【目的】研究固氮施氏假单胞菌(Pseudomonas stutzeri)A1501亚硝酸盐还原酶结构基因nir S的转录调控机制及其在反硝化过程中的功能。【方法】构建nir S-lac Z融合载体,利用三亲本结合法将其导入野生型A1501,通过β-半乳糖苷酶活性的测定,分析不同供氧状况、不同浓度的硝酸盐、亚硝酸盐对nir S基因表达的影响;同时将该载体导入rpo N突变株中,研究氮代谢调控因子Rpo N对nir S基因转录影响。通过同源重组方法构建nir S突变株,通过生化表型测定明确nir S在反硝化过程中的功能。【结果】启动子活性测定表明,nir S基因厌氧条件下高水平表达,是好氧条件下表达水平的4倍;nir S的表达受硝酸盐诱导,但不受亚硝酸盐的诱导;Rpo N突变株中,nir S的表达活性为野生型的1/4,nir S启动子未发现Rpo N的保守结合位点,表明nir S的表达受Rpo N间接调控。表型测定显示以硝酸盐为电子受体时Δnir S的反硝化能力降低了约20%;以亚硝酸盐为电子受体时Δnir S仅有微弱的反硝化能力,并且nir S的突变使得菌体在反硝化条件下利用亚硝酸盐的能力显著减弱。nir S突变提高了菌体在亚硝酸为电子受体的反硝化条件下的固氮酶活。【结论】A1501中nir S基因的转录受外界氧及硝酸盐的影响,同时受氮代谢Sigma因子Rpo N的调控。nir S在A1501菌反硝化过程中起关键作用,参与了亚硝酸盐的转化。
[Objective] To study the transcriptional regulation of nirS encoding nitrite reductase and the function of n irS involved in the denitrification process of Pseudomonas stutzeri A 1501. [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 nits 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 nitS 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 AnirS was reduced by about 20% compared to the wild type when nitrate was used as the sole electron receptor, while the AnirS had little denitrification with nitrite as the sole electron receptor, thus the utilization of nitrite was apparently decreased in AnirS. Compared to the wild type, the nitrogenase activity of AnirS was increased under anaerobic conditions with nitrite. [Conclusion] The transcription of nitS 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.