A concentration-dependent switch in the bacterial response to temperature

We observed that bacteria grown below a critical concentration, in batch-mode cultures, swim towards warm regions when subjected to a temperature gradient. Above that concentration, they swim towards colder regions. Our findings indicate that the secreted intercellular signal, glycine, mediates this switch through methylation of Tsr receptors. At high bacterial concentration, the switch is reinforced by an inversion of the Tar/Tsr expression ratio.     

Cooperative signaling among bacterial chemoreceptors

Four chemoreceptors in Escherichia coli mediate responses to chemicals in the environment. The receptors self-associate and localize to the cell poles. This aggregation implies that interactions among receptors are important parameters of signal processing during chemotaxis. We examined this phenomenon using a receptor-coupled in vitro assay of CheA kinase activity. The ability of homogeneous populations of the serine receptor Tsr and the aspartate receptor Tar to stimulate CheA was directly proportional to the ratio of the receptor to total protein in cell membranes up to a fraction of 50%. Membranes containing mixed populations of Tar and Tsr supported an up to 4-fold greater stimulation of CheA than expected on the basis of the contributions of the individual receptors. Peak activity was seen at a Tar:Tsr ratio of 1:4. This synergy was observed only when the two proteins were expressed simultaneously, suggesting that, under our conditions, the fundamental "cooperative receptor unit" is relatively static, even in the absence of CheA and CheW. Finally, we observed that inhibition of receptor-stimulated CheA activity by serine or aspartate required significantly higher concentrations of ligand for membranes containing mixed Tsr and Tar populations than for membranes containing only Tsr (up to 10(2)-fold more serine) or Tar (up to 10(4)-fold more aspartate). Together with recent analyses of the interactions of Tsr and Tar in vivo, our results reveal the emergent properties of mixed receptor populations and emphasize their importance in the integrated signal processing that underlies bacterial chemotaxis.     

Chemotactic Adaptation Is Altered by Changes in the Carboxy-Terminal Sequence Conserved among the Major Methyl-Accepting Chemoreceptors

In Escherichia coli and Salmonella typhimurium, methylation and demethylation of receptors are responsible for chemotactic adaptation and are catalyzed by the methyltransferase CheR and the methylesterase CheB, respectively. Among the chemoreceptors of these species, Tsr, Tar, and Tcp have a well-conserved carboxy-terminal motif (NWET/SF) that is absent in Trg and Tap. When they are expressed as sole chemoreceptors, Tsr, Tar, and Tcp support good adaptation, but Trg and Tap are poorly methylated and supported only weak adaptation. It was recently discovered that CheR binds to the NWETF sequence of Tsr in vitro. To examine the physiological significance of this binding, we characterized mutant receptors in which this pentapeptide sequence was altered. C-terminally-mutated Tar and Tcp expressed in a receptorless E. coli strain mediated responses to aspartate and citrate, respectively, but their adaptation abilities were severely impaired. Their expression levels and attractant-sensing abilities were similar to those of the wild-type receptors, but the methylation levels of the mutant receptors increased only slightly upon addition of attractants. When CheR was overproduced, both the adaptation and methylation profiles of the mutant Tar receptor became comparable to those of wild-type Tar. Furthermore, overproduction of CheR also enhanced adaptive methylation of wild-type Trg, which lacks the NWETF sequence, in the absence of any other chemoreceptor. These results suggest that the pentapeptide sequence facilitates effective adaptation and methylation by recruiting CheR.     

Effects of population density and chemical environment on the behavior of Escherichia coli in shallow temperature gradients

In shallow temperature gradients, changes in temperature that bacteria experience occur over long time scales. Therefore, slow processes such as adaptation, metabolism, chemical secretion and even gene expression become important. Since these are cellular processes, the cell density is an important parameter that affects the bacteria's response. We find that there are four density regimes with distinct behaviors. At low cell density, bacteria do not cause changes in their chemical environment; however, their response to the temperature gradient is strongly influenced by it. In the intermediate cell-density regime, the consumption of nutrients becomes significant and induces a gradient of nutrients opposing the temperature gradient due to higher consumption rate at the high temperature. This causes the bacteria to drift toward low temperature. In the high cell-density regime, interactions among bacteria due to secretion of an attractant lead to a strong local accumulation of bacteria. This together with the gradient of nutrients, resulted from the differential consumption rate, creates a fast propagating pulse of bacterial density. These observations are a result of classical nonlinear population dynamics. At extremely high cell density, a change in the physiological state of the bacteria is observed. The bacteria, at the individual level, become cold seeking. This appears initially as a result of a change in the methylation level of the two most abundant sensing receptors, Tsr and Tar. It is further enforced at an even higher cell density by a change in the expression level of these receptors.     

Phenol sensing by Escherichia coli chemoreceptors: a nonclassical mechanism

The four transmembrane chemoreceptors of Escherichia coli sense phenol as either an attractant (Tar) or a repellent (Tap, Trg, and Tsr). In this study, we investigated the Tar determinants that mediate its attractant response to phenol and the Tsr determinants that mediate its repellent response to phenol. Tar molecules with lesions in the aspartate-binding pocket of the periplasmic domain, with a foreign periplasmic domain (from Tsr or from several Pseudomonas chemoreceptors), or lacking nearly the entire periplasmic domain still mediated attractant responses to phenol. Similarly, Tar molecules with the cytoplasmic methylation and kinase control domains of Tsr still sensed phenol as an attractant. Additional hybrid receptors with signaling elements from both Tar and Tsr indicated that the transmembrane (TM) helices and HAMP domain determined the sign of the phenol-sensing response. Several amino acid replacements in the HAMP domain of Tsr, particularly attractant-mimic signaling lesions at residue E248, converted Tsr to an attractant sensor of phenol. These findings suggest that phenol may elicit chemotactic responses by diffusing into the cytoplasmic membrane and perturbing the structural stability or position of the TM bundle helices, in conjunction with structural input from the HAMP domain. We conclude that behavioral responses to phenol, and perhaps to temperature, cytoplasmic pH, and glycerol, as well, occur through a general sensing mechanism in chemoreceptors that detects changes in the structural stability or dynamic behavior of a receptor signaling element. The structurally sensitive target for phenol is probably the TM bundle, but other behaviors could target other receptor elements.     

Precision Sensing by Two Opposing Gradient Sensors: How Does Escherichia coli Find its Preferred pH Level?

It is essential for bacteria to find optimal conditions for their growth and survival. The optimal levels of certain environmental factors (such as pH and temperature) often correspond to some intermediate points of the respective gradients. This requires the ability of bacteria to navigate from both directions toward the optimum location and is distinct from the conventional unidirectional chemotactic strategy. Remarkably, Escherichia coli cells can perform such a precision sensing task in pH taxis by using the same chemotaxis machinery, but with opposite pH responses from two different chemoreceptors (Tar and Tsr). To understand bacterial pH sensing, we developed an Ising-type model for a mixed cluster of opposing receptors based on the push-pull mechanism. Our model can quantitatively explain experimental observations in pH taxis for various mutants and wild-type cells. We show how the preferred pH level depends on the relative abundance of the competing sensors and how the sensory activity regulates the behavioral response. Our model allows us to make quantitative predictions on signal integration of pH and chemoattractant stimuli. Our study reveals two general conditions and a robust push-pull scheme for precision sensing, which should be applicable in other adaptive sensory systems with opposing gradient sensors.     

Responses of Escherichia coli Bacteria to Two Opposing Chemoattractant Gradients Depend on the Chemoreceptor Ratio

Escherichia coli chemotaxis has long served as a simple model of environmental signal processing, and bacterial responses to single chemical gradients are relatively well understood. Less is known about the chemotactic behavior of E. coli in multiple chemical gradients. In their native environment, cells are often exposed to multiple chemical stimuli. Using a recently developed microfluidic chemotaxis device, we exposed E. coli cells to two opposing but equally potent gradients of major attractants, methyl-aspartate and serine. The responses of E. coli cells demonstrated that chemotactic decisions depended on the ratio of the respective receptor number of Tar/Tsr. In addition, the ratio of Tar to Tsr was found to vary with cells’ growth conditions, whereby it depended on the culture density but not on the growth duration. These results provide biological insights into the decision-making processes of chemotactic bacteria that are subjected to multiple chemical stimuli and demonstrate the importance of the cellular microenvironment in determining phenotypic behavior.In their natural environment, both prokaryotic and eukaryotic cells are exposed to multiple chemical stimuli. It is thus important to learn how cells make a decision when confronted with complex chemical stimuli. Escherichia coli bacteria have long served as a model system for chemotaxis studies due to their known and simple genetic makeup. Signaling in bacterial chemotaxis is comparatively well understood (3, 18, 19). To summarize it briefly, there are five types of chemoreceptors in E. coli, of which Tar and Tsr are the most abundant. The basic functional chemosensing unit is a ternary complex that consists of transmembrane chemoreceptors, a linker molecule, CheW, and a histidine kinase, CheA. Within each functional receptor complex, the receptors are known to function in a cooperative manner (9, 12, 16). Upon the binding of attractant molecules, this sensory complex undergoes a conformational change that suppresses the autophosphorylation activity of CheA. This response is then transmitted to the flagellar motor via a regulator protein, CheY. As a result, the run time of an E. coli bacterium is lengthened when swimming toward a high-chemoattractant-concentration region (4).While the molecular mechanisms governing bacterial chemotaxis in a single gradient have been investigated extensively both in experiments and in theory (see reference 8 and references therein), very little is known about how bacteria behave in the presence of dual chemical gradients (1, 17). Early work by Adler and Tso explored the chemotactic responses of E. coli cells in the presence of both attractant and repellent gradients by using a microcapillary chemotaxis assay (1). Twenty years later, Strauss et al. (17) revisited the problem by using a stop-flow chamber. Both investigations concluded that bacteria sum the chemical signals to provide a coordinated output to control flagellar rotation. However, the molecular mechanisms responsible for this calculation have not yet been explored.In this paper, we investigated the molecular mechanism that underlies the bacterial decision-making processes in two opposing attractant gradients that are sensed by the two most abundant E. coli receptors, Tar and Tsr, respectively. By varying the relative expression levels of Tar and Tsr, we demonstrated that the receptor ratio defines the attractant preference in dual gradients of their ligands. The Tar-to-Tsr ratio itself depends on the cell culture density but not on the duration of growth.     

Thermosensing ability of Trg and Tap chemoreceptors in Escherichia coli.

The thermosensing ability of the Trg and Tap chemoreceptors in Escherichia coli was investigated after amplifying these receptors in a host strain lacking all four known chemoreceptors (Tar, Tsr, Trg, and Tap). Cells with an increased amount of either Trg or Tap showed mostly smooth swimming and no response to thermal stimuli. However, when the smooth-swimming bias of the cells was reduced by adding Trg- or Tap-mediated repellents, the cells showed clear changes in the swimming pattern upon temperature changes; Trg-containing cells showed tumbling at 23 degrees C but mostly smooth swimming at 32 degrees C, while Tap-containing cells showed smooth swimming at 20 degrees C but tumbling at 32 degrees C. These results indicate that although both Trg and Tap have the ability to sense thermal stimuli, Trg functions as a warm receptor, as reported previously for Tar and Tsr, while Tap functions as a cold receptor.     

Differential activation of Escherichia coli chemoreceptors by blue-light stimuli

Enteric bacteria tumble, swim slowly, and are then paralyzed upon exposure to 390- to 530-nm light. Here, we analyze this complex response in Escherichia coli using standard fluorescence microscope optics for excitation at 440 +/- 5 nm. The slow swimming and paralysis occurred only in dye-containing growth media or buffers. Excitation elicited complete paralysis within a second in 1 muM proflavine dye, implying specific motor damage, but prolonged tumbling in buffer alone. The tumbling half-response times were subsecond for onset but more than a minute for recovery. The response required the chemotaxis signal protein CheY and receptor-dependent activation of its kinase CheA. The study of deletion mutants revealed a specific requirement for either the aerotaxis receptor Aer or the chemoreceptor Tar but not the Tar homolog Tsr. The action spectrum of the wild-type response was consistent with a flavin, but the chromophores remain to be identified. The motile response processed via Aer was sustained, with recovery to either step-up or -down taking more than a minute. The response processed via Tar was transient, recovering on second time scales comparable to chemotactic responses. The response duration and amplitude were dependent on relative expression of Aer, Tar, and Tsr. The main response features were reproduced when each receptor was expressed singly from a plasmid in a receptorless host strain. However, time-resolved motion analysis revealed subtle kinetic differences that reflect the role of receptor cluster interactions in kinase activation-deactivation dynamics.     

Mutational analysis of N381, a key trimer contact residue in Tsr, the Escherichia coli serine chemoreceptor

Chemoreceptors such as Tsr, the serine receptor, function in trimer-of-dimer associations to mediate chemotactic behavior in Escherichia coli. The two subunits of each receptor homodimer occupy different positions in the trimer, one at its central axis and the other at the trimer periphery. Residue N381 of Tsr contributes to trimer stability through interactions with its counterparts in a central cavity surrounded by hydrophobic residues at the trimer axis. To assess the functional role of N381, we created and characterized a full set of amino acid replacements at this Tsr residue. We found that every amino acid replacement at N381 destroyed Tsr function, and all but one (N381G) of the mutant receptors also blocked signaling by Tar, the aspartate chemoreceptor. Tar jamming reflects the formation of signaling-defective mixed trimers of dimers, and in vivo assays with a trifunctional cross-linking reagent demonstrated trimer-based interactions between Tar and Tsr-N381 mutants. Mutant Tsr molecules with a charged amino acid or proline replacement exhibited the most severe trimer formation defects. These trimer-defective receptors, as well as most of the trimer-competent mutant receptors, were unable to form ternary signaling complexes with the CheA kinase and with CheW, which couples CheA to receptor control. Some of the trimer-competent mutant receptors, particularly those with a hydrophobic amino acid replacement, may not bind CheW/CheA because they form conformationally frozen or distorted trimers. These findings indicate that trimer dynamics probably are important for ternary complex assembly and that N381 may not be a direct binding determinant for CheW/CheA at the trimer periphery.     

Ligand-specific activation of Escherichia coli chemoreceptor transmethylation

Adaptation in the chemosensory pathways of bacteria like Escherichia coli is mediated by the enzyme-catalyzed methylation (and demethylation) of glutamate residues in the signaling domains of methyl-accepting chemotaxis proteins (MCPs). MCPs can be methylated in trans, where the methyltransferase (CheR) molecule catalyzing methyl group transfer is tethered to the C terminus of a neighboring receptor. Here, it was shown that E. coli cells exhibited adaptation to attractant stimuli mediated through either engineered or naturally occurring MCPs that were unable to tether CheR as long as another MCP capable of tethering CheR was also present, e.g., either the full-length aspartate or serine receptor (Tar or Tsr). Methylation of isolated membrane samples in which engineered tethering and substrate receptors were coexpressed demonstrated that the truncated substrate receptors (trTsr) were efficiently methylated in the presence of tethering receptors (Tar with methylation sites blocked) relative to samples in which none of the MCPs had tethering sites. The effects of ligand binding on methylation were investigated, and an increase in rate was produced only with serine (the ligand specific for the substrate receptor trTsr); no significant change in rate was produced by aspartate (the ligand specific for the tethering receptor Tar). Although the overall efficiency of methylation was lower, receptor-specific effects were also observed in trTar- and trTsr-containing samples, where neither Tar nor Tsr possessed the CheR binding site at the C terminus. Altogether, the results are consistent with a ligand-induced conformational change that is limited to the methylated receptor dimer and does not spread to adjacent receptor dimers.     

Transmembrane signalling by the chimeric chemosensory receptors of Escherichia coli Tsr and Tar with heterologous membrane-spanning regions

The serine and aspartate chemosensory receptors (Tsr and Tar) of Escherichia coli have two membrane-spanning regions TM1 and TM2. To investigate their roles in transmembrane signalling, we constructed two chimeric receptors from Tsr and Tar with heterologous combinations of TM1 and TM2: the N-terminus of one receptor, including TM1 and the periplasmic domain, was fused to the C-terminus of the other, beginning with TM2. Both of the chimeric receptor genes rescued the chemotactic defect of a receptorless E. coli strain, indicating that the chimeric receptors are functional. Their apparent affinities for the specific ligands were the same as those of Tsr or Tar. Therefore, as far as transmembrane signalling abilities are concerned, the TW2 regions of Tsr and Tar are interchangeable, suggesting that sequence-specific interaction between TM1 and TM2 may not be required for the signal transmission across the membrane. The cells expressing either of the chimeric receptors, however, showed ‘smooth’, biased, basal swimming patterns. Moreover, they adapted quickly after stimulation with the repellent glycerol. This rapid adaptation was observed even in the methyltransferase-defective strain. Therefore, exchange of TM2 might impose structural constraints on the chimeric receptors that stabilize conformations which elicit smooth swimming.     

Conditional inversion of the thermoresponse in Escherichia coli.

Mutants in Escherichia coli having defects in one of the methyl-accepting chemotaxis proteins, Tsr protein, which is the chemoreceptor and transducer for L-serine, showed a reduced but similar type of thermoresponse compared with wild-type strains; the cells showed smooth swimming upon temperature increase and tumbling upon temperature decrease. However, when the mutant cells were adapted to attractants such as L-aspartate and maltose, which are specific to another methyl-accepting chemotaxis protein, Tar protein, the direction of the thermoresponse was found to be inverted; a temperature increase induced tumbling and a temperature decrease induced smooth swimming. Consistent with this, the mutant cells showed inverted changes in the methylation level of Tar protein upon temperature changes. Wild-type strains but not Tar protein-deficient mutants exhibited the inverted thermoresponse when the cells were simultaneously adapted to L-aspartate and L-serine, indicating that Tar protein has a key role in the inversion of the thermoresponse. Thus, besides Tsr protein, Tar protein has a certain role in thermoreception. A simple model for thermoreception and inversion of the thermoresponse is also discussed.     

Organization of the receptor-kinase signaling array that regulates Escherichia coli chemotaxis

Motor behavior in prokaryotes is regulated by a phosphorelay network involving a histidine protein kinase, CheA, whose activity is controlled by a family of Type I membrane receptors. In a typical Escherichia coli cell, several thousand receptors are organized together with CheA and an Src homology 3-like protein, CheW, into complexes that tend to be localized at the cell poles. We found that these complexes have at least 6 receptors per CheA. CheW is not required for CheA binding to receptors, but is essential for kinase activation. The kinase activity per mole of bound CheA is proportional to the total bound CheW. Similar results were obtained with the E. coli serine receptor, Tsr, and the Salmonella typhimurium aspartate receptor, Tar. In the case of Tsr, under conditions optimal for kinase activation, the ratio of subunits in complexes is approximately 6 Tsr:4 CheW:1 CheA. Our results indicate that information from numerous receptors is integrated to control the activity of a relatively small number of kinase molecules.     

Transmembrane Signaling in Chimeras of the Escherichia coli Aspartate and Serine Chemotaxis Receptors and Bacterial Class III Adenylyl Cyclases

The Escherichia coli chemoreceptors for serine (Tsr) and aspartate (Tar) and several bacterial class III adenylyl cyclases (ACs) share a common molecular architecture; that is, a membrane anchor that is linked via a cytoplasmic HAMP domain to a C-terminal signal output unit. Functionality of both proteins requires homodimerization. The chemotaxis receptors are well characterized, whereas the typical hexahelical membrane anchor (6TM) of class III ACs, suggested to operate as a channel or transporter, has no known function beyond a membrane anchor. We joined the intramolecular networks of Tsr or Tar and two bacterial ACs, Rv3645 from Mycobacterium tuberculosis and CyaG from Arthrospira platensis, across their signal transmission sites, connecting the chemotaxis receptors via different HAMP domains to the catalytic AC domains. AC activity in the chimeras was inhibited by micromolar concentrations of l-serine or l-aspartate in vitro and in vivo. Single point mutations known to abolish ligand binding in Tar (R69E or T154I) or Tsr (R69E or T156K) abrogated AC regulation. Co-expression of mutant pairs, which functionally complement each other, restored regulation in vitro and in vivo. Taken together, these studies demonstrate chemotaxis receptor-mediated regulation of chimeric bacterial ACs and connect chemical sensing and AC regulation.     

Changing the specificity of a bacterial chemoreceptor

The methyl-accepting chemotaxis proteins are a family of receptors in bacteria that mediate chemotaxis to diverse signals. To explore the plasticity of these proteins, we have developed a simple method for selecting cells that swim to target attractants. The procedure is based on establishing a diffusive gradient in semi-soft agar plates and does not require that the attractant be metabolized or degraded. We have applied this method to select for variants of the Escherichia coli aspartate receptor, Tar, that have a new or improved response to different amino acids. We found that Tar can be readily mutated to respond to new chemical signals. However, the overall change in specificity depended on the target compound. A Tar variant that could detect cysteic acid still showed a strong sensitivity to aspartate, indicating that the new receptor had a broadened specificity relative to wild-type Tar. Tar variants that responded to phenylalanine or N-methyl aspartate, or that had an increased sensitivity to glutamate showed a strong decrease in their response to aspartate. In at least some of the cases, the maximal level of sensitivity that was obtained could not be attributed solely to substitutions within the binding pocket. The new tar alleles and the techniques described here provide a new approach for exploring the relationship between ligand binding and signal transduction by chemoreceptors and for engineering new receptors for applications in biotechnology.     

Inversion of aerotactic response in Escherichia coli deficient in cheB protein methylesterase.

Mutants of Escherichia coli and Salmonella typhimurium that were deficient in protein methylesterase activity encoded by cheB had an inverted response to oxygen; they were repelled by concentrations of oxygen that attract wild-type bacteria. Normal responses to oxygen and phosphotransferase substrates were observed in mutants that were deficient in protein methyltransferase (CheR) and the methyl-accepting transducing proteins (Tsr, Tar, Trg). However, the methylation-independent response to oxygen was modified by the loss of esterase activity. The inversion was apparently effected by the amidated Tsr protein present in cheB tsr+ mutants because aerotaxis was normal in cheB tsr strains. Chemotaxis to phosphotransferase sugars was normal in cheB mutants provided the extreme clockwise bias of the flagellar motors was modified to increase the probability of counterclockwise rotation.     

Ligand specificity determined by differentially arranged common ligand-binding residues in bacterial amino acid chemoreceptors Tsr and Tar

Escherichia coli has closely related amino acid chemoreceptors with distinct ligand specificity, Tar for l-aspartate and Tsr for l-serine. Crystallography of the ligand-binding domain of Tar identified the residues interacting with aspartate, most of which are conserved in Tsr. However, swapping of the nonconserved residues between Tsr and Tar did not change ligand specificity. Analyses with chimeric receptors led us to hypothesize that distinct three-dimensional arrangements of the conserved ligand-binding residues are responsible for ligand specificity. To test this hypothesis, the structures of the apo- and serine-binding forms of the ligand-binding domain of Tsr were determined at 1.95 and 2.5 Å resolutions, respectively. Some of the Tsr residues are arranged differently from the corresponding aspartate-binding residues of Tar to form a high affinity serine-binding pocket. The ligand-binding pocket of Tsr was surrounded by negatively charged residues, which presumably exclude negatively charged aspartate molecules. We propose that all these Tsr- and Tar-specific features contribute to specific recognition of serine and aspartate with the arrangement of the side chain of residue 68 (Asn in Tsr and Ser in Tar) being the most critical.     

Opposite responses by different chemoreceptors set a tunable preference point in Escherichia coli pH taxis

In bacterial habitats, the ability to follow spatial gradients of environmental factors that affect growth and survival can be largely advantageous. The bacterial strategy for unidirectional chemotactic movement in gradients of typical attractants or repellents, such as nutrients or toxins, is well understood. Optimal levels of other factors, however, may be found at intermediate points of a gradient and thus require a bidirectional tactic movement towards the optimum. Here we investigate the chemotactic response of Escherichia coli to pH as an example of such bidirectional taxis. We confirm that E. coli uses chemotaxis to avoid both extremes of low and high pH and demonstrate that the sign of the response is inverted from base‐seeking to acid‐seeking at a well‐defined value of pH. Such inversion is enabled by opposing pH sensing by the two major chemoreceptors, Tar and Tsr, such that the relative strength of the response is modulated by adaptive receptor methylation. We further demonstrate that the inversion point of the pH response can be adjusted in response to changes in the cell density.     

Cross-linking evidence for motional constraints within chemoreceptor trimers of dimers

Chemotactic behavior in bacteria relies on the sensing ability of large chemoreceptor clusters that are usually located at the cell pole. In Escherichia coli, chemoreceptors exhibit higher-order interactions within those clusters based on a trimer-of-dimers organization. This architecture is conserved in a variety of other bacteria and archaea, implying that receptors in many microorganisms form trimer-of-dimer signaling teams. To gain further insight into the assembly and dynamic behavior of receptor trimers of dimers, we used in vivo cross-linking targeted to cysteine residues at various positions that define six different levels along the cytoplasmic signaling domains of the aspartate and serine chemoreceptors, Tar and Tsr, respectively. We found that the cytoplasmic domains of these receptors are close to each other near the trimer contact region at the cytoplasmic tip and lie farther apart as the receptor dimers approach the cytoplasmic membrane. Tar and Tsr reporter sites within the same or closely adjacent levels readily formed mixed cross-links, whereas reporters located different distances from the tip did not. These findings indicate that there are no significant vertical displacements of one dimer with respect to the others within the trimer unit. Attractant stimuli had no discernible effect on the cross-linking efficiency of any of the reporters tested, but a strong osmotic stimulus reproducibly enhanced cross-linking at most of the reporter sites, indicating that individual dimers may move closer together under this condition.