The transcriptional regulation and functional identification of nitrite reductase nirS in Pseudomonas stutzeri A1501

Abstract:

[Objective] To study the transcriptional regulation of nirS encoding nitrite reductase and the function of nirS involved in the denitrification process of Pseudomonas stutzeri A1501. [Methods] The nirS-lacZ fusion vector was constructed and transformed to A1501 and rpoN mutant strains by triparental conjugation. The β-galactosidase activity was detected to analyze the expression of the nirS gene in A1501 under different concentrations of oxygen, nitrate and nitrite. The fusion vector was also transformed to the rpoN mutant to investigate the effect of RpoN on the transcription of the nirS gene through β-galactosidase activity analysis. Furthermore, we constructed the nirS mutant strain by homologous recombination and investigated the function of nirS as it is involved in the denitrification process. [Results] Expressional activity of the A1501 nirS promoter under anaerobic conditions was four-fold higher than that under aerobic conditions. Nitrate significantly induced the expression of nirS, while nitrite showed only slight induction of the nirS promoter. Compared to the wild type, one fourth of the nirS expression was observed in the rpoN mutant. No conserved RpoN binding sites were found in the nirS promoter region, suggesting that RpoN regulates nirS expression through an indirect pattern. The denitrification capability of ΔnirS was reduced by about 20% compared to the wild type when nitrate was used as the sole electron receptor, while the ΔnirS had little denitrification with nitrite as the sole electron receptor, thus the utilization of nitrite was apparently decreased in ΔnirS. Compared to the wild type, the nitrogenase activity of ΔnirS was increased under anaerobic conditions with nitrite. [Conclusion] The transcription of nirS in A1501 was influenced not only by anaerobic conditions and nitrate, but also under the control of RpoN. The nirS played a key role in the denitrification process of A1501, which is involved in nitrite metabolism.