Koki Horikoshi

Phylogenetic identification of hypermastigotes, Pseudotrichonympha, Spirotrichonympha, Holomastigotoides, and parabasalian symbionts in the hindgut of termites.

Abstract The phylogenetic diversity of parabasalian flagellates was examined based on the sequences of small subunit ribosomal RNA genes amplified directly from the mixed population of flagellates in the hindgut of lower termites. In total, 33 representative sequences of parabasalids were recovered from eight termite species. Fluorescent-labeled oligonucleotide probes specific for certain sequences were designed and used for the in situ identification of parabasalian species by whole-cell hybridization. The hypermastigotes, Pseudotrichonympha grassii, Spirotrichonympha leidyi, and Holomastigotoides mirabile in the hindgut of Coptotermes formosanus, and Spirotrichonympha sp. and Trichonympha spp. in Hodotermopsis sjoestedti were identified. In the phylogenetic tree constructed, the sequences from the termites were dispersed within the groups of known members of parabasalids, reflecting the presence of diverse parabasalids in the hindgut of termites. There were three paraphyletic lineages of hypermastigotes represented by Pseudotrichonympha, Trichonympha, and Spirotrichonympha, in agreement with the morphology-based taxonomic groups. The analysis of the tree-root suggested that the Pseudotrichonympha group is the most probable ancient lineage of parabasalids and that the Trichonympha group is the secondly deep-branching lineage. The Spirotrichonympha group and the Trichomonadida may have emerged later.

Development of the enzyme reactor system with an amperometric detection and application to estimation of the incipient stage of spoilage of chicken

Abstract An enzyme reactor system with an amperometric detection was developed for the determination of diamines, total concentration of putrescine, cadaverine and spermidine, and applied to the detection of the incipient stage of spoilage of chicken during storage at 5 and 15°C. The system was prepared from the combination of an enzyme reactor and an amperometric electrode for detection of hydrogen peroxide by the enzyme reaction. The reactor was packed with putrescine oxidase immobilized on chitosan porous beads. Response of the system was obtained for the three substrates, putrescine, cadaverine (97% of the response to putrescine) and spermidine (96%). The limit of detection is 0.01xa0mM for the three substrates, the relative standard deviation is 2.5% (n=100) and the response is linear up to 1xa0mM (R2=0.992). One assay could be completed within 3xa0min. The immobilized enzyme was sufficiently stable for at least 8 months at 4°C and usable for at least 300 assays. As the saprophytic microorganisms were reached 107/g of chicken, generally recognized as the indication of the initial stage of putrefaction, the current response increased very rapidly because of the formation of diamines by microorganisms. These results suggest that the proposed reactor system provides a simple, rapid and economical method for the determination of incipient stage of spoilage.

Surface plasmon resonance of electrochemically deposited Au-black

Au-black, a kind of gold nanostructure, was electrochemically deposited on a Au-vapor-deposited glass plate, and its optical properties were evaluated with a surface plasmon resonance (SPR) measurement system. The Au-black was found to exhibit an extraordinary high resonance angle (minimum reflectance angle) compared to that of the Au-vapor-deposited glass plate. We proposed a five-layer model, which consists of glass, Cr, Au, Au-black, and solution layers, to explain the high resonance angle of the Au-black plate. The calculation was based on the Fresnel formulas extended for multilayers. When the Bruggeman formula was used to obtain a refractive index of the Au-black layer, the numerical simulation could qualitatively explain the experimental observations. The curve fitting method by the five-layer model enabled us to simultaneously estimate the thickness of the Au-black layer and the volume fraction of Au in Au-black.

Natronoarchaeum mannanilyticum gen. nov., sp. nov., an aerobic, extremely halophilic archaeon isolated from commercial salt

Strain YSM-123(T) was isolated from commercial salt made from Japanese seawater in Niigata prefecture. Optimal NaCl and Mg(2+) concentrations for growth were 4.0-4.5 M and 5 mM, respectively. The isolate was a mesophilic and slightly alkaliphilic haloarchaeon, whose optimal growth temperature and pH were 37 °C and pH 8.0-9.0. Phylogenetic analysis based on 16S rRNA gene sequence analysis suggested that strain YSM-123(T) is a member of the phylogenetic group defined by the family Halobacteriaceae, but there were low similarities to type strains of other genera of this family (≤90u200a%); for example, Halococcus (similarity <89u200a%), Halostagnicola (<89u200a%), Natronolimnobius (<89u200a%), Halobiforma (<90u200a%), Haloterrigena (<90u200a%), Halovivax (<90u200a%), Natrialba (<90u200a%), Natronobacterium (<90u200a%) and Natronococcus (<90u200a%). The G+C content of the DNA was 63 mol%. Polar lipid analysis revealed the presence of phosphatidylglycerol, phosphatidylglycerophosphate methyl ester, disulfated diglycosyl diether and an unknown glycolipid. On the basis of the data presented, we propose that strain YSM-123(T) should be placed in a new genus and species, Natronoarchaeum mannanilyticum gen. nov., sp. nov. The type strain of Natronoarchaeum mannanilyticum is strain YSM-123(T) (=JCM 16328(T) =CECT 7565(T)).

Cloning and characterization of the gene encoding RNA polymerase sigma factor σ54 of deep-sea piezophilic Shewanella violacea

We have recently reported that a sigma(54)-like factor recognizes a DNA element, designated as region A, upstream of a pressure-regulated operon in piezophilic Shewanella violacea strain DSS12 (Nakasone et al., FEMS Microbiology Lett. 176 (1999) 351-356). In this study, we isolated and characterized the rpoN gene of this piezophilic bacterium. The rpoN gene was found to encode a putative protein consisting of 492 amino acid residues with a predicted molecular mass of 55359 Da. Significant homology was evident comparing the rpoN sequence of S. violacea with that of Escherichia coli (62.8% identity), Vibrio anguillarum (61.7% identity) and Pseudomonas putida (57.0% identity). The DNA-binding domain at the C-terminus of sigma(54) is well conserved in the case of the S. violacea rpoN gene product and the helix-turn-helix motif and the RpoN box are also present. In addition, the conserved glutamine-rich domain is present at the N-terminus. sigma(54) in S. violacea was expressed at a relatively constant level under various growth conditions as determined by both primer extension and Western blotting analyses. By means of a recombinant plasmid, a hexahistidine-tagged derivative of the sigma(54) from strain DSS12 was overexpressed in Escherichia coli and purified to near homogeneity. An electrophoretic mobility shift assay demonstrated that the purified sigma(54) protein specifically recognizes region A in the above-mentioned pressure-regulated operon.

Salarchaeum japonicum gen. nov., sp. nov., an aerobic, extremely halophilic member of the Archaea isolated from commercial salt

Strain YSM-79(T) was isolated from commercial salt made from seawater in Yonaguni island, Okinawa, Japan. The strain is an aerobic, Gram-negative, chemo-organotrophic and extremely halophilic archaeon. Cells are short rods that lyse in distilled water. Growth occurs at 1.5-5.3 M NaCl (optimum 2.5-3.0 M), pH 5.0-8.8 (optimum pH 5.2-6.3) and 20-55 °C (optimum 40 °C). Mg²⁺ is required for growth, with maximum growth at 200-300 mM Mg²⁺. Polar lipid analysis revealed the presence of phosphatidylglycerol, phosphatidylglycerophosphate methyl ester, sulfated diglycosyl diether-1 and five unidentified glycolipids. The G+C content of the DNA was 64 mol%. On the basis of 16S rRNA gene sequence analysis, strain YSM-79(T) was determined to be a member of the family Halobacteriaceae, with the closest related genus being Halobacterium (94u200a% sequence identity). In addition, the rpoB gene sequence of strain YSM-79(T) had <88u200a% sequence similarity to those of other members of the family Halobacteriaceae. The results of phenotypic, chemotaxonomic and phylogenetic analysis suggested that strain YSM-79(T) should be placed in a new genus, Salarchaeum gen. nov., as a representative of Salarchaeum japonicum sp. nov. The type strain is YSM-79(T) (u200a=u200aJCM 16327(T) u200a=u200aCECT 7563(T)).

Ureases of extreme halophiles of the genus Haloarcula with a unique structure of gene cluster.

We searched for urease activities in 71 strains of extreme halophiles by a urea-phenol red-agar plate method. Positive strains were further investigated by measuring the ammonia released from urea in cell-free extracts. Only 4 strains of the genus Haloarcula, Har. aidinensis, Har. hispanica, Har. japonica, and Har. marismortui were finally shown as the urease producers. A partially purified urease from Har. hispanica was a typical halophilic enzyme in that it showed maximum activity at 18–23% NaCl and lost the activity irreversibly in the absence of NaCl. Partial genes (1596 bp) of the urease encoding from upstream of the β subunit down to the N-terminal 139 amino acids of the α subunit, were PCR amplified from the four strains, as well as from five urease-negative Haloarcula strains. Strains of other genera, which were urease-negative, did not yield PCR products. The deduced amino acid sequences of the β subunit and partial α subunit were similar to each other (92–100% similarities) and to those from other organisms. Analysis of the draft genome sequence of Har. marismortui, however, suggested that the order of the genes encoding the three subunits (with the total number of amino acids of 834) and four accessory proteins was β-α-γ-UreG-UreD-UreE-UreF. This order is quite unique, since in other microorganisms the order is γ-β-α-UreE-UreF-UreG-UreD in most cases. No open reading frames were detected in the PCR-amplified upstream of the β subunit, suggesting that all Haloarcula species have the same unique structure of the urease gene cluster.

Production of trehalose by a dual enzyme system of immobilized maltose phosphorylase and trehalose phosphorylase

Abstract Maltose phosphorylase (MP) and trehalose phosphorylase (TP) in the crude extract from a strain of Plesiomonas were adsorbed simultaneously on an anion-exchange resin to examine the continuous production of trehalose from maltose. The activity recoveries of both enzymes after immobilization were about 97% for MP, 33% for TP, and 39% for the trehalose-forming activity (TFA). The immobilized enzyme(s) expressed by TFA was stable at nearly neutral pH and showed about 96% of the original activity after a 1-h incubation at 55°C and pH 7.0 after which the nonimmobilized enzymes completely lost their activities. About 10 m m or more of inorganic phosphate (Pi) was required for TFA of the immobilized enzyme to achieve its full activity, and apparent values of the Michaelis constant (Km) and maximum velocity (Vmax) for Pi were about 0.2 m m and 94.2 μmol trehalose min−1 mg−1 protein at pH 6.2 and 50°C, respectively. The maximum yield of trehalose from maltose after removing small amounts of glucose-1-phosphate (G-1-P) which was formed concomitantly during the reaction was about 60%. The optimum flow rate achieving the maximum productivity of trehalose was about 1.0 h−1 of the space velocity (SV) at 20% ( w v ) maltose concentration at which the productivity was 252 mg trehalose h−1 g−1. Half-lives of the immobilized enzyme in continuous operation at 0.2 h−1 of SV and pH 6.2 in the presence of 10 m m Pi with 30% ( w v ) corn syrup containing about 84% as dry basis of maltose as a substrate were calculated at about 164 days at 55°C, 28 days at 60°C, and 8 days at 65°C, respectively. Using the method reported here, the mother liquor for preparing crystalline trehalose from maltose can be produced conveniently on an industrial scale.

Organic Solvent Tolerance of Halophilic Archaea

Organic solvent tolerance was tested in type strains of type species of the sixteen genera of Halobacteriaceae, the halophilic archaea. Most of the strains were observed to grow in the presence of hexylether (log P ow=5.1), but none grew in the presence of n-octane (log P ow=4.9) except Halogeometricum borinquense JCM 10706T and Halorubrum saccharovorum JCM 8865T. On the other hand, two strains, Haloarcula spp. OHF-1 and 2 isolated from a French solar salt were found to show stronger tolerance even to isooctane (log P ow=4.8). Growth of some strains was retarded by the presence of n-decane but reached to the same cell densities at late stationary phase. Final cell densities of some strains were greatly repressed by the presence of the solvent.

Superoxide dismutase is involved in high tolerance to copper in the deep-sea yeast, Cryptococcus sp. N6

The activity and expression of superoxide dismutase (SOD) was analyzed in a copper-tolerant yeast, Cryptococcus sp. N6. Using cell extracts, two distinct bands exhibiting SOD activity appeared on native PAGE: one band, with higher mobility, appeared when the cells were grown without CuSO4, and the other band appeared when the cells were grown with 10 mM CuSO4. Cells grown with 3 mM CuSO4 produced both SOD isoforms. Western blot analysis, using a monoclonal antibody against human SOD-1, showed that SOD protein was expressed in the absence of CuSO4 and that the expression level increased when the cells were grown with 3 or 10 mM CuSO4. The molecular weight of SOD from strain N6 was approx. 18 kDa. Treatment of the cells with the protein synthesis inhibitor, cycloheximide at 0.5 μg ml−1, did not affect cell growth in the absence of CuSO4 but significantly inhibited growth in the presence of 10 mM CuSO4 and inhibited expression of SOD protein. This suggests that SOD may play a role in cell growth in the presence of high concentrations of CuSO4.