Alexander J. Ninfa

Modular cell biology: retroactivity and insulation

Modularity plays a fundamental role in the prediction of the behavior of a system from the behavior of its components, guaranteeing that the properties of individual components do not change upon interconnection. Just as electrical, hydraulic, and other physical systems often do not display modularity, nor do many biochemical systems, and specifically, genetic networks. Here, we study the effect of interconnections on the input–output dynamic characteristics of transcriptional components, focusing on a property, which we call ‘retroactivity’, that plays a role analogous to non‐zero output impedance in electrical systems. In transcriptional networks, retroactivity is large when the amount of transcription factor is comparable to, or smaller than, the amount of promoter‐binding sites, or when the affinity of such binding sites is high. To attenuate the effect of retroactivity, we propose a feedback mechanism inspired by the design of amplifiers in electronics. We introduce, in particular, a mechanism based on a phosphorylation–dephosphorylation cycle. This mechanism enjoys a remarkable insulation property, due to the fast timescales of the phosphorylation and dephosphorylation reactions.

PII signal transduction proteins

PII proteins, found in Bacteria, Archaea and plants, help coordinate carbon and nitrogen assimilation by regulating the activity of signal transduction enzymes in response to diverse signals. Recent studies of bacterial PII proteins have revealed a solution to the signal transduction problem of how to coordinate multiple receptors in response to diverse stimuli yet permit selective control of these receptors under various conditions and allow adaptation of the system as a whole to long-term stimulation.

Role of the GlnK signal transduction protein in the regulation of nitrogen assimilation in Escherichia coli

Two structurally similar but functionally distinct PII‐like 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 phosphorylation state of the transcriptional activator NRI∼P (NtrC∼P, 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− 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 NRI∼P during conditions of severe nitrogen starvation, and by so doing to contribute to the regulation of certain Ntr genes.

Dimerization Is Required for the Activity of the Protein Histidine Kinase CheA That Mediates Signal Transduction in Bacterial Chemotaxis

The histidine protein kinase CheA plays an essential role in stimulus-response coupling during bacterial chemotaxis. The kinase is a homodimer that catalyzes the reversible transfer of a γ-phosphoryl group from ATP to the N-3 position of one of its own histidine residues. Kinetic studies of rates of autophosphorylation show a second order dependence on CheA concentrations at submicromolar levels that is consistent with dissociation of the homodimer into inactive monomers. The dissociation was confirmed by chemical cross-linking studies. The dissociation constant (CheA2 ↔ 2CheA; KD = 0.2-0.4 μM) was not affected by nucleotide binding, histidine phosphorylation, or binding of the response regulator, CheY. The turnover number per active site within a dimer (assuming 2 independent sites/dimer) at saturating ATP was approximately 10/min. The kinetics of autophosphorylation and ATP/ADP exchange indicated that the dissociation constants of ATP and ADP bound to CheA were similar (KD values ≈ 0.2-0.3 mM), whereas ATP had a reduced affinity for CheA∼P (KD ≈ 0.8 mM) compared with ADP (KD≈ 0.3 mM). The rates of phosphotransfer from bound ATP to the phosphoaccepting histidine and from the phosphohistidine back to ADP seem to be essentially equal (kcat ≈ 10 min−).

Characterization of the GlnK protein of Escherichia coli

The GlnK and PII signal transduction proteins are paralogues that play distinct roles in nitrogen regulation. Although cells lacking GlnK appear to have normal nitrogen regulation, in the absence of PII, the GlnK protein controls nitrogen assimilation by regulating the activities of the PII receptors glutamine synthetase adenylyltransferase (ATase) and the kinase/phosphatase nitrogen regulator II (NRII or NtrB), which controls transcription from nitrogen‐regulated promoters. Here, the wild‐type GlnK protein and two mutant forms of GlnK were purified, and their activities were compared with those of PII using purified components. GlnK and PII were observed to have unique properties. Both PII and GlnK were potent activators of the phosphatase activity of NRII, although PII was slightly more active. In contrast, PII was approximately 40‐fold more potent than GlnK in the activation of the adenylylation of glutamine synthetase by ATase. While both GlnK and PII were readily uridylylated by the uridylyltransferase activity of the signal‐transducing uridylyltransferase/uridylyl‐removing enzyme (UTase/UR), only PII∼UMP was effectively deuridylylated by the UR activity of the UTase/UR. Finally, there were subtle differences in the regulation of GlnK activity by the small molecule effector 2‐ketoglutarate compared with the regulation of PII activity by this effector. Altogether, these results suggest that GlnK is unlikely to play a significant role in the regulation of ATase in wild‐type cells, and that the main role of GlnK may be to contribute to the regulation of NRII and perhaps additional, unknown receptors in nitrogen‐starved cells. Also, the slow deuridylylation of GlnK∼UMP by the UTase/UR suggests that rapid interconversion of GlnK between uridylylated and unmodified forms is not necessary for GlnK function. One mutant form of GlnK, containing the alteration R47W, was observed to lack specifically the ability to activate the NRII phosphatase in vitro ; it was able to be uridylylated by the UTase/UR and to activate the adenylylation activity of ATase. Another mutant form of GlnK, containing the Y51N alteration at the site of uridylylation, was not uridylylated by the UTase/UR and was defective in the activation of both the NRII phosphatase activity and the ATase adenylylation activity.

Integration of antagonistic signals in the regulation of nitrogen assimilation in Escherichia coli

Publisher Summary To maintain balanced metabolism, Escherichia coli must coordinate the assimilation of nitrogen with the assimilation of carbon and other essential nutrients. This coordination is accomplished in part by a signal transduction system that measures the signals of carbon and nitrogen status and regulates the activity of glutamine synthetase (GS) and the transcription of nitrogen-regulated (Ntr) genes, whose products facilitate the use of poor nitrogen sources. The key sensory components of this signal transduction system are the uridylyltransferase/ uridylyl-removing enzyme (UTase/UR), PII protein, and adenylyltransferase (ATase) that regulates GS by reversible adenylylation. This chapter discusses the current state of understanding of these signal-transducing proteins and the mechanisms by which they detect and transduce the signals of nitrogen and carbon status. It also discusses the physiology of the response to nitrogen and carbon availability and presents an overview of the signal transduction system.

Activation of the glnA, glnK, and nac promoters as Escherichia coli undergoes the transition from nitrogen excess growth to nitrogen starvation

The nitrogen-regulated genes and operons of the Ntr regulon of Escherichia coli are activated by the enhancer-binding transcriptional activator NRI approximately P (NtrC approximately P). Here, we examined the activation of the glnA, glnK, and nac promoters as cells undergo the transition from growth on ammonia to nitrogen starvation and examined the amplification of NRI during this transition. The results indicate that the concentration of NRI is increased as cells become starved for ammonia, concurrent with the activation of Ntr genes that have less- efficient enhancers than does glnA. A diauxic growth pattern was obtained when E. coli was grown on a low concentration of ammonia in combination with arginine as a nitrogen source, consistent with the hypothesis that Ntr genes other than glnA become activated only upon amplification of the NRI concentration.

Signaling properties of a covalent modification cycle are altered by a downstream target

We used a model system of purified components to explore the effects of a downstream target on the signaling properties of a covalent modification cycle, an example of retroactivity. In the experimental system used, a bifunctional enzyme catalyzed the modification and demodification of its substrate protein, with both activities regulated by a small molecule stimulus. Here we examined how a downstream target for one or both forms of the substrate of the covalent modification cycle affected the steady-state output of the system, the sensitivity of the response to the stimulus, and the concentration of the stimulus required to provide the half-maximal response (S50). When both the modified and unmodified forms of the substrate protein were sequestered by the downstream target, the sensitivity of the response was dramatically decreased, but the S50 was only modestly affected. Conversely, when the downstream target only sequestered the unmodified form of the substrate protein, significant effects were observed on both system sensitivity and S50. Behaviors of the experimental systems were well approximated both by simple models allowing analytical solutions and by a detailed model based on the known interactions and enzymatic activities. Modeling and experimentation indicated that retroactivity may result in subsensitive responses, even if the covalent modification cycle displays significant ultrasensitivity in the absence of retroactivity. Thus, we provide examples of how a downstream target can alter the signaling properties of an upstream signal transduction covalent modification cycle.

Physiological role of the GlnK signal transduction protein of Escherichia coli : survival of nitrogen starvation

Escherichia coli contains two PII‐like signal trans‐duction proteins, PII and GlnK, involved in nitrogen assimilation. We examined the roles of PII and GlnK in controlling expression of glnALG , glnK and nac during the transition from growth on ammonia to nitrogen starvation and vice versa. The PII protein exclusively controlled glnALG expression in cells adapted to growth on ammonia, but was unable to limit nac and glnK expression under conditions of nitrogen starvation. Conversely, GlnK was unable to limit glnALG expression in cells adapted to growth on ammonia, but was required to limit expression of the glnK and nac promoters during nitrogen starvation. In the absence of GlnK, very high expression of the glnK and nac promoters occurred in nitrogen‐starved cells, and the cells did not reduce glnK and nac expression when given ammonia. Thus, one specific role of GlnK is to regulate the expression of Ntr genes during nitrogen starvation. GlnK also had a dramatic effect on the ability of cells to survive nitrogen starvation and resume rapid growth when fed ammonia. After being nitrogen starved for as little as 10 h, cells lacking GlnK were unable to resume rapid growth when given ammonia. In contrast, wild‐type cells that were starved immediately resumed rapid growth when fed ammonia. Cells lacking GlnK also showed faster loss of viability during extended nitrogen starvation relative to wild‐type cells. This complex phenotype resulted partly from the requirement for GlnK to regulate nac expression; deletion of nac restored wild‐type growth rates after ammonia starvation and refeeding to cells lacking GlnK, but did not improve viability during nitrogen starvation. The specific roles of GlnK during nitrogen starvation were not the result of a distinct function of the protein, as expression of PII from the glnK promoter in cells lacking GlnK restored the wild‐type phenotypes.

Building biological memory by linking positive feedback loops

A common topology found in many bistable genetic systems is two interacting positive feedback loops. Here we explore how this relatively simple topology can allow bistability over a large range of cellular conditions. On the basis of theoretical arguments, we predict that nonlinear interactions between two positive feedback loops can produce an ultrasensitive response that increases the range of cellular conditions at which bistability is observed. This prediction was experimentally tested by constructing a synthetic genetic circuit in Escherichia coli containing two well-characterized positive feedback loops, linked in a coherent fashion. The concerted action of both positive feedback loops resulted in bistable behavior over a broad range of inducer concentrations; when either of the feedback loops was removed, the range of inducer concentrations at which the system exhibited bistability was decreased by an order of magnitude. Furthermore, bistability of the system could be tuned by altering growth conditions that regulate the contribution of one of the feedback loops. Our theoretical and experimental work shows how linked positive feedback loops may produce the robust bistable responses required in cellular networks that regulate development, the cell cycle, and many other cellular responses.