The nac (Nitrogen Assimilation Control) Gene from Escherichia coli

Abstract

ABSTRACT The nitrogen assimilation control gene, nac , was detected in Escherichia coli but not in Salmonella typhimurium by Southern blotting, using a probe from the Klebsiella aerogenes nac ( nac K ) gene. The E. coli nac gene ( nac E ) was isolated from a cosmid clone by complementation of a nac mutation in K. aerogenes. nac E was fully functional in this complementation assay. DNA sequence analysis showed considerable divergence between nac E and nac K , with a predicted amino acid sequence identity of only 79% and most of the divergence in the C-terminal half of the protein sequence. The total predicted size of NAC E is 305 amino acids, the same as for NAC K . A null mutation, nac-28 , was generated by reverse genetics. Mutants bearing nac-28 have a variety of phenotypes related to nitrogen metabolism, including slower growth on cytosine, faster growth on arginine, and suppression of the failure of an Ntr-constitutive mutant to grow with serine as sole nitrogen source. In addition to a loss of nitrogen regulation of histidase formation, nac-28 mutants also showed a loss of a weak repression of glutamate dehydrogenase formation. This repression was unexpected because it is balanced by a NAC-independent activation of glutamate dehydrogenase formation during nitrogen-limited growth. Attempts to purify NAC E by using methods established for NAC K failed, and NAC E appears to be degraded with a half-life at 30°C as short as 15 min during inhibition of protein synthesis.