Methyl-Accepting Chemotaxis Protein III and Transducer Gene Trg GERALD L

JOURNAL OF BACTERIOLOGY, Jan. 1981, p. 43-49 Vol. 145, No. 1 0021-9193/81/010043-07$02.00/0 Methyl-Accepting Chemotaxis Protein III and Transducer Gene trg GERALD L. HAZELBAUER,t* PETER ENGSTROM,t AND SHIGEAKI HARAYAMAt The Membrane Group, The Wallenberg Laboratory, University of Uppsala, 751 22 Upsala, Sweden A comparison ofthe two-dimensional gel patterns ofmethyl-3H- and 35S-labeled membrane proteins from trg+ and trg null mutant strains of Escherichia coli indicated that the product of trg is probably methyl-accepting chemotaxis protein III. Like the other known methyl-accepting chemotaxis proteins, the trg product is a membrane protein that migrates as more than one species in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, implying that it too is multiply methylated. It appears likely that all chemoreceptors are linked to the tumble regulator through a single class of membrane protein transducers which are methyl-accepting proteins. Three transducers are coded for by genes tsr, tar, and, probably, trg. Another methyl-accepting protein, which is not related to any of these genes, was observed.

Chemotactic bacteria respond to temporal tsr mutants do not respond to gradients of changes in their chemical environment (2). Fa- serine, some other amino acids, and some repel- vorable changes (attractant increases, repellent lents, whereas tar mutants do not respond to decreases) result in counterclockwise rotation of gradients of aspartate, maltose, and some differ- bacterial flagella, and unfavorable changes result ent repellents (23, 26). The two mutant classes in clockwise rotation. The former mode produces define two pathways of signal transduction from smooth, forward swimming, whereas the latter groups of receptors to the tumble regulator (25, produces uncoordinated tumbling and random 26). It appears that the linkage between receptor reorientation. Modulation of the balance be- and transducer may be a direct physical inter- tween swimming and tumbling by the bacterial action between the ligand-occupied receptor and sensory system allows cells to make net progress the appropriate transducer MCP (17). Neither along spatial gradients. tsr nor tar mutations reduce responses to most The bacterial response to stimuli is transient. sugar attractants (maltose is the only exception) A temporal gradient results in immediate sup- (23, 26), and recent work from this laboratory pression or induction of tumbles. However, after (14) has indicated that the pathways for excit- a time ranging from a few seconds to several atory signals from sugar receptors to the tumble minutes (depending on the compound and the regulator are separate from the pathways de- magnitude of the gradient), the cells resume fined by tsr and tar. Mutations in the trg gene their initial pattern of swimming and tumbling eliminate responses to gradients ofgalactose and even though the active compound is still present ribose and thus define a transductional pathway at a new concentration. There is a convincing for signals from the two receptors for these correlation between this adaptation and meth- sugars (11, 15, 19). The responses mediated by ylation or demethyltion of the methyl-accepting enzyme II receptors are not reduced by any of chemotaxis proteins (MCPs) (10, 13, 27) which the three known types of transducer mutations, are found in the cytoplasmic membrane (24). so there should be at least a fourth pathway to The methylation reactions are catalyzed by a accomodate these receptors (14, 15). specific methyltransferase (28), which transfers Stimuli transduced through the tsr and tar the methyl group from S-adenosylmethionine to pathways are adapted to by changes in the levels glutamyl residues to form glutamyl methyl es- of methylation of the respective gene products ters (16, 30), and by a demethylase (29). Two (25, 26). Reports from several laboratories, in- MCPs, MCP I and MCP II, have been studied cluding our own, have indicated that these extensively (10, 13, 27) and are known to be the MCPs have several methyl-accepting sites per products of genes tsr and tar, respectively (25, polypeptide chain (5-7, 9). MCP I and MCP II 26). both appear in sodium dodecyl sulfate-polyacrylamide gels as a set of bands in the area t Present address: Biochemistry/Biophysics Program, Washington State University, Pullman, WA 99164. around 60,000 apparent molecular weight. At f Present address: Laboratory of Genetics, Faculty of Sci- least some of this heterogeneity is thought to ence, University of Tokyo, Hongo, Tokyo, Japan. result from a slight reduction in apparent molec- 43 44 HAZELBAUER, ENGSTROM, AND HARAYAMA J. BACTrERIOL. ular weight on gels upon acceptance of each B14, the parent of strain T49-5H, appears to exhibit a additional methyl group by the MCP (5-7, 9), high frequency of mutation (11), and this may be the although other factors may also be involved source of the mutation tsr-49. (14a). Media. The growth media used, tryptone broth, and Hi minimal salts have been described previously If the Trg protein constitutes an independent (1). transductional pathway, then, in analogy with Chemicals. L-[methyl-3HJmethionine (15 mCi/ we the Tsr and Tar proteins, would expect it to mmol) and L-[rS]methionine (>800 Ci/mmol) were be involved in adaptation to the stimuli trans- purchased from The Radiochemical Centre, Amer- duced, presumably by methylation. Thus, we sham, England. Ampholytes were from LKB, Stock- looked for a third methylated protein. Soon after holm, Sweden. we observed such an MCP, Kondoh et al. (19) Methods. Growth and labeling of cells with reported their studies of an MCP III, which was [methyl-3H`]- or [3S]methionine, sodium dodecyl sul- methylated upon adaptation of trg+ cells to fate-polyacrylamide gel electrophoresis (20), two-di- stimuli of galactose and ribose. Their data mensional electrophoresis (3, 21), autoradiography, fluorography (4), and densitometry were as described showed that methylation of MCP III requires an previously (9). In the two-dimensional system ampho- active trg gene, but since the trg product was lytes were 1.2% pH 4 to 6,0.4% pH 3.5 to 10, and 0.4% not identified, they could not establish whether pH 6 to 8 or 1.4% pH 4 to 6 and 0.6% pH 6 to 8. The the methylated protein was the gene product. concentration of polyacrylamide in the second dimen- We report here a further characterization of sion was 10 or 12.5%. MCP III and present evidence that this MCP is RESULTS the product of trg. A by-product of these studies was the observation of a previously undetected MCP appears as a double band on gels. MCP, independent of tsr, tar, and trg. As reported by Kondoh et al. (19), a minor methyl-accepting protein is visible at approxi- MATERLALS AND METHODS mately 55,000 apparent molecular weight on Bacterial strains. The strains used in this work gels, just below the region of the predominant are all derivatives of Escherichia coli K12 (Table 1). MCPs. In our gel system this MCP III was The RP strains are all related to strain B275 (22) and resolved into two methylated bands, both of thus are closely related to each other. tar-52A1 is a which appeared only in trg+ cells and were un- deletion constructed in vitro (25), which does not affected by null mutations in tsr (MCP I-) and affect of the che genes. Strain expression T49-5H, tar (MCP I-) (Fig. 1). Thus, MCP II resembled which carries a TnlO insertion, and thus is tetracycline MCP I and MCP II in exhibitng a pattern of resistant, was found to be phenotypically Tsr and was originally thought to be tsr.:TnlO (14); thus, the tsr multiple bands on sodium dodecyl sulfate-poly- mutation in strain T49-5H was used to construct mu- acrylamide gels. Only two receptors, the well- tant strains null for tsr. After such strains were shown characterized galactose- and ribose-binding pro- to be missing the tsr product, we discovered (14a) that teins, are linked to MCP HI, raising the possi- the TnlO insertion was near, but not in, tsr. Strain bility that the two MCP III bands might be TABLE 1. Bacterial strains Strain Relevant description Origin/reference RP437 F- thi thr leu his met eda rpsL J. S. Parkinson (22) RP4673 Like RP437, except met' cheR203a J. S. Parkinson (22) RP4679 Like RP437, except met' cheB287 J. S. Parkinson (22) HB233 RP437 met' RP437 x P1(B14) Met+ transductant HB234 HB233 trg-1::Tn5 HB233 x P1(UH23A) Kmn transductant (11) HB235 HB233 rg.2:T1lOb HB233 x P1(UH23B) TetT transductant (11) HB237 HB233 itt-l::TnlO tsr-49 HB233 x P1(T49-5H) Tet, Tsr tranaductant HB238 HB233 eda+ tar-52A1 HB233 x P1(RP4375) glucuronic acid+, Tar transductant HB239 HB233 itt-l::TnlOb tsr-49 trg-1::Tn5 HB237 x P1(UH23A) Kmr transductant HB240 HB233 eda+ tar-52A1 trg-1::Tn5 HB238 x P1(UH23A) Kmr tranaductant HB243 HB233 eda+ itt-l::TnlOb tsr-49 tar-52A1 HB238 x P1(T49-5H) Tete, Tsr tranaductant HB244 HB233 eda+ itt-l::TnlOb tsr-49 tar-52A1 HB240 x P1(T49-5H) Tetr, Tar tranaductant trg-1::Tn5 HB261 HB233 flaI HB233 x P1(159A fal) glucuronic acid+, Fla- transductant a In an attempt to standardize the nomenclature of che genes in E. coli and Salnwnella typhimurium (D. E. Koshland, Jr., personal communication), the E. coli gene formerly termed cheX (23) has been renamed cheR, the corresponding gene in S. tyhimurium (8). The names of all other che genes of E. coli are unchanged. b itt, insertion transposon ten (see text). VOL. 145, 1981 MCP III AND trg 45 related to these two different receptors. We in- TABLE 2. Increase in methylation ofMCP III vestigated this possibility by examining the rel- bands upon adaptation to trg-related stimulia ative increases in methylation of the two MCP % Increase in methylation III bands after adaptation of cells to maximal Stimulus stimuli of galactose and its non-metabolizable Upper band Lower band analog fucose or of ribose and its non-metabo- Ribose 40 33 lizable analog allose. Within the limits of quan- Allose 81 87 titation of the densities of these minor bands on Galactose 24 57 fluorograms, methylation of both bands in- Fucose 69 57 creased in the same general pattern upon adap- a Methylation was quantitated by using densitom- tation to stimuli of either class of attractants eter tracings of fluorograms similar to those in Fig. 1. (Table 2). Thus, there does not appear to be a Values were normalized to the 45,000-dalton methyl- "galactose receptor band" and a "ribose receptor ated band, as described previously (9). Allose and band," but rather methylation of both bands is fucose are non-metabolizable analogs of ribose and involved in adaptation to both types of stimuli. galactose, respectively. Smaller increases upon adaptation to ribose and galactose than upon adaptation to This would be expected if the multiple MCP III their analogs may be a function of metabolic destruc- bands are a reflection of multiple methyl-action of the former sugars during the experiment (19). cepting sites on the MCP III polypeptide chain, as is the case for MCP I and MCP II (5-7, 9). Two-dimensional gel patterns of methyl- consistent difference in the banding patterns, 'H-labeled proteins. We approached identifi- whether total membrane proteins or only cyto- cation of the trg product by using well-charac- plasmic membrane proteins were compared. terized transposon insertions into trg (11). This is not surprising if the minor amount of Strains carrying the trg-l::Tn5 or trg-2::TnlO methyl-labeled MCP III reflects a small number mutation should not produce an intact trg prod- of copies per cell. uct, and thus a comparison of the complements To increase resolution, we separated mem- of protein obtained from these strains with a brane proteins on two-dimensional gels (3, 21). wild-type complement should reveal the trg pro- By using appropriate mutants, we identified tein. However, a comparison of the sodium do- methyl-3H-labeled MCP III as two streaks begin- decyl sulfate gel patterns of seS-labeled proteins ning at a pH of about 6.4 and trailing toward from wild-type and trg strains did not reveal a neutral. Representative gels from these studies are shown in Fig. 2. Often, spots at the basic side of two-dimensional gels appeared to begin at a

:..z pH where the spots would otherwise focus and trail toward neutral. MCP III consistently streaked in this manner. methyl-3H-labeled MCP I and MCP II also often appear as streaks (12, 17a, 25), but on favorable gels these proteins are resolved as a series of spots falling on diagonal lines from higher-apparent-molecular- weight, acidic areas toward lower-apparent-molecular-weight, basic areas (9, 14a). This pattern is consistent with a progressive addition of methyl groups, each neutralizing the negative charge of a glutamyl residue and also shifting w.t. tar tsr trg tar tsr the apparent molecular weight ofthe MCP poly- trg trg peptide to a slightly lower value. Unfortunately, FIG. 1. Sodium dodecyl sulfate gel patterns of we did not obtain well-resolved patterns of methyl-3H-labeled proteins. The two MCP III bands methyl-3H-labeled MCP III, but if the acidic are indicated by arrows. Only a portion of the fluo- ends of the two streaks represented the respec- rogram of a 9% polyaciylamide gel is shown. The tive pl's, then the two MCP III species are MCP I bands are those missing in tsr mutants, and related along a diagonal in the same way as the MCP II bands are those missing in tar mutants. other MCPs. MCPs are located at apparent molecular weights of Two-dimensional 55,000 to 65,000. The methyl-'H-labeled band at the gel patterns of 36S-la- bottom of the figure, which is not related to chemo- beled protein. If MCP III were the trg product, taxis (9), is at about 45,000 apparent molecular then all MCP III spots should be missing in trg weight. The strains used were HB233 (wild-type null mutants, whereas if MCP III were only [w.t.]), HB238 (tar), HB237 (tsr), HB235 (trg), HB240 dependent on the trg product for methylation, (tar trg), and HB239 (tsr trg). then an unmethylated form should be present in 46 HAZELBAUER, ENGSTROM, AND HARAYAMA J. BACTERIOL. a tsr is reduced drastically (14, 19), and so we used a tsr tar double mutant, hoping that the 35S-la- -.11 --milf" beled MCP III would be predominantly unmeth- N\ ylated and thus migrate as a single spot, which could be clearly identified as MCP III. A mod- estly streaked spot corresponding to MCP III (probably in its unmethylated form) was clearly present in the patterns from the tsr tar protein b tsrtrg (Fig. 3c and 4a), but it was completely missing *__ mmb.W in the patterns ofprotein from a tsr tar trgstrain (Fig. 3d and 4b), as well as from a flaI strain (Fig. 4c). A corresponding spot was present in the patterns from a cheR (methyltransferase) mutant (Fig. 5a) and absent in the patterns from a chieB (demethylase) mutant (Fig. 5b). In the cheB material, a fully methylated MCP III would be expected to migrate at a position below and to the left of the urnmethylated spot, probably at the position of the spot which is darker in the cheB patternthan in the cheR pattern (see arrow, Fig. 5b). Another MCP. In two-dimensional patterns d tartrg __t ~ of methyl-3H-labeled proteins from a tar trg c- ) _mutant, which was missing MCP II and MCP Im, there were two spots in the MCP II region (Fig. 2d, brackets). These spots were less intense but still discernible in the pattem of the tar _AW, single mutant (Fig. 2c, brackets). In protein patterns of tar' cells, the region containing the FIG. 2. Two-dimensiontal gel patterns of methyl-. minor spots was dominated by MCP II, but 'H-labeled membraneproiteins. MCP Iis indicated in there was a spot (Fig. 2a and 2b, circles) at the (c), MCP II is indicate(d in (a), and MCP III is same position as in the patterns of tar cells. indicated by arrows in ('a) and (c). The methylated Since the minor spots apparently occurred in- spots circled (a and b) or bracketed (c and d) repre- dependently of the activities of tsr, tar, and trg, sent the new MCP. Only'Ptions of of an the gels are shown. Isoelk fluorograms they are candidates for MCP separate from acidic was from left to;right, and sodiuim dodecyl MCP I, MCP II, and MCP IH. This possibility sulfate-polyacrylamide (1!0%) gel electrophoresisw was supported by the absence of the spots in from top to bottom. MCP'I focused at a pI of 5.3 to patterns of flal cells (data not shown) and the 5.4, and MCP II focused at 5.8 to 5.9. In (b), MCP II positioning of the two spots on a diagonal line, is artifactualy smeared in both the acidic and the which was reminiscent of the distribution of the basic directions. The bre-ak in MCP I in (d) is a gel spots of the known MCPs. artifact. Bacterial strains were as in Fig. 1. trg null mutants. In analogy with the behavior DISCUSSION ofthe other MCPs (14a), an unmethylated MCP We identified MCP m in two-dimensional gel III should appear at a position slightly more patterns of MS-labeled membrane protens as a acidic and slightly higher in the molecular minor, streaked spot, which corresponded to weight dimension than the least methylated imilar streaks of methyl-3H-labeled MCP III. In form. If the low intensity of methyl-3H-labeled a two-dimensional pattern ofthe 'S-labeled pro- MCP III reflected a small amount of the protein teins from a cheR mutant that was unable to per cell, then it might be difficult to visualize methylate MCPs, MCP Im appeared as a more such a minor species of 'S-labeled protein, par- distinct spot. In trg transposon insertion mu- ticularly if the material were distributed in one tants, neither the methylated nor the unmeth- or more streaks. In fact, we observed a faint ylated form of MCP III is found, indicating that streak in the position of MCP Im only on some the product of trg is required for the appearance gels of 'S-labeled membrane proteins from trg+ in cells of the MCP III polypeptide chain and cells, although we never observed it in protein not simply for methylation of the protein. We patterns from trg cells (Fig. 3a and b). In most favor the simplest interpretation of these obser- tsr tar double mutants, methylation of MCP m vations, namely, that trg codes for MCP III; VOL. 145, 1981 MCP III AND trg 47

a .:...:.-:::%--k" :. ".9I'm li". .. 51 -.0 I_ v Mz1-*

.__ 6. _

w w.t.

~4b =~ [ ** ; e_,trg_

bIrtar 6 AL.... _~

400 tsrtartrgt FIG. 3. Two-dimensional gelpatterns ofMS-labeled membrane proteins. The positions ofMCP I and MCP HI are indicated by arrows labeled I and II, respectively. Brackets mark the position of the MCP III streak, which is visible in (a) and (c) but missing in (b) and (d). Only portions of the fluorograms are shown, corresponding approximately to the regions shown in Fig. 2. Condition-s were as9 described in the legend to Fig. 2, except UWthan the second dimension the polyacrylamide concentration was 12.5%. Bacterial 8trains wereasinFig. 1. however, the formal possibility remains that the MCP I and MCP II. In terms of response time structural gene for MCP Ill is not trg, but in- per 103 occupied receptors, a maltose stimulus stead the MCP III gene is dependent on trg for mediated by maltose-binding protein and MCP expression. II is about six times more effective than ribose In a previous study (14) we argued that trg and galactose stimuli mediated by the respective represented an independent pathway for trans- binding proteins and MCP III (18). This differ- duction of tactic signals from the galactose and ence may simply reflect the number of trans- ribose receptors to the tumble regulator and that ducer molecules per cell. There appear to be a MCP Im is thus a transducer analogous to MCP few thousand MCP I and MCP II molecules per I and MCP II. That analogy is strengthened by cell (14a). If the content of methionine is about the observations that, like MCP I and MCP II, equivalent for all three transducers, then the MCP m is localized in the membrane, its pro- relative intensities of the 3'S-labeled proteins duction is dependent on flal activity, and it is indicate that there are probably only a few distributed among multiple, methylated forms. hundred MCP III molecules per cell. As a transducer, MCP III is not as effective as As indicated above, there should be at least a 48 HAZELBAUER, ENGSTROM, AND HARAYAMA J. BACTrERIOL. fourth pathway, besides the pathways defined a by the tsr, tar, and trg mutations, to accomodate the enzyme II receptors. Presumably, this pathway(s) would consist of additional transducer MCPs. The new methyl-accepting species observed in the two-dimensional patterns may be such a transducer for enzyme II receptors or other receptors not linked to known transducers. / It may correspond to one of the minor methylated bands observed in some tsr tar trg triple mutants by Kondoh et al. (19). Hayashi and co- workers (12, 17a) have observed another enve- che R lope-associated methyl-accepting protein of about 45,000 molecular weight. It is clearly dif- b ferent from the three well-characterized MCPs,

a _.

che B FIG. 5. Two-dimensional gel patterns of 35S-labeled membrane proteins from mutants defective in tsrtar methylation or demethylation ofMCPs: detail of the region around MCP III. Arrows mark probable MCP III spots that are unmethylated (a) or maximally methylated (b). Conditions were as described in the legend to Fig. 3. The bacterial strains used were RP4673 (cheR) and RP4779 (cheB). Similar patterns

_ were observed for strains RP4672 (cheR) and RP4788 _~mm (cheB).

c 4NOW~~~~~~~~~~~~:' as well as from the 55,000-molecular-weight methyl-accepting protein described here, and it may be involved in adaptation to stimuli medi- tsrtartrg ated by some enzyme II receptors. While preparing this manuscript, we learned that Koiwai and co-workers (17a) had also in- vestigated MCP III and concluded that it is the product of trg. ACKNOWLEDGMENT This work was supported by grants from the Swedish f:w < .^s:4- > Natural Sciences Research Council. LITERATURE CITD _ :. .. ,q. , _ -,B, He Wdkii-I. 1. Adler, J. 1973. A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis by Escherichia coli. J. Gen. Microbiol. 74: fla I 77-91. FIG. 4. Two-dimensional gel patterns of 35S-la- 2. Adler, J. 1975. Chemotaxis in bacteria. Annu. Rev. Bio- membrane detail the around chem. 44:341-356. beked proteins: of region 3. Ames, G. F.-L., and K. Nikaido. 1976. Two-dimensional MCP III. (a) and (b) are enlargements ofFig. 3c and gel electrophoresis of membrane proteins. Biochemistry d, respectively. Brackets mark the position of the 15:616-623. MCP III streak, which is visible in (a) but missing in 4. Bonner, W. M., and R. A. Laskey. 1974. A film detection (b) and (c). Conditions were as described in the legend method for tritium-labeiled proteins and nucleic acids to Fig. 3. The flaI mutant used was strain HB261. in polyacrylamide gels. Eur. J. Biochem. 46:83-88. VOL. 145, 1981 MCP III AND trg 49

5. Boyd, A., and M. L. Simon. 1980. Multiple electropho- membrane-bound chemosensing component. J. Bio- retic forms of methyl-accepting chemotaxis proteins chem. (Tokyo) 86:27-34. generated by stimulus-elicited methylation in Esche- 17a.Koiwai, O., S. Minoshima, and H. Hayashi. 1980. richia coli. J. Bacteriol. 143:809-815. Studies on bacterial chemotaxis. V. Possible involve- 6. Chel8ky, D., and R. W. Dahlquist. 1980. Structural ment offour species ofthe methyl-accepting chemotaxis studies of methyl-accepting chemotaxis proteins of protein in chemotaxis of Escherichia coli. J. Biochem. Escherichia coli: evidence for multiple methylation. (Tokyo) 87:1365-1370. sites. Proc. Natl. Acad. Sci. U.SA. 77:2434-2438. 18. Koman, A., S. Harayama, and G. L. Hazelbauer. 1979. 7. De Franco, A. L., and D. E. Koshland, Jr. 1980. Mul- Relation of chemotactic response to the amount of tiple methylation in the processing of sensory signals receptor: evidence for different efficiencies of signal during bacterial chemotaxis. Proc. Natl. Acad. Sci. transduction. J. Bacteriol. 138:739-747. U.SA. 77:2429-2433. 19. Kondoh, H., C. B. BalL and J. Adler. 1979. Identifica- 8. De Franco, A. L, J. S. Parkinson, and D. E. Kosh- tion of a methyl-accepting chemotaxis protein for the land, Jr. 1979. Functional homology of chemotaxis ribose and galactose chemoreceptors of Escherichia genes inEscherichia coli and Salmonella typhimurium. coli. Proc. Natl. Acad. Sci. U.SA. 76:260-264. J. Bacteriol. 139:107-114. 20. Laemmli, U. K. 1970. Cleavage of structural proteins 9. Engstriim, P., and G. L Hazelbauer. 1980. Multiple during the asembly of the head of bacteriophage T4. methylation of methyl-accepting chemotaxis proteins Nature (London) 227:680-685. during adaptation ofEscherichia coli to chemical stim- 21. O'Farrell, P. 1975. High resolution two-dimensional elec- uli. Cell 20:165-171. trophoresis of proteins. J. Biol. Chem. 250:4007 4021. 10. Goy, M. F., and M. S. Springer. 1978. In search of the 22. Parkinson, J. S. 1978. Complementation analysis and linkage between receptor and response: the role of a deletion mapping ofEscherichia coli mutants defective protein methylation reaction in bacterial chemotaxis, p. in chemotaxis. J. Bacteriol. 135:45-53. 1-34. In G. L Hazelbauer (ed.), Taxis and behavior. 23. Reader, R. W., W.-W. Tao, M. S. Springer, M. F. Goy, Chapman and Hall, London. and J. Adler. 1979. Pleiotropic aspartate taxis and 11. Harayama, S., E. T. Palva, and G. L Hazelbauer. seine taxis mutants ofEscherichia coli. J. Gen. Micro- 1979. Transposon-insertion mutants ofEscherichia coli biol. 111:363-374. K12 defective in a component common to galactose and 24. Ridgeway, H. F., M. Silverman, and M. I. Simon. 1977. ribose chemotaxis. Mol. Gen. Genet. 171:193-203. Iocalization of proteins controlling motility and chem- 12. Hayashi, H., 0. Koiwad, and M. Kozuka. 1979. Studies otaxis in Ewcherichia coli. J. Bacteriol. 132:657-665. on bacterial chemotaxis. II. Effect of cheB and cheZ 25. Silverman, M., and M. Simon. 1977. Chemotaxis in mutations on the methylation of methyl-accepting Escherichia coli: methylation of che gene products. chemotaxis protein of Escherichia coli. J. Biochem. Proc. Natl. Acad. Sci. U.S.A. 74:3317-3321. (Tokyo) 35:1213-1223. 26. Springer, M. S., KL F. Goy, and J. Adler. 1977. Sensory 13. Hazelbauer, G. L 1979. Bacterial chemotaxis and protein transduction in Escherichia coli: two complementary carboxymethylation. Nature (London) 279:18-19. pathways of information processing that involve meth- 14. Hazelbauer, G. L., and P. Engstr6m. 1980. Parallel ylated proteins. Proc. Natl. Acad. Sci. U.S.A. 74:3312- pathways for transduction of chemotactic signals in 3316. Escherichia coli. Nature (London) 283:98-100. 27. 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