JOURNAL OF BACTERIOLOGY, Sept. 1990, p. 5218-5224 Vol. 172, No. 90021-9193/90/095218-07$02.00/0Copyright © 1990, American Society for Microbiology

Repellents for Escherichia coli Operate Neither by ChangingMembrane Fluidity nor by Being Sensed by Periplasmic

Receptors during ChemotaxisMICHAEL EISENBACH,* CONSTANTINOS CONSTANTINOU,t HAMUTAL ALONI, AND MEIR SHINITZKY

Department of Membrane Research, The Weizmann Institute of Science, 76100 Rehovot, Israel

Received 2 May 1990/Accepted 2 July 1990

A long-standing question in bacterial chemotaxis is whether repellents are sensed by receptors or whetherthey change a general membrane property such as the membrane fluidity and this change, in turn, is sensedby the chemotaxis system. This study addressed this question. The effects of common repellents on themembrane fluidity of Escherichia coli were measured by the fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene in liposomes made of lipids extracted from the bacteria and in membrane vesicles.Glycerol, indole, and L-leucine had no significant effect on the membrane fluidity. NiSO4 decreased themembrane fluidity but only at concentrations much higher than those which elicit a repellent response in intactbacteria. This indicated that these repellents are not sensed by modulating the membrane fluidity. Aliphaticalcohols, on the other hand, fluidized the membrane, but the concentrations that elicited a repellent responsewere not equally effective in fluidizing the membrane. The response of intact bacteria to alcohols was monitoredin various chemotaxis mutants and found to be missing in mutants lacking all the four methyl-acceptingchemotaxis proteins (MCPs) or the cytoplasmic che gene products. The presence of any single MCP wassufficient for the expression of a repellent response. It is concluded (i) that the repellent response to aliphaticalcohols can be mediated by any MCP and (ii) that although an increase in membrane fluidity may take partin a repellent response, it is not the only mechanism by which aliphatic alcohols, or at least some of them, areeffective as repellents. To determine whether any of the E. coli repellents are sensed by periplasmic receptors,the effects of repellents from various classes on periplasm-void cells were examined. The responses to all therepellents tested (sodium benzoate, indole, L-leucine, and NiSO4) were retained in these cells. In a controlexperiment, the response of the attractant maltose, whose receptor is periplasmic, was lost. This indicates thatthese repellents are not sensed by periplasmic receptors. In view of this finding and the involvement of theMCPs in repellent sensing, it is proposed that the MCPs themselves are low-affinity receptors for the repellents.

The nonstimulated swimming of peritrichously flagellatedbacteria is a random walk consisting of swimming in ratherstraight lines interrupted occasionally by brief episodes oftumbling. Smooth swimming results from counterclockwiserotation of the flagella, and tumbling results from clockwiserotation and from nonsynchronized pauses in the rotation.Attractants increase the counterclockwise bias of the flagellaand thus prolong the smooth swimming. Repellents increasethe clockwise bias and the pausing frequency, resulting inmore frequent tumbles (2, 4, 7, 10, 21, 22, 27, 28, 47, 58).Since the discovery of repellents for Escherichia coli andSalmonella typhimurium (58, 59), the mechanism of functionof only one group of repellents has been revealed. These areweak organic acids, such as formic, acetic, benzoic, andsalicylic acid, which act by lowering the intracellular pH (16,40). This change in pH is detected by Tsr and Tap (19, 49,62), two of the four methyl-accepting chemotaxis proteins(MCPs), membrane proteins which function as receptors forsome attractants as well as sensory transducers (see refer-ences 26 and 53 and M. Eisenbach, Signal Transduction inBacterial Chemotaxis, in press, for recent reviews). TheseMCPs elicit a repellent response, whereas the other twoMCPs, Tar and Trg, elicit a weaker, attractant response (19,62). The other group or groups of repellents include aminoacids such as leucine and valine, indole and its derivatives,

* Corresponding author.t Present address: Department of Genetics, University of Witwa-

tersrand, Johannesburg, South Africa.

alcohols and polyalcohols, heavy cations such as Co2" andNi2+, and others (58, 59). The response to any of theserepellents involves one or more of the four MCPs (30, 51,62).

Initially, on the basis of competition and additivity exper-iments, it was assumed that repellent receptors exist (58, 59).However, since, with time, receptors for repellents have notbeen identified, and in view of the relatively high repellentconcentrations (in the millimolar range) needed to elicit aresponse, the mere existence of repellent receptors has beenquestioned. An alternative that has been raised is that one ormore of the membrane properties, e.g., the membrane lipidfluidity, is affected nonspecifically by repellents (25, 33, 44).Indeed, the lack of stereospecificity for the repellent re-sponse is in line with this alternative. (For example, thepotencies of D- and L-leucine or D- and L-phenylalanine asrepellents are comparable [59].)The only studies of membrane fluidity of bacteria in

relation to chemotaxis that have been published so farexamined the effects of membrane fluidity on the response toattractants and on the unstimulated tumbling frequency (24,29). Although an apparent requirement for a fluid membranewas initially found in capillary assays (24), subsequentstudies found no significant differences in tumble frequencyor in the response time to attractants between cells having afluid or rigid membrane (29). No direct measurements of theeffect of repellents on the membrane fluidity have beenpublished. As a matter of fact, many of the repellents for E.coli (59), e.g., unsaturated fatty acids or aliphatic alcohols,

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TABLE 1. Bacterial strains used in this study

Strain Relevant genotype Parent Source or reference

RP437 Wild type RP477 36OWl Wild type AW574 32RP5695 /tsr RP437 5RP5700 Atsr RP437 1RP3839 Afar RP437 48RP3840 Atar RP437 48RP3525 Atap RP437 J. S. ParkinsonCP167 Atrg HB233 (RP437 G. L.

Met+) HazelbauerCP177 Atrg OWl 34RP5543 Atsr Atar RP437 5RP5872 Atsr Afar Atap RP437 J. S. ParkinsonAW660 tsr tar trg OWl 18CP362 Atsr Atar Atap Atrg RP437 35RP1091 A(cheA-cheZ) RP437 37

apparently may induce a disorder in the lipid domain,expressed as an increase in the membrane fluidity (45).The aim of the present study was to determine which of

the possibilities raised above is correct; namely, to deter-mine whether there are receptors for repellents or whetherrepellents affect general membrane properties such as themembrane fluidity.

MATERIALS AND METHODSChemicals. Antibody against E. coli flagellin (serotype

H48) was a gift from the National Center for Enterobac-teriaceae, Central Laboratories, Ministry of Health, Jerusa-lem, Israel. 1,6-Diphenyl-1,3,5-hexatriene (DPH) of highpurity and indole were obtained from Fluka, and tetraethyl-ene pentamine (Tetren) was obtained from Merck. Otherchemicals were of the highest purity grade.

Bacteria. Strains of E. coli used in this study are listed inTable 1. E. coli cells were grown at 35°C either in H-1minimal medium (15) supplemented with glycerol (0.5%[vol/vol]) or in tryptone broth as described previously (6, 8).When indicated, the cells were permeabilized by the Tris-EDTA technique of Leive (23) as modified by Szmelcmanand Adler (55). The efficiency of the permeabilization wasdetermined as described previously (6). The permeabilizedcells were finally suspended in a chemotaxis medium (pH7.0) consisting of potassium phosphate buffer (10 mM, pH7.0), Tetren (0.1 mM) and L-methionine (0.1 mM). All othercells were washed twice and resuspended in the chemotaxismedium without permeabilization.

Preparations. Penicillin-treated cells were prepared byincubating growing cells sequentially with penicillin for 45min at 35°C and with chloramphenicol for 30 min as de-scribed previously (39). The resulting periplasm-void 'cellswere suspended in potassium phosphate buffer (10 mM, pH7.0), sucrose (20%), MgSO4 (1 mM), and EDTA (0.1 mM).Membrane vesicles were prepared from intact bacteria by a

French pressure cell as described previously (17). Bacteriallipids were extracted from intact bacteria by isopropanol andchloroform as described previously (42). Liposomes fromthese lipids were prepared by dispersing the lipids in aque-ous chemotaxis medium detailed above (1 mg/ml) and thensonicating for 1 min with a tip sonifier (Branson model 350)at 100 W.Chemotaxis assays. Temporal assays (27) were performed

at room temperature as previously described (11, 52). Thedirection of flagellar rotation was determined by tethering

assays (47). They were carried out in a flow chamber (3) asdescribed previously (38). The flow medium was the chemo-taxis medium detailed above, supplemented with stimulantsas indicated.Membrane microviscosity. Lipid microviscosity was deter-

mined by steady-state fluorescence polarization of DPH asdescribed previously (46). The probe was allowed to equili-brate with the membrane preparations for 45 min before theexperiment. The value of the microviscosity (Xq) was calcu-lated from the empirical relation -q = 2P/(0.46 - P) where Pis the degree of fluorescence polarization, obtained directlyfrom the fluorometer used (46). The lipid fluidity was definedas 1/j. In this method, changes of more than 1% in -q areconsidered significant (46).

RESULTS

Membrane labeling. Lipid microviscosity of biologicalmembranes is commonly measured with DPH as a probe(46). The use ofDPH with bacteria is limited to cases wherethe probe is permeant through the outer membrane. Thus,the cytoplasmic membrane is accessible to DPH in E. colicells derived from strain B/r (63). To evaluate labelingconditions, we first permeabilized the outer membrane ofour strain of study by Tris-EDTA treatment and then deter-mined the fluorescence signal resulting from uptake of'DPHby the membrane. The initial fluorescence signal was rela-tively small (about twofold that in the absence of bacteria),and it monotonically but very slowly increased with time,reaching a ninefold signal in an hour. When the same batchof bacteria was sonicated in the presence of DPH so as tomake their cytoplasmic membrane accessible to DPH, arapid increase in fluorescence was recorded, reaching a34-fold signal within 9 min and leveling off at a 40-fold signalafter 35 min. This indicates that in our strains, permeabili-zation of the outer membrane by the Tris-EDTA method isnot sufficient for making the cytoplasmic membrane acces-sible to DPH. To circumvent the accessibility difficulty, weused two other preparations, complementary to each other,which are commonly used in bacterial membrane studies (57,60). The first preparation consisted of liposomes made of thelipid extract from the wild-type strain of study, RP437. Theother preparation consisted of membrane vesicles made byFrench press from the same strain. The vesicle preparationhas the advantage that it contains vesicles from the naturalmembrane, but it also includes fragments of the outermembrane. The liposome preparation, on the other hand, isa pure system, but it does not contain the membrane proteinswhich may affect the membrane properties. DPH uptake wassignificant by both systems (79- and 37-fold signal increase inliposomes and vesicles, respectively). In view of the advan-tages and disadvantages of each system, we used bothsystems in all of our subsequent measurements.

Effects of repWlIents on membrane fluidity. We determinedthe effect of four classes of repellents, whose mode of actionis not understood, on membrane fluidity of both prepara-tions. For this purpose, suspensions of liposomes and mem-brane vesicles in the presence of DPH were titrated witheach of the repellents. In parallel, we determined the mini-mal concentration of each repellent that caused tumbling intemporal assays and 10 to 20 s of clockwise rotation intethering assays (denoted as threshold concentration). Asshown in Fig. 1, three of the repellents tested (glycerol,indole, and L-leucine) had no significant effect on the mem-

brane microviscosity (Fig. 1A through C). The thresholdconcentrations of these repellents (marked by arrows in the

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FIG. 1. The effect of common repellents on membrane microviscosity. Liposomes (closed circles) and membrane vesicles (open circles)were labeled with DPH as described in Materials and Methods. The degree of fluorescence polarization was then recorded, and microviscositywas calculated. The results are presented as relative microviscosity. The arrow in each panel marks the minimal repellent concentration whichelicited a chemotactic response in intact bacteria (clockwise rotation of tethered cells and tumbling of free-swimming cells). Theseconcentrations were 500, 0.2, 5, and 0.2 mM for glycerol, indole, L-leucine, and NiSO4, respectively (A to D).

figure) were not effective in changing the membrane fluidity.The fourth repellent tested, NiSO4, increased the membranemicroviscosity (i.e., decreased the membrane fluidity), butits threshold concentration as a repellent was 0.2 mM, muchbelow the concentration at which an increase in the mem-brane microviscosity was detectable (Fig. ID). It is thuspossible to conclude that these repellents do not act byaffecting the bulk membrane fluidity. At this stage, thisconclusion should be taken with caution because the deter-mination of membrane microviscosity and the behavioralstudies were not carried 'out with the same preparation.However, it does not seem likely that this difference in thepreparations should affect the conclusion. Even if the acces-sibility of the repellent to the cytoplasmic membrane wasdifferent in each preparation, it was probably smallest in thepreparation of intact bacteria. Yet, the response of intactbacteria to repellents was at concentrations much belowthose which elicited effects on the other preparations.Among the repellents that we have examined, only one

class, aliphatic alcohols, actually increased the membranefluidity (shown as a decrease in membrane microviscosity inFig. 2). The threshold concentrations of the repellents thatelicited a behavioral response in intact bacteria varied from20 to 200 mM. These concentrations were also effective inincreasing the membrane fluidity. However, as shown in Fig.3, there was no consistent membrane microviscosity valueamong the alcohols at which a repellent response was

observed. It seems, therefore, that even for the case of

aliphatic alcohols, their mechanism of action is at best onlypartially due to increasing of membrane fluidity.As a control, similar experiments were carried out with

the potent attractants L-serine (0 to 200 mM) and L-aspartate(0 to 150 mM). No change in membrane fluidity could bedetected throughout this range, while a behavioral responsewas already observed at concentrations lower than 1 mM.

Identification of the MCP(s) that mediates the response toalcohols. To determine whether alcohols use the regularchemotaxis machinery for the response or whether theyhave a direct effect on the motor, we studied the alcoholresponse of a mutant which lacks a large part of the che-motaxis machinery owing to a deletion of the genes fromcheA to cheZ, RP1091. As shown in Table 2, which includesresults with three representative alcohols, RP1091 did notrespond to any of them, unlike its wild-type parent, RP437.This indicates that alcohol sensing is processed by theregular chemotaxis machinery.A number of mediators which transduce the chemotactic

signal across the cytoplasmic membrane are currentlyknown (see M. Eisenbach, in press, for a recent review). Allthe repellents that have been so far investigated, excludinghigh oxygen concentrations, are sensed by the MCPs. Todetermine which, if any, of the MCPs mediate the responseto the various alcohols, we studied mutants'having variouscombinations of mutations, mostly deletions of the genesthat code for these MCPs (Table 1). As shown in Table 2,only a deletion of all four genes that code for the MCPs led

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FIG. 2. The effect of aliphatic alcohols on membrane microviscosity of liposomes of bacterial lipids and vesicles. Microviscosity was

calculated as in Fig. 1. The symbols used are +, ethanol; O, propanol; *, isopropanol; 0, butanol; and *, isobutanol.

to loss of the responsiveness to the repellents (strain CP362in the table). Deletions or mutations in 1 to 3 of these genes

were not sufficient to cause loss of response to alcohols-thepresence of even one of the four MCPs was sufficient forthe repellent response (i.e., strains RP5872 and AW660). Itseems, therefore, that the response to alcohols can bemediated by any single MCP, similarly to what was previ-ously found with glycerol and ethylene glycol (30, 31).

Effects of stimuli on penicillin-treated bacteria. In view ofthe above observations, we wished to determine whetherrepellents are detected by periplasmic receptors. For thispurpose we studied the response of penicillin-treated cells tochemotactic stimuli. Such cells, unlike spheroplasts, areprepared by a relatively short incubation with penicillin,

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FIG. 3. Membrane microviscosity at which a repellent responseis observed. See text for details. Black and gray columns representmembrane vesicles and liposomes, respectively. The thresholdconcentrations of the alcohols were 20 mM (isobutanol), 50 mM(propanol, isopropanol, and butanol), and 200 mM (ethanol).

only until a crack in the cell wall is formed. Consequently,they have the same shape as intact bacteria. Chloramphen-icol is added to prevent further swelling of the cells. Such atreatment yields periplasm-void, motile cells (39). Becauseof their fragility, these cells cannot be tethered (39). There-fore, only temporal assays were performed with them. Thepenicillin-treated cells retained the responsiveness to all therepellents tested (Table 3). At the same time, they lost theresponsiveness to the control attractant, maltose, whosereceptor, the maltose-binding protein, is periplasmic (12). Asanother control, we examined the response to the attractantsL-serine and L-aspartate, whose receptors are the MCPs Tsrand Tar, respectively (13, 54, 61). As shown in Table 3, theresponse to these attractants was retained. On the basis ofthese observations it is possible to conclude, with a highdegree of confidence, that repellents are not sensed byperiplasmic receptors.

DISCUSSIONThe aim of this study was to determine whether repellents

affect a general membrane property that influences thebehavior of bacteria or, by way of elimination, whether theyare detected by specific receptors. General membrane prop-erties which may, in principle, affect bacterial behavior arethe membrane potential and the membrane fluidity. Theformer has been previously ruled out (50), as no consistentchanges in the membrane potential in response to repellentswere observed (see reference 9 for a review on membranepotential measurements). The present study appears to ruleout also the possibility of membrane fluidity: (i) most of therepellents tested caused a typical repellent response atconcentrations much below those at which any change in themembrane fluidity could be detected (Fig. 1), and (ii) whenthe membrane was fluidized by alcohols (Fig. 2), there was

no consistent value of membrane microviscosity underwhich a repellent response was elicited (Fig. 3). Theseobservations are in line with those of Miller and Koshland(29), who found that an increase in the membrane fluidity byaltering the lipid composition had no effect on the tumblingfrequency of the bacteria. It is, therefore, reasonable toconclude that repellents do not affect bacterial behavior by

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TABLE 2. The responsiveness of chemotaxis mutants to alcohols

Gene products present Responsea toStrain

Tsr Tar Tap Trg CheA-CheZ Ethanol Isopropanol Isobutanol

RP437 + + + + + ++ ++ ++owl + + + + + ++ ++ ++RP5695 - + + + + + + + +RP5700 - + + + + + + + + + +RP3839 + - + + + ++ + ++RP3840 + - + + + + + + + + +RP3525 + + - + + + + + + +CP167 + + + - + ++ ++ ++CP177 + + + - + + + + + + +RP5543 - - + + + + + + + +RP5872 - - - + + + + + +AW660 - - + - + + + + +CP362 - - - - + -RP1091 + - + + - - -

a The response was determined by the tethering technique as described in Materials and Methods. + + indicates that a large majority of the cells respondedto the alcohols by an increase in the clockwise bias of the rotation; + indicates that about a half or less of the cells responded; - indicates no response. Isobutanolcaused a gradual loss of rotation; the responsiveness to this alcohol was determined by the change in the direction of rotation prior to the motility loss. Theconcentrations of ethanol, isopropanol, and isobutanol were 200, 100, and 100 mM, respectively. More than 30 tethered cells of each strain were examined witheach alcohol.

modulating general membrane properties and, therefore, byway of elimination, that they are likely to be detected byreceptors. These receptors should be associated with thecytoplasmic membrane because the release of periplasmdoes not affect the response of bacteria to repellents (Table3). Since the response to most, if not all, of the repellentsother than high oxygen concentrations is mediated by spe-cific MCPs (16, 40, 51, 59, 62), and since the mechanism ofencounter between this putative receptor and the appropri-ate MCP through lateral diffusion is highly unlikely (9, 43), itis reasonable to assume that the receptors are the MCPsthemselves. The recent findings of Yamamoto et al. (62),which indicate that the repellent phenol has a specificrecognition site on Trg and that the phenol-sensing site byTsr is on its periplasmic domain, are in line with thisproposition.How can this conclusion be reconciled with the lack of

stereospecificity, with the relatively high concentrations ofrepellents required for eliciting a behavioral response, andwith the lack of positive identification of receptors? Appar-ently, the affinity of the receptors, presumably the MCPs, torepellents is low. Receptors with such low affinity requirehigh concentrations of ligand for binding, they are difficult toidentify, and their specificity is rather low. Analogous casesare those of the olfactory and taste systems in eucaryotes. Alarge repertoire of odorants are detected with very lowaffinities by the olfactory system, causing a debate of

whether or not olfactory receptors exist (20). In the tastesystem, on the other hand, the existence of receptors isunquestionable. Nevertheless, the affinity of the yet-uniden-tified taste receptors to their ligands is very low. Forexample, the concentration of sugar needed to elicit ahalf-maximal response is in a range as high as 0.5 M (14, 56).

In this study we have found that aliphatic alcohols belongto a group of repellents, the response to which is mediatedby any one of the four MCPs (Table 2). Other members ofthis group are glycerol and ethylene glycol (30, 31). Themechanism by which the alcohols are sensed is not known.The aliphatic alcohols, unlike glycerol, fluidize the mem-brane (Fig. 2). However, their threshold concentrations arenot equal and they fluidize the membrane to different extents(Fig. 3). It seems that alcohols interact with the MCPsdirectly albeit in a nonspecific manner. The result of thisinteraction is a clockwise signal, sent to the flagella via theregular chemotaxis machinery, as is evident from the loss ofthis response in a mutant deleted for the cytoplasmic chegene products (Table 2).

In line with the findings presented in this study, it might beproposed that in the case of aliphatic alcohols, the mem-brane lipid layer may be looked upon as a dense array oflow-affinity binding sites in which the low-affinity receptor,presumably the MCP, is embedded. The repellent action ofthe alcohol may be initiated by partitioning within the lipidlayer, which can then tunnel the alcohol through two-

TABLE 3. Effects of stimuli on penicillin-treated cellsa

ResponseStimulant Receptor location

Intact bacteriab Penicillin treated

Maltose (10 mM)c Periplasm Smooth swimming NoneL-serine (1 mM) + L-aspartate (10 puM)C Membrane Smooth swimming Smooth swimmingBenzoate (12.5 mM) Membrane Tumbling TumblingIndole (0.6 mM) Unknown Tumbling TumblingL-leucine (30 mM) Unknown Tumbling TumblingNiSO4 (6 mM) Unknown Tumbling Tumbling

a Prepared as described in Materials and Methods from cells growing in tryptone broth supplemented with 0.1% maltose.b The bacteria were from the same batch used for preparing penicillin-treated cells.c Since unstimulated cells swim rather smoothly, attractant responses were measured by first making the cells tumbly by addition of benzoate (12.5 mM). Only

then, the attractant, mixed with 12.5 mM benzoate, was added.

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dimensional diffusion or shuttling into the specific site on theMCP. Such a tunneling mechanism can increase the effec-tiveness of the alcohol with respect to the ambient concen-tration by orders of magnitude (41).

ACKNOWLEDGMENTS

We thank R. M. Macnab for comments on the manuscript.This study was supported by Public Health Service grant A124675

from the National Institute of Allergy and Infectious Diseases.

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