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Effect of Ammonia on the Synthesis and Function of the N₂-Fixing Enzyme System in Clostridium pasteurianum

Department of Biological Sciences, Purdue University, Lafayette, Indiana 47907

ABSTRACT

The N₂-fixing system of Clostridium pasteurianum operates under regulatory controls; no activity is found in cultures growing on excess NH₃. The conditions which are necessary for the synthesis and function of this system were studied in whole cells by using acetylene reduction as a sensitive assay for the presence of the N₂-fixing system. Nitrogenase of N₂-fixing cultures normally can fix twice as much N₂ as is needed to maintain the growth rate. When cultures that have grown for four or more generations on NH₃ exhaust NH₃ from the medium, a diauxic lag of about 90 min ensues before growth is resumed on N₂. Neither N₂-fixing nor acetylene reduction activity can be detected before growth is resumed on N₂. N₂ is not a necessary requirement for this synthesis since under argon that contains less than 10^–8M N₂, the N₂-fixing system is made. If NH₃ is added to N₂-dependent cultures, synthesis of the enzyme system is abruptly stopped, but the enzyme already present remains stable and functional for at least 6 hr (over three generations). Cultures grown under argon in a chemostat controlled by limiting ammonia have derepressed nitrogenase synthesis. If the argon is removed and replaced by N₂, partial repression of nitrogenase occurs.

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