Mariette R. Atkinson

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.

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.

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.

Context-Dependent Functions of the PII and GlnK Signal Transduction Proteins in Escherichia coli

Two closely related signal transduction proteins, PII and GlnK, have distinct physiological roles in the regulation of nitrogen assimilation. Here, we examined the physiological roles of PII and GlnK when these proteins were expressed from various regulated or constitutive promoters. The results indicate that the distinct functions of PII and GlnK were correlated with the timing of expression and levels of accumulation of the two proteins. GlnK was functionally converted into PII when its expression was rendered constitutive and at the appropriate level, while PII was functionally converted into GlnK by engineering its expression from the nitrogen-regulated glnK promoter. Also, the physiological roles of both proteins were altered by engineering their expression from the nitrogen-regulated glnA promoter. We hypothesize that the use of two functionally identical PII-like proteins, which have distinct patterns of expression, may allow fine control of Ntr genes over a wide range of environmental conditions. In addition, we describe results suggesting that an additional, unknown mechanism may control the cellular level of GlnK.

Governor of the glnAp2 promoter of Escherichia coli

Low‐affinity sites for the activator NRI∼P (NtrC∼P) that map between the enhancer and the glnAp2 promoter were responsible for limiting promoter activity at high concentrations of NRI∼P in intact cells and in an in vitro transcription system consisting of purified bacterial components. That is, the low‐affinity sites constitute a ‘governor’, limiting the maximum promoter activity. As the governor sites are themselves far from the promoter, they apparently act either by preventing the formation of the activation DNA loop that brings the enhancer‐bound activator and the promoter‐bound polymerase into proximity or by preventing a productive interaction between the enhancer‐bound activator and polymerase. The combination of potent enhancer and governor sites at the glnAp2 promoter provides for efficient activation of the promoter when the activator concentration is low, while limiting the maximum level of promoter activity when the activator concentration is high.

Using Two‐Component Systems and Other Bacterial Regulatory Factors for the Fabrication of Synthetic Genetic Devices

Synthetic biology is an emerging field in which the procedures and methods of engineering are extended living organisms, with the long-term goal of producing novel cell types that aid human society. For example, engineered cell types may sense a particular environment and express gene products that serve as an indicator of that environment or affect a change in that environment. While we are still some way from producing cells with significant practical applications, the immediate goals of synthetic biology are to develop a quantitative understanding of genetic circuitry and its interactions with the environment and to develop modular genetic circuitry derived from standard, interoperable parts that can be introduced into cells and result in some desired input/output function. Using an engineering approach, the input/output function of each modular element is characterized independently, providing a toolkit of elements that can be linked in different ways to provide various circuit topologies. The principle of modularity, yet largely unproven for biological systems, suggests that modules will function appropriately based on their design characteristics when combined into larger synthetic genetic devices. This modularity concept is similar to that used to develop large computer programs, where independent software modules can be independently developed and later combined into the final program. This chapter begins by pointing out the potential usefulness of two-component signal transduction systems for synthetic biology applications and describes our use of the Escherichia coli NRI/NRII (NtrC/NtrB) two-component system for the construction of a synthetic genetic oscillator and toggle switch for E. coli. Procedures for conducting measurements of oscillatory behavior and toggle switch behavior of these synthetic genetic devices are described. It then presents a brief overview of device fabrication strategy and tactics and presents a useful vector system for the construction of synthetic genetic modules and positioning these modules onto the bacterial chromosome in defined locations.

A Synthetic Biology Approach to Understanding Biological Oscillations: Developing a Genetic Oscillator for Escherichia coli

Our goals are to construct a simple genetic clock that will stably oscillate in Escherichia coli and to identify the design principles and parameters responsible for oscillations. We previously described a simple genetic circuit of linked activator and repressor operons that produced damped oscillations. Here, we altered the repression of the activator operon and identified an oscillator that produces improved oscillations over our initial system. We also explored mathematical models of the oscillator. Toy models were used to investigate the behaviors that may be obtained from our clock circuitry. Depending on parameters, the circuitry produced a wide array of oscillatory systems, including sinusoidal and relaxation oscillators. We also attempted to explicitly model all known interactions that affect the oscillator, producing a 32-dimensional ODE model. This model can produce results similar to those obtained in experiments, and we have begun attempts to fit experimental data to the model.