The psychrophilic, moderately piezophilic bacterium Shewanella violacea strain DSS12 is a deep-sea isolate from a sediment sample collected at the Ryukyu Trench (depth: 5,110 m), which grows optimally at 30 MPa and 8°C but also grows at atmospheric pressure (0.1 MPa) and 8°C. We have examined this strain to elucidate the molecular basis for gene and protein regulations at different pressure conditions because this strain is useful as a model bacterium for comparing the various features of bacterial physiology under pressure conditions. Proteins, from such deep-sea adapted piezophiles, could be active under high-pressure conditions in general. Actually atmospheric pressure adapted proteins can be inactive under higher-pressure conditions. For bio-processing under pressure, people are looking for pressure-tolerant enzymes, thus, “piezophilic proteins” would be focus on such industrial applications. In the case of respiratory proteins, cell divisional protein FtsZ, RNA polymerase subunit structure, and dihydrofolate reductase (DHFR), piezophilic proteins were unique for adaptation to high-pressure environment and some of them were more stable and active under higher pressure conditions.

The method of electrophoretic mobility shift assay under high-pressure conditions was improved using a high-pressure electrophoresis apparatus with capillary narrow-tube gel. It was found that the protein–DNA complex in the gel was stained as a high-resolution spot with ethidium bromide. Using this method, it was found that the behavior under high-pressure conditions of the protein–DNA complex composed of NtrC protein and its target promoter DNA is important for the pressure-regulated transcription process, and it was confirmed that the complex was dissociated above a pressure of 70 MPa.

RNA polymerase from cells of the deep-sea bacterium Shewanella violacea DSS12 was purified using three chromatographic steps. An in vitro transcription assay indicated that the purified enzyme was σ⁷⁰ containing RNA polymerase. The enzyme activity was inhibited in the presence of rifampicin when the sensitive domain was targeted. The rpoBC genes encoding for the β and β′ subunits of RNA polymerase were cloned and their nucleotide sequences determined. Expression plasmids, designated pQSVB and pQSVC, to overproduce these proteins were constructed, and the proteins were purified using a Ni²⁺ affinity column. In vitro reconstitution using all proteins for the holoenzyme (α, β, β′, σ⁷⁰) was carried out and the activity of the recombinant RNA polymerase was detected.

NtrC protein of piezophilic Shewanella violacea was overexpressed and purified, to confirm the protein-DNA interaction. An electrophoretic mobility shift assay demonstrated that the NtrC recognizes the sequence for NtrC binding within the region upstream of the glnA operon. Western blot analysis also showed that the NtrC is expressed at a higher level under high-pressure conditions than under atmospheric pressure conditions.

The primary structure of the chicken ribosomal protein L30 was deduced from the sequence of nucleotide in a recombinant cDNA. Chicken ribosomal protein L30 has 115 amino acids and a molecular weight of 12,814 including initiator methionine. Hybridization of the cCDNA to the restriction enzyme digests of chicken liver DNA suggests that L30 gene is a single copy. Chicken L30 is homologous to ribosomal proteins from other eukaryotes and archaebacteria but not to those from eubacteria.

平成15年度文部科学省サイエンス・パートナーシッププログラム採択事業についての研究成果を報告した。 動機付け教育として実験、実習が効果的であり、生徒・学生の学習活動が継続的に行なわれれば、自学自習によって高度な教育が可能であることが示唆された。同時に実験・実習内容の精査やプログラム以前での実験技術に拘わる事前実習やティーティングアシスタントの養成が重要であることを指摘した。

窒素固定菌Paenibacillus azotofixansのニトロゲナーゼ鉄タンパク質をコードする遺伝子nifHを含む2つのオペロンのクローニングを行い、塩基配列の決定によりそれら遺伝子群の構造を明らかにした。

全ゲノム解析が進行している深海微生物 Shewanella violacea のリボソーム関連遺伝子群の構造解析を行い、 報告した。

高度好塩古細菌 Haloarcula japonica の 16SrDNA のコピー数と多型性に関する系統解析を行い、 Haloarcula 属の進化について考察した。

深海微生物 Shewanella violacea の全ゲノム解析による応用の可能性について、 特に環境修復技術開発の観点から論じた。

窒素固定菌 Paenibacillus azotofixans （IFO16645） のニトロゲナーゼ鉄タンパク質をコードする遺伝子 niflt を含む 2 つのオペロンのクローニングを行い、 それら遺伝子群の構造を明らかにした。

高圧下における転写メカニズムを明らかにする目的で、深海由来好圧性細菌より転写酵素 RNA ポリメラーゼをコードする遺伝子をクローン化し、それらを試験管内にて蛋白合成を行い、本酵素の再構成を試み、それに成功した。

Awamori is distilled liquor made from Indica-rice in Okinawa, and Aspergillus luchuensis and Saccharomyces cerevisiae are involved. Awamori flavors is characterized by vanillin generated from 4-vinylguaiacol (4-VG). In the fermentation, the microbial phenolic acid decarboxylase decarboxylates ferulic acid with production of 4-VG. Thus, the enzyme are important for the awamori flavors and the sources of the enzyme were unclear. In this study, the gene was detected on the genome of A. luchuensis. Besides two microbes, importance of other microbes such as lactic acid bacteria as a contaminant in the moromi are suggested, because the awamori brewery is an open system from outside. A lactic acid bacterium was isolated from the moromi, and the padC gene was also detected, suggesting both A. luchuensis and the bacterium may contribute the production of the flavor of 4-VG and vanillin. Biochemical characterization of these enzymes are carried out for future application of the flavor.

In this research funding area, researches on probing several gene and genome resources and its industrial applications by post-genomic approaches were carried out, using deep-sea isolate, Shewanella violacea strain DSS 12 which is already sequenced whole genome (gene candidates ; 4300). Several studies using deep-sea isolate, S. violacea strain DSS12 were included such as 1) development of a software searching efficiently upstream promoters interested in from genome sequence, 2) construction of gene database for several industrially useful enzymes, 3) biochemical analyses of alkaline phosphatases useful in the field of genetic engineering, in terms of high pressure and low temperature. The software is being developed in Perl language, and uses Tk modules for the user interface. It was initially developed to look for high-pressure-response sigma 54 promoter sequences in S. violacea genome, using a fuzzy search algorithm, but it can be used to find other promoter sequences such as sigma 32 or sigma 70 promoter sequences, or for example NtrC binding sites. The database for several industrially useful enzymes will be opened after publication of the genome analyses. In biochemical analyses of alkaline phosphatases, three enzymes whose gene is independently distributed on the genome and have different homology each other, were expressed in E. coli after construction expression plasmids. Each enzyme purified has different optimal temperature in dephosphrylation activity, showing its diversity of the enzymes and suggesting adaptation to deep-sea environments. Trying to isolate industrially useful new deep-sea microbes from deep-sea mud of Japan Sea, was also performed. From the several screening procedures, some protease-producing and amylase producing bacteria were isolated. These were suggested to be psychrotolerant isolates. Moreover, spore-forming nitrogen-fixing bacterium (Genus Paenibacillus), first example from deep-sea environment, was isolated. Phylogenetic analyses based on 16S rDNA of these isolates, showed that several bacteria seem to be new species.

Raft houses, similar to other human settlements, form a water-based community with distinguish characteristics. The physical features of the raft houses. There are many roof styles such as gable, manila or hip. Wall types are also varied. Originally they were made of different natural lightweight materials such as woven bamboo mats or acrew pines. The wall can be made as a lifted up panel or a sliding partition wall, which can be removed and stored away. However, the major difference between the raft house and the traditional Thai house is the foundation. The raft house has no pillar. It sits on a raft supports the whole weight of the house. There are two kinds of raft : Luffashaped rafts and Rectangular pontoon. Moreover, the raft house structure is not tightly fixed together. Economic aspects of the raft house are also analyzed. The inner part of the houses is generally used for living and sleeping while the outer part is used as commercial apace like a conventional shop-house with removable walls and panels. Social futures of the raft house are also revealed. The raft house community is based on self-reliance, sharing labour, affordability sustainability and community cohesiveness. Moreover, inhabitants have closely associated with water. Watercourses are used for bathing, washing and watering plantations. The study also finds the coexistence among the raft houses, water environment and urban-rural activities in various locations. In Pitsanulok, the location of the raft house settlement is in the urban area. The inhabitants have changed their life style to suit the environment. They are no longer working in agricultural sector.New careers such as general service worker or land-baced vendor are more preferable. The raft house settlement is affected by this phenomenon. It is gradually disappearing to give way to urban modernization.

光触媒の抗菌作用については多くの研究例が報告されているが,いずれも光触媒を機能させるためには紫外線しか利用できないので,抗菌作用について紫外線の効果と光触媒の効果とを区別して議論することは不可能であった。本研究では我々のグループが開発した可視光応答性酸化チタン光触媒を用いることにより,上記の問題点の解決を図った。検討項目は以下の3項目である。 (1)微生物の影響を調査するのに好ましい光触媒の薄膜化 (2)原核微生物として大腸菌を対象に可視光-光触媒の効果を確認 (3)真核微生物として酵母を対象に可視光-光触媒の効果を確認 (1)については,ゾルゲル法をベースとする方法で可視光応答性酸化チタン光触媒の薄膜化に成功した。具体的には,硫酸チタンを原料としてアンモニアで加水分解後,過酸化水素を加えて加水分解物を解コウして得た過酸化チタン前駆体ゾルを得た。薄膜は前駆体ゾルにアンモニアを加え,パイレックスガラス基材表面にスピンコーターでコーティングし,乾燥後,350℃の温度で1時間焼成することで成膜した。(特許出願) (2)可視光応答型酸化チタン光触媒をコーティングしたパイレックスガラス基材表面に大腸菌(E.coli from)を含む水滴を滴下し,4℃で水分が蒸発しない条件で青色および緑色LEDを照射し,光照射による影響を生菌数によって評価した。その結果,LEDを照射した場合のcell numberは,青色LEDでは0.12×10^2,緑色LEDでは0.23×10^2,と光を照射しないときの4.13×10^2と比較すると明らかに減少し,光触媒の効果が確認された。(3)酵母を対象に(2)と同様の実験を行ったところ,光触媒の効果はほとんど見られなかったことから,光触媒の作用は原核細胞には有効であるが,真核細胞には効果は薄いことが示唆された。