Functional Analysis of Nine Putative Chemoreceptor Proteins in Sinorhizobium meliloti

The genome of the symbiotic soil bacterium Sinorhizobium meliloticontains eight genes coding for methyl-accepting chemotaxisproteins (MCPs) McpS to McpZ and one gene coding for a transducer-likeprotein, IcpA. Seven of the MCPs are localized in the cytoplasmicmembrane via two membrane-spanning regions, whereas McpY andIcpA lack such hydrophobic regions. The periplasmic regionsof McpU, McpV, and McpX contain the small-ligand-binding domainCache. In addition, McpU possesses the ligand-binding domainTarH. By probing gene expression with lacZ fusions, we haveidentified mcpU and mcpX as being highly expressed. Deletionof any one of the receptor genes caused impairments in the chemotacticresponse toward most organic acids, amino acids, and sugarsin a swarm plate assay. The data imply that chemoreceptor proteinsin S. meliloti can sense more than one class of carbon sourceand suggest that many or all receptors work as an ensemble.Tactic responses were virtually eliminated for a strain lackingall nine receptor genes. Capillary assays revealed three importantsensors for the strong attractant proline: McpU, McpX, and McpY.Receptor deletions variously affected free-swimming speed andattractant-induced chemokinesis. Noticeably, cells lacking mcpUwere swimming 9% slower than the wild-type control. We inferthat McpU inhibits the kinase activity of CheA in the absenceof an attractant. Cells lacking one of the two soluble receptorswere impaired in chemokinetic proficiency by more than 50%.We propose that the internal sensors, IcpA and the PAS domaincontaining McpY, monitor the metabolic state of S. meliloti.

INTRODUCTION

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Introduction

The process of chemotaxis enables many motile bacterial speciesto sense their environment and move in a beneficial direction.The underlying signaling pathway for responding to changes inthe concentrations of chemical attractants or repellents hasbeen most intensely studied for Escherichia coli and Salmonellaenterica serovar Typhimurium (for reviews, see references 24and 69). In the absence of a chemical stimulus, E. coli showsa random swimming pattern consisting of alternating runs andtumbles. The addition of an attractant or the removal of a repellentpromotes counterclockwise flagellar rotation and therefore straightruns. Ergo, the cell is directed to a more advantageous environment.The signal transduction pathway to the flagellar motor consistsof chemoreceptor proteins and a two-component signaling system.E. coli uses four membrane-bound methyl-accepting chemotaxisproteins (MCPs)—Tar for aspartate and maltose, Tsr forserine, Trg for ribose and galactose, and Tap for dipeptides—aswell as the membrane-bound Aer as an oxygen sensor (16, 24).MCP molecules typically consist of a periplasmic ligand-bindingdomain, two transmembrane helices, and a highly conserved cytoplasmicsignaling domain (24, 67). To enable high sensitivity over arange of attractant concentrations, adaptational modificationsare introduced at specific glutamate residues in two methylationhelices, MH1 and MH2 (38). Methyl groups are transferred fromS-adenosylmethionine by the methyltransferase CheR (72), whiletheir removal is accomplished by the methylesterase CheB (33,68). The highly abundant major receptors in E. coli, Tsr andTar, have an NWETF pentapeptide sequence at the C terminus,which serves as a docking site for CheR and CheB (12, 25, 77).

Recent studies suggest that chemotaxis in other bacteria departsfrom the E. coli model by involving more che genes and chemoreceptors(4, 7, 17, 59, 70). The nitrogen-fixing plant symbiont Sinorhizobiummeliloti, a member of the alpha subgroup of proteobacteria (52),differs from the enterobacterial (gamma-subgroup) behavioralscheme in its modes of flagellar rotation, signal processing,and gene regulation (59). The rigid complex flagellar filamentsconsist of four related flagellin subunits, and interflagellinbonds lock the filaments into right-handedness (21, 29, 60).Hence, S. meliloti cells are propelled by flagella that rotateexclusively clockwise, and swimming cells respond to tacticstimuli by modulating their rotary speed (8, 58). In E. coli,tactic signals are processed by a single response regulator,CheY, and a phosphatase, CheZ. In contrast, signal processingin S. meliloti involves a retrophosphorylation loop with tworesponse regulators, CheY1 and CheY2, but no phosphatase (64,65). CheY2 is the main regulator of motor function, causinga decrease in the rotary speed of the unidirectional clockwise-rotatingflagellar motor (59). It has been reported previously that S.meliloti exhibits positive chemotactic responses toward a widerange of substances such as amino acids, sugars, and exudatesfrom roots of legume host plants (20, 22, 29, 32, 45). S. melilotihas nine putative chemoreceptors to sense the concentrationsof these attractants (26, 48). In order to elucidate the rolesof individual chemoreceptor proteins in chemotaxis, we introducedsingle and multiple gene disruptions and analyzed the chemotacticabilities of the resulting mutant strains toward nutrients.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains and plasmids. Derivatives of E. coli K-12 and S. meliloti MV II-1 (37) andthe plasmids used are listed in Table 1.

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TABLE 1. Bacterial strains and plasmids

Media and growth conditions. E. coli strains were grown in Luria broth (43) at 37°C.S. meliloti strains were grown in TYC (0.5% tryptone, 0.3% yeastextract, 0.13% CaCl₂·6H₂O [pH 7.0]) at 30°C (60).Motile cells prepared for swimming velocity analysis and capillaryassays were grown for 2 days in TYC with streptomycin, dilutedfirst 1:5 in 3 ml of TYC with streptomycin for 24 h and then1:500 in 10 ml RB minimal medium (29), layered on Bromfieldagar plates (64), and incubated at 30°C for 15 h to an opticaldensity at 600 nm (OD₆₀₀) of 0.1. The following antibioticswere used at the indicated final concentrations: for E. coli,kanamycin at 50 mg/liter and tetracycline at 10 mg/liter; forS. meliloti, neomycin at 120 mg/liter, streptomycin at 600 mg/liter,and tetracycline at 10 mg/liter.

Swarm assays. Swarm plates containing Bromfield medium and 0.3% Bacto agarand swarm plates containing RB minimal medium complemented witha carbon source at a concentration of 10^–4 M and 0.27%Bacto agar were inoculated with 3-µl droplets of the testculture and incubated at 30°C for 3 to 4 days.

Computerized motion analysis of free-swimming cells. The speed of free-swimming cells at an OD₆₀₀ of 0.1 was measuredby using the computerized motion analysis of the Hobson BacTrackersystem (Hobson Tracking Systems, Sheffield, United Kingdom)as previously described (64). Cells were observed with a Zeissstandard 14 phase-contrast microscope (magnification, x400)at a constant room temperature of 22°C. The effects of chemoattractantson the motile behavior of wild-type and mutant cell populationswere determined within 20 s of their addition to the cell samples.

Capillary assays. Capillary assays were performed essentially as described byAdler (2) with minor modifications according to Götz etal. (30). Cells grown to an OD₆₀₀ of 0.1 were centrifuged at2,000 x g for 5 min at room temperature and resuspended in RBminimal medium without a carbon source to an OD₆₀₀ of 0.1. ClosedU-shaped tubes (bent from 65-mm micropipettes; Drummond ScientificCo., Broomall, PA) were placed between two glass plates andfilled with 0.4 ml of the bacterial suspension. Capillary tubes(1-µl disposable micropipettes; DESAGA GmbH, Wiesloch,Germany) were sealed at one end and filled with an attractantdissolved in RB minimal medium. The capillaries were inserted,open end first, into the bacterial pond and incubated for 2h in a thermostat chamber at 30°C. Capillaries were removed,the sealed end was cut off, and the complete contents were transferredto 1 ml RB minimal medium. Dilutions were plated in duplicateon TYC plates containing streptomycin. After incubation for3 days at 30°C, colonies were counted.

Genetic manipulations and reporter gene assay. Deletion mutants of S. meliloti (listed in Table 1) were generatedin vitro by overlap extension PCR as described by Higuchi (34).Constructs containing the mutation were cloned into the mobilizablesuicide vector pK18mobsacB, used to transform E. coli S17-1,and conjugally transferred to S. meliloti by filter matingsaccording to the method of Simon et al. (62). Allelic replacementwas achieved by sequential selections on neomycin and 10% sucroseas described previously (64). Confirmation of allelic replacementand elimination of the vector was obtained by PCR with gene-specificprimers, DNA sequencing, and Southern blotting. The broad-host-rangeplasmid pPHU234 and its derivatives pPHU235 and pPHU236 servedas vectors for translational fusions of the seven mcp promotersand the promoters of the two che operons. The resulting lacZfusion plasmids were used to transform E. coli S17-1 and thenwere transferred conjugally to RU11/001 by streptomycin-tetracyclinedouble selection, as described by Labes et al. in 1990 (41).

DNA methods. S. meliloti DNA was isolated and purified as described previously(64). Plasmid DNA was purified with NucleoSpin (Macherey Nagel,Düren, Germany), and DNA fragments or PCR products werepurified from agarose gels using a QiaEx DNA purification kit(QIAGEN, Hilden, Germany) and a GFX PCR and gel band purificationkit (Amersham Biosciences). PCR amplification of chromosomalDNA and Southern blotting were carried out according to publishedprotocols (66).

ß-Galactosidase assays. Cultures of S. meliloti containing lacZ fusions were sampled,diluted 1:1 in Z buffer (46), permeabilized with 1 drop of toluene,and assayed for ß-galactosidase activity by the methodof Miller (46).

RESULTS

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Results

The S. meliloti genome contains eight mcp genes and one atypical chemoreceptor gene. The chemotaxis operon (che operon) of S. meliloti is composedof the orf1, orf2, cheY1, cheA, cheW, cheR, cheB, cheY2, cheD,and orf10 genes (31, 66). The gene product of orf1 has beenclassified as a transducer-like protein based on the presenceof a signaling domain homologous to E. coli MCPs (24, 59). However,the absence of Glu or Gln residues that would serve as methyl-acceptingsites, as well as the lack of hydrophobic transmembrane andperiplasmic receptor regions, prompted us to name this atypicalchemoreceptor IcpA (for internal chemotaxis protein A) (59).IcpA is also listed as McpE of S. meliloti strain 1021 in GenBank.PCR analysis using degenerate primers flanking the conservedsignaling domain and Southern blot hybridization combined witha plasmid rescue approach were performed to identify seven chemoreceptorgenes in the chromosome. They were named mcpT, mcpU, mcpV, mcpW,mcpX, mcpY, and mcpZ, since their gene products exhibit typicalfeatures of E. coli MCPs (39, 48). The majority of these genes,except for mcpW, which is cotranscribed with a putative cheWgene, have a monocistronic organization and are scattered throughoutthe genome. The genome sequencing project of S. meliloti strain1021 revealed the presence of an additional, eighth mcp gene,mcpS. It is localized on the symA plasmid and is organized asthe third gene in a putative chemotaxis operon (the che2 operon)containing cheR, cheW, mcpS, cheA, and cheB (14, 26).

The derived receptor polypeptide sequences yield proteins withmolecular masses between 56 and 74 kDa. They can be classifiedas transmembrane and soluble receptors according to their domainorganization. We utilized the MiST (microbial signal transduction)(73), Pfam (protein families database of alignments) (15), PSORTb(27), and SMART (simple modular architecture research tool)(42) databases for a complete domain architecture of all ninereceptors as depicted in Fig. 1. Seven of the receptor proteinshave two hydrophobic membrane-spanning (transmembrane) regionsand are consequently localized in the cytoplasmic membrane witha large periplasmic sensing domain and a large cytoplasmic signalingdomain. The sizes of the extracytoplasmic ligand-binding domainsof McpT, McpV, and McpW are similar to that of the E. coli receptorsTar and Tsr, comprising 160 amino acid residues. In contrast,McpU, McpX, and McpZ are distinguished by extended periplasmicdomains with 250 to 390 amino acid residues. The cytoplasmicdomain of all MCPs contains the highly conserved region commonto all chemosensory transducers across bacterial and archaealspecies, consisting of a methylation helix (MH1), the signalingdomain, and a second methylation helix (MH2) (69). Another conserveddomain in MCPs is the HAMP (histidine kinases, adenylyl cyclases,MCPs, and phosphatases) domain, which is thought to participatein signal transmission from the periplasmic sensing domain tothe cytoplasmic signaling domain of the transducer (18). InS. meliloti, a HAMP domain is present adjacent to the secondtransmembrane region of all transmembrane receptors. Five receptorshave an additional HAMP domain in front of MH1 (Fig. 1).

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FIG. 1. Domain organization of chemoreceptor proteins from S. meliloti according to the MiST (73), Pfam (15), and SMART (42) databases. GI (gene identification) numbers for each protein are listed after the name. Conserved domains are symbolized by identical shading. TM1 and TM2, transmembrane regions 1 and 2; HAMP, conserved signal transduction domain in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases (6); H1 and H2, helices 1 and 2 lacking the conserved methylation sites in IcpA; signaling, MCP signaling domain and interaction site for CheW and CheA; B/R, interaction site for CheB and CheR; TarH, four-helix ligand-binding domain (74); Cache, acronym formed from the names of proteins in which these signaling domains were recognized (animal Ca²⁺ channel subunits and prokaryotic chemotaxis receptors) (5); PAS, acronym formed from the names of the proteins in which imperfect repeat sequences were first recognized (the Drosophila period clock protein [PER], the vertebrate aryl hydrocarbon receptor nuclear translocator [ARNT], and the Drosophila single-minded protein [SIM] (49, 54).

In addition to the classical transmembrane receptors, S. melilotipossesses two receptors, McpY and IcpA, which lack obvious hydrophobic,membrane-spanning regions as predicted by the databases usedin this study. This prediction gives rise to the suspicion thatthey are localized in the cytosol. McpY is distinguished bythe presence of a tandem repeat of PAS domains in its N-terminalpart (Fig. 1). PAS domains are known to function as sensorsfor oxygen, energy, and light (71). The signaling domain andthe methylation helices, MH1 and MH2, including the positionof the methylated sites, are highly conserved in McpY, whereasIcpA has only modest similarity to MH1 and MH2. Conspicuously,the methylated sites characteristic of classical MCPs are absentin both helices (H1 and H2) of IcpA. Nevertheless, the highlyconserved signaling domain allows its classification as a transducerprotein in chemotaxis. The Pfam data bank analysis also detectedthe presence of a Cache signaling domain, which is known tobind small molecules such as amino acids (5), in the periplasmicregions of McpU, McpV, and McpX. An additional conserved domainin the periplasmic region of McpU is TarH, homologous to thefour-helix bundle, ligand-binding domains in E. coli transmembraneMCPs (74).

In E. coli, the high-abundance receptors, Tar and Tsr, differfrom low-abundance receptors by the presence of a conservedcarboxyl-terminal pentapeptide sequence, NWETF, that enhancesadaptational covalent modification (11, 13). Four of the S.meliloti MCPs, namely, McpT (DWEEF), McpW (NWEEF), McpX (NWEEF),and McpY (DWENF), contain a modified NWETF motif with the consensussequence (N/D)WEEF. Residues critical for the binding of CheR,namely, Trp in the second position and Phe in the fifth position,are conserved (61).

Contribution of individual receptors to motility on Bromfield swarm plates. Single chemoreceptor genes were deleted in frame by allelicexchange (57). An initial insight into the function of theirgene products was provided by a comparison of swarm diametersobtained on Bromfield soft-agar plates with the wild-type standard(RU11/001). Unlike E. coli, which forms three distinct chemotacticbands on semisolid nutrient agar (1), S. meliloti does not formindividual swarm rings. The formation of uniform swarm ringshas also been reported for other bacterial species such as Caulobactercrescentus (23) and Vibrio parahaemolyticus (56). When motileproficiency was assessed, the ${Delta}$ mcpS mutant showed no decreasein swarm size and the ${Delta}$ mcpT mutant exhibited a decrease of only8% in swarm diameter (Fig. 2). Three knockout mutants, the ${Delta}$ mcpV, ${Delta}$ mcpY, and ${Delta}$ mcpZ mutants, generated swarms 55 to 70% of the wild-typeswarm size. The greatest impairment was observed for strainslacking mcpU, mcpW, mcpX, or icpA, with a reduction in swarmsize greater than 50%. In addition to the single gene disruptions,we deleted all nine receptor genes successively to create strainRU13/149, also named ${Delta}$ 9 (Table 1). When its behavior on Bromfieldswarm plates was compared to that of the wild-type control (RU11/001),swarming was reduced by about 70%. A cheA deletion strain (RU11/310),which is chemotaxis deficient due to the lack of kinase activity,is similarly impaired (Fig. 3). Therefore, the phenotype ofthe ${Delta}$ 9 strain can be defined as nontactic.

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FIG. 2. Histogram of swarm sizes on Bromfield agar of 11 chemotaxis mutants relative to that of wild-type cells. Strain designations: ${Delta}$ S to ${Delta}$ Z, in-frame deletion of mcpS (RU13/148), mcpT (RU11/838), mcpU (RU11/828), mcpV (RU11/830), mcpW (RU11/803), mcpX (RU11/805), mcpY (RU11/804), and mcpZ (RU11/818), respectively; ${Delta}$ icpA, in-frame deletion of icpA (RU11/815); ${Delta}$ 9, in-frame deletion of mcpS, mcpT, mcpU, mcpV, mcpW, mcpX, mcpY, mcpZ, and icpA (RU13/149); ${Delta}$ cheA, in-frame deletion of cheA (RU11/310). Percentages of the wild-type swarm diameter (after subtraction of the 7-mm diameter of a nonmotile fla mutant) on 0.3% Bromfield agar are the means of 15 replicates.

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FIG. 3. Swarm test of wild-type S. meliloti RU11/001 (wt), the ${Delta}$ 9 receptor mutant RU13/149, and the cheA mutant RU11/310 ( ${Delta}$ cheA). Strains to be tested were transferred by micropipette (3 µl) onto Bromfield swarm plates and incubated at 30°C for 2 days. The diameter of a swarm ring reflects the motile and tactic proficiency of a given strain.

Sugars, amino acids, and organic acids provoke chemokinesis responses. A multitude of compounds are released into the soil by plants,including numerous sugars, amino acids, and organic acids, whichcan be utilized as carbon sources by soil microorganisms (75).Previous studies reported positive chemotaxis of S. melilotitoward a number of organic compounds (20, 29, 45, 55). We testedthe growth and swarming proficiencies of an S. meliloti wild-typestrain (RU11/001) by using semisolid plates containing minimalmedium with single carbon sources. Typically, swarming was optimalat concentrations of 10^–4 M, whereas higher concentrationssuppressed swarming. Organic acids of the tricarboxylic acidcycle (citrate, malate, succinate, and fumarate) were good carbonsources and attractants. Of the 20 L-amino acids tested, theformation of distinct swarm rings was observed only in the presenceof glutamate, glutamine, histidine, lysine, or proline. We alsoscreened six D-sugars and one sugar alcohol, all of which servedas good carbon sources and attractants.

As a response to attractant stimuli, S. meliloti increases itsswimming speed, a phenomenon called chemokinesis (8, 64). Toassess the potency of organic acids, amino acids, and sugarsas attractants to induce chemokinesis, we used computerizedmotion analysis to monitor and average the free-swimming speedsof cell populations. Table 2 lists the free-swimming speedsof the S. meliloti wild-type strain (RU11/001) observed in theabsence and the presence of chemoattractants. All organic acidsexcept succinate were good attractants, eliciting an increaseof about 4% in swimming speed. Responses to amino acids variedwidely. Glutamate and glutamine were weak attractants, whereashistidine, lysine, and proline were potent attractants. Allsugars were proven to provoke a very strong response (4 to 6.5%).This is in line with the findings of Malek (45), who reportedthat compound sugars were better chemoattractants than aminoacids. However, other groups showed that sugars were weakerattractants than amino acids (20, 29). In essence, it is anextensive and problematic task to compare these studies, becausedifferent strains, cell culture growth conditions, and chemotaxisassays have been used.

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TABLE 2. Free-swimming speeds of the S. meliloti wild-type strain (RU11/001) without and with stimulation by chemoattractants

Contributions of individual receptors to motility on swarm plates with single carbon sources. The swarming proficiencies of the wild-type strain, 10 receptordeletion strains, and a cheA deletion strain on organic acids(Fig. 4A), amino acids (Fig. 4B), and sugars (Fig. 4C) as solecarbon sources and attractants were analyzed. Swarm rings thatformed on plates containing organic acids or amino acids hadless-distinct outlines than those that formed on plates containingsugars. The blurred edges of swarms hampered analysis of swarmring diameters. The ${Delta}$ 9 mutant strain (RU13/149), which lacksall nine receptor genes, and the cheA deletion strain were stillable to generate swarms with 30 to 50% of the wild-type swarmdiameter, a behavior similar to that observed on Bromfield swarmplates (Fig. 2).

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FIG. 4. Histogram of swarm sizes on single carbon sources of 11 chemotaxis mutants relative to that of wild-type cells. For strain designations, see the legend to Fig. 2. Shown are results for swarm behavior on organic acids (A), L-amino acids (B), and D-sugars (C). All carbon sources were used at a concentration of 10^–4 M. Percentages of the wild-type swarm diameter (after subtraction of the 7-mm diameter of a nonmotile fla mutant) on 0.27% agar are the means of six replicates.

On swarm plates containing one of four different organic acids,most deletion strains generated swarms at 80 to 100% of thesize of the wild-type swarm standard. Only strains with deletionsin mcpU, mcpW, mcpX, and icpA formed swarms at 70 to 80% ofthe wild-type control size (Fig. 4A). Swarms generated on platescontaining amino acids were more variable in size than thosegenerated on organic acids or on sugars. Also noted as a result,swarm sizes were strongly reduced. In particular, swarms producedby strains lacking mcpU, mcpW, mcpY, and icpA were 30 to 75%of the wild-type swarm size (Fig. 4B). Most interestingly, theswarm size of the mcpU deletion strain generated on histidine,lysine, or proline was comparable to the ${Delta}$ 9 swarm size. We inferthat McpU is a major sensor for these three amino acids. Onswarm plates containing different sugars, swarms produced bymost strains were 85 to 100% of the size of the wild-type control.Only strains with deletions in mcpU, mcpW, mcpX, and icpA generatedswarms that were 70 to 80% of the wild-type swarm size. In conclusion,none of the single receptor deletion strains, except ${Delta}$ mcpU, showeda total loss of swarming proficiency on any of the substancesassayed. However, most of the strains exhibited impaired responsestoward all substances. This result suggests that (i) receptorproteins can sense more than one carbon source and (ii) onecarbon source is detected by more than one receptor.

Contributions of individual receptors to swimming speed and chemokinesis. The swarm ring provides an indirect measure of motility andchemotaxis. Hence, as an additional assay of motility, the free-swimmingspeeds of cell populations were analyzed. Table 3 lists theswimming speeds of mutant and wild-type cells in both the absenceand the presence of the strong chemoattractant proline. TheS. meliloti wild-type strain (RU11/001) typically had a free-swimmingspeed of 37.4 µm/s, whereas the cheA deletion mutant (RU11/310)and a mutant lacking all nine receptor genes (RU13/149) had8% and 10% increases in swimming speed, respectively. This resultis in agreement with the behavior of E. coli cells lacking bothof the high-abundance chemoreceptors (Tsr and Tar). These cellsswim smoothly, because flagellar motors rotate exclusively counterclockwisedue to the low basal activity of CheA (19, 25, 40). When eitherTsr or Tar is expressed as a sole chemoreceptor, a normal rotationalbias and concomitant run-tumble behavior are maintained (25,40). In analogy, we expected only minute changes in swimmingspeed when individual receptors were missing. The absence ofa receptor, exerting a stimulatory effect on kinase activity,is likely to cause an increase in swimming velocity. Such behaviorwas observed for six receptor deletion strains, with the ${Delta}$ mcpTstrain swimming 7% faster than the wild-type strain. Surprisingly,we noticed a decrease in swimming speed for three of the deletionstrains, ${Delta}$ mcpU, ${Delta}$ mcpY, and ${Delta}$ mcpZ. As one possible explanation forthis behavior, we suggest that these three receptors inhibitthe kinase activity of CheA in the absence of an attractant.The loss of mcpU had the most detrimental effect, reducing free-swimmingspeed by 9% from that of the wild-type control. Next, the chemokinesisresponses of mutant strains were compared to the behavior ofthe wild-type control. Wild-type S. meliloti reacted to theaddition of the attractant proline by a 7.5% increase in swimmingspeed. The cheA deletion strain and the ${Delta}$ 9 strain suffered severelosses of chemokinesis response. They swam only 0.5% fasterupon addition of proline, which is 10% of the wild-type increase.Six of the nine single-deletion strains were diminished in theirchemokinesis responses to various degrees. We observed the mostpronounced decrease in chemokinesis for strains lacking mcpYor icpA, with a reduction of 66% or 55%, respectively. Interestingly,a mutant strain missing both internal receptor genes (RU13/107[Table 1]) lost 74% of its chemokinesis proficiency. It shouldbe noted that strains lacking mcpZ, mcpU, or mcpX exhibiteda chemokinesis response that was increased 4% to 19% over thatof the wild-type control.

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TABLE 3. Free-swimming speeds of the S. meliloti wild-type strain and 12 chemotaxis mutants without and with stimulation by proline

How do free-swimming speed and chemokinesis correlate with thebehavior of mutant strains on swarm plates? A balanced chemokinesisis most important for efficient swarming. Generally speaking,mutant strains with extremely weak or extremely strong chemokinesis,e.g., ${Delta}$ icpA and ${Delta}$ mcpX strains, generate smaller swarm rings thanmutants with chemokinesis responses close to that of the wild-typecontrol, e.g., ${Delta}$ mcpS and ${Delta}$ mcpZ strains. A decrease in free-swimmingspeed ( ${Delta}$ mcpU) is more devastating for swarming than an increasein speed ( ${Delta}$ mcpT).

Contributions of individual receptors to the reaction to proline in capillary assays. A standard for quantitative assessment of chemotaxis is thecapillary tube assay established by Adler in 1973 (2). In thisassay, the number of bacteria attracted into a capillary tubecontaining an attractant is measured. We determined concentration-responsecurves for the strong chemoattractant proline for the wild-typestrain and 11 mutant strains. Wild-type cells responded optimallyto proline at a concentration of 100 mM. Compared to that ofwild-type cells, the taxis of the cheA deletion strain and the ${Delta}$ 9 strain was almost completely abolished (Fig. 5A to C). Theattraction of strains lacking mcpS, mcpW, or mcpZ to prolinewas about 10 to 20% weaker than that of the wild-type control.Interestingly, maximum response was shifted to lower concentrationsof proline (10 mM) for the ${Delta}$ mcpS and ${Delta}$ mcpW strains. A distinctreduction in the tactic response to proline was observed forstrains with deletions in mcpT, mcpV, and icpA, with a 40 to50% decreased sensitivity compared to the wild-type standard.Again, maximum response for the ${Delta}$ icpA strain was shifted to 10mM proline. The group of deletion strains with the weakest responsecomprises the ${Delta}$ mcpU, ${Delta}$ mcpX, and ${Delta}$ mcpY strains. In this group, taxistoward proline was reduced by 65 to 75%. In conclusion, thecapillary assays confirmed the significant role of McpU forproline taxis, as already observed on swarm plates. They alsorevealed two additional important receptors for proline sensing:McpX and McpY (Fig. 4B). The importance of McpY for prolinesensing is verified by the weak chemokinesis effect (Table 3).The weak response of the mcpX deletion strain, however, wasunexpected according to the swarm plate assays (Fig. 4B), althoughchemotaxis on swarm plates also involves cell growth and division,which in this case may have disguised the effect.

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FIG. 5. Concentration-response curves for wild-type and chemotaxis mutant strains. Results for capillary assays performed with the wild-type ( ${blacksquare}$ ), ${Delta}$ 9 (•), and ${Delta}$ cheA ( ${blacktriangleup}$ ) strains, connected by dashed lines, are given as references in all graphs. Shown are curves for the ${Delta}$ mcpS ( ${blacktriangledown}$ ), ${Delta}$ mcpT ( ${blacklozenge}$ ), and ${Delta}$ mcpU ( ${circ}$ ) strains (A), for the ${Delta}$ mcpV ( ${diamond}$ ), ${Delta}$ mcpW (•), and ${Delta}$ mcpX ( ${blacktriangleleft}$ ) strains (B), and for the ${Delta}$ mcpZ ( ${blacktriangleup}$ ), ${Delta}$ icpA ( ${square}$ ) strains (C). Each curve represents the mean of two experiments, each in triplicate, after background subtraction (0.4 x 10⁵ to 2.3 x 10⁵ bacteria in capillaries with buffer).

How do the results from the capillary assays correlate withchemokinesis? Mutant strains that swam as fast as the wild-typestrain were only weakly impaired in the capillary assay ( ${Delta}$ mcpS, ${Delta}$ mcpW, ${Delta}$ mcpZ). However, mutants with strongly reduced chemokineticcapability ( ${Delta}$ mcpY, ${Delta}$ icpA), mutants with extremely reduced swimmingspeed ( ${Delta}$ mcpU), and mutants that swam faster than the wild-typecontrol after stimulation with proline ( ${Delta}$ mcpT, ${Delta}$ mcpX) were stronglyimpaired in the capillary assay. Apparently, fast swimmers havedifficulties entering the opening of the capillary. From theseresults, it is evident that the three different chemotaxis assaysused in this study complement one another.

Energy taxis is not a dominant determinant of chemotaxis in S. meliloti. Receptors mediating the chemotactic behavior of S. melilotican sense a wide range of chemical substances and even classesof substances (Fig. 4). We therefore asked the question whetherenergy taxis, a mechanism described for a related alpha-proteobacterium,is involved in the tactic response of S. meliloti. In Azospirillumbrasilense, energy taxis is the dominant determinant of chemotaxistoward most chemoeffectors. Energy taxis is metabolism dependent,and behavioral responses are triggered by changes in the electrontransport system (3). We thus compared the stimulation of chemokinesisby five different substrates with the stimulation caused bytheir nonmetabolizable analogs (Fig. 6). Clearly, in the casesof succinate, alanine, and proline, the chemokinesis responsesof wild-type S. meliloti cells to the correspondent analogs,itaconic acid, ${alpha}$ -amino-isobutyrate, and azetidine-2-carboxylate,are equal to or even stronger than those to the substrates themselves.This result confirms the findings of the 1993 study by Robinsonand Bauer (55), who used itaconic acid as an attractant in capillaryassays. For the two sugars tested, glucose and lactose, theresponses to the analogs, 2-methylglucoside and isopropylthiogalactoside,were weaker but still significant. We therefore conclude thatthe responses to organic acids and amino acids are not mediatedby the mechanism of energy taxis. However, further investigationsare needed to rule out this possibility for sugar sensing.

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FIG. 6. Effects of substrate analogs on free-swimming speed of wild-type S. meliloti. Substrates were diluted from concentrated stock solutions in RB minimal medium, which was buffered with 10 mM phosphate (pH 7.0). The horizontal line marks the swimming speed of unstimulated cells. For each substrate, 1,000 individual tracks from at least five independent cell populations were analyzed by the Hobson BacTracker system.

Chemoreceptor genes are transcribed at different levels. Transcription from chemoreceptor gene promoters was probed byusing plasmid-borne lacZ fusions in the wild-type background.Table 4 lists the positions and lengths of the constructs relativeto the start codon of each gene. ß-Galactosidase activitieswere determined from cells grown to exponential phase in RBminimal medium layered on Bromfield agar plates. Seven of thenine reporter constructs induced significant enzyme activity,while the PmcpV construct was inactive and PmcpS (Pche2) hadonly minimal activity. Nevertheless, transcription of thesegenes was anticipated, because deletions in their coding regionshad distinct effects in our chemotaxis assays (Fig. 2, 4, and5; Table 3). At this point, we can only speculate that the upstreamregion, fused to the reporter gene, was too short or that thereal start codon is further downstream of the one originallypredicted. Thus, the reasons for the inactivity of these constructsremain unclear. The activities of PmcpW, PmcpZ, and PicpA wereabout five times higher than those of the low-expression genesmcpT and mcpY. However, the promoters of mcpX and mcpU wereclearly the strongest. Together they comprise more than 50%of the combined promoter activities, underscoring their importancefor the chemotactic response of S. meliloti.

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TABLE 4. In vivo mcp promoter activity

DISCUSSION

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Discussion

The increasing number of genome sequences reveals the diversityand complexity of chemotactic signaling pathways in prokaryotes.Most motile bacterial species with available genome sequenceshave multiple homologs of che genes and more receptor genesthan the five present in the well-studied enterobacterial species.For example, in the alpha subgroup of proteobacteria, Agrobacteriumtumefaciens has 20, C. crescentus has 18, and Rhodobacter sphaeroideshas 13 MCPs or transducer-like proteins. Some of these receptorproteins have no periplasmic domains, owing to the lack of obvioustransmembrane regions (28, 44, 50). The motility system of thealphaproteobacterium S. meliloti deviates from the enterobacterialparadigm in its mode of flagellar rotation and in the abilityto increase its swimming speed as a reaction toward attractants(8, 58, 64).

In this study, we analyzed the roles of seven transmembraneand two cytosolic receptor proteins in chemotaxis and chemokinesis.Each of the cytosolic receptor proteins has distinctive molecularfeatures. McpY possesses two PAS domains, which are typicallyinvolved in sensing redox potential, oxygen, or light (71),while IcpA lacks the otherwise highly conserved methylated residues(Fig. 1). A possible role for these internal receptors willbe discussed below.

Four of the MCP polypeptides have conserved C-terminal pentapeptidesrelated to the one found for E. coli high-abundance receptors.For E. coli MCPs, this motif serves as a docking site for enzymesinvolved in adaptation. It remains to be experimentally investigatedif the same holds true for the (N/D)WEEF pentapeptide in McpS,McpW, McpX, and McpY. Unlike the situation for E. coli, however,there is no correlation between high expression levels and thepresence of the motif in S. meliloti MCPs.

We were unable to detect promoter activity for the 5' upstreamregion of mcpV. However, two lines of evidence are in favorof expression of this gene: (i) deletion in the coding regionresulted in reduced chemotactic proficiency on swarm plates(Fig. 4) and in capillary assays (Fig. 5); (ii) cells carryinga chromosomal 3' gfp fusion to mcpV fluoresce at the cell poles(data not shown).

S. meliloti strains lacking all nine receptors or the histidinekinase CheA were still able to form swarm rings with 30% ofthe size of a swarm ring generated by the wild-type control(Fig. 2 and 4). In contrast, E. coli cells that fail to tumble,e.g., due to the lack of receptor proteins, are nearly incapableof moving from the inoculation point. Mutant cells that swimsmoothly inevitably collide with the agar walls and get trappedin the agar (76). The intrinsic speed modulation in the flagellarmotor of S. meliloti presumably enables cells to back away fromobstructions in the agar. This behavior can explain the largerswarm sizes generated by mutant cells. According to the presentdata, the possibility that the residual chemokinesis activityof mutant strains accounts for this behavior as well cannotbe excluded (Table 3).

We can only speculate what factor(s) could trigger the minorincrease in swimming speed of the ${Delta}$ 9 or ${Delta}$ cheA strain when stimulatedby an attractant. The chromosome of S. meliloti contains a totalof two cheA (one in each chemotaxis operon) and four cheW genes.However, participation of CheA2 in the chemotactic responseis unlikely, because a cheA1 cheA2 double-deletion strain showedthe same residual chemokinesis activity (data not shown). Twoadditional pathways seem plausible: (i) there are other two-componentregulatory systems that might target the flagellar motor eitherdirectly or by cross talk with CheA and/or CheY2; (ii) fumarateor its metabolites might directly modulate rotary speed. Fumaratehas been reported as a regulator of motor switching for E. coli(10, 47).

In the absence of an attractant, E. coli MCPs stimulate thekinase activity of CheA and consequently increase the phosphorylatedCheY concentration, resulting in a higher tumbling bias. Thesame effect is seen for S. meliloti; disruptions in six of thereceptor genes result in increases in swimming speed to variousdegrees. This reaction was most pronounced for a strain lackingmcpT. Conspicuously, three receptor deletion strains exhibiteddecreases in free-swimming speed. This behavior was most distinctlyobserved for a strain lacking mcpU (Table 3). It is possiblethat McpU inhibits the kinase activity of CheA in the absenceof an attractant, thereby causing an increase in swimming speed.The effect of McpU on kinase activity will be explored usingphosphorylation assays in vitro. Besides McpU, McpX and McpYwere also major players in proline sensing (Fig. 5). Interestinglyenough, the periplasmic region of McpU contains a TarH domain,which is homologous to the four-helix, ligand-binding domainin E. coli transmembrane MCPs. The complex domain structuresupports our experimental findings of McpU being a major chemoreceptor.The function of the TarH domain needs to be further elucidated,as well as that of the Cache_1 domain in McpU and McpX.

Receptors mediating the chemotactic behavior of S. melilotican sense a wide range of chemical substances and even classesof substances (Fig. 4). It also becomes apparent from our studythat the functions of receptors in S. meliloti are redundant.That is to say that more than one receptor contributes to detectingchemical stimuli and to producing an output signal that controlsthe speed of the flagellar motor. In conclusion, heterologousreceptors all work as a team to generate a collaborative signalingbehavior, ensuring the functionality and robustness of the chemotaxissystem in S. meliloti.

It is evident that the two cytoplasmic receptors are importantfor the chemokinesis response of S. meliloti (Table 3). We speculatethat IcpA and McpY are sensors for the metabolic state of thecell. Currently, we are investigating the roles of McpY andIcpA in chemokinesis to various attractants. What is the functionof the PAS domains in McpY? Up to now, no data have revealedredox, aerotactic, or phototactic behavior of S. meliloti (78).In addition, deleting either of the two PAS domain-encodingDNA regions had no effect on the chemotactic behavior of theresulting mutant strains on swarm plates (data not shown). Thus,unlike for E. coli Aer (16) and Halobacterium salinarum HemAT(35), it is improbable that the PAS domains of McpY bind cofactorssuch as flavin adenine dinucleotide or heme. Further experimentsare needed to determine whether McpY and IcpA are indeed localizedin the cytosol or whether they are attached to the membrane.If they are membrane bound, do they colocalize with the transmembranereceptors? These and other questions will be addressed in ourfuture studies.

ACKNOWLEDGMENTS

This work was supported by grant Scha914/2-1/2 from the DeutscheForschungsgemeinschaft.

We thank Alexandre Cruz and Wolfdieter Springer for help withthe setup of reporter assays, Akira Tabuchi for constructingstrains RU11/803, RU11/804, and RU11/805, and Thomas Barth forassistance with swarm plate assays. We are indebted to SabineSchneider for help with EndNote and Earl J. Sheehan, Jr., forcritical review.

FOOTNOTES

* Corresponding author. Mailing address: Lehrstuhl für Genetik, Universität Regensburg, D-93040 Regensburg, Germany. Phone: 49 (941) 9433170. Fax: 49 (941) 9433163. E-mail: birgit.scharf{at}biologie.uni-regensburg.de.

Published ahead of print on 22 December 2006.

Dedicated to Rüdiger Schmitt on the occasion of his 70thbirthday.

Present address: Promega GmbH, 68199 Mannheim, Germany.

REFERENCES

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