We have isolated insertion and deletion mutants in glnB, the structural gene of PI,, a member of the adenylylation system for glutamine synthetase of Escherichia coli, to study the role of PI, in the regulation of the synthesis of glutamine synithetase and of histidase in response to nitrogen deprivation or excess. We have studied the effects of this mutation alone and combined with nul mutations resulting from the insertion of transposons or from a deletion in the other genes affecting this regulation, ginD, glnF (ntrA), ginG (ntrC), and glnL (ntrB). Our rqsults confirm that only the products of glnF and ginG are essential for this regulation. In cells of the wild type, the response is mediated by the products of ginD and glnB via the product of glnL, In the condition of nitrogen excess, PII, the product of glnB, appears to convert the product of glnL to a form that prevents the activation of transcription of the structural gekes for glutamine synthetase and for histidase by the products of glnF and ginG. During nitrogen deprivation, uridylyltransferase, the product of ginD, is activated

Escherichia coli rpoN mutants lack or4 and are therefore unable to initiate the transcription of ginA at glnAp2, which is required for the production of a high intracellular concentration of glutamine synthetase. We have found that the dependence on a54 can be overcome by iutations that have apparently created a new r70-dependent promoter. The position -35 RNA polymerase contact site of this new promoter overlaps glnAp2.The initiation of transcription at the new promoter is inhibited by r54-RNA polymerase even in the absence of nitrogen regulator I-phosphate, the activator required for the initiation of transcription at glnAp2. The results suggest that in cells growing with an excess of nitrogen and therefore lacking nitrogen regulator I-phosphate, V54-RNA polymergse is bound at glnAp2.Glutamine synthetase is essential for the assimilation of ammonia when Escherichia coli and other species of enteric bacteria are grown in a nitrogen-limited medium. The level of glutamine synthetase and the level of proteins that facilitate the transport and degradation of nitrogen-containing compounds (Ntr systems, nitrogen regulated) are elevated during nitrogen-limited growth (8). The transcription of Ntr genes requires RNA polymerase containing the r54 protein, the product of rpoN (glnF ntrA) rather than the abundant cro protein. We have previously referred to o5 as u6O; however, it has been shown that the molecular weight of this protein based on the amino acid sequence predicted by the nucleotide sequence of rpoN is actually 54,000 (11). The promoters at which transcription is initiated by Cr54 RNA polymerase have the consensus sequence CTGGYAYRN4TTGCA (1,6,7,19).The transcription at these promoters is initiated when the cells experience nitrogen deprivation. The agents responsible for the initiation of transcription are the proteins nitrogen regulators I and II (NRI and NRII), the products, respectively, of glnG (ntrC) and glnL (ntrB). NRII is a kinase responsible for the conversion of NRI to NRI-phosphate, the actual activator of transcription (6,7,12,18). The phosphorylation of NRI is blocked by PI,, the product of glnB. Consequently, transcription requires the removal of PI,. This removal consists in its conversion to the uridylylated form P11-UMP. This reaction is catalyzed by uridylyl transferase, the product of gInD, activated by 2-ketoglutarate. Conversely, the same enzyme is activated by glutamine to remove the uridylyl group from P11-UMP (8,12). As a result of these interactions, the intracellular concentration of PI, and consequently the intracellular concentration of NRIphosphate, the transcriptional activator, is regulated by the intracellular ratio of glutamine to 2-ketoglutarate, a sensitive barometer of the availability of nitrogen. A high intracellular 2-ketoglutarate/glutamine ratio indicating nitrogen defi-* Corresponding author. ciency results in the initiation of transcription at Ntr promoters; a high intracellular glutamine/2-ketoglutarate ratio indicating nitrogen excess prevents the initiation of tra...

In Klebsiella aerogenes, the product of a his-linked gene, nac, appears to play a crucial role in tying the synthesis of enzymes activated or repressed by ammonia deprivation, such as histidase and glutamate dehydrogenase, to the known regulators of nitrogen assimilation, the products of glnG and glnF.

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