JOURNAL OF BACTERIOLOGY, July 2011, p. 3332–3341
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 193, No. 13
CheY3 of Borrelia burgdorferi Is the Key Response Regulator Essential
for Chemotaxis and Forms a Long-Lived Phosphorylated Intermediate?
M. A. Motaleb,1,2* Syed Z. Sultan,1Michael R. Miller,3Chunhao Li,2# and Nyles W. Charon2*
Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27834,1
and Departments of Microbiology, Immunology, and Cell Biology2and Biochemistry,3Health Sciences Center,
West Virginia University, Morgantown, West Virginia 26506-9177
Received 15 March 2011/Accepted 21 April 2011
Spirochetes have a unique cell structure: These bacteria have internal periplasmic flagella subterminally
attached at each cell end. How spirochetes coordinate the rotation of the periplasmic flagella for chemotaxis
is poorly understood. In other bacteria, modulation of flagellar rotation is essential for chemotaxis, and
phosphorylation-dephosphorylation of the response regulator CheY plays a key role in regulating this rotary
motion. The genome of the Lyme disease spirochete Borrelia burgdorferi contains multiple homologues of
chemotaxis genes, including three copies of cheY, referred to as cheY1, cheY2, and cheY3. To investigate the
function of these genes, we targeted them separately or in combination by allelic exchange mutagenesis.
Whereas wild-type cells ran, paused (flexed), and reversed, cells of all single, double, and triple mutants that
contained an inactivated cheY3 gene constantly ran. Capillary tube chemotaxis assays indicated that only those
strains with a mutation in cheY3 were deficient in chemotaxis, and cheY3 complementation restored chemo-
tactic ability. In vitro phosphorylation assays indicated that CheY3 was more efficiently phosphorylated by
CheA2 than by CheA1, and the CheY3-P intermediate generated was considerably more stable than the CheY-P
proteins found in most other bacteria. The results point toward CheY3 being the key response regulator
essential for chemotaxis in B. burgdorferi. In addition, the stability of CheY3-P may be critical for coordination
of the rotation of the periplasmic flagella.
Spirochetes are a group of motile bacteria that have a
unique morphology and means of motility. On the surface of
the spirochete is an outer membrane, which is often referred to
as the outer membrane sheath. Within this outer membrane
are the cell cylinder and the periplasmic flagella. The periplas-
mic flagella reside between the outer membrane and the cell
cylinder. Because of its medical importance and recent ad-
vances in genetic manipulation, we have focused on the Lyme
disease spirochete Borrelia burgdorferi to analyze spirochete
motility and chemotaxis (for recent reviews, see references 10,
26, 34, and 68). This spirochete is relatively long (10 to 20 ?m)
and thin (0.31 ?m) and has a flat-wave morphology, and mo-
tility is generated by rotation of the periplasmic flagella (11, 14,
24, 25, 33, 42). Approximately 7 to 11 periplasmic flagella are
subterminally attached at each cell end (30), and recent elec-
tron cryotomography analysis indicates that these periplasmic
flagella form elegant ribbons that wrap clockwise (CW) around
the cell cylinder (11). Not only are the periplasmic flagella
involved in motility, but these organelles have a skeletal func-
tion that in part dictates the flat-wave shape of the cell (10, 14,
26, 35, 40, 53, 68). Thus, mutants that lack periplasmic flagella
are nonmotile and have a rod-shaped morphology (35, 40, 53).
Motility is accomplished by backward-moving flat waves along
the cell body. These waves are generated by the coordinated
rotation of the rigid periplasmic flagella as they exert force on
the relatively flexible cell cylinder (10, 11, 14, 26, 33).
The motile behavior of B. burgdorferi and other spirochetes
is unique and complex (10, 26, 34). Tracking of B. burgdorferi
swimming reveals three different swimming modes: run, flex,
and reverse (1, 10, 25, 33, 42). Runs occur when the periplas-
mic flagellar motors at one end rotate in the direction opposite
that of the motors at the other end. Thus, the periplasmic
flagella of the anterior ribbon rotates counterclockwise (CCW)
and those of the posterior end rotate CW (as a frame of
reference, a periplasmic flagellum is viewed from its distal tip
along the filament toward insertion into the motor) (10, 11, 14,
26, 33, 34). The flex is a nontranslational mode and is often
associated with bending in the cell center with a distorted
appearance (25, 42). The spirochete flex is thought to be equiv-
alent to the Escherichia coli and Salmonella enterica serovar
Typhimurium tumble (6, 10, 16, 26, 33, 34, 42). During the flex,
the motors at both ends rotate in the same direction (10, 16, 26,
33, 34, 42); i.e., both rotate either CW or CCW. The final
mode, cell reversal, occurs in translating cells when the motors
at each end reverse their direction of rotation (10, 16, 33, 42).
In B. burgdorferi, this reversal can last less than 300 ms (N.
Charon, unpublished data).
Chemotaxis is defined as movement toward or away from a
chemical stimulus. Bacteria undergo a biased random walk
during chemotaxis, and this walk is achieved by modulating the
direction of rotation of the flagella or, in some cases, the speed
of flagellar rotation (50, 60). A two-component system medi-
ates the direction or speed of flagellar rotation. In the para-
* Corresponding author. Mailing address for M. A. Motaleb: De-
partment of Microbiology and Immunology, East Carolina University
School of Medicine, Greenville, NC 27834. Phone: (252) 744-3129.
Fax: (252) 744-3535. E-mail: email@example.com. Mailing address for
N. W. Charon: Department of Microbiology, Immunology, and Cell
Morgantown, WV 26506-9177. Phone: (304) 293-4170. Fax: (304) 293-
7823. E-mail: firstname.lastname@example.org.
# Present address: Department of Oral Biology, State University of
New York at Buffalo, Buffalo, NY 14214.
?Published ahead of print on 29 April 2011.
digm chemotaxis model of E. coli and S. enterica, the response
regulator phosphorylated CheY (CheY-P) shuttles between
methyl-accepting chemotaxis protein (MCP) receptor signal
complexes and flagellar motors. CheY is phosphorylated by
the histidine protein kinase CheA, which forms part of the
signal complexes, which are located preferentially at or near
the cell poles (8, 39, 50, 60, 61, 63, 70, 72). CheY-P diffuses
through the cytoplasm and interacts with the flagellar switch
protein FliM, causing the motor rotational biases to shift
from the default rotation of CCW to CW. Dephosphoryla-
tion of CheY-P, which restores the default CCW behavior, is
dramatically enhanced by the action of the CheY-P-specific
phosphatase CheZ (49, 73).
Chemotaxis in B. burgdorferi and other spirochetes is differ-
ent from the well-studied paradigms of E. coli and S. enterica.
For spirochetes to swim toward an attractant, the organisms
must be able to coordinate the rotation of the motors at the
two ends of the cell; these motors (32, 36) are located at a
considerable distance from one another (often greater than 10
?m) (10, 33, 70). One of the long-standing questions related to
spirochete chemotaxis is how the organisms are able to achieve
this coordination (10, 16, 27, 33). To begin to understand this
process, we have used allelic exchange mutagenesis to identify
specific genes involved in chemotaxis. Genomic analysis indi-
cates that B. burgdorferi is similar to the majority of bacteria in
having multiple copies of chemotaxis genes (10, 18, 26, 39, 49,
69). For example, it has two cheA (cheA1 and cheA2), three
cheW (cheW1, cheW2, and cheW3), and three cheY (cheY1,
cheY2, and cheY3) genes. It does not have cheZ but instead
possesses cheX, which is a CheY-P-specific phosphatase prev-
alent in several species of bacteria (42, 44, 46, 47). In addition,
it has two homologs of the switch protein FliG rather than one
(18, 35). We have shown that cheA2, but not cheA1, and cheX
are involved in chemotaxis (33, 42). cheA2 mutants constantly
run and fail to reverse or flex; thus, in the default state, the
motors at each end of the cell rotate in opposite directions
(33). In contrast, cheX mutants constantly flex and are also
nonchemotactic (42). In this communication, we examine the
roles of cheY1, cheY2, and cheY3 in chemotaxis by inactivating
these genes separately or in combination. We found that only
cheY3 is involved in chemotaxis. Furthermore, biochemical
assays indicate that CheY3 is more efficiently phosphorylated
by CheA2 than by CheA1. Finally, CheY3 is unique compared
to most other bacterial chemotaxis proteins, as it forms a
relatively stable, long-lived CheY3-P intermediate in the ab-
sence of CheX phosphatase. The results point toward CheY3
being the key response regulator for chemotaxis, and the sta-
bility of CheY3-P may be critical for coordinating the rotation
of the periplasmic flagellar motors located at both cell ends.
MATERIALS AND METHODS
Bacterial strains and growth conditions. High-passage, avirulent B. burgdorferi
sensu stricto strain B31A and nonmotile flaB mutant strain MC-1 have been
described previously (7, 40). Cells were grown in BSK-II medium at 34°C in a
2.5% CO2humidified incubator (41).
Construction of cheY mutants. Inactivation of cheY1 (gene locus bb0551; gene
length, 381 bp), cheY2 (bb0570; gene length, 375 bp), and cheY3 (bb0672; gene
length, 441 bp) was achieved essentially as described previously (33, 40–42).
Briefly, cheY1, cheY2, and cheY3 plus flanking DNA were, respectively, amplified
by PCR with the following primers (5?-3?): for cheY1, CheY1-F (ATTTGCAG
TTGTTTTATGAC) and CheY1-R (GCAAATCAAGATCATAAACC); for
cheY2, CheY2-F (TCTGCTAGGTTTCAAAATAT) and CheY2-R (TGGACT
TACCCTTTACATAG); and for cheY3, CheY3-F (GGGGAGCTGATTGTTT
GGAAG) and CheY3-R (ACAGTCCCAGTGAATATAGAG). The PCR prod-
ucts were ligated into the pGEM-T Easy vector (Promega Inc.), yielding
pCheY1-Easy, pCheY2-Easy, and pCheY3-Easy, respectively. Antibiotic resis-
tance cassettes were similarly amplified by PCR with restriction sites at both ends
(see below). The cheY1, cheY2, and cheY3 genes were inactivated by inserting
erythromycin (ermC), synthesized modified coumermycin A1 (gyrB), and kana-
mycin (PflgB-kan; aphI) resistance cassettes, respectively (7, 15, 33, 40, 52, 55).
The cheY1 inactivation plasmid was constructed by inserting an ermC cassette
into the HindIII site (18 bp downstream from the cheY1 translation start site).
Inactivation plasmids for cheY2 and cheY3 were constructed with gyrB and aphI
cassettes, respectively, and inserted at the unique EcoRV sites within cheY2 (177
bp downstream from the translation start site) and cheY3 (317 bp downstream
from the translation start site). Linear, PCR-amplified DNA containing cheY1-
ermC, cheY2-gyrB, or cheY3-aphI was electroporated separately into competent
B31A cells to obtain individual single mutants (7, 41). Transformants were
selected with 0.06 ?g/ml erythromycin (for cheY1::ermC), 2.5 ?g/ml coumermy-
cin A1 (cheY2::gyrB), and 350 ?g/ml kanamycin (cheY3::aphI). The cheY1 and
cheY2 genes were also inactivated using the aphI cassette as described above.
Double mutants were constructed by electroporating linear DNA containing
one inactivated cheY gene into cells containing a different cheY mutation. A
cheY1::ermC cheY2::gyrB cheY3::aphI triple mutant was obtained in a similar
manner by introducing cheY2-gyrB DNA into a cheY1 cheY3 double mutant, and
colonies were selected on plates containing erythromycin, coumermycin A1, and
Construction of complementation vehicle. The previously described shuttle
vector pKSSF1, which carries the streptomycin cassette, was used to complement
the cheY3::aphI mutant (17). The B. burgdorferi flgB promoter (7, 23) and cheY3
gene sequences were amplified with primers containing HindIII and NdeI re-
striction enzyme sites (5?-3? and 3?-5?, respectively) and inserted into the NdeI
site (the 3? end of the promoter fragment and the 5? end of the cheY3 gene) to
yield pFlgBCheY3. The primers used were as follows (5?-3?): for flgB, FlgB-Hind
(AAGCTTTAATACCCGAGCTTCAAG) and FlgB-Nde (CATATGGAAACC
TCCCTCAT); for cheY3, CheY3-Nde (CATATGATTCAAAAGACTAC)
and CheY3-Hind (AAGCTTTAACAAATACAGACATTAC). Underlined se-
quences indicate restriction sites. The flgB cheY3 DNA was then ligated into the
HindIII site of pKSSF1 to yield pCheY3.com. Approximately 10 ?g of purified
pCheY3.com plasmid was used to transform competent cheY3:: aphI mutant cells
by electroporation as described above. Transformants were selected with 350
?g/ml kanamycin plus 100 ?g/ml streptomycin. To confirm that the plasmid was
complementing in trans, the pCheY3.com shuttle vector was rescued from com-
plemented cheY3?cells, transformed, and then purified from E. coli and the
integrity of the flgB-cheY3 construct was verified by restriction digestion.
Reverse transcription (RT)-PCR and RNA ligase-mediated (RLM) rapid am-
plification of cDNA ends (RACE). To determine the operon structure and pro-
moter in genes contained in the cheY1 cluster, exponentially growing cells (2 ?
107/ml) were treated with RNAprotect bacterial reagent and then total RNA was
isolated using the RNeasy mini kit (Qiagen Inc.). Contaminating DNA in the
RNA samples was removed by RNase-free Turbo DNase I (Ambion Inc.) diges-
tion for 3 h at 37°C, followed by RNeasy mini purification. For RT-PCR, cDNA
was prepared from purified RNA using Superscript III reverse transcriptase
according to the manufacturer’s protocol (Invitrogen Inc.). RT-PCR primer
sequences are not shown but can be obtained upon request. To determine the
transcription start site (TSS) of the cheY1 operon, 5? RLM-RACE was per-
formed using 2 ?g purified total RNA and the cheY1 gene-specific primer
5?-CTTGGGCTTCTAAAAATTCT-3? according to the manufacturer’s protocol
(Ambion Inc.). RLM-RACE PCR products were cloned into the pGEM-T Easy
vector (Promega Inc.); this was followed by DNA sequencing. As a positive
control, we determined the TSS of the monocistronically transcribed flaB gene
operon previously reported (22) using a flaB-specific primer (5?-CTTCATTTA
Protein preparation and antibody production. Recombinant CheY1, CheY2,
CheY3, CheA1, and CheA2 proteins (rCheY1, rCheY2, rCheY3, rCheA1, and
rCheA2, respectively) were synthesized by PCR amplification of the sequences of
the genes that encode them, without the translation initiation ATG/TTG codon.
Amplified DNA fragments of cheY1 and cheY2 were ligated into the pQE30
expression vector (Qiagen Inc.) using restriction sites SphI and KpnI. PCR-
amplified cheY3 DNA was inserted into the pQE30 expression vector restricted
with BamHI and HindIII. PCR primers (5?-3?) were as follows: for CheY1,
RY1-F (GCATGCGATAAAAGGAGTGCTAGT) and RY1-R (GGTACCCT
AATCCAAAAGTTTAAT); for CheY2, RY2-F (GCATGCAAAAAAAGAAT
TTTGGTT) and RY2-R (GGTACCTTAAAATATCTTTGAGAT); for CheY3,
VOL. 193, 2011THE cheY GENES OF BORRELIA BURGDORFERI3333
RY3-F (GGATCCATTCAAAAGACTACAATTGC) and RY3-R (AAGCTTT
TTAACAAATACAGACATTAC). Vectors containing cheY1, cheY2, and cheY3
were transformed into an E. coli expression strain containing M15 (pREP4)
(Qiagen Inc.) and overexpressed using 1 mM isopropyl-?-D-thiogalactopyrano-
side. Proteins were affinity purified according to the manufacturer’s protocol and
dialyzed against phosphate-buffered saline. Amino-terminally His-tagged CheA1
and CheA2 were prepared using similar procedures and are described elsewhere
(33, 41, 42). Rats (for CheY3) and rabbits (for CheY1 and CheY2) were immu-
nized with 200 to 400 ?g of dialyzed protein to raise specific antiserum using
standard methods (Alpha Diagnostic International Inc.).
Gel electrophoresis and Western blot analysis. Sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE) and Western blotting with an en-
hanced chemiluminescence detection method (ECL; Amersham Pharmacia)
were carried out as previously reported (21). The protein concentration in the
cell lysates was determined with a Bio-Rad protein assay kit. Unless noted
otherwise, 10 ?g of lysates was loaded into each lane of an SDS-PAGE gel and
subjected to immunoblotting using specific antibodies. Monoclonal and poly-
clonal antibodies that were kindly provided by other investigators included the
following: anti-FlaB (H9724) by A. G. Barbour (University of California, Irvine,
CA), anti-FlaA by B. Johnson (Centers for Disease Control and Prevention,
Atlanta, GA), anti-DnaK by J. Benach (SUNY, Stony Brook, NY), and poly-
clonal anti-CheA by P. Matsumura (University of Illinois, Chicago, IL). The
specific reactivities of those antibodies to B. burgdorferi FlaB, FlaA, DnaK, and
CheA2, respectively, have been reported previously (1, 13, 33, 40, 43). The
intensity of the CheY3 protein bands in wild-type and complemented cheY3?
was measured using Alpha View spot densitometry (Version 18.104.22.168; Alpha
Capillary tube chemotaxis assays. Capillary tube chemotaxis assays were per-
formed as previously described (1, 33, 41, 42), and flow cytometry to quantify
cells was carried out as previously reported (1, 41). A positive chemotactic
response was defined as at least twice as many cells entering the attractant-filled
tubes as the buffer-filled tubes (1).
Microscopy and computer-assisted motion analysis. B. burgdorferi cells were
observed using dark-field microscopy (Zeiss Axioskop 2 microscope; Carl Zeiss
Inc.) (1, 41). Cells were centrifuged at 2,500 ? g and resuspended in a motility
buffer containing 1% bovine serum albumin and 1% methylcellulose (400 mesh;
Sigma-Aldrich Co.). Cells were tracked on a temperature-controlled stage (34°C)
using Volocity (Perkin-Elmer) (1, 41). For each strain, at least 10 individual cells
were recorded for up to 1 min. Because the cheY3 mutant cells constantly ran,
they exited the microscopic field within several seconds, precluding 1-min re-
cording periods. Results are expressed as the total distance the center of each cell
traveled/number of seconds the cell was tracked. The resulting velocity (?m/s) is
a minimal velocity, as cells do not translate during flexes. Cell reversal frequency
(number of reversals per minute) was also determined; reversals in direction
were often accompanied by an intervening flex of less than 0.5 s. For conve-
nience, a prolonged flex interval is defined as lasting 0.5 to 2 s with the cell
stopped and having a twisted morphology.
Phosphorylation assays. Phosphorylation of B. burgdorferi CheA1 and CheA2
(6?His-tagged rCheA1 and rCheA2) and phosphate transfer to rCheY3 were
carried out as previously described (41, 42). [?-32P]ATP (6,000 Ci/mmol) was
purchased from MB Biomedicals. Micro Bio-Spin6 columns were obtained from
Bio-Rad; all other reagents were obtained from Sigma-Aldrich Chemical Co.
rCheA1 and rCheA2 (2 ?M) were incubated in 50 mM Tris (pH 8.5)–50 mM
KCl,–5 mM MgCl2–0.3 mM ATP–1 ?Ci [?-32P]ATP (6,000 Ci/mmol)/?l for 30
min. Reactions were initiated by the addition of ATP plus [?-32P]ATP. Auto-
phosphorylated rCheA reaction mixtures were applied to Bio-Spin6 columns
according to the manufacturer’s instructions to remove unincorporated
[32P]ATP. To measure phosphotransfer from rCheA-P to CheY, 2 ?M32P-
labeled rCheA was incubated with 14.8 ?M rCheY3 for the indicated times at
25°C, reactions were stopped by the addition of 4? stop buffer (0.2 M Tris-HCl,
pH 6.8, 0.4 M dithiothreitol, 8% SDS, 0.4% bromophenol blue, 40% glycerol,
20% 2-mercaptoethanol), and reaction products were electrophoresed on 15%
SDS polyacrylamide gels. Dried gels were exposed to a phosphorimaging screen
(usually for 1 h) and analyzed with a Molecular Dynamics Storm 820; the
intensity of phosphorylated proteins was calculated using ImageQuant v2003.02
software and is expressed as relative “volume.”
Functional residues of chemotaxis response regulators are
conserved in CheY1, CheY2, and CheY3. To initially investi-
gate the extent to which the three putative B. burgdorferi CheY
proteins might be functional, the deduced amino acid se-
quences of B. burgdorferi CheY1, CheY2, and CheY3 were
aligned and compared with those of E. coli and Bacillus subtilis
CheY (Fig. 1). B. burgdorferi CheY1, CheY2, and CheY3 share
32%, 38%, and 25% amino acid sequence identity with E. coli
CheY, respectively. B. burgdorferi CheY1, CheY2, and CheY3
share 41%, 33%, and 47% sequence identity with B. subtilis
CheY; thus, based on sequence identity, B. burgdorferi CheY2
is more similar to CheY of E. coli and CheY1 and CheY3 are
more similar to CheY of B. subtilis. As pointed out below, an
analysis of specific key residues in each of the three CheY
proteins supports this proposition. All of the functional resi-
dues of a CheY response regulator were found to be conserved
in CheY1, CheY2, and CheY3, suggesting that they all are
potential chemotaxis response regulators (57, 67) (Fig. 1, cir-
cles). For example, E. coli CheY and B. burgdorferi CheY
proteins have a (??)5 “Rossman fold” where E. coli residues
D12 and D13 are located on the loop connecting ?1 with ?1,
?3 ends with D57, ?4 ends at T/S87, and ?5 ends at K109 (Fig.
1) (57, 67). In addition, the aromatic residue Y106 of E. coli
that rotates upon phosphorylation is conserved in CheY1 (cir-
cle). The nonconserved active-site residues of E. coli CheY,
N59 and E89, which participate in CheY autodephosphoryla-
tion (57, 58), are conserved in B. burgdorferi CheY2 but not in
CheY1 or CheY3 (Fig. 1, arrowheads). E89 is also involved in
CheZ dephosphorylation in E. coli CheY (57, 58, 73). How-
ever, as determined by the analysis of the B. burgdorferi cocrys-
tal CheY3-CheX, amino acid residue T81 of CheY3 (Fig. 1,
diamond) participates in the CheX-mediated dephosphoryla-
tion of CheY3-P (47). T81 is also conserved in B. burgdorferi
CheY1 and B. subtilis CheY but not in E. coli CheY or B.
burgdorferi CheY2. These results are consistent with the known
dephosphorylation activity of CheX on B. burgdorferi CheY3-P
and that of the CheX homolog CheC on B. subtilis CheY-P (42,
Transcription of cheY1, cheY2, and cheY3. The three B. burg-
dorferi chromosomal cheY genes are located in different oper-
ons, and two of the three cheY genes (cheY2 and cheY3) map
at the distal end of the operons (18, 19, 33). The operon
structures housing CheY2 (cheW2 operon) and CheY3 (flaA
operon) have been characterized. The cheY2 operon, consist-
ing of cheW2, orf566, cheA1, cheB2, orfO569, and cheY2, was
shown to be initiated by a ?70promoter (33). The operon that
contains cheY3 was shown to be initiated by either of two ?70
promoters, one upstream of flaA (flaA, cheA2, cheW3, cheX,
and cheY3) and the other upstream of ami (ami, bb0665,
bb0666, bb0667, flaA, cheA2, cheW3, cheX, and cheY3) (20, 65,
71). The gene cluster housing cheY1 consists of cheY1, fliS
(formerly flaJ), bb0549, polA, coaE, and bb0546, but whether
these genes are coordinately transcribed has not been re-
ported. RT-PCR indicated that these genes are transcribed as
a polycistronic mRNA (Fig. 2a and b, lanes 2 to 6). The region
between cheY1 and gene bb0552 failed to be amplified, which
is consistent with bb0552 being divergently transcribed and not
part of the cheY1 operon (Fig. 2b, lane 1).
To determine if expression of the cheY1 operon is also me-
diated by a ?70promoter, the TSS of the cheY1 operon was
determined using RLM-RACE. A TSS with a predicted ?70-
like promoter directly upstream of cheY1 was identified (TTG
TCG-N20-TAGAAT-N7-A) (Fig. 2c and d). To validate the
3334MOTALEB ET AL.J. BACTERIOL.
RACE result, we determined the TSS of the flaB (bb0147)
gene as a control, as the TSS of the flaB monocistronic operon
has previously been determined using primer extension assays
(22). Our RACE analysis produced the expected results (TT
CTTT-N17-TATTCT-N7-A) (not shown; see reference 22).
Thus, the cheY1 operon, like the operons that contain cheY2 or
cheY3, is initiated by a ?70-like promoter and is polycistroni-
Western blot analysis of single, double, and triple cheY mu-
tants and the complemented cheY3 mutant. The response reg-
ulator CheY plays a key role in the chemotactic signaling in
bacteria. Western blot analysis was used to determine if all
three CheY proteins were expressed in B. burgdorferi and to
determine the extent to which these CheY proteins participate
in chemotaxis. cheY1, cheY2, and cheY3 were inactivated singly
or in combination using targeted mutagenesis with different
antibiotic resistance cassettes as described in Materials and
Methods. PCR results of specific clones verified that the anti-
biotic resistance cassettes were appropriately inserted into
the cheY1 (cheY1::ermC), cheY2 (cheY2::gyrB), and cheY3
(cheY3::aph1) genes, as expected (not shown). Western blot
analyses indicated that CheY1, CheY2, and CheY3 were ex-
pressed in wild-type cells but not in the corresponding mutant
cells (Fig. 3a), and as expected, the masses of the reactive
proteins varied from 11 to 14 kDa. trans complementation of
cheY3 (cheY3?) restored CheY3 synthesis, albeit at a higher
level (3.5 times by densitometry) than in the wild-type cells
(Fig. 3a). We also constructed all combinations of cheY double
mutants (cheY1 cheY3, cheY2 cheY3, and cheY1 cheY2) and the
triple cheY mutant (cheY1 cheY2 cheY3) (Fig. 3b and not
shown). Western blot analysis indicated that, as with the single
mutants, the appropriate cheY genes were specifically inacti-
vated (Fig. 3b). For example, in the cheY1 cheY2 double mu-
tant, the expression of CheY1 and CheY2 was abolished with-
out affecting the expression of CheY3 (Fig. 3b, lanes cheY1Y2).
As expected, Western blot analysis also demonstrated that
none of the CheY proteins were expressed in the triple cheY
(cheY1 cheY2 cheY3) mutant (Fig. 3b, lanes cheY1Y2Y3).
Chemotactic behavior of single cheY mutants. In other spe-
cies that have multiple cheY homologs, often only one partic-
ipates in chemotaxis. However, in some species, more than one
cheY homolog is necessary for full chemotaxis (31, 49–51, 59,
62). Accordingly, we tested if the three cheY genes functioned
in chemotaxis using capillary tube assays (Fig. 4). We found
that the cheY1 and cheY2 mutants had a chemotactic response
indistinguishable from that of the wild type. In contrast, the
cheY3 mutant failed to significantly respond to the attractant,
but complementation restored chemotaxis to a level equivalent
to that of the wild type (Fig. 4a). Thus, these results indicate
that cheY3, but not cheY1 or cheY2, is involved in chemotaxis.
FIG. 1. Amino acid sequence alignments of B. burgdorferi CheY proteins with E. coli and B. subtilis CheY using T-Coffee (45). Conserved
residues that are essential for function in E. coli are indicated by solid circles. The nonconserved active-site residues of E. coli CheY, N59 and E89,
that are conserved in B. burgdorferi CheY2, but not in CheY1 or CheY3, are identified by arrowheads. T81 of CheY3, which is involved in CheX
dephosphorylation of CheY3-P and is also conserved in B. burgdorferi CheY1 and in B. subtilis CheY, is identified by a diamond. Secondary
structure elements as determined for E. coli CheY and predicted for B. burgdorferi CheY proteins are shown above the sequence alignment. The
E. coli and B. subtilis CheY proteins are identified as Ec-CheY and Bsu-CheY, respectively. Amino acid residue numbers are indicated on the left.
The last amino acid residue number of each CheY sequence is shown on the right.
VOL. 193, 2011 THE cheY GENES OF BORRELIA BURGDORFERI3335
In addition, the lack of a detectable defect on the chemotaxis
phenotype in cheY1 and cheY2 mutants was not due to the use
of the ermC and gyrB cassettes, as identical results were ob-
tained when each was inactivated with the aphI cassette (not
Chemotactic behavior of double and triple cheY mutants.
The results in Fig. 1 and 3 indicate that all three CheY proteins
have the necessary domains for chemotaxis and are expressed
in wild-type cells. We next asked whether CheY3, in combina-
tion with either CheY1 or CheY2, was essential for che-
motaxis. Conceivably, CheY1 and CheY2 could overlap in
function to augment chemotaxis mediated by CheY3 in a man-
ner analogous to CheY3 or CheY4 promoting CheY6-medi-
ated chemotaxis in Rhodobacter sphaeroides (49–51). To test
this possibility, we constructed a cheY1 cheY2 double mutant
and measured its ability to undergo chemotaxis. Capillary tube
chemotaxis assays indicated that the cheY1 cheY2 double mu-
tant had no detectable alteration in its chemotactic response
(Fig. 4b). In contrast, the cheY1 cheY3 and cheY2 cheY3 double
mutants, as well as the cheY1 cheY2 cheY3 triple mutant, were
deficient in chemotaxis (Fig. 4b). Taken together, these results
FIG. 2. cheY1 (bb0551) operon structure. (a) Schematic represen-
tation of the cheY1 operon. Arrowheads indicate specific primer pairs
that amplify regions between genes. (b) Agarose gel showing the RT-
PCR products. The number above each lane represents a number
between arrowheads in panel a. Lane 1 did not amplify a product, as
cheY1 and bb0552 are divergently transcribed. A no-RT control reac-
tion is represented by a minus sign. (c) DNA sequencing chromato-
gram of an RLM-RACE analysis that resulted in the identification of
the TSS of the cheY1 operon (right-angled arrow). The cheY1 trans-
lation start TTG codon is boxed. A horizontal line with arrowheads
represents the 5? RACE adapter sequence (provided in the kit). (d)
Predicted ?35 and ?10 promoter sequences with the TSS (right-
angled arrow) of cheY1 operon (top) and a typical ?70promoter se-
quence (bottom) are shown (28).
FIG. 3. Western blot analyses of CheY1, CheY2, and CheY3 in
wild-type and cheY mutant B. burgdorferi cells. Approximately 10 ?g
protein of cell lysates from the wild-type strain and the indicated cheY
mutant strains was subjected to Western blotting and probed with
antibodies specific to the CheY proteins (?CheY1, ?CheY2, and
?CheY3). Samples containing 3 ?g lysates were used for the loading
control (?DnaK, panel a, rightmost). The approximate molecular
masses of CheY1 (11 kDa), CheY2 (10 kDa), CheY3 (14 kDa), and
DnaK (72 kDa) were determined based on the masses of marker
proteins (not shown). In panel a, lysates from wild-type; cheY1, cheY2,
or cheY3 single mutant; and complemented cheY3?B. burgdorferi cells
were probed with the indicated CheY antisera. In panel b, lysates from
wild-type, cheY1 cheY2 double mutant (cheY1Y2), and cheY1 cheY2
cheY3 triple mutant B. burgdorferi cells (cheY1Y2Y3) were probed with
CheY1, CheY2, and CheY3 antisera.
FIG. 4. Chemotaxis assays of cheY mutants. (a and b) Capillary
tube chemotaxis assays coupled with flow cytometry were performed
with wild-type (WT) and mutant strains. The results are expressed as
n-fold increases in the number of cells entering capillary tubes con-
taining glucosamine attractant relative to the number of cells entering
tubes containing a no-attractant control (buffer alone). A 2-fold in-
crease (horizontal line) compared to the buffer control is considered
significant (1). Results are expressed as the mean ? standard error
from three independent experiments.
3336 MOTALEB ET AL.J. BACTERIOL.
indicate that cheY1 and cheY2 are not essential for chemotaxis
and that cheY3 is the sole response regulator required for B.
burgdorferi chemotaxis under our assay conditions.
Swimming behavior of the cheY mutants. One possible ex-
planation for the above results is that the cheY3 mutation
resulted in a defect in motility and not in chemotaxis, as found
in some species of bacteria. For example, in Rhodospirillum
centenum, cheY3 mutants fail to synthesize flagella and are
nonmotile (2). To test for this possibility, we analyzed individ-
ual cells using a computer-assisted cell tracker coupled with
video microscopy. Because the periplasmic flagella influence
cell shape (10, 14, 26, 35, 40, 53), we first analyzed cell struc-
ture by dark-field microscopy. We found that the morphology
of the mutants was indistinguishable from that of the wild type
(not shown). Furthermore, the swimming velocity of each of
the cheY mutants, including those containing the cheY3 muta-
tion, was similar to that of the wild type (Table 1). The veloc-
ities ranged from 8 to 11 ?m/s. These results indicate that the
cheY3 mutation did not result in a defect in motility.
However, the swimming behavior of the mutants carrying
the cheY3 mutation was different. The wild-type and cheY1,
cheY2, and cheY1 cheY2 mutant cells reversed 18 to 22 times
per minute, whereas the mutants carrying the cheY3 mutation
failed to reverse, even when these cells were observed for
several minutes. As expected, wild-type swimming behavior
was restored when the cheY3 single mutant was complemented
in trans, albeit with a slightly higher rate of reversal (Table 1).
In addition, when the cheY3?cells reversed, the reversal was
consistently accompanied by an intervening prolonged flex
which lasted longer than 0.5 s. In contrast, when the wild type
reversed, only 6 to 10 times out of 20 was there an accompa-
nying intervening prolonged flex lasting more than 0.5 s. These
results suggest that cheY3?had a higher rate of flexing than the
wild type; this result could be related to the finding that the
cheY3?mutant produces ?3.5 times more CheY3 than
the wild type (Fig. 3a).
Synthesis of motility and chemotaxis proteins. We tested
whether the cheY3 mutation caused altered expression of other
motility and chemotaxis genes that resulted in a constant run-
ning phenotype. Western blot analysis indicated that the level
of expression of the major and minor periplasmic flagellar
proteins FlaB and FlaA was similar in the cheY3 single mutant,
cheY triple mutant, and wild-type cells (Fig. 5), indicating that
mutations in cheY3 did not alter the expression of these fla-
gellar proteins. In addition, because cheA2 mutants have also
been shown to run constantly, we examined whether cheY3
mutants influenced CheA2 expression (1, 33). We found that
the level of CheA2 expression was unaltered in the cheY3
mutant or the triple mutant cells (Fig. 5). Thus, the altered
swimming and defective chemotactic behaviors observed in
cheY3 strains were not due to the altered expression of CheA2.
Phosphorylation of CheY3. The genetic-phenotypic analysis
indicates that only CheY3 is involved in chemotaxis. Previous
work in our laboratory indicated that CheA2, but not CheA1,
is involved in chemotaxis (1, 33). cheA2 and cheY3 are also in
the same operon; we therefore asked if CheY3 is preferentially
phosphorylated by CheA2 compared to CheA1. We also as-
sessed the stability of the CheY3-P intermediate relative to
CheY-P proteins of other bacteria. We first autophosphory-
lated CheA1 and CheA2 with [32P]ATP. CheA1-32P and
CheA2-32P were found to be stable for at least 60 min, with no
detectable loss of phosphate (not shown). The addition of an
?7-fold molar excess of CheY3 resulted in the loss of phos-
phate from CheA1-32P and CheA2-32P and transfer to
CheY3-P (Fig. 6). Phosphotransfer from CheA2-32P to CheY3
was markedly more efficient than that from CheA1-32P. Spe-
cifically, complete phosphotransfer occurred within 10 s with
CheA2-32P, whereas it took 90 s with CheA1-32P. We also
found that CheY3-32P had a half-life of greater than 10 min,
which makes it markedly more stable than CheY-P of E. coli
and most other bacteria (29, 48, 59). These results are consis-
tent with those we and our colleagues recently obtained using
a photometric assay for CheY3-P autodephosphorylation (47).
Taken together, our results indicate that CheY3 is more effi-
ciently phosphorylated by CheA2 than by CheA1, which agrees
FIG. 5. Western blot analysis of motility and chemotaxis proteins
among mutants. Western blot analysis using B. burgdorferi monoclonal
anti-FlaB, -FlaA, and -DnaK and E. coli polyclonal anti-CheA anti-
bodies. Cell lysates (10 ?g protein) from wild-type and cheY3 and
cheY1 cheY2 cheY3 mutant (cheY1Y2Y3) cells were probed with the
indicated antibodies. For FlaB, 3 ?g was loaded in each lane, and for
Dnak, 2 ?g of lysate was loaded in each lane.
TABLE 1. Swimming behavior of B. burgdorferi cheY mutants
(?m/s) ? SDa
Mean no. of
reversals/min ? SDa
cheY1 cheY2 mutant
cheY1 cheY3 mutant
cheY2 cheY3 mutant
cheY1 cheY2 cheY3 mutant
10.0 ? 0.8
8.6 ? 1.8
11.0 ? 1.4
10.5 ? 1.7
9.5 ? 0.8
10.8 ? 2.3
11.7 ? 2.9
10.1 ? 1.8
11.7 ? 1.8
20.0 ? 1.2b
18.5 ? 1.5
19.0 ? 2.1
25.0 ? 1.5d
21.0 ? 3.0
aStandard deviations were calculated from data obtained from at least 10
individual tracked cells of each strain.
bIn the 20 reversals that occurred in the wild type during a 1-min interval, a
prolonged flex lasting more than 0.5 s occurred 6 to 10 times.
cCells ran in only one direction and did not reverse.
dIn the cheY3?mutant, all 25 reversals were accompanied by a flex lasting
more than 0.5 s.
VOL. 193, 2011THE cheY GENES OF BORRELIA BURGDORFERI 3337
with the in vivo results that only CheA2 is involved in che-
motaxis (1, 33) and that CheY3-P has a markedly weak au-
The three cheY genes of B. burgdorferi are distributed
throughout the chromosome. Two of the cheY genes (cheY2
and cheY3) reside in operons with other chemotaxis gene
homologs, and the third cheY gene (cheY1) is in an operon
with a motility gene and other genes of unknown function.
B. burgdorferi is not unusual in possessing multiple homologs
of chemotaxis genes in its genome. A recent genomic bioin-
formatic analysis of over 450 bacteria indicates that more
than 50% of those with chemotaxis gene homologs have
more than one copy of these genes (69). These homologs
have been shown to participate not only in flagellum-medi-
ated chemotaxis but also in type 4 pilus-based motility (4,
38), polysaccharide biosynthesis associated with pilus-based
gliding motility (5), and flagellar morphogenesis (2). In
many cases, genetic analysis has not been successful in sort-
ing out the function of these chemotaxis gene homologs
In this study, we determined the role of the three B.
burgdorferi cheY genes in chemotaxis and motility. All three
CheY proteins were expressed in B. burgdorferi (Fig. 3), and
sequence analysis indicates that all three cheY genes contain
the conserved functional residues associated with response
regulatory proteins that form phosphorylated intermediates
(Fig. 1). Consistent with this analysis, CheY3 was phosphor-
ylated by both CheA1 and CheA2 (Fig. 6), and recent results
suggest that both CheY1 and CheY2 can also be phosphor-
ylated by B. burgdorferi CheA proteins (M. Motaleb, M.
Miller, and N. Charon, unpublished data). These results
agree with previous reports that CheY3 can be phosphory-
lated by CheA2 (41, 42). We found that when the three cheY
genes were inactivated singly or in combination, only muta-
tions in cheY3 led to a nonchemotactic, constantly running
phenotype; complementation of cheY3 restored the chemo-
tactic response, but such cells had an increase in flexing (Fig.
4; Table 1). Thus, we conclude that only CheY3 is essential
for chemotaxis under the conditions tested. We used an
avirulent strain, and it is conceivable that in a virulent strain,
CheY1 and CheY2 could participate in chemotaxis under
conditions that best mimic those in the tick or the mammal
hosts (10, 64). However, the identical cheY3::aphI mutation,
when introduced into virulent strain B31-A3, resulted in
cells with a constant running phenotype similar to that
found with the avirulent strain (M. Motaleb and N. Charon,
unpublished data). These results indicate that cheY3 is in-
volved in chemotaxis in a virulent strain as well.
cheY3 is in a cluster with other genes (cheA2, cheW3,
cheX, and cheY3) that are known to participate in che-
motaxis (33, 42). Specifically, mutations in cheA2, cheX, and
now cheY3 all result in a nonchemotactic phenotype (33,
42). Preliminary experiments with cheW3 mutants indicate
that this gene is also involved in chemotaxis (K. Zhang, C.
Li, and N. Charon, unpublished data). This chemotaxis gene
cluster is well conserved in B. burgdorferi along with the
spirochetes Treponema denticola and Treponema pallidum in
both gene order and sequence identity (56). Because the
treponemes have only one homolog of these genes in their
genomes, these clusters also likely function in chemotaxis.
In support of this proposition, analysis of a cheA mutant in
T. denticola revealed it to be nonchemotactic (37).
FIG. 6. Transfer of phosphate from CheA1-32P (top panels) and CheA2-32P (bottom panels) to CheY3. CheA1 and CheA2 were autophos-
phorylated with [32P]ATP, and unincorporated [32P]ATP was removed by centrifugation on Bio-Spin6 columns. Recovered CheA1-32P and
CheA2-32P (2 ?M each) were incubated with 14.8 ?M CheY3 for the indicated periods of time. Reactions were stopped, and products were
analyzed as described in the text. Phosphor images are shown in the left panels, and relative intensities (PI “volumes”) are shown in the right panels.
CheA1-32P and CheA2-32P are represented as solid lines and squares, and CheY3-P is represented as circles and dashed lines.
3338MOTALEB ET AL. J. BACTERIOL.
Phosphorylation assays revealed that CheA1 and CheA2
transferred phosphate to CheY3. CheA2 was considerably
more efficient than CheA1 (Fig. 6). These results are ex-
pected, as genetic analysis indicates that both CheA2 and
CheY3, but not CheA1, are involved in chemotaxis (1, 33)
and both CheA2 and CheY3 reside in the same operon.
Although biochemical assays indicate that CheA1-P trans-
ferred its phosphate to CheY3 in vitro, there was no evi-
dence of cross talk in which CheA1 could substitute for
CheA2 in vivo (33).
The other noteworthy biochemical reaction is that
CheY3-P is unusually stable, with a half-life of approxi-
mately 10 min. E. coli CheY-P is considerably less stable,
with a half-life measured in seconds (29). To our knowledge,
the only reported CheY-P homolog that is more stable is
DifD of Myxococcus xanthus (5), with a half-life of 30 min.
DifD is involved in both polysaccharide biosynthesis associ-
ated with social motility and chemotaxis. This stability has
been related to the slow gliding movement of M. xanthus and
its prolonged adaptation time for chemotaxis (5). At this
time, we do not know why CheY3-P has such a prolonged
half-life; however, it points to CheX, besides CheA2, playing
a critical role in modulating CheY-P concentration.
The results presented here and previous results from our
laboratory point toward CheY3 being the key response reg-
ulator controlling the direction of flagellar rotation in B.
burgdorferi. In E. coli and S. enterica, CheY-P binding to
motor proteins promotes CW rotation of flagella, resulting
in tumbles; attractants reduce levels of CheY-P, generating
longer runs (49, 50, 54, 60). Two conditions should lead to
high CheY3-P concentrations in B. burgdorferi. One is inac-
tivation of the cheX phosphatase, and the other is overpro-
duction of CheY3 (12, 42). Both of these conditions result in
an increase in flexing frequency, with cheX mutants con-
stantly flexing (Table 1; Fig. 3a), (42). In contrast, mutations
in either cheA2 or cheY3 should lead to negligible intracel-
lular concentrations of CheY3-P. We found that mutations
in either of these genes result in cells that constantly run (1,
33). These results suggest that CheY3-P affects the rotation
of flagellar rotors in a manner similar to that found in E.
coli: Both species constantly run under low CheY-P concen-
trations. However, the situation is more complex in B. burg-
dorferi and is presently not understood, as during its run, the
periplasmic flagella at both ends rotate in opposite direc-
tions, not in the same direction as in E. coli (10, 33).
It is too early to understand the basis of periplasmic
flagellar coordination that results in chemotaxis in B. burg-
dorferi. Recent results suggest that the MCPs are subpolarly
located, form clusters at both ends, and are in close prox-
imity to the flagellar motors (8, 70). CheA2 likely forms a
complex via CheW3 with these MCPs, as CheA, CheW, and
MCPs form complex arrays in other bacteria (3, 49). Thus,
CheA2 is also likely to be subpolarly localized. Based on
these assumptions and the evidence that CheY3 is the key
response regulator essential for chemotaxis, two possible
models of flagellar coordination are evident. One model
states that if an attractant is bound to MCPs at one end of
the cell, a signal in the form of a change in the CheY3-P
concentration is generated at that end that affects flagellar
rotation not only at the proximal end but also at the distal
cell end. Given B. burgdorferi’s velocity of ?10 ?m/s (Table
1), a change in CheY3-P concentration must be rapidly
sensed at the distal end of the cell by this model. Diffusion
of CheY3-P to the distal end is unlikely to be responsible for
this coordination, as it is too slow (using t ? L2/D, where L
is a cell length of 10 ?m and D is 10 ?m2/s, t is 10 s). Thus,
it would take at least 10 s for a change in CheY3-P concen-
tration generated at one end to diffuse to the other end (66).
Perhaps CheY3-P is transported to the distal end through a
presently unknown internal cytoskeletal structure, and the
relative stability we find in CheY3-P is essential for this to
occur (9). For example, the gliding motion of M. xanthus is
related to intracellular movement of AglZ, and this move-
ment is mediated by the cell skeletal protein MreB (38).
Because transport of AglZ from one cell end to the other is
on the order of several minutes, a CheY3 transport system
in B. burgdorferi would, by necessity, have to be considerably
faster. Our results do not rule out the possibility that
CheY3-P acts at another, unknown, site such that the mem-
brane potential is altered when the cell is undergoing che-
As an alternative, perhaps there is no internal signal that
coordinates the motors at both cell ends. According to this
model, flagellar coordination and chemotaxis are achieved
by the attractant binding to either one or both of the MCP
clusters at the cell ends. The change in CheY3-P concentra-
tion generated by this binding specifically affects the direc-
tion of rotation of the motors that are adjacent to those
MCPs. For example, consider the following scenario. If the
attractant binds the MCPs at one cell end, it causes the
motors only at that end of the cell to change their direction
of rotation, and perhaps the cell flexes. In contrast, if at-
tractant molecules simultaneously bind to the MCPs at both
cell ends, the motors at both ends change directions and the
cell runs. Now that we know which compounds serve as
attractants (1) and that CheY3 is the response regulator, we
are finally in a position to determine how the rotation of
periplasmic flagella in these spirochetes is coordinated for
We thank A. Barbour, J. Benach, B. Johnson, and P. Matsumura for
sharing antibodies, P. Stewart and P. Rosa for sharing plasmids, and A.
Cockburn, K. Miller, M. James, U. Pal, B. Schraf, and R. Silversmith
This research was supported by National Institutes of Health grant
AI29743 to N.W.C. and an East Carolina University Research and
Development start-up grant to M.A.M.
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