Conserved Amplification of Chemotactic Responses through Chemoreceptor Interactions

Many bacteria concentrate their chemoreceptors at the cell poles.Chemoreceptor location is important in Escherichia coli, sincechemosensory responses are sensitive to receptor proximity.It is not known, however, whether chemotaxis in other bacteriais similarly regulated. To investigate the importance of receptor-receptorinteractions in other bacterial species, we synthesized saccharide-bearingmultivalent ligands that are designed to cluster relevant chemoreceptors.As has been shown with E. coli, we demonstrate that the behaviorsof Bacillus subtilis, Spirochaete aurantia, and Vibrio furnissiiare sensitive to the valence of the chemoattractant. Moreover,in B. subtilis, chemotactic responses to serine were increasedby pretreatment with saccharide-bearing multivalent ligands.This result indicates that, as in E. coli, signaling informationis transferred among chemoreceptors in B. subtilis. These resultssuggest that interreceptor communication may be a general mechanismfor modulating chemotactic responses in bacteria.

INTRODUCTION

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Introduction

Chemotaxis is a well-studied process that has been exploredin diverse bacteria, including Escherichia coli, Salmonellaenterica serovar Typhimurium, and Bacillus subtilis (11, 14,18, 62, 76). Chemotaxis is mediated by a series of chemoreceptorsthat transform chemosensory information into a behavioral responsethrough a two-component system. E. coli serves as the canonicalmodel, and in this species, the two-component signaling systemcomprises the receptor-associated histidine kinase CheA andthe cytoplasmic response regulator CheY (22, 65). Changes inchemoreceptor occupancy modulate the kinase activity of CheA,which in turn controls the concentrations of phosphorylatedCheY and CheB. Phospho-CheY can interact with the flagellarmotor protein FliM (74), thereby influencing the rotation ofthe flagella and the behavior of the cell (9). The cell is returnedto its prestimulatus behavior by the methyltransferase CheR,an enzyme that transfers methyl groups from S-adenosylmethionineto glutamate residues on the cytoplasmic domains of the chemoreceptors(71, 75). Phospho-CheB regulates this adaptation through itsmethylesterase activity (78). Consequently, the chemoreceptorsare termed methyl-accepting chemotaxis proteins (MCPs).

Self-association of the MCPs is important for their function.The structures of the MCPs from both E. coli and Salmonellaserovar Typhimurium have been investigated by X-ray crystallographyand directed cross-linking. The MCPs from both species are stablehomodimers (12, 19, 40, 55, 56, 77). The receptor units arehighly helical throughout their periplasmic and cytoplasmicdomains. At their cytoplasmic ends, the homodimers associatein a four-helix bundle of two coiled-coils connected by hairpinturns. A recent crystallographic study (40) of the E. coli serinereceptor demonstrates that MCPs can oligomerize through theassociation of their cytoplasmic domains to form a trimer ofdimers. In addition, Ames et al. (6) recently provided geneticand biochemical evidence that suggests that the E. coli Tsrand Tar MCPs interact in "signaling teams" larger than dimers.Thus, data from diverse types of experiments suggest a rolefor higher-order oligomers in MCP function. In addition to thesedimeric and oligomeric states, the chemoreceptors are concentratedat the cell poles in patches in E. coli (47), in other bacteria(5, 24, 32, 41), and in an archaeon (24). MCP localization isalso observed in elongated cells of E. coli, Vibrio parahaemolyticus,and Proteus mirabilis, where, although the MCPs are not restrictedto the poles, they remain concentrated in patches (24, 48).Thus, there is evidence that MCPs have three levels of self-association:helical-hairpin dimers (stable protein-protein contacts), trimersof dimers (higher-order assemblies), and localization in micrometer-sizedpatches or arrays.

One potential function for MCP-MCP interactions is modulationof responses to chemoeffectors. Models of oligomeric MCP signalingarrays have suggested such a function (13, 17, 67-69), and recentexperiments provide evidence that higher-order oligomers playa role in signaling (6, 23, 44). E. coli senses small (1 to10%) changes in the concentrations of stimuli over large dynamicranges (as many as 6 orders of magnitude) (3, 35, 39, 53, 54,66). Detection of low chemoeffector concentrations requiressubstantial signal amplification to generate a behavioral responseequivalent to that induced by a high ligand concentration (35,70, 72). Bray and others have suggested that interactions betweenMCPs could account for the necessary signal amplification (13,17, 67-69). They propose that a functional "lattice" of chemoreceptorscan transmit signals within a higher-order complex of chemoreceptors.Recently, experimental support in E. coli for this hypothesishas emerged (6, 23, 26, 46). A full complement of MCPs is requiredfor maximum amplification of chemotactic responses, and thedata indicate that clustering of the MCPs may be functionallyimportant. It is not known, however, whether communication amongMCPs functions is a general mechanism for chemotactic responseamplification in other bacteria.

Although many aspects of the chemotactic machinery are conservedamong bacteria, variations are common (8, 11). For example,the number of MCP family members varies among species; Vibriocholerae has 45 MCP-like open reading frames (33), while E.coli has only five MCP-family receptors (Tsr, Tar, Trg, Tap,and Aer) (30). Other examples of differences between E. coliand other species include the importance of membrane depolarizationin chemotactic signaling in Spirochaeta aurantia (28, 29), themechanism of adaptation in B. subtilis, which is mediated bydemethylation rather than methylation of the MCPs (11), andthe presence of multiple types of flagellar structures, as seenin V. parahaemolyticus (52). Additionally, in some bacteria,signaling components not found in E. coli, such as the proteinCheV (21, 51), are necessary for chemotaxis.

In contrast to these differences, the localization of the MCPsis highly conserved. This suggested to us that, despite thedifferences among chemotactic systems in bacteria, the interreceptorcommunication observed in E. coli is a conserved mechanism bywhich chemotactic responses are amplified. This hypothesis issupported not only by the conserved localization of the MCPs(5, 24, 32, 41, 47) but also by the similar requirements amongbacteria for sensitive sensory responses. Like E. coli, manytypes of bacteria, including B. subtilis (58, 60) and S. aurantia(31), respond to changes in chemoeffector concentration overranges spanning 4 to 6 orders of magnitude. This sensitivitysuggests that they also require a means of signal amplification.Understanding the general mechanisms by which chemotactic responsesare amplified may lead to a better understanding of signalingin chemotaxis and other two-component systems.

Synthetic multivalent chemoattractants have provided insightinto the role of MCP clustering in amplifying chemotactic responsesin E. coli (23, 26). Multivalent ligands can be used to manipulatereceptor proximity and elucidate the importance of this parameterin output responses (Fig. 1) (37). We report here the design,synthesis, and use of multivalent ligands to explore the mechanismof chemotactic response amplification in a range of bacteria.Using multivalent attractants, we demonstrate that bacteriaother than E. coli, including those as divergent (15) as B.subtilis, amplify chemotactic responses through interreceptorcommunication.

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FIG. 1. Reorganization of polar MCPs by synthetic multivalent ligands. (Top) Micrograph of an E. coli cell labeled with a fluorescent antibody against the MCPs. As has been observed previously (24, 47), the MCPs are concentrated at the poles. (Bottom left) Diagram of a collection of MCPs within the polar array. The MCPs are Tsr, the E. coli serine receptor, Tar, the aspartate receptor, and Trg, the receptor for glucose, galactose, and ribose. For simplicity the receptors are initially shown as isolated dimers, but a similar model would apply to oligomers. (Bottom right) Addition of a galactose-bearing multivalent ligand stabilizes the association between MCPs (23, 26). These multivalent ligands therefore serve as probes of processes that require receptor-receptor interactions. This figure depicts three levels of MCP self-association: dimers, oligomers, and polar arrays.

MATERIALS AND METHODS

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Materials and Methods

Bacteria and growth conditions. The bacterial strains used in these studies include E. coliAW405, B. subtilis OI1085, S. aurantia J1, and Vibrio furnissiiSR1514. For E. coli, S. aurantia, and V. furnissii, motile cellswere obtained by collecting cells from Luria-Bertani (LB) swimplates (0.25% agar) incubated at 30°C. For B. subtilis,motility was obtained on T broth plates (0.3% agar, 1% tryptone,0.2 mM MgCl₂, 0.5% NaCl, 0.01 mM MnCl₂, 10 mM glucose, and 0.5%glycerol) grown at 37°C. Liquid cultures inoculated fromthese plates were supplemented with a 1 mM concentration ofthe monosaccharide relevant for the subsequent experiments.

Synthesis of chemoattractants. Compounds 1 to 11 were synthesized as described previously (26,36, 73). Briefly, monomers 1, 5, and 8 were generated by standardprocedures utilized in ring-opening metathesis polymerization(ROMP) reactions to synthesize polymers 2 to 4, 6 and 7, and9 to 11 (36). Termination of the polymerization reaction witha bifunctional capping agent (27, 61) provided the means toattach fluorophores. Compounds 12 to 14 were generated by thissynthetic process. The valences (n) of compounds 2 to 4, 6 and7, and 9 to 14 are reported here as the ratio between the monomerand the initiator used in the polymerization. This value canbe used to predict the lengths of the resulting polymers (36,50). Gel permeation chromatography (GPC) can be used to determinethe degree of polymerization (DP), which is another estimationof the average number of monomer units incorporated into polymers.Reported DPs for these types of compounds are approximately18 (n = 10) and 45 (n = 25) (73). The polydispersity indices(a measure of the heterogeneity of the polymer lengths produced)of polymers prepared by this method are on the order of 1.1to 1.2 (a single compound has a polydispersity index of 1) (36).The lengths, and therefore the molecular masses, of these ligandswere controlled to allow penetration of the outer membrane (ingram-negative bacteria). For example, compound 4 has an averagemolecular mass of 17 kDa. Globular proteins as large as 10 kDahave been shown to penetrate the outer membrane of E. coli (16).

Fluorescence microscopy. Cells were prepared for fluorescence microscopy as describedpreviously (24, 26). Compounds 12 to 14 were used at 500 µM.Concentrations are reported as the molar concentration of thesaccharide (or saccharide residue), not the polymer concentration.

Capillary chemotaxis assay. Capillary accumulation assays were performed as previously described(2, 10, 26, 31, 59). The buffer employed in experiments withE. coli was 10 mM potassium phosphate, pH 7.0, with 10 µMEDTA (2). For experiments with S. aurantia, a 10 mM potassiumphosphate buffer, pH 7.0, with 0.2 mM cysteine was used (31).For V. furnissii experiments, a 10 mM phosphate buffer, pH 7.2,with 0.1 mM EDTA and 340 mM NaCl was employed (10, 64). ForB. subtilis chemotaxis experiments, a 10 mM phosphate buffer,pH 7.0, with 10 µM EDTA, 0.05% glycerol, and 0.3 mM (NH₄)₂SO₄was used (59). Capillary assays were performed at 30°C forE. coli, 37°C for B. subtilis, and 25°C for S. aurantiaand V. furnissii. Results are averages from at least three experimentsperformed in duplicate. The error was approximately 20%.

Motion analysis. The behavior of B. subtilis was quantitated by analysis of bacterialmotion, as described previously for E. coli (26, 63). The similarlocomotion of these bacteria allowed the use of identical instrumentation.For experiments measuring the response of B. subtilis to compounds5 to 7, cells were washed three times with chemotaxis bufferand resuspended to an optical density at 550 nm (OD₅₅₀) of 0.01.A sample of this solution was placed under a coverslip and allowedto equilibrate for 1 to 2 min prior to addition of the attractant.After addition of the attractant, 2 min of bacterial behaviorat 25°C was recorded. The paths of the cells were determinedby using the ExpertVision system (Motion Analysis Corporation).Experiments measuring the response of B. subtilis to serinewere performed similarly to those described above. After equilibrationfor 1 to 2 min, cells were treated with either a buffer or compounds5 to 7 at a glucose residue concentration of 10 µM. Priorto treatment with serine, cells were incubated for 120 s, whichallowed full adaptation to attractants 5 to 7. Serine was addedto a final concentration of 10 µM, and motion was recordedfor 60 s. The angular velocity was used as a measure of chemotacticresponse, but analysis of the linear velocity yielded similarresults (data not shown). In contrast to experiments with E.coli (26), no EDTA treatment was required.

RESULTS

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Results

Design and synthesis of chemoattractants. In our studies, we targeted E. coli, S. aurantia, V. furnissii,and B. subtilis to represent a range of gram-negative and gram-positivespecies (15). Each type of bacteria recognizes a distinct setof chemoattractants. For example, galactose is a potent chemoattractantfor E. coli (3), but it is a poor attractant for B. subtilis(60). To investigate chemotactic responses in diverse bacteria,we sought to synthesize multivalent chemoattractants possessinga range of saccharides to provide access to reagents with broadutility. Toward this goal, the galactose-, glucose-, and mannose-bearingmultivalent ligands 2 to 4, 6 and 7, and 9 to 11 were generatedby ROMP (Fig. 2). Monomers 1, 5, and 8 were also tested as attractantsfor comparison. Such experiments were critical to verify thatthe modification of the attractant for incorporation into thepolymer did not abolish its activity (25). In addition, fluorescentderivatives 12 to 14 were generated for colocalization studieswith the MCPs (27, 61).

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FIG. 2. Chemical structures of saccharide-bearing compounds.

Fluorescent multivalent chemoattractants colocalize with the chemoreceptors. To investigate whether synthetic multivalent chemoattractantsbind specifically to the MCPs, we examined the colocalizationof fluorescently labeled polymers 12 to 14 with fluorescentanti-MCP antibodies by microscopy. Our experiments are basedon the observation by Hazelbauer and coworkers that antibodiesraised against the E. coli chemoreceptors are cross-reactivewith putative MCPs from other bacteria (4, 57). Examinationby fluorescence microscopy of bacteria labeled with both fluorescentanti-MCP antibodies and compounds 12 to 14 revealed that thepolymers colocalize with the anti-MCP antibodies in E. coli,B. subtilis, S. aurantia, and V. furnissii (Fig. 3). Importantly,when the saccharide moiety displayed from the ligand was nota chemoattractant for the species, no specific labeling wasobserved (data not shown). Additionally, high concentrationsof unlabeled monosaccharide prevented binding of the fluorescentligands (data not shown). These results suggest that syntheticsaccharide-bearing polymers interact specifically with the MCPs.We therefore investigated the utility of the synthetic ligandsas chemoattractants.

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FIG. 3. Binding of fluorescent multivalent ligands to the MCPs. (a) E. coli treated with an anti-MCP antibody and fluorescent galactose-bearing polymer 12. (b) B. subtilis treated with an anti-MCP antibody and fluorescent glucose-bearing polymer 13. (c) S. aurantia treated with an anti-MCP antibody and fluorescent glucose-bearing polymer 13. (d) V. furnissii treated with an anti-MCP antibody and mannose-bearing polymer 14.

Chemoattractant valence influences chemotactic response. It has been shown previously that multivalent chemoattractantsof sufficient length are capable of clustering the MCPs of E.coli (23, 26). Additionally, the concentration of a multivalentligand required for chemotactic responses decreases with increasingligand valence (26). To extend these findings to evolutionarilydiverse bacteria, we conducted capillary assays on cells ofB. subtilis, S. aurantia, and V. furnissii. We used the ligandsat concentrations normalized to the amount of saccharide derivativeto allow comparisons between attractants of various lengths.Addition of compounds 1 to 11 to these cells revealed that chemotacticresponses were dependent on the valence of the attractant (Table1). As the valence of the ligand increased, its chemotacticpotency also increased. These data suggest that chemotaxis inthese bacteria may be influenced by MCP proximity. Consistentwith our fluorescence microscopy results, only those ligandsdisplaying saccharides that serve as chemoattractants for theindividual bacterial species were effective in this assay.

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TABLE 1. Effect of chemoattractant valence on chemotactic responses

The results of capillary assays can sometimes be difficult tointerpret. Complicating factors include chemoeffector diffusion,accessibility, and turnover (1, 63). To examine chemotacticactivity using a different assay, B. subtilis chemotactic responseswere investigated by motion analysis (26, 63). In this assay,an increase in attractant concentration is expected to reducedirectional changes (angular velocity) and enhance swimmingspeed (translational velocity). In agreement with the resultsobtained from capillary assays, the behavior of B. subtiliswas dependent on the valence of the chemoattractant (Fig. 4).The highest-valence glucose-bearing ligand 7 generated an angularvelocity that was decreased by 11% relative to that caused bythe monovalent ligand 5 at an identical glucose concentration.This result indicates that the multivalent ligand 7 is a morepotent chemoattractant than its monovalent counterpart. Thisassay also provided information about the adaptation of B. subtilisto compounds 5 to 7. Unlike that in E. coli (23), adaptationin B. subtilis was not significantly affected by ligand valence;times for adaptation to compounds 5 to 7 were all approximately110 to 130 s.

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FIG. 4. Effects of synthetic ligands on chemotactic response in B. subtilis. (a) Time course for monitoring of the angular velocities of B. subtilis after treatment at time zero with compounds 5 to 7 at 10 µM. Ligand concentrations are calculated based on the glucose residue concentration. The angular velocity of the cells prior to treatment is shown. Data points are averages of 10-s intervals. Approximately 2 s were lost during movement of the microscope objective prior to and after addition of ligand. Data are from a single experiment that is representative of three replicates. (b) The average angular velocities of B. subtilis during the first 60 s after treatment with compounds 5 to 7 at 10 µM. Data are averages from three independent experiments performed in triplicate. Error bars, single standard deviations.

Clustering of one chemoreceptor type influences responses mediated through another. In previous experiments using E. coli (23, 26), we found thathigh-valence galactose-bearing ligands were capable of stabilizingclusters of MCPs. These clusters contained copies of the MCPsthat mediate responses to both galactose (Trg) and serine (Tsr).Responses to serine were potentiated in the presence of a multivalentligand that could stabilize these clusters. The potentiationof serine responses within clusters strongly supports the involvementof interreceptor interactions in signaling. To examine the sameissue in other species, we added attractants 5 to 7 to B. subtilisand then allowed the cells to adapt to the synthetic ligandsfor various times. The subsequent response to serine was monitoredby motion analysis (Fig. 5). The highest-valence ligand, compound7, caused a 20% increase in the response to serine. No potentiationwas observed upon pretreatment with the monovalent ligand 5,and only a minor effect was observed with the shorter oligovalentligand 6. These results suggest that stabilizing inter-MCP interactionsenhances the amplification of chemotactic responses mediatedby receptors within the clusters.

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FIG. 5. Behavioral response of B. subtilis to serine after pretreatment with compounds 5 to 7. (a) Cells were treated either with a buffer or with compounds 5 to 7 at 10 µM. After 120 s, serine was added to a final concentration of 10 µM. Data points are the average angular velocity of 5-s intervals. Results are representative of four independent experiments performed in triplicate. (b) First 15 s of the response of B. subtilis to serine after pretreatment with a buffer or compounds 5 to 7. Results are averages from four independent experiments performed in triplicate. Error bars, standard deviations.

DISCUSSION

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Discussion

In E. coli, there is evidence that interreceptor communicationwithin a polar lattice is important in the amplification ofchemotactic responses (6, 13, 23). We have investigated whetherthis mechanism is conserved in other bacteria. Using syntheticmultivalent ligands, we found that, as in E. coli, chemotacticresponses of B. subtilis, V. furnissii, and S. aurantia aresensitive to ligand valence. Our results focus on chemotacticbehavior, and this behavior is directly related to signaling.Thus, the data suggest that changes in MCP localization influencethe phosphorylation and methylation of the relevant componentsof the chemotactic signaling system. These results suggest thatcommunication between MCPs is evolutionarily conserved, as isthe importance of receptor-receptor interactions in the amplificationof chemotactic responses. Our data provide a functional rolefor MCP localization in the many bacteria that localize theseproteins.

In addition to their similarities, there are differences betweenthe chemotactic responses and signaling mechanisms of variousbacteria. Our results indicate that variation within the chemotacticmachinery of a particular species can be illuminated with multivalentligands. Specifically, it had been found previously that increasingthe valence of a chemoattractant increased the adaptation timeof E. coli approximately twofold (23). In our studies with B.subtilis, however, we observed no effect of ligand valence onadaptation times (Fig. 4). This result is consistent with thedifferent mechanisms for adaptation in E. coli and B. subtilis(11). Additionally, the sensitivity of S. aurantia to ligandvalence was far greater than that of E. coli. In S. aurantia,a 100-fold increase in potency was observed for the multivalentligands, and by comparison, a maximum 10-fold increase was observedin experiments with E. coli (Table 1). This variation couldbe related to the differences between the chemotactic systemsof these organisms. Chemotaxis in S. aurantia involves membranedepolarization (28, 29), but in E. coli it does not. Alternatively,differences in sensitivity could be related to structural differencesbetween the MCPs in these organisms. For instance, multivalentligands might cluster the chemoreceptors more effectively inone species than in another. Conversely, changes in chemoreceptorclustering might have different effects on kinase or methyltransferaseactivities in different species. Thus, these synthetic reagentsmay serve as sensitive probes of differences in chemotacticmachinery.

There are several mechanisms by which the multivalent ligandsmight potentiate chemotactic responses. The synthetic attractantsare designed to stabilize interreceptor interactions (Fig. 1),and they have been shown to be capable of this function (26).An alternative mode of action for these materials, however,is potentiation of responses via increased avidity. Multivalentligands often have higher avidity for their target than monovalentderivatives (37, 38, 43, 49). Although a multivalent chemoattractantcan exhibit an increase in binding through avidity (25), thismechanism does not account for all of the data. If the enhancedchemotactic response were due to avidity alone, saccharide-bearingligands should not influence B. subtilis responses to serine(Fig. 5). The potentiation of a response through the serine-sensingreceptor, which does not directly bind the synthetic multivalentchemoattractants, provides evidence of communication betweenthe glucose- and serine-sensing receptors. Therefore, our datasupport a mechanism that involves MCP clustering.

Changes in MCP clustering could influence the structure of thesignaling complex and/or the stoichiometry of signaling componentswithin it. Specifically, clustering may favor direct protein-proteincontacts between glucose- and serine-sensing MCPs, similar tothose observed in vitro in dimers and oligomers (40, 44). Thus,changes in conformation induced by chemoeffector binding wouldbe relayed to other MCP members of the complex and ultimatelyto the signaling components. Moreover, MCP clustering may serveto concentrate or exclude cytoplasmic components, such as CheAor CheB (70). Additional experiments to further elucidate themolecular mechanisms of signal amplification are ongoing.

Two-component systems mediate many prokaryotic and some eukaryoticresponses to stimuli (7, 34, 42, 45). Additionally, two-componentsystems, including those governing chemotaxis (20), have beenimplicated in virulence and pathogenicity. We suggest that syntheticmolecules designed to control interreceptor proximity may serveto uncover relationships between receptor location and signalingin a range of physiologically and medically important two-componentsystems.

ACKNOWLEDGMENTS

Bacterial strains were provided by J. Adler (E. coli AW405),G. Ordal (B. subtilis OI1085), E. P. Greenberg (S. aurantiaJI), and L. McCarter (V. furnissii SR1514). For supplying antibodieswe thank G. Hazelbauer (anti-Trg), J. S. Parkinson (anti-Tsr),and G. Ordal (anti-McpB). S. Bednarek supplied access to fluorescencemicroscopy equipment. We thank F. Boehm for providing syntheticintermediates.

This research was supported in part by a grant from the NIH(GM 55984). A.C.L. acknowledges the NSF predoctoral fellowshipprogram for support. J.E.G. thanks the NIH Biotechnology TrainingProgram for a predoctoral fellowship (GM 08349). L.E.S. wassupported by an NIH predoctoral fellowship (GM 18750). R.M.O.was supported by a Pharmacia-Upjohn fellowship and a fellowshipfrom Eastman Chemical Company.

FOOTNOTES

* Corresponding author. Mailing address: Departments of Chemistry and Biochemistry, University of Wisconsin at Madison, 1101 University Ave., Madison, WI 53706. Phone: (608) 262-0541. Fax: (608) 265-0764. E-mail: kiessling{at}chem.wisc.edu.

Present address: Quintessence Biosciences, Madison, WI 53719.

REFERENCES

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