Involvement Of Ammonium Transporter AmtB In The Control Of Nif Gene Expression In Pseudomonas Stutzeri A1501

Pseudomonas stutzeri A1501was isolated from rice field in South China, which colonized tightly on rhizosphere of the host plants or invaded the roots of plants for growth and nitrogen fixation and belonged to y subgroup of Proteobaeteria. This strain can fix nitrogen under nitrogen-limited and microaerobic conditions in the free-living state. Ammonium transporter (AmtB) has an important role in balancing the nitrogen source of the cell, mainly in ammonium transport and ammonium sensing. The nitrogen-fixing Pseudomonas stutzeri strain A1501contains two ammonium transporter genes, amtBl and amtB2, located downstream of glnK and co-transcribed with glnK.1. Deletion of the amtB1amtB2region was performed by homologous recombination according to the strategy of PCR-based fusions. Gene disruption resulted from the integration of recombination plasmid after homologous recombination into the target gene by triparental conjugation. The mutant strain was named AamtB. The complementary strain pLamtB was also constructed.2. Growth of an amtBl-amtB2double deletion mutant strain was not impaired as compared to that of the wild type under any conditions tested and it was still capable of taking up ammonium ions at nearly wild-type rates, which demonstrates that growth and ammonium uptake in stutzeri A1501can occur in the absence of AmtB and diffusion of NH3is sufficient for optimal growth at low concentration of ammonium.3. Disruption of both amtB genes did not prevent derepression of nitrogenase. Nitrogenase activity of the wild-type A1501was higher than mutant strain AamtB. Nitrogenase activity was repressed in the wild-type A1501in response to addition of ammonium; but nitrogenase activity was only partially impaired in the amtBl and amtB2double mutant, suggesting that the two AmtB proteins are involved in regulating the expression of nitrogenase or its activity in response to ammonium.4. Quantitative RT-PCR analysis of nifH and nifA expression in the wild-type A1501and amtB mutant strain1568was performed under conditions of nitrogen fixation and ammonium shock. After addition of ammonium, nifH and nifA transcription decreased by20and10-fold, respectively in the wild-type strain whereas in the amtB deletion strain, a much smaller response to ammonium was observed, with transcription of nifH and nifA maintained to60%and80%, respectively of the levels prior to ammonium shock.5. Significant quantities of ammonium were excreted by the amtB mutant strain with constitutive expression of nifA gene under nitrogen fixation conditions while no ammonium was detected in the wild type strain, indicating that AmtB proteins play a role in controlling the internal pool of ammonium within the cell.6. For the heterologous expression of AmtB from stutzeri, the amtB gene was PCR amplified, resequenced, cloned into the expression vector pET28a, and introduced into E. coli BL21(DE3). The purified protein will be used to be made antibody and further verify the function of AmtB protein.7. A model that AmtB invovles in the regulation of nitrogenase activity is proposed. Under conditions of nitrogen limitation, GlnK possibily in its uridylylated form from the cytoplasmic interacts with the inhibitory NifL-NifA complex, resulting in its dissociation and allowing NifA to activate nif gene transcription. After addition of ammonium, GlnK (deuridylylated) may be sequestered to the cytoplasmic membrane in an AmtB-dependent manner, effectively blocking ammonium transport into the cytoplasm. Removal of a substantial amount of GlnK proteins from the cytoplasm allows NifL to form a complex with NifA, shutting down the nif gene transcription.