| Associative nitrogen-fixing bacteria can colonize on rhizoplane of the host crops for growth and nitrogen fixation. Associative nitrogen-fixing bacteria could supply host plants with nitrogen, but the supply is not sufficient. Pseudomonas stutzeri A1501, isolated from rhizosphere of rice, is an associative nitrogen-fixing bacterium. Recently, the genome sequence and annotation of A1501 have been completed. On the basis of the genetic information and the previous study on expression and regulation of nitrogen fixation systems in P. stutzeri A1501, we construct, identify and characterize an ammonium-excreting strain by improving the ability of nitrogen fixation of P. stutzeri A1501.The complete nifA gene amplified from A1501 genomic DNA was ligated into the plasmid pVK100, which encodes the key activator NifA of nif gene expression, and then the resulting plasmid, pVA3, was transformed into the wild type strain A1501 and the ammonium transporter AmtBl/AmtB2 deficient mutant (1561) by tri-parental conjugation, to generate the strain A1501/pVA3 and 1561/pVA3, respectively. The data showed that the overexpression of nifA gene enhanced the nitrogenase activity (7.3U/mg·protein) in P. stutzeri under nitrogen fixation conditions and remained nitrogenase activity (1.53U/mg·protein) under non-nitrogen-fixing conditions. But no ammonium was detected in the culture of A1501 or 1501/pVA3; no significant differences of growth rate and nitrogenase activity were observed between the strain 1561 and the wild type strain. However, when the strain 1561 was incubated in the N-free medium containing 8 or 12 mM NO3- for 33h, the ammoniumn (0.60 or 1.32mM) in the extracellular milieu was detected, suggesting that disruption of ammonium transporter AmtB1/AmtB2 caused the cells leakage of ammoniumn.The nitrogenase activity of the strain 1561/pVA3 was about as 1.95-fold as that of the wild type strain under nitrogen fixation conditions, and maintained 30% of nitrogenase activity in presence of 10mM ammonium, which was higher than that of the strain 1501/pVA3. After 15-day incubation in the N-free medium, the medium pH became slightly alkaline, and also the ammoniumn concentration was as high as 5.15 mM. The results showed that the overexpression of nifA and disruption of ammonium transporter could make nitrogenase activity enhanced and amount of ammonium released into the medium.Rice plant was inoculated with strain 1561/pVA3. The plant height (19.4 cm), dry weights of shoot and root (12.85mg and 2.96mg) of the rice plant treated with 1561/pVA3 were significnatly higher than those of the wild type strain (14.8cm, 11.70mg and 2.11mg). 1561/pVA3 showed a strongly enhanced competitive root-colonizing ability compared with wild type strain. These results indicated that 1561/pVA3 strain obviously improved rice plant growth.To further understand the ammonium-excretion of the strain 1561/pVA3, ammonium shock experiments were performed, and the concentrations of ammonium, nitrate, amino acid and GS activities were measured. These results indicated that 1) constitutive plasmid of nifA, pVA3, can enhance the expression of nif A and nifH; 2) disruption of AmtB1/B2 deprives function of transporting ammonium, leads to ammonium release into extracellular culture; 3) AmtB1AmtB2 might be involved in post-translational modification of nitrogenase and control of nif gene expression.
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