Distinctive Light and CO 2 -Fixation Requirements of Nitrate and Ammonium Utilization by the Cyanobacterium Anacystis nidulans

Abstract

I The effect of light intensity on the rates of ammonium and nitrate uptake and of CO2 fixation has been determined in intact Anacystis nidulans cells. Ammonium uptake became saturated at photon flux values of about 60 microeinsteins per square meter per second, whereas both nitrate uptake and CO2 fixation reached saturation at about 250 microein-steins per square meter per second, the rates of the two latter processes being tightly correlated at any light intensity assayed. Inhibition of ammonium assimilation resulted in the loss of correlation between CO2 fixation and nitrate uptake, the latter process exhibiting then a reduced light requirement. The results establish a clear distinction between am-monium utilization and nitrate utilization with regard to their light requirement and to the nature of their dependence upon CO2 fixation. Nitrate and ammonium are the forms of combined inorganic nitrogen most widely used by cyanobacteria in natural environments. The utilization of either nitrogen source is strictly dependent on light and on the availability of CO2. The light requirement seems to be derived from the requirement for pho-tosynthetically generated reductant and ATP exhibited by the corresponding assimilatory processes, whereas the CO2 requirement arises from different interactions between carbon and nitrogen metabolism (5, 7, 13). In Anacystis, as well as in other cyanobacteria, the utilization of nitrate includes: (a) the entrance of the anion into the cell, apparently mediated by an active transport system; (b) the 8-electron reduction of nitrate to am-monium catalyzed by the Fd-dependent enzymes nitrate and nitrite reductases; and (c) the incorporation of ammonium to carbon skeletons via the ATP-dependent glutamine synthetase and the Fd-dependent glutamate synthase enzyme system (5, 7). The utilization of ammonium is simpler and includes the entrance of the cation into the cell, apparently mediated by an energy-requiring transport system (2), and the subsequent incorporation of intracellular ammonium to carbon skeletons. Nitrate utilization in cyanobactena seems to be subject to feedback regulation by some organic nitrogen product(s) resulting from its assimilation (4, 5, 7), a situation that also applies to eukaryotic microalgae (13). It has recently been shown that treatment ofAnacystis cells with inhibitors of ammonium assim-' Supported by grant 0045/84 from Comisi6n Asesora de Investigaci6n (Spain). ilation, such as MSX2 or azaserine, releases nitrate uptake from both the inhibition by ammonium and the requirement for active CO2 fixation (4, 6, 9, 1 1). Based on these and other facts, a model has been proposed for the regulation of nitrate utilization, involving the concerted participation of assimilation products of both nitrogen and carbon (6, 11). Little is known, however, about the nature of the CO2 requirement of ammonium utilization except that CO2 is required for sustained ammonium uptake (2). In this communication we report on the effects of varying light intensity on the rates of CO2 fixation and on those of nitrate and ammonium utilization. The obtained results establish a clear distinction between ammonium utilization and nitrate utilization with regard to the nature of their CO2 dependence. MATERIALS AND METHODS Organism and Culture Conditions. Anacystis nidulans (Syne-chococcus leopoliensis 1402-1, Gottingen University, F.R.G.) was grown photoautotrophically with nitrate as the sole nitrogen source as previously described (8). Cells were harvested by cen-trifugation after 24 h growth (15-20 ,ug Chl a ml-'), washed with 25 mm Tricine-NaOH/KOH buffer (pH 8.3), and resuspended in the same buffer. Chl a was estimated after extraction with methanol using the extinction coefficient given by McKinney (10). Nitrate and Ammonium Uptake. The assays were carried out at 40°C with continuous shaking and illumination at different intensities of white light in air-opened conical flasks. The assay medium contained, in a volume of 4 ml: 100 gmol Tricine-NaOH/KOH buffer (pH 8.3), 40 ,umol NaHCO3, 1 ,umol of either KNO3 or NH4C1, and an amount of cells equivalent to 40 ,ig Chl. The reaction was started by simultaneously switching on the light and adding NaHCO3 and, in some experiments, KNO3 or NH4C1. At regular time intervals, 0.5 ml aliquots were withdrawn and, after rapid removal ofthe cells by filtration (Millipore HA 0.45 um pore size filter), nitrate or ammonium was determined in the filtrates. Nitrate was determined by optical absorption at 210 nm in acid solution (3), and ammonium by the glutamate dehydrogenase method (1). Incident photon fluxes of photosynthetically active radiation (400-700 nm) were measured with a Li-Cor integrating quantum/radiometer/photometer provided with a LI-190SB quantum sensor cell. Treatment with MSX was performed by adding 4 jmol of the compound to the illuminated cell suspensions 15 min before starting the experiment. 2Abbreviation: MSX, L-methionine D,L-SUlfoximine. 686