The smaller of two overlapping cheA gene products is not essential for chemotaxis in Escherichia coli.

The cheA locus of Escherichia coli encodes two similar proteins, CheAL (654 amino acids) and CheAS (557 amino acids), which are made by initiating translation from different in-frame start sites [start(L) and start(S)]. CheAL plays an essential role in chemotactic signaling. It autophosphorylates at a histidine residue (His-48) and then donates this phosphate to response regulator proteins that modulate flagellar rotation and sensory adaptation. CheAS lacks the first 97 amino acids of CheAL, including the phosphorylation site at His-48. Although it is unable to autophosphorylate, CheAS can form heterodimers with mutant CheAL subunits to restore kinase function and chemoreceptor control of autophosphorylation activity. To determine whether these or other activities of CheAS are important for chemotaxis, we constructed cheA lesions that abrogated CheAS expression. Mutants in which the CheAS start codon was changed from methionine to isoleucine (M98I) or glutamine (M98Q) retained chemotactic ability, ranging from 50% (M98Q) to 80% (M98I) of wild-type function. These partial defects could not be alleviated by supplying CheAS from a specialized transducing phage, indicating that the lesions in CheAL--not the lack of CheAS--were responsible for the reduced chemotactic ability. In other respects, the behavior of the M98I mutant was essentially normal. Its flagellar rotation pattern was indistinguishable from wild type, and it exhibited wild-type detection thresholds and peak positions in capillary chemotaxis assays. The lack of any substantive defect in this start(S) mutant argues that CheAS makes a negligible contribution to chemotactic ability in the laboratory. Whether it has functional significance in other settings remains to be seen.     

Coexpression of the long and short forms of CheA, the chemotaxis histidine kinase, by members of the family Enterobacteriaceae.

CheA is the histidine protein kinase of a two-component signal transduction system required for bacterial chemotaxis. Motile cells of the enteric species Escherichia coli and Salmonella typhimurium synthesize two forms of CheA by utilizing in-frame initiation sites within the gene cheA. The full-length protein, CheAL, plays an essential role in the chemotactic signaling pathway. In contrast, the function of the short form, CheAs, remains elusive. Although CheAs lacks the histidine residue that becomes phosphorylated in CheAL, it exhibits both kinase activity and the ability to interact with and enhance the activity of CheZ, a chemotaxis protein that accelerates dephosphorylation of the two-component response regulator CheY. To determine whether other members of the family Enterobacteriaceae express CheAs and CheZ, we analyzed immunoblots of proteins from clinical isolates of a variety of enteric species. All motile, chemotactic isolates that we tested coexpressed CheAL, CheAs, and CheZ. The only exceptions were closely related plant pathogens of the genus Erwinia, which expressed CheAL and CheZ but not CheAs. We also analyzed nucleotide sequences of the cheA loci from isolates of Serratia marcescens and Enterobacter cloacae, demonstrating the presence of in-frame translation initiation sites similar to those observed in the cheA loci of E. coli and S. typhimurium. Since coexpression of CheAs and CheZ appears to be limited to motile, chemotactic enteric bacteria, we propose that CheAs may play an important role in chemotactic responses in some environmental niches encountered by enteric species.     

Genetic analysis of the catalytic domain of the chemotaxis-associated histidine kinase CheA.

Escherichia coli cells express two forms of CheA, the histidine kinase associated with chemotaxis. The long form, CheA(L), plays a critical role in chemotactic signal transduction by phosphorylating two chemotaxis-associated response regulators, CheY and CheB. CheA(L) first autophosphorylates amino acid His-48 before its phosphoryl group is transferred to these response regulators. The short form, CheA(S), lacks the amino-terminal 97 amino acids of CheA(L) and therefore does not possess the site of phosphorylation. The centrally located transmitter domain of both forms of CheA contains four regions, called N, G1, F, and G2, highly conserved among histidine kinases of the family of two-component signal transduction systems. On the basis of sequence similarity to highly conserved regions of certain eukaryotic kinases, the G1 and G2 regions are purported to be involved in the binding and hydrolysis of ATP. We report here that alleles mutated in the G1, G2, or F region synthesize CheA variants that cannot autophosphorylate in vitro and which cannot support chemotaxis in vivo. We also show that in vitro, the nonphosphorylatable CheA(S) protein mediates transphosphorylation of a CheA(L) variant defective in both G1 and G2. In contrast, CheA(L) variants defective for either G1 or G2 mediate transphosphorylation of each other poorly, if at all. These results are consistent with a mechanism by which the G1 and G2 regions of one protomer of a CheA dimer form a unit that mediates transphosphorylation of the other protomer within that dimer.     

pH dependence of CheA autophosphorylation in Escherichia coli.

Chemotaxis by cells of Escherichia coli and Salmonella typhimurium depends upon the ability of chemoreceptors called transducers to communicate with switch components of flagellar motors to modulate swimming behavior. This communication requires an excitatory pathway composed of the cytoplasmic signal transduction proteins, CheAL, CheAS, CheW, CheY, and CheZ. Of these, the autokinase CheAL is most central. Modifications or mutations that affect the rate at which CheAL autophosphorylates result in profound chemotactic defects. Here we demonstrate that pH can affect CheAL autokinase activity in vitro. This activity exhibits a bell-shaped dependence upon pH within the range 6.5 to 10.0, consistent with the notion that two proton dissociation events affect CheAL autophosphorylation kinetics: one characterized by a pKa of about 8.1 and another exhibiting a pKa of about 8.9. These in vitro results predict a decrease in the rate of CheAL autophosphorylation in response to a reduction in intracellular pH, a decrease that should cause increased counterclockwise flagellar rotation. We observed such a response in vivo for cells containing a partially reconstituted chemotaxis system. Benzoate (10 mM, pH 7.0), a weak acid that when undissociated readily traverses the cytoplasmic membrane, causes a reduction of cytoplasmic pH from 7.6 to 7.3. In response to this reduction, cells expressing CheAL, CheAS, and CheY, but not transducers, exhibited a small but reproducible increase in the fraction of time that they spun their flagellar motors counterclockwise. The added presence of CheW and the transducers Tar and Trg resulted in a more dramatic response. The significance of our in vitro results, their relationships to regulation of swimming behavior, and the mechanisms by which transducers might affect the pH dependence of CheA autokinase activity are discussed.     

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.     

Genetic evidence for interaction between the CheW and Tsr proteins during chemoreceptor signaling by Escherichia coli.

This study presents two lines of genetic evidence consistent with the premise that CheW, a cytoplasmic component of the chemotactic signaling system of Escherichia coli, interacts directly with Tsr, the membrane-bound serine chemoreceptor. (i) We demonstrated phenotypic suppression between 10 missense mutant CheW proteins and six missense mutant Tsr proteins. Most of these mutant proteins had leaky chemotaxis defects and were partially dominant, implying relatively minor functional alterations. Their suppression pattern was allele specific, suggesting that the mutant proteins have compensatory conformational changes at sites of interactive contact. (ii) We isolated five partially dominant CheW mutations and found that four of them were similar or identical to the suppressible CheW mutant proteins. This implies that there are only a few ways in which CheW function can be altered to produce dominant defects and that dominance is mediated through interactions of CheW with Tsr. The amino acid replacements in these mutant proteins were inferred from their DNA sequence changes. The CheW mutations were located in five regularly spaced clusters in the first two-thirds of the protein. The Tsr mutations were located in a highly conserved region in the middle of the cytoplasmic signaling domain. The hydrophobic moments, overall hydrophobicities, and predicted secondary structures of the mutant segments were consistent with the possibility that they are located at the surface of the CheW and Tsr molecules and represent the contact sites between these two proteins.     

Constitutively signaling fragments of Tsr, the Escherichia coli serine chemoreceptor.

Tsr, the serine chemoreceptor of Escherichia coli, has two signaling modes. One augments clockwise (CW) flagellar rotation, and the other augments counterclockwise (CCW) rotation. To identify the portion of the Tsr molecule responsible for these activities, we isolated soluble fragments of the Tsr cytoplasmic domain that could alter the flagellar rotation patterns of unstimulated wild-type cells. Residues 290 to 470 from wild-type Tsr generated a CW signal, whereas the same fragment with a single amino acid replacement (alanine 413 to valine) produced a CCW signal. The soluble components of the chemotaxis phosphorelay system needed for expression of these Tsr fragment signals were identified by epistasis analysis. Like full-length receptors, the fragments appeared to generate signals through interactions with the CheA autokinase and the CheW coupling factor. CheA was required for both signaling activities, whereas CheW was needed only for CW signaling. Purified Tsr fragments were also examined for effects on CheA autophosphorylation activity in vitro. Consistent with the in vivo findings, the CW fragment stimulated CheA, whereas the CCW fragment inhibited CheA. CheW was required for stimulation but not for inhibition. These findings demonstrate that a 180-residue segment of the Tsr cytoplasmic domain can produce two active signals. The CCW signal involves a direct contact between the receptor and the CheA kinase, whereas the CW signal requires participation of CheW as well. The correlation between the in vitro effects of Tsr signaling fragments on CheA activity and their in vivo behavioral effects lends convincing support to the phosphorelay model of chemotactic signaling.     

CheW binding interactions with CheA and Tar. Importance for chemotaxis signaling in Escherichia coli

The initial signaling events underlying the chemotactic response of Escherichia coli to aspartic acid occur within a ternary complex that includes Tar (an aspartate receptor), CheA (a protein kinase), and CheW. Because CheW can bind to CheA and to Tar, it is thought to serve as an adapter protein in this complex. The functional importance of CheW binding interactions, however, has not been investigated. To better define the role of CheW and its binding interactions, we performed biochemical characterization of six mutant variants of CheW. We examined the ability of the purified mutant CheW proteins to bind to CheA and Tar, to promote formation of active ternary complexes, and to support chemotaxis in vivo. Our results indicate that mutations which eliminate CheW binding to Tar (V36M) or to CheA (G57D) result in a complete inability to form active ternary complexes in vitro and render the CheW protein incapable of mediating chemotaxis in vivo. The in vivo signaling pathway can, however, tolerate moderate changes in CheW-Tar and CheW-CheA affinities observed with several of the mutants (G133E, G41D, and 154ocr). One mutant (R62H) provided surprising results that may indicate a role for CheW in addition to binding CheA/receptors and promoting ternary complex formation.     

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.     

Assembly of an MCP receptor, CheW, and kinase CheA complex in the bacterial chemotaxis signal transduction pathway.

We examined the binding interactions of the methylation-dependent chemotaxis receptors Tsr and Tar with the chemotaxis-specific protein kinase CheA and the coupling factor CheW. Receptor directly bound CheW, but receptor-CheA binding was dependent upon the presence of CheW. These observations in combination with our previous identification of a CheW-CheA complex suggest that CheW physically links the kinase to the receptor. The ternary complex of receptor, CheW, and CheA is both kinetically and thermodynamically stable at physiological concentrations. Stability is not significantly altered by changes associated with attractant or repellent binding to the receptor. Such binding greatly modulates the kinase activity of CheA. Our results demonstrate that modulation of the kinase activity does not require association-dissociation of the ternary complex. This suggests that the receptor signal is transduced through conformational changes in the ternary complex rather than through changes in the association of the kinase CheA with receptor and/or CheW.     

Methylation segments are not required for chemotactic signalling by cytoplasmic fragments of Tsr, the methyl-accepting serine chemoreceptor of Escherichia coli

The serine chemoreceptor Tsr and other methyl-accepting chemotaxis proteins (MCPs) control the swimming behaviour of Escherichia coli by generating signals that influence the direction of flagellar rotation. MCPs produce clockwise (CW) signals by stimulating the autophosphorylation activity of CheA, a cytoplasmic histidine kinase, and counter-clockwise signals by inhibiting CheA. CheW couples CheA to chemoreceptor control by promoting formation of MCP/CheW/CheA ternary complexes. To identify MCP structural determinants essential for CheA stimulation, we inserted fragments of the tsr coding region into an inducible expression vector and used a swimming contest called 'pseudotaxis' to select for transformant cells carrying CW-signalling plasmids. The shortest active fragment we found, Tsr (350–470), stimulated CheA in a CheW-dependent manner, as full-length Tsr molecules do. It spans a highly conserved 'core' (370–420) that probably specifies the CheA and CheW contact sites and other determinants needed for stimulatory control of CheA. Tsr (350–470) also carries portions of the left and right arms flanking the core, which probably play roles in regulating MCP signalling state. However, this Tsr fragment lacks all of the methylation sites characteristic of MCP molecules, indicating that methylation segments are not essential for generating receptor output signals.     

CheA kinase and chemoreceptor interaction surfaces on CheW

Chemotactic responses of Escherichia coli to aspartic acid are initiated by a ternary protein complex composed of Tar (chemoreceptor), CheA (kinase), and CheW (a coupling protein that binds to both Tar and CheA and links their activities). We used a genetic selection based on the yeast two-hybrid assay to identify nine cheW point mutations that specifically disrupted CheW interaction with CheA but not with Tar. We sequenced these single point mutants and purified four of the mutant CheW proteins for detailed biochemical characterizations that demonstrated the weakened affinity of the mutant CheW proteins for CheA, but not for Tar. In the three-dimensional structure of CheW, the positions affected by these mutations cluster on one face of the protein, defining a potential binding interface for interaction of CheW with CheA. We used a similar two-hybrid approach to identify four mutation sites that disrupted CheW binding to Tar. Mapping of these "Tar-sensitive" mutation sites and those from previous suppressor analysis onto the structure of CheW defined an extended surface on a face of the protein that is adjacent to the CheA-binding surface and that may serve as an interface for CheW binding to Tar.     

Tandem translation starts in the cheA locus of Escherichia coli.

The cheA locus of Escherichia coli encodes two protein products, CheAL and CheAS. The nucleotide sequences of the wild-type cheA locus and of two nonsense alleles confirmed that both proteins are translated in the same reading frame from different start points. These start sites were located on the coding sequence by direct determination of the amino-terminal sequences of the two CheA proteins. Both starts are flanked by inverted repeats that may play a role in regulating the relative expression rates of the CheA proteins through alternative mRNA secondary structures.     

The roles of the multiple CheW and CheA homologues in chemotaxis and in chemoreceptor localization in Rhodobacter sphaeroides

Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in two major operons and other, unlinked, loci. These include cheA1 and cheW1 (che Op1) and cheA2, cheW2 and cheW3 (che Op2). We have deleted each of these cheA and cheW homologues in-frame and examined the chemosensory behaviour of these strains on swarm plates and in tethered cell assays. In addition, we have examined the effect of these deletions on the polar localization of the chemoreceptor McpG. In E. coli, deletion of either cheA or cheW results in a non-chemotactic phenotype, and these strains also show no receptor clustering. Here, we demonstrate that CheW2 and CheA2 are required for the normal localization of McpG and for normal chemotactic responses under both aerobic and photoheterotrophic conditions. Under aerobic conditions, deletion of cheW3 has no significant effect on McpG localization and only has an effect on chemotaxis to shallow gradients in swarm plates. Under photoheterotrophic conditions, however, CheW3 is required for McpG localization and also for chemotaxis both on swarm plates and in the tethered cell assay. These phenotypes are not a direct result of delocalization of McpG, as this chemoreceptor does not mediate chemotaxis to any of the compounds tested and can therefore be considered a marker for general methyl-accepting chemotaxis protein (MCP) clustering. Thus, there is a correlation between the normal localization of McpG (and presumably other chemoreceptors) and chemotaxis. We propose a model in which the multiple different MCPs in R. sphaeroides are contained within a polar chemoreceptor cluster. Deletion of cheW2 and cheA2 under both aerobic and photoheterotrophic conditions, and cheW3 under photoheterotrophic conditions, disrupts the cluster and hence reduces chemotaxis to any compound sensed by these MCPs.     

Mutational analysis of the chemoreceptor-coupling domain of the Escherichia coli chemotaxis signaling kinase CheA

During chemotactic signaling by Escherichia coli, autophosphorylation of the histidine kinase CheA is coupled to chemoreceptor control by the CheW protein, which interacts with the C-terminal P5 domain of CheA. To identify P5 determinants important for CheW binding and receptor coupling control, we isolated and characterized a series of P5 missense mutants. The mutants fell into four phenotypic groups on the basis of in vivo behavioral and protein stability tests and in vitro assays with purified mutant proteins. Group 1 mutants exhibited autophosphorylation and receptor-coupling defects, and their CheA proteins were subject to relatively rapid degradation in vivo. Group 1 mutations were located at hydrophobic residues in P5 subdomain 2 and most likely caused folding defects. Group 2 mutants made stable CheA proteins with normal autophosphorylation ability but with defects in CheW binding and in receptor-mediated activation of CheA autophosphorylation. Their mutations affected residues in P5 subdomain 1 near the interface with the CheA dimerization (P3) and ATP-binding (P4) domains. Mutant proteins of group 3 were normal in all tests yet could not support chemotaxis, suggesting that P5 has one or more important but still unknown signaling functions. Group 4 mutant proteins were specifically defective in receptor-mediated deactivation control. The group 4 mutations were located in P5 subdomain 1 at the P3/P3' interface. We conclude that P5 subdomain 1 is important for CheW binding and for receptor coupling control and that these processes may require substantial motions of the P5 domain relative to the neighboring P3 and P4 domains of CheA.     

The carboxy-terminal portion of the CheA kinase mediates regulation of autophosphorylation by transducer and CheW.

The CheA kinase is a central protein in the signal transduction network that controls chemotaxis in Escherichia coli. CheA receives information from a transmembrane receptor (e.g., Tar) and CheW proteins and relays it to the CheB and CheY proteins. The biochemical activities of CheA proteins truncated at various distances from the carboxy terminus were examined. The carboxy-terminal portion of CheA regulates autophosphorylation in response to environmental signals transmitted through Tar and CheW. The central portion of CheA is required for autophosphorylation and is also presumably involved in dimer formation. The amino-terminal portion of CheA was previously shown to contain the site of autophosphorylation and to be able to transfer the phosphoryl group to CheB and CheY. These studies further delineate three functional domains of the CheA protein.     

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.     

Role of the CheW protein in bacterial chemotaxis: overexpression is equivalent to absence.

The cheW gene from Escherichia coli has been cloned an inducible promoter, and the effects of the overproduction of the CheW protein on chemotactic behavior and receptor covalent modification have been examined. Plasmids that contain the cheW gene behind a regulatable promoter complement a cheW mutation when the CheW protein is produced at low levels. However, when the CheW protein is greatly overproduced in either a wild-type strain or a cheW mutant, chemotaxis is greatly inhibited, cheW null mutant cells swim smoothly as if they were constantly responding to an attractant. Surprisingly, cells in which the CheW protein is overproduced also swim smoothly. The behavioral defect produced by overproduction of the CheW protein does not require the presence of the cheR, cheB, or cheZ gene. Receptor demethylation is also inhibited by overproduction of the CheW protein, as it is by a mutation in the cheW gene or a response to an attractant. In all respects, therefore, overproduction of the CheW protein has the same consequences as does a mutation in the cheW gene or a response to an attractant. A model involving two states of the CheW protein is proposed to explain its role in bacterial chemotaxis.     

Site-specific and synergistic stimulation of methylation on the bacterial chemotaxis receptor Tsr by serine and CheW

Background  

Specific glutamates in the methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli are modified during sensory adaptation. Attractants that bind to MCPs are known to increase the rate of receptor modification, as with serine and the serine receptor (Tsr), which contributes to an increase in the steady-state (adapted) methylation level. However, MCPs form ternary complexes with two cytoplasmic signaling proteins, the kinase (CheA) and an adaptor protein (CheW), but their influences on receptor methylation are unknown. Here, the influence of CheW on the rate of Tsr methylation has been studied to identify contributions to the process of adaptation.     

Stabilization of polar localization of a chemoreceptor via its covalent modifications and its communication with a different chemoreceptor

In the chemotaxis of Escherichia coli, polar clustering of the chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW is thought to be involved in signal amplification and adaptation. However, the mechanism that leads to the polar localization of the receptor is still largely unknown. In this study, we examined the effect of receptor covalent modification on the polar localization of the aspartate chemoreceptor Tar fused to green fluorescent protein (GFP). Amidation (and presumably methylation) of Tar-GFP enhanced its own polar localization, although the effect was small. The slight but significant effect of amidation on receptor localization was reinforced by the fact that localization of a noncatalytic mutant version of GFP-CheR that targets to the C-terminal pentapeptide sequence of Tar was similarly facilitated by receptor amidation. Polar localization of the demethylated version of Tar-GFP was also enhanced by increasing levels of the serine chemoreceptor Tsr. The effect of covalent modification on receptor localization by itself may be too small to account for chemotactic adaptation, but receptor modification is suggested to contribute to the molecular assembly of the chemoreceptor/histidine kinase array at a cell pole, presumably by stabilizing the receptor dimer-to-dimer interaction.