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

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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.