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

为了利用DNA重组技术培育好氧反硝化细菌，我们克隆了反硝化细菌的基因，并测定了NirS位点的DNA序列。本研究测定了NIRS下游侧翼区（6.7kbp的SmaI-BamHI片段）的DNA序列。为此，将DNA分成3段分别克隆到pUC 118和pUC 119载体上，测定了每个片段的DNA序列，并尝试测定了NirS上游侧翼区的序列。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。我们发现了2个额外的开放阅读框，命名为ORF 4和6。ORF 6位于NirS基因下游4kbp处，与吡咯喹啉醌合成酶在氨基酸水平上有30.2%的同源性。ORF 4位于NirS基因下游5 kbp处（与ORF 6相邻），与Ps的NirC（40%）和NirM（25%）有部分同源性。铜绿假单胞菌的氨基酸水平。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，位于铜绿假单胞菌NirS基因下游4- 5 kbp的位点可能编码电子传递所必需的蛋白质。

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.