The Mechanism Of RpoN Modulates Nitrate Assimilation In Nitrogen Fixing Pseudomonas Stutzeri A1501

RpoN(σ54) widely modulates gene regulation in various bacteria metabolic pathways, such as carbon and nitrogen source utilization, motility, bioflim formation and ion transport. Pseudomonas stutzeri A1501 is a versatile soil bacteria that exhibits the unusual feature, for a Pseudomonas, of being capable of nitrogen fixation and endophytic association with rice plants. Our previous studies on different metabolic systems that affect nitrogen-fixing capability of A1501 had shown that, RpoN influence those systems as an important regulator in different extent. In order to make comprehensive understanding of RpoN regulatory role, we made comparative transcriptome analysis of A1501 wild type and rpoN mutant, and on the basis of this, we made further study on the mechanism of RpoN modulates nitrate assimilation in A1501. The main research results are described as follows:1. High-throughput sequencing technology was used to analyze transcript profiles in A1501 wild type and the rpoN mutant. The results showed that transcript levels of 563 genes were significantly changed in the rpoN mutant compared to the wild type, of which 305 genes with decreased transcript levels and 258 genes with increased transcript levels. Most dominant categories identified by this method were genes related to flagellar and pili biosynthesis, carbon catabolite repression, iron and amino acid transport and nitrate assimilation. Through homologous comparison, 11 genes were identified to be nitrate assimilation genes(NAS) in A1501 genome, such as regulator coding genes nasR and nasST, nitrate and nitrite reductase coding genes nasBCG and glutamine synthetase coding gene glnA. Products of these genes work together to transport extracellular nitrate into cytoplasm and step by step to reduce the transform of intracellular nitrate to organic nitrogen source. It is proposed that RpoN directly or indirectly affect nitrate assimilation process as 11 nitrate assimilation genes showed decreased transcript levels in the rpoN mutant. Online tools were used to analyze the promoter of the 11 genes, all of them contained a putative RpoN binding site GG-N10-GC. Therefore, RpoN may directly modulate nitrate assimilation process by regulating genes expression.2. Nitrate or nitrite was used as sole nitrogen source to investigate the regulatory role of RpoN in nitrate assimilation process. The absence of rpoN caused impaired nitrate uptake and utilization. Besides, nasST, nasR, nasF, nasB and glnA showed decreased transcript levels in rpoN mutant when these genes were induced by the presence of nitrate in the wild type. Therefore, it is proposed that RpoN directly or indirectly controls the expression of nasST, nasR, nasF, nasB and glnA. The nasR, nasT mutant were constructed to understand the function of these two regulators and the nitrate assimilation regulatory system they involved in. The mutation of nasT caused loss of nitrate utilization ability in A1501, but nitrate uptake efficiency remained. Compared with A1501 wild type, the nitrate uptake efficiency and utilization ability drop by 60% and 40% in nasR mutant, respectively. In A1501 genome, nasR located upstream of nitrate transport complex coding genes nasFED, nasT located upstream of nitrate and nitrite reductase coding genes nasBCG. Gene locations and phenotype of nasR, nasT mutant indicate that NasR modulates the expression of nasFED to form the nitrate transport complex while NasT modulates the expression of nasBCG to form the nitrate and nitrite reductase. Besides, in Klebsiella oxytoca and Azotobacter vinelandii studies, NasT and NasR work as anti-terminators to control the expression of down-stream genes. Further research is needed to confirm the same regulatory mechanism of NasR and NasT in A1501 nitrate assimilation process.3. Putative RpoN binding site was found in nasR and nasT promoter sequence, however, there have not been any related studies in RpoN initializing nasR and nasT transcription by now. His-Tag was used to purify RpoN protein, DIG was used to label nasR and nasT promoter DNA. The specific binding of RpoN-nasR/ nasT promoter, or σ54-RNAP-nasR/nasT promoter was detectd by electrophoretic mobility shift assay. EMSA results showed that σ54-RNAP complex or σ54 alone can directly bind to nasR and nasT promoter regions, this was taken as proof that RpoN can directly regulate nasR and nasT by initializing their transcriptions.Global regulatory protein RpoN in Pseudomonas stutzeri A1501 directly modulates transcriptional initiation of nasR and nasT. Then these two anti-terminators regulate genes coding nitrate transport complex and reductases to control A1501 extracellular nitrate utilization.