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Receptor Interactions Through Phosphorylation And Proc. Natl. Acad. Sci. USA Vol. 85, pp. 8425-8429, November 1988 Biochemistry Receptor interactions through phosphorylation and methylation pathways in bacterial chemotaxis (second messenger/feedback/excitation/memory/adaptation) DAVID AVRAM SANDERS AND DANIEL E. KOSHLAND, JR. Department of Biochemistry, University of California, Berkeley, CA 94720 Contributed by Daniel E. Koshland, Jr., August 5, 1988 ABSTRACT The effects of messages initiated by one re- process (21-24), but the chemistry of the excitation was ceptor on the covalent modification of a second receptor were unknown until recently. In vivo experiments by Simon and studied by use of a technique for rapidly separating the co-workers (25, 26) strongly suggest that phosphorylation is receptors. Methylation of the bacterial-chemotactic serine involved in the second-messenger system ofbacteria chemo- receptor increases as a result of aspartate binding to the tactic signaling, as in that ofeukaryotic cells (27). The protein aspartate receptor. The aspartate-induced methylation on the that serves as a kinase in chemotaxis is the CheA protein, serine receptor is absent in a strain that lacks cheA and cheW which had previously been identified as part of the signaling genes and is not the result of physical interaction, such as the system between receptors and flagella, and the CheA protein formation of heterodimers between the aspartate and serine is believed to modify the CheY protein, which is a strong receptors, or of alterations in the affmiity ofthe serine receptor candidate for the response regulator. for the methyltransferase and the methylesterase. Serine- Previous work suggested a feedback signal generated at the induced methylation ofthe serine receptor did not require cheA receptor could inhibit the methylesterase (28, 29). However, and cheW. A model is presented in which the receptor direct investigation of the consequences of the regulation of methylation level depends on the combination of (i) a ligand- the methylesterase for receptor methylation levels had not induced conformational change on the receptor substrate ofthe been attempted. The cheA and cheW genes are implicated in methylation enzymes and (ii) an indirect cytoplasmic signal these feedback effects (30-32); these same genes had also that operates through the methylesterase. been implicated in signaling from the receptor to the flagella. Such indirect effects could be produced through three pos- sible mechanisms: (i) feedback through some cytoplasmic Organisms, tissues, and cells experience a large variety of signal that reacted independently with either receptor, (ii) the stimulatory events and must not only respond to each one presence of heterodimers in which a conformational change individually but also integrate their responses. Stimuli, such in an aspartate-receptor subunit induced a conformational as light, hormones, or neurotransmitters, are sensed by change in a serine-receptor subunit, and (iii) an effect of transmembrane receptors that activate particular second- differential binding ofmethyltransferase and/or methylester- messenger systems (1). The second messengers are respon- ase to the directly stimulated receptor, which could thereby sible for producing the appropriate response to the stimulus alter the rates and levels ofmethylation ofthe other receptor. and coordinating the responses to different stimuli. For To establish the mechanism of the effect and its quantitative example, in bacterial chemotaxis a response regulator that contribution to the excitation and adaptation systems re- integrates responses from a variety of bacterial chemotaxis quired a method ofseparating two very similar receptors (33, receptors has been postulated as a device that controls output 34) so that we could measure accurately the methylation level of the cells and provides short-term memory (2). of each receptor. A technique that involved localized muta- Bacterial chemotaxis is an attractive system for the inves- genesis and that could be applied to other difficult-to-separate tigation of second messengers because of the power of the proteins was used to accomplish this task. available genetic and molecular biological tools. Escherichia coli and Salmonella typhimurium modulate their swimming behavior in reaction to changes in the concentrations of MATERIALS AND METHODS certain chemicals that act as attractants or repellents (3). Bacterial Strains. E. coli strains RP437 (wild type for Their motility pattern results from an integration of informa- chemotaxis), RP4372 (Atar-tap, tsr) and RP2898 (AcheA- tion obtained through a variety of receptors (4, 5). Aspartate cheW-tar-tap) were obtained from J. S. Parkinson (Univer- and serine, for example, bind to specific cell-surface chemo- sity of Utah) (35). taxis receptors, which produce signals that alter migration of Cloning of tars-S517C and tsrE onto a Single Plasmid, the sensing bacterium (6-8). These receptors are methylated pSK140, and Functionality Test. The EcoRl fragment of the on specific glutamate residues by the CheR protein, a plasmid pAB100 (33) containing the tsrE (E. coli serine methyltransferase (9-12). The reaction is reversible, because receptor) gene was ligated into the EcoRI site of plasmid hydrolysis of the methyl groups with the consequent produc- pFK139 (36), which encodes tars-S517C (S. typhimurium tion of methanol is catalyzed by the CheB protein, a methyl- aspartate receptor with Ser-517 -+ Cys mutation) and ampi- esterase (13, 14). Both aspartate and serine increase the rate cillin resistance, to create plasmid pSK140. Strains trans- of methylation and decrease the rate of demethylation of the formed with this plasmid and control plasmids were tested for receptor to which they bind, in vivo and in vitro (15-19). their ability to exhibit chemotaxis on swarm plates (37). The theory of a response regulator suggested that there Colonies were inoculated into the center of0.35% agar plates were two steps, a fast excitation step and a slow adaptation containing Vogel-Bonner citrate medium (38) supplemented process (20). Methylation was identified with the adaptation with 1% glycerol and histidine, methionine, leucine, threo- The publication costs of this article were defrayed in part by page charge Abbreviations: tsrE, Escherichia coli serine receptor gene; tars- payment. This article must therefore be hereby marked "advertisement" S517C, Salmonella typhimurium aspartate receptor gene with Ser- in accordance with 18 U.S.C. §1734 solely to indicate this fact. 517 Cys mutation. 8425 Downloaded by guest on October 2, 2021 8426 Biochemistry: Sanders and Koshland Proc. Natl. Acad. Sci. USA 85 (1988) nine, and thiamine (each at 50 Ag/ml) and, in some cases, the background in the dimer band were divided by 0.6 to obtain attractants 100 ,tM aspartate or 100 ttM serine; either aspartate-receptor methylation, and the remaining cpm were ampicillin (100 ,g/ml) or tetracycline (8 ,g/ml) was also in attributed to the serine receptor. the medium. After an initial lag, the rate of swarming, as When receptor demethylation was being examined, non- measured by the increase in diameter per hr, was linear. The radioactive methionine (final concentration, 250'g/ml) was plasmid pSK140 conferred chemotaxis toward both aspartate added as a chase to the cultures that had been incubated for and serine on a strain (RP4372) that was defective in both 30 min with radioactive methionine as described in the first traits. Plasmids pFK139 and pAB100 conferred chemotaxis paragraph of this section. The specific activity of the me- toward only aspartate or serine, respectively (data not thionine was thereby reduced 50-fold. Receptor methylation shown). was determined at various time points before and after Separation and Measurement of Methylation of Receptors. attractant addition. Seven minutes of incubation with the Bacteria were grown to an OD650 of 0.7-1.2 units at 30'C in nonradioactive methionine preceded the addition of attrac- Vogel-Bonner citrate medium supplemented with 1% glyc- tant to ensure that the S-adenosylmethionine pools were erol and histidine, methionine, leucine, threonine, and thia- equilibrated. mine (each at 50 ,ug/ml) with ampicillin (250 /.g/ml) or tetracycline (8 pug/ml) ifrequired. The cells were collected by centrifugation at 5000 x g for 10 min, washed twice with the RESULTS same medium and supplements-except that the concentra- Stimulation of Methylation ofthe Serine Receptor During an tion of methionipne was 5 ,g/ml, and then resuspended at an Aspartate Response Requires the Presence of the Aspartate OD650 of 1.0 in the same medium containing an additional 350 Receptor. To examine whether a response generated by one ,uCi of L-[methyl-3H]methionine per ml (85 Ci/mmol; 1 Ci = receptor affected the methylation of a second receptor 37 GBq). The cultures were incubated at 30°C for 30 min required a special technique for measurement of the meth- before attractant addition. ylation level of each receptor separately. Two-dimensional At various times after attractant addition, duplicate 200-,ul polyacrylamide gels did not separate the aspartate and serine aliquots were precipitated in 1 ml of ice-cold 10% trichloro- receptors sufficiently to allow for accurate quantification of acetic acid. The pellet from one aliquot was extracted into 40 their methylation levels. To make a clean separation of the ,l of 2x sodium dodecyl sulfate (SDS) buffer (750 mM two receptors, a cysteine residue was introduced by localized Tris HCl, pH 8.8/20% glycerol (wt/vol)/0.004%
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