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Regulation of the direction of flagellar rotation is central to the mechanism of bacterial chemotaxis. The transitions between counterclockwise and clockwise rotation are controlled by a "switch complex" composed of three proteins (FUG, FHlM, and FUN) CheY (8,9). In this way, the flux of information from the receptors is integrated into a common form: phosphorylated CheY. Changes in the phosphorylation level ofCheY are believed to be sensed by a group ofproteins located on the cytoplasmic face of the flagellar motor known as the "switch complex" (10), which causes the motor to adopt either a clockwise bias (if levels of phosphorylated CheY are high) or a counterclockwise bias (when phosphorylated CheY is low) (11). The mechanism of switch function is not known. Overproduction of CheY in a strain lacking all other chemotaxis proteins (12-14) or insertion of purified CheY into cell envelopes devoid of cytoplasm (15) MATERIALS AND METHODSPreparation of CheY Beads. CheY beads and control beads were prepared in parallel under identical conditions. Two samples (1-g dry weight each) of CNBr-activated Sepharose 4B beads (Pharmacia) were washed five times in 14 ml of 1 mM HCl and then three times in coupling buffer (0.1 M NaHC03/0.5 M NaCl, pH 8.3). CheY [5-45 mg, determined from absorbance at 280 nm by using a molar extinction coefficient of 6970 M-1"cm-1 (19)] purified from Escherichia coli (11) was dialyzed twice against 2 liters of coupling buffer and added to one tube. An equal volume of coupling buffer was added to the other tube (containing control beads). Both tubes were mixed end-over-end overnight at 4°C. In this way 95-99% ofthe CheY became covalently immobilized. Bovine serum albumin (BSA, 20 mg), previously dialyzed against coupling buffer, was then added to both tubes in order to block unreacted cyano groups, and the incubation with mixing continued for 5-10 hr at 4°C. To complete the coupling, additional BSA (20 mg), this time in 0.1 M Tris HCl (pH 7.9), was added to both tubes and incubated further for 5-10 hr. Finally the beads were washed in 50 mM Tris-HCl (pH 7.9) and stored at 4°C. Prior to use in the binding assay, the beads were washed and resuspended in 8-12 ml of Tris buffer. CheY57DE was produced and isolated as described earlier (20)

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The Tar chemoreceptor of Escherichia coli is a membrane-bound sensory protein that facilitates bacterial chemotaxis in response to aspartate. The EnvZ molecule has a membrane topology similar to Tar and is a putative osmosensor that is required for osmoregulation of the genes for the major outer membrane porin proteins, OmpF and OmpC. The cytoplasmic signaling domain of Tar was replaced with the carboxyl portion of EnvZ, and the resulting chimeric receptor activated transcription of the ompC gene in response to aspartate. The activation of ompC by the chimeric receptor was absolutely dependent on OmpR, a transcriptional activator for ompF and ompC.

The nature of the biochemical signal that is involved in the excitation response in bacterial chemotaxis is not known. However, ATP is required for chemotaxis. We have purified all of the proteins involved in signal transduction and show that the product of the cheA gene is rapidly autophosphorylated, while some mutant CheA proteins cannot be phosphorylated. The presence of stoichiometric levels of two other purified components in the chemotaxis system, the CheY and CheZ proteins, induces dephosphorylation. We suggest that the phosphorylation of CheA by ATP plays a central role in signal transduction in chemotaxis.Bacteria can respond to chemical changes in their environment by altering their pattern of motility, resulting in swimming toward higher concentrations of attractants and away from repellents. The chemotaxis response is mediated by a series of transmembrane receptor-transducer proteins that bind specific ligands and transmit information about changes in ligand concentration as a function of time to the bacterial flagellar apparatus (for reviews, see refs. 1, 2, and 3). The components involved in the intracellular signal transduction pathway for chemotaxis in Escherichia coli and Salmonella have been identified by genetic techniques. Four genes, cheA, cheW, cheY, and cheZ elaborate products that are required for the integration and transduction of information. Two other gene products encoded by cheR and cheB are responsible for adaptation to wide ranges of ligand concentration. They reversibly methylate specific glutamic acid residues on the cytoplasmic portion of the receptor-transducer, modulating its function.While a great deal is known about the components of the information-processing system, little is known about the biochemical nature of the chemotactic signal. A number of laboratories have found that ATP is required for signal transduction (4-7). However, the exact nature of its involvement was not clear. Indirect experiments have led to the formulation of models for the function of the chemotaxis proteins and their interaction with ATP (2, 8). To measure these interactions directly, we purified all of the proteins known to be involved in the central pathway for information transduction. In this paper we show that the cheA gene product can autophosphorylate with ATP. We can isolate a phosphorylated CheA intermediate and show that the CheY and CheZ proteins can influence the course of CheA phosphorylation. Furthermore, mutations that eliminate chemotaxis and map within the cheA gene result in proteins that are defective in the phosphorylation reaction.MATERIALS AND METHODS Protein Purifications. CheA and CheW were overexpressed from a plasmid, pDV4 (P.M., unpublished data), containing the cheA and cheW genes. The plasmid was maintained in an E. coli W3110 derivative SVS402 AtrpE-A, recAl, tna-2, bglR, obtained from R. Bauerle (University of Virginia, Charlottesville, VA). CheA was purified by a protocol including the use of dye-ligand chromatography and gel filtration and will be describe...