Receptor sensitivity in bacterial chemotaxis

Abstract

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...

Universal architecture of bacterial chemoreceptor arrays

“…Tightly coupled, communicating chemoreceptor arrays are thought to enable the main features of the signaling mechanism: heightened sensitivity (28), signal gain (29), cooperativity (30,31), and adaptation (32,33). The universal hexagonal architecture and secondary structure of chemoreceptor arrays we observed in diverse bacterial species therefore implies that the trimer-of-dimers arrangement and the underlying signaling mechanism are preserved over long evolutionary distances (Fig.…”

Spatiotemporal modelling of CheY complexes in Escherichia coli chemotaxis

“…This simple description has been used by a number of authors as described in Albert et al (2004) to link the extracellular concentration of an attractant ligand to the change in autophosphorylation rate of CheA. In what follows we take K ¼ 2.6mM and h ¼ 1.2 (Sourjik and Berg, 2002b) and consider the bacterial response to an attractant of concentration L ¼ 10 mM. The ligand concentration is described by L ¼ 10 mM;…”

Bacterial chemoreceptors: high-performance signaling in networked arrays

“…By contrast, Nanodisc-segregated dimers were ineffective at kinase activation, which required more than one dimer per disc [40]. Peak kinase activation occurred in preparations averaging almost three dimers per disc, and this activation was inhibited by ligand, as it is in intact cells [10]. An optimum for ligand-controlled kinase activation at approximately three dimers per disc is tantalizing because it correlates activation with potential formation of trimers-of-dimers, although these results cannot rule out activation by other oligomer sizes.…”

“…a 1% change in receptor occupancy elicits a 50% change in the rotational bias of the flagellar motors [9], a minimal estimate because receptors act in intermixed arrays [10]). Elegant measurements of CheA kinase activity in vivo using fluorescence resonance energy transfer (FRET) showed that much of this amplification (36-fold) occurs at the signaling complex [10]. In many signaling systems the mechanism of gain involves enzyme activation, generating many downstream signaling molecules from an occupancy change at one receptor.…”