Bacterial community analysis of Indonesian hot springs

We report the first attempts to describe thermophilic bacterial communities in Indonesia's thermal springs using molecular phylogenetic analyses. 16S rRNA genes from laboratory cultures and DNA directly amplified from three hot springs in West Java were sequenced. The 22 sequences obtained were assignable to the taxa Proteobacteria, Bacillus and Flavobacterium, including a number of clades not normally associated with thermophily.     

Recovery of ionizing-radiation damage after high doses of gamma ray in the hyperthermophilic archaeon Thermococcus gammatolerans

The recently discovered hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans is of great interest to compare and contrast the impact of its physiology on radioresistance and its ability to repair damaged chromosomes after exposure to gamma irradiation with radioresistant bacteria. We showed that, in contrast to other organisms, cell survival was not modified by the cellular growth phase under optimal growth conditions but nutrient-limited conditions did affect the T. gammatolerans radioresistance. We determined the first kinetics of damaged DNA recovery in an archaeon after exposure to massive doses of gamma irradiation and compared the efficiency of chromosomal DNA repair according to the cellular growth phase, nutrient availability and culture conditions. Chromosomal DNA repair kinetics showed that stationary phase cells reconstitute disrupted chromosomes more rapidly than exponential phase cells. Our data also revealed that this radioresistant archaeon was proficient to reconstitute shattered chromosomes either slowly or rapidly without any loss of viability. These results suggest that rapid DNA repair is not required for the extreme radioresistance of T. gammatolerans. Angels Tapias and Christophe Leplat contributed equally to this work.     

Hyper-thermostability of CutA1 protein, with a denaturation temperature of nearly 150 degrees C

We found that the CutA1 protein, from Pyrococcus horikoshii (PhCutA1), has an extremely high denaturation temperature (T(d)) of nearly 150 degrees C, which exceeds the highest record determined by DSC by about 30 degrees C. To elucidate the mechanism of the ultra-high stability of PhCutA1, we analyzed the crystal structures of CutA1 proteins from three different sources, P. horikoshii, Thermus thermophilus, and Escherichia coli, with different growth temperatures (98, 75, and 37 degrees C). This analysis revealed that the remarkably increased number of ion pairs in the monomeric structure contributes to the stabilization of the trimeric structure and plays an important role in enhancing the T(d), up to 150 degrees C, for PhCutA1.     

The hyperthermophilic bacterium Thermotoga neapolitana possesses two isozymes of the 3-phosphoglycerate kinase/triosephosphate isomerase fusion protein

Abstract The phosphoglycerate kinase ( pgk ), triosephosphate isomerase ( tpi ), and enolase ( eno ) genes from Thermotoga neapolitana have been cloned and expressed in Escherichia coli . In high copy number, the pgk gene complemented an E. coli pgk ⁻ strain. In T. neapolitana , the pgk and tpi genes appear to be fused and eno is near those genes. Like T. maritima , T. neapolitana produces phosphoglycerate kinase as both an individual enzyme and a fusion protein with triosephosphate isomerase, and triosephosphate isomerase activity is not found without associated phosphoglycerate kinase activity. Unlike T. maritima , which forms only a 70-kDa fusion protein, T. neapolitana expresses both 73-kDa and 81-kDa isozymes of this fusion protein. These isozymes are present in both T. neapolitana cells and in E. coli cells expressing T. neapolitana genes.     

An Uncharacterized Member of the Ribokinase Family in Thermococcus kodakarensis Exhibits myo-Inositol Kinase Activity

Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high K_m values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater k_cat and much lower K_m value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in k_cat/K_m values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate.     

Biochemical and phylogenetic characterization of a novel terrestrial hyperthermophilic archaeon pertaining to the genus <Emphasis Type="Italic">Pyrococcus</Emphasis> from an Algerian hydrothermal hot spring

A hyperthermophilic anaerobic archeon, strain HT3, was isolated from hydrothermal hot spring in Northeast Algeria. The strain is a regular coccus, highly motile, obligatory anaerobic, heterotrophic. It utilizes proteinaceous complex media (peptone, tryptone or yeast extract). Sulfur is reduced to Hydrogen sulfide and enhances growth. It shares with other Pyrococcus species the heterotrophic mode of nutrition, the hyperthermophily, the ability to utilize amino acids as sole carbon and nitrogen sources and the ether lipid composition. The optimal growth occurs at 80–85°C, pH 7.5 and 1.5% NaCl. The G + C content was 43 mol%. Considering its morphology, physiological properties, nutritional features and phylogenetic analyses based on 16S rRNA gene sequencing, this strain is described as a new terrestrial isolate pertaining to the genus Pyrococcus.     

DNA Repair Systems in Archaea: Mementos from the Last Universal Common Ancestor?

DNA repair in the Archaea is relevant to the consideration of genome maintenance and replication fidelity in the last universal common ancestor (LUCA) from two perspectives. First, these prokaryotes embody a mix of bacterial and eukaryal molecular features. Second, DNA repair proteins would have been essential in LUCA to maintain genome integrity, regardless of the environmental temperature. Yet we know very little of the basic molecular mechanisms of DNA damage and repair in the Archaea in general. Many studies on DNA repair in archaea have been conducted with hyperthermophiles because of the additional stress imposed on their macromolecules by high temperatures. In addition, of the six complete archaeal genome sequences published so far, five are thermophilic archaea. We have recently shown that the hyperthermophile Pyrococcus furiosus has an extraordinarily high capacity for repair of radiation-induced double-strand breaks and we have identified and sequenced several genes involved in DNA repair in P. furiosus. At the sequence level, only a few genes share homology with known bacterial repair genes. For instance, our phylogenetic analysis indicates that archaeal recombinases occur in two paralogous gene families, one of which is very deeply branched, and both recombinases are more closely related to the eukaryotic RAD51 and Dmc1 gene families than to the Escherichia coli recA gene. We have also identified a gene encoding a repair endo/exonuclease in the genomes of several Archaea. The archaeal sequences are highly homologous to those of the eukaryotic Rad2 family and they cluster with genes of the FEN-1 subfamily, which are known to be involved in DNA replication and repair in eukaryotes. We argue that there is a commonality of mechanisms and protein sequences, shared between prokaryotes and eukaryotes for several modes of DNA repair, reflecting diversification from a minimal set of genes thought to represent the genome of the LUCA.     

Membrane-associated redox activities in Thermotoga neapolitana

Elemental sulfur reduction by the hyperthermophilic bacterium Thermotoga neapolitana provides an alternative to hydrogen evolution during fermentation. Electrons are transferred from reduced cofactors (ferredoxin and NADH) to sulfur by a series of unknown steps. One enzyme that may be involved is an NADH:methyl viologen oxidoreductase (NMOR), an activity that in other fermenting organisms is associated with NADH:ferredoxin oxidoreductase. We found that 83% of NMOR activity was contained in the pellet fraction of cell extracts subjected to ultracentrifugation. This pellet fraction, presumably containing cell membranes, was required for electron transfer to NAD⁺ from ferredoxin-dependent pyruvate oxidation. However, the NMOR activity in this fraction used neither Thermotoga nor clostridial ferredoxins as substrates. NMOR activity was also detected in aerobically prepared vesicles. By comparison with ATPase activities, NMOR was found primarily on the cytoplasmic face of these vesicles. During these studies, an extracytoplasmic hydrogenase activity was discovered. In contrast to the soluble hydrogenase, this hydrogenase activity was completely inhibited when intact cells were treated with cupric chloride and was present on the extracytoplasmic face of vescides. In contrast to a soluble hydrogenase reported in Thermotoga maritima, this activity was air-stable and was inhibited by low concentrations of nitrite. Received: 28 May 1998 / Accepted: 23 June 1998     

A stress protein is induced in the deep-sea barophilic hyperthermophile Thermococcus barophilus when grown under atmospheric pressure

The whole-cell protein inventory of the deep-sea barophilic hyperthermophile Thermococcus barophilus was examined by one-dimensional SDS gradient gel electrophoresis when grown under different pressure conditions at 85°C (T _opt). One protein (P60) with a molecular mass of approximately 60 kDa was prominent at low pressures (0.3 MPa hydrostatic pressure and 0.1 MPa atmospheric pressure) but not at deep-sea pressures (10, 30, and 40 MPa). About 17 amino acids were sequenced from the N-terminal end of the protein. Sequence homology analysis in the GenBank database showed that P60 most closely resembled heat-shock proteins in some sulfur-metabolizing Archaea. A high degree of amino acid identity (81%–93%) to thermosome subunits in Thermococcales strains was found. Another protein (P35) with molecular mass of approximately 35.5 kDa was induced at 40 MPa hydrostatic pressure but not under low-pressure conditions. No amino acid sequence homology was found for this protein when the 40 amino acids from the N-terminal end were compared with homologous regions of proteins from databases. A PTk diagram was generated for T. barophilus. The results suggest that P _habitat is about 35 MPa, which corresponds to the in situ pressure where the strain was obtained. Received: May 14, 1999 / Accepted: July 30, 1999