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Cloning and structure analyzes of genes from Paracoccus denitrificans related to denitrification

反硝化副球菌反硝化相关基因的克隆及结构分析

基本信息

批准号：
05454036
负责人：
IIJIMA Shinji
金额：
$ 4.22万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454036/
关键词：
Environment Waste water treatment Denitrification Nitrous oxide 窒素化合物

项目摘要

In order to bread denitrifying bacterium which acts under aerobic conditions by recombinant DNA technology, we cloned genes for denitrification and determined the DNA sequence of NirS locus. In this year, we determined the DNA sequence of NirS downstream flanking region (6.7 kbp SmaI-BamHI fragment). For this, DNA was cloned as 3 pieces in pUC 118 and 119 vectors and DNA sequences of each fragment were determined.We also tried to determin the sequence of NirS upstream flanking region. Although we have not yet completely determined the entire sequence, a part of the lucus was homologous to norB gene of Ps. aeruginosa which encodes a subunit of nitrous oxide reductase.We found 2 additional open reading frames designated as ORFs 4 and 6. ORF6 which located 4 kbp downstream of NirS gene was partly homologous to pyrolquinoline quinone synthetase in amino acid level (30.2%). Furthermore, ORF4 which located 5 kbp downstream from NirS gene (just adiacent to ORF6) showed partial homology to NirC (40%) and NirM (25%) of Ps. aeruginosa in amino acid level. NirC and Nir M genes of Ps. aeruginosa encoded cytochrome C and cytochrome C-551, respectively. Therefore, it is possible that the locus located 4-5kbp downstream of NirS gene of P.denitrificans encoded a proteins which are essential for electron transfer.

为了通过重组DNA技术培育出在好氧条件下发挥作用的反硝化细菌，我们克隆了反硝化基因，并确定了NirS位点的DNA序列。今年，我们确定了NirS下游侧翼区的DNA序列（6.7 kbp SmaI-BamHI片段）。为此，将 DNA 在 pUC 118 和 119 载体中克隆为 3 段，并确定每个片段的 DNA 序列。我们还尝试确定 NirS 上游侧翼区域的序列。虽然我们还没有完全确定整个序列，但lucus的一部分与Ps的norB基因同源。铜绿假单胞菌编码一氧化二氮还原酶亚基。我们发现了两个额外的开放阅读框，分别命名为ORF 4和6。ORF6位于NirS基因下游4 kbp处，与吡咯喹啉醌合成酶在氨基酸水平上部分同源(30.2%)。此外，位于 NirS 基因下游 5 kbp 的 ORF4（紧邻 ORF6）与 Ps 的 NirC（40%）和 NirM（25%）显示出部分同源性。铜绿假单胞菌的氨基酸水平。 Ps 的 NirC 和 Nir M 基因。铜绿假单胞菌分别编码细胞色素C和细胞色素C-551。因此，位于脱氮假单胞菌NirS基因下游4-5kbp的位点可能编码电子传递必需的蛋白质。