Computer analysis of the binding reactions leading to a transmembrane receptor-linked multiprotein complex involved in bacterial chemotaxis.

The chemotactic response of bacteria is mediated by complexes containing two molecules each of a transmembrane receptor and the intracellular signaling proteins CheA and CheW. Mutants in which one or the other of the proteins of this complex are absent, inactive, or expressed at elevated amounts show altered chemotactic behavior and the phenotypes are difficult to interpret for some overexpression mutants. We have examined the possibility that these unexpected phenotypes might arise from the binding steps that lead to active complex formation. A limited genetic algorithm was used to search for sets of binding reactions and associated binding constants expected to give mutant phenotypes in accord with experimental data. Different sets of binding equilibria and different assumptions about the activity of particular receptor complexes were tried. Computer analysis demonstrated that it is possible to obtain sets of binding equilibria consistent with the observed phenotypes and provided a simple explanation for these phenotypes in terms of the distribution of active and inactive complexes formed under various conditions. Optimization methods of this kind offer a unique way to analyze reactions taking place inside living cells based on behavioral data.

Computer simulation of the phosphorylation cascade controlling bacterial chemotaxis.

We have developed a computer program that simulates the intracellular reactions mediating the rapid (nonadaptive) chemotactic response of Escherichia coli bacteria to the attractant aspartate and the repellent Ni2+ ions. The model is built from modular units representing the molecular components involved, which are each assigned a known value of intracellular concentration and enzymatic rate constant wherever possible. The components are linked into a network of coupled biochemical reactions based on a compilation of widely accepted mechanisms but incorporating several novel features. The computer motor shows the same pattern of runs, tumbles and pauses seen in actual bacteria and responds in the same way as living bacteria to sudden changes in concentration of aspartate or Ni2+. The simulated network accurately reproduces the phenotype of more than 30 mutants in which components of the chemotactic pathway are deleted and/or expressed in excess amounts and shows a rapidity of response to a step change in aspartate concentration similar to living bacteria. Discrepancies between the simulation and real bacteria in the phenotype of certain mutants and in the gain of the chemotactic response to aspartate suggest the existence of additional as yet unidentified interactions in the in vivo signal processing pathway.

Throwing the switch in bacterial chemotaxis.

In Escherichia coli chemotaxis, the switch from counterclockwise to clockwise rotation of the flagella occurs as a result of binding of the phosphorylated CheY protein to the base of the flagellum. Analysis of CheY variants has provided a picture of the surface of CheY that undergoes conformational shifts, as a result of phosphorylation, to interact directly with the flagellum. Whether phospho-CheY binding and flagellar switching are sequential steps or can occur in a concerted fashion has yet to be determined.

Protein phosphorylation in the bacterial chemotaxis system.

Bacterial chemotaxis involves the detection of changes in concentration of specific chemicals in the environment of the cell as a function of time. This process is mediated by a series of cell surface receptors that interact with and activate intracellular protein phosphorylation. Five cytoplasmic proteins essential for chemotaxis have been shown to be involved in a coupled system of protein phosphorylation. Ligand binding to cell surface receptors affects the rate of autophosphorylation of the CheA protein. In the absence of an attractant bound to receptor and in the presence of the CheW protein, the rate of CheA autophosphorylation is markedly increased. Phosphorylated CheA can transfer phosphate to the CheY or CheB proteins; phosphorylation of these "effector" proteins may increase their activity. The CheY protein is thought to regulate flagellar rotation and thus control swimming behavior. The CheB protein modifies the cell surface receptor and thus regulates receptor function. Finally, another chemotaxis protein, CheZ, acts to specifically dephosphorylate CheY-phosphate. This system shows marked similarity to the 2-component sensor-regulator systems found to control specific gene expression in a variety of bacteria.

Lysogenization of Escherichia coli him+, himA, and himD hosts by bacteriophage Mu.

The possible outcomes of infection of Escherichia coli by bacteriophage Mu include lytic growth, lysogen formation, nonlysogenic surviving cells, and perhaps simple killing of the host. The influence of various parameters, including host himA and himD mutations, on lysogeny and cell survival is described. Mu does not grow lytically in or kill him bacteria but can lysogenize such hosts. Mu c+ lysogenizes about 8% of him+ bacteria infected at low multiplicity at 37 degrees C. The frequency of lysogens per infected him+ cell diminishes with increasing multiplicity of infection or with increasing temperature over the range from 30 to 42 degrees C. In him bacteria, the Mu lysogenization frequency increases from about 7% at low multiplicity of infection to approach a maximum where most but not all cells are lysogens at high multiplicity of infection. Lysogenization of him hosts by an assay phage marked with antibiotic resistance is enhanced by infection with unmarked auxiliary phage. This helping effect is possible for at least 1 h, suggesting that Mu infection results in formation of a stable intermediate. Mu immunity is not required for lysogenization of him hosts. We argue that in him bacteria, all Mu genomes which integrate into the host chromosome form lysogens.

Intermediates in bacteriophage Mu lysogenization of Escherichia coli him hosts.

Characterization of a putative intermediate in the Mu lysogenization pathway is possible in a variant Escherichia coli himD strain which exhibits greatly diminished lysogen formation. In this strain, most infecting Mu genomes form stable, transcribable, nonreplicating structures. Many of these genomes can be mobilized to form lysogens by a second Mu infection, which can be delayed by at least 100 min. This intermediate structure can be formed in the absence of Mu A or B function. We suggest that the inferred intermediate could be the previously reported protein-linked circular form of the Mu genome. Providing Mu B function from a plasmid enhances Mu lysogenization in this him strain, and the enhancement is much greater when both Mu A and B functions are provided.

Proposed signal transduction role for conserved CheY residue Thr87, a member of the response regulator active-site quintet.

CheY serves as a structural prototype for the response regulator proteins of two-component regulatory systems. Functional roles have previously been defined for four of the five highly conserved residues that form the response regulator active site, the exception being the hydroxy amino acid which corresponds to Thr87 in CheY. To investigate the contribution of Thr87 to signaling, we characterized, genetically and biochemically, several cheY mutants with amino acid substitutions at this position. The hydroxyl group appears to be necessary for effective chemotaxis, as a Thr-->Ser substitution was the only one of six tested which retained a Che+ swarm phenotype. Although nonchemotactic, cheY mutants with amino acid substitutions T87A and T87C could generate clockwise flagellar rotation either in the absence of CheZ, a protein that stimulates dephosphorylation of CheY, or when paired with a second site-activating mutation, Asp13-->Lys, demonstrating that a hydroxy amino acid at position 87 is not essential for activation of the flagellar switch. All purified mutant proteins examined phosphorylated efficiently from the CheA kinase in vitro but were impaired in autodephosphorylation. Thus, the mutant CheY proteins are phosphorylated to a greater degree than wild-type CheY yet support less clockwise flagellar rotation. The data imply that Thr87 is important for generating and/or stabilizing the phosphorylation-induced conformational change in CheY. Furthermore, the various position 87 substitutions differentially affected several properties of the mutant proteins. The chemotaxis and autodephosphorylation defects were tightly linked, suggesting common structural elements, whereas the effects on self-catalyzed and CheZ-mediated dephosphorylation of CheY were uncorrelated, suggesting different structural requirements for the two dephosphorylation reactions.

Synthesis and characterization of a stable analog of the phosphorylated form of the chemotaxis protein CheY.

The bacterial chemotaxis protein CheY is activated in vivo by the covalent phosphorylation of a single aspartate residue at position 57. However, this phosphate linkage is unstable (t1/2 approximately 20 s at room temperature), thereby precluding many biochemical analyses. Here we present a synthetic scheme to prepare an analog of CheY-phosphate (Che Y-P) with chemical stability of the phosphate linkage enhanced by several orders of magnitude relative to the native protein. Starting with CheY D57C, a site-specific mutant of CheY with a unique cysteine residue in place of the aspartate at position 57, two sequential disulfide exchange reactions were performed to form the final product 'CheY D57C-SPO3' with a thiophosphate moiety covalently bonded to the protein in a disulfide linkage. Mass spectral analysis showed that the desired analog was present at 70-80% of the total protein. The disulfide linkage had a t1/2 of 8 days at 4 degrees C. Biochemical characterization of CheY D57C-SPO3 included assessment of conformational properties using tryptophan fluorescence, evaluation of metal binding properties and measurement of binding interactions with the chemotaxis proteins CheZ and FliM. Despite possessing a phosphoryl group at a nearly identical location as native CheY-phosphate, the analog was unable to emulate CheY-phosphate function, thereby supporting the idea that there are very precise geometric requirements for successful CheY activation.

Activation of CheY mutant D57N by phosphorylation at an alternative site, Ser-56.

The site of phosphorylation of the chemotaxis response regulator CheY is aspartate 57. When Asp-57 is replaced with an asparagine, the resultant protein can be phosphorylated at an alternative site. We report here that phosphorylation of this mutant protein, CheY D57N, at the alternative site affords the protein activity in vivo in the absence of CheZ. Using a direct phosphopeptide mapping approach, we identified the alternate phosphorylation site as serine 56. Introduction of a Ser-->Ala substitution at this position in wild-type CheY had no effect on function. However, replacement of Ser-56 with Ala in CheY D57N abrogated the activity seen in vivo for the CheY D57N single mutant protein, and no phosphorylation of the CheY S56A/D57N double mutant protein was observed in vitro. Construction and analysis of double mutants CheY D57N/T87A and CheY D57N/K109R, which were both inactive, suggested that phosphorylation at Ser-56 or Asp-57 may activate the protein by similar mechanisms. In contrast to CheY D57N, mutant CheY D57E displayed no activity in vivo, despite its ability to be phosphorylated in vitro. Acid-base stability analysis indicated that CheY D57E phosphorylates on an acidic residue, presumably Glu-57. These data suggest that a key determinant of the ability of a phosphoryl group to activate CheY is proximity to the hydrophobic core of the protein, with consequent opportunity to reposition key residues, irrespective of the chemical nature of the linkage attaching the phosphoryl group to CheY.

Intermolecular complementation of the kinase activity of CheA.

CheA is a dimeric autophosphorylating protein kinase that plays a critical role in the signal transduction network controlling chemotaxis in Escherichia coli. The autophosphorylation reaction was analysed using mutant proteins defective in kinase and regulatory functions. Proteins in which the site of autophosphorylation was mutated (CheA48HQ) or missing (CheAs) were found to phosphorylate the kinase-defective mutant, CheA470GK. The kinetics of this reaction support the hypothesis that autophosphorylation is the result of trans-phosphorylation within a dimer. The carboxy-terminal portion of CheA was previously shown to be dispensable for autophosphorylation, but required for regulation in response to environmental signals transmitted through a transducer and CheW. Mixing of CheA48HQ or CheA470GK with a truncated protein lacking this regulatory domain demonstrated that regulated autophosphorylation requires the presence of both carboxy-terminal portions in a CheA dimer. These results indicate that the dimeric form of CheA plays an integral role in signal transduction in bacterial chemotaxis.

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.

Histidine phosphorylation and phosphoryl group transfer in bacterial chemotaxis.

A cascade of protein phosphorylation, initiated by autophosphorylation of the CheA protein, may be important in the signal transduction pathway of bacterial chemotaxis. A proteolytic fragment of CheA cannot autophosphorylate, but can still transfer phosphate to proteins that generate excitation and adaptation signals. The site of CheA phosphorylation is His 48; mutants altered at this position are non-chemotactic. Similar mechanisms of transient protein phosphorylation and phosphoryl group transfer seem to be involved in processing sensory data and in activating specific gene expression.

Isolation and characterization of nonchemotactic CheZ mutants of Escherichia coli.

The Escherichia coli CheZ protein stimulates dephosphorylation of CheY, a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism. Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization. To identify putative regions of functional importance in CheZ, we subjected cheZ to random mutagenesis and isolated 107 nonchemotactic CheZ mutants. Missense mutations clustered in six regions of cheZ, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene. Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ(1-189) peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer. Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically. All of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ. There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate. In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein.

Acetylation at Lys-92 enhances signaling by the chemotaxis response regulator protein CheY.

When Escherichia coli cells lacking all chemotaxis proteins except the response regulator CheY are exposed to acetate, clockwise flagellar rotation results, indicating the acetate stimulus has activated signaling by CheY. Acetate can be converted to acetyl-CoA by either of two different metabolic pathways, which proceed through acetyl phosphate or acetyl-AMP intermediates. In turn, CheY can be covalently modified by either intermediate in vitro, leading to phosphorylation or acetylation, respectively. Either pathway is sufficient to support the CheY-mediated response to acetate in vivo. Whereas phosphorylation of Asp-57 is a recognized mechanism for activation of CheY to stimulate clockwise flagellar rotation, acetylation of CheY is less well characterized. We found evidence for multiple CheY acetylation sites by mass spectrometry and directly identified Lys-92 and Lys-109 as acetylation sites by Edman degradation of peptides from [14C]acetate-labeled CheY. Replacement of CheY Lys-92, the preferred acetylation site, with Arg has little effect on chemotaxis but completely prevents the response to acetate via the acetyl-AMP pathway. Thus acetylation of Lys-92 activates clockwise signaling by CheY in vivo. The mechanism by which acetylation activates CheY apparently is not simple charge neutralization, nor does it involve enhanced binding to the FliM flagellar switch protein. Thus acetylation probably affects signal generation by CheY at a step after switch binding.

Conserved aspartate residues and phosphorylation in signal transduction by the chemotaxis protein CheY.

The CheY protein is phosphorylated by CheA and dephosphorylated by CheZ as part of the chemotactic signal transduction pathway in Escherichia coli. Phosphorylation of CheY has been proposed to occur on an aspartate residue. Each of the eight aspartate residues of CheY was replaced by using site-directed mutagenesis. Substitutions at Asp-12, Asp-13, or Asp-57 resulted in loss of chemotaxis. Most of the mutant CheY proteins were still phosphorylated by CheA but exhibited modified biochemical properties, including reduced ability to accept phosphate from CheA, altered phosphate group stability, and/or resistance to CheZ-mediated dephosphorylation. The properties of CheY proteins bearing a substitution at position 57 were most aberrant, consistent with the hypothesis that Asp-57 is the normal site of acyl phosphate formation. Evidence for an alternate site of phosphorylation in the Asp-57 mutants is presented. Phosphorylated CheY is believed to cause tumbling behavior. However, a dominant mutant CheY protein that was not phosphorylated in vitro caused tumbling in vivo in the absence of CheA. This phenotype suggests that the role of phosphorylation in the wild-type CheY protein is to stabilize a transient conformational change that can generate tumbling behavior.

Conformational coupling in the chemotaxis response regulator CheY.

CheY, a response regulator protein in bacterial chemotaxis, serves as a prototype for the analysis of response regulator function in two-component signal transduction. Phosphorylation of a conserved aspartate at the active site mediates a conformational change at a distal signaling surface that modulates interactions with the flagellar motor component FliM, the sensor kinase CheA, and the phosphatase CheZ. The objective of this study was to probe the conformational coupling between the phosphorylation site and the signaling surface of CheY in the reverse direction by quantifying phosphorylation activity in the presence and absence of peptides of CheA, CheZ, and FliM that specifically interact with CheY. Binding of these peptides dramatically impacted autophosphorylation of CheY by small molecule phosphodonors, which is indicative of reverse signal propagation in CheY. Autodephosphorylation and substrate affinity, however, were not significantly affected. Kinetic characterization of several CheY mutants suggested that conserved residues Thr-87, Tyr-106, and Lys-109, implicated in the activation mechanism, are not essential for conformational coupling. These findings provide structural and conceptual insights into the mechanism of CheY activation. Our results are consistent with a multistate thermodynamic model of response regulator activation.

Catalytic mechanism of phosphorylation and dephosphorylation of CheY: kinetic characterization of imidazole phosphates as phosphodonors and the role of acid catalysis.

Kinetic and equilibrium measurements of phosphotransfer events involving CheY carried out over a range of pH conditions elucidated several features of the phosphotransfer mechanism. Using tryptophan fluorescence intensity measurements as a monitor of phosphorylation, we showed that phosphorylation using small molecule phosphodonors occurred by fast association of CheY with the phosphodonor, followed by rate-limiting phosphotransfer. Two previously uncharacterized phosphodonors, monophosphoimidazole and diphosphoimdazole, were able to phosphorylate CheY at a concentration about 6-fold lower than that of the previously described phosphodonors acetyl phosphate and phosphoramidate. This was shown to be due to tighter binding of the imidazole phosphates to CheY and implied the presence of binding interactions between CheY and the imidazole group. The ability of CheY to autophosphorylate through the pH range of 5-10 differed for various phosphodonors. Acetyl phosphate and diphosphoimidazole were unaffected by pH over this range, whereas phosphoramidate and monophosphoimidazole showed a steep dependence on pH with a loss of phosphorylation ability at about pH 7.4 (midpoint) for monophosphoimidazole and pH 7.8 (midpoint) for phosphoramidate. This behavior correlated with the loss of the positive charge on the nitrogen atom in the nitrogen-phosphorus bond in both monophosphoimidazole and phosphoramidate and implied that CheY was not capable of donating a proton to the leaving group in phosphotransfer with small molecules. The rate of phosphotransfer from [32P]CheA-phosphate to wild type CheY also decreased markedly (> 150 times) between pH 7.5 and 10. Because the mutant CheY proteins K109R and T87A showed the same pH dependence as the wild type, the loss of activity in the alkaline range could not be attributed to deprotonation of either of these active site residues. This observation, combined with the moderate decreases in phosphotransfer rates for these mutants relative to that of wild type CheY, indicated that it is unlikely that either Thr87 or Lys109 plays a direct role in the catalysis of phosphotransfer. Finally, we showed that the rate of autodephosphorylation of CheY was independent of pH over the range of 4.5-11. Together, these studies led to a model with CheY playing a largely entropic role in its own phosphorylation and dephosphorylation.

Purification and characterization of Bacillus subtilis CheY.

Amino acid sequence comparison suggests that numerous proteins are common to the signal transduction pathways controlling chemotaxis in Bacillus subtilis and Escherichia coli. However, previous work has indicated several differences between the two systems. We have undertaken a comparative study of the roles of the CheY protein in chemotaxis by B. subtilis and E. coli. Although CheY from the two species share only 36% amino acid sequence identity, purified B. subtilis CheY was phosphorylated in vitro by E. coli CheA, and dephosphorylation of CheY-P was enhanced by E. coli CheZ. Alteration of the putative site of phosphorylation in B. subtilis CheY, Asp54, eliminated chemotaxis in vivo, further confirming that phosphorylation is important for B. subtilis chemotaxis. Loss of CheY function resulted in tumbling behavior in B. subtilis. Introduction of positively charged residues in place of Asp10 of B. subtilis CheY abolished function, whereas the corresponding changes in E. coli CheY apparently result in constitutive activation. The B. subtilis CheY Asp10 mutant proteins also failed to cause tumbling in E. coli, consistent with a different interaction between CheY and the flagellar switch in the two species. Finally, B. subtilis adapted more rapidly to positive stimuli than negative stimuli, whereas the opposite is true of E. coli. We conclude that B. subtilis regulates its response to positive chemotactic stimuli by enhancing phosphorylation of chemotaxis proteins, whereas E. coli reduces phosphorylation in the same circumstance.

Mutations in the chemotactic response regulator, CheY, that confer resistance to the phosphatase activity of CheZ.

CheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway. The concentration of phospho-CheY is thought to determine the swimming behaviour of the cell: high levels of phospho-CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phosphorylated form of the protein allow counter-clockwise rotation of the flagella and smooth swimming. The phosphorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphorylation of phospho-CheY. The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho-CheY and by the CheZ protein, which accelerates dephosphorylation. We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY proteins. Two point mutations were identified which were 50-fold and 5-fold less sensitive to the activity of CheZ than was the wild-type protein. Nonetheless, the phosphorylation and autodephosphorylation rates of these mutants. CheY23ND and CheY26KE, were observed to be identical to those of wild-type CheY in the absence of CheZ. These are the first examples of cheY mutations that reduce sensitivity to the phosphatase activity of CheZ without being altered in terms of their intrinsic phosphorylation and autodephosphorylation rates. Interestingly, the residues Asn-23 and Lys-26 are located on a face of CheY far from the phosphorylation site (Asp-57), distinct from the previously described site of interaction with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch.