Purification of a Crenarchaeal ATP Synthase in the Light of the Unique Bioenergetics of Ignicoccus Species.

In this study, the ATP synthase of Ignicoccus hospitalis was purified, characterized, and structurally compared to the respective enzymes of the other Ignicoccus species, to shed light on energy conservation in this unique group of archaea. The crenarchaeal genus Ignicoccus comprises three described species, i.e., I. hospitalis and Ignicoccus islandicus from hot marine sediments near Iceland and Ignicoccus pacificus from a hydrothermal vent system in the Pacific Ocean. This genus is unique among all archaea due to the unusual cell envelope, consisting of two membranes that enclose a large intermembrane compartment (IMC). I. hospitalis is the best studied member of this genus, mainly because it is the only known host for the potentially parasitic archaeon Nanoarchaeum equitansI. hospitalis grows chemolithoautotrophically, and its sole energy-yielding reaction is the reduction of elemental sulfur with molecular hydrogen, forming large amounts of hydrogen sulfide. This reaction generates an electrochemical gradient, which is used by the ATP synthase, located in the outer cellular membrane, to generate ATP inside the IMC. The genome of I. hospitalis encodes nine subunits of an A-type ATP synthase, which we could identify in the purified complex. Although the maximal in vitro activity of the I. hospitalis enzyme was measured around pH 6, the optimal stability of the A1AO complex seemed to be at pH 9. Interestingly, the soluble A1 subcomplexes of the different Ignicoccus species exhibited significant differences in their apparent molecular masses in native electrophoresis, although their behaviors in gel filtration and chromatography-mass spectrometry were very similar.IMPORTANCE The Crenarchaeota represent one of the major phyla within the Archaea domain. This study describes the successful purification of a crenarchaeal ATP synthase. To date, all information about A-type ATP synthases is from euryarchaeal enzymes. The fact that it has not been possible to purify this enzyme complex from a member of the Crenarchaeota until now points to significant differences in stability, possibly caused by structural alterations. Furthermore, the study subject I. hospitalis has a particular importance among crenarchaeotes, since it is the only known host of N. equitans The energy metabolism in this system is still poorly understood, and our results can help elucidate the unique relationship between these two microbes.

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.

[Capacity of hyperthermophilic Crenarchaeota for decomposition of refractory protiens (α- and ß-keratins)].

Anaerobic thermophilic archaea of the genera Thermogladius and Desulfurococcus capable of a- and P3-keratin decomposition were isolated from hot springs of Kamchatka and Kunashir Island. For two of them (strains 2355k and 3008g), the presence of high-molecular mass, cell-bound endopeptidases active against nonhydrolyzed and partially hydrolyzed proteins at high values of temperature and pH was shown. Capacity for ß-keratin decomposition was also found in collection strains (type strains of Desulfurococcus amylolyticus subsp. amylolyticus, D. mucosus subsp. mobilis, and D. fermentans).

Complete genome sequence of Desulfurococcus fermentans, a hyperthermophilic cellulolytic crenarchaeon isolated from a freshwater hot spring in Kamchatka, Russia.

Desulfurococcus fermentans is the first known cellulolytic archaeon. This hyperthermophilic and strictly anaerobic crenarchaeon produces hydrogen from fermentation of various carbohydrates and peptides without inhibition by accumulating hydrogen. The complete genome sequence reported here suggested that D. fermentans employs membrane-bound hydrogenases and novel glycohydrolases for hydrogen production from cellulose.

Complete genome sequence of the hyperthermophilic cellulolytic crenarchaeon "Thermogladius cellulolyticus" 1633.

Strain 1633, a novel member of the genus Thermogladius, isolated from a freshwater hot spring, is an anaerobic hyperthermophilic crenarchaeon capable of fermenting proteinaceous and cellulose substrates. The complete genome sequence reveals genes for protein and carbohydrate-active enzymes, the Embden-Meyerhof pathway for glucose metabolism, cytoplasmic NADP-dependent hydrogenase, and several energy-coupling membrane-bound oxidoreductases.

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)).

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.

[Detection of hyperthermophilic archaea of the genus Desulforococcus by hybridization with oligonucleotide probes]. / Detektsiia gipertermofil'nykh arkhei roda Desulfurococcus putem gibridizatsii s oligonukleotidnymi zondami.

Based on the analysis of nucleotide sequences of 16S rRNA, oligonucleotide probes were designed for the detection and identification of representatives of the genus Desulfurococcus (kingdom Crenarchaeota of the domain Archaea). The detection procedure included obtaining of PCR products on DNA isolated from pure cultures, enrichments, or natural samples with a Crenarchaeota-specific primer pair designed: Cren 7F (5'-TTCCGGTTGATCCYGCCGGACC-3') and Cren 518R (5'-GCTGGTWTTACCGCGGCGGCTGA-3'). The PCR products were hybridized with Dig-11-dUTP-labeled oligonucleotide probes targeting the genus Desulfurococcus (Dco 198, 5'-CGTTAACYCCYGCCACACC-3) and its species D. mobilis (Dco_mob 198, 5'-CGTTAACCCCTGCCACACC-3') and D. amylolyticus (Dco_amy 198, 5'-CGTTAACCCCCGCCACACC-3'). With the use of these primers and probes, four new strains isolated from hydrotherms of Kamchatka and Kunashir Island were identified as members of the species Desulfurococcus amylolyticus. Desulfurococcus representatives were detected in several natural samples, including a sample taken from a marine hydrotherm at the Kunashir Island; this demonstrates that representatives of this genus occur not only in terrestrial but also in marine environments.

The ultrastructure of Ignicoccus: evidence for a novel outer membrane and for intracellular vesicle budding in an archaeon.

A novel genus of hyperthermophilic, strictly chemolithotrophic archaea, Ignicoccus, has been described recently, with (so far) three isolates in pure culture. Cells were prepared for ultrastructural investigation by cultivation in cellulose capillaries and processing by high-pressure freezing, freeze-substitution and embedding in Epon. Cells prepared in accordance with this protocol consistently showed a novel cell envelope structure previously unknown among the Archaea: a cytoplasmic membrane; a periplasmic space with a variable width of 20 to 400 nm, containing membrane-bound vesicles; and an outer sheath, approximately 10 nm wide, resembling the outer membrane of gram-negative bacteria. This sheath contained three types of particles: numerous tightly, irregularly packed single particles, about 8 nm in diameter; pores with a diameter of 24 nm, surrounded by tiny particles, arranged in a ring with a diameter of 130 nm; and clusters of up to eight particles, each particle 12 nm in diameter. Freeze-etched cells exhibited a smooth surface, without a regular pattern, with frequent fracture planes through the outer sheath, indicating the presence of an outer membrane and the absence of an S-layer. The study illustrates the novel complex architecture of the cell envelope of Ignicoccus as well as the importance of elaborate preparation procedures for ultrastructural investigations.

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).

Stetteria hydrogenophila, gen. nov. and sp. nov., a novel mixotrophic sulfur-dependent crenarchaeote isolated from Milos, Greece.

A new hyperthermophilic, strictly anaerobic crenarchaeote, Stetteria hydrogenophila DSM11227 representing a new genus within the family of Desulfurococcaceae, was isolated from the sediment of a marine hydrothermal system at Paleohori Bay in Milos, Greece. Cells are gram-negative irregular and disc-shaped cocci, 0.5-1.5 microm in diameter, which are flagellate and can form cytoplasmatic protrusions up to 2 microm in length. The strain grew optimally at 95 degrees C at pH 6.0 and at a NaCl concentration of 3%. The organism grew mixotrophically on peptide substrates. It required elemental sulfur as an external electron acceptor, and in addition, its growth was completely dependent on the presence of molecular hydrogen. Sulfur could be replaced by thiosulfate. H2S, CO2, acetate, and ethanol were identified as products of metabolism. The G + C content of DNA was 65 mol%. Analysis of its phylogenetic position by sequence analysis of 16S rRNA placed this organism in the family of Desulfurococcaceae. The dependence of this organism on both hydrogen and sulfur during growth on peptide substrates distinguishes Stetteria from all previously described species of Crenarchaeota.