Phylogenetic and functional diverse ANME-1 thrive in Arctic hydrothermal vents.

The methane-rich areas, the Loki's Castle vent field and the Jan Mayen vent field at the Arctic Mid Ocean Ridge (AMOR), host abundant niches for anaerobic methane-oxidizers, which are predominantly filled by members of the ANME-1. In this study, we used a metagenomic-based approach that revealed the presence of phylogenetic and functional different ANME-1 subgroups at AMOR, with heterogeneous distribution. Based on a common analysis of ANME-1 genomes from AMOR and other geographic locations, we observed that AMOR subgroups clustered with a vent-specific ANME-1 group that occurs solely at vents, and with a generalist ANME-1 group, with a mixed environmental origin. Generalist ANME-1 are enriched in genes coding for stress response and defense strategies, suggesting functional diversity among AMOR subgroups. ANME-1 encode a conserved energy metabolism, indicating strong adaptation to sulfate-methane-rich sediments in marine systems, which does not however prevent global dispersion. A deep branching family named Ca. Veteromethanophagaceae was identified. The basal position of vent-related ANME-1 in phylogenomic trees suggests that ANME-1 originated at hydrothermal vents. The heterogeneous and variable physicochemical conditions present in diffuse venting areas of hydrothermal fields could have favored the diversification of ANME-1 into lineages that can tolerate geochemical and environmental variations.

Discovery of a Thermostable GH10 Xylanase with Broad Substrate Specificity from the Arctic Mid-Ocean Ridge Vent System.

A two-domain GH10 xylanase-encoding gene (amor_gh10a) was discovered from a metagenomic data set, generated after in situ incubation of a lignocellulosic substrate in hot sediments on the sea floor of the Arctic Mid-Ocean Ridge (AMOR). AMOR_GH10A comprises a signal peptide, a carbohydrate-binding module belonging to a previously uncharacterized family, and a catalytic glycosyl hydrolase (GH10) domain. The enzyme shares the highest sequence identity (42%) with a hypothetical protein from a Verrucomicrobia bacterium, and its GH10 domain shares low identity (24 to 28%) with functionally characterized xylanases. Purified AMOR_GH10A showed thermophilic and halophilic properties and was active toward various xylans. Uniquely, the enzyme showed high activity toward amorphous cellulose, glucomannan, and xyloglucan and was more active toward cellopentaose than toward xylopentaose. Binding assays showed that the N-terminal domain of this broad-specificity GH10 binds strongly to amorphous cellulose, as well as to microcrystalline cellulose, birchwood glucuronoxylan, barley ß-glucan, and konjac glucomannan, confirming its classification as a novel CBM (CBM85).IMPORTANCE Hot springs at the sea bottom harbor unique biodiversity and are a promising source of enzymes with interesting properties. We describe the functional characterization of a thermophilic and halophilic multidomain xylanase originating from the Arctic Mid-Ocean Ridge vent system, belonging to the well-studied family 10 of glycosyl hydrolases (GH10). This xylanase, AMOR_GH10A, has a surprisingly wide substrate range and is more active toward cellopentaose than toward xylopentaose. This substrate promiscuity is unique for the GH10 family and could prove useful in industrial applications. Emphasizing the versatility of AMOR_GH10A, its N-terminal domain binds to both xylans and glycans, while not showing significant sequence similarities to any known carbohydrate-binding module (CBM) in the CAZy database. Thus, this N-terminal domain lays the foundation for the new CBM85 family.

Comparison of isocitrate dehydrogenase from three hyperthermophiles reveals differences in thermostability, cofactor specificity, oligomeric state, and phylogenetic affiliation.

With the aim of gaining insight into the molecular and phylogenetic relationships of isocitrate dehydrogenase (IDH) from hyperthermophiles, we carried out a comparative study of putative IDHs identified in the genomes of the eubacterium Thermotoga maritima and the archaea Aeropyrum pernix and Pyrococcus furiosus. An optimum for activity at 90 degrees C or above was found for each IDH. PfIDH and ApIDH were the most thermostable with a melting temperature of 103.7 and 109.9 degrees C, respectively, compared with 98.3 and 98.5 degrees C for TmIDH and AfIDH, respectively. Analytical ultracentrifugation revealed a tetrameric oligomeric state for TmIDH and a homodimeric state for ApIDH and PfIDH. TmIDH and ApIDH were NADP-dependent (K(m)((NADP)) of 55.2 and 44.4 microm, respectively) whereas PfIDH was NAD-dependent (K(m)((NAD)) of 68.3 microm). These data document that TmIDH represents a novel tetrameric NADP-dependent form of IDH and that PfIDH is a homodimeric NAD-dependent IDH not previously found among the archaea. The homodimeric NADP-IDH present in A. pernix is the most common form of IDH known so far. The evolutionary relationships of ApIDH, PfIDH, and TmIDH with all of the available amino acid sequences of di- and multimeric IDHs are described and discussed.

Differences in the oligomeric states of the LDH-like L-MalDH from the hyperthermophilic archaea Methanococcus jannaschii and Archaeoglobus fulgidus.

L-Malate (MalDH) and L-lactate (LDH) dehydrogenases belong to the same family of NAD-dependent enzymes. To gain insight into molecular relationships within this family, we studied two hyperthermophilic (LDH-like) L-MalDH (proteins with LDH-like structure and MalDH enzymatic activity) from the archaea Archaeoglobus fulgidus (Af) and Methanococcus jannaschii (Mj). The structural parameters of these enzymes determined by neutron scattering and analytical centrifugation showed that the Af (LDH-like) L-MalDH is a dimer whereas the Mj (LDH-like) L-MalDH is a tetramer. The effects of high temperature, cofactor binding, and high phosphate concentration were studied. They did not modify the oligomeric state of either enzyme. The enzymatic activity of the dimeric Af (LDH-like) L-MalDH is controlled by a pH-dependent transition at pH 7 without dissociation of the subunits. The data were analyzed in the light of the crystallographic structure of the LDH-like L-MalDH from Haloarcula marismortui. This showed that a specific loop at the dimer-dimer contact regions in these enzymes controls the tetramer formation.

Desulfobulbus rhabdoformis sp. nov., a sulfate reducer from a water-oil separation system.

A mesophilic, Gram-negative, rod-shaped, marine, propionate-oxidizing sulfate reducer (strain M16T) was isolated from a water-oil separation system on a North Sea oil platform. The optimum conditions for growth were 31 degrees C, pH 6.8-7.2 and 1.5-2.0% (w/v) NaCl and 0.1-0.3% (w/v) MgCl2.6H2O in the medium. The growth yield with sulfate was 4.6 g cell biomass (mol propionate oxidized)-1. Strain M16T is nutritionally related to members of the genus Desulfobulbus, but differs in that it has no vitamin requirement and is able to utilize fumarate and malate as carbon and energy sources. Hydrogenase activity measured as hydrogen uptake was mainly membrane-bound and varied with the growth substrate. Highest activity [28 mumol min-1 (mg protein)-1] was found in cells grown with hydrogen and lowest [50 nmol min-1 (mg protein)-1] in cells grown with propionate as electron donors for sulfate reduction. Desulforubidin, menaquinone-5(H2) and cytochromes of the c- and b-type were present. The fatty acid pattern was similar to that found for Desulfobulbus propionicus. The DNA base composition was 50.6 mol% G + C. Strain M16T is equidistantly related to D. propionicus and Desulfobulbus elongatus with 96.1% 16S rDNA similarity. On the basis of differences in genotypic, phenotypic and immunological characteristics, strain M16T (= DSM 8777T) is proposed as the type strain of a new species, Desulfobulbus rhabdoformis.

Purification and characterization of a monomeric isocitrate dehydrogenase from the sulfate-reducing bacterium Desulfobacter vibrioformis and demonstration of the presence of a monomeric enzyme in other bacteria.

NADP(+)-specific isocitrate dehydrogenase (EC 1.1.1.42) was purified to homogeneity from the sulfate-reducing bacterium Desulfobacter vibrioformis, and shown to be a monomeric protein with a molecular mass of 80 kDa. The pH and temperature optima were 8.5 and 45 degrees C, respectively. The N-terminal amino acid sequence (Thr, Glu, Thr, Ile, Arg, Trp, Thr, X, Thr, Asp, Glu, Ala, Pro, Leu, Leu, Ala, Thr) showed similarity with that of other known monomeric isocitrate dehydrogenases. Catalytically active isocitrate dehydrogenase from D. vibrioformis was obtained by activity staining after SDS-PAGE and removal of SDS from the gel. This technique revealed a NADP(+)-dependent monomeric enzyme in other Desulfobacter spp., Desulfuromonas acetoxidans and Chlorobium tepidium. These findings imply that monomeric isocitrate dehydrogenases are present in distantly related bacteria and indicate an early evolution of monomeric isocitrate dehydrogenases in the bacterial lineage.

Biochemical and phylogenetic characterization of isocitrate dehydrogenase from a hyperthermophilic archaeon, Archaeoglobus fulgidus.

A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE. Following separation by SDS-PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining. The enzyme showed dual coenzyme specificity with a high preference for NADP+. Optimal temperature for activity was 90 degrees C or above, and a half-life of 22 min was found for the enzyme when incubated at 90 degrees C in a 50 mM Tricine-KOH buffer (pH 8.0). Based on the N-terminal amino acid sequence, the gene encoding the isocitrate dehydrogenase was cloned. DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined for the purified enzyme. The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli. All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E. coli isocitrate dehydrogenase are conserved in the enzyme from A. fulgidus. The primary structure of A. fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea. Multiple alignment of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows that they can be divided into three distinct phylogenetic groups.

Properties and primary structure of a thermostable L-malate dehydrogenase from Archaeoglobus fulgidus.

A thermostable l-malate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was isolated and characterized, and its gene was cloned and sequenced. The enzyme is a homodimer with a molecular mass of 70 kDa and catalyzes preferentially the reduction of oxaloacetic acid with NADH. A. fulgidus L-malate dehydrogenase was stable for 5 h at 90 degrees C, and the half-life at 101 degrees C was 80 min. Thus, A. fulgidus L-malate dehydrogenase is the most thermostable L-malate dehydrogenase characterized to date. Addition of K2HPO4 (1 M) increased the thermal stability by 40%. The primary structure shows a high similarity to L-lactate dehydrogenase from Thermotoga maritima and gram-positive bacteria, and to L-malate dehydrogenase from the archaeon Haloarcula marismortui and other L-lactate-dehydrogenase-like L-malate dehydrogenases.

Purification and properties of an extremely thermostable NADP+-specific glutamate dehydrogenase from Archaeoglobus fulgidus.

NADP+-specific glutamate dehydrogenase (EC 1.4.1.4) was purified to homogeneity from the extremely thermophilic, strictly anaerobic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324. The native enzyme (263 kDa) is composed of subunits of mol. mass 46 kDa, suggesting a hexameric structure. The temperature optimum for enzyme activity was > 95 degrees C. The enzyme was highly thermostable, having a half-life of 140 min at 100 degrees C. Potassium phosphate, KCl, and NaCl enhanced the thermal stability and increased the rate of activity three- to fourfold. The N-terminal 26-amino-acid sequence showed a high degree of similarity to glutamate dehydrogenases from Pyrococcus spp. and Thermococcus spp.