Thenac gene of E. coli and its role in nitrogen regulation.

The nac (nitrogen assimilation control) gene isolated from Escherichia coli complemented nac mutants in Klebsiella aerogenes. The nucleotide sequence of the gene was 70% identical to the nac gene from K. aerogenes. E. coli mutants deleted for the nac gene failed to activate a heterologous hutH gene from K. aerogenes. Primer extension experiments showed that nac transcription initiated at a unique adenine residue and was regulated by nitrogen availability in an NTR-dependent manner. NAC regulates two targets in E. coli. NAC binds the codBA operon promoter region and activates transcription both in vivo and in vitro. NAC binds at -59 bp from the start of transcription in this promoter, in contrast to the binding at -64 bp seen at NAC activated operons in Klebsiella aerogenes. Mutations in the NAC binding site of the codBA promoter that eliminated binding also eliminated NAC regulation. A second target, the gltBDF operon (which encodes glutamate synthase) was repressed by NAC. NAC binds weakly to the gltBDF promoter but the repression seen here may be indirect as NAC regulation persisted after deletion of the apparent NAC binding site. NAC$\sp{E}$ was insoluble in salt concentrations from 25 mM to 1.2 M. N-terminal fusions to a 56 amino acid leader or the maltose binding protein (MBP) resulted in soluble but transcriptionally inactive proteins still able to bind DNA. MBP-NAC fusions activated transcription following removal of the MBP tag, but degraded quickly. Addition of a 6 histidine tag to the C-terminus of NAC$\sp{E}$ increased activity in vivo, but not solubility in vitro. Carboxy terminal deletions of the NAC protein retaining as few as 99 N-terminal amino acids (of 305) retained significant in vivo activity. The properties of NAC proteins retaining 100, 120 and 129 N-terminal amino acids suggest that the N-terminal 100 amino acids of NAC contain domains for DNA binding, activation and dimerization that are locked in an activation competent conformation.PhDBiological SciencesGeneticsMicrobiologyMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/130077/2/9712046.pd