An extremely thermophilic archaeon, strain AL662T, was isolated from a deep-sea hydrothermal vent located on the East Pacific Rise at a latitude of 21"N. This strain is a strictly anaerobic coccus, and its cells range from 0.8 to 2 pm in diameter. The optimum temperature, pH, and Sea Salt concentration for growth are 85"C, 6, and 20 to 40 g/liter, respectively. Strain AL662T grows preferentially on proteolysis products, on a mixture of 20 amino acids, and on maltose in the presence of elemental sulfur. The membrane lipids consist of di-and tetraether glycerol lipids. The DNA G+C content is 58 mol%. Sequencing of the 16s rRNA gene showed that strain AL662T belongs to the genus Thermococcus. On the basis of hybridization results, we propose that this strain should be placed in a new species, Thermococcus hydrothermalis.Since the discovery of hyperthermophilic archaea, strains belonging to the order Thermococcales have been isolated from both terrestrial and marine hot environments and described. Species belonging to the genus Thermococcus have been isolated from various deep-sea and shallow marine hydrothermal areas and described. All of the strains have quite similar physiological characteristics and can be divided into the following two groups on the basis of their G + C contents: (i) a group of strains with high G + C contents (50 to 58 mol%), including strains belonging to two species from shallow marine environments, Therrnococcus celer (34) and Thermococcus stetteri (20), and three species from deep-sea environments, Thermococcus profundus (14), "Thermococcus peptonophilus" (S), and Thermococcusfumicolans (7); and (ii) a group of strains with low G+C contents (38 to 47 mol%), including strains belonging to two species from deep-sea environments, "Thermococcus chitonophagus" (1 1) and "Thermococcus alcaliphilus" (13), and an organism from a shallow marine environment, "Thermococcus litoralis" (22). All of these species are able to use proteinaceous carbon substrates in the absence or presence of sulfur. Some of these strains are able to grow in the absence of elemental sulfur, but sulfur greatly enhances growth. In this paper, we describe a new hyperthermophilic heterotrophic archaeal species that was isolated from a deep-sea hydrothermal vent on the East Pacific Rise at a latitude of 21"N. This organism was selected on the basis of the results of a preliminary screening for alcohol dehydrogenase (ADH) activity in 71 hyperthermophilic heterotrophic strains that were isolated at 80 and 95°C from various deep-sea hydrothermal vent areas (15). MATERIALS AND METHODSReference strains. Thermococcus celer DSM 2476', "Thermococcus litoralis" DSM 5474T, Thermucoccus stetteri DSM 5262.", Sulfolobus acidocaldarius DSM 639", and Thermotugu maritima DSM 310gT were obtained from the Deutsche Sammlung von Mikroorganismen, Braunschweig-Stockeim, Germany. Other strains were directly obtained from researchers. DT5432T was provided by Tetsuo Kobayashi (RIKEN, Wako, Japan). Thermococcus fumicolans CIP104680~" was isolated in our...

A new thermophilic, anaerobic rod-shaped bacterium, strain B1429T was isolated from the gills of a deep-sea vent hydrothermal mussel, Bathymodiolus brevior, from the Lau Basin (Southwestern Pacific Ocean). Phenotypically, this isolate exhibited characteristics similar to those described for members of the order Thermotogales. This organism was identified as a member of the genus Thermusipho on the basis of the presence of the typical outer sheath-like structure (toga), its 16s rRNA sequence, and its ability to grow on carbohydrates (sucrose, starch, glucose, maltose, lactose, cellobiose, and galactose). The cells of this organism were gram negative and rod shaped and generally occurred singly or in pairs, rarely occurring as chains with a maximum of five rods. At the optimum temperature for growth (70°C), optimum pH (6.5), and optimum salinity (30 g of NaCl per liter), the doubling time was 100 min. In spite of the high percentage of similarity of its 16s rRNA sequence with that of Thermosipho africanus (98.6%), the weak level of DNA-DNA reassociation with this strain (2%) and particular physiological characteristics allowed us to differentiate this new organism from the sole species of the genus Thermosipho previously described (7'. aficanus). On the basis of these observations, we propose that the new organism should be described as a new species, Thermosipho melanesiensis. The type strain of T. melanesiensis is BI429.Members of the order Thermotogales thrive within extreme geological environments. They occur in shallow marine hydrothermal systems as well as in continental springs and in petroleum reservoirs. Up to now the Thermotogales have been represented by five genera. The genus Thermotoga is represented by the species Thermotoga maritima (ll), found so far only within marine coastal hydrothermal systems; Thermotoga neapolitana (15, 37), isolated from both marine and low-salt continental hot springs; Thennotoga thermarum (37), obtained exclusively from continental hot springs; and Thermotoga subterranea (16) and Thermotoga elfii (26), isolated from continental oil reservoirs. The genus Thermosipho is represented only by the species Thermosipho aficanus (12, 28), isolated from a marine coastal hydrothermal system in Djibouti. The genus Fervidobacterium is represented by the species F. nodosum (22), isolated from a hot spring in New Zealand; F. islandicum (13), isolated from an Icelandic hot spring; and F. gondwanense, isolated from an artesian basin in Australia (1). The two last genera, Geotoga and Petrotoga, with the species G. petraea, G. subterranea, and P. miotherma (4), were isolated from brines collected from oil fields in Oklahoma and Texas.In this work, we describe the isolation and characterization of a new strain of thermophilic bacterium isolated from the gills of a deep-sea vent hydrothermal mussel and present evidence for its phylogenetic position within the Thermotogales. Based on phenotypical and phylogenetical analysis, this new strain can be described as a new species of Thermosipho that ...

A NADP-dependent group III alcohol dehydrogenase (ADH) was purified from the hyperthermophilic strictly anaerobic archaeon Thermococcus hydrothermalis, which grows at an optimum temperature of 85 8C and an optimum pH of 6. The gene encoding this enzyme was cloned, sequenced, and over-expressed in Escherichia coli. The recombinant enzyme was purified, characterized and compared with the native form of the enzyme. The enzyme structure is pH-dependent, being a 197-kDa tetramer (subunit of 45 kDa) at pH 10.5, the pH optimum for alcohol oxidation, and a 80.5-kDa dimer at pH 7.5, the pH optimum for aldehyde reduction. The kinetic parameters of the enzyme show that the affinity of the enzyme is greater for the aldehyde substrate and NADPH cofactor, suggesting that the dimeric form of the enzyme is probably the active form in vivo. The ADH of T. hydrothermalis oxidizes a series of primary aliphatic and aromatic alcohols preferentially from C 2 to C 8 but is also active towards methanol and glycerol and stereospecific for monoterpenes. T. hydrothermalis ADH is the first Thermococcale ADH to be cloned and overproduced in a mesophilic heterologous expression system, and the recombinant and the native forms have identical main characteristics.Keywords: alcohol dehydrogenase; NADP-dependent; over-expression; pH-dependent structure; thermophilic.Alcohol dehydrogenases (ADH) are present in all organisms. The NAD-or NADP-dependent dehydrogenases can be divided into three major groups [19]. Only the N-terminal amino acid sequences are available for these latter two ADHs.The genes encoding five archaeal ADHs have been identified: the genes for the two closely related zinc-containing ADHs from Sulfolobus spp. have recently been cloned and over-expressed in E. coli [12], the gene encoding the ironcontaining ADH from T. zilligii [16], and the genes for the two ADHs from P. furiosus [14] have also been cloned and overexpressed in E. coli.An ADH activity was found [20] in crude extracts of the hyperthermophilic archaeon T. hydrothermalis [21] growing at an optimal temperature of 85 8C as part of a search for new ADHs with different catalytic properties involved in bioconversion. This report describes the purification of the native enzyme, the design of an oligonucleotidic probe corresponding to the N-terminal amino acid sequence of the protein, and the cloning of its gene. The recombinant enzyme was overexpressed in E. coli to compare the main characteristics of the native and recombinant enzymes and to define the substrate specificity, regulatory properties and thermostability of the recombinant enzyme. M A T E R I A L S A N D M E T H O D SOrganisms, plasmids and chemicals T. hydrothermalis [21] is a hyperthermophilic archaeon isolated from a chimney wall sample collected on the East Pacific Rise at latitude 218N. This strain is a strictly anaerobic coccus, that grows at an optimum temperature of 85 8C, a pH of 6.0 and a sea salt concentration of 20±40 g´L 21. Elemental sulfur greatly enhances the growth of T. hydrothermalis, but ca...

We report on a probe modification of a quartz crystal microbalance (QCM) DNA-biosensor that permits to reversibly change the DNA sequence detected. A QCM DNA-biosensor was designed by immobilization of a 20-base DNA-disulfide probe on the gold-covered quartz surface of a 27 MHz microbalance (9 MHz, third overtone). After immobilization on the gold covered quartz surface, this probe was modified by hybridization with a 45-base DNA that includes the complementary 20-base sequence, a 5-base spacer and a non-complementary 20-base sequence. The non-complementary sequence constitutes a new probe, different from the DNA-disulfide probe, that permits the detection of a new DNA target. As this 45 bases DNA is changeable by dehybridization, successive different DNA targets can be detected. Kinetics and thermodynamic studies of the DNA-disulfide and modified biosensors indicate that the modified biosensor is as sensitive, selective, fast, renewable and reproducible as the DNA-disulfide biosensor, but with a higher hybridization ratio. This modification method offers wider investigation field and practical and economic advantages to DNA-biosensors based on irreversible immobilization of DNA probes on solid substrate.

We report the immobilization on a gold surface of a 20-base DNA probe labeled with disulfide group and on the selective hybridization with the complementary 20-base DNA strand. The oligonucleotide probe is the complementary strand of a partial sequence of the gene encoding for a large ribosomal RNA sub-unit which is a coding sequence of Alexandrium minutum DNA, a microalgae that produces neurotoxins responsible for paralytic shellfish poisoning on European and Asian coasts. The kinetics of DNA probe immobilization and hybridization were monitored in situ by using a 27 MHz quartz crystal microbalance under controlled hydrodynamic conditions. The frequency of the setup is stable to within a few hertz, corresponding to the nanogram scale, for 3h and makes it possible to follow frequency change from immobilization of the probe to hybridization of the complementary DNA target. This setup constitutes a biosensor, which is sensitive and selective, and the hybridization ratio between hybridized complementary DNA and immobilized DNA probes is 47%.