Aritsune Uchida

Aeropyrum pernix gen. nov., sp. nov., a novel aerobic hyperthermophilic Archaeon growing at temperatures up to 100°C

A novel aerobic hyperthermophilic archaeon was isolated from a coastal solfataric vent at Kodakara-Jima Island, Japan. The new isolate, strain K1, is the first strictly aerobic organism growing at temperatures up to 100°C. It grows optimally at 90 to 95°C, pH 7.0, and a salinity of 3.5%. The cells are spherical shaped and 0.8 to 1.2 μm in diameter. Various proteinaceous complex compounds served as substrates during aerobic growth. Thiosulfate stimulates growth without producing H2S. The core lipids consist solely of C25-isopranyl archaeol(glycerol diether). The G+C content of the genomic DNA is 67 mol%. Phylogenetic analysis based on 16S rRNA sequence indicates that strain K1 is a new member of Crenarchaeota. On the basis of our results, the name Aeropyrum pernix gen. nov., sp. nov. is proposed (type strain: K1; JCM 9820).

Rhodothermus obamensis sp. nov., a Modern Lineage of Extremely Thermophilic Marine Bacteria

A novel extremely thermophilic bacterium was isolated from a shallow marine hydrothermal vent environment (depth, 22 m) in Tachibana Bay, Nagasaki Prefecture, Japan. The cells of this organism were gram-negative rods. Growth occurred at temperatures between 50 and 85 degrees C (optimum temperature, 80 degrees C; doubling time at optimum temperature, 90 min), at pH 5.5 and 9.0 (optimum pH, 7.0), and in the presence of 1 and 5% NaCl (optimum NaCl concentration, 3%). The new isolate was an aerobic heterotroph which utilized the following compounds as sole energy and carbon sources: yeast extract, peptone, starch, casein, Casamino Acids, a variety of sugars, some carboxylic acids, and amino acids. As determined by a sequence analysis of the 16S rRNA, the new isolate belongs to the genus Rhodothermus and represents a modern lineage of extreme thermophiles within the domain Bacteria. On the basis of the physiological and molecular properties of the new isolate, we describe a new species, Rhodothermus obamensis. The type strain of R. obamensis is strain OKD7 (= JCM 9785).

PURIFICATION AND CHARACTERIZATION OF A SULFOTRANSFERASE SPECIFIC TO N-21 OF SAXITOXIN AND GONYAUTOXIN 2+3 FROM THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM (DINOPHYCEAE)

A sulfotransferase (ST) specific to N‐21 of saxitoxin (STX) and gonyautoxin 2+3 (GTX2+3) designated as N‐ST was purified to homogeneity from the cytosolic fraction of clonal‐axenic vegetative cells of the toxic dinoflagellate Gymnodinium catenatum Graham GC21V, which causes paralytic shellfish poisoning. The enzyme transferred a sulfate group from 3′‐phosphoadenosine 5′‐phosphosulfate (PAPS) to N‐21 in the carbamoyl group of STX and GTX2+3 to produce GTX5 and C1+2, respectively. The molecular mass of the purified enzyme was determined by SDS‐PAGE to be 59 kDa. Gel filtration chromatography showed a native molecular mass of 65 kDa, indicating that the N‐ST is a monomeric enzyme. The N‐ST was specific to only N‐21 of STX and GTX2+3, and O‐22 sulfation was not observed. Moreover, the N‐ST was not active toward neo STX and GTX1+4, which differed from STX and GTX2+3, respectively, in only N‐1 hydroxylation. When various compounds previously reported to be substrates for STs in other organisms and paralytic shellfish poisoning toxins other than STX and GTX2+3 were added to the reaction mixture, N‐ST activity was not decreased. The enzyme required PAPS as the sole source of sulfate. The enzyme was optimally active at pH 6.0 and 25° C, and its activity was enhanced by Mg2+ and Co2+. The Km values of the N‐ST for STX and GTX2+3 were 16.1 μM and 29.8 μM, respectively.

FLUORESCENCE IN SITU HYBRIDIZATION USING rRNA-TARGETED PROBES FOR SIMPLE AND RAPID IDENTIFICATION OF THE TOXIC DINOFLAGELLATES ALEXANDRIUM TAMARENSE AND ALEXANDRIUM CATENELLA1

The toxic marine dinoflagellates Alexandrium tamarense (Lebor) Balech and A. catenella (Whedon and Kofoid) Taylor have been mainly responsible for paralytic shellfish poisoning in Japan. Rapid and precise identification of these algae has been difficult because this genus contains many morphologically similar toxic and nontoxic species. Here, we report a rapid, precise, and quantitative identification method using three fluorescent, rRNA‐targeted, oligonucleotide probes for A. tamarense (Atm1), A. catenella (Act1), and the nontoxic A. affine (Inoue et Fukuyo; Aaf1). Each probe was species specific when applied using fluorescence in situ hybridization (FISH). None of the probes reacted with three other Alexandrium spp., A. lusitanicum Balech, A. ostenfeldii (Paulsen) Balech & Tangen, and A. insuetum Balech, or with eight other microalgae, including Gymnodinium mikimotoi Miyake et Kominami ex Oda and Heterosigma akashiwo (Hada) Hara et Chihara, suggesting that the species specificity of each probe was very high. Cells labeled with fluorescein 5‐isothiocyanate–conjugated probes showed strong green fluorescence throughout the whole cell except for the nucleus. FISH could be completed within 1 h and largely eliminated the need for identifying species based on key morphological criteria. More than 80% of targeted cells of both species could be identified by microscopy and quantified during growth up to the early stationary phase; more than 70% of cells could be detected in the late stationary phase. The established FISH protocol was found to be a specific, rapid, precise, and quantitative method that might prove to be a useful tool to distinguish and quantify Alexandrium cells collected from Japanese coastal waters.

Pyrobaculum oguniense sp. nov., a novel facultatively aerobic and hyperthermophilic archaeon growing at up to 97 degrees C.

A novel hyperthermophilic, heterotrophic, rod-shaped archaeon was isolated from a terrestrial hot spring at Oguni-cho, Kumamoto Prefecture, Japan. The new isolate, strain TE7T, grew under aerobic, microaerobic and anaerobic conditions. Isolate TE7T grew optimally at 90-94 degrees C and pH 7.0-7.5 (adjusted at 25 degrees C) under atmospheric air with vigorous shaking. Strain TE7T cells were motile rods 2-10 microm in length and covered with a surface-layer lattice. Cell yields at 90 degrees C under aerobic conditions were twice that under anaerobic conditions. Under aerobic conditions, growth was inhibited by elemental sulfur, but thiosulfate stimulated growth. Under anaerobic conditions, no growth was observed in the presence of nitrate and nitrite, but elemental sulfur, thiosulfate, L-cystine and oxidized glutathione stimulated growth. The 16S rDNA sequence of TE7T exhibited a close relationship to the sequences of Pyrobaculum aerophilum and Thermoproteus neutrophilus, which belong to the cluster of the genus Pyrobaculum. DNA-DNA hybridization analysis showed a low level of DNA similarity between TE7T and previously described Pyrobaculum species. As TE7T is phenotypically and phylogenetically different from the other members of this genus, it is described as a new species named Pyrobaculum oguniense (type strain TE7T = JCM 10595T = DSM 13380T).

Molecular cloning and nucleotide sequence analysis of psbA from the dinoflagellates: Origin of the dinoflagellate plastid

Cloning and sequencing of psbA, the gene encoding D1 protein of photosystem II, from six species of dinoflagellates harboring a peridinin type plastid [Prorocentrum micans Ehrenberg, Amphidinium carterae Hulburt, Heterocapsa triquetra Stein, Lingulodinium polyedra (Dodge) Stein, Alexandrium tamarense (Lebour) Balech and Alexandrium catenella (Whedon et Kofoid) Balech] is reported. Using the polymerase chain reaction technique, the psbA gene was detected in a satellite DNA band isolated from total DNA of A. catenella by CsCl‐Hoechst 33258 gradient ultracentrifugation. This finding suggests that in dinoflagellates psbA is encoded in the plastid genome. The deduced amino acid sequences of D1 from the dinoflagellates did not reveal a typical ‘C‐terminus extension’, which should be removed by proteolytic cleavage from the D1 precursor. Molecular phylogenetic analysis based on the deduced amino acid sequences of D1 revealed that the six species of dinoflagellates are monophyletic and also showed that dinoflagellates cluster with rhodophytes, a cryptophyte and heterokonts. These results support the hypothesis that the peridinin type plastid in dinoflagellates originated from an engulfed red alga.

Preliminary phylogenetic analysis of plastid‐encoded genes from an anomalously pigmented dinoflagellate Gymnodinium mikimotoi (Gymnodiniales, Dinophyta)

We cloned and sequenced three plastid‐encoded genes, psbA (encoding D1 protein), psaA (encoding P700 chlorophyll a apoprotein) and the small‐subunit ribo‐somal RNA (pl‐SSU rRNA) from an anomalously pigmented dinoflagellate, Gymnodinium mikimotoi Miyake et Kominami ex Oda, with a plastid containing 19′‐hexanoyloxyfucoxanthin, 19′‐butanoyloxyfucoxanthin and fucoxanthin instead of peridinin as the major carot‐enoids. Molecular phylogenetic trees based on the deduced amino acid sequences of D1 and P700 chlorophyll a apoprotein and nucleotide sequence of pl‐SSU rRNA were then constructed separately. In the D1 tree, G. mikimotoi and typically pigmented dinofl age Nates harboring a peridinin type plastid were monophyletic and G. mikimotoi was positioned most basally within the dinoflagellate lineage. The dinoflagellate lineage was the sister group of heterokonts and the dinoflagellates/heterokonts lineage was clustered with the rhodophytes/cryptophyte lineage. In the P700 chlorophyll a apoprotein phylogenetic tree, G. mikimotoi was clustered with a rhodo‐phyte, a cryptophyte and a heterokont. In the pl‐SSU rRNA tree, G. mikimotoi and haptophytes constituted a monophyletic group associated with rhodophytes and heterokonts. These results, derived from the three phylogenetic analyses, support the hypothesis that the plastid of G. mikimotoi belongs to the rhodoplast lineage. Although we have previously demonstrated that D1 from peridinin type dinofl age Nates lacks a ‘C‐terminus extension’ (which should be removed by proteolytic cleavage from the D1 precursor), the D1 from G. mikimotoi revealed a C‐terminus extension that is different from those of other photosynthetic organisms with respect to the length of the amino acid residues.

Geographic differences in paralytic shellfish poisoning toxin profiles among Japanese populations of Alexandrium tamarense and A. catenella (Dinophyceae)

To reconsider whether toxin profile could be used as a marker for populations from different geographical areas, clonal isolates of the toxic dinoflagellates Alexandrium tamarense (Lebour) Balech and Alexandrium catenella (Whedon et Kofoid) Balech from Ofunato Bay (Iwate Prefecture), Atsumi Bay (Aichi Prefecture), Tanabe Bay (Wakayama Prefecture), Harima‐Nada (Kagawa Prefecture), Uranouchi Bay (Kochi Prefecture), Hiroshima Bay (Hiroshima Prefecture) and Yamakawa Bay (Kagoshima Prefecture), which were identified on the basis of morphotaxonomy, immunological and molecular biological techniques, were subjected to analysis of paralytic shellfish poisoning toxins by high performance liquid chromatography‐fluorometric method. All the isolates except A. tamarense OF152 from Ofunato Bay contained mainly N‐sulfocarbamoyl toxins (C1 +2) with various amounts of derivatives, and a typical north‐to‐south trend of decreasing toxicity was observed. In both A. tamarense and A. catenella, toxin profiles were rather constant within a geographical area and divergent among different geographical areas. The toxin profiles of A. tamarense from Harima‐Nada were well conserved among different bloom years. Toxin profile showed that isolates of A. tamarense from Ofunato Bay, A. tamarense from Harima‐Nada isolated in 1988 and A. catenella from Uranouchi Bay were heterogeneous. However, only two or three groups of isolates with different toxin profiles were observed in a geographical region, suggesting that several representative isolates express the genotype in a given region. These observations confirmed that toxin composition could be used as a marker to discriminate different geographical populations of these species.

A NEW METHOD FOR IDENTIFICATION OF INTER‐AND INTRA‐SPECIES OF THE RED TIDE ALGAE CHATTONELLA ANTIQUA AND CHATTONELLA MARINA (RAPHIDOPHYCEAE) BY MEANS OF MONOCLONAL ANTIBODIES

Four monoclonal antibodies AT‐83, AT‐86, MR‐18 and MR‐21 were obtained from culture supernatants of hybridomas which had been established by cell fusion between myeloma and spleen cells of mice immunized with the raphidophycean algae Chattonella antiqua (H) O NIES‐83 and 86 and Chattonella marina (S) H et C NIES‐118 and 121, respectively. Antibody AT‐86 was reactive with all ten strains of Chattonella antiqua tested but not with those of Chattonella marina. On the other hand, MR‐18 was reactive with those of Chattonella marina but not with those of Chattonella antiqua. Species can thus be identified using these two antibodies. Antibody MR‐21 was reactive with some of the strains of Chattonella antiqua and all six strains of Chattonella antiqua and all six strains of Chattonella marina. AT‐83 was reactive with some strains of Chattonella antiqua By the selective reactivities of these four monoclonal antibodies, strains of the two species could be separated into two groups. AT‐83 could distinguish between Chattonella marina isolated from Kagoshima prefecture (reactive) and the Seto Inland Sea region (non‐reactive). Reactivities of MR‐21 suggested that at least two groups of Chattonella antiqua caused red tides in Harima‐Nada during 1977 and 1978. Monoclonal antibodies appear to be useful for the identification of inter‐species and grouping of intra‐species of these organisms.

Purification and characterization of phosphoenolpyruvate carboxylase from the hyperthermophilic archaeon Methanothermus sociabilis

Phosphoenolpyruvate carboxylase (PEPC) was purified for the first time from hyperthermophilic archaeon Methanothermus sociabilis, growing autotrophically with an optimum at 88°C. The optimum temperature for enzyme activity was similar to that for growth and was 85°C. The native enzyme was a homotetramer of 240 kDa molecular mass and the subunit displayed an apparent molecular mass of 60 kDa. The archaeal PEPC was insensitive to various metabolites which are known as allosteric effectors for most bacterial and eucaryal counterparts. The enzyme showed extreme thermostability such that there remained 80% of the enzyme activity after incubation for 2 h at 80°C. These results implied that archaeal PEPC was significantly different from bacterial and eucaryal entities.