Analysis of the system design principles of signaling systems requires model systems where all components and regulatory interactions are known. Components of the Lac and Ntr systems were used to construct genetic circuits that display toggle switch or oscillatory behavior. Both devices contain an "activator module" consisting of a modified glnA promoter with lac operators, driving the expression of the activator, NRI. Since NRI activates the glnA promoter, this creates an autoactivated circuit repressible by LacI. The oscillator contains a "repressor module" consisting of the NRI-activated glnK promoter driving LacI expression. This circuitry produced synchronous damped oscillations in turbidostat cultures, with periods much longer than the cell cycle. For the toggle switch, LacI was provided constitutively; the level of active repressor was controlled by using a lacY mutant and varying the concentration of IPTG. This circuitry provided nearly discontinuous expression of activator.

Nitrogen regulation of transcription in Escherichia coli requires sensation of the intracellular nitrogen status and control of the dephosphorylation of the transcriptional activator NRI-P. This dephosphorylation is catalyzed by the bifunctional kinase/phosphatase NRII in the presence of the dissociable PII protein. The ability of PII to stimulate the phosphatase activity of NRII is regulated by a signal transducing uridylyltransferase/uridylyl-removing enzyme (UTase/UR), which converts PII to PII-UMP under conditions of nitrogen starvation; this modification prevents PII from stimulating the dephosphorylation of NRI approximately P. We used purified components to examine the binding of small molecules to PII, the effect of small molecules on the stimulation of the NRII phosphatase activity by PII, the retention of PII on immobilized NRII, and the regulation of the uridylylation of PII by the UTase/UR enzyme. Our results indicate that PII is activated upon binding ATP and either 2-ketoglutarate or glutamate, and that the liganded form of PII binds much better to immobilized NRII. We also demonstrate that the concentration of glutamine required to inhibit the uridylyltransferase activity is independent of the concentration of 2-ketoglutarate present. We hypothesize that nitrogen sensation in E. coli involves the separate measurement of glutamine by the UTase/UR protein and 2-ketoglutarate by the PII protein.

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SummaryTwo structurally similar but functionally distinct PIIlike proteins, PII and GlnK, regulate nitrogen assimilation in Escherichia coli. Studies with cells indicated that both PII (the glnB product) and GlnK (the glnK product) acted through the kinase/phosphatase NRII [NtrB, the glnL (ntrB ) product] to reduce transcription initiation from Ntr promoters, apparently by regulating the phosphor ylation state of the transcriptional activator NRIϳP (NtrCϳP, the phosphorylated form of the glnG (ntrC ) product). Both GlnK and PII also acted through adenylyltransferase (ATase, the glnE product) to regulate the adenylylation state of glutamine synthetase (GS). The activity of both GlnK and PII was regulated by the signal-transducing uridylyltransferase/ uridylyl-removing enzyme (UTase/UR, glnD product). Our experiments indicate that either PII or GlnK could effectively regulate ATase, but that PII was required for the efficient regulation of NRII required to prevent expression of glnA , which encodes GS. Yet, GlnK also participated in regulation of NRII. Although cells that lack either PII or GlnK grew well, cells lacking both of these proteins were defective for growth on nitrogen-rich minimal media. This defect was alleviated by the loss of NRII, and was apparently due to unregulated expression of the Ntr regulon. Also, mutations in glnK, designated glnK *, were obtained as suppressors of the Ntr ¹ phenotype of a double mutant lacking PII and the UTase/UR. These suppressors appeared to reduce, but not eliminate, the ability of GlnK to prevent Ntr gene expression by acting through NRII. We hypothesize that one role of GlnK is to regulate the expression of the level of NRIϳP during conditions of severe nitrogen starvation, and by so doing to contribute to the regulation of certain Ntr genes.

Transcription of the Ntr regulon is controlled by the two-component system consisting of the response regulator NRI (NtrC) and the kinase/phosphatase NRI, (NtrB), which both phosphorylates and dephosphorylates NRI. Even though in vitro transcription from nitrogen-regulated promoters requires phosphorylated NRI, NRII-independent activation of NRI also occurs in vivo. We show here that this activation likely involves acetyl phosphate; it is eliminated by mutations that reduce synthesis of acetyl phosphate and is elevated by a mutation expected to cause accumulation of acetyl phosphate. With purified components, we investigated the mechanism by which acetyl phosphate stimulates glutamine synthetase synthesis. Acetyl phosphate, carbamyl phosphate, and phosphoramidate but not ATP or phosphoenolpyruvate acted as substrates for the autophosphorylation of NRI in vitro. Phosphorylated NRI produced by this mechanism exhibited the properties associated with NRI phosphorylated by NRn, including the activated ATPase activity of the central domain of NRI and the ability to activate transcription from the nitrogen-regulated glutamine synthetase ginAp2 promoter.The Ntr regulon of enteric bacteria is a collection of genes and operons that are regulated by the availability of ammonia and whose products facilitate survival under nitrogenlimiting growth conditions. The most important enzyme for the assimilation of ammonia under nitrogen-limiting conditions is glutamine synthetase (GS), the product of the glnA gene. Under nitrogen-excess conditions, a low intracellular concentration of this enzyme results from transcription initiated at a o70-dependent promoter known as gbnApl. Under nitrogen-limiting conditions, a much higher intracellular concentration of this enzyme results from transcription from a nitrogen-regulated promoter known asglnAp2. The activation of transcription from the glnAp2 promoter of enteric bacteria and other nitrogen-regulated promoters in intact cells and by purified components has been studied in some detail (reviewed in references 12, 13, 19, and 32). Transcription from theglnAp2 promoter requires RNA polymerase containing the alternative sigma factor a54 (8,9). This polymerase binds tightly to the glnAp2 promoter sequence, but it lacks the capacity to melt the DNA in the region surrounding the transcription start site (26,29). The formation of an open complex by or" RNA polymerase requires a transcriptional activator; for the glnAp2 promoter, this activator is the phosphorylated form of NR, (NtrC [21]). The efficient action of phosphorylated NR, (NR,-P) in bringing about the activation of transcription is facilitated by high-affinity binding sites on the template; these sites can be located far from the promoter and are functionally analogous to enhancer sequences (23, 28). The enhancers serve to increase the local concentration of NRI-P near the promoter (38). NRI-P, bound to its enhancer, interacts with &rI4 RNA polymerase at the promoter by means of a DNA loop (33) and, by so doing, somehow brings abou...