Repellent response functions of the Trg and Tap chemoreceptors of Escherichia coli.

The chemoreceptors responsible for the repellent response of Escherichia coli to phenol were investigated. In the absence of all four known methyl-accepting chemoreceptors (Tar, Tsr, Trg, and Tap), cells showed no response to phenol. However, when Trg, which mediates the attractant response to ribose and galactose, was introduced via a plasmid, the cells acquired a repellent response to phenol. About 1 mM phenol induced a clear repellent response; this response was suppressed by 1 mM ribose. Thus, Trg mediates the repellent response to phenol. Mutant Trg proteins with altered sensing for ribose and galactose showed a normal response to phenol, indicating that the interaction site for phenol differs from that for the ribose- and galactose-binding proteins. Tap, which mediates the attractant response to dipeptides, mediated a weaker repellent response to phenol. Tsr, which mediates the attractant response to serine, mediated an even weaker response to phenol. Trg and Tap were also found to function as intracellular pH sensors. Upon a pH decrease, Trg mediated an attractant response, whereas Tap mediated a repellent response. These results indicate that all the receptors in E. coli have dual functions, mediating both attractant and repellent responses.     

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.     

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.     

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.     

Chimeric Chemoreceptors in Escherichia coli: Signaling Properties of Tar-Tap and Tap-Tar Hybrids

The Tap (taxis toward peptides) receptor and the periplasmic dipeptide-binding protein (DBP) of Escherichia coli together mediate chemotactic responses to dipeptides. Tap is a low-abundance receptor. It is present in 5- to 10-fold-fewer copies than high-abundance receptors like Tar and Tsr. Cells expressing Tap as the sole receptor, even from a multicopy plasmid at 5- to 10-fold-overexpressed levels, do not generate sufficient clockwise (CW) signal to tumble and thus swim exclusively smoothly (run). To study the signaling properties of Tap in detail, we constructed reciprocal hybrids between Tap and Tar fused in the linker region between the periplasmic and cytoplasmic domains. The Tapr hybrid senses dipeptides and is a good CW-signal generator, whereas the Tarp hybrid senses aspartate but is a poor CW-signal generator. Thus, the poor CW signaling of Tap is a property of its cytoplasmic domain. Eighteen residues at the carboxyl terminus of high-abundance receptors, including the NWETF sequence that binds the CheR methylesterase, are missing in Tap. The Tart protein, created by removing these 18 residues from Tar, has diminished CW-signaling ability. The Tapl protein, made by adding the last 18 residues of Tar to the carboxyl terminus of Tap, also does not support CW flagellar rotation. However, Tart and Tapl cross-react well with antibody directed against the conserved cytoplasmic region of Tsr, whereas Tap does not cross-react with this antibody. Tap does cross-react, however, with antibody directed against the low-abundance chemoreceptor Trg. The hybrid, truncated, and extended receptors exhibit various levels of methylation. However, Tar and Tapl, which contain a consensus CheR-binding motif (NWETF) at their carboxyl termini, exhibit the highest basal levels of methylation, as expected. We conclude that no simple correlation exists between the abundance of a receptor, its methylation level, and its CW-signaling ability.     

Uropathogenic Escherichia coli strains generally lack functional Trg and Tap chemoreceptors found in the majority of E. coli strains strictly residing in the gut

The prevalence and function of four chemoreceptors, Tsr, Tar, Trg, and Tap, were determined for a collection of uropathogenic, fecal-commensal, and diarrheagenic Escherichia coli strains. tar and tsr were present or functional in nearly all isolates. However, trg and tap were significantly less prevalent or functional among the uropathogenic E. coli strains (both in 6% of strains) than among fecal-commensal strains (both in > or =50% of strains) or diarrheal strains (both in > or =75% of strains) (P < 0.02).     

Multiple covalent modifications of Trg, a sensory transducer of Escherichia coli

The sensory transducers of Escherichia coli are integral membrane proteins that mediate the tactic response of cells to chemical stimuli. Adaptation to environmental stimuli is correlated with methylation of the transducer proteins. Two transducer genes, tsr and tar, exhibit extensive homologies while no homology has been detected between a third transducer, trg, and those genes. The Tsr and Tar proteins have been shown to contain multiple sites for methylation as well as two sites for another modification that requires an active cheB gene product and is designated the CheB-dependent modification. In this study, covalent modifications of the Trg protein were characterized by analysis of tryptic peptides. We found that methylation occurred at several sites on the Trg protein and that the protein contained at least three sites for CheB-dependent modification, two of which were located on a tryptic peptide that contains both methionine and lysine. This tryptic peptide is analogous to the methionine- and lysine-containing methyl-accepting peptides isolated from the Tsr and Tar proteins and like those peptides may contain several methyl-accepting sites. We estimated the pKa of the group created by the CheB-dependent modification on the methionine- and lysine-containing peptide of Trg to be between pH 2.2 and 5.8. This result supports the idea that the CheB-dependent modification is an enzymatic deamidation of glutamine to glutamic acid.     

Aberrant regulation of methylesterase activity in cheD chemotaxis mutants of Escherichia coli

The adaptation process in several cheD chemotaxis mutants, which carry defects in tsr, the serine transducer gene, was examined. cheD mutants are smooth swimming and generally nonchemotactic; the defect is dominant to the wild-type tsr gene (J. S. Parkinson, J. Bacteriol. 142:953-961, 1980). All classes of methyl-accepting chemotaxis proteins synthesized in unstimulated cheD strains are overmethylated relative to the wild type. We found that the steady-state rate of demethylation in cheD mutants was low; this may explain their overmethylated phenotype. In addition, all cheD mutants showed diminished responsiveness of methylesterase activity to attractant and repellent stimuli transduced by either the Tsr or Tar protein, and they did not adapt. These results suggest that the dominant nature of the cheD mutations is manifested as a general defect in the regulation of demethylation. Some of these altered properties of methylesterase activity in cheD mutants were exhibited in wild-type cells that were treated with saturating concentrations of serine. The mutant Tsr protein thus seems to be locked into a signaling mode that suppresses tumbling and inhibits methylesterase activity in a global fashion. We found that the Tar and mutant Tsr proteins synthesized in cheD strains were methylated and deamidated at the correct sites and that the mutations were not located in the methylated peptides. Thus, the signaling properties of the transducers may be controlled at sites distinct from the methyl-accepting sites.     

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.     

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.     

Adaptational assistance in clusters of bacterial chemoreceptors

Sensory adaptation of low-abundance chemoreceptors in Escherichia coli requires assistance from high-abundance receptors, because only high-abundance receptors carry the carboxyl-terminal pentapeptide sequence NWETF that enhances adaptational covalent modification. Using membrane vesicles containing both high-abundance receptor Tar and low-abundance receptor Trg, we observed effective assistance in vitro for all three adaptational modifications: methylation, demethylation and deamidation. These results demonstrated that adaptational assistance involves not only the previously documented assistance for methylation but also assistance for the two CheB-catalysed reactions. We determined rates of assisted methylation and demethylation at many ratios of assisting to assisted receptor. Analysis by a model of assistance indicated one Tar dimer could assist seven Trg dimers in methylation or five in demethylation, defining assistance neighbourhoods. These neighbourhoods were larger than a trimer of homodimers, required only receptors and were minimally affected by formation of signalling complexes. Time courses of assisted Trg methylation in membranes with low amounts of Tar showed that assisting receptors did not diffuse beyond initial neighbourhoods for at least two hours. Taken together, these observations indicate that chemoreceptors can form stable neighbourhoods larger than trimers in the absence of other chemotaxis proteins. Such interactions are likely to occur in natural receptor clusters in vivo.     

Comparison In Vitro of a High- and a Low-Abundance Chemoreceptor of Escherichia coli: Similar Kinase Activation but Different Methyl-Accepting Activities

In Escherichia coli, high-abundance chemoreceptors are present in cellular amounts approximately 10-fold greater than low-abundance chemoreceptors. Cells containing only low-abundance receptors exhibit abnormally low tumble frequencies and do not migrate effectively in spatial gradients. These defects reflect an inherent activity difference between the two receptor classes. We used in vitro assays to investigate this difference. The low-abundance receptor Trg mediated an ∼100-fold activation of the kinase CheA, only twofold less than activation by the high-abundance receptor Tar. In contrast, Trg was less than 1/20 as active as Tar for in vitro methylation. As observed for high-abundance receptors, kinase activation by Trg varied with the extend of modification at methyl-accepting sites; low methylation corresponded to low kinase activation. Thus, in Trg-only cells, low receptor methylation would result in low kinase activation, correspondingly low content of phospho-CheY, and a decreased dynamic range over which attractant binding could modulate kinase activity. These features could account for the low tumble frequency and inefficient taxis exhibited by Trg-only cells. Thus, the crucial functional difference between the receptor classes is likely to be methyl-accepting activity. We investigated the structural basis for this functional difference by introducing onto the carboxy terminus of Trg a CheR-binding pentapeptide, usually found only at the carboxy termini of high-abundance receptors. This addition enhanced the in vitro methyl-accepting activity of Trg 10-fold.Transmembrane, methyl-accepting receptor proteins mediate chemotaxis by Escherichia coli (13, 18, 37). Many related proteins have been detected in other eubacterial and archaeal species by antigenic cross-reaction (30) or sequence comparisons (23, 49). These proteins define an extensive family of sensory receptors that mediate bacterial and archaeal taxis. The hallmarks of the family are a highly conserved region (∼50 residues) crucial for intracellular signaling and adjacent regions containing methyl-accepting glutamyl residues that are covalently modified in the process of sensory adaptation. In E. coli and Salmonella typhimurium, the highly conserved regions have been shown to be involved in the control of a noncovalently associated histidine kinase, CheA (reviewed in reference 37), which is a well-characterized member of a large family of homologous histidine kinases that serve not only taxis systems but also a vast array of two-component, environment-sensing systems in eubacteria, archaea, and eukaryotes (1, 3).The influence of a chemoreceptor on an associated kinase has two distinct aspects: (i) basal activation and (ii) modulation of activity in response to changes in receptor occupancy (6–8, 32). Both require formation of a ternary complex consisting of a receptor, CheA, and an accessory protein, CheW (17, 41). This complex is stable over times relevant for sensory response and adaptation (17). Basal activation of CheA by an interacting receptor establishes a steady-state activity of the kinase that determines the cellular content of the phosphorylated response regulator CheY (phospho-CheY). Phospho-CheY interacts with the flagellar switch to induce clockwise (CW) rotation of an otherwise counterclockwise (CCW)-rotating motor. Proper basal activation establishes a phospho-CheY content in a normal cell that creates a balance between CCW and CW flagellar rotation, which produces a corresponding alternation between smooth swimming and tumbling. The pattern causes a swimming cell to move in a random walk. Modulation of kinase activity from its level of basal activation is effected by receptors that have experienced a change in ligand occupancy but have not yet adapted. The modulation results in an altered content of phospho-CheY, which changes the CCW-to-CW balance and thus the tumble frequency, biasing the random walk to direct the cell in a favorable direction.The four well-characterized receptors of E. coli have a common organization (see references 13 and 18 for details and specific references). In each monomer of a receptor homodimer, an amino-terminal periplasmic domain of ∼150 residues and a carboxy-terminal cytoplasmic domain of ∼300 residues are connected by two transmembrane segments. Residue identity among the aligned sequences of the four periplasmic domains is minimal but is nearly 60% for the cytoplasmic domain. The cytoplasmic domain includes the highly conserved region and the methyl-accepting sites. The receptors Tsr, Tar, Trg, and Tap mediate taxis toward serine, aspartate and maltose, ribose and galactose, and dipeptides, respectively. A recently discovered fifth receptor, Aer, lacks a substantial periplasmic domain but mediates responses to oxygen and to perturbations of membrane energetics by utilizing a bound flavin (4, 40). Among the four extensively characterized receptors, two high-abundance chemoreceptors, Tsr and Tar, are present in cellular amounts 5- to 10-fold greater than two low-abundance receptors, Trg and Tap (19). In the absence of high-abundance receptors, cells exhibit abnormally low tumble frequencies and greatly compromised abilities to migrate in spatial gradients of attractants recognized by the remaining low-abundance receptors (14, 20, 45, 46). These defects are not corrected by increasing cellular amounts of the low-abundance receptor (14, 46). Thus high-abundance and low-abundance receptors are distinguished not simply by different amounts in a wild-type cell but also by an inherent difference in activity. Characterization of hybrids between a high-abundance receptor and a low-abundance receptor, either Tsr and Trg (14) or Tar and Tap (46), revealed that this inherent difference in activity resides in the cytoplasmic domain, even though it is in this domain that residue identity among receptors is most conserved.What is the nature of the difference between high-abundance and low-abundance receptors that allows the former but not the latter to establish a physiologically useful tumble frequency and to mediate effective taxis as the sole receptor in a cell? A clear possibility is that low-abundance receptors are ineffective in basal activation of the kinase CheA. Ineffective activation would mean a low level of steady-state phosphorylation that would result in a low cellular concentration of phospho-CheY and thus a low tumble frequency. Also, low basal activation of CheA could affect signaling by reducing the dynamic range over which kinase activity could be modulated in response to increases in receptor occupancy. A difference in kinase activation would be consistent with the observation that the inherent difference between high- and low-abundance receptors resides in the cytoplasmic domain. Thus, we undertook the study of kinase activation by the low-abundance receptor Trg, using in vitro assays for phosphorylation. In these assays Trg was almost as effective as the high-abundance receptor Tar in activating CheA, implying that kinase activation was unlikely to be the crucial functional difference between the two receptor types. However, we observed a striking difference in vitro in the methyl-accepting activities of the two receptors. The low-abundance receptor Trg was significantly less effective than the high-abundance receptor Tar as an acceptor for in vitro methylation. This appears to be the central functional difference between the two receptor types.     

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.     

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.     

Chemoreceptor Gene Loss and Acquisition via Horizontal Gene Transfer in Escherichia coli

Chemotaxis allows bacteria to more efficiently colonize optimal microhabitats within their larger environment. Chemotaxis in Escherichia coli is the best-studied model system, and a large number of E. coli strains have been sequenced. The Escherichia/Shigella genus encompasses a great variety of commensal and pathogenic strains, but the role of chemotaxis in their association with the host remains poorly understood. Here we show that the core chemotaxis genes are lost in many, but not all, nonmotile strains but are well preserved in all motile strains. The genes encoding the Tar, Tsr, and Aer chemoreceptors, which mediate chemotaxis to a broad spectrum of chemical and physical cues, are also nearly uniformly conserved in motile strains. In contrast, the clade of extraintestinal pathogenic E. coli strains apparently underwent an ancestral loss of Trg and Tap chemoreceptors, which sense sugars, dipeptides, and pyrimidines. The broad range of time estimated for the loss of these genes (1 to 3 million years ago) corresponds to the appearance of the genus Homo.     

Cloning of trg, a gene for a sensory transducer in Escherichia coli.

Clones of trg, a gene which codes for a chemotactic transducer, were isolated linked to ColE1 and pBR322 vectors. Studies with the hybrid plasmids demonstrated unequivocally that trg is the structural gene for methyl-accepting chemotaxis protein III. The Trg protein was found to be structurally complex, electrophoresing as a series of seven bands on high-resolution sodium dodecyl sulfate-polyacrylamide gels. The multiplicity of bands is a function of the activity of cheR, which codes for a methyltransferase, and of cheB, which codes for a demethylase. It appears that Trg, a quantitatively minor transducer, resembles the two major transducer proteins, Tsr and Tar, in that all three are multiply methylated and also multiply modified in a second way which requires an active cheB gene. However, preliminary analysis of the Trg protein indicated that it is significantly less related structurally to the Tsr or Tar protein than those two transducers are to each other. This implies that the features of multiple methylation and cheB-dependent modification are likely to be critical for the common physiological functions in chemotactic excitation and adaptation performed by all three transducers.     

Precision and Variability in Bacterial Temperature Sensing

In Escherichia coli, the ratio of the two most abundant chemoreceptors, Tar/Tsr, has become the focus of much attention in bacterial taxis studies. This ratio has been shown to change under various growth conditions and to determine the response of the bacteria to the environment. Here, we present a study that makes a quantitative link between the ratio Tar/Tsr and the favored temperature of the cell in a temperature gradient and in various chemical environments. From the steady-state density-profile of bacteria with one dominant thermo-sensor, Tar or Tsr, we deduce the response function of each receptor to temperature changes. Using the response functions of both receptors, we determine the relationship between the favored temperature of wild-type bacteria with mixed clusters of receptors and the receptor ratio. Our model is based on the assumption that the behavior of a wild-type bacterium in a temperature gradient is determined by a linear combination of the independent responses of the two receptors, factored by the receptor’s relative abundance in the bacterium. This is confirmed by comparing our model predictions with measurements of the steady-state density-profile of several bacterial populations in a temperature gradient. Our results reveal that the density-profile of wild-type bacteria can be accurately described by measuring the distribution of the ratio Tar/Tsr in the population, which is then used to divide the population into groups with distinct Tar/Tsr values, whose behavior can be described in terms of independent Gaussian distributions. Each of these Gaussians is centered about the favored temperature of the subpopulation, which is determined by the receptor ratio, and has a width defined by the temperature-dependent speed and persistence time.     

Chemosensory and thermosensory excitation in adaptation-deficient mutants of Escherichia coli

Methyl-accepting chemotaxis protein-methyltransferase-deficient mutants, cheR mutants, of Escherichia coli showed a tumble response to repellents only at low temperatures, and the resultant tumbling lasted unless the condition was changed. The swimming pattern of the repellent-treated cells was different at different temperatures, indicating that the absolute temperature is a determinant of the tumbling frequency of those cells. The tumbling of those cells was also suppressed by the addition of attractants. Under a suitable repellent concentration, the tumbling frequency of the cells was found to be simply determined by the ligand occupancy of chemoreceptors for many attractants. In a methyl-accepting chemotaxis protein-methylesterase-deficient mutant, a cheB deletion mutant, the tumbling frequency was also determined by receptor occupancy of some attractants. These results indicate that in the adaptation-deficient mutants, sensory signals are produced in proportion to the amount of ligand-bound or of thermally altered receptors and transmitted to the flagellar motors without any modification. Thus, it is concluded that the adaptation system, namely, the methylation-demethylation system of methyl-accepting chemotaxis proteins, is not concerned with the step of chemosensory or thermosensory excitation. A simple model is proposed to explain how the swimming pattern of the adaptation-deficient mutants is determined.     

Evolution of chemotactic-signal transducers in enteric bacteria.

The methyl-accepting chemotactic-signal transducers of the enteric bacteria are transmembrane proteins that consist of a periplasmic receptor domain and a cytoplasmic signaling domain. To study their evolution, transducer genes from Enterobacter aerogenes and Klebsiella pneumoniae were compared with transducer genes from Escherichia coli and Salmonella typhimurium. There are at least two functional transducer genes in the nonmotile species K. pneumoniae, one of which complements the defect in serine taxis of an E. coli tsr mutant. The tse (taxis to serine) gene of E. aerogenes also complements an E. coli tsr mutant; the tas (taxis to aspartate) gene of E. aerogenes complements the defect in aspartate taxis, but not the defect in maltose taxis, of an E. coli tar mutant. The sequence was determined for 5 kilobases of E. aerogenes DNA containing a 3' fragment of the cheA gene, cheW, tse, tas, and a 5' fragment of the cheR gene. The tse and tas genes are in one operon, unlike tsr and tar. The cytoplasmic domains of Tse and Tas are very similar to those of E. coli and S. typhimurium transducers. The periplasmic domain of Tse is homologous to that of Tsr, but Tas and Tar are much less similar in this region. However, several short sequences are conserved in the periplasmic domains of Tsr, Tar, Tse, and Tas but not of Tap and Trg, transducers that do not bind amino acids. These conserved regions include residues implicated in amino-acid binding.     

Construction of a genetic multiplexer to toggle between chemosensory pathways in Escherichia coli

Many applications require cells to switch between discrete phenotypic states. Here, we harness the FimBE inversion switch to flip a promoter, allowing expression to be toggled between two genes oriented in opposite directions. The response characteristics of the switch are characterized using two-color cytometry. This switch is used to toggle between orthogonal chemosensory pathways by controlling the expression of CheW and CheW*, which interact with the Tar (aspartate) and Tsr* (serine) chemoreceptors, respectively. CheW* and Tsr* each contain a mutation at their protein-protein interface such that they interact with each other. The complete genetic program containing an arabinose-inducible FimE controlling CheW/CheW* (and constitutively expressed tar/tsr*) is transformed into an Escherichia coli strain lacking all native chemoreceptors. This program enables bacteria to swim toward serine or aspartate in the absence or in the presence of arabinose, respectively. Thus, the program functions as a multiplexer with arabinose as the selector. This demonstrates the ability of synthetic genetic circuits to connect to a natural signaling network to switch between phenotypes.