Questioning the radiation limits of life: Ignicoccus hospitalis between replication and VBNC.

Radiation of ionizing or non-ionizing nature has harmful effects on cellular components like DNA as radiation can compromise its proper integrity. To cope with damages caused by external stimuli including radiation, within living cells, several fast and efficient repair mechanisms have evolved. Previous studies addressing organismic radiation tolerance have shown that radiotolerance is a predominant property among extremophilic microorganisms including (hyper-) thermophilic archaea. The analysis of the ionizing radiation tolerance of the chemolithoautotrophic, obligate anaerobic, hyperthermophilic Crenarchaeon Ignicoccus hospitalis showed a D10-value of 4.7 kGy, fourfold exceeding the doses previously determined for other extremophilic archaea. The genome integrity of I. hospitalis after γ-ray exposure in relation to its survival was visualized by RAPD and qPCR. Furthermore, the discrimination between reproduction, and ongoing metabolic activity was possible for the first time indicating that a potential viable but non-culturable (VBNC) state may also account for I. hospitalis.

Life on the edge: functional genomic response of Ignicoccus hospitalis to the presence of Nanoarchaeum equitans.

The marine hyperthermophilic crenarchaeon Ignicoccus hospitalis supports the propagation on its surface of Nanoarchaeum equitans, an evolutionarily enigmatic archaeon that resembles highly derived parasitic and symbiotic bacteria. The cellular and molecular mechanisms that enable this interarchaea relationship and the intimate physiologic consequences to I. hospitalis are unknown. Here, we used concerted proteomic and transcriptomic analyses to probe into the functional genomic response of I. hospitalis as N. equitans multiplies on its surface. The expression of over 97% of the genes was detected at mRNA level and over 80% of the predicted proteins were identified and their relative abundance measured by proteomics. These indicate that little, if any, of the host genomic information is silenced during growth in the laboratory. The primary response to N. equitans was at the membrane level, with increases in relative abundance of most protein complexes involved in energy generation as well as that of several transporters and proteins involved in cellular membrane stabilization. Similar upregulation was observed for genes and proteins involved in key metabolic steps controlling nitrogen and carbon metabolism, although the overall biosynthetic pathways were marginally impacted. Proliferation of N. equitans resulted, however, in selective downregulation of genes coding for transcription factors and replication and cell cycle control proteins as I. hospitalis shifted its physiology from its own cellular growth to that of its ectosymbiont/parasite. The combination of these multiomic approaches provided an unprecedented level of detail regarding the dynamics of this interspecies interaction, which is especially pertinent as these organisms are not genetically tractable.

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).

Ignicoccus hospitalis and Nanoarchaeum equitans: ultrastructure, cell-cell interaction, and 3D reconstruction from serial sections of freeze-substituted cells and by electron cryotomography.

Ultrastructure and intercellular interaction of Ignicoccus hospitalis and Nanoarchaeum equitans were investigated using two different electron microscopy approaches, by three-dimensional reconstructions from serial sections, and by electron cryotomography. Serial sections were assembled into 3D reconstructions, for visualizing the unusual complexity of I. hospitalis, its huge periplasmic space, the vesiculating cytoplasmic membrane, and the outer membrane. The cytoplasm contains fibres which are reminiscent to a cytoskeleton. Cell division in I. hospitalis is complex, and different to that in Euryarchaeota or Bacteria. An irregular invagination of the cytoplasmic membrane is followed by separation of the two cytoplasms. Simultaneous constriction of cytoplasmic plus outer membrane is not observed. Cells of N. equitans show a classical mode of cell division, by constriction in the mid-plane. Their cytoplasm exhibits two types of fibres, elongated and ring-shaped. Electron micrographs of contact sites between I. hospitalis and N. equitans exhibit two modes of interaction. One is indirect and mediated by thin fibres; in other cells the two cell surfaces are in direct contact. The two membranes of I. hospitalis cells are frequently seen in direct contact, possibly a prerequisite for transporting metabolites or substrates from the cytoplasm of one cell to the other. Rarely, a transport based on cargo vesicles is observed between I. hospitalis and N. equitans.

Nanoarchaeum equitans and Ignicoccus hospitalis: new insights into a unique, intimate association of two archaea.

Nanoarchaeum equitans and Ignicoccus hospitalis represent a unique, intimate association of two archaea. Both form a stable coculture which is mandatory for N. equitans but not for the host I. hospitalis. Here, we investigated interactions and mutual influence between these microorganisms. Fermentation studies revealed that during exponential growth only about 25% of I. hospitalis cells are occupied by N. equitans cells (one to three cells). The latter strongly proliferate in the stationary phase of I. hospitalis, until 80 to 90% of the I. hospitalis cells carry around 10 N. equitans cells. Furthermore, the expulsion of H2S, the major metabolic end product of I. hospitalis, by strong gas stripping yields huge amounts of free N. equitans cells. N. equitans had no influence on the doubling times, final cell concentrations, and growth temperature, pH, or salt concentration ranges or optima of I. hospitalis. However, isolation studies using optical tweezers revealed that infection with N. equitans inhibited the proliferation of individual I. hospitalis cells. This inhibition might be caused by deprivation of the host of cell components like amino acids, as demonstrated by 13C-labeling studies. The strong dependence of N. equitans on I. hospitalis was affirmed by live-dead staining and electron microscopic analyses, which indicated a tight physiological and structural connection between the two microorganisms. No alternative hosts, including other Ignicoccus species, were accepted by N. equitans. In summary, the data show a highly specialized association of N. equitans and I. hospitalis which so far cannot be assigned to a classical symbiosis, commensalism, or parasitism.

Composition of the lipids of Nanoarchaeum equitans and their origin from its host Ignicoccus sp. strain KIN4/I.

The contents and nature of the membrane lipids of Nanoarchaeum equitans and Ignicoccus sp. strain KIN4/I, grown at 90 degrees C, and Ignicoccus sp. strain KIN4/I, cultivated at its lowest and highest growth temperatures (75 degrees C and 95 degrees C) were analyzed. Both organisms contained very simple and qualitatively identical assemblages of glycerol ether lipids, showing only differences in the amounts of certain components. LC-MS analyses of the total lipid extracts revealed that archaeol and caldarchaeol were the main core lipids. The predominant polar headgroups consisted of one or more sugar residues attached either directly to the core lipid or via a phosphate group. GC-MS analyses of hydrolyzed total lipid extracts revealed that the co-culture of N. equitans and Ignicoccus sp. strain KIN4/I, as well as Ignicoccus sp. strain KIN4/I grown at 90 degrees C, contained phytane and biphytane in a ratio of approximately 4:1. Purified N. equitans cells and Ignicoccus sp. strain KIN4/I cultivated at 75 degrees C and 95 degrees C had a phytane to biphytane ratio of 10:1. Sugar residues were mainly mannose and small amounts of glucose. Consistent 13C fractionation patterns of isoprenoid chains of N. equitans and its host indicated that the N. equitans lipids were synthesized in the host cells.

Influence of temperature on tRNA modification in archaea: Methanococcoides burtonii (optimum growth temperature [Topt], 23 degrees C) and Stetteria hydrogenophila (Topt, 95 degrees C).

We report the first study of tRNA modification in psychrotolerant archaea, specifically in the archaeon Methanococcoides burtonii grown at 4 and 23 degrees C. For comparison, unfractionated tRNA from the archaeal hyperthermophile Stetteria hydrogenophila cultured at 93 degrees C was examined. Analysis of modified nucleosides using liquid chromatography-electrospray ionization mass spectrometry revealed striking differences in levels and identities of tRNA modifications between the two organisms. Although the modification levels in M. burtonii tRNA are the lowest in any organism of which we are aware, it contains more than one residue per tRNA molecule of dihydrouridine, a molecule associated with maintenance of polynucleotide flexibility at low temperatures. No differences in either identities or levels of modifications, including dihydrouridine, as a function of culture temperature were observed, in contrast to selected tRNA modifications previously reported for archaeal hyperthermophiles. By contrast, S. hydrogenophila tRNA was found to contain a remarkable structural diversity of 31 modified nucleosides, including nine methylated guanosines, with eight different nucleoside species methylated at O-2' of ribose, known to be an effective stabilizing motif in RNA. These results show that some aspects of tRNA modification in archaea are strongly associated with environmental temperature and support the thesis that posttranscriptional modification is a universal natural mechanism for control of RNA molecular structure that operates across a wide temperature range in archaea as well as bacteria.

Inhibitory effect of Maillard reaction products on growth of the aerobic marine hyperthermophilic archaeon Aeropyrum pernix.

It was found that the growth of Aeropyrum pernix was severely inhibited in a medium containing reducing sugars and tryptone due to the formation of Maillard reaction products. The rate of the Maillard browning reaction was markedly enhanced under aerobic conditions, and the addition of Maillard reaction products to the culture medium caused fatal growth inhibition.

Minimal sulfur requirement for growth and sulfur-dependent metabolism of the hyperthermophilic archaeon Staphylothermus marinus.

Staphylothermus marinus is an anaerobic hyperthermophilic archaeon that uses peptides as carbon and energy sources. Elemental sulfur (S(o)) is obligately required for its growth and is reduced to H2S. The metabolic functions and mechanisms of S(o) reduction were explored by examining S(o)-dependent growth and activities of key enzymes present in this organism. All three forms of S(o) tested--sublimed S(o), colloidal S(o) and polysulfide--were used by S. marinus, and no other sulfur-containing compounds could replace S(o). Elemental sulfur did not serve as physical support but appeared to function as an electron acceptor. The minimal S(o) concentration required for optimal growth was 0.05% (w/v). At this concentration, there appeared to be a metabolic transition from H2 production to S reduction. Some enzymatic activities related to S(o)-dependent metabolism, including sulfur reductase, hydrogenase, glutamate dehydrogenase and electron transfer activities, were detected in cell-free extracts of S. marinus. These results indicate that S(o) plays an essential role in the heterotrophic metabolism of S. marinus. Reducing equivalents generated by the oxidation of amino acids from peptidolysis may be transferred to sulfur reductase and hydrogenase, which then catalyze the production of H2S and H2, respectively.

Expression of two kinds of recombinant glutamate dehydrogenase from Aeropyrum pernix with different N-terminal sequence length in Escherichia coli.

Two recombinant Aeropyrum pernix glutamate dehydrogenase (GDH) enzymes with different length N-termini were cloned and expressed in Escherichia coli: sGDH begins with the amino acid sequence of the extracted native enzyme (M-Q-P-T-D-P-L-E-E), whereas lGDH begins with the sequence of the predicted ORF (M-E-V-L-A-L-Q-P-T-D) and is longer than sGDH by five amino acids (M-E-V-L-A). Purified recombinant lGDH was more stable than sGDH, indicating that the N-terminal extension, containing mostly hydrophobic residues, affected the overall stability of recombinant lGDH. This stabilising effect of extending the N-terminal sequence on an oligomeric enzyme such as GDH is novel; factors affecting stabilisation have previously only been discussed in the context of the contribution of internal amino acids.

Characterization of native glutamate dehydrogenase from an aerobic hyperthermophilic archaeon Aeropyrum pernix K1.

Glutamate dehydrogenase (GDH) was purified and characterized from an aerobic hyperthermophilic archaeon Aeropyrum pernix (A. pernix) K1. The enzyme has a hexameric structure with a native molecular mass of about 285 +/- 15 kDa. It was specific for NADP and thermostable (74% activity was remained after 5 h incubation at 100 degrees C). The activity of the enzyme increased in the presence of polar water-miscible organic solvents such as acetonitrile, methanol, and ethanol. The N-terminal sequence of GDH is Met-Gln-Pro-Thr-Asp-Pro-Leu-Glu-Glu-Ala. This sequence, except for the methionine, corresponds to amino acids 7-15 of the open reading frame (ORF) encoding the predicted GDH (ORF APE 1386). In the ORF nucleotide sequence, the codon TTG appears at the position of the methionine, suggesting that the leucine codon might be recognized as an initiation codon and translated to methionine in A. pernix GDH.

Physiological aspects involved in production of xylanolytic enzymes by deep-sea hyperthermophilic archaeon Pyrodictium abyssi.

Xylanases (EC 3.2.1.8) catalyze the hydrolysis of xylan, the major constituent of hemicellulose. The use of these enzymes could greatly improve the overall economics of processing lignocellulosic materials for the generation of liquid fuels and chemicals. The hyperthermophilic archaeon Pyrodictium abyssi, which was originally isolated from marine hot abyssal sites, grows optimally at 97 degrees C and is a prospective source of highly thermostable xylanase. Its endoxylanase was shown to be highly thermostable (over 100 min at 105 degrees C) and active even at 110 degrees C. The growth of the deep-sea archaeon P. abyssi was investigated using different culture techniques. Among the carbohydrates used, beech wood xylan, birch wood glucuronoxylan and the arabinoxylan from oats pelt appeared to be good inducers for endoxylanase and beta-xylosidase production. The highest production of arabinofuranosidase, however, was detected in the cell extracts after growth on xylose and pyruvate, indicating that the intermediate of the tricarboxylic acid cycle acted as a nonrepressing carbon source for the production of this enzyme. Electron microscopic studies did not show a significant difference in the cell surface (e.g., xylanosomes) when P. abyssi cells were grown on different carbohydrates. The main kinetic parameters of the organism have been determined. The cell yield was shown to be very low owing to incomplete substrate utilization, but a very high maximal specific growth rate was determined (micromax = 0.0195) at 90 degrees C and pH 6.0. We also give information on the problems that arise during the fermentation of this hyperthermophilic archaeon at elevated temperatures.

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).

In vivo observation of cell division of anaerobic hyperthermophiles by using a high-intensity dark-field microscope.

To study growth and cell division of anaerobic hyperthermophilic archaea in vivo, a cultivation technique using glass capillaries was developed. At temperatures of 90 to 98 degrees C, at least 10 successive cell divisions of Pyrodictium abyssi TAG 11 were documented. Cells divide by binary fission. Visualized under a modified dark-field microscope, the formation of cannulae, which finally connected all cells, was observed. The cannulae elongated at 1.0 to 1.5 micrometers/min and reached final lengths of between 30 and 150 micrometers. A "snapping division"-like mode of cell fission was discovered for Thermoproteus tenax.

Purification and properties of an extremely thermostable membrane-bound sulfur-reducing complex from the hyperthermophilic Pyrodictium abyssi.

The chemolithoautotrophic archaeon Pyrodictium abyssi isolate TAG 11 gains energy by reducing sulfur with H2 to H2S. From this hyperthermophile, a sulfur-reducing complex catalyzing this reaction was purified 13.5-fold. The native complex exhibited a brownish-yellow colour and showed an apparent molecular mass of 520 kDa. SDS/PAGE revealed the presence of nine different major polypeptides with apparent molecular masses of 82, 72, 65, 50, 47, 42, 40, 30 and 24 kDa. The native complex contained 50-55 mol acid-labile sulfur, 50-55 mol iron, 1.6 mol nickel, 1.2 mol copper, 2.8 mol cytochrome b and 0.3 mol cytochrome c (all per mol native complex). The temperature optimum of the H2:sulfur oxidoreductase complex was 100 degrees C, which is consistent with the physiological growth optimum of the native organism. The complex is extremely heat stable. During 5 h incubation at 100 degrees C, no decrease in H2S-forming activity could be observed.

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.