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Analysis of thermophilic Bacillus sp.Strain TB-90 urease gene and its product

嗜热芽孢杆菌TB-90脲酶基因及其产物分析

基本信息

批准号：
04453131
负责人：
UOZUMI Takeshi
金额：
$ 3.84万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

The thermophilic Bacillus sp. TB-90 urease is composed of three subunits with molecular masses of 61, 12 and 11 kDa. By using synthetic oligonucleotide probes based on N-terminal amino acid sequences of each subunit, we cloned a 3.2-kb EcoRI-digested fragment of TB-90 genomic DNA.Moreover, we cloned other two DNA fragments by gene walking starting from this fragment. Finally, we in vitro reconstructed a 6.2-kb DNA fragment which expressed catalytically active urease in E.coli by combining these three DNA fragments. The nucleotide sequencing analysis revealed that the urease gene complex consists of nine genes which were designated ureA, ureB, ureC, ureE, ureF, ureG, ureD, ureH and ureI in order of arrangement. The structural genes, ureA, ureB and ureC, encode the 11- 12- and 61-kDa subunits, respectively. The deduced amino acid sequences of UreD, UreE, UreF and UreG, the gene products of four accessory genes, and homologous to those of the corresponding Ure proteins of Kkebsiella aerogenes. The UreD, UreF and UreG were essential for expression of urease activity in E coli and were suggested to play important roles in the maturation step of the urease in co- and/or post-translational manners. On the other hand, UreH and UreI exhibited no significant similarity to the known accessory proteins of other bacteria. However, UreH showed a similarity of 23% amino acid identity to Alcaligenes eutrophus HoxN protein, a high-affinity nickel transporter.

嗜热芽孢杆菌TB-90脲酶由3个亚基组成，分子量分别为61、12和11 kDa。利用人工合成的基于每个亚基n端氨基酸序列的寡核苷酸探针，克隆了一个3.2 kb ecori -酶切的TB-90基因组DNA片段。此外，我们从该片段开始，通过基因行走克隆了另外两个DNA片段。最后，我们将这三个DNA片段结合，在体外构建了一个表达催化活性脲酶的6.2 kb DNA片段。核苷酸测序分析表明，该脲酶基因复合物由9个基因组成，依次为ureA、ureB、ureC、ureE、ureF、ureG、ureD、ureH和ureI。结构基因尿素、ureB和ureC分别编码11- 12和61-kDa亚基。推导出4个辅助基因产物UreD、UreE、UreF和UreG的氨基酸序列，并与产气克雷伯菌对应的Ure蛋白序列同源。在大肠杆菌中，UreF、UreG和UreF是表达脲酶活性所必需的，并且被认为在脲酶的共翻译和/或翻译后成熟过程中发挥重要作用。另一方面，UreH和UreI与其他已知细菌的辅助蛋白没有明显的相似性。然而，UreH与富营养菌高亲和镍转运蛋白HoxN蛋白的氨基酸相似性为23%。