Azotobacter vinelandii, grown with NH4' as nitrogen source, was shown to possess an active transport system which can take up NH4' against a concentration gradient of 58-fold. The properties of the NH4' uptake system were investigated with the NH4' analog CH3NH3'. The use of this analog was justified on the basis of the conclusion that the uptake of NH4+ and CH3NH3+ involves a common binding site, as shown by the competitive inhibition of CH3NH3+ uptake by NH4+ (Ki approximately 3 ,uM). A

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... aver-Burk plot for CH3NH3+ uptake revealed a biphasic curve, suggesting the existence of two CH3NH3+ (NH4+) uptake systems with apparent Km's for CH3NH3+ equal to 61 ,M and 661 ,M. The uptake of CH3NH3+ was inhibited by arsenate, as well as by cyanide or carbonyl cyanide-mchlorophenyl hydrazone, indicating that phosphate bond energy is required. All free-living N2-fixing bacteria are known to respond to the presence of fixed nitrogen in the environment by shutting off synthesis of nitrogenase. The regulatory mechanism which represented elsewhere [R. A. Moore and J. K.