JOURNAL OF BACTERIOLOGY, Sept. 1973, p. 824-847Copyright © 1973 American Society for Microbiology

Vol. 115, No. 3Printed in U.S.A.

Chemotaxis Toward Sugars in Escherichia coliJULIUS ADLER, GERALD L. HAZELBAUER, AND M. M. DAHL

Departments of Biochemistry and Genetics, College of Agricultural and Life Sciences, University ofWisconsin-Madison, Madison, Wisconsin 53706

Received for publication 30 April 1973

Using a quantitative assay for measuring chemotaxis, we tested a variety ofsugars and sugar derivatives for their ability to attract Escherichia coli bacteria.The most effective attractants, i.e., those that have thresholds near 10-5 M or

below, are N-acetyl-D-glucosamine, 6-deoxy-D-glucose, D-fructose, D-fucose,1-D-glycerol-,f-D-galactoside, galactitol, D-galactose, D-glucosamine, D-glucose,a-D-glucose-1-phosphate, lactose, maltose, D-mannitol, D-mannose, methyl-f-D-galactoside, methyl-fl-D-glucoside, D-ribose, D-sorbitol, and trehalose. Lactose,and probably n-glucose-i-phosphate, are attractive only after conversion to thefree monosaccharide, while the other attractants do not require breakdown fortaxis. Nine different chemoreceptors are involved in detecting these various at-tractants. They are called the N-acetyl-glucosamine, fructose, galactose, glucose,maltose, mannitol, ribose, sorbitol, and trehalose chemoreceptors; the specificityof each was studied. The chemoreceptors, with the exception of the one for n-glu-cose, are inducible. The galactose-binding protein serves as the recognition com-ponent of the galactose chemoreceptor. E. coli also has osmotically shockablebinding activities for maltose and n-ribose, and these appear to serve as therecognition components for the corresponding chemoreceptors.

What is the repertoire of behavioral responsesin an organism? For studies of chemotaxis, it isnecessary to know which chemicals attract anorganism and how these chemicals are detected.In this report we survey a number of sugars andsugar analogues for their ability to attractEscherichia coli bacteria, and we classify theattractants into nine groups, each detected by adifferent chemoreceptor. Portions of this workhave been described earlier (1, 18, 19). Aprevious paper reports on the ability of variousamino acids and amino acid analogues to at-tract E. coli (32).

MATERIALS AND METHODSChemotaxis assay. The details of' this procedure,

including growth and preparation of bacteria, havebeen described recently (2). In brief, it consists of thefollowing. A capillary (1-,4liter disposable mi-cropipette) containing a solution of attractant isplaced into a 0.2-ml suspension of about 107 bacteriaon a slide. The medium (10-2 M potassium phosphateat pH 7, and 10-i M ethylenediaminetetraacetate)allows motility and chemotaxis but not growth; thebacteria rely on their endogenous energy source. Afterincubation at 30 C for 1 h, the capillary is removedand the number of bacteria inside the capillary isdetermined by plating its contents and countingcolonies the next day. The standard deviation forreplicate determinations is 9% (2).

82.

A typical concentration response curve is shown inthe top curve of Fig. 1A. From such a curve or, better,from a double logarithmic plot of' the same data, onecan extrapolate to a threshold concentration foraccumulation inside the capillary, in this case about10 -6 M. There is a peak concentration where theresponse (the accumulation in the capillary) is maxi-mal, in this case about 10 -3M. At the highestconcentrations, so much attractant diffuses out thatthe bacteria are saturated outside the capillary and donot enter in the time allowed.The threshold concentration, the peak concentra-

tion, and the saturating concentration all depend onthe affinity between the attractant and the chemore-ceptor (R. Mesibov et al., J. Gen Physiol., in press).Furthermore, these values can vary over a 100-f'oldrange depending on the rate of' use of' the chemical.The less use and, hence, the less destruction of' theattractant gradient, the lower these concentrations(compare Fig. 4 and 13 in reference 1).

Bacteria. Unless otherwise indicated, Escherichiacoli K-12 strain B14 (2), a wild-type streptomycin-resistant bacterium, was used. We previously ref'erredto this strain as W3110 (1). The galactose chemore-ceptor mutants have been described previously (18,19). Other mutants, all E. coli K-12 strains, aredescribed in the text and in Table 2, together with areference to indicate their source and f'urther proper-ties.Medium. The growth medium, containing mineral

salts (Hi medium) and one of' various carbon andenergy sources (0.05 M for glycerol, 0.03 M for

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CHEMOTAXIS TOWARD SUGARS IN E. COLI

monosaccharides, 0.015 M for disaccharides), wasdescribed previously (2).

Chemicals. The sugars and sugar analogues wereobtained from various commercial sources, except for1-D-glycerol-fl-D-galactoside and 2-glycerol-fl-D-galac-toside, which had been synthesized and purified byand were gifts of W. Boos, and gymnemic acid (salts)(7), which was a gift of G. P. Dateo.

Galactitol and D-mannitol were purified by beingwashed with water, in which they are less soluble thanmost sugars, including D-glucose and D-sorbitol.

Purification of the other compounds was carriedout by descending chromatography on Whatman no.40 paper, using solvents that would remove anycontaminating D-galactose or D-glucose. The proce-dures were described previously (1) for 2-deoxy-D-glucose, D-fucose, D-galactose, methyl-a-D-glucoside,and L-sorbose. L-Arabinose, 2-deoxy-D-galactose, lac-tose, D-mannose, methyl-fl-D-galactoside, methyl-fl-thio-D-galactoside, D-ribose, and D-xylose were chro-matographed in a 1-butanol-ethanol-water (5:1:4)system (21) for 28 h, except 97 h was used forD-mannose. D-Lyxose and maltose were chromato-graphed in an isopropanol-water (4: 1) system (37) for28 h. D-Fructose was chromatographed in a 1-butanol-ethanol-water (10: 1: 2) system (40) for 98 h. We thankB. E. Butterworth for carrying out most of thesepurifications.

It should be emphasized that in numerous cases(for example D-fructose, D-mannose, and maltose)purification was necessary to obtain the results re-ported here.

Chromatography of D-glucose, melibiose, and tre-halose showed no detectable (<0.1%) contaminatingsugar, and for D-galactosamine, D-glucosamine, andisopropyl-fl-thio-D-galactoside there was no detect-able (<0.1%) D-galactose or 1-glucose. The 6-deoxy-1-glucose used was chromatographically pure andcontained no detectable D-galactose, 1-glucose, ormethyl-a-D-glucoside, according to W. Epstein.Hence, these compounds were not purified.

Other chemicals used in this study were not testedfor purity and were not purified.

RESULTSSurvey of sugars and related compounds.

Chemotaxis was measured by determining howmany bacteria are attracted into capillariescontaining the test chemical at various concen-trations. We report the resulting concentration-response curve (example: top curve of Fig. 1A)either by (i) showing it, (ii) listing the thresholdconcentration and the concentration and size ofthe peak response, or (iii) measuring the areaunder the curve.Table 1 presents a survey of various sugars

and sugar analogues for their ability to attractE. coli. Also shown is the ability of thesechemicals to serve as sole carbon and energysource for the growth of the bacteria.The compounds that attract the bacteria

most effectively, i.e., with thresholds of about

10-5 M or less, are shown in capital letters inTable 1 and are listed in the abstract. All butfour of these had been tested for purity andpurified if necessary (see footnote c in Table 1).These 19 compounds include representativesfrom a variety of types of sugars (pentoses,hexoses, disaccharides, amino sugars, deoxysugars, and sugar alcohols), and there is nocorrelation with ability to serve for growth.Many compounds attracted the bacteria less

well, i.e., with a higher threshold. Such attrac-tion could result from a small impurity of ahighly attractive sugar. Thus, only purifiedcompounds of this class can be confidentlyconsidered attractants. It is also possible thatchemicals which attract weakly or not at allcontain inhibitors of chemotaxis and are, infact, excellent attractants. To test this, most ofthe inactive chemicals were tested for inhibitionof taxis toward a good attractant, and noinhibition was found.

In addition to the chemicals listed in Table 1,three synthetic sweeteners were tested andfound to be inactive at concentrations of 10- 7 to10'-I M: benzoic sulfimide, sodium salt (sodiumsaccharin); cyclohexanesulfamic acid, sodiumsalt (sodium cyclamate); and p-phenetylurea(sucrol or dulcin). Gymnemic acid blocks sweetsensation in man (7) but not in arthropods (28).When tested at 3 mg/ml, it failed to affect taxistoward D-glucose in E. coli.How many chemoreceptors? We have pre-

sented evidence that bacteria detect attractantsby means of "chemoreceptors" (1). A chemore-ceptor is defined as a sensing device that signalschanges in the concentration of a chemicalwithout requiring extensive metabolism of thatchemical. How this device works is still un-known.How many different chemoreceptors are in-

volved in detecting the various sugar attract-ants? The criteria used to determine whether achemical is detected by a given chemoreceptorare as follows.

(i) We determined that the chemical itself,not a metabolic product of it, is the attractant.This can be done by showing that the firstproduct of metabolism is not an attractant, orby using a mutant that is blocked at an earlystep (preferably the first step) in the metabo-lism of the chemical.

(ii) Mutants are now available that are mis-sing a particular chemoreceptor (18, 19). Allattractants that are inert in such a mutant arepresumably detected by the missing chemore-ceptor. Attractants that are still active in such amutant must be detected by other chemorecep-tors.

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TABLE 1. A survey of sugars and sugar analoguesa

Chemotaxis

Growth Maximum responseSugars and analogues5 (h for doubling)Threshold (M) No. of

(M) bacteriaattracted

A. Three-carbonD-Glyceraldehyde NG >10-Dihydroxyacetone NG >10-Glycerol 1.8 >10-L-a-Glycerophosphate 1.8 10- 4 10- 2 25,000DL-Glycerate 2.8 >10-

B. Four-carbonD-Erythrose NG 10-3 10-1 50,000Erythritol NG >10-

C. Five-carbonAldosugars

D-Arabinose NG 10- 2 10-1 7,000L-Arabinosec 1.2 10- 4 10-1 115,000D-Lyxosec NG >10-'D-RIBOSEc 1.8 7 x 10-6 1lo- 70,000D-Xylosec 1.3 10- 4 10- 2 118,000L-Xylose NG 10- 2 10-1 35,000

KetosugarsD-Ribulose NG 10- 4 10-1 61,000

Deoxysugars2-Deoxy-r)-ribose NG 10- 2 10-1 1o,000

Sugar alcoholsD-Arabitol NG >10-'L-Arabitol NG 3 x 10- 3 10- 2 13,000Ribitol NG > 10-1Xylitol NG 10-1

Sugar acidsD-ribonate NG >10-

Ribosides and ribotidesAdenosine > 10-3Cytidine >10-1Guanosine > 10-3Uridine > 10-3Adenosine-5'-monophosphate > 10-2Adenosine-3', 5'-cyclic monophosphate > 10-2Adenosine triphosphate >10-2

D. Six-carbonAldosugarsD-GALACTOSEc 2.6 10-6 10-3 120,000L-Galactose NG 10-2 10-1 20,000D-GLUCOSEc 1.2 10-6 10-3 140,000L-Glucose NG 10-3 10' 35,000D-MANNOSEc 2.2 10-5 10-3 57,000L-Mannose NG 3 x 10-3 10-1 87,000

KetosugarsD-FRUCTOSEc 1.7 10-5 10-2 52,000L-Sorbosec NG 10-4 10-2 120,000

Deoxy sugars2-Deoxy-D-galactosec NG 10-3 1o-1 20,0006-DEOXY-D-GALACTOSE (D-FUCOSE)c NG 2 x 10-5 10-2 290,0006-Deoxy-L-galactose (L-fucose) 2.0 >10-1

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TABLE 1-Continued

Chemotaxis

Sugarsand analogues" Growth Maximum responseSugarsandanalogues' ~(h for doubling)Threshold (M) No. of

(M) bacteriaattracted

D. Six-carbon-Continued2-Deoxy-D-glucosec NG 10- 4 10-1 122,0006-DEOXY-D-GLUCOSEC 3 x 10' 10-' 305,0006-Deoxy-L-mannose (L-rhamnose) 1.8 3 x 10-3 101- 24,000

Amino sugarsN-ACETYL-D-GLUCOSAMINE 1.8 10-6 10- 2 63,000N-Acetyl-D-mannosamine NG 10-2 10-1 10,000D-GLUCOSAMINEc 2.3 10- 5 10-1 178,000D-Galactosaminec NG 3 x 10-' 10'_ 128,000D-Mannosamine NG 10-2 10-' 67,000

Sugar alcoholsGALACTITOL (DULCITOL)c NG 2 x 10-5 10-3 34,000D-SORBITOL (D-GLUCITOL) 1.9 10-5 10-2 45,000D-MANNITOLc 1.3 7 x 10- 10-3 38,000Myo-inositol NG 3 x 10-3 10-1 23,000

Sugar acidsD-Gluconate 1.1 >10-1D-Glucuronate 1.1 >10-1D-Galactonate 2.0 10-1 10-' 5,000D-Galacturonate 1.7 10- 10- 37,000

Phosphorylated sugarsD-Fructose-1-phosphate 8.5 10-1 10-1 5,000D-Fructose-6-phosphate 1.5 >10-1D-Fructose- 1, 6-diphosphate NG >10-1a-D-Galactose-1-phosphate 10 2 x 10-4 10-2 145,000D-Galactose-6-phosphate NG 3 x 10-3 10-1 30,000a-D-GLUCOSE-1-PHOSPHATE 3.1 2 x 10-5 10-2 125,000D-Glucose-6-phosphate 1.4 10-' 10-2 15,0002-Deoxy-D-glucose-6-phosphate NG >10-D-Mannose-6-phosphate >8 10-1 10-1 5,000

Methylated sugars3-o-Methyl-D-glucose NG >10-

Alkyl glycosidesIsopropyl-fl-thio-D-galactosidec NG 10-2 10-1 24,0001-D-GLYCEROL-l-D-GALACTOSIDEc 10- 6 10-4 60,0002-Glycerol-f-D-galactoside" >10-2

METHYL-t-D-GALACTOSIDEc 5.5 2 x 10' 10-2 37,000Methyl-a-D-glucosidec NG 10-4 10-1 210,000METHYL-fl-D-GLUCOSIDE 9 3 x 10-1 10-2 149,000Methyl-a-D-mannoside NG >10-1Methyl-#-thio-D-galactosidec NG 10-3 10-1 40,000

E. Seven-carbonD-Gala-L-mannoheptose NG 3 x 10-3 10'_ 26,000Glucoheptose NG >10-1D-Mannoheptulose NG >10-1Sedoheptulosan NG >10-1

F. DisaccharidesCellobiose, (4-o-,6-D-glucosyl-D-glucose) NG 3 x 102 10-1 18,000LACTOSEc (4-o-O-D-galactosyl-D-glucose) 1.3 10-' 10-3 56,000MALTOSEC (4-o-a-D-glucosyl-D-glucose) 1.5 3 x 10-6 10-' 120,000Melibiosec (6-o-a-D-galactosyl-D-glucose) 2.5 10-' 10-2 14,000Sucrose (fl-D-fructosyl-a-D-glucoside) NG >10-1TREHALOSEC (a-D-glucosyl-a-D-glucoside) 2.0 6 x 10-6 10-2 120,000

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828 ADLER, HAZELBAUER, AND DAHL

TABLE 1-Continued

J. BACTERIOL.

Chemotaxis

Sugars and analoguesb Growth Maximum responseSugarsandanalogue ~~(h for doubling)Threshold (M) No. of

(M) bacteriaattracted

G. PolysaccharidesInulin (poly-D-fructose) NG >10-1Cyclohexaamylose (Schardinger a-dextrin) > 10-2Cycloheptaamylose (Schardinger 0-dextrin) NG 3 x 10 - 2 10-1 13,000

a"The chemotaxis assays were carried out for 1 h at 30 C as described in Materials and Methods. Chemicalswere usually tested over the range 10-7 to 10-1 M (except for a few that were not soluble at the highestconcentration or were not readily available). (In the case of the polysaccharides, molarity refers to themonomer.) Threshold concentrations were determined as described in Materials and Methods; the error is atleast + threefold since they were determined with a small number of points. Further, threshold concentrationsare lower than the ones listed if the chemicals are utilizable (see Materials and Methods). No response over theentire range tested is indicated in the threshold column by > 10-1 (or > the highest concentration tried).Maximal response means the number of bacteria attracted into a capillary in 1 h at the peak concentration ofattractant. A background value, i.e., no attractant in the capillary, of about 3,000 bacteria has been subtracted.Each chemical that supported growth with a doubling time of less than 3 h was tested for taxis with bacteriagrown on that chemical as sole carbon and energy source and also with bacteria grown on D-galactose. Theformer result usually gave the better taxis and is reported in the table, but for some chemicals (L-arabinose,D-fructose-6-phosphate, D-glucose, D-glucose-6-phosphate, D-gluconate, D-glucuronate, DL-glycerate, D-man-nitol, and D-xylose) growth on that chemical as sole carbon and energy source resulted in bacteria that werepoorly motile and gave reduced chemotaxis even to D-glucose, a control response included in each experiment.In these cases, the results reported are for bacteria grown on D-galactose, except for D-fructose-6-phosphatewhere D-fructose-grown cells were used. For lactose bacteria grown on D-galactose plus 10-3 M methyl-fl-thio-D-galactoside were used. For chemicals that did not support growth with a doubling time of less than 3 h, bacteriagrown on D-galactose were used, with the following exceptions. Bacteria grown on D-fructose were used forD-fructose-1-phosphate and D-fructose-1, 6-diphosphate; bacteria grown on D-mannose were used for N-acetyl-D-mannosamine, D-mannosamine, D-mannose-6-phosphate, and methyl-a-D-mannoside; bacteria grown onD-ribose were used for 2-deoxy-D-ribose, D-erythrose, erythritol, ribitol, D-ribonate, and D-ribulose. For all theseexcept the hexose phosphates just mentioned, bacteria grown on D-galactose were also tested, and these gaveessentially the same results. No growth (NG) indicates that the bacteria failed to double in 18 h. Empty spacein this column means the chemical was not tested for growth. The bacteria used were E. coli strain B14, exceptthat strain RV, a mutant with the lactose operon deleted, was used for all chemotaxis studies with1-D-glycerol-l-D-galactoside and methyl-B-D-galactoside to prevent hydrolysis to D-galactose by ,B-galactosi-dase; otherwise the response might be attributable to the D-galactose produced.

b Capital letters indicate those compounds that attract most effectively, i.e., with thresholds of about 10-5 Mor less.

c These chemicals had been tested for purity and purified if necessary; see details in Materials and Methods.

(iii) All the attractants detected by one chem-oreceptor compete with each other, but theydo not compete with an attractant detected by adifferent chemoreceptor (1). In such "competi-tion" or "cross-inhibition" experiments, oneattractant, at various concentrations, is in thecapillary tube and another attractant, at aconcentration of 10-2 M, is present in both thecapillary and the bacterial suspension. If thetwo attractants are recognized by one and thesame chemoreceptor, the response should beinhibited; if they are not, the response shouldnot be affected. Inhibition could result fromother causes too, but absence of inhibition isstrong evidence that the two attractants usedifferent chemoreceptors.One needs to appreciate that the degree of

inhibition is proportional to the relative affini-ties of the "competitor" and "attractant" forthe chemoreceptor and to their relative concen-trations.

In the case of inducible taxes it is important touse cells that are already induced for taxistoward the attractant in competition experi-ments, and this has been done in all the studiesreported here. Otherwise, the inhibition by asecond attractant could result from inhibition ofinduction, possibly by inhibition of the trans-port needed to bring about induction.

(iv) Some chemoreceptors are inducible, i.e.,their activity requires prior exposure to theattractant. Taxis toward all attractants de-tected by that induced chemoreceptor will beincreased over the uninduced level, but taxis

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toward attractants detected by a different,uninduced chemoreceptor, will not be in-creased.

Evidence from most, but not all, of thesecriteria will be presented for each of the bestattractants. Using these criteria, we have foundthat nine different chemoreceptors can accountfor taxis toward nearly all these sugar attract-ants. Where feasible, the experimental resultswill be presented in the sequence just outlined.

D-Galactose taxis and a galactosechemoreceptor. D-Galactose attracts E. coli(Table 1 and top curve of Fig. 1A). We will showthat a distinct chemoreceptor, the "galactosechemoreceptor," is responsible for its detection.

(i) Galactose itself is the attractant. Mu-tants that are more than 99% blocked in theoxidation of D-galactose, owing to the absence ofthe first three enzymes needed for its metabo-lism, are attracted normally to D-galactose(Table 2 and reference 1). Furthermore, a mu-tant, 20SOK-, that is 99.5% blocked in theuptake of D-galactose at 10-6 M, as well as beingdefective in the first enzyme of its metabolism,galactokinase, is attracted to it well (Table 2and reference 1). As expected, if degradation ofD-galactose is not required for taxis, a mutant,DF2000, that is blocked in the use of theresulting D-glucose-6-phosphate also shows nor-mal D-galactose taxis (Table 2).

(ii) Galactose chemoreceptor mutants.These mutants (18, 19) fail to carry out taxistoward D-galactose and seven additional at-tractants (Table 3, column A, top 8 lines).Hence, these chemicals are detected only by thegalactose chemoreceptor. The galactose chemo-receptor mutants show a reduced chemotac-tic response to D-glucose and four of its ana-logues (Table 3, column A, lines 9-13), and thusthese sugars are detected by both the galactosechemoreceptor and a "glucose chemoreceptor."The galactose chemoreceptor mutants shownormal taxis toward the other sugars tested(Table 3, column A), so these sugars must beadequately detected by some chemoreceptorsother than the one for D-galactose.

(iii) Galactose competition experiments. InFig. 1, competition experiments are reported indetail for two examples; to save space, allsubsequent competition experiments will bepresented in table form only (Table 4). Theexperiment of Fig. 1A shows that D-glucoseabolishes taxis toward D-galactose, as expectedif D-glucose is detected by the galactose chem-oreceptor.Table 4 shows, as expected from the specific-

ity of the galactose chemoreceptor describedabove, that D-galactose abolishes taxis toward

the chemicals detected only by that chemore-ceptor (the first eight chemicals listed) andpartially inhibits taxis toward the chemicalsdetected by both the galactose and glucosechemoreceptors (lines 9-13 and Fig. 1B). D-Glucose completely inhibits taxis toward allthese compounds (Table 4, lines 1-13). Somereciprocal competition experiments for attract-ants detected by the galactose chemoreceptorare reported in line 1 of Table 4. More detailedstudies of the inhibition of D-galactose taxis bythe substrates of the galactose chemoreceptorhave been reported elsewhere (18). Chemicalsthat are not attractants, for example 2-glycerol-f,-D-galactoside (Table 1), fail to inhibit D-galactose taxis.The partial inhibition of D-ribose taxis by

D-galactose and the partial inhibition by D-ribose of attractants detected weakly by thegalactose chemoreceptor (Table 4) suggest thatD-ribose may be weakly detected by the galac-tose chemoreceptor. (See "Trehalose taxis" forinhibition of D-galactose taxis by trehalose.)

(iv) Inducibility of galactose taxis. Figure2A shows that bacteria grown on D-galactosegive a greater response to D-galactose thanbacteria grown on glycerol. When chloramphen-icol is present during the assay, the differencebetween the D-galactose-grown cells and theglycerol-grown cells becomes more pronounced(Fig. 2B). Chloramphenicol inhibits the re-sponse of the glycerol-grown cells presumablyby preventing the synthesis of galactose chemo-receptor during the chemotaxis assay. Theuninduced level of D-galactose taxis (glycerol-grown cells in the presence of chloramphenicol)is significant, but this is also the case for theenzymes of D-galactose metabolism (24). Chlor-amphenicol does not markedly inhibit the re-sponse of the galactose-grown cells; this indi-cates that motility and chemotaxis do notrequire continued protein synthesis. In somestrains, growth on D-mannose serves to induceo-galactose taxis (as for DF2000, Table 2).As one would expect from the specificity of

the galactose chemoreceptor, bacteria inducedfor D-galactose taxis are simultaneously inducedfor taxis toward D-fucose (Fig. 3), L-arabinose,and D-xylose. This study has not been extendedto the other attractants detected by the galac-tose chemoreceptor. The contrast between D-galactose-grown and glycerol-grown cells isgreater for D-fucose taxis than for D-galactosetaxis (compare Fig. 2A and 3), perhaps becauseD-fucose can not serve as an energy source forinduction of galactose chemoreceptor during thechemotaxis assay.

Figures 2 and 3 serve as examples of experi-

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830 ADLER, HAZELBAUER, AND DAHL J. BACTERIOL.

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832 ADLER, HAZELBAUER, AND DAHL

TABLE 3. Chemotaxis by chemoreceptor mutantsa

J. BACTERIOL.

A. Galactose B. Mutant in C. Mannitol D. Maltosechemoreceptor both galactose chemoreceptor chemoreceptor

mutant and glucose mutant mutantchemoreceptorsChemoreceptor Attractant Fold Fold Fold Fold

name increase increase increase increaseIovere Parental over Parental over Parental oere Parental

parental response parental response parental response parental response

thresh- (%) thresh- ( thresh- (%) thresh -old old old old

D-Galactose 104 lb > 105 0 1 97c 1 163cl-D-Glycerol-/- >3 x 104 0

D-galactoside6-Deoxy-D-glu- > 105 0

coseD-Fucose > 104 ob

Galactose Methyl-O-D- > 103 0galactoside

L-Arabinose > 104 Ob

D-Xylose > 104 0L-Sorbose > 2 x 102 Ob

D-Glucose 10 44bdd > 104 0 1 109 1 83D-Glucosamine 10 37e 102 82-Deoxy-D-glu- 1 8 > 104 0

coseGlucose Methyl-,8-D- 102 13 >3 x 10-2 0

glucosideMethyl-a-D- 10 6 > 103 0glucoside>10D-Mannose 1 74 >103 0

N-Acetyl N-Acetyl-D- 1 147glucosamine glucosamine

Fructose D-Fructose 1 100" 1 140 1 lOO1

Mannitol D-Mannitol 1 94 103 109, 409

Sorbitol D-Sorbitol 1 154 1 75hGalactitol 1 162

Ribose D-Ribose 1 100" 1 132 1 59

Maltose Maltose 1 34 1 74 104 1

Trehalose Trehalose 10 56 1 105 10 40Lactose 102 10' > 103 0

a Threshold is expressed as fold increase over the parent. For the actual threshold, see the B14 data of Table1, since the threshold data are the same for the parent used and for B14. An exception to this is column C (seebelow). Percentage of parental response was calculated from measurements of areas under the concentration-response curves, as described in the footnote Table 4. The peak concentration was the same for mutant, parent,and B14 (Table 1), except in cases noted by footnotes. Chemotaxis was studied up to 10-1 M, except that forsome of the purified chemicals 2 x 10-2 M was the highest concentration tried. Empty space indicates theexperiments were not done. Column A, Except for lines 10 to 14 the galactose chemoreceptor mutant is AW520(18, 19) and its response is compared with its galactose chemoreceptor revertant, AW521 (18, 19) (referred tohere as the "parent"; the true parent is unknown). For lines 10 to 14 the galactose chemoreceptor mutant isAW543 and its parent is B275 (18); qualitatively similar results were obtained with AW520. The strains weregrown on D-galactose for studies of chemotaxis toward attractants detected by the galactose and glucosechemoreceptors, and on the respective sugars for the remaining taxes, except glycerol plus 10- 3 Mmethyl-fl-thio-D-galactoside for lactose taxis. For 1-D-glycerol-fO-D-galactoside and methyl-O-D-galactoside,strains lacking f-galactosidase were used. RV (Tables 1 and 2) for the parent and AW526 (18) for the mutant.Column B, The strain missing both the galactose and glucose chemoreceptors is AW581, produced by

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ments on the inducibility of sugar taxis. To savespace, subsequent experiments on inducibilitywill be presented in table form only (Table 6).Growth on D-fructose or lactose yields motile

cells showing little or no D-galactose taxis. Thiscould result from repression of the galactosechemoreceptor, or from competition by anyresulting D-glucose or D-galactose. (See also"Lactose taxis" and "Effect of growth in glucoseon taxis.")

(v) Galactose-binding protein. Evidencehas been presented elsewhere (18, 23) that thegalactose-binding protein (5, 8, 24) is the part ofthe galactose chemoreceptor that recognizes thechemicals detected by that chemoreceptor. This

FIG. 1. A, Effect of D-glucose on taxis towardD-galactose by E. coli strain B14. Symbols: 0, with-out D-glUcose; 0, with 10-2 M D-glucose. B, Effect ofD-galactose on taxis toward D-glucose by E. coli strainB14. Symbols: *, without D-galactose; 0, with 10-2M D-galactose.

protein is also required for the transport ofD-galactose by the methyl-galactoside permease(5, 8, 23). The protein, which can be removedfrom cells by a mild osmotic shock (20), has twodissociation constants for D-galactose, about10-7 and 10-5 M (9). In shock fluid fromuninduced (glycerol-grown) or induced (D-galactose-grown) E. coli B14, it was present at240 or 1510 pmol/mg of protein, respectively, asdetermined by procedures published previously(18). The specificity for binding is similar to thespecificity of the galactose chemoreceptor (18).

D-Glucose taxis and a glucosechemoreceptor. D-Glucose is an attractant forE. coli (Table 1 and top curve of Fig. 1B), and itis detected by both the galactose chemoreceptorand a "glucose chemoreceptor."

(i) Glucose itself is the attractant. D-

Glucose-6-phosphate, the first product of me-tabolism of D-glucose (15, 26, 27), is a poorattractant (Table 1) even in cells induced forthe transport of this phosphorylated sugar.Further, a mutant DF2000 that is 97% blockedin the oxidation of D-glucose is attracted to itperfectly well (Table 2 and reference 1).

(ii) Chemoreceptor mutants. The galactosechemoreceptor mutants are still attracted toD-glucose, although at threshold and peak con-centrations that are 10-fold higher (Table 3,column A) than wild-type bacteria (Table 1).Thus, besides the galactose chemoreceptor,there is another chemoreceptor that can detectD-glucose, and this "glucose chemoreceptor"

introducing the AW543 mutation (18) for the galactose chemoreceptor into ZSC103a, which lacks both of theenzymes II for D-glucose. The "parent," AW579, is an isogenic galactose chemoreceptor mutant produced byintroducing the same galactose chemoreceptor mutation into ZSC71t, a strain closely related to ZSC103a but inwhich the two enzymes II for D-glucose are present. The ZSC strains were isolated and characterized by S. J.Curtis (Ph.D. thesis, University of Chicago, 1973). Exceptions: For D-galactose and lactose, the parent wasZSC71t, since AW579 already has a galactose chemoreceptor mutation and hence shows no D-galactose taxisand practically no lactose taxis; and for maltose, the "parent" was B14 since AW579 fails to grow on maltose.Growth was on glycerol for attractants detected by the glucose chemoreceptor (lines 9-14) and on the respectivesugars for the remaining attractants, except that for lactose growth was on glycerol plus 10- 3M methyl-B-thio-D-galactoside. Column C, The mannitol chemoreceptor mutant is AW592 (isolated by G. W. Ordal and R. W.Reader), and its parent is AW591, a strain that is wild type for chemotaxis. Strains were grown on the respectivesugars, except 1-glucose taxis was studied with cells grown on n-galactose. Doubling time for three mannitoltaxis mutants and their parent was the same on glycerol or D-galactose; on D-mannitol it was 1.7 to 2.0 h and 1.4h, respectively; on D-sorbitol 1.8 to 1.9 h and 1.6 h, respectively. Column D, The maltose chemoreceptor mutantis AW470 (for derivation see text), and its parent is B14. The strains were grown on glycerol plus 10-2 M malt-ose, except for trehalose taxis they were grown on trehalose, and for D-galactose and D-glucose taxis the parentwas grown on glycerol.

bConcentration-response curve is shown in reference 19.c The peak concentrations were 10-I M for parent and mutant.d The peak concentrations were 10-3 M for the parent and 10-3 to 10-2 M for the mutant.eThe peak concentrations were 10- I M for the parent and 10-2 M for the mutant.tThe peak concentrations were 10-I M for parent and mutant.' The peak concentrations were 10-3 M for the parent and 10-l M for two mutants, AW592 and AW593.h The peak concentrations were 10-2 M for the parent and 10-3 M for the mutant.'The peak concentrations were 10-2 M for parent and mutant.

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ADLER, HAZELBAUER, AND DAHL

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CHEMOTAXIS TOWARD SUGARS IN E. COLI

FIG. 2. D-Galactose taxis by E. coli strain B14grown on D-galactose (closed circles) or on glycerol(open circles). A, Chloramphenicol was not added. B,Chloramphenicol (200 ug/ml) was present during, aswell as 20 min before, the chemotaxis assay.

GALACTOSE

0

~0 f

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<' ~~~~D-FUCOSE MOLARITY

XX FIG. 3. D-Fucose taxis by E. coli strain B14grownoo on D-galactose (-) or on glycerol (O).

._o

E, has a lower affinity for D-glucose than does the,,< galactose chemoreceptor. Likewise, the galac-

0 tose chemoreceptor mutants are still attracted,y although less well than wild-type bacteria, to

,, the D-glucose analogues shown in lines 10 to 14c>g of Table 3. The glucose chemoreceptor must not^co be able to detect the compounds shown in lines

O s= 1 to 8 of Table 3, since taxis toward these isC)= _ absent when the galactose chemoreceptor is

GLOmissing(Table 3, column A).Recently, we have obtained a mutant that is

missing the glucose chemoreceptor (J. Adler

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ADLER, HAZELBAUER, AND DAHL

and W. Epstein, manuscript in preparation). Asexpected, D-galactose taxis was normal in thisstrain, but D-glucose taxis was one-half normal,showed a peak at 10 - I M instead of 10-3 M, andwas completely eliminated by 10-2 M galactose.To study chemotaxis in a glucose chemore-

ceptor mutant free of the galactose chemorecep-tor, we introduced a galactose chemoreceptormutation into this mutant. Table 3, column B,shows that certain chemicals are no longerattractants for this strain; hence these are

detected by either or both of these two chemore-ceptors. Taxis toward other sugars tested in thisstrain is normal or near normal (Table 3, col-umn B); hence these must be detected bychemoreceptors other than the galactose andglucose chemoreceptors.

(iii) Glucose competition studies. Consist-ent with the view that wild-type bacteria havetwo chemoreceptors for D-glucose, only one ofwhich (the galactose chemoreceptor) also de-tects D-galactose, is the fact that the response toD-glucose by wild-type bacteria is only abouthalf inhibited by D-galactose at 10-2 M (Fig.1B), or even 10-1 M, while the response toD-galactose is abolished by the presence of 10-2M D-glucose (Fig. IA). The residual response toD-glucose has about the same threshold (10-sM) as that shown by the mutant lacking thegalactose chemoreceptor. As expected, the re-

sponses of wild-type bacteria to the other chem-icals detected by both the galactose and glucosecht moreceptors are abolished by the presence ofD-[lucose but only partly inhibited by D-galac-tose (Table 4, lines 10-13).

Taxis toward D-glucose by wild-type bacteriawas affected little or none by nearly all the otherattractants tested (Table 4). However, if any ofthese sugars were detected by the glucosechemoreceptor, they might not severely affectD-glucose taxis, since D-glucose would still bedetected by the more sensitive galactosechemoreceptor. Therefore, inhibition of D-glu-cose taxis was studied in a mutant lacking thegalactose chemoreceptor (Table 5). A number ofthe attractants had little or no effect, but therewas inhibition by N-acetyl-D-glucosamine, D-mannose, D-sorbitol, D-mannitol, maltose, andD-glucosamine. Therefore, the glucose chemore-ceptor appears to detect these in that decreas-ing order. However, the inhibition by maltoseprobably resulted from some breakdown toD-glucose and, since the N-acetyl-D-glucosa-mine and D-sorbitol used were not purified,inhibition by them could have resulted fromcontaminating D-glucose or other substrates.

(iv) Effect of growth in glucose on taxis.Owing to catabolite repression, wild-type E. coli

grown on D-glucose make few flagella and henceare poorly motile (3, 41), so their chemotaxis isexpected to be poor. Such bacteria show essen-tially no taxis toward D-fucose or D-galactose(data not shown), but they do carry out slighttaxis toward D-glucose (Table 6). This D-glucosetaxis has a threshold at 10 - 5 M, similar toD-glucose taxis in mutants lacking the galactosechemoreceptor. This result is further evidencefor a glucose chemoreceptor and suggests thatgrowth on D-glucose represses the galactosechemoreceptor but not the glucose chemorecep-tor.The glucose chemoreceptor appears to be

constitutive. Wild-type bacteria grown onglycerol show excellent D-glucose taxis. This isnot largely due to induction by D-glucose duringthe chemotaxis assay, since chloramphenicol inthe assay did not inhibit strongly (Table 6).Inducibility of the glucose chemoreceptor isbetter studied in a galactose chemoreceptormutant, where the glucose chemoreceptor hasprimary responsibility for detecting D-glucose.We have a galactose chemoreceptor mutant(AW579) whose flagella synthesis happens to beresistant to repression by D-glucose. Whethergrown on D-glucose or on glycerol, taxis towardD-glucose by this mutant was inhibited to aboutthe same extent, and not severely, by chloram-phenicol (Table 6).We did not find a binding activity for D-

glucose in shock fluid from a galactose chemore-

TABLE 5. Competition experiments for D-glucosetaxis in a galactose chemoreceptor mutanta

Remaining responseCompetitor (10-2 M) (percent response

without competitor)

N-Acetyl-D-glucosamine 15D-Fructose 69Galactitol 82D-Galactose 67D-GlucOsamine 53D-Glucose 4Lactose 100Maltose 42D-Mannitol 38D-Mannose 17D-Ribose 110D-Sorbitol 17Trehalose 104

a The E. coli galactose chemoreceptor mutantAW520 was used, except that in the case of lactose anE. coli galactose chemoreceptor mutant, AW526,unable to metabolize lactose, was used to preventhydrolysis of lactose. The bacteria were grown onD-galactose. The procedure is otherwise the same asdescribed in footnote a, Table 4.

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CHEMOTAXIS TOWARD SUGARS IN E. COLI

ceptor mutant (AW520, grown on D-galactose).D-Mannose taxis. D-Mannose, an attractant

for E. coli (Table 1), is detected primarily bythe glucose chemoreceptor. The evidence fol-lows.

(i) Mannose itself is the attractant. Taxistoward D-mannose is not the result of taxistoward D-glucose or other metabolites producedfrom the D-mannose since (i) the first product ofmetabolism, D-mannose-6-phosphate (26, 27,31), attracts bacteria little or not at all (Table1), even in cells induced for its transport; and(ii) a mutant MC118 that is missing an enzyme

needed for the metabolism of D-mannose and forits conversion to D-fructose and D-glucose (31) isattracted normally by D-mannose (Table 2).

(ii) Chemoreceptor mutants. D-Mannosetaxis is only slightly decreased in a galactosechemoreceptor mutant (Table 3, column A). Inthe mutant missing the glucose chemoreceptoronly, D-mannose taxis is very weak (15,000bacteria at 10-3 M) and is inhibited by D-galac-tose. Thus, the galactose chemoreceptor ap-

pears to respond very weakly to D-mannose. Inthe strain lacking both the galactose and glu-cose chemoreceptors, D-mannose taxis is absent(Table 3, column B). Therefore the glucosechemoreceptor is primarily responsible for thedetection of D-mannose.

(iii) Mannose competition experiments.Taxis toward D-mannose is not inhibited byD-galactose in wild-type bacteria, and D-man-nose does not inhibit D-galactose taxis (Table4). This indicates that the contribution of thegalactose chemoreceptor in the detection ofD-mannose by wild-type bacteria is not signifi-cant. In contrast, taxis toward D-mannose iseliminated by D-glucose (Table 4). D-Mannosetaxis is strongly inhibited by trehalose andD-glucosamine, and weakly inhibited by galac-titol, D-sorbitol, and N-acetyl-D-glucosamine(Table 4). However, the inhibition by trehalosecould have resulted from its breakdown toD-glucose, and the D-sorbitol and N-acetyl-D-glucosamine used were not purified and maycontain traces of D-glucose or D-mannose.D-Mannose has a strong inhibitory effect on

D-glucose taxis in the galactose taxis mutant(Table 5). In wild-type bacteria, D-mannoseinhibits D-glucose taxis only slightly (Table 4).In the latter case, inhibition of the glucosechemoreceptor by D-mannose might be ex-

pected to have little effect since the galactosechemoreceptor, which detects D-glucose even

better than does the glucose chemoreceptor, isstill functioning, and its ability to detect D-mannose is weak. D-Mannose strongly inhibitstaxis toward another substrate of the glucose

TABLsE 6. Inducibility of sugar taxes a

A B(Response (Percent inhibition by

by attractant- chloramphenicol)Attractant grown cells/

response Attractant- Glycerol-by glycerol- grown growngrown cells) cells cells

N-Acetyl-D- 0.7 8 87glucosamine

D-Fructose 5.6D-Fucose 7.7Galactitol 92cD-Galactose 1.9 22 70D-Glucose O.1d 2d 29D-Glucosee 0.6 28 35Lactose 6.3'Maltose 8.7D-Mannitol 0.3 15 93D-Ribose 1.8 38 82C6-Sorbitol 1.9 52 88Trehalose 3.9

aThe accumulation of bacteria in capillaries con-taining attractant between 10-6 and 10-l M wasdetermined at the end of 1 h with and without 200 ggof chloramphenicol per ml present in both the capil-lary and bacterial suspension. The chloramphenicolwas added 20 min before the start of the assay. Thecurves were then plotted (see Fig. 2 to 5 for examples).After subtraction of the background (the value ob-tained with no attractant present), the area undereach curve was measured with a planimeter; this areais called the "response." A standard deviation of 4.5%in the response was found when a single experimentwas repeated for 8 days. For actual size of the responseby bacteria grown on the attractant, see Table 1.Column A reports experiments where chlorampheni-col was absent. The column lists the ratios of responseby attractant-grown cells to response by glycerol-grown cells. Ratios below 1.0 result from poor motilityowing to catabolite repression of flagella synthesis.Column B compares the response in the presence andabsence of chloramphenicol. Part of the inhibition bychloramphenicol can be accounted for by a small, butdefinite, inhibition of motility. This is evident frommicroscope observations and also from reduction ofthe background by 25% (average of 28 values). Thebacteria used were E. coli strain B14. See footnote efor exception. "Attractant-grown cells" refers to bac-teria grown on the attractant listed in the first column,with exceptions given in footnotes below. Emptyspaces mean the experiment was not done.

b For D-fucose taxis growth was on D-galactose.c Growth was on D-galactose instead of on glycerol.d For D-glucose taxis with B14, growth was on

D-galactose to prevent catabolite repression of flagellasynthesis by D-glucose.

e A galactose chemoreceptor mutant AW579 wasused, which could be grown on 1-glucose withoutincurring much catabolite repression of flagella syn-thesis.

' For lactose taxis growth was on glycerol plus 10-3M methyl-B-thio-D-galactoside.

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ADLER, HAZELBAUER, AND DAHL

chemoreceptor, D-glucosamine, but not towardthe other sugars tested (Table 4).D-Glucosamine taxis. D-Glucosamine at-

tracts E. coli (Table 1). According to the follow-ing evidence, it is detected primarily by theglucose chemoreceptor but also by the galactosechemoreceptor.

(i) Chemoreceptor mutants. D-Glucosaminetaxis is less in a galactose chemoreceptor mu-tant than in its parent (Table 3, column A).D-Glucosamine taxis is substantial in the mu-tant missing the glucose chemoreceptor only(threshold 10- 3 M, 130,000 bacteria at 10- I M),and D-galactose inhibits 76%, a strong effect.(D-Glucosamine was found to contain less than0.1% D-glucose or D-galactose and was notpurified. The above responses, however, couldbe due to a contamination by some othersubstrate of the galactose chemoreceptor.) Inthe strain missing both the galactose and glu-cose chemoreceptors, D-glucosamine taxis isbarely detectable (Table 3, column B).

(ii) Glucosamine competition experiments.Taxis towards D-glucosamine is only slightlyinhibited by D-galactose in the wild-type strain,and D-glucosamine does not inhibit D-galactosetaxis (Table 4). On the other hand, taxis towardD-glucosamine is strongly inhibited by D-glucoseand D-mannose (Table 4), substrates of theglucose chemoreceptor. It is apparent that,when cells are grown on D-glucosamine, theglucose chemoreceptor is primarily responsiblefor its detection, and the galactose chemorecep-tor does not play a significant role. Inhibition byN-acetyl-D-glucosamine (Table 4) could haveresulted from impurities such as D-glucosaminesince this chemical was not purified.D-Glucosamine inhibits D-glucose taxis in the

galactose chemoreceptor mutant (Table 5), butit apparently is not detected as well as D-glucoseor D-mannose. In wild-type bacteria grown onD-galactose, D-glucose taxis is not much affectedby D-glucosamine (Table 4), as expected sincethe galactose chemoreceptor remains to detectD-glucose, and this chemoreceptor has a highthreshold for D-glucosamine. D-Mannose taxis,however, is strongly inhibited by D-glucosamine(Table 4).

It is unlikely that taxis toward D-glucosamineis due to production of D-fructose or D-glucose,since D-glucosamine does not inhibit D-fructoseor D-galactose taxis (Table 4).N-Acetyl-D-glucosamine and its chemo-

receptor. N-Acetyl-D-glucosamine, an attrac-tant for E. coli (Table 1), has its own chemo-receptor, according to the following evidence.

(i) Chemoreceptor mutants. The mutantmissing both the galactose and glucose chemo-receptors, grown on N-acetyl-D-glucosamine,

is attracted to N-acetyl-D-glucosamine nor-mally (Table 3, column B). Hence these twochemoreceptors are not primarily responsiblefor its detection.

(ii) N-Acetyl-glucosamine competitionexperiments. In the wild-type strain, taxistoward N-acetyl-D-glucosamine is inhibitedpoorly or not at all by any of nine sugars tested,including D-glucosamine (Table 4). Also, N-acetyl-D-mannosamine fails to inhibit (data notshown). This supports the notion of a separateand highly specific chemoreceptor for N-acetyl-D-glucosamine.

Conversely, in the wild-type strain, N-acetyl-D-glucosamine does not inhibit taxis towardD-fructose, D-galactose, or D-glucose (Table 4).Inhibition of taxis toward D-glucosamine andD-mannose in the wild-type strain (Table 4) andinhibition of D-glucose taxis in the galactosechemoreceptor mutant (Table 5) could be ex-plained either by recognition of N-acetyl-D-glucosamine by the glucose chemoreceptor, orby contamination with some D-glucosamine orD-glucose.

(iii) Inducibility. N-Acetyl-D-glucosaminetaxis is inducible (Table 6, see inhibition bychloramphenicol).D-Fructose taxis and a fructose chemo-

receptor. D-Fructose is an attractant forE. coli (Table 1). All the evidence points to adistinct chemoreceptor for D-fructose.

(i) Fructose itself is the attractant. Thefirst products of D-fructose metabolism (14, 16,26, 27), D-fructose- 1-phosphate, D-fructose-6-phosphate, and D-fructose-1, 6-diphosphate,have little or no activity (Table 1) even in cellsinduced for their transport.

(ii) Chemoreceptor mutants. The galactosechemoreceptor mutant, grown on D-fructose,shows normal D-fructose taxis (Table 3, columnA), as does the strain missing both the galactoseand glucose chemoreceptors (Table 3, columnB). Thus, neither the galactose nor the glucosechemoreceptor is responsible for D-fructose tax-is.

(iii) Fructose competition experiments. Inagreement with the evidence from the chemore-ceptor mutants, D-fructose has little effect onD-galactose or D-glucose taxis by wild-type E.coli grown on D-galactose (Table 4), or onD-glucose taxis by galactose chemoreceptor mu-tants (Table 5).As expected for a distinct chemoreceptor,

fructose taxis by E. coli grown on D-fructose isinhibited little or not at all by the other hexoses,D-galactose, D-glucose, and D-mannose, or byany of the other chemicals shown in Table 4.Sucrose is not an attractant (Table 1) nor doesit inhibit D-fructose taxis. Thus, the fructose

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chemoreceptor shows a high degree of specific-ity for D-fructose.

(iv) Inducibility of fructose taxis. Bacteriagrown on glycerol (Table 6) or on D-galactose(data not presented) show very little D-fructosetaxis. To carry out taxis toward D-fructoseeffectively, the bacteria must first be grown onD-fructose. As mentioned under D-galactose tax-is, bacteria grown on D-fructose show very littleD-galactose taxis. These results on inductionand repression again show that the chemorecep-tor for D-fructose is distinct from the one forD-galactose, and that it is inducible.No binding activity for D-fructose was found

in shock fluid from E. coli strain B14 grown onD-fructose.Hexose phosphate taxis. Of nine hexose

phosphates tested (Table 1), only two are fairlygood attractants: a-D-galactose-1-phosphateand a-D-glucose-l-phosphate. Induction of thehexose phosphate transport system by growthfor one generation in the presence of D-glucose-6-phosphate or 2-deoxy-glucose-6-phosphate(11 and references cited there) does not result inimproved taxis toward any of the hexose phos-phates.

Taxis toward D-galactose-1-phosphate is to-tally inhibited by D-galactose (10-2 M) and isabsent in a galactose chemoreceptor mutant.Taxis toward D-glucose-1-phosphate is 50% in-hibited by D-galactose and 94% inhibited byD-glucose (both 10 - 2 M); it is present in agalactose chemoreceptor mutant but absent inthe strain lacking both the galactose and glu-cose chemoreceptors. Most likely, the attractiontoward the two sugar phosphates results fromtaxis toward the free sugars produced by theperiplasmic hexose phosphatase (11, 12), orpossibly the sugar phosphates are themselvesdetected by the respective sugar chemorecep-tors, or there may be some contamination bythe free hexoses. The first possibility is sup-ported by the fact that growth of the bacteria onglycerol (instead of D-galactose), known to re-press synthesis of the hexose phosphatase (12),results in 80 to 90% reduction of taxis towardthe two sugar phosphates.

D-Mannitol taxis and a mannitol chemo-receptor. Three six-carbon sugar alcohols areattractants for E. coli (Table 1): D-mannitol,D-sorbitol (D-glucitol), and galactitol. Evidenceis now presented that D-mannitol is detectedprimarily by a chemoreceptor, distinct fromthose mentioned so far, that may also detectD-sorbitol and, poorly, D-glucose, but not galac-titol.

(i) Chemoreceptor mutants. The mutantmissing both the galactose and glucose chem-oreceptors, grown on D-mannitol, is attracted

normally to D-mannitol (Table 3, column B),and hence these two chemoreceptors could notbe primarily responsible for D-mannitol taxis.

George W. Ordal and Robert W. Reader haveisolated three D-mannitol taxis mutants. Allshow D-mannitol taxis only at unusually highconcentrations when grown on D-mannitol, butnormal taxis, as good as that of the parent,toward D-glucose, D-sorbitol, and all other sug-ars tested (Table 3, column C). These mutantsare therefore regarded as defective specificallyin a mannitol chemoreceptor.

(ii) Mannitol competition experiments.Taxis toward D-mannitol by wild-type E. coligrown on D-mannitol is eliminated by D-sorbitol(Table 4). This might indicate that the man-nitol chemoreceptor can detect D-sorbitol; how-ever, this is uncertain since commercial D-sor-bitol can contain up to 2% D-mannitol (29) andthe D-sorbitol used was not purified. There ispartial inhibition by D-glucose but not by D-mannose, galactitol, or several other sugarstried (Table 4). The stimulation by some of thesugars (Table 4) is not explained. Except for thepossible recognition of D-sorbitol and a veryweak recognition of D-glucose, the mannitolchemoreceptor thus appears to be specific forD-mannitol.

D-Galactose and D-glucose taxis by wild-typeE. coli grown on D-galactose is not affected byD-mannitol (Table 4). D-Glucose taxis by thegalactose chemoreceptor mutant is partiallyinhibited by D-mannitol (Table 5). Thus, thegalactose chemoreceptor does not detect D-man-nitol, and the glucose chemoreceptor may de-tect it somewhat. D-Mannitol does not inhibittaxis toward any of the other sugars shown inTable 4 (except D-sorbitol) (see next section)and hence is not detected by the chemoreceptorsresponsible for those taxes.

(iii) Inducibility of mannitol taxis. Chlor-amphenicol inhibits D-mannitol taxis of glyce-rol-grown cells much more than it inhibits thistaxis by D-mannitol-grown cells (Table 6);hence D-mannitol taxis appears to be inducedby D-mannitol during the course of the assay.Without chloramphenicol present, glycerol-grown (Table 6) or D-galactose-grown cells showgreater D-mannitol taxis than D-mannitol-growncells because the latter are poorly motile, owingto severe repression of flagella synthesis.No binding activity for D-mannitol was found

in shock fluid from E. coli strain B14 grown onD-mannitol.

D-Sorbitol taxis and a sorbitol chemo-receptor. D-Sorbitol (D-glucitol) attracts E. coli(Table 1). As reported above, it may possiblybe detected by the glucose and mannitol chemo-receptors, but in addition it is detected by a

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distinct chemoreceptor, based on the followingevidence.

(i) Chemoreceptor mutants. The strainmissing both the galactose and glucose chem-oreceptors shows normal D-sorbitol taxis whengrown on D-sorbitol (Table 3, column B); hencethese two chemoreceptors could not be primar-ily responsible for the detection of D-sorbitol.This D-sorbitol taxis was completely inhibitedby 10-2 M D-mannitol but was unaffected by102 M D-glucose.The mannitol chemoreceptor mutants show

good D-sorbitol taxis when grown on D-sorbitol(Table 3, column C). Therefore, there mustexist a chemoreceptor, other than the mannitolchemoreceptor, that can detect D-sorbitol.Grown on glycerol, these mutants show normalD-glucose taxis, as expected if the glucosechemoreceptor is constitutive, but such cellsfail to carry out D-sorbitol taxis. Thus the glu-cose chemoreceptor is not responsible here forthe detection of D-sorbitol, and there must be aseparate sorbitol chemoreceptor inducible byD-sorbitol. (Induction during the assay wasprevented by omission of essential aminoacids.) Inducibility is also indicated for thewild-type strain B14 (Table 6).

(ii) Sorbitol competition experiments. D-Sorbitol taxis by the mannitol chemoreceptormutant AW592 grown on D-sorbitol was 90%inhibited by 10-2 M mannitol, which indicatessome recognition of D-mannitol or there couldbe residual D-sorbitol in the purified D-mannitolused. It was completely inhibited by 10-2 MD-glucose, contrary to the result cited abovewith the strain missing the galactose and glu-cose chemoreceptors, and this difference is notunderstood.

D-Sorbitol taxis by wild-type E. coli grown onD-sorbitol (Table 4) is eliminated by D-glucoseand strongly inhibited by D-mannitol. It isweakly inhibited by D-mannose but not bygalactitol or the other sugars tried (Table 4).The sorbitol chemoreceptor thus may be able todetect, besides D-sorbitol, D-glucose, D-man-nitol, and D-mannose, in that decreasing order.

Galactitol taxis. E. coli grown on D-galactoseare attracted to galactitol (Table 1), but chlor-amphenicol largely eliminates this taxis (Table6). This indicates that the chemoreceptor thatdetects galactitol is induced during the assayand that it is not the galactose chemoreceptor.The strain of E. coli used here does not grow ongalactitol; a mutant was isolated that does growon galactitol, but its motility was poor and itwas not studied further. We were unable toprepare motile cells preinduced for galactitoltaxis; hence competition experiments could

not be done in induced cells and are missingfrom Table 4. The chemoreceptor that detectsgalactitol has, therefore, not been identified.The strain missing both the galactose and

glucose chemoreceptors, grown on D-galactose,does show galactitol taxis (Table 3, column B).Hence these two chemoreceptors could not beprimarily responsible for taxis toward galac-titol.

Galactitol fails to inhibit taxis toward any ofthe chemicals listed in Table 4, except D-man-nose, so it is not detected well by any of thechemoreceptors responsible for those taxes.Since D-mannose is detected by the glucosechemoreceptor, the partial inhibition of D-man-nose taxis could indicate that this chemorecep-tor detects galactitol weakly, relative to D-man-nose. Galactitol does not appreciably inhibitD-glucose taxis in the galactose chemoreceptormutant (Table 5), so the glucose chemoreceptormust detect it very weakly relative to D-glucose.D-Ribose taxis and a ribose chemoreceptor.

E. coli are attracted to D-ribose (Table 1), andthere is a separate chemoreceptor for D-riboseaccording to the following evidence.

(i) Ribose itself is the attractant. A mutantBJ503 that fails to grow on D-ribose or tometabolize it past the pentose stage, owing to adefect in transketolase (22), shows normal D-ribose taxis (Table 2). Absence of transketolasestill allows the formation of the pentose isomersL-arabinose, D-ribulose, D-xylose, and D-xylulose(22), but three of these attract E. coli with athreshold higher than for D-ribose (Table 1);D-xylulose was not available for testing.

(ii) Chemoreceptor mutants. There was nor-mal D-ribose taxis in the mutants missing thegalactose chemoreceptor, the galactose and glu-cose chemoreceptors, and the mannitol chem-oreceptor, all grown on D-ribose (Table 3,columns A-C). Hence these three chemorecep-tors could not be primarily responsible for thedetection of D-ribose.

(iii) Ribose competition experiments. Taxistoward D-ribose by wild-type E. coli grown onD-ribose is not much affected by the presence of'any of the sugars tested (Table 4). Conversely,D-ribose inhibits poorly or not at all taxis towardthese sugars except for the cases mentionedabove where the suggestion is made that thegalactose chemoreceptor may detect D-riboseweakly. D-glucose taxis by the galactose chem-oreceptor mutant is unaffected by the pres-ence of D-ribose (Table 5). Thus we concludethat D-ribose is detected primarily by a separateribose chemoreceptor.With regard to the specificity of the ribose

chemoreceptor, D-ribulose attracts E. coli,

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though not as well as does D-ribose (Table 1).D-Ribulose (10-2 M) eliminates taxis towardD-ribose, and the converse is also true. However,it must be emphasized that contamination ofthe D-ribulose by D-ribose was not checked.L-Arabinose and D-xylose, detected by the ga-lactose chemoreceptor, inhibit D-ribose taxisonly 35 and 25%, respectively, when present at aconcentration of 10-2 M. Other five-carbonsugars and sugar alcohols listed in Table 1either were not attractants or had thresholdsconsiderably higher than for D-ribose.

(iv) Inducibility of ribose taxis. Table 6shows that growth on D-ribose results in betterD-ribose taxis than prior growth on glycerol or(data not shown) on D-galactose, but inducibil-ity is more clearly demonstrated with chloram-phenicol (Table 6). D-Ribose taxis is absent incells grown on D-glucose (data not shown),owing presumably to catabolite repression ofthe ribose chemoreceptor.

(v) Ribose-binding protein. We have foundan osmotically shockable binding activity forD-ribose, with a dissociation constant for D-ribose of about 2 x 10-6 M (18). In shock fluidfrom uninduced (glycerol-grown) or induced(D-ribose-grown) E. coli B14 cells, it was presentat 150 or 1720 pmol/mg of protein, respectively.The binding of D-ribose was not inhibited by thepresence of D-galactose, D-glucose, or maltose(18). This activity may well be the componentof the ribose chemoreceptor that recognizes theD-ribose, since the inducibility and specificity ofthe binding activity resemble those of the chem-oreceptor. Aksamit and Koshland (4) havepurified a ribose-binding protein from Sal-monella typhimurium and have presented evi-dence for its role in D-ribose taxis.Maltose taxis and a maltose chemorecep-

tor. E. coli are attracted to maltose (4-o-a-D-glucosyl-D-glucose) (Table 1 and top curve ofFig. 4) and, based on the following evidence,they have a specific chemoreceptor for thatsugar.

(i) Maltose itself is the attractant. A mu-tant MB11 (MPE2) that lacks uptake of mal-tose (34) and a mutant MQ7 that lacks the firstenzyme of maltose metabolism, amylomaltase(17), are attracted perfectly well to maltose(Table 2). (In these mutants maltose taxis isconstitutive [see Table 2] as are the enzymes formaltose metabolism.) As expected if maltosedegradation is not required for taxis, a mutantDF2000 that is blocked in the use of theresulting D-glucose-6-phosphate also shows nor-mal maltose taxis (Table 2). Below we presentfurther evidence that maltose taxis is not pri-marily due to production of D-glucose, a first

product of maltose metabolism.(ii) Maltose chemoreceptor mutant. One

maltose metabolism mutant we have investi-gated is not attracted to maltose, and we maytherefore regard it as a "maltose chemoreceptormutant," even though its defect is not limitedto the chemoreceptor. This is AW470 (Table 2),a derivative of B14 prepared by us by selectingfor resistance to X phage and lack of maltosemetabolism, i.e., it is a malT mutant (17). Thisstrain fails to be attracted to maltose, whethergrown on glycerol or glycerol plus maltose(Table 2; Table 3, column D). This may bebecause maltose cannot enter the cells to inducethe maltose chemoreceptor or because the genefor the maltose chemoreceptor may be shut offalong with the genes for maltose metabolism.

This mutant shows nomal taxis toward D-galactose and D-glucose (Table 3, column D).This provides support for the conclusion thatthe chemoreceptor for maltose is different fromthe galactose and glucose chemoreceptors.

Further support for this conclusion comesfrom the finding that the strain missing boththe galactose and glucose chemoreceptors stillshows maltose taxis (Table 3, column B). Thereduced response to maltose may be explained ifa portion of the attraction to maltose in wild-type bacteria grown on maltose is due to taxistoward the D-glucose produced.

(iii) Maltose competition experiments.Taxis toward maltose by wild-type bacteriagrown on maltose is not inhibited by D-galac-tose, D-glucose, or any of the other sugars tried(Table 4). The maltose chemoreceptor thus failsto detect any of these sugars, or detects themextremely poorly. Conversely, the presence ofmaltose inhibits little, or not at all, taxis towardD-galactose or D-glucose in wild-type E. coligrown on D-galactose (Table 4), and it inhibitsonly partially taxis toward D-glucose in thegalactose chemoreceptor mutant grown on D-galactose (Table 5). Thus the galactose andglucose chemoreceptors detect maltose not atall or weakly.

(iv) Inducibility of maltose taxis. Wild-typeE. coli grown on glycerol or D-galactose areattracted to maltose only slightly, but whengrown on glycerol plus maltose, or on maltose,they are attracted strongly (Fig. 4 and Table 6).Thus, the galactose and glucose chemorecep-tors, present in the galactose-grown cells, detectmaltose weakly or not at all.The requirement for maltose in the growth

medium to bring about maltose taxis is ex-plained by the hypothesis that maltose inducesthe synthesis of a specific maltose chemorecep-tor. An alternative hypothesis, that maltose

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O 10-6 10- 10-4 10-3 10-2MALTOSE MOLARITY

FIG. 4. Maltose taxis by E. coli strain B14 g

on glycerol plus 10-2 M maltose (0) or on glyonly (0).

metabolism must be induced to producereal attractant, D-glucose, is ruled out byfinding cited above. (i) The studies on ma

mutants show that uptake and metabolismaltose are not required. (ii) The mutant r

ing both the galactose and glucose chenceptors, grown on maltose, is still attractEmaltose. (iii) D-Glucose fails to inhibit mataxis.

Wild-type E. coli strain B14 grown on ma

shows poor or no taxis toward the other dcharides cellobiose, lactose, melibiose, suc

or trehalose (data not shown). Maltose taxthese cells was inhibited slightly or not at a

lactose or trehalose (Table 4) or by cellobmelibiose, or sucrose (data not shown). TIfore the maltose chemoreceptor is specifithis disaccharide.

(v) Maltose-binding protein. We have ftan osmotically shockable binding activit3maltose, with a dissociation constant fortose of about 5 x 10 - 6 M (18). Unind(glycerol-grown) or induced (maltose-grishock fluid from E. coli B14 cells containedand 880 pmol/mg of protein, respectively.binding of maltose was not inhibited by D-gtose or D-glucose (18). Since the induciland specificity of this activity resemble thothe maltose chemoreceptor, this binding a

ity probably serves as the maltose-recogncomponent of the maltose chemoreceptor.

cently 0. Kellermann has purified this proteinand has identified it as a product of the Jl geneof the malB region of the E. coli chromosome(Dipl6me d'Etudes Approfondies-UniversityParis VII, 1972).Trehalose taxis and a trehalose

chemoreceptor. Trehalose (a-D-glucosyl-a-D-glucoside) is an attractant for E. coli (Table 1).Evidence will now be presented that there is aseparate chemoreceptor for trehalose, but inaddition the D-glucose produced is detected bythe galactose and glucose chemoreceptors; inwild-type strains, capable of carrying out D-

glucose taxis, the latter process predominates.(i) Trehalose taxis in mutant unable to

carry out glucose taxis. The strain missingboth the galactose and glucose chemoreceptors,grown on trehalose, still carries out trehalosetaxis (Table 3, column B). Hence there must bea chemoreceptor, different from the galactoseand glucose chemoreceptors, that can detect

o__ trehalose. The threshold for trehalose taxis inthis strain is 10-s M, and the peak response is at

rown102 M with 50,000 bacteria accumulating per h.

rcerol Trehalose taxis in the strain missing both thegalactose and glucose chemoreceptors is notinhibited by any of the other sugars tried,

the including maltose (Table 7). The trehalosethe chemoreceptor is thus distinct from the other

ltose chemoreceptors.,m of (ii) Specificity of trehalose chemoreceptor.miss- This chemoreceptor is different from the onlynore- other disaccharide chemoreceptor, the one forad to maltose, since: (i) maltose does not inhibit, as

ltose just stated; (ii) trehalose does not appreciablyinhibit maltose taxis (Table 4); (iii) wild-type

ltose cells induced for maltose taxis show very slightlisac- trehalose taxis; (iv) the maltose chemoreceptorrose, mutant carries out good trehalose taxis after(is of

byiose,here-c for

oundy formal-ucedown)<30The

,alac-)ility)se ofLctiv-izingRe-

TABLE 7. Competition experiments for trehalose taxisin mutant missing both the galactose and glucose

chemoreceptorsa

Remaining responseCompetitor (10 - 2 M) (percent response

without competitor)

N-Acetyl-D-glucosamine 44D-Fructose 123D-Galactose 200D-Glucose 255Maltose 190D-Mannitol 148D-Ribose 117D-Sorbitol 140a E. coli strain AW581, lacking both the galactose

and glucose chemoreceptors, was used. It was grownon trehalose. The procedure was otherwise the sameas described in footnote a, Table 4.

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growth on trehalose (Table 3, column D).The other disaccharides, cellobiose, lactose,

melibiose, and sucrose, must also be detectedweakly or not at all by the trehalose chemore-ceptor since there is little or no taxis towardthem in wild-type E. coli grown on trehalose,and the trehalose taxis by such cells is notappreciably inhibited by them at 10-2 M. Suchcells show a full level of maltose taxis (200,000bacteria per h at 10-3 M) that is not inhibitedby 10-2 M trehalose; apparently trehalose in-duces the maltose chemoreceptor, and it isknown to induce the enzymes of maltose metab-olism (35).

Trehalose taxis is inducible (Table 6).(iii) Trehalose taxis due to glucose produc-

tion in glucose tactic strains. The amount ofD-glucose produced from trehalose, and possiblyaccumulating in the periplasmic space or ex-creted, will depend on the activity of trehalase.(See "Lactose taxis" for a discussion of how thehexose produced might lead to taxis.) This en-zyme and its inducibility have not been studiedin E. coli to our knowledge, and we have not di-rectly tested for the accumulation of D-glucose.Wild-type bacteria grown on trehalose show

trehalose taxis that is strongly inhibited byD-glucose but is inhibited little by D-galactose(Table 4). This is expected if the bacteria areattracted by the D-glucose that they producefrom the trehalose. (See "D-glucose taxis" forpartial inhibition of D-glucose taxis by D-galac-tose.) Inhibition by maltose (Table 4) can resultfrom conversion of maltose to D-glucose sincethe trehalose-grown cells are known to be in-duced for maltose metabolism (35).

Trehalose nearly abolishes D-galactose taxisand strongly inhibits D-glucose and D-mannosetaxis in wild-type bacteria grown on trehalose(data not shown), D-galactose, or D-mannose(Table 4). This is expected if a level of D-glucoseis produced that is high enough to inhibit thegalactose and glucose chemoreceptors.The glucose chemoreceptor must not recog-

nize trehalose, since the galactose taxis mutant,grown on D-galactose, shows D-glucose taxisthat is not inhibited by trehalose (Table 5).These cells do not carry out trehalose taxis.Grown on trehalose, these bacteria do show1-glucose taxis that is inhibited (50-75%) by10- 2 M trehalose, and they are attracted to tre-halose (Table 3, column A). Apparently in thisstrain growth on D-galactose fails to producetrehalase and the trehalose chemoreceptor.No binding activity for trehalose was found in

shock fluid from E. coli strain B14 grown ontrehalose. If trehalase were periplasmic andpresent in the shock fluid, the trehalose might

have been degraded during the binding studies.Lactose taxis. Lactose (4-o-fl-D-galactosyl-D-

glucose) is not itself an attractant for E. coli.However, it attracts bacteria quite well underconditions where it can be broken down (Table1 and top curve of Fig. 5). It is the D-galactose(and also some D-glucose) which accumulatesthat accounts for this attraction. The evidencefor this follows.

(i) Inducibility of lactose taxis. Wild-typeE. coli grown on glycerol (Fig. 5) or D-galactose(data not shown) are attracted little or not at allto lactose. Furthermore, lactose fails to inhibitD-galactose or D-glucose taxis in wild-type E.coli grown on D-galactose (Table 4) or D-glucosetaxis in the galactose chemoreceptor mutants(Table 5). Hence the galactose and glucosechemoreceptors must be unable to detect lac-tose.

Cells grown on glycerol or D-galactose mediato which methyl-fl-thio-D-galactoside has beenadded show fairly good attraction to lactose(Fig. 5 and Table 1). Thus lactose taxis isinducible. Cells grown on lactose show little orno lactose taxis. This can be explained by thefact that the galactose chemoreceptor of suchcells fails to function, perhaps because theresulting monosaccharides inhibit it. Thus, cellsgrowing on lactose under natural conditionsmight not be attracted to it.The requirement for methyl-fl-thio-D-galacto-

side in the growth medium is explained by thehypothesis that bacteria are attracted to theD-galactose (and also some D-glucose) producedfrom the lactose, and induction of lactose me-

FIG. 5. Lactose taxis by E. coli strain B14 grown onglycerol plus 10-s M methyl-,3-thio- D-galactoside(TMG) (-) or on glycerol only (0).

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tabolism is therefore a prerequisite for thistaxis. An alternate hypothesis, that methyl-f-thio-D-galactoside is required for induction ofa specific lactose chemoreceptor, is made un-likely by some results described below. (i)3-Galactosidase was found essential for lactose

taxis; (ii) lactose taxis is nearly abolished by thepresence of D-galactose or D-glucose; and (iii)absence of the galactose and glucose chemore-ceptors results in absence of lactose taxis.A mutant that is constitutive for lactose

metabolism, RV/F'i -, is attracted perfectlywell to lactose when grown in the absence ofmethyl-f-thio-D-galactoside (Table 2).

(ii) Lactose metabolism mutants. Mutantsthat are blocked in the breakdown of lactose failto be attracted to lactose, even when methyl-/-thio-galactoside is present in the growth me-dium. This is true for mutants that have thelactose operon deleted, such as strain RV, ormutants that lack just 3-galactosidase, for ex-ample 20SOK- (Table 2). A mutant lackingonly the permease, 230U, is attracted to lactoseabout one-half as well as wild-type bacteria,and the response is shifted to higher concentra-tions (Table 2), presumably where entry oflactose can proceed in the absence of thispermease. Thus both f-galactosidase and lac-tose permease are needed for good attraction tolactose. This is interpreted to mean that D-

galactose and/or D-glucose produced from thelactose are actually responsible for the attrac-tion. An alternative interpretation, that bacte-ria are attracted well to lactose only when theycan metabolize the resulting D-galactose andD-glucose and can get energy from this, wasruled out by the following experiment. A mu-tant DF2000, which cannot metabolize D-galac-tose or D-glucose past D-glucose-6-phosphatebecause of mutations in the genes for phospho-glucose isomerase and glucose-6-phosphate-dehydrogenase but does have the genes forlactose permease and 3-galactosidase, is at-tracted to lactose well when grown in thepresence of methyl-f-thio-D-galactoside (Table2).

(iii) Lactose competition experiments. Lac-tose taxis by wild-type E. coli grown on glycerolplus methyl-/3-thio-D-galactoside is abolishedby D-glucose and almost completely abolishedby D-galactose (Table 4). (The very slightamount of D-galactose-resistant taxis can repre-sent attraction to small amounts of D-glucoseaccumulating from the breakdown of lactose;D-glucose would be preferentially used by thecells.) In these bacteria, lactose completelyabolishes taxis toward both D-galactose andD-glucose (data not shown). Since D-galactosecannot completely eliminate D-glucose taxis,

some amount of D-glucose would have to haveaccumulated from the breakdown of lactose.

(iv) Chemoreceptor mutants. As expected ifthe response to lactose is primarily due toD-galactose produced, the lactose-positivechemoreceptor mutant A520 grown on glycerolplus methyl-f-thio-D-galactoside shows onlyslight lactose taxis and only at high concentra-tions (.10-s M) compared to a wild-type re-sponse by its galactose chemoreceptor revertantAW521 (Table 3, column A). This residual taxisin AW520 may represent attraction to theD-glucose produced. The strain missing both thegalactose and glucose chemoreceptors fails toshow lactose taxis when grown on glycerol plusmethyl-f-thio-D-galactoside (Table 3, columnB).The excretion of D-galactose (6) and D-glucose

(P. Wayne and L. Shapiro, unpublished data)by E. coli metabolizing lactose has been demon-strated by others. Some f-galactosidase mayleak out of the cells and cleave lactose to thehexoses, or the accumulated hexoses might beexcreted; then the hexoses can attract otherbacteria. It is also possible that the hexoses donot have to leave the cells; in this case thebacteria would be guided by the concentrationof intracellular or periplasmic hexose, whichwould reflect the concentration of lactose exter-nally. Indeed, Macnab and Koshland (30) havereported that bacteria sense time gradients ofattractants: a change in concentration of hexoseat the chemoreceptor sites, as the bacteriumswims about, should evoke a chemotactic re-sponse.

DISCUSSIONWe have surveyed various sugars and sugar

derivatives for their ability to attract Esche-richia coli (Table 1). Although many com-pounds were found to be attractive, only 19showed a high degree of attractiveness (i.e.,with thresholds of about 10-5 M or lower).

Previously, we demontrated that bacteriadetect attractants by means of chemorecep-tors-specific sensing devices that signalchanges in concentration' of chemicals withoutrequiring uptake or metabolism of the chemi-cals, or energy production from them (1).The present results confirm and extend this

conclusion that the attractants themselves aredetected, i.e., that breakdown of the attract-ants, resulting in extensive metabolism andproduction of energy, is not required for taxis.Table 1 includes attractants that wild-typebacteria do not metabolize, and Table 2 showsthat chemicals remain attractants after theiruptake (in the case of D-galactose and maltose)and metabolism are blocked by mutation. The

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only exceptions encountered are lactose andprobably D-glucose-1-phosphate, where break-down to the free hexose is required; but evenhere, metabolism past the hexose stage is notneeded for taxis.Using criteria presented in Results we have

been able to classify most of these 19 best sugar

attractants into nine groups, each handled by adifferent chemoreceptor. We wish to cautionthat the evidence is not equally strong for eachof the chemoreceptors. Thus only for the galac-tose, glucose, maltose, and mannitol chemore-ceptors have chemoreceptor mutants been iso-lated so far. To establish more securely the

TABLE 8. Summary of sugar chemoreceptors and their specificities

Chemoreceptor 1 Chemicals detected | Analogues not detected or detected poorlyname Jhemicals_detected_(thresholds> 10-3 M)

N-Acetyl-D-glucosamine

D-Fructose

D-Galactose and D-glucose best. Alsodetected: 1-D-glycerol-j,-D-galacto-side, 6-deoxy-D-glucose, methyl-fl-D-glucoside, D-fucose, methyl-fl-D-galac-toside, methyl-fl-D-glucoside, L-

arabinose, D-xylose, L-sorbose, 2-deoxy-D-glucose, and D-glucosamine,in that approximate order of decreas-ing effectiveness.0 Possibly D-man-nose is detected very weakly.

Detects D-glucose, but not as well as

galactose chemoreceptor does. De-tects D-mannose. Along with galactosechemoreceptor, detects D-glucosa-mine, 2-deoxy-D-glucose, methyl-,6-D-glucoside, and methyl-a-D-glucoside,in that approximate order of decreas-ing effectiveness'

Maltose

D-Mannitol, possibly D-sorbitol

D-Ribose, possibly D-ribulose

D-Sorbitol, possibly D-mannitol

Trehalose

N-Acetyl-D-mannosamine, D-gl'ucosamine.

Other common hexoses not detected. Analogues ofD-fructose that are poor attractants for wild-type:D-fructose- 1-phosphate, D-fructose-6-phosphate,D-fructose-1,6-diphosphate; sucrose, inulin.

D-Fructose. Maltose, trehalose. Analogues of D-galac-tose that are poor attractants for wild-type: D-

galactonate, L-galactose, D-galactose-6-phosphate,2-deoxy-D-galactose, D-galacturonate, D-gala-L-mannoheptose, 2-glycerol-O-D-galactoside, isopro-pyl-fl-thio-D-galactoside, lactose (as such), andmethyl-fl-thio-D-galactoside. Galactitol detectedby different chemoreceptor. Analogues of D-glu-cose that are poor attractants for wild-type: see

below.

D-Fructose, D-galactose. Maltose, trehalose. Ana-logues of D-glucose that are poor attractants forwild-type: glucoheptose, D-gluconate, L-glucose,D-glucose-6-phosphate, 2-deoxy-D-glucose-6-phos-phate, 3-o-methyl-D-glucose, D-glucuronate, cel-lobiose, sucrose, Schardinger dextrins. Analoguesof D-mannose that are poor attractants for wild-type: N-acetyl-D-mannosamine, D-mannohep-tulose, D-mannosamine, L-mannose, D-mannOse-

6-phosphate, and methyl-a-D-mannoside.

Cellobiose, lactose, melibiose, sucrose, trehalose.Monosaccharides not detected.

D-Arabitol, the five-carbon analogue; galactitol;D-mannose.

L-Arabinose, D-xylose. Analogues of D-ribose that are

poor attractants for wild-type: 2-deoxy-D-ribose;D-arabinose, n-lyxose, L-xylose; D- or L-arabitol,ribitol, xylitol; D-ribonate; all ribosides and ribo-tides tested; D-erythrose (four-carbon analogue ofD-ribose) and erythritol. See also Askamit andKoshland (4). Hexoses not detected.

Xylitol, the five-carbon analogue; galactitol; D-

glucose uncertain.

Cellobiose, lactose, maltose, melibiose, sucrose.

Monosaccharides not detected.

a Based on thresholds (see Table 1) and competition experiments (Table 4 and reference 17).

b Based on thresholds in the galactose chemoreceptor mutants.

N-Acetyl-glu-cosamine

Fructose

Galactose

Glucose

Maltose

Mannitol

Ribose

Sorbitol

Trehalose

r r-T

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ADLER, HAZELBAUER, AND DAHL

existence and specificity for each chemorecep-tor, it will be necessary to find mutants for eachof the remaining chemoreceptors.Table 8 lists the nine chemoreceptors and

summarizes their specificity.We have presented evidence (18) that the

part of the galactose chemoreceptor which rec-ognizes the attractants is the galactose-bind-ing protein (5, 8, 23). This material is releasedfrom the cells by osmotic shock and presumablyresides in the space between the cell membraneand the cell wall (the periplasmic space) (20).This protein also plays a role in the transport ofD-galactose by the methyl-galactoside permease(5, 8, 23). The finding that the galactose-bind-ing protein is the recognition component of thegalactose chemoreceptor led us to test for shock-able binding proteins for other sugar chemore-ceptors (18). Such activities were -found formaltose (18; 0. Kellermann, unpublished data)and D-ribose (4, 18), and these presumably serve

as recognition components for the respectivechemoreceptors. Where sought, binding activi-ties were not detected for the other chemorecep-tors (D-fructose, D-glucose, D-mannitol, treha-lose). This could mean that the affinity for thesubstrates was too low to allow measurement bythe assay used, that the substrates were alteredby enzymes present, or that the recognitioncomponents of these chemoreceptors are notreleased from the cells by osmotic shock.

Recently we have found evidence that theenzyme II complex of the phosphotransferasesystem (26) can serve as recognition componentfor chemoreceptors that detect attractantstransported by that system. In fact, the "glu-cose chemoreceptor mutant" described in Re-sults lacks the two known (13, S. J. Curtis,Ph.D. thesis, University of Chicago, 1973) en-

zymes II for D-glucose. Preliminary experimentssuggest that each of these glucose enzymes II

can act as a recognition component but withdifferent specificities-one detects D-glucose,2-deoxy-D-glucose, D-glucosamine, and D-man-nose, while the other detects D-glucose andmethyl-a-D-glucoside; only when both are mis-sing is there no receptor activity for D-glucose(J. Adler and W. Epstein, in press). Separateenzymes II are known in E. coli for D-glucose(13, 27), D-fructose (14, 16, 27), N-acetyl-D-glucosamine (40), D-mannitol (38), and D-sor-bitol (29). This list includes nearly all of thechemoreceptors for which shockable bindingactivities were not found. The enzymes II are

known to be firmly bound to the cytoplasmicmembrane (27) and are apparently not releasedby osmotic shock (25). Preliminary results withmutants lacking the enzyme II for D-fructose

(14) or D-mannitol (38) indicate that they fail tocarry out those taxes and, hence, that theenzymes II are components of the respectivechemoreceptors. Yet there must be additionalcomponents for a chemoreceptor since the D-mannitol taxis mutants show a near-normalgrowth rate on D-mannitol (see footnote a,Table 3).There is, thus, a very close relationship be-

tween chemotaxis and transport; the two proc-esses share certain components. However,transport itself is not required, at least for thegalactose chemoreceptor (1) and the maltosechemoreceptor. The enzyme II mutants whichlack taxis also lack transport; for these systemsit still has to be determined if transport isrequired for taxis. The mechanism by whichinteraction of an attractant with its transportsystem brings about a chemotactic responseremains to be elucidated. Although for all theattractants one or more transport systems areknown, the converse is not true-there arechemicals with known transport systems thatare not attractants.Why only these particular sugars are detected

by E. coli is unknown. Sugar taxis must providea selective advantage for bacteria by allowingthem to find an energy and carbon source.Presumably, during the evolution of these bac-teria certain sugars were present in their envi-ronment, and thus selection for the develop-ment of chemoreceptors for those sugars wasfavored. No doubt other kinds of bacteria thatlive in different environments will be found tohave a different set of chemoreceptors.

ACKNOWLEDGMENTSThis research was supported by Public Health Service

grant AI-08746 from the National Institute of' Allergy andInfectious Diseases.We thank George W. Ordal and Robert W. Reader for

isolation of the mannitol taxis mutants, and the variouspeople cited in Table 2 for generously providing metabolismmutants. We are indebted to R. W. Reader for helping tomake this paper a little more readable.

LITERATURE CITED1. Adler, J. 1969. Chemoreceptors in bacteria. Science

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4. Aksamit, R., and D. E. Koshland, Jr. 1972. A ribosebinding protein of' Salmonella typhimurium. Biochem.Biophys. Res. Commun. 48:1348-1353.

5. Anraku, Y. 1968. Transport of' sugars and amino acids inbacteria. I. Purification and specit'icity of the galactose-and leucine-binding proteins. J. Biol. Chem.243:3116-3122.

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17. Hatfield, D., M. Hofnung, and M. Schwartz. 1969.Genetic analysis of the maltose A region in Escherichiacoli. J. Bactetiol. 98:559-567.

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19. Hazelbauer, G. L., R. E. Mesibov, and J. Adler. 1969.Escherichia coli mutants defective in chemotaxis to-ward specific chemicals. Proc. Nat. Acad. Sci. U.S.A.64:1300-1307.

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28. Larimer, J., and B. Oakley. 1968. Failure of Gymnemaextract to inhibit the sugar receptors of two inverte-brates. Comp. Biochem. Physiol. 25:1091-1097.

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