Repression of glutamate dehydrogenase formation in Klebsiella aerogenes requires two binding sites for the nitrogen assimilation control protein, NAC.

In Klebsiella aerogenes, the gdhA gene codes for glutamate dehydrogenase, one of the enzymes responsible for assimilating ammonia into glutamate. Expression of a gdhAp-lacZ transcriptional fusion was strongly repressed by the nitrogen assimilation control protein, NAC. This strong repression (>50-fold under conditions of severe nitrogen limitation) required the presence of two separate NAC binding sites centered at -89 and +57 relative to the start of gdhA transcription. Mutants lacking either or both of these sites lost the strong repression. The distance between the two sites was less important than the face of the helix on which they lay. Insertion or deletion of 10 bp between the sites had little effect on the strong repression, but insertion of 5 bp or deletion of either 5 or 15 bp decreased the repression significantly. We propose that the strong repression of gdhAp-lacZ expression requires an interaction between the NAC molecules bound at the two sites. A weaker repression of gdhAp-lacZ expression (about threefold) required only the NAC site centered at -89. This weaker repression appears to result from NAC's ability to prevent the action of a positive effector the target of which overlaps the NAC binding site centered at -89. Point mutations and deletions of this region result in the same threefold reduction in gdhAp-lacZ expression as the presence of NAC at this site.