Identification and Enzymatic Analysis of an Archaeal ATP-Dependent Serine Kinase from the Hyperthermophilic Archaeon Staphylothermus marinus.

Serine kinase catalyzes the phosphorylation of free serine (Ser) to produce O-phosphoserine (Sep). An ADP-dependent Ser kinase in the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-SerK) is involved in cysteine (Cys) biosynthesis and most likely Ser assimilation. An ATP-dependent Ser kinase in the mesophilic bacterium Staphylococcus aureus is involved in siderophore biosynthesis. Although proteins displaying various degrees of similarity with Tk-SerK are distributed in a wide range of organisms, it is unclear if they are actually Ser kinases. Here, we examined proteins from Desulfurococcales species in Crenarchaeota that display moderate similarity with Tk-SerK from Euryarchaeota (42 to 45% identical). Tk-serK homologs from Staphylothermus marinus (Smar_0555), Desulfurococcus amylolyticus (DKAM_0858), and Desulfurococcus mucosus (Desmu_0904) were expressed in Escherichia coli. All three partially purified recombinant proteins exhibited Ser kinase activity utilizing ATP rather than ADP as a phosphate donor. Purified Smar_0555 protein displayed activity for l-Ser but not other compounds, including d-Ser, l-threonine, and l-homoserine. The enzyme utilized ATP, UTP, GTP, CTP, and the inorganic polyphosphates triphosphate and tetraphosphate as phosphate donors. Kinetic analysis indicated that the Smar_0555 protein preferred nucleoside 5'-triphosphates over triphosphate as a phosphate donor. Transcript levels and Ser kinase activity in S. marinus cells grown with or without serine suggested that the Smar_0555 gene is constitutively expressed. The genes encoding Ser kinases examined here form an operon with genes most likely responsible for the conversion between Sep and 3-phosphoglycerate of central sugar metabolism, suggesting that the ATP-dependent Ser kinases from Desulfurococcales play a role in the assimilation of Ser. IMPORTANCE Homologs of the ADP-dependent Ser kinase from the archaeon Thermococcus kodakarensis (Tk-SerK) include representatives from all three domains of life. The results of this study show that even homologs from the archaeal order Desulfurococcales, which are the most structurally related to the ADP-dependent Ser kinases from the Thermococcales, are Ser kinases that utilize ATP, and in at least some cases inorganic polyphosphates, as the phosphate donor. The differences in properties between the Desulfurococcales and Thermococcales enzymes raise the possibility that Tk-SerK homologs constitute a group of kinases that phosphorylate free serine with a wide range of phosphate donors.

A new symbiotic nanoarchaeote (Candidatus Nanoclepta minutus) and its host (Zestosphaera tikiterensis gen. nov., sp. nov.) from a New Zealand hot spring.

Three thermophilic Nanoarchaeota-Crenarchaeota symbiotic systems have been described. We obtained another stable anaerobic enrichment culture at 80°C, pH 6.0 from a New Zealand hot spring. The nanoarchaeote (Ncl-1) and its host (NZ3T) were isolated in co-culture and their genomes assembled. The small (∼200nm) flagellated cocci were often attached to larger cocci. Based on 16S rRNA gene similarity (88.4%) and average amino acid identity (52%), Ncl-1 is closely related to Candidatus Nanopusillus acidilobi. Their genomes both encode for archaeal flagella and partial glycolysis and gluconeogenesis pathways, but lack ATP synthase genes. Like Nanoarchaeum equitans, Ncl-1 has a CRISPR-Cas system. Ncl-1 also relies on its crenarchaeotal host for most of its biosynthetic needs. The host NZ3T was isolated and grows on proteinaceous substrates but not on sugars, alcohols, or fatty acids. NZ3T requires thiosulfate and grows best at 82°C, pH 6.0. NZ3T is most closely related to the Desulfurococcaceae, Ignisphaera aggregans (∼92% 16S rRNA gene sequence similarity, 45% AAI). Based on phylogenetic, physiological and genomic data, Ncl-1 and NZ3T represent novel genera in the Nanoarchaeota and the Desulfurococcaceae, respectively, with the proposed names Candidatus Nanoclepta minutus and Zestosphaera tikiterensis gen. nov., sp. nov., type strain NZ3T (=DSMZ 107634T=OCM 1213T).

Reclassification of Desulfurococcus mobilis as a synonym of Desulfurococcus mucosus, Desulfurococcus fermentans and Desulfurococcus kamchatkensis as synonyms of Desulfurococcus amylolyticus, and emendation of the D. mucosus and D. amylolyticus species descriptions.

Representatives of the crenarchaeal genus Desulfurococcus are strictly anaerobic hyperthermophiles with an organotrophic type of metabolism. Since 1982, five Desulfurococcus species names have been validly published: Desulfurococcus mucosus, D. mobilis, D. amylolyticus, D. fermentans and D. kamchatkensis. Recently, the genomic sequences of all five species became available, promoting the refinement of their taxonomic status. Analysis of full-length high-quality 16S rRNA gene sequences shows that the sequences of D. mobilis and D. mucosus are 100 % identical and differ by 2.2 % from those of D. amylolyticus, D. fermentans and D. kamchatkensis. The latter three sequences differ from each other by 0.1-0.3 % (99.9 % similarity in the D amylolyticus-D. kamchatkensis pair and 99.7 % in the pairs involving D. fermentans). In silico prediction of DNA-DNA hybridization (DDH) values by comparison of genomes using ggdc 2.0 blast+ at http://ggdc.dsmz.de/ produced results that correlated with the 16S rRNA gene sequence similarity values. In the D. mucosus-D. mobilis and D. amylolyticus-D. kamchatkensis pairs, the predicted DDH values were 99 and 92 %, respectively, much higher than the recommended 70 % species-delimiting DDH value. Between members of different pairs, these values were no higher than 20 %. For D. fermentans, its predicted DDH values were around 70 % with D. amylolyticus and D. kamchatkensis and no higher than 20 % with D. mobilis and D. mucosus. These results indicated that D. mobilis should be reclassified as a synonym of D. mucosus, whereas D. kamchatkensis and D. fermentans should be reclassified as synonyms of D. amylolyticus.

The unusual cell biology of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis.

The Crenarchaeon Ignicoccus hospitalis is an anaerobic, obligate chemolithoautotrophic hyperthermophile, growing by reduction of elemental sulfur using molecular hydrogen as electron donor. Together with Nanoarchaeum equitans it forms a unique, archaeal biocoenosis, in which I. hospitalis serves as host for N. equitans. Both organisms can be cultivated in a stable coculture which is mandatory for N. equitans but not for I. hospitalis. This strong dependence is affirmed by the fact that N. equitans obtains its lipids and amino acids from the host. I. hospitalis cells exhibit several unique features: they can adhere to surfaces by extracellular appendages ('fibers') which are not used for motility; they use a novel CO(2) fixation pathway, the dicarboxylate/4-hydroxybutyrate pathway; and they exhibit a unique cell envelope for Archaea consisting of two membranes but lacking an S-layer. These membranes form two cell compartments, a tightly packed cytoplasm surrounded by a weakly staining intermembrane compartment (IMC) with a variable width from 20 to 1,000 nm. In this IMC, many round or elongated vesicles are found which may function as carriers of lipids or proteins out of the cytoplasm. Based on immuno-EM analyses and immuno-fluorescence experiments it was demonstrated recently that the A(1)A(O) ATP synthase, the H(2):sulfur oxidoreductase complex and the acetyl-CoA synthetase (ACS) of I. hospitalis are located in its outermost membrane. Therefore, this membrane is energized and is here renamed as "outer cellular membrane" (OCM). Among all prokaryotes possessing two membranes in their cell envelope, I. hospitalis is the first organism with an energized outermost membrane and ATP synthesis outside the cytoplasm. Since DNA and ribosomes are localized in the cytoplasm, energy conservation is separated from information processing and protein biosynthesis in I. hospitalis. This raises questions concerning the function and characterization of the two membranes, the two cell compartments and of a possible ATP transfer to N. equitans.

Thermogladius shockii gen. nov., sp. nov., a hyperthermophilic crenarchaeote from Yellowstone National Park, USA.

A hyperthermophilic heterotrophic archaeon (strain WB1) was isolated from a thermal pool in the Washburn hot spring group of Yellowstone National Park, USA. WB1 is a coccus, 0.6-1.2 µm in diameter, with a tetragonal S-layer, vacuoles, and occasional stalk-like protrusions. Growth is optimal at 84°C (range 64-93°C), pH 5-6 (range 3.5-8.5), and <1 g/l NaCl (range 0-4.6 g/l NaCl). Tests of metabolic properties show the isolate to be a strict anaerobe that ferments complex organic substrates. Phylogenetic analysis of the 16S rRNA gene sequence places WB1 in a clade of previously uncultured Desulfurococcaceae and shows it to have ≤ 96% 16S rRNA sequence identity to Desulfurococcus mobilis, Staphylothermus marinus, Staphylothermus hellenicus, and Sulfophobococcus zilligii. The 16S rRNA gene contains a large insertion similar to homing endonuclease introns reported in Thermoproteus and Pyrobaculum species. Growth is unaffected by the presence of S(0) or SO(4)(2-), thereby differentiating the isolate from its closest relatives. Based on phylogenetic and physiological differences, it is proposed that isolate WB1 represents the type strain of a novel genus and species within the Desulfurococcaceae, Thermogladius shockii gen. nov., sp. nov. (RIKEN = JCM-16579, ATCC = BAA-1607, Genbank 16S rRNA gene = EU183120).

Desulfurococcus kamchatkensis sp. nov., a novel hyperthermophilic protein-degrading archaeon isolated from a Kamchatka hot spring.

A novel obligately anaerobic, hyperthermophilic, organotrophic archaeon, designated strain 1221n(T), was isolated from a hot spring of Uzon Caldera (Kamchatka Peninsula, Russia). Cells of strain 1221n(T) were non-motile regular cocci, 0.6-1 mum in diameter. The temperature range for growth at pH 6.5 was 65-87 degrees C, with an optimum at 85 degrees C. The pH range for growth at 85 degrees C was 5.5-7.5, with an optimum at pH 6.5. Growth was not observed at or below 6 degrees C or at or above 90 degrees C, as well as at or below pH 5.0 and at or above pH 8.0. The isolate fermented a wide range of substrates including proteins: alpha-keratin, albumin and gelatin. Elemental sulfur was not essential for growth, but stimulated growth. Strain 1221n(T) synthesized 40 and 120 kDa proteinases localized on the cell envelope. The G+C content of the DNA was 44.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparison indicated that strain 1221n(T) was affiliated with the genus Desulfurococcus. The level of 16S rRNA gene sequence similarity with other Desulfurococcus species was 96.7-98.1 %, and Desulfurococcus amylolyticus was found to be the most closely related organism. Based on the data from the phylogenetic analysis and the physiological properties of the novel isolate, strain 1221n(T) should be classified as representing a novel species, for which the name Desulfurococcus kamchatkensis sp. nov. is proposed. The type strain is 1221n(T) (=DSM 18924(T)=VKM B-2413(T)).

The complete genome sequence of Staphylothermus marinus reveals differences in sulfur metabolism among heterotrophic Crenarchaeota.

BACKGROUND: Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes. RESULTS: The 1.57 Mbp genome of the hyperthermophilic crenarchaeote Staphylothermus marinus has been completely sequenced. The main energy generating pathways likely involve 2-oxoacid:ferredoxin oxidoreductases and ADP-forming acetyl-CoA synthases. S. marinus possesses several enzymes not present in other crenarchaeotes including a sodium ion-translocating decarboxylase likely to be involved in amino acid degradation. S. marinus lacks sulfur-reducing enzymes present in the other two sulfur-reducing crenarchaeotes that have been sequenced -- Thermofilum pendens and Hyperthermus butylicus. Instead it has three operons similar to the mbh and mbx operons of Pyrococcus furiosus, which may play a role in sulfur reduction and/or hydrogen production. The two marine organisms, S. marinus and H. butylicus, possess more sodium-dependent transporters than T. pendens and use symporters for potassium uptake while T. pendens uses an ATP-dependent potassium transporter. T. pendens has adapted to a nutrient-rich environment while H. butylicus is adapted to a nutrient-poor environment, and S. marinus lies between these two extremes. CONCLUSION: The three heterotrophic sulfur-reducing crenarchaeotes have adapted to their habitats, terrestrial vs. marine, via their transporter content, and they have also adapted to environments with differing levels of nutrients. Despite the fact that they all use sulfur as an electron acceptor, they are likely to have different pathways for sulfur reduction.

Complete genome sequence of the anaerobic, protein-degrading hyperthermophilic crenarchaeon Desulfurococcus kamchatkensis.

Desulfurococcus kamchatkensis is an anaerobic organotrophic hyperthermophilic crenarchaeon isolated from a terrestrial hot spring. Its genome consists of a single circular chromosome of 1,365,223 bp with no extrachromosomal elements. A total of 1,474 protein-encoding genes were annotated, among which 205 are exclusive for D. kamchatkensis. The search for a replication origin site revealed a single region coinciding with a global extreme of the nucleotide composition disparity curve and containing a set of crenarchaeon-type origin recognition boxes. Unlike in most archaea, two genes encoding homologs of the eukaryotic initiator proteins Orc1 and Cdc6 are located distantly from this site. A number of mobile elements are present in the genome, including seven transposons representing IS607 and IS200/IS605 families and multiple copies of miniature inverted repeat transposable elements. Two large clusters of regularly interspaced repeats are present; none of the spacer sequences matches known archaeal extrachromosomal elements, except one spacer matches the sequence of a resident gene of D. kamchatkensis. Many of the predicted metabolic enzymes are associated with the fermentation of peptides and sugars, including more than 30 peptidases with diverse specificities, a number of polysaccharide degradation enzymes, and many transporters. Consistently, the genome encodes both enzymes of the modified Embden-Meyerhof pathway of glucose oxidation and a set of enzymes needed for gluconeogenesis. The genome structure and content reflect the organism's nutritionally diverse, competitive natural environment, which is periodically invaded by viruses and other mobile elements.

Ignicoccus hospitalis sp. nov., the host of 'Nanoarchaeum equitans'.

A novel chemolithoautotrophic and hyperthermophilic member of the genus Ignicoccus was isolated from a submarine hydrothermal system at the Kolbeinsey Ridge, to the north of Iceland. The new isolate showed high similarity to the two species described to date, Ignicoccus islandicus and Ignicoccus pacificus, in its physiological properties as well as in its unique cell architecture. However, phylogenetic analysis and investigations on the protein composition of the outer membrane demonstrated that the new isolate was clearly distinct from I. islandicus and I. pacificus. Furthermore, it is the only organism known so far which is able to serve as a host for 'Nanoarchaeum equitans', the only cultivated member of the 'Nanoarchaeota'. Therefore, the new isolate represents a novel species of the genus Ignicoccus, which we name Ignicoccus hospitalis sp. nov. (type strain KIN4/I(T)=DSM 18386(T)=JCM 14125(T)).

Desulfurococcus fermentans sp. nov., a novel hyperthermophilic archaeon from a Kamchatka hot spring, and emended description of the genus Desulfurococcus.

An obligately anaerobic, hyperthermophilic, organoheterotrophic archaeon, strain Z-1312(T), was isolated from a freshwater hot spring of the Uzon caldera (Kamchatka Peninsula, Russia). The cells were regular cocci, 1-4 microm in diameter, with one long flagellum. The cell envelope was composed of a globular layer attached to the cytoplasmic membrane. The temperature range for growth was 63-89 degrees C, with an optimum between 80 and 82 degrees C. The pH range for growth at 80 degrees C was 4.8-6.8, with an optimum at pH 6.0. Strain Z-1312(T) grew by hydrolysis and/or fermentation of a wide range of polymeric and monomeric substrates, including agarose, amygdalin, arabinose, arbutin, casein hydrolysate, cellulose (filter paper, microcrystalline cellulose, carboxymethyl cellulose), dextran, dulcitol, fructose, lactose, laminarin, lichenan, maltose, pectin, peptone, ribose, starch and sucrose. No growth was detected on glucose, xylose, mannitol or sorbitol. Growth products when sucrose or starch were used as the substrate were acetate, H(2) and CO(2). Elemental sulfur, thiosulfate and nitrate added as potential electron acceptors for anaerobic respiration did not stimulate growth when tested with starch as the substrate. H(2) at 100 % in the gas phase did not inhibit growth on starch or peptone. The G+C content of the DNA was 42.5 mol%. 16S rRNA gene sequence analysis placed the isolated strain Z-1312(T) as a member of the genus Desulfurococcus, where it represented a novel species, for which the name Desulfurococcus fermentans sp. nov. (type strain Z-1312(T) = DSM 16532 (T) = VKM V-2316(T)) is proposed.

Phylogenetic diversity analysis of subterranean hot springs in Iceland.

Geothermal energy has been harnessed and used for domestic heating in Iceland. In wells that are typically drilled to a depth of 1,500 to 2,000 m, the temperature of the source water is 50 to 130 degrees C. The bottoms of the boreholes can therefore be regarded as subterranean hot springs and provide a unique opportunity to study the subterranean biosphere. Large volumes of geothermal fluid from five wells and a mixture of geothermal water from 50 geothermal wells (hot tap water) were sampled and concentrated through a 0.2-microm-pore-size filter. Cells were observed in wells RG-39 (91.4 degrees C) and MG-18 (71.8 degrees C) and in hot tap water (76 degrees C), but no cells were detected in wells SN-4, SN-5 (95 to 117 degrees C), and RV-5 (130 degrees C). Archaea and Bacteria were detected by whole-cell fluorescent in situ hybridization. DNAs were extracted from the biomass, and small-subunit rRNA genes (16S rDNAs) were amplified by PCR using primers specific for the Archaea and Bacteria domains. The PCR products were cloned and sequenced. The sequence analysis showed 11 new operational taxonomic units (OTUs) out of 14, 3 of which were affiliated with known surface OTUs. Samples from RG-39 and hot tap water were inoculated into enrichment media and incubated at 65 and 85 degrees C. Growth was observed only in media based on geothermal water. 16S rDNA analysis showed enrichments dominated with Desulfurococcales relatives. Two strains belonging to Desulfurococcus mobilis and to the Thermus/Deinococcus group were isolated from borehole RG-39. The results indicate that subsurface volcanic zones are an environment that provides a rich subsurface for novel thermophiles.

Ignicoccus gen. nov., a novel genus of hyperthermophilic, chemolithoautotrophic Archaea, represented by two new species, Ignicoccus islandicus sp nov and Ignicoccus pacificus sp nov. and Ignicoccus pacificus sp. nov.

Two species of novel, chemolithoautotrophic, sulfidogenic micro-organisms were isolated from submarine hydrothermal systems in the Atlantic (at the Kolbeinsey Ridge north of Iceland) and in the Pacific (at 9 degrees N, 104 degrees W). The coccoid cells grew within a temperature range of 70-98 degrees C (optimum around 90 degrees C). They gained energy by reduction of elemental sulfur using molecular hydrogen as the electron donor. 165 rDNA-based sequence comparisons revealed that the organisms are members of the crenarchaeal branch of the Archaea. They represent a new, deeply branching lineage within the family of the Desulfurococcaceae. In DNA-DNA hybridization experiments both strains exhibited low levels of hybridization to each other and to further representatives of this family. Therefore, they represent a new genus, for which the name Ignicoccus gen. nov. is proposed. At present it consists of two new species, Ignicoccus islandicus sp. nov. (type strain is Kol8T = DSM 13165T = ATCC 700957T) and Ignicoccus pacificus sp. nov. (type strain is LPC33T = DSM 13166T = ATCC 700958T).

Thermococcus aegaeicus sp. nov. and Staphylothermus hellenicus sp. nov., two novel hyperthermophilic archaea isolated from geothermally heated vents off Palaeochori Bay, Milos, Greece.

Two novel, hyperthermophilic, anaerobic, heterotrophic archaea were isolated from shallow hydrothermal vents off Palaeochori Bay, Milos, Greece. Strain P5T (BK17S6-3-b2T) is an irregular coccus, with a single polar flagellum, growing optimally at 90 degrees C, pH 6 and 2% NaCl. The DNA G+C content was 45 mol%. Due to its morphology, phylogenetic analyses based on 16S rRNA gene sequencing, DNA-DNA hybridization experiments, physiological properties and nutritional features, this strain represents a new species within the genus Thermococcus for which the name Thermococcus aegaeicus is proposed. The type strain is P5T (= DSM 12767T = JCM 10828T). Strain p8T (BK20S6-10-b1T) is a coccus that forms aggregates. It grew optimally at 85 degrees C, pH 6 and 3% NaCl. The DNA G+C content was 38 mol%. Physiological properties and sequence analysis of the 165 rRNA gene, as well as DNA-DNA hybridization experiments, indicate that this strain is a new species belonging to the genus Staphylothermus for which the name Staphylothermus hellenicus is proposed. The type strain is P8T (= DSM 12710T = JCM 10830T).

Sulfur-inhibited Thermosphaera aggregans sp. nov., a new genus of hyperthermophilic archaea isolated after its prediction from environmentally derived 16S rRNA sequences.

Recently, a new procedure was developed which allowed for the first time the isolation of a hyperthermophilic archaeum tracked by 165 rRNA analysis from a terrestrial hot solfataric spring ('Obsidian Pool', Yellowstone National Park, WY, USA). This novel isolate is characterized here. Cells are round cocci with a diameter of 0.2-0.8 micron, occurring singly, in pairs, short chains and in grape-like aggregates. The aggregates exhibit a weak bluish-green fluorescence under UV radiation at 420 nm. The new isolate is an anaerobic obligate heterotroph, using preferentially yeast extract for growth. The metabolic products include CO2, H2, acetate and isovalerate. Growth is observed between 65 and 90 degrees C (optimum: 85 degrees C), from pH 5.0 to 7.0 (optimum: 6.5) and up to 0.7% NaCl. The apparent activation energy for growth is about 149 kJ mol-1. Elemental sulfur or hydrogen inhibits growth. The core lipids consist mainly of acyclic and cyclic glycerol diphytanyl tetraethers. The cell envelope contains a cytoplasmic membrane covered by an amorphous layer of unknown composition; there is no evidence for a regularly arrayed surface-layer protein. The G + C content is 46 mol%. On the basis of 165 rRNA sequence comparisons in combination with morphological, physiological and biochemical properties, the isolate represents a new genus within the Desulfurococcaceae, which has been named Thermosphaera. The type species is Thermosphaera aggregans, the type strain is isolate M11TLT (= DSM 11486T).