Energy-dependence of the Assimilatory Nitrate Uptake in Azotobacter chroococcum

Abstract

Nitrate assimilation by suspensions of Azotobacter chroococcum, as determined by the disappearance of the ion from the external medium, displayed saturation kinetics, was inhibited by nitrite, and exhibited an affinity for nitrate higher than that of nitrate reductase. This suggests that the entry of nitrate into the cell is mediated by a specific transporter. Nitrate assimilation required a readily utilizable carbon source and aerobic conditions and was blocked by the uncouplers carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 2,4-dinitrophenol (DNP) but not by N, N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of ATPases. The inhibition of nitrate assimilation in the absence of an appropriate carbon source was overcome by the non-physiological energy source ascorbate plus N-methylphenazinium methylsulphate (PMS), a substrate combination that allowed respiration. Though an ATP-dependent nitrate uptake mechanism cannot be ruled out, these data suggest that transport of nitrate into the cell is directly dependent on the proton electrochemical gradient across the cytoplasmic membrane.