JOURNAL OF BACTERIOLOGY, Dec. 2005, p. 8403–8410
0021-9193/05/$08.00⫹0 doi:10.1128/JB.187.24.8403–8410.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 187, No. 24

Only One of the Five CheY Homologs in Vibrio cholerae
Directly Switches Flagellar Rotation
Akihiro Hyakutake,1 Michio Homma,1 Melissa J. Austin,3 Markus A. Boin,3Claudia C. Häse,3 and Ikuro Kawagishi1,2*
Division of Biological Science, Graduate School of Science,1 and Institute for Advanced Research,2Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan, and Department of Microbiology,
Oregon State University, 220 Nash Hall, Corvallis, Oregon 973313
Received 4 August 2004/Accepted 15 September 2005

Vibrio cholerae has three sets of chemotaxis (Che) proteins, including three histidine kinases (CheA) and four
response regulators (CheY) that are encoded by three che gene clusters. We deleted the cheY genes individuallyor in combination and found that only the cheY3 deletion impaired chemotaxis, reinforcing the previous
conclusion that che cluster II is involved in chemotaxis. However, this does not exclude the involvement of
the other clusters in chemotaxis. In other bacteria, phospho-CheY binds directly to the flagellar motor tomodulate its rotation, and CheY overexpression, even without CheA, causes extremely biased swimming
behavior. We reasoned that a V. cholerae CheY homolog, if it directly controls flagellar rotation, should also
induce extreme swimming behavior when overproduced. This was the case for CheY3 (che cluster II). However,no other CheY homolog, including the putative CheY (CheY0) protein encoded outside the che clusters, affected
swimming, demonstrating that these CheY homologs cannot act directly on the flagellar motor. CheY4 veryslightly enhanced the spreading of an Escherichia coli cheZ mutant in semisolid agar, raising the possibility thatit can affect chemotaxis by removing a phosphoryl group from CheY3. We also found that V. cholerae CheY3
and E. coli CheY are only partially exchangeable. Mutagenic analyses suggested that this may come fromcoevolution of the interacting pair of proteins, CheY and the motor protein FliM. Taken together, it is likelythat the principal roles of che clusters I and III as well as cheY0 are to control functions other than chemotaxis.
Vibrio cholerae, the causative agent of cholera, inhabits river,
estuary, and other aquatic environments, where it actively
swims by rotating its polar flagellum as a screw-like propeller.
When introduced into a host with contaminated foods or water, the bacterium reduces its swimming motility and colonizes
the epithelium of the small intestine, where it produces cholera
toxin (CT), which causes severe diarrhea, a diagnostic symptomof cholera (27). It has been reported that some chemotaxisrelated genes are involved in the pathogenicity of V. cholerae(1, 3, 9, 15, 20). A recent study showed that some nonchemotactic mutants outcompete the wild-type strain during infection(6, 7). Thus, studying the mechanisms of motility and chemotaxis is of vital importance to understanding the pathogenicity
and survival strategies of this bacterium.
Signal transduction in chemotaxis, which involves control offlagellar rotation in response to environmental stimuli, hasbeen best studied with Escherichia coli (for reviews, see references 2, 10, and 32). Central to this pathway is a two-component regulatory system that consists of the histidine kinase
CheA and the response regulator CheY. CheA is activatedwhen coupled to an unliganded chemoreceptor (also known as
methyl-accepting chemotaxis protein [MCP]). CheA phosphorylates itself and transfers a phosphate group to CheY.
Binding of phospho-CheY to the switch complex of the flagellar motor, which otherwise rotates counterclockwise, induces

its clockwise (CW) rotation, resulting in reorientation of thecell and hence an abrupt change in its swimming direction.
The genome sequence of V. cholerae El Tor predicts that thebacterium has three sets of Che proteins and 45 MCP-like proteins (hereafter called MLPs) (16). Each set of che genes forms
clusters as follows: che cluster I contains cheY1 (VC1395), cheA1(VC1397), cheY2 (VC1398), cheR1 (VC1399), cheB1 (VC1401),
and the putative gene cheW (VC1402); cluster II contains cheW1(VC2059), cheB2 (VC2062), cheA2 (VC2063), cheZ (VC2064),
and cheY3 (VC2065); and cluster III contains cheB3 (VCA1089),
cheD (VCA1090), cheR3 (VCA1091), cheW2 (VCA1093), cheW3(VCA1094), cheA3 (VCA1095), and cheY4 (VCA1096). cheR2(VC2201) is in a fla gene cluster located just next to che cluster II.
Clusters I and II are located on chromosome I, and cluster III islocated on chromosome II. In addition, chromosome I contains apair of genes encoding a non-CheA histidine kinase (VC1315)
and a putative CheY protein (VC1316) outside of any che or fla
gene cluster.
Several lines of evidence suggest that at least some of thesechemotaxis-related genes are involved in virulence. An in vivo
screen and further characterization revealed that several chemotaxis genes (mcpX [VC2161], cheZ, cheA2, and cheY3) are
required for the production of CT or its positive regulator
ToxT upon infection of mice, although the production of CT in
vitro does not require any of these genes except cheZ (20).
Nonchemotactic mutants with counterclockwise- and clockwise-biased flagellar rotation were reported to show enhanced
and attenuated infectivity, respectively (6). In an in vitro study(3), cells lacking or overproducing CheY4 exhibited a decreased or increased production of CT, respectively, and CheY4* Corresponding author. Mailing address: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku,
Nagoya 464-8602, Japan. Phone: 81 52 789 2993. Fax: 81 52 789 3001.
E-mail: i45406a@cc.nagoya-u.ac.jp.
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was suggested to be involved in attachment to host cell layers
and migration toward ingredients of the intestine. It was also
reported that two MLPs (TcpI and AcfB) are involved incolonization (9, 15) and that another MLP (HlyB) is involvedin the secretion of hemolysin (1, 18).
In this context, it is important to examine whether only one
che cluster is involved in the chemotaxis of V. cholerae, assuggested earlier (12). The deletion of cheA2, but not cheA1 or
cheA3, abolishes chemotaxis, and only cheA2 complements thedeletion of cheA2. A deletion of cheY3 was also reported to
impair chemotaxis (6, 7). These results demonstrate that cluster II containing cheA2 and cheY3 is involved in chemotaxis.
However, the results do not exclude the possible involvementof clusters I and III in chemotaxis, since the che genes of theseclusters (or any other genes required for the functions of thecheA genes) might not be expressed sufficiently under the conditions tested.
In this study, we focused on the V. cholerae CheY homologs
that are predicted from the genome sequence. Overproductionof the E. coli CheY protein (CheYE) induces CW rotation offlagella even in a gutted E. coli strain, which is deleted for cheA
and all other che genes. The D13K mutation of CheYE induces
CW flagellar rotation without involving phosphorylation (5). In
Bacillus subtilis, the situation is a little different: the overproduction of CheY (CheYB) causes incessant smooth swimming,and the corresponding D-to-K mutation fails to activate
CheYB (4). We therefore reasoned that the overproduction of
any of the CheY homologs that are involved in the chemotaxisof V. cholerae should induce biased flagellar rotation and extreme swimming behavior in V. cholerae. We generated several
specific deletion mutants, cloned all of the predicted cheY
genes from V. cholerae, and substituted Lys for the aspartate
residues corresponding to Asp-13 of CheYE. We then examined the effects of these wild-type or DK mutant CheY proteins on the swimming behavior of V. cholerae as well as E. coli.
Among all five V. cholerae CheY proteins, only CheY3 affectedchemotaxis, suggesting that this is the only CheY protein directly controlling flagellar rotation. Among the other CheY
homologs, CheY4 very slightly improved spreading in semisolid agar of an E. coli cheZ mutant strain, raising the possibility that it can indirectly affect chemotaxis by receiving aphosphoryl group from CheY3.
MATERIALS AND METHODS
Bacterial strains. V. cholerae classical strain O395N1 has wild-type chemotaxis.
Strain VcheA123 (12), derived from O395N1, lacks all three cheA genes. The
O395N1 derivative strains VcheY1, VcheY2, VcheY3, and VcheY4, each ofwhich has one cheY gene deleted, were constructed as described below. All of theE. coli strains used are derivatives of K-12. Strain RP437 has wild-type chemotaxis (25). Strain HCB1262 [⌬(cheA-Z):: zeoR thr leu his met rpsL136], lacking allche genes, tar, and tap, was used as a nontumbling strain (H. C. Berg, personalcommunication). Strain RP1616, lacking cheZ, was used as a constantly tumblingstrain (J. S. Parkinson, personal communication).
Plasmids. The vector pFLAG-CTC (Sigma), derived from plasmid pBR322,
carries the lacI and bla genes and encodes a FLAG M2 tag sequence downstreamof the tac promoter. Plasmid pBAD33 (14), containing the p15A replicon and thearaBAD promoter, is compatible with pFLAG-CTC in the same cell. The suicide
vector pWM91 (22), containing the R6K origin but lacking the pir gene required
for replication and the sacB genes required for sucrose counterselection, wasused to disrupt the cheY genes by homologous recombination.
Cloning of cheY genes. Each cheY gene was amplified by PCR, with chromosomal DNA of V. cholerae strain O395N1 or E. coli strain W3110 as the template,
to introduce XhoI and KpnI sites at the 5⬘ and 3⬘ ends of the coding region,

J. BACTERIOL.
respectively. The resulting DNA fragments were digested with XhoI and KpnIand cloned into the vector plasmid pFLAG-CTC so that the FLAG tag was fusedto the carboxyl terminus of each CheY protein.
The D-to-K mutations of the CheY homologs corresponding to the constitutively active D13K mutation of CheYE were introduced by site-directed mutagenesis using mutant forward primers. The charge-reversing mutations of
CheYE and CheY3 were introduced similarly, using mutant reverse primers. The
D58K mutant of CheY4 was constructed by a two-step PCR method as described
previously (34). The resulting fragments of the mutant cheY genes were cloned
into pFLAG-CTC.
The fragment encoding CheY1-FLAG was amplified by PCR to introduce a
SacI site and a Shine-Dalgarno (SD) sequence at the 5⬘ end and a PstI site at the3⬘ end of the fragment. The resulting DNA fragment was digested with SacI and
PstI and cloned into pBAD33.
Deletion of cheY genes. In-frame deletions in the cheY genes were constructedby overlap extension PCR (17, 35), using four primers for each gene. A region of
DNA from the V. cholerae chromosome spanning from approximately 1 kb
upstream to the 5⬘ end of the desired gene was PCR amplified using primers
1 and 2. A second region, from approximately 1 kb downstream to the 3⬘ end of
the gene, was PCR amplified using primers 3 (complementary to a portion ofprimer 2) and 4. A mixture of these two DNA fragments (100 ng each) was usedas the template in a third PCR amplification using primers 1 and 4. The productof the third amplification contained an approximately 2-kb fragment with an
in-frame deletion in the desired gene, with engineered restriction sites at its 5⬘
and 3⬘ ends that allowed subcloning of the DNA fragment into the suicide vector
pWM91. A protocol for introducing cloned mutations into the chromosome of V.
cholerae by homologous recombination using a suicide vector system followed bysucrose selection was used (8).
Nucleotide sequencing. Nucleotide sequences of the cloned genes were determined by the dideoxy chain termination method, using a BigDye Terminator v3.1
cycle sequencing kit (Applied Biosystems).
Analyses of amino acid sequences and three-dimensional structures of proteins. Amino acid sequences were aligned using Clustal W, version 1.4. Sequencehomology and similarity were calculated using the BLAST 2 sequence program(33). Three-dimensional structures of proteins were drawn and analyzed by using
Swiss PDB Viewer, version 3.7b1.
Immunoblotting. Immunoblotting was carried out essentially as described
previously (24). An overnight culture of E. coli in TG (1% tryptone, 0.5% NaCl,
0.5% glycerol) was diluted 1:40 in fresh TG supplemented with IPTG (isopropyl␤-D-thiogalactopyranoside), if necessary. Cells were grown with vigorous shaking, harvested at late exponential phase, and washed with MLM buffer (10 mM
potassium phosphate [pH 7.0], 0.1 mM EDTA, 10 mM sodium lactate, 100 ␮Mmethionine). An overnight culture of V. cholerae in TG was diluted 1:100 in fresh
TG supplemented with IPTG, if necessary. Cells were grown with vigorous
shaking, harvested at late exponential phase, and washed with TM buffer (50 mM
Tris-HCl [pH 7.4], 5 mM glucose, 5 mM MgCl2). The samples were subjected
to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE;
15% polyacrylamide) followed by immunoblotting with anti-FLAG M2 antibody(Sigma).
Swarm assay. Swarm assays were performed using tryptone semisolid agar
(1% tryptone, 0.5% NaCl, and 0.3% agar). When necessary, 50 ␮g ml⫺1 ampicillin and/or 0.1 or 1 mM IPTG was added. An aliquot (1 ␮l) of overnight culture
in TG was spotted onto a plate, which was then incubated at 30°C for 6 to 24 h.
Observation of swimming behavior. Free-swimming cells were observed essentially as described previously (19, 23). Cells were grown as described above.
V. cholerae cells were resuspended in TMN (50 mM Tris-HCl [pH 7.4], 5 mM
glucose, 5 mM MgCl2, 100 mM NaCl), and E. coli cells were resuspended in
MLM buffer. After incubation at room temperature for 1 h, cells were observed
under a dark-field microscope (Nikon 74257). Swimming tracks of the cells weretraced using an Argus-10 image processor (Hamamatsu Photonics). The reorientation frequency of V. cholerae cells was defined as the number of changes ofswimming direction per second and was calculated from the swimming tracks of
20 cells. The smooth-swimming fraction of E. coli was defined as the percentage
of smooth-swimming cells (i.e., cells running without reorientation for 2 s)among the total swimming cells (⬎100 cells), not including cells attached to theglass surface.
Nucleotide sequence accession numbers. The sequence data for the cheY
homologs of O395N1 have been deposited in the DDBJ database under thefollowing accession numbers: cheY0, AB185929; cheY1, AB185930; cheY2,
AB185931; cheY3, AB185932; and cheY4, AB185933.

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FIG. 1. Sequence alignment of CheY homologs of V. cholerae with CheYE. Amino acid sequences of the CheY homologs deduced from the
DNA sequences of the cloned genes from V. cholerae O395N1 were aligned using Clustal W (version 1.4). CheY0 indicates the putative CheY
homolog used throughout this paper. Particularly important residues that have been identified in CheY of E. coli (CheYE) are marked with boxes
with the following colors: black, phosphorylation site; white, sites for function as a response regulator (36); gray, sites interacting with FliM (21,28, 30). Although the cheY1 gene of V. cholerae El Tor biotype strain N16961 is deduced to encode a truncated product (up to Glu-94 of the productdeduced from the database [the corresponding residue of the deduced product from the cloned gene is indicated by an arrow], with nine foreign
residues) because of a frameshift, the cheY1 gene of the V. cholerae classical biotype strain O395N1 was deduced to encode a full-length protein.

RESULTS
Cloning of the five cheY homologs of a classical V. cholerae
strain and alignment of the deduced amino acid sequences of
their products with that of CheYE. The five V. cholerae cheY
genes were cloned and sequenced as described in Materials
and Methods. The deduced amino acid sequences of the different CheY homologs were aligned using Clustal W, version
1.4 (Fig. 1). The published genome sequence of the V. cholerae
El Tor biotype strain predicts that its CheY1 protein is truncated because of a frameshift in the coding region. However,the cheY1 gene cloned from the chromosome of V. cholerae
classical biotype strain O395N1 had an insertion of a guanine
nucleotide between guanine 282 and cytosine 283 of the published cheY1 sequence of El Tor strain N16961, leading toretention of the full-length reading frame (encoding 126 aminoacid residues).
The deduced amino acid sequences of the V. cholerae
CheY0, CheY1, CheY2, CheY3, and CheY4 proteins showed
26%, 38%, 33%, 57%, and 36% identity and 42%, 57%, 52%,
74%, and 53% similarity, respectively, to that of CheYE. The
site of phosphorylation (Asp-57) and the other residues criticalfor phosphorylation (Asp-12, Asp-13, and Lys-109) in CheYE(36) were conserved perfectly in all of the V. cholerae CheY
homologs (Fig. 1). The FliM-interacting residues were identified by characterizations of suppressors (28, 30) and by X-ray
crystallography of a complex of CheYE and the N-terminal
fragment of FliM (21). These FliM-interacting residues werebest conserved in CheY3.

Detection of cheY gene products. The substitution of Lys for
Asp-13 of CheYE results in a constitutively active phenotype,
inducing CW rotation of the flagellar motor independent ofphosphorylation (5). In addition, the corresponding D11K mutant of CheB also exhibits a constitutively active phenotype(31). Similar D-to-K mutants of the V. cholerae CheY proteins
corresponding to D13K of CheYE were constructed to examine whether they induce extreme swimming behavior (Fig. 2A).
The five cheY coding regions of V. cholerae O395N1 werecloned into pFLAG-CTC so that a FLAG peptide was fused tothe carboxyl terminus of each CheY protein (Fig. 2A). The
D-to-K mutations were introduced into the cloned genes, andthe nucleotide sequences of all constructs were determined.
The resulting plasmids encoding the wild-type or mutant
version of each CheY-FLAG protein were introduced into
V. cholerae O395N1 (Che⫹) cells. The CheY-FLAG proteins
were induced in V. cholerae with 1 mM IPTG and were detected by immunoblotting using anti-FLAG M2 antibody(Fig. 2B). All of the wild-type and mutant CheY-FLAG proteins were expressed at similar levels, except the wild-type andmutant CheY0-FLAG proteins, which were expressed atslightly higher levels. In the absence of IPTG, no CheY-FLAG
protein was detected. In E. coli cells, all of the CheY-FLAG
proteins were expressed at similar levels, except for the
CheY0-FLAG proteins, which were expressed at slightly
higher levels (data not shown).
Construction and characterization of cheY deletion strains.
In-frame deletion mutants with deletions of the V. cholerae

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HYAKUTAKE ET AL.

J. BACTERIOL.

FIG. 2. Immunoblotting of CheY-FLAG proteins. (A) Schematic diagram of the CheY-FLAG proteins constructed for this study. The
V. cholerae cheY coding regions were cloned into plasmid pFLAG-CTC so that the FLAG tag was fused to the carboxyl terminus of each CheY
protein (wild type [WT]). The “MKLLE” and “GTRSV” sequences were derived from the vector. A D-to-K mutant of each CheY-FLAG protein,corresponding to the constitutively active D13K mutant of CheYE, and a dominant negative mutant of CheY4-FLAG, corresponding to thenonphosphorylatable D57N mutant of CheYE, were also constructed. (B) Expression of CheY-FLAG proteins in V. cholerae. The constructed
plasmids were introduced into the Che⫹ strain O395N1. Lysates of cells grown in the absence (⫺) or presence (⫹) of 1 mM IPTG were subjected
to SDS-PAGE (15%) followed by immunoblotting with anti-FLAG M2 antibody. (C) Coexpression of CheY1 and CheY2-FLAG proteins in
V. cholerae. The constructed plasmids were introduced into the Che⫹ strain O395N1. Lysates of cells grown in the presence of 1 mM IPTG and
5 mM arabinose were subjected to SDS-PAGE (15%) followed by immunoblotting with anti-FLAG M2 antibody.

O395N1 cheY1, cheY2, cheY3, and cheY4 genes were constructed, and the resulting strains were tested for chemotaxis.
Only the ⌬cheY3 strain was defective in chemotactic spreading
in semisolid agar (Table 1) and in reorientation in liquid medium (Fig. 3A). The phenotype of the ⌬cheY3 strain is consistent with those of previously reported cheY3 mutant strains
derived from the El Tor strain (6, 20). The single and multiple
cheY deletion mutations in cheY1, cheY2, and cheY4 did notaffect swimming behavior (Fig. 3A; Table 1). Among the fivecheY homologs, only the cheY3 gene complemented the defects
in the cheY3 strain in spreading (Table 2) and reorientation(data not shown). To analyze the effect of coexpression of
CheY1 and CheY2, we constructed pBAD33-based plasmids
encoding the wild-type or D-to-K mutant version of CheY1FLAG that are compatible with pCTC-FLAG and its derivatives. The coexpression of CheY1 and CheY2 (Fig. 2C) did notcomplement the defects of the cheY3 mutant, either (data notshown). The cheYE gene only partially complemented defects

TABLE 1. Spreading in semisolid agar of V. cholerae cells
lacking single or multiple cheY genes
StrainRelative diameter a

Wild type .............................................................................. 1.0 ⫾ 0.14
⌬cheY1 .................................................................................. 1.1 ⫾ 0.09
⌬cheY2 ..................................................................................0.96 ⫾ 0.07
⌬cheY3 ..................................................................................0.14 ⫾ 0.02
⌬cheY4 ..................................................................................0.98 ⫾ 0.04
⌬cheY12 ................................................................................1.01 ⫾ 0.02
⌬cheY14 ................................................................................1.01 ⫾ 0.02
⌬cheY24 ................................................................................0.98 ⫾ 0.02
⌬cheY124 ..............................................................................1.01 ⫾ 0.02aOvernight cultures were spotted onto tryptone–semisolid 0.3% agar, which
was then incubated at 30°C for 7 h, and the diameters of the swarm rings were
measured. The relative diameter of a swarm ring of a particular strain was
defined as the ratio of the diameter to that of the wild-type strain. The averagevalues with standard deviations from a triplicate assay are shown.

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TABLE 2. Spreading in semisolid agar of V. cholerae ⌬cheY3
cells expressing each CheY homolog
CheY homolog

Mutation

Relative diameter a

None
CheYE

b

0.18 ⫾ 0.02
0.43 ⫾ 0.05
0.33 ⫾ 0.11
0.18 ⫾ 0.01
0.15 ⫾ 0.02
0.17 ⫾ 0.00
0.17 ⫾ 0.01
0.20 ⫾ 0.01
0.17 ⫾ 0.01
1.0 ⫾ 0.05
1.08 ⫾ 0.06
0.20 ⫾ 0.03
0.17 ⫾ 0.01
1.37 ⫾ 0.03

CheY0
CheY1
CheY2
CheY3
CheY4
CheYE

NA
None
D13K
None
D11K
None
D10K
None
D10K
None
D16K
None
D15K
E117K

a
The relative diameter of a swarm ring was measured as described in Table 1,
except that the average diameter of the triplicate swarm rings of wild-type cells
expressing CheY3 was defined as 1.0, tryptone–semisolid 0.3% agar was supplemented with 0.1 mM IPTG, and the incubation time at 30°C was 12 h.
bNA, not applicable.

FIG. 3. Effects of CheY homologs on free swimming of V. cholerae
cells in liquid medium. (A) Reorientation frequencies of wild-type andcheY knockout strains. Overnight cultures of cells in LB containing
ampicillin (50 ␮g ml⫺1) were diluted in fresh medium and grown withvigorous shaking. The cells were harvested at late logarithmic phase,
washed, resuspended in TMN at room temperature, and observed
under a dark-field microscope. The average reorientation frequency(s⫺1) of each strain was determined by analyzing swimming tracks.
(B) Reorientation of CheY-expressing VcheY3 (⌬cheY3) strain.
VcheY3 cells carrying plasmids encoding wild-type or mutant CheY3
or CheYE were grown in the presence of 0.1 mM IPTG. Cells wereobserved and analyzed as described above. ⫹, frequency is above zero.

of the cheY3 mutant strain VcheY-3 in spreading (Table 2) andreorientation (⬍0.5 s⫺1).
Effects of CheY homologs on swimming behavior of chemotactic V. cholerae cells. To determine whether any CheY homolog other than CheY3 can act directly on the flagellar motor, we examined the effects of overproduction of the CheY
homologs in chemotactic (Che⫹) V. cholerae cells (O395N1)(Table 3). Cells overexpressing wild-type or mutant CheY3
spread much slower than cells carrying the vector. The wildtype and mutant CheYE proteins had milder effects than the
CheY3 proteins. The expression of any other V. cholerae CheY
homolog had no significant effect. Free-swimming cells expressing wild-type or mutant CheY3 reoriented more frequently than cells carrying the vector (Fig. 4A). Cells expressing the D-to-K version of CheY3 reoriented more frequently
than those expressing the wild-type version, indicating that theformer version is more active than the latter in V. cholerae cells(Fig. 4A). The coexpression of CheY1 and CheY2 (Fig. 2C)
did not affect spreading in semisolid agar (data not shown) or
the reorientation frequency (Fig. 4B). A triple cheY mutant
strain lacking cheY1, cheY2, and cheY4 was also used to exclude
any possible interaction between the CheY homologs thatmight suppress the effect of a particular CheY homolog, but
again no CheY homolog other than CheY3 showed any detectable effect on spreading or swimming when overexpressed(data not shown). These results suggest that only CheY3 of thefive V. cholerae CheY homologs can control the flagellar motor

and that the D-to-K substitution in CheY3, like the corresponding mutation in CheYE, causes a constitutively active
phenotype.
Effects of CheY homologs on swimming behavior of V. cholerae cells lacking any CheA homolog. To exclude any possible
involvement of the three CheA homologs encoded by V. cholerae, we examined the effects of the CheY homologs on theswimming behavior of VcheA123 cells, a V. cholerae derivativeof O395N1 lacking all three cheA genes (12). Althoughnonchemotactic (Che⫺) cells cannot form a chemotactic ringin semisolid agar, those with an unregulated but appropriate
run/reorientation bias can spread significantly, albeit very
slowly (37). Cells expressing wild-type or mutant CheY3
spread significantly in tryptone semisolid agar, whereas noother CheY homolog induced spreading (Table 4). Free-swimming cells expressing each CheY protein were also observed by
microscopy, and only cells expressing wild-type or mutant
CheY3 reoriented. Cells expressing the D-to-K version reori-

TABLE 3. Spreading in semisolid agar of wild-type V. cholerae
cells expressing each CheY homolog
CheY homolog

Mutation

Relative diameter a

None
CheYE

NAb
None
D13K
None
D11K
None
D10K
None
D10K
None
D16K
None
D15K

1.0 ⫾ 0.03
0.67 ⫾ 0.03
0.45 ⫾ 0.05
0.84 ⫾ 0.05
0.88 ⫾ 0.05
0.96 ⫾ 0.03
0.88 ⫾ 0.02
1.06 ⫾ 0.03
0.96 ⫾ 0.07
0.55 ⫾ 0.03
0.42 ⫾ 0.02
1.00 ⫾ 0.07
0.92 ⫾ 0.03

CheY0
CheY1
CheY2
CheY3
CheY4

a
The relative diameters of swarm rings were measured as described in Table 1,
except that the average diameter of the triplicate swarm rings of wild-type cells withthe vector was defined as 1.0, tryptone–semisolid 0.3% agar was supplemented with
0.1 mM IPTG, and the incubation time at 30°C was 6 h.
bNA, not applicable.

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FIG. 4. Effects of CheY homologs on free swimming of V. cholerae
cells in liquid medium. (A) Effects on chemotactic V. cholerae(O395N1) cells. (B) Effects of coexpression of CheY1 and CheY2 on
chemotactic V. cholerae (O395N1) cells. (C) Effects of wild-type andmutant versions of CheY3 on nonchemotactic V. cholerae VcheA123(⌬cheA123) cells. Overnight cultures of O395N1 or VcheA123 cells
carrying plasmids were grown in the presence of 1 mM IPTG (A and
C). CheY-coexpressing cells were grown in 1 mM IPTG and 0.5 mMarabinose (B). Cells were observed and analyzed as described in thelegend to Fig. 3.

ented more frequently than those expressing the wild-typeversion (Fig. 4C). These results support the previous conclusion that only CheY3 of the five CheY homologs can induce
CW rotation of the flagellar motor of V. cholerae.
Effects of CheY homologs on swimming behavior of E. coli
cells. Unlike CheY3, both the wild-type and D-to-K mutant
versions of CheYE could hardly promote the reorientation of
V. cholerae cells. To test the exchangeability between E. coli and
V. cholerae, the V. cholerae CheY homologs as well as CheYE
were expressed in Che⫹ E. coli cells (RP437) or cells lacking all
Che proteins (⌬Che; HCB1262), and spreading of the resulting
cells in tryptone semisolid agar was examined in the presence of
0.1 mM IPTG. The wild-type or mutant version of CheY3, but noversion of the other CheY homologs, could induce CW rotationof the flagellar motor of E. coli (data not shown).
It has been reported that five of the six CheY homologs of
Rhodobacter sphaeroides can rescue the chemotactic defect(tumbling) of an E. coli cheZ mutant strain (26, 29). We therefore examined the effects of the V. cholerae CheY homologs on
spreading in semisolid agar of the E. coli cheZ mutant strain
RP1616. Most of the CheY homologs, including CheY3,
showed no detectable effect, but cells expressing the wild-typeor D-to-K version of CheY4 formed a slightly larger ring withblurred edges indicative of an improved run/tumble bias. This
increased spreading was not seen for cells expressing CheY4D58N, in which the putative phosphorylatable aspartate residue was replaced (Fig. 5), although the mutant protein was
expressed at a level similar to those of the wild-type and D-

J. BACTERIOL.

to-K versions (data not shown). Thus, the small effect of
CheY4 on the E. coli cheZ strain is significant and may involve
the phosphorylation of CheY4. These results suggest that
CheY4 might affect flagellar rotation indirectly by receiving aphosphoryl group from CheY3 under some conditions.
Effects of substitutions at the interface between CheY andthe flagellar motor protein FliM. Interestingly, CheYE only
partially functioned in V. cholerae, whereas the V. cholerae
CheY3 protein could act efficiently on the flagellar motor of E.
coli. This asymmetry might be explained by the following
amino acid substitutions in the interface between CheY andthe flagellar switch protein FliM. The N-terminal sequence of
FliM is highly conserved, but the V. cholerae FliM protein(FliMV; MTDLLSQDEIDALLHGV) has Asp-8 in place of
Ala-9 of the E. coli FliM protein (FliME; MGDSILSQAEIDALLNGD). Glu-117 of CheYE and Ala-9 of FliME are inclose proximity in the complex of CheYE and the N-terminal
16-residue peptide of FliME (21). The former residue corresponds to Lys-120 of the CheY3 protein. Therefore, it waspredicted that FliMV would disfavor CheYE due to an electric
repulsion between Asp-8 and Glu-117, whereas an inverse
combination of CheY3 and FliME would be more tolerant.
To test this hypothesis of coevolution of the CheY-FliM
pair, we introduced a Glu-117-to-Lys (E117K) mutation into
CheYE and a Lys-120-to-Glu (K120E) mutation into CheY3.
Immunoblotting revealed that the mutant proteins were expressed at levels similar to those of the wild-type versions (datanot shown). cheY3 knockout cells expressing CheY3-K120E
reoriented less than cells expressing the wild-type version(Fig. 3B). On the other hand, cells expressing CheYE-E117K
spread (Table 2) and reoriented more frequently than thoseexpressing the wild-type version (Fig. 3B). These results are
consistent with the notion that residue Lys-120 of CheY3 isinvolved in switching of the flagellar motor of V. cholerae.

TABLE 4. Spreading in semisolid agar of V. cholerae ⌬cheA123
cells expressing each CheY homolog
CheY homolog

Mutation

Relative diameter a

None
CheYE

NAb
None
D13K
None
D11K
None
D10K
None
D10K
None
D16K
None
D15K

0.53 ⫾ 0.06
0.53 ⫾ 0.02
0.59 ⫾ 0.05
0.50 ⫾ 0.02
0.50 ⫾ 0.06
0.59 ⫾ 0.05
0.55 ⫾ 0.02
0.53 ⫾ 0.02
0.50 ⫾ 0.06
1.00 ⫾ 0.05
1.03 ⫾ 0.08
0.50 ⫾ 0.02
0.62 ⫾ 0.08

CheY0
CheY1
CheY2
CheY3
CheY4

a
The relative diameters of swarm rings were measured as described inTable 1, except that the average diameter of the triplicate swarm rings of⌬cheA123 cells expressing CheY3 was defined as 1.0, tryptone–semisolid 0.3%
agar was supplemented with 0.1 mM IPTG, and the incubation time at 30°C was
24 h.
b
NA, not applicable.

V. CHOLERAE CheY HOMOLOG SWITCHES FLAGELLAR ROTATION

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FIG. 5. Effects of CheY homologs on spreading of E. coli cheZ mutant cells in semisolid agar. Overnight cultures of RP1616 (cheZ) cells
expressing each CheY-FLAG protein were spotted onto tryptone semisolid agar supplemented with 50 ␮g ml⫺1 ampicillin and 0.1 mM IPTG at
30°C for 12 h.

DISCUSSION
In this study, we examined the roles of the five different
CheY homologs encoded in the genome of V. cholerae inswimming behaviors to determine which of the three che clusters are involved in chemotaxis. Deletion of the cheY3 gene,but not that of the cheY1, cheY2, and cheY4 genes, resulted ina defect in chemotaxis. This is consistent with a previous study
looking at the requirement of the CheA homologs (12). In
R. sphaeroides, all six CheY homologs, including those that are
not involved in chemotaxis, bind to the motor switch protein
FliM (11). Therefore, to assess whether each CheY homolog
can control flagellar rotation directly, we examined the effectof CheY overexpression on swimming behavior rather thanassaying the in vitro binding of CheY to FliM. Overproductionof the CheY3 protein, but not that of the other four CheY
homologs, induced reorientation, indicating that only CheY3
directly controls flagellar rotation. Therefore, we concluded
that only che cluster II is directly responsible for the (flagellum-mediated) chemotaxis of V. cholerae and that che clusters
I and III as well as CheY0 might be involved in some physiological functions other than chemotaxis.
Among the CheA, CheB, and CheR homologs of V. cholerae, the most similar to those of E. coli are CheA3, CheB3,
and CheR3, which are encoded in che cluster III (data notshown). In contrast, among the CheY homologs, the mostsimilar to that of E. coli is CheY3, which is encoded in che cluster
II. In particular, the FliM-interacting residues of CheYE are best
conserved in CheY3, consistent with the finding that this is the
only CheY homolog of V. cholerae that can regulate flagellar
rotation.

We could not find phenotypes of V. cholerae cheY knockouts
and overproducers, except for the cheY3 mutant. Growth rates
and cell morphologies of cells overproducing each CheY homolog were similar to those of wild-type cells (data not shown).
Since CheY has no recognizable “output” domain (such as a
DNA-binding domain), we suspect that the CheY homologs
exert their functions by interacting with their target proteins.
We failed to find a nonflagellar V. cholerae protein from thedatabase with significant homology to the N-terminal aminoacid sequence of the V. cholerae flagellar motor FliM proteinthat is deduced to serve as the target of CheY3. The V. cholerae
CheY homologs other than CheY3 might interact with target
sequences distinct from that of FliM.
It has been reported that duplication and deletion ofcheY4 in V. cholerae classical biotype strain 569B (serotype
Inaba) increase and decrease spreading in LB semisolid agar
as well as migration toward isolated intestinal mucus layers,respectively (3). This apparent discrepancy with our study
might be accounted for by the difference in genetic background of the bacterial strains used or the polar effects ofduplication and deletion of cheY4. Alternatively, cheY4
might modulate chemotaxis indirectly. The effect of CheY4
on the cheZ mutant raises the intriguing possibility that
CheY4 can affect chemotaxis though interaction with andphosphotransfer from CheY3. Another possibility is that themodulated chemotaxis is due to varied gene expression regulated by CheY4. Indeed, the duplication of cheY4 increases
the expression levels of ctxAB and tcpA (3). It has also beenreported that motility and the expression of ctxAB are inversely regulated by bile (13). Therefore, it is possible that

8410

HYAKUTAKE ET AL.

CheY4 regulates the expression of a certain factor(s) that inturn affects chemotaxis under some condition(s). These possibilities should be tested experimentally.
Other genes in che clusters I and III might also contributeindirectly to the flagellum-mediated chemotaxis of V. cholerae
by modulating the CheY3 function. For example, it is possiblethat CheY3 receives signals from noncognate CheA proteins
under conditions that have not yet been identified. However,our data argue that flagellar rotation is modulated exclusively
by CheY3. It has been reported that che genes in cluster II
are involved in the expression of ctxAB and toxT genes (20).
This suggests a link between chemotaxis itself and virulence.
V. cholerae might be attracted by a factor in the intestine whichfunctions as an inducer of expression of CT and Tcp genes, orche genes in cluster II might play a role in virulence gene
expression. Chemotaxis might rather interfere with infection,although motility is required, since nonchemotactic mutants
with counterclockwise biases of flagellar rotation outcompete
the wild-type strain during mouse infections (6, 7). On theother hand, the MCP-like proteins TcpI and AcfB are also
implicated in virulence (9, 15). Their roles in chemotaxis might
be indirect unless they regulate the activity of CheA2, which
seems unlikely from sequence comparisons (data not shown).
Clearly, comprehensive genetic and biochemical analyses ofthis complex system are needed to clarify these issues.
ACKNOWLEDGMENTS
This work was supported in part by a grant from the Ellison Medical
Foundation (to C.C.H.) and by grants-in-aid for scientific research fromthe Japan Society for the Promotion of Science (to M.H. and I.K.).
We thank Andrew Camilli of Tufts University School of Medicine
and Toshihiro Yakushi of Nagoya University for helpful discussions
and encouragement.
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