Repression of Glutamate Dehydrogenase Formation in Klebsiella aerogenes Requires Two Binding Sites for the Nitrogen Assimilation Control Protein, NAC

Enteric bacteria such as Klebsiella aerogenes and Escherichia coli use two routes to assimilate ammonia from the growth medium into organic material (16). When ammonia is abundant, glutamate dehydrogenase (GDH) can reductively aminate α-ketoglutarate in an NADPH-dependent reaction that yields glutamate directly. When the concentration of ammonia is too low to be efficiently fixed by GDH, which has a Km for ammonia in the millimolar range (17), enteric bacteria use the other route for ammonia fixation via the combined action of glutamine synthetase and glutamate synthase. Either route is effective when ammonia is abundant, although GDH confers a distinct growth advantage under conditions of energy limitation (9). However, under conditions of nitrogen limitation, GDH serves little or no function in ammonia fixation. The formation of GDH is subject to complex regulation. In K. aerogenes, GDH is strongly repressed under conditions of nitrogen-limited growth (16). This repression requires the well-studied Ntr system and is mediated by the nitrogen assimilation control protein, NAC, the synthesis of which is under direct Ntr control (14). Under conditions of nitrogen-limited growth, the Ntr system activates nac gene expression, and the resulting NAC accumulation represses gdhA expression. Under conditions of nitrogen excess, the Ntr system fails to activate nac gene expression, and in the absence of NAC, gdhA expression is not repressed. Mutants that lack NAC do not repress GDH formation under conditions of nitrogen-limited growth, and in mutants in which NAC expression is inducible by isopropyl-β-d-thiogalactopyranoside (IPTG), GDH expression is repressed by IPTG, independent of the quality of the nitrogen source (26). It is clear that NAC is both necessary and sufficient for the repression of GDH formation in vivo, but the mechanism of this repression is unknown. In addition, GDH formation is repressed about threefold when lysine is present in the growth medium (11). Little is known about this phenomenon, but the target for the effector appears to overlap a region where NAC binds (21). Finally, GDH expression in K. aerogenes is also repressed about 20-fold by growth in rich medium. However the focus of the experiments described here is the nitrogen regulation of GDH expression by NAC. NAC is a homodimeric protein with a subunit molecular weight of 32,759 (25). It is a member of a large family of transcriptional regulatory proteins known as the LysR family, many of which are homotetramers (24). In vitro transcription experiments have shown that purified NAC is able to activate transcription of the hut operons and repress the nac gene of K. aerogenes (5, 6). Experiments with the nac gene under the control of an IPTG-inducible promoter have shown that NAC is an activator of the ureDABCEFG (26), putP (14), codBA (W. B. Muse and R. A. Bender, unpublished observations), and dadAB operons (10), as well as a repressor of gdhA (26) in K. aerogenes. NAC activates transcription by binding to a site on the DNA near the RNA polymerase binding site, often at −64 relative to the start of transcription. The sites to which NAC binds contain the nucleotides ATA-N9-TAT, and these have been shown to be important for NAC binding and for NAC's ability to activate transcription (22). Point mutations affecting these six nucleotides reduce, but do not generally abolish, the ability of NAC to bind and activate (22). Promoters in which NAC represses transcription do not contain this sequence, but do have a sequence that is similar, ATA-N9-GAT (or its equivalent on the opposite strand, ATC-N9-TAT), which is thought to serve as a binding site for NAC in these promoters (5; unpublished observation). As part of our long-term interest in the role of NAC in bacterial nitrogen metabolism, we began a study of NAC-mediated repression of GDH formation in K. aerogenes and found that the mechanism is complex and involves an interaction between two separate NAC binding sites near the promoter of the gdhA gene coding for GDH.