JOURNAL Op BACTERIOLOGY, Dec. 1983, p. 1228-12350021-9193/83/121228-08$02.00/0Copyright © 1983, American Society for Microbiology

Vol. 156, No. 3

Requirement of the cheB Function for Sensory Adaptation inEscherichia coli

HIROYUKI YONEKAWA,1 HIROSHI HAYASHI,W AND JOHN S. PARKINSON2*Institute ofMolecular Biology, Nagoya University, Nagoya, Japan, ' and Biology Department, University of

Utah, Salt Lake City, Utah 841122

Received 18 July 1983/Accepted 27 September 1983

The chemotactic behavior of Escherichia coli mutants defective in cheBfunction, which is required to remove methyl esters from methyl-acceptingchemotaxis proteins, was investigated by subjectitng swimming or antibody-tethered cells to various attractant chemicals. Two cheB point mutants, onemissense and one nonsense, exhibited stimulus response times much longer thandid the wild type, but they eventually returned to the prestimulus swimmingpattern, indicating that they were not completely defective in sensory adaptation.In contrast, strains deleted for the cheB function showed no evidence ofadaptation ability after stimulation. The crucial difference between these strainsappeared to be the residual level of cheB-dependent methylesterase activity theycontained. Both point mufants showed detectable levels of methanol evolutiondue to turnover of methyl groups on methyl-accepting chemotaxis proteinmolecules, whereas the cheB deletion mutant did not. In addition, it was possibleto incorporate the methyl label into the methyl-accepting chemotaxis proteins ofthe point mutants but not into those of the cheB deletion strain. These findingsindicate that cheB function is essential for sensory adaptation in Escherichia coli.

Escherichia coli swims by rotating its flagellarfilaments. Counter-clockwise (CCW) rotationproduces smooth swimming, whereas clockwise(CW) rotation causes abrupt turning movementsor tumbles (1, 12, 24). In the absence of chemo-tactic stimuli, the cells alternate CCW and CWrotational episodes, enabling them to move ran-domly through their environment. Chemotacticmovements are mediated by modulating the di-rection offlagellar rotation in response to chemi-cal stimuli (12). For example, attractant in-creases or repellent decreases cause enhancedCCW flagellar rotation. Conversely, attractantdecreases and repellent increases cause en-hanced CW rotation. The cells respond onlytransiently to temporal changes in attractant orrepellent concentration. In static chemical envi-ronments they undergo sensory adaptation andreturn to their unstimulated swimming pattern(2, 13, 30).

Genetic and biochemical studies of bacterialchemotaxis have identified several methyl-ac-cepting chemotaxis proteins (MCPs) that playkey roles in both the excitatory and adaptivephases of chemotactic responses (11, 28). MCPmolecules appear to span the cytoplasmic mem-brane. At their outer face they interact withsmall molecules (6, 34) or with liganded bindingproteins (10, 20) to trigger changes in swimmingbehavior (excitation). At their cytoplasmic face

they undergo methylation or demethylation re-actions that culminate in sensory adaptation (3).Adaptation to CCW rotation-enhancing stimuliis correlated with an increase in the methylationstate of the MCP molecules engaged in excitato-ry signaling, whereas adaptation to CW rotation-enhancing stimuli is accompanied by a decreasein the MCP methylation state.The methyl groups on MCP molecules are

derived from methionine (by means of S-adeno-sylinethionine) and are in the form of a y-glutamyl methylester (9, 33). Two enzymes con-trol MCP methylation state: the cheR geneproduct which is a methyltransferase (29) andthe cheB gene product which is a methylesterase(5, 31). Mutants defective in cheR function canrespond to certain stimuli but cannot adapt,presumably, because they are unable to addmethyl groups to their MCP molecules (4, 9).

If there is a causal relationihip betweenchanges in MCP methylation state and sensoryadaptation, as is imphed by the properties ofcheR mutants, loss of cheB function should alsoresult in adaptation defects. This prediction hasnot been confirmed in previous studies of cheBmutants. Mutants defective in cheB functionexhibit CW flagellar biases, but they can stillrespond to stimuli that enhance CCW flagellarrotation (16, 17). If the stimuli are relativelyweak, the responses are short lived, suggesting

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ADAPTATION DEFECTS IN cheB MUTANTS 1229

that adaptation has occurred. With somewhatstronger stimuli, cheB mutants show a dispro-portionate increase in response times (22). Thispotentiation effect could be due to a partialdefect in adaptation ability.We suspected that the main reason cheB mu-

tants failed to show dramatic adaptation defectswas that the mutants used in previous studiesmay have been phenotypically leaky. In thisreport we show that the adaptation ability ofcheB strains is related to their residual methyles-terase activity. We also demonstrate that a cheBdeletion mutant is completely unable to adapt tochemotactic stimuli, confirming the notion thatchanges in the MCP methylation state are obli-gately coupled to the process of sensory adapta-tion in E. coli. The implications of these findingswith respect to the role of cheB function inmodulating MCP signaling behavior are alsodiscussed.

MATERIALS AND METHODSBacterial strains. All bacterial strains used in this

work were derivatives of E. coli K-12. The cheBmutants and wild-type control strains are listed inTable 1. The construction of the cheB deletion strainsis described below. The flaI mutant YK4136 (F- flalthi his pyrC thyA araD lac nalA rpsL) was obtainedfrom Y. Komeda (National Institute of Genetics, Mi-shima, Japan) and was used as a negative control inmethylesterase assays.Growth conditions. All of the experimental methods

described below utilized cells grown in tryptone brothor 3% Casamino Acids (Difco Laboratories). The cellswere grown at 30 or 35° C to densities of 5 x 108 to 6 x108/ml and harvested by centrifugation. The pelletedcells were washed three times at room temperature inmotility buffer (10 mM potassium phosphate [pH 7.5],0.1 mM potassium EDTA) and then suspended inmotility buffer at ca. 5 x 108 cells per ml for subse-quent use.

Flagellar rotation. Flagella were sheared from thecells by treatment in a Waring blender, as described by

Khan et al. (8), or in a Teflon homogenizer. Thedeflagellated cells were pelleted by centrifugation,resuspended in motility buffer, and tethered to micro-scope slides or cover slips with antiflagellar antibodyas described previously (16). For quantitative analy-ses, individual rotating cells were each examined for15 s and the proportion of time spent rotating CCWand CW was measured. At least 100 cells of each strainunder study were examined and classified as reversingor as exclusively CCW or CW during the 15-s observa-tion period.Swimming behavior. Swimming cells in motility

buffer were subjected to chemotactic stimuli by mix-ing with attractant solution and were then observedby phase-contrast microscopy. In most cases ex-periments were videotaped; however, quantitativeanalyses were carried out with "swimming tracks"recorded on Kodak Tri-X film with a dark-field photo-microscope (15).

Demethylation assay. Turnover of MCP methylgroups was assessed by measuring methanol produc-tion essentially as described by Toews et al. (32).Washed cells were suspended at 109/ml in motilitybuffer containing 10 mM potassium DL-lactate, 10 mMpotassium nitrate, and 10 ,uM methionine. Cell sam-ples were incubated at 30° C for 10 min, and then[methyl-3H]methionine (12 Ci/mmol; New EnglandNuclear Corp.) was added to a final concentration of 3,Ci/ml. At various times, 0.5-ml samples were with-drawn, quenched by the addition of 0.5 ml of 10%otrichloroacetic acid, and then placed in the outer wellof a Conway microdiffusion cell, with 1 ml of water inthe center well. The diffusion cells were left for 3 h atroom temperature, and then, 0.5 ml of solution wasremoved from the center well and counted for radioac-tivity with scintillant ACS-II (Amersham Corp.).

Methylation assay. For the methylation assay, thecell preparation, labeling conditions, and incubationtimes were identical to those used in the demethylationassays described above through the trichloroaceticacid quenching step. At that point, cell pellets werecollected by centrifugation and dissolved in electro-phoresis sample buffer and subjected to electrophore-sis in sodium dodecyl sulfate-containing polyacryl-amide gels as previously described (14). The MCP

TABLE 1. Properties of cheB strainsFlagellar rotation Response thresholds

Strain Chemotaxis genotype (% rotating cells) (M) for:CCW reversing CW Serine Aspartate

cheB point mutantsRP4753 cheB274 14 35 51 10-6 10-4RP4751 cheB294 3 47 50 10-6 5 x 10-4

cheB deletion mutantsRP1063 A(tap-cheB)2241 (X i434, X 4 8 88 10-6 10-3

che22A11-A37)RP1075 A(tap-cheB)2241 (X che22All-A37) 2 9 89 10-6 10-3RP5096 A(tar-cheB)2234 (X che22A25-A37) 4 10 86 lo-6 10-3

cheB+ control strainsRP437 Wild type 7 93 0 5 x 10-7 5 x l0-7RP477 Wild type 9 91 0RP1041 A(tap-cheB)2241 (X che22) 3 94 3RP1049 A(tar-cheB)2234 (A che22) 11 88 1

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1230 YONEKAWA, HAYASHI, AND PARKINSON

regions of the gels were removed and subjected toalkaline treatment to hydrolyze the MCP methyl estergroups, and the volatile radioactivity was counted inACS-II scintillant to determine the amount of methyllabel incorporated into the MCP molecules.

RESULTSConstruction of cheB deletion strains. The

cheB gene is located in an operon of chemotaxis-related genes (Fig. 1). To construct strains thatcompletely lacked cheB function yet retained allthe other chemotaxis functions of the operon,two different deletion mutations were used. Oneof the deletions (A2241) inactivates the tap,cheR, and cheB genes; the other deletion(A2234) inactivates the same three genes as wellas the tar gene (Fig. 1). Neither deletion had anydetectable polar effects on expression of thedownstream cheY and cheZ genes (18). Hoststrains carrying these deletions were lysoge-nized with specialized k transducing phages thatfurnished all of the missing chemotaxis functionsexcept cheB. The A2241 host was lysogenizedwith k che22All-A37, which expresses tap andcheR function; the A2234 host was lysogenizedwith k che22A25-A37, which expresses tar, tap,and cheR functions (Fig. 1). Neither phage hasthe A site-specific intergration system, so stablelysogens were constructed by using either theundeleted portion of the host che region or aresident heteroimmune prophage to provide ho-mology for integrative exchanges. In addition, itshould be noted that the tap and cheR genescarried by K che22A11-437 are expressedthrough transcriptional fusion to the motA pro-moter (Fig. 1). Since the motA and tar promot-ers have similar, if not identical, efficiencies, thelevel of expression of the tap and cheR functions

in strains carrying this phage should be essen-tially normal. In fact, we found that all of ourcheB deletion strains exhibited very similar be-havior, regardless of their method of construc-tion. Most of the experiments to be describeddeal with strain RP1075 (Table 1).

Flagelar rotation patterns of cheB mutants.The unstimulated flagellar rotation patterns ofthe strains used in this study are listed in Table1. RP4753 carries the cheB274 allele, which ismost likely a missense mutation. RP4751 carriescheB294, an amber mutation that maps in thecarboxy-terminal coding region of the cheB gene(Fig. 1) and has no detectable polar effect on theexpression of the che Y and cheZ functions (17).RP1063, RP1075, and RP5096 are cheB deletionstrains whose construction was describedabove. In the absence of chemotactic stimuli, allof these cheB mutants had a pronounced CWrotational bias and, consequently, a lower rever-sal frequency than did $train RP437 or the otherwild-type controls (Table 1). The rotational pat-terns of the two point mutants were similar butless CW-biased than those of the deletionstrains. The more extreme CW bias exhibited bythe AcheB strains suggests that the point mu-tants could be somewhat leaky.Response thresholds of cheB mutants. The re-

sponse thresholds of cheB mutants to aspartateand serine, the two most potent amino acidattractants of E. coli, were measured by micro-scopic observations of the swimming cells. Ex-amples of the experimental findings with strainRP1075 AcheB are shown in Fig. 2. Beforestimulation, the cells had a high tumbling fre-quency, and consequently, their swimmingpaths were very short (Fig. 2A). After stimula-tion with 0.02 mM serine, the tumbling frequen-

motA motBI l

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_ I II I I Xche22A25-t:37FIG. 1. Genetic map of the cheB region. Gene sizes are drawn approximately to scale based on gene product

sizes (25) and physical mapping studies (26). The relative positions of the two point mutations, cheB274 andcheB294, and the endpoints of the deletions, A2241 and A2234, are based on deletion mapping data (S. Houts, P.Talbert, and J. Parkinson, unpublished data). The genetic content of X transducing phages used in this work isshown at the bottom of the figure. Arrows above the map indicate the orientation and the extent oftranscriptional units. Thick lines indicate deletions, thin lines indicate chromosomal material, and wavy linesindicate A arm material.

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ADAPTATION DEFECTS IN cheB MUTANTS

cy decreased and the path lengths increased(Fig. 2B). When stimulated with 0.01 mM eachof serine and aspartate (Fig. 2C), the cells swamvery smoothly with essentially no tumbling epi-sodes. The threshold concentrations needed toelicit smooth swimming responses in the cheBmutants are summarized in Table 1. These mea-surements were made simply by adding theattractant to a drop of cells and visually observ-ing changes in swimming pattern. Under theseconditions, the wild-type control exhibited a 2-to 3-min response to either aspartate or serineconcentrations of 5 x 10-7 M. The cheB mutantsbegan to show significant serine responses atabout 10-6 M. The aspartate responses of themutants were much poorer and rather variable.Typically, strain cheB274 began responding atabout 10-5 M, strain cheB294 at about 5 x 10-4M, and the AcheB strains at about 1o-3 M, if atall.The high response thresholds of cheB mutants

to aspartate have also been noted in previouswork (16). It does not appear that this defect inaspartate responses is simply due to a loss ofcheB function because strains deleted for cheBand cheR responded well to aspartate stimuli.For example, strains containing the A2241 dele-tion (and no X prophages) responded to aspartateat concentrations of 5 x 10-6 M (data notshown). This suggests that the cheB function isnot required for aspartate responses, but ratherthat the loss of cheB activity may alter theaspartate sensing or signaling machinery undersome conditions. Possible reasons for the seem-ingly specific threshold defects of cheB mutantsare considered below.

Adaptation behavior of cheB mutants. Theresponse time of cheB mutants to various stimuliwere examined by recording the swimming be-havior of stimulated cells with dark-field photo-microscopy. The responses of AcheB strainstypically persisted for longer than 30 min. Forexample, the smooth swimming response elicit-ed by the combination of 0.01 mM serine andaspartate (Fig. 2C) lasted longer than 24 h. Theresponse of wild-type strains to the same stimu-lus lasted for about 5 min, demonstrating thatstrain RP1075 is extremely defective in adapta-tion ability. Swimming responses in the twocheB point mutants were also much longer thanin the wild type, but they gradually decayed tothe prestimulus tumbling rate. This apparentadaptation was evidently not caused by metabo-lism of the stimulating attractants because, evenwhen the attractant concentration was main-tained by dialysis, the cells regained tumblingbehavior over time (data not shown).The difference in behavior of the cheB point

mutants and cheB deletion strain RP1075 isillustrated in Fig. 3. In this experiment, swim-

ming behavior was recorded immediately afterstimulation and again 30 min later. Relativetumbling frequencies were determined by mea-suring the straight-line distance between succes-sive tumbles in the swimming tracks. Althoughthis method was less exact than counting theactual number of turns, it was much more con-venient, and the discrepancy between the twomethods was not great. The responses to 0.02mM serine (shown in the top panels of Fig. 3)indicate that strain cheB274 had fully adapted by

A

FIG. 2. Swimming responses of strain RP1075 toattractant stimuli. The AcheB strain RP1075 waswashed and suspended in motility buffer containingattractant, and the swimming response was recorded30 min later by a 2-s exposure under dark-field illumi-nation. (A) Buffer (control); (B) buffer containing 20,uM serine; (C) buffer containing 10 ,uM serine plus 10,uM aspartate.

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1232 YONEKAWA, HAYASHI, AND PARKINSON

30 min, the cheB294 strain had partially adapted,and the AcheB strain had not adapted at all. Thesame general pattern was observed with a 0.02mM aspartate stimulus; however, in this casethe mutants responded very poorly due to theirhigh thresholds for aspartate (middle panels ofFig. 3). Even the dramatic response elicited by amixture of several attractants had decayed in thecheB point mutants by 30 min (bottom panels ofFig. 3). These results demonstrate that cheBpoint and deletion mutants are defective in adap-tation, but that the point mutants might retainsome methylesterase activity because they ex-hibit slow adaptation to stimuli.

Residual methylesterase activity in cheB mu-tants. We suspected that the slow adaptationexhibited by cheB point mutants was due toleakiness. To test this idea, we examined MCPdemethylation activity in different cheB mu-tants. The methylesterase assay we used wassimilar to that described by Toews et al. (32);this assay measures the rate of production ofvolatile, radioactive compounds from cells givenmethyl-labeled methionine. Under these condi-tions, most of the volatile radioactivity producedby the cells was methanol (H. Hayashi and H.Yonekawa, Abstr. Annu. Meet. Japn. Biophys.Soc. 1982, Seibutsubutsuri 22:5213 [in Japa-nese]). Aflal strain which does not synthesizeany chemotaxis-related proteins exhibited verylittle signal (Fig. 4). Thus, nearly all of the

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methanol signal observed in flal+ strains repre-sents methanol produced by a chemotaxis-relat-ed mechanism. Since the signal given by thecheB deletion mutant was nearly identical to thatof the flaI control strain (Fig. 4), the assayappears to be a good measure of cheB-depen-dent methylesterase activity.The rate of methanol production by the

cheB274 and cheB294 strains was significantlygreater than in the control (Fig. 4), suggestingthat these two point mutants are somewhatleaky. Consistent with this conclusion is the factthat some of the MCP molecules in the cheBpoint mutants could be labeled with radioactivemethyl donors (Fig. 5). In the latter experiment,the cells were grown in the presence of attrac-tants, so their MCPs would be as completelymethylated as possible. If cheB function werecompletely absent, there should be no turnoverof these methyl groups and therefore no oppor-tunity to incorporate the methyl label into theMCP. This was the case for the cheB deletionstrain; however, the cheB274 strain incorporat-ed 8% of the wild-type amount of the label, andthe cheB294 strain incorporated 3% of the wild-type amount, indicating that a few of the MCPmolecules in these mutants were capable ofaccepting labeled methyl groups. The most rea-sonable explanation of this result is that residualmethylesterase activity in the mutants led to aslow turnover of MCP methyl groups during the

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FIG. 3. Adaptation properties of cheB mutants. Washed cells were suspended in motility buffer containingthe indicated concentrations of serine (top panels), aspartate (middle panels), or an equimolar mixture of serine(Ser), aspartate (Asp), ribose (rib), and galactose (gal) (bottom panels). Swimming tracks were recorded with 3-sexposures at 30 s after stimulation (0) and again at 30 min after stimulation (0). For each data point, the end-to-end distances of 100 swimming tracks were measured and averaged.

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ADAPTATION DEFECTS IN cheB MUTANTS 1233

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10 minFIG. 4. Rate of methanol evolution in cheB mu-

tants. The demethylation assay described in the textwas employed to assess methylesterase activity incheB mutants. Symbols: A, flaI control; A, cheBdeletion strain RP1075; 0, cheB274 strain; *, wild-type control strain RP437. The vertical scale is offsetto facilitate comparison of the rates. The wild-typerate corresponds to ca. 200 molecules of methanol percell per min.

course of the labeling period. Alternatively, itcould be argued that a few methyl-acceptingsites were available at the outset of the experi-ment. Once again, however, this would implythe existence of a low-level methylesterase ac-tivity in the point mutants.

DISCUSSIONMCP methylation state and sensory adaptation.

Extensive studies of the role of MCP methyl-ation-demethylation reactions have led to thefollowing picture of chemotaxis in E. coli. Toadapt to stimuli that induce CCW flagellar re-sponses (e.g., attractant increases), methylgroups are added to the MCP molecules engagedin flagellar signaling; to adapt to stimuli thatelicit CW flagellar responses (e.g., repellentincreases), methyl groups are removed from theMCP molecules engaged in signaling. Theseobservations imply that MCP molecules canexist in two alternative states or forms: one thatcorresponds to CCW flagellar rotation and onethat corresponds to CW flagellar rotation. Addi-tion of methyl groups favors the CW form,whereas removal of methyl groups favors theCCW form. Interconversion of these two formscan evidently be brought about either by chemi-cal stimuli or by changes in methylation state.These two modes of modulating MCP signalingbehavior-chemoreception versus methylationand demethylation-can operate independentlyof one another. For example, cheR mutants,which cannot change their MCP methylationstate, are nevertheless capable of detecting andresponding to chemical stimuli-they just don'tadapt.The cheB product appears to be an MCP-

specific methylesterase that is essential for re-moval of MCP methyl groups (5, 31). Mutants

lacking this activity are able to add methylgroups to MCP but cannot remove them. De-pending on the number of MCP sites still avail-able for methylation, cheB mutants might beexpected to show a limited capacity for adapta-tion, for example, to small stimuli that do notrequire much increase in the methylation state tocounteract the MCP signal induced by the stimu-lus. However, cheB mutants should not be ableto adapt to large stimuli that exceed their re-maining methylation capacity. Previous behav-ioral work failed to demonstrate convincing ad-aptation defects in cheB point mutants for tworeasons. First, relatively small stimuli were em-ployed (16). Second, the mutations were evi-dently leaky. The work of Rubik and Koshland(22) provided the first hint that cheB mutantsmight have adaptation defects. They demon-strated that relatively large stimuli caused dis-proportionately long responses, although themutants did recover in time. We have nowshown that this adaptation ability of cheB mu-tants is due to phenotypic leakiness. Deletionstrains with no detectable methylesterase activi-ty exhibited complete adaptation defects; cheBpoint mutants with residual demethylation abili-ty exhibited less severe defects in adaptationability.

Role of cheB function in chemotaxis. The mainfunction of the cheB product appears to be toregulate, through its methylesterase activity, theMCP methylation state of the cell. Modulationof MCP methylation is in turn crucial for sensoryadaptation, an essential component of efficientchemotactic responses. Recently, the cheBproduct was found to be involved as well inanother post-translational modification of MCP

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FIG. 5. Incorporation of methyl label into MCPs ofcheB mutants. Cells were grown and prepared forlabeling as described in the text. After washing, thecells were suspended in chemotaxis buffer containing[methyl-3H]methionine and serine at the indicatedconcentrations. The net incorporation of methyl labelinto MCP molecules was determined after a 10-minincubation period as described in the text.

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1234 YONEKAWA, HAYASHI, AND PARKINSON

molecules (21, 23). This cheB-dependent modifi-cation is irreversible, unlike demethylation, andseems to involve deamidation of several gluta-mine residues in the MCP molecules (7). Thisdeamidation reaction changes the signalingproperties of MCP in the same manner as de-methylation, but its functional significance is notyet known.Some of the characteristic behavioral proper-

ties of cheB mutants are probably in large partdue to the undeamidated condition of their MCPmolecules. For example, the CW-biased flagel-lar rotation seen in cheB mutants is not strictlycorrelated with the MCP methylation state be-cause cheR cheB deletion strains also exhibitCW flagellar biases even though their MCP istotally unmethylated (18). The relatively pooraspartate responses of cheB mutants may alsobe a consequence of the failure to deamidateMCP molecules. Aspartate stimuli are detectedand processed by MCPII, the tar gene product;serine stimuli are handled by MCPI, the tsr geneproduct (27). It is possible that methylated,undeamidated MCPII molecules function poorlyeither as a chemoreceptor or as a signaler,whereas MCPI molecules function almost nor-mally under the same conditions. The methyl-ation-demethylation and cheB-dependent deami-dation reactions of MCP molecules provide avaluable biochemical handle with which to in-vestigate and eventually understand these com-plex signal-transducing molecules.

ACKNOWLEDGMENTSThe portions of this work performed at the University of

Utah were supported by U.S. Public Health Service GrantGM-19559 from the National Institute of General MedicalSciences.

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2. Berg, H. C., and P. M. Tedesco. 1975. Transient responseto chemotactic stimuli in Escherichia coli. Proc. NatI.Acad. Sci. U.S.A. 72:3235-3239.

3. Goy, M. F., M. S. Springer, and J. Adler. 1977. Sensorytransduction in Escherichia coli: role of a protein methyl-ation reaction in sensory adaptation. Proc. Natl. Acad.Sci. U.S.A. 74:4964-4968.

4. Goy, M. F., M. S. Springer, and J. Adler. 1978. Failure ofsensory adaptation in bacterial mutants that are defectivein a protein methylation reaction. Cell 15:1231-1240.

5. Hayashi, H., 0. Koiwal, and M. Kozuka. 1979. Studies onbacterial chemotaxis II. Effect of cheB and cheZ muta-tions on the methylation of methyl-accepting chemotaxisprotein of Escherichia coli. J. Biochem. (Tokyo) 85:1213-1223.

6. Hedblom, M. L., and J. Adler. 1980. Genetic and bio-chemical properties of Escherichia coli mutants withdefects in serine chemotaxis. J. Bacteriol. 144:1048-1060.

7. Kehry, M. R., F. W. Dahlquist, and M. W. Bond. 1983.Bacterial chemotaxis: the chemical properties of thecheB-dependent modification, p. 533-549. In H. Sund andC. Veeger (ed.), Mobility and recognition in cell biology.

Walter de Gruyter, New York.8. Khan, S., R. M. Macnab, A. L. DeFranco, and D. E.

Koshland, Jr. 1978. Inversion of a behavioral response inbacterial chemotaxis: explanation at the molecular level.Proc. NatI. Acad. Sci. U.S.A. 75:4150-4154.

9. Kleene, S. J., M. L. Toews, and J. Adler. 1977. Isolationof glutamic acid methyl ester from an Escherichia colimembrane protein involved in chemotaxis. J. Biol. Chem.252:3214-3218.

10. Koiwal, O., and H. Hayashi. 1979. Studies on bacterialchemotaxis IV. Interaction of maltose receptor with amembrane-bound chemosensing component. J. Biochem.(Tokyo) 86:27-34.

11. Kort, E. N., M. F. Goy, S. H. Larsen, and J. Adler. 1975.Methylation of a protein involved in bacterial chemotaxis.Proc. Natl. Acad. Sci. U.S.A. 72:3939-3940.

12. Larsen, S. H., R. W. Reader, E. N. Kort, W. W. Tso, andJ. Adler. 1974. Change in the direction of flagellar rotationis the basis of the chemotactic response in Escherichiacoli. Nature (London) 249:74-77.

13. Macnab, R. W., and D. E. Koshland, Jr. 1972. The gradi-ent-sensing mechanism in bacterial chemotaxis. Proc.Natl. Acad. Sci. U.S.A. 69:2509-2512.

14. Minoshima, S., M. Ohba, and H. Hayashi. 1981. An invitro study of the methylation of methyl-accepting chemo-taxis protein of Escherichia coli. Construction of thesystem and effect of mutant protein on the system. J.Biochem. (Tokyo) 89:411-420.

15. Ohba, M., and H. Hayashi. 1979. Studies on bacterialchemotaxis. III. Effect of methyl esters on the chemotac-tic response of Escherichia coli. J. Biochem. (Tokyo)85:1331-1338.

16. Parkinson, J. S. 1976. cheA, cheB, and cheC genes ofEscherichia coli and their role in chemotaxis. J. Bacteriol.126:758-770.

17. Parkinson, J. S. 1978. Complementation analysis and de-letion mapping of Escherichia coli mutants defective inchemotaxis. J. Bacteriol. 135:45-53.

18. Parkinson, J. S., and S. E. Houts. 1982. Isolation andbehavior of Escherichia coli deletion mutants lackingchemotaxis functions. J. Bacteriol. 151:106-113.

19. Parkinson, J. S., and P. T. Revello. 1978. Sensory adapta-tion mutants of E. coli. Cell 15:1221-1230.

20. Richarme, G. 1982. Interaction of the maltose-bindingprotein with membrane vesicles of Escherichia coli. J.Bacteriol. 149:662-667.

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