into the nature of host-virus and virus-virus interactions, within hydrothermal plumes. By reducing the complexity of the viral community through targeted sequencing of prominent dsDNA viral fractions, this method has selectively mimicked virus dominance approaching that hitherto achieved only through culturing, thus enabling bioinformatic analysis to locate a lambdoid viral "needle" within the greater viral community "haystack". Such targeted analyses have great potential for accelerating the extraction of biological knowledge from diverse and poorly understood environmental viral communities.

The complex structures of isocitrate dehydrogenase from Clostridium thermocellum and Desulfotalea psychrophila suggest a new active site locking mechanism.

Isocitrate dehydrogenase (IDH) catalyzes the oxidative NAD(P)(+)-dependent decarboxylation of isocitrate into α-ketoglutarate and CO2 and is present in organisms spanning the biological range of temperature. We have solved two crystal structures of the thermophilic Clostridium thermocellum IDH (CtIDH), a native open apo CtIDH to 2.35 Å and a quaternary complex of CtIDH with NADP(+), isocitrate and Mg(2+) to 2.5 Å. To compare to these a quaternary complex structure of the psychrophilic Desulfotalea psychrophila IDH (DpIDH) was also resolved to 1.93 Å. CtIDH and DpIDH showed similar global thermal stabilities with melting temperatures of 67.9 and 66.9 °C, respectively. CtIDH represents a typical thermophilic enzyme, with a large number of ionic interactions and hydrogen bonds per residue combined with stabilization of the N and C termini. CtIDH had a higher activity temperature optimum, and showed greater affinity for the substrates with an active site that was less thermolabile compared to DpIDH. The uncompensated negative surface charge and the enlarged methionine cluster in the hinge region both of which are important for cold activity in DpIDH, were absent in CtIDH. These structural comparisons revealed that prokaryotic IDHs in subfamily II have a unique locking mechanism involving Arg310, Asp251' and Arg255 (CtIDH). These interactions lock the large domain to the small domain and direct NADP(+) into the correct orientation, which together are important for NADP(+) selectivity.

New insight into stratification of anaerobic methanotrophs in cold seep sediments.

Methane seepages typically harbor communities of anaerobic methane oxidizers (ANME); however, knowledge about fine-scale vertical variation of ANME in response to geochemical gradients is limited. We investigated microbial communities in sediments below a white microbial mat in the G11 pockmark at Nyegga by 16S rRNA gene tag pyrosequencing and real-time quantitative PCR. A vertical stratification of dominating ANME communities was observed at 4 cmbsf (cm below seafloor) and below in the following order: ANME-2a/b, ANME-1 and ANME-2c. The ANME-1 community was most numerous and comprised single or chains of cells with typical rectangular morphology, accounting up to 89.2% of the retrieved 16S rRNA gene sequences. Detection rates for sulfate-reducing Deltaproteobacteria possibly involved in anaerobic oxidation of methane were low throughout the core. However, a correlation in the abundance of Candidate division JS-1 with ANME-2 was observed, indicating involvement in metabolisms occurring in ANME-2-dominated horizons. The white microbial mat and shallow sediments were dominated by organisms affiliated with Sulfurovum (Epsilonproteobacteria) and Methylococcales (Gammaproteobacteria), suggesting that aerobic oxidation of sulfur and methane is taking place. In intermediate horizons, typical microbial groups associated with methane seeps were recovered. The data are discussed with respect to co-occurring microbial assemblages and interspecies interactions.

Thermodynamic and kinetic stability of a large multi-domain enzyme from the hyperthermophile Aeropyrum pernix.

The multi-domain enzyme isocitrate dehydrogenase from the hyperthermophile Aeropyrum pernix was studied by denaturant-induced unfolding. At pH 7.5, changes in circular dichroism ellipticity and intrinsic fluorescence showed a complex unfolding transition, whereas at pH 3.0, an apparently two-state and highly reversible unfolding occurred. Analytical ultracentrifugation revealed the dissociation from dimer to monomer at pH 3.0. The thermodynamic and kinetic stability were studied at pH 3.0 to explore the role of inter-domain interactions independently of inter-subunit interplay on the wild type and R211M, a mutant where a seven-membered inter-domain ionic network has been disrupted. The unfolding and folding transitions occurred at slightly different denaturant concentrations even after prolonged equilibration time. The difference between the folding and the unfolding profiles was decreased in the mutant R211M. The apparent Gibbs free energy decreased approximately 2 kcal/mol and the unfolding rate increased 4.3-fold in the mutant protein, corresponding to a decrease in activation free energy of unfolding of 0.86 kcal/mol. These results suggest that the inter-domain ionic network might be responsible for additional stabilization through a significant kinetic barrier in the unfolding pathway that could also explain the larger difference observed between the folding and unfolding transitions of the wild type.

The phylogeny of the aromatic amino acid hydroxylases revisited by characterizing phenylalanine hydroxylase from Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum contains only one aromatic amino acid hydroxylase (AAAH) gene compared to at least three in metazoans. As shown in this work this gene codes for a phenylalanine hydroxylase (DictyoPAH) and phylogenetic analysis places this enzyme close to the precursor AAAHs, aiding to define the evolutionary history of the AAAH family. DictyoPAH shows significant similarities to other eukaryote PAH, but it exhibits higher activity with tetrahydrodictyopterin (DH4) than with tetrahydrobiopterin (BH4) as cofactor. DH4 is an abundant tetrahydropterin in D. discoideum while BH4 is the natural cofactor of the AAAHs in mammals. Moreover, DictyoPAH is devoid of the characteristic regulatory mechanisms of mammalian PAH such as positive cooperativity for L-Phe and activation by preincubation with the substrate. Analysis of the few active site substitutions between DictyoPAH and mammalian PAH, including mutant expression analysis, reveals potential structural determinants for allosteric regulation.