Studies on probing genome resources and its application from deep-sea microorganism by post-genomic approaches

在本研究资助领域，利用已完成全基因组测序的紫希瓦氏菌深海分离株DSS 12（候选基因；4300），开展了后基因组方法探测多种基因和基因组资源及其工业应用的研究。本文介绍了紫茎甘蓝深海分离菌株DSS12的研究进展，包括：1)开发了从基因组序列中高效搜索上游启动子的软件；2)构建了几种工业用酶的基因数据库；3)从高压和低温两个方面对基因工程领域中有用的碱性磷酸酶进行了生化分析。本软件采用Perl语言开发，用户界面采用Tk模块。该方法最初是利用模糊搜索算法在紫百合基因组中寻找高压响应的sigma 54启动子序列，但也可用于寻找其他启动子序列，如sigma 32…More或sigma 70启动子序列，或例如NtrC结合位点。基因组分析结果公布后，将开放几种工业上有用的酶的数据库。在碱性磷酸酶的生化分析中，构建表达质粒后，在大肠杆菌中表达了三种基因独立分布于基因组且同源性不同的酶。每一种纯化酶的去磷酸化活性的最适温度不同，显示出酶的多样性，表明其对深海环境的适应性。并尝试从日本海的深海泥浆中分离出工业上有用的新型深海微生物。经过多次筛选，分离出一些产蛋白酶菌和产淀粉酶菌。这些菌株被认为是耐寒菌株。此外，首次从深海环境中分离到芽孢固氮菌（Paenibacillus属）。基于16S rDNA的系统发育分析表明，其中一些细菌似乎是新种。少

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 … More 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. Less