丙酮酸对浑球红假单胞菌突变株(GOGAT^-Nif^-)固氮酶活性的调节

Regulation of Nitrogenase Activity by Pyruvate in Glutamate Synthetase Mutant (GOGAT^-Nif^-) of Rhodobacter sphaeroides

固氮无效的浑球红假单胞菌GOGAT突变株经丙酮酸诱导产生固氮酶活性。固氮酶比活随丙酮酸浓度增加而提高,同时依赖于蛋白质合成的菌体生长。丙酮酸产生固氮酶时氮源Glu的浓度高达60 mmol/L。液相色谱分析表明,丙酮酸诱导固氮酶活性的形成与胞内Gln耗竭有关而与Asn无关。用丙酮酸代替苹果酸诱导,突变株向胞外分泌的游离氨大大减少。丙酮酸诱导时变种谷氨酰胺酶比活较高,它可能参与胞内Gln的分解。

A GOGAT^- mutant 204 of Rhodobacter sphaeroides showed a Nif^- phenotype in RCVBN medium containing glutamate as the sole nitrogen source and malate as the sole carbon source. It caused the appearence of nitrogenase activity while shifting the cells to a pyruvate-containing medium without malate. Nitrogenase levels as measured by acetylene reduction and hydrogen production were strongly correlated with the concentratin of pyruvate (Fig. 1) and with the growth of cells (Fig. 2). The mutant also displayed nitrogenase activity under very high concentration of glutamate (10～60 mmol/L) in the culture supplemented with pyruvate (20 mmol/L)(Fig. 5). Much less ammonia was excreted into the medium when the mutant was cultured with pyruvate than with malate (Table 1). The analyses by High Performance Liquid Chromatography (HPLC) revealed that the GOGAT minus mutant in RCVBN midium showed high content of intracellular Gln because the break of GOGAT reaction and nitrogenase activity occurred concomittantly with the depletion of intracellular Gln in the mutant subjected to pyruvaate induction (Table 2). Moreover, the glutaminase activity was found in Rb. Sphaeroides cells. The specific activity of glutaminase was higher in the mutant induced by pyruvate than by malate (Table 3). The enzyme, which was inhibited by DON but not MSX, may be involved in the decomposition of intracellular Gln during the pyruvate induction.