Study On Nitrite Reductase Encoding Gene NirS In Pseudomonas Stutzeri A1501

Denitrification by prokaryotes is an important part of the global nitrogen cycle. Pseudomonas stutzeri A1501 was isolated from rice paddies in the southern area of China. Bacterial denitrification is expressed in response to the singal of low-oxygen tension. In this study, we present physiological and molecular data on denitrification in Pseudomonas stutzeri A1501.Then nirS was cloned into pSUP202 suicide plasmid vector, which was capable of conjugal self-transfer to a wide variety of Gram-negative bacteria. Then km-lacZ fragment from pKOK5 was introduced into the encoded region of nirS, and nirS-Km-LacZ fusion plasmid(pSUP202-TCK) was constructed. Then the recombinant plasmid was transfered into the wild type strain A1501 by tri-parental mating assay and the nirS mutant(M3) was obtained by insertion of the km cassette into the genome by homologous recombination in the host genome.Denitrification between nirS mutant and wild type A1501 had distinct difference. When nitrate was used as terminal electron acceptor, denitrification ability discreased. When nitrite was used as terminal electron acceptor, when wild type A1501 and nirS mutant were incubated 10 hours, nitrite in the culture could be absolutely consumed for A1501, compared to 20% for nirS mutant. It is concluded that there exists another approach different from NirS denitrification process and low matabolization activity in A1501.By designing the specific primers, the nirS promoter was cloned by PCR method. Then it was cloned into pGD926 plasmid vector which was its extensive host ranges and was capable of conjugal self-transfer to a wide variety of Gram-negative bacteria. The fusion vector was transferred into wild type strain A1501and rpoN by conjugation, and then B-galactosidase activity was assayed. The result showed that the expression of nirS was repressed by oxygen, and denitrification activity under anaerobic conditions was higher. Under different nitrite concentration, the expression of nirS was low, and the highest activity arrived to 2600 units. The expression of nirS was induced by nitrate. Nitrogenase activity was highest in the presense of 1.0mmol/L nitrate concentration, about 12900 Units, hi the presence of too high nitrate concentration (10 mmol/L) or too low nitrate concentration (0.1 mmol/L), nitrogenase activity is about 3500 units. When nitrate was used as terminal electron acceptor under anaerobic condition, the expression of nirS in rpoN was lower than in wild type, was about one fourth in wild type. It showed that nirSp was a induced σ⁵⁴-dependent promoter. There was no characteristic a54 sequence in nirS promoter origin, so it was supposed that there exists other a54 characteristic sequence in nirS promoter origin.Also colonization on the surface of rice root of nirS mutant was studied.