In vivo energetics and control of nitrogen fixation: changes in the adenylate energy charge and adenosine 5'-diphosphate/adenosine 5'-triphosphate ratio of cells during growth on dinitrogen versus growth on ammonia

Abstract

The effects of the intracellular energy balance and adenylate pool composition on N2 fixation were examined by determining changes in the energy charge (EC) and the ADP/ATP (D/T) ratio of cells in chemostat and batch cultures of Clostridium pasteurianum, Klebsiella pneumoniae, and Azotobacter vinelandii. When cells of C. pasteurianum, K. pneumoniae, and A. vinelandii in sucrose-limited chemostats were examined, in all cases the EC increased greater than or equal to 15% when the nitrogen source was switched from N2 to NH3 and decreased greater than or equal to 15% when the nitrogen source was switched from NH3 to N2. The D/T ratio of the same cultures decreased greater than or equal to 70% when they were switched from N2 to NH3. In such cultures the adenylate pools remained constant when the cells were grown on either NH3 or N2. In nitrogen (NH3)-limited cultures, the adenylate pool was two- to threefold higher than the adenylate pool in sucrose-limited cultures, and the nitrogenase content of such cells was two- to threefold greater than the nitrogenase content of sucrose-limited N2-fixing cells. The EC and D/T ratio of cells from batch cultures of C. pasteurianum growing on NH3 in the presence of N2 were 0.82 and 0.83, respectively, but when the NH3 was consumed and the cells were switched to a nitrogen-fixing metabolism, the EC and D/T ratio changed to 0.70 and 0.90, respectively. Conversely, when NH3 was added to N2-fixing cultures the EC and D/T ratio changed within 1.5 h the EC and D/T ratio of NH3-grown cells. The nitrogen content of N2-fixing cells to which NH3 was added decreased at a rate greater could be accounted for by cell growth in the absence of further synthesis. This decay of nitrogenase activity (with a half-life about 1.2 to 1.4 h) suggests that some type of inactivation of nitrogenase occurs during repression. The nitrogenase of whole cells was estimated to be operating at about 32% of its theoretical maximum activity during steady-state N2-fixing conditions. Similarities in the data from chemostat and batch cultures of both aerobic and anaerobic N2-fixing organisms suggest that low EC and high D/T ratio are normal manifestations of an N2-fixing physiology.