Chemoreceptors in Escherichia coli are coupled to the flagella by a labile phosphorylated intermediate, CheYϳP. Its activity can be inferred from the rotational bias of flagellar motors, but motor response is stochastic and limited to a narrow physiological range. Here we use fluorescence resonance energy transfer to monitor interactions of CheYϳP with its phosphatase, CheZ, that reveal changes in the activity of the receptor kinase, CheA, resulting from the addition of attractants or repellents. Analyses of cheR and͞or cheB mutants, defective in receptor methylation͞demethylation, show that response sensitivity depends on the activity of CheB and the level of receptor modification. In cheRcheB mutants, the concentration of attractant that generates a half-maximal response is equal to the dissociation constant of the receptor. In wild-type cells, it is 35 times smaller. This amplification, together with the ultrasensitivity of the flagellar motor, explains previous observations of high chemotactic gain.Escherichia coli ͉ fluorescence ͉ fluorescence resonance energy transfer B acteria move up spatial gradients of chemical attractants in a biased random walk, now running smoothly, now tumbling randomly, extending runs that carry them in a favorable direction (1). The central point of regulation in the chemotaxis signal transduction pathway is the level of phosphorylation of the diffusible signaling protein CheY (2). CheY is phosphorylated by the kinase CheA that is coupled to specific receptors (3). CheYϳP binds to FliM, a component of the switch complex, at the cytoplasmic face of the flagellar motor and modulates the direction of motor rotation (4-6). The phosphatase CheZ binds to CheYϳP and accelerates its dephosphorylation (7,8). The receptor is thought to exist in two states, active (activating CheA) and inactive (inhibiting CheA) (9). Attractant binding lowers the probability of the active state, decreasing the level of CheYϳP. Attractant removal raises the probability of the active state, increasing the level of CheYϳP. Adaptation is by receptor methylation by CheR and demethylation by CheB (10-12). Receptor methylation compensates for attractant binding, with the modified receptor more likely to be in the active state. Thus, CheR promotes adaptation to increasing levels of attractants, and CheB promotes adaptation to decreasing levels of attractants. Two major chemoreceptors exist in Escherichia coli: the aspartate receptor, Tar, and serine receptor, Tsr. Tar has four methylation sites, which are glutamates; however, two of these are expressed as glutamines, deamidated by CheB. The glutamines are thought to be physiologically equivalent to methylated glutamates (13).We used fluorescence resonance energy transfer (FRET) to measure intracellular changes in phosphorylation-dependent interactions of CheY upon chemotactic stimulation. FRET, which relies on the distance-dependent transfer of energy from an excited donor fluorophore to an acceptor fluorophore, is one of the few tools available for monitoring prot...

Bacterial chemotaxis is a model system for signal transduction, noted for its relative simplicity, high sensitivity, wide dynamic range and robustness. Changes in ligand concentrations are sensed by a protein assembly consisting of transmembrane receptors, a coupling protein (CheW) and a histidine kinase (CheA). In Escherichia coli, these components are organized at the cell poles in tight clusters that contain several thousand copies of each protein. Here we studied the effects of variation in the composition of clusters on the activity of the kinase and its sensitivity to attractant stimuli, monitoring responses in vivo using fluorescence resonance energy transfer. Our results indicate that assemblies of bacterial chemoreceptors work in a highly cooperative manner, mimicking the behaviour of allosteric proteins. Conditions that favour steep responses to attractants in mutants with homogeneous receptor populations also enhance the sensitivity of the response in wild-type cells. This is consistent with a number of models that assume long-range cooperative interactions between receptors as a general mechanism for signal integration and amplification.

Adaptation is the essential process by which an organism becomes better suited to its environment. The benefits of adaptation are well documented, but the cost it incurs remains poorly understood. Here, by analysing a stochastic model of a minimum feedback network underlying many sensory adaptation systems, we show that adaptive processes are necessarily dissipative, and continuous energy consumption is required to stabilize the adapted state. Our study reveals a general relation among energy dissipation rate, adaptation speed and the maximum adaptation accuracy. This energy-speed-accuracy relation is tested in the Escherichia coli chemosensory system, which exhibits near-perfect chemoreceptor adaptation. We identify key requirements for the underlying biochemical network to achieve accurate adaptation with a given energy budget. Moreover, direct measurements confirm the prediction that adaptation slows down as cells gradually de-energize in a nutrient-poor medium without compromising adaptation accuracy. Our work provides a general framework to study cost-performance tradeoffs for cellular regulatory functions and information processing.

Bacteria swim by means of rotating flagella that are powered by ion influx through membrane-spanning motor complexes. Escherichia coli and related species harness a chemosensory and signal transduction machinery that governs the direction of flagellar rotation and allows them to navigate in chemical gradients. Here, we show that Escherichia coli can also fine-tune its swimming speed with the help of a molecular brake (YcgR) that, upon binding of the nucleotide second messenger cyclic di-GMP, interacts with the motor protein MotA to curb flagellar motor output. Swimming velocity is controlled by the synergistic action of at least five signaling proteins that adjust the cellular concentration of cyclic di-GMP. Activation of this network and the resulting deceleration coincide with nutrient depletion and might represent an adaptation to starvation. These experiments demonstrate that bacteria can modulate flagellar motor output and thus swimming velocity in response to environmental cues.