Cloning and sequencing of a gene encoding nitrite reductase from Paracoccus denitrificans and expression of the gene in Escherichia coli

A structural gene for nitrite reductase (nirS) was cloned from a denitrifying bacterium Paracoccus denitrificans into Escherichia coli MV1184. The coding sequence of nirS consisted of 1788 nucleotides and the value of the G+C content was 68%. This gene seemed to be in an operon structure. The size of nitrite reductase (NIR) was predicted to be 65.5 kDal from the amino acid sequence, which was similar to the value determined with purified NIR by SDS-polyacrylamide gel electrophoresis analysis. From hydrophathy analysis, the NIR from P. denitrificans seemed to be a periplasmic enzyme. Crude extract from the recombinant E. coli harboring nirS and about 10 kbp of its downstream flanking region had significant activity of NO and N₂O formation from nitrite (NO₂^-), whereas crude extract from E. coli harboring only nirS had only weak activity. This result suggested that the downstream region includes the gene responsible for the protein which involved in NIR activation. In addition, the downstream region seemed to have a NO reductase gene, because NO to N₂O conversion activity was also detected in the crude extract from the recombinant E. coli harboring nirS and about 10 kbp of its downstream flanking region.