土壤杆菌固氮生理特性研究

STUDIES ON PHYSIOLOGICAL CHARACTERISTIES OF NITROGEN FIXATION BY AGROBACTERIUM TUMEFACIENS

对根癌土壤杆菌C58/pGV3850菌株的固氮生理特性研究结果表明,该菌具有自生固氮活性,其固氮活性的最适pH为8.0,温度为30℃.固氮活性在对数生长后期(14h)最高,延缓期和静止期乙炔还原活性较低.该菌株好氧,通过氧呼吸和吸氢酶的作用达到避氧固氮.当通入氧气超过呼吸耗氧时,对固氮活性产生抑制作用.在培养过程中增加NH_4^+浓度,固氮活性会降低,当达到15mmol/L时完全丧失固氮活性,表现NH_4^+阻遏固氮酶蛋白合成.在乙炔还原测定系统中加入NH_4^+并不影响乙炔还原反应,说明没有NH_4^+关闭现象.培养过程中加入MSX(2.5mg/ml)能解除10mmol/L NH_4^+对固氮酶合成的阻遏作用.固定的游离氮不能以NH_4^+形式分泌于胞外.固氮过程中放出大量氢气,培养16小时产氢量达65μmol H_2/mg蛋白.在限碳条件下(0.2%蔗糖)其吸氢酶活力可达520nmol H_2·ml^(-1)·h^(-1).

Under free-living condition Agrobacterium tumefaciens C58/pGV3850 can fix nitrogen in modified Burk's medium and the optimal conditions for nitrogenase activity are pH 8. 0 and 30 C. The nitrogenase activity of C58/pGV3850 was lower in lag and stationary phases, and the maximum amount acetylene reduction rate was observed in late exponential phase (14h), but no acetylene reduction was measured at 48 hours of cultivation. As an aerobic organism, C58/pGV3850 can synthesize nitrogenase protein while oxygen was consumpted by respiration to establish an anaerobic environment within the cell, when oxygen was supplied over consumpted, the nitrogenase activity would be inhibited. The nitrogenase protein synthesis was repressed by ammonia presented in growth medium, the more ammonia the less acetylene reduction untill no nitrogenase activity with 15 mmol/L ammonia present in medium. On the other hand, in acetylene reduction assay system no effection of ammonia on nitrogenase activity was observed even the ammonia concentration was up to 40 mmol/L. These results suggest that ammonia switch-off of nitrogenase activity is not operative in C58/pGV3850. The relief effect of MSX (2. 5 mg/ml) on nitrogenase protein synthesis repressed by ammonia (10 mmol/ L) was observed in experiment. There is no ammonia secreating outside the cells during nitrogen fixation process. Large amount of hydrogen (65μmol H2/mg cellprotein) was evoluted in nitrogen fixation process, and the hydrogenase activity was 520 nmol H2 uptake·ml-1·h-1, while C58/pGV3850 was incubated in the medium of limited carbon source (0. 2% sucrose) and induced with 10% H2. Cultivited in the medium of 2% sucrose, the nitrogenase activity was increased from 728 to 919 nmol C2H4·mg cell protein-1 ·h-1 by means of adding 5% H2 in acetylene reduction assay system. Thus, nitrogen fixation was supported by molecular hydrogen even with sufficient carbon source. It may be concluded that reversible hydrogenase was present from the effect that hydrogen evolution still taken place, although no nitrogenase activity was determined in 40 mmol/ L ammonia growth condition.