Two CheW coupling proteins are essential in a chemosensory pathway of Borrelia burgdorferi

Article (PDF Available)inMolecular Microbiology 85(4):782-94 · July 2012with22 Reads
DOI: 10.1111/j.1365-2958.2012.08139.x · Source: PubMed
  • 16.81 · University at Buffalo, SUNY
  • 36.31 · University of Texas Health Science Center at Houston
  • 15.82 · Soochow University (PRC)
  • 30.4 · University at Buffalo, The State University of New York
In the model organism Escherichia coli, the coupling protein CheW, which bridges the chemoreceptors and histidine kinase CheA, is essential for chemotaxis. Unlike the situation in E. coli, Borrelia burgdorferi, the causative agent of Lyme disease, has three cheW homologues (cheW(1) , cheW(2) and cheW(3) ). Here, a comprehensive approach is utilized to investigate the roles of the three cheWs in chemotaxis of B. burgdorferi. First, genetic studies indicated that both the cheW(1) and cheW(3) genes are essential for chemotaxis, as the mutants had altered swimming behaviours and were non-chemotactic. Second, immunofluorescence and cryo-electron tomography studies suggested that both CheW(1) and CheW(3) are involved in the assembly of chemoreceptor arrays at the cell poles. In contrast to cheW(1) and cheW(3) , cheW(2) is dispensable for chemotaxis and assembly of the chemoreceptor arrays. Finally, immunoprecipitation studies demonstrated that the three CheWs interact with different CheAs: CheW(1) and CheW(3) interact with CheA(2) whereas CheW(2) binds to CheA(1) . Collectively, our results indicate that CheW(1) and CheW(3) are incorporated into one chemosensory pathway that is essential for B. burgdorferi chemotaxis. Although many bacteria have more than one homologue of CheW, to our knowledge, this report provides the first experimental evidence that two CheW proteins coexist in one chemosensory pathway and that both are essential for chemotaxis.


Two CheW coupling proteins are essential in a chemosensory
pathway of
Borrelia burgdorferi
Kai Zhang,1Jun Liu,3Youbin Tu,4Hongbin Xu,1
Nyles W. Charon5and Chunhao Li1,2*
Departments of 1Oral Biology and 2Microbiology and
Immunology, the State University of New York at
Buffalo, Buffalo, NY 14214, USA.
3Department of Pathology and Laboratory Medicine,
University of Texas Medical School at Houston,
Houston, TX 77030, USA.
4Department of Bioinformatics, Soochow University,
Suzhou 215123, China.
5Department of Microbiology, Immunology and Cell
Biology, Health Sciences Center, West Virginia
University, Morgantown, WV 26506, USA.
In the model organism Escherichia coli, the coupling
protein CheW, which bridges the chemoreceptors and
histidine kinase CheA, is essential for chemotaxis.
Unlike the situation in E. coli,Borrelia burgdorferi, the
causative agent of Lyme disease, has three cheW
homologues (cheW1,cheW2and cheW3). Here, a com-
prehensive approach is utilized to investigate the roles
of the three cheWs in chemotaxis of B. burgdorferi.
First, genetic studies indicated that both the cheW1
and cheW3genes are essential for chemotaxis, as the
mutants had altered swimming behaviours and were
non-chemotactic. Second, immunofluorescence and
cryo-electron tomography studies suggested that
both CheW1and CheW3are involved in the assembly of
chemoreceptor arrays at the cell poles. In contrast to
cheW1and cheW3,cheW2is dispensable for chemot-
axis and assembly of the chemoreceptor arrays.
Finally, immunoprecipitation studies demonstrated
that the three CheWs interact with different CheAs:
CheW1and CheW3interact with CheA2whereas CheW2
binds to CheA1. Collectively, our results indicate
that CheW1and CheW3are incorporated into one
chemosensory pathway that is essential for B. burg-
dorferi chemotaxis. Although many bacteria have
more than one homologue of CheW, to our knowledge,
this report provides the first experimental evidence
that two CheW proteins coexist in one chemosensory
pathway and that both are essential for chemotaxis.
Chemotaxis allows motile bacteria to swim towards a
favourable environment or away from one that is toxic.
The signalling transduction system controlling bacterial
chemotaxis has been extensively studied in two model
organisms, Escherichia coli and Salmonella enterica (for
recent reviews see Wadhams and Armitage, 2004; Hazel-
bauer et al., 2008; Sourjik and Armitage, 2010). The core
structural unit in the chemotaxis signalling pathway con-
sists of a ternary complex of chemoreceptors (often
referred to as methyl-accepting chemotaxis proteins,
MCPs), a histidine autokinase CheA and a coupling
protein CheW (Liu and Parkinson, 1989; Gegner et al.,
1992). CheW is a single-domain cytoplasmic protein
(Griswold and Dahlquist, 2002).
MCPs sense various environmental signals, which
control the activity of CheA. Activated CheA (CheA-P)
transfers its phosphoryl group to CheY, a response regu-
lator that controls the rotational direction of flagellar
motors. The phosphorylated CheY (CheY-P) diffuses from
the core complex to the flagellar motors, where it binds
motor-switch complex proteins to promote a switch in the
rotational direction from counterclockwise (CCW) to clock-
wise (CW). CCW rotation results in smooth swimming
(also referred to as run), and CW rotation leads to tum-
bling. Cells responding to a positive response (binding of
an attractant to MCPs) lengthen the intervals between
tumbling events and hence have longer runs that allow the
bacteria to swim preferentially towards higher concentra-
tions of attractants (Sourjik and Armitage, 2010; Porter
et al., 2011). In the enteric bacteria, there are single homo-
logues of cheA,cheW and cheY, and null mutations in any
of these genes cause cells to run constantly and to
become deficient in chemotaxis (Parkinson, 1977; Parkin-
son and Houts, 1982).
Borrelia burgdorferi, the causative agent of Lyme
disease (Burgdorfer et al., 1982), is highly motile and
shows chemotactic responses to several attractants pro-
duced by the hosts (Charon and Goldstein, 2002; Shih
et al., 2002; Bakker et al., 2007). Our recent study
shows that chemotaxis is involved in the pathogenicity of
Accepted 14 June, 2012. *For correspondence. E-mail cli9@buffalo.
edu; Tel. (+1) 716 829 6014; Fax (+1) 716 829 3942.
Molecular Microbiology (2012) 85(4), 782–794 doi:10.1111/j.1365-2958.2012.08139.x
First published online 11 July 2012
© 2012 Blackwell Publishing Ltd
B. burgdorferi (Sze et al., 2012). Chemotaxis in B. burg-
dorferi differs from that of E. coli and S. enterica in several
important respects (for recent reviews, see Charon and
Goldstein, 2002; Charon et al., 2012). B. burgdorferi cells
are relatively long (10–20 mm in length) and thin (0.3 mmin
diameter), and two flat ribbons of periplasmic flagella (PFs)
arise in the subpolar region at each cell end (Charon
et al., 2009; Liu et al., 2009). Motility is powered by the
co-ordinated rotation of the PFs. This architecture requires
that the swimming behaviour of spirochaetes is very differ-
ent from that of the peritrichously flagellated enteric bacte-
ria (Goldstein et al., 1994; Li et al., 2002; Motaleb et al.,
2005; 2011b; Dombrowski et al., 2009; Yang et al., 2011;
Harman et al., 2012). B. burgdorferi has three swimming
modes: run, flex and reversal. A run occurs when the
bundle of PFs at the anterior end rotates CCW and that at
the posterior end rotates CW. A reversal happens when
both bundles change their rotational direction nearly simul-
taneously.A flex represents a non-translational mode when
the two bundles of PFs rotate in the same direction (both
CCW or both CW).
During a chemotaxis response, the spirochaetes must
co-ordinate the rotation of the motors at the two ends of
cells (i.e. repressing the time spent in flexing and revers-
ing, and increasing the time spent in running). A long-
standing question about the spirochaete chemotaxis is
how the cells achieve this co-ordination (Charon and
Goldstein, 2002; Li et al., 2002; Charon et al., 2012). In
the spirochaetes, the motors at the two ends of the cells
are located at a considerable distance from one another
(at least 10 mm), and the MCPs form clusters that are in
close proximity to the motors (Briegel et al., 2009; Charon
et al., 2009; Liu et al., 2009; Xu et al., 2011). It would
seem too slow to transmit signals from one end of the cell
to the other simply by diffusion of CheY-P (Sarkar et al.,
2010; Motaleb et al., 2011b; Porter et al., 2011).
Unlike E. coli and S. enterica,B. burgdorferi contains
more than one homologue of cheA,cheW and cheY: two
cheAs (cheA1and cheA2), three cheWs (cheW1,cheW2
and cheW3) and three cheYs (cheY1,cheY2and cheY3)
(Fraser et al., 1997; Charon and Goldstein, 2002). Many of
these genes reside within two gene clusters: the flaA
operon (flaA-cheA2-cheW3-cheX-cheY3) and the cheW2
operon (cheW2-bb0566-cheA1-cheB2-bb0569-cheY2) (Ge
and Charon, 1997; Li et al., 2002). We have recently
identified several genes that are essential for the chemot-
axis of B. burgdorferi, including cheA2,cheY3and cheX (an
analogue of cheZ from E. coli). The cheA2and cheY3
mutants fail to reverse and constantly run, whereas the
cheX mutant constantly flexes. None of these mutants is
able to carry out chemotaxis (Li et al., 2002; Motaleb et al.,
2005; 2011b; Bakker et al., 2007; Sze et al., 2012).
In contrast to the flaA operon, the genes studied to
date in the cheW2operon are not required for the
chemotaxis of B. burgdorferi, e.g. the cheA1and cheY2
mutants have a chemotaxis phenotype that is similar to
wild type (Li et al., 2002; Motaleb et al., 2011b). It has
been speculated that B. burgdorferi may possess two
chemotaxis pathways that function in different hosts
during the infection cycle (Charon and Goldstein, 2002;
Li et al., 2002; Sze et al., 2012). For example, the
chemotaxis genes (cheA2-cheW3-cheX-cheY3)intheflaA
operon may form a pathway that executes chemotaxis in
mammalian hosts, whereas the genes in the cheW2
operon (cheW2-cheA1-cheY2) may constitute a pathway
that controls chemotaxis in the tick vector. In E. coli,
CheW interacts with both MCPs and CheA and plays a
pivotal role in chemotaxis and formation of the MCP–
CheW–CheA ternary complexes (Liu and Parkinson,
1989; Gegner et al., 1992; Boukhvalova et al., 2002b; Vu
et al., 2012). Thus, elucidating the roles of the three
CheWs of B. burgdorferi in chemotaxis will help us
determine whether this organism has two different
chemotaxis pathways.
In this report, the three cheW genes of B. burgdorferi
were separately inactivated by allelic exchange mutagen-
esis, and their roles in chemotaxis and chemoreceptor
assembly were investigated by an approach consisting of
computer-based bacterial tracking analysis, swim plate
and capillary assays, immunofluorescence assay (IFA)
and cryo-electron tomography (cryo-ET). Furthermore,
the interactions between the two CheAs and three CheWs
were studied by co-immunoprecipitation (co-IP). The
results support the idea that B. burgdorferi has two
different chemosensory pathways: CheW1/CheW3-
CheA2-CheY3, which form a pathway that is essential
for chemotaxis under the tested in vitro conditions;
CheW2-CheA1-CheY2and/or CheY1, which form another
pathway that either is required for chemotaxis under other
conditions or is involved in a different signalling pathway.
Conservation of functionally important residues in
CheW1, CheW2and CheW3
Among the three cheW genes, cheW2(bb0565) is the first
gene in the cheW2operon, cheW3(bb0670) is the third
gene in the flaA operon, and cheW1(bb0312) is located in
a gene cluster where no other putative chemotaxis or
motility genes are evident (Fraser et al., 1997; Charon and
Goldstein, 2002; Li et al., 2002). CheW1consists of 176
amino acids (aa) with a predicted molecular weight (MW) of
20 kDa. CheW2is 180 aa in length with a predicted MW of
21 kDa. CheW3contains 466 aa, and its predicted MW is
53 kDa. A BLAST search showed that the N-terminus of
CheW3is a conserved CheW domain (aa 26–165) and that
its C-terminus (aa 196–466) contains a CheR-like domain
Borrelia burgdorferi
© 2012 Blackwell Publishing Ltd, Molecular Microbiology,85, 782–794
(Fig. S1) (Djordjevic and Stock, 1997; 1998; Shiomi et al.,
2002). The E. coli CheA contains a CheW-like domain, P5,
which mediates the interaction between CheA and CheW
(Bilwes et al., 1999; Park et al., 2006). Sequence align-
ment showed that the three CheW proteins also share
certain similarity to the P5 domains from the CheA proteins
of E. coli and B. burgdorferi (Fig. S2).
The function of CheW has been extensively studied in
E. coli, and the key residues involved in the CheW/MCP
and CheW/CheA interactions have been identified (Liu and
Parkinson, 1989; 1991; Boukhvalova et al., 2002a,b;
Cardozo et al., 2010; Vu et al., 2012). B. burgdorferi
CheWs share 28% (CheW1), 28% (CheW2) and 30% (the
CheW domain in CheW3) sequence identity with E. coli
CheW (CheWEc). Sequence alignment disclosed that the
majority of the residues essential for the function of CheWEc
are conserved among the three CheWs (Fig. 1), including
I33, E38, G57, R62, G63, G99, V108 and G133. A few
residue variations were also observed (e.g. V36/I in
CheW1, V88/M and V105/I in CheW3; see Fig. 1). These
similarities suggest that all three CheWs may function like
CheW1and CheW3have more structural similarities with
The structure of Thermotoga maritima CheW (designated
as CheWTm) has been determined by nuclear magnetic
resonance (NMR) (Griswold and Dahlquist, 2002; Park
et al., 2006), and CheWEc and CheWTm appear to share a
very similar 3D structure (Li et al., 2007). To reveal the
structural features of CheW1, CheW2and CheW3, homol-
ogy modelling analysis was conducted using CheWTm as a
structure template. Like CheWTm, all three CheW proteins
are predicted to contain two b-sheet domains (domain 1
and domain 2), and each domain consists of a five-
stranded b-barrel (Fig. 2). In addition, five highly variable
regions (HVR) were identified (Fig. 2). Structural alignment
revealed that the root-mean-square deviations (RMSD) of
backbone atoms between CheWTm (blue) and CheW1
(yellow), CheW2(orange) or the N-terminal CheW domain
of CheW3(red) were 0.566 Å, 1.617 Å and 0.347 Å respec-
tively. In contrast to CheW1and CheW3, CheW2had a long
loop inserted near the N-terminus of bstrand 6 in domain 2
(Fig. 2B), within the binding interface predicted for CheA
(Griswold and Dahlquist, 2002; Park et al., 2006). These
structural features suggest that CheW1and CheW3are
more structurally similar to CheWEc and CheWTm than is
Immunoblot analysis of cheW mutants and their cognate
complemented strains
As a coupling protein, CheW interacts with both MCPs and
CheA. In E. coli, CheW plays a critical role in chemotaxis;
acheW null mutant constantly runs and is deficient in
Fig. 1. Sequence comparison between E. coli CheW and the three CheWs of B. burgdorferi. The numbers show the positions of residues in
E. coli CheW and B. burgdorferi CheW1, CheW2, and the CheW domain of CheW3. Dots represent functionally important residues identified in
E. coli CheW (Liu and Parkinson, 1989; Boukhvalova et al., 2002a,b; Alexandre and Zhulin, 2003). The black dots represent residues
conserved in all four CheWs, and grey dots represent residues that are different in one or more of the three CheWs of B. burgdorferi. The
boxes represent conserved residues of CheWs. The alignments were performed using the program MacVector 10.6.
K. Zhang
et al
© 2012 Blackwell Publishing Ltd, Molecular Microbiology,85, 782–794
chemotaxis (Parkinson, 1977; Liu and Parkinson, 1989;
1991). To investigate the roles of CheW1, CheW2and
CheW3in chemotaxis, the genes encoding these three
proteins were inactivated by allelic exchange mutagenesis
(described in Experimental procedures). A PCR analysis
showed that the individual cheW genes were targeted by
the antibiotic-resistant makers as expected (Fig. S3).
A single clone representing each mutation (DW1,DW2
and DW3, which represent the cheW1,cheW2and cheW3
mutants respectively) was selected for further character-
izations. Immunoblot analyses using anti-CheW antisera
(designated as aCheW1,aCheW2and aCheW3) showed
that CheW1, CheW2and CheW3were all detected in the
wild-type strain B31A but not in the corresponding mutant
clones (Fig. 3). Among these three mutants, as DW1
and DW3had altered chemosensory behaviours, these
two mutants were complemented using the vectors
CheW1/pBSV2G and CheW3/pBSV2G, which were con-
structed as described in Experimental procedures. Immu-
noblot analyses showed that the complementation of
cheW1(DW1+) and cheW3(DW3+) by the corresponding
wild-type genes restored the synthesis of CheW1(Fig. 3A)
and CheW3(Fig. 3C).
The cheW1and cheW3mutants are defective in
Chemotaxis in the DW1,DW2and DW3mutants was char-
acterized using swim plate and capillary assays. In the
swim plate assay, the DW2mutant formed similar-sized
colonies as the B31A strain (Fig. 4B). However, the DW1
and DW3mutants formed considerably smaller rings that
were similar to that of a DflaB strain (Fig. 4A and C, and
Fig. S4), a previously documented non-motile mutant
(Motaleb et al., 2000). Thus, cheW1and cheW3, but not
cheW2, are critical for chemotaxis under the tested con-
ditions. Consistent with the results of swim plate assay,
the capillary assay demonstrated that DW1and DW3do
not respond to GlcNAc as an attractant (Fig. 4D and F),
whereas the DW2 mutant showed the same response to
GlcNAc as the wild-type strain (Fig. 4E). The cognate
complemented strains, DW1+and DW3+, exhibited spread-
ing on the swim plates and chemotactic responses to
GlcNAc at wild-type levels (Fig. 4A, C, D and F). Collec-
tively, these results indicate that cheW1and cheW3are
required for B. burgdorferi chemotaxis, whereas cheW2is
dispensable for chemotaxis.
Fig. 2. Homology modelling of B. burgdorferi CheWs.
A. Structure alignment of CheW1(yellow), CheW3(red), E. coli CheW (green) and T. maritima CheW (blue).
B. Structure alignment of CheW2(orange), E. coli CheW and T. maritima CheW.
The N-terminal regions ahead of bstrand were removed for better visualization. T. maritima CheW (Griswold and Dahlquist, 2002) (Protein
Data Bank ID: 1K0S) was selected as the basis for structural modelling using the program Modeller 9v7 (Sali and Blundell, 1993). All
structures were analysed and visualized in PyMol. The numbers represent the highly variable regions (HVR) identified. This figure is available
in colour online at
Fig. 3. Immunoblot analysis of the three cheW mutants and their complemented strains.
A. Immunoblot analysis of the cheW1mutant (DW1) and its complemented strain (DW1+) using aCheW1.
B. Immunoblot analysis of the cheW2mutant (DW2) using aCheW2.
C. Immunoblot analysis of the cheW3mutant (DW3), its complemented strain (DW3+), and the mutant complemented with the N-terminal CheW
domain (aa 1–210) of CheW3(DW3N+) using aCheW3.
The predicted molecular weights of CheW1, CheW2, CheW3and the N-terminal CheW domain of CheW3are approximately 20 kDa, 21 kDa,
53 kDa and 24 kDa respectively.
Borrelia burgdorferi
© 2012 Blackwell Publishing Ltd, Molecular Microbiology,85, 782–794
The cheW1and cheW3mutants show an altered
swimming behaviour
Non-chemotactic mutants often show altered swimming
behaviours, e.g. the cheA2and cheY3mutants of B. burg-
dorferi fail to reverse and constantly run (Li et al., 2002;
Motaleb et al., 2011b). The tracking analysis using a
computer-assisted cell tracker coupled with video micros-
copy disclosed that the DW2mutant had swimming behav-
iour indistinguishable from the wild type (Videos S1 and
S2, Table 1), whereas the DW1and DW3mutants had
altered swimming behaviours. The DW3mutant failed to
reverse and constantly ran in one direction (Video S3,
Table 1), like the cheA2and cheY3mutants of B. burgdor-
feri. The behaviour of the DW1mutant is mixed (Video S4):
approximately half of the cells (21 out of 50) failed to
reverse and swam exclusively in one direction. The
remainder of the cells (29 out of 50) reversed, but at a lower
reversal frequency (9 reversals min-1) compared with the
wild type (23 reversals min-1). A similar pattern was
observed in a reconstructed DW1mutant, suggesting that
the observed mixed phenotype is stochastic and not
caused by genetic heterogeneity. The complemented
mutants (DW3+and DW1+) had a similar swimming behav-
iour as the wild type (Videos S3A and S4A, and Table 1). All
three cheW mutants had similar swimming velocities as the
wild type (Table 1), ranging from 9 to 12 mms
-1. Thus, none
of the cheW mutations causes a decrease in the propulsive
force generated by the flagella.
The CheR-like domain in CheW3is not required for
CheW3possesses a CheR-like domain at its C-terminus
(Fig. S1). In E. coli, CheR functions as a methyltransferase
that is involved in chemoreceptor adaptation (Djordjevic
and Stock, 1997; 1998; Porter et al., 2011). Searching
large sets of CheW homologues from microbial genome
databases revealed that only CheWs from some spirocha-
ete species have a similar domain composition as CheW3,
including CheW1(TP_0364) of Treponema pallidum and
CheW1(TDE_1492) of Treponema denticola (Fraser et al.,
1998; Seshadri et al., 2004). To determine whether the
CheR-like domain is required for normal chemotaxis, the
DW3mutant was complemented with a plasmid producing
Fig. 4. B. burgdorferi cheW1and cheW3mutants are non-chemotactic. Swim plate (A) and capillary (D) assays of the cheW1mutant (DW1)
and its complemented strain (DW1+). Swim plate (B) and capillary (E) assays of the cheW2mutant (DW2). Swim plate (C) and capillary (F)
assays of the cheW3mutant (DW3) and its complemented strains (DW3+and DW3N+). The swim plate and capillary assays were carried out as
previously described (Motaleb et al., 2000; Li et al., 2002; Bakker et al., 2007). For the swim-plate assay, DflaB, a previously constructed
non-motile mutant (Motaleb et al., 2000), was used as a control to determine the size of non-spreading colonies on the plates. For the
capillary assay, N-acetyl-D-glucosamine (GlcNAc) was used as an attractant. Results are expressed as the means SEM from five plates or
capillary tubes. The asterisk (*) represents a Pvalue <0.01.
Table 1. Effects of CheWs on swimming behaviours of
B. burgdorferi.
Mean velocity
Mean number of
reversals min-1SEMa
B31A 10.0 0.8 23.3 4.2
DW112.3 1.5 9.0 6.0b
DW210.0 1.4 20.0 3.2
DW311.0 0.8 0.0c
DW1+9.8 2.2 25.0 2.6
DW3+9.7 1.8 26.0 1.9
DW3N+9.0 1.7 24.0 2.1
a. Standard errors of the means were calculated from data obtained
from at least 30 individual tracked cells of each strain.
b. Approximately one half of the cells ran in one direction and did not
reverse; the other half of the cells did reverse and the mean reversal
frequency was calculated from this group of the cells.
c. Cells ran in one direction and did not reverse.
K. Zhang
et al
© 2012 Blackwell Publishing Ltd, Molecular Microbiology,85, 782–794
only the N-terminal CheW domain of CheW3(aa 1–210).
Immunoblotting using aCheW3showed that the expression
of the N-terminal CheW domain was restored in the
complemented clone (DW3N+) (Fig. 3C). The swim plate
(Fig. 4C), capillary (Fig. 4F) and tracking (Table 1) assays
demonstrated that chemotaxis in the DW3N+strain was
indistinguishable from that of the wild-type and DW3+
strains, indicating that deletion of the CheR-like domain
does not affect the chemotactic function of CheW3under
the conditions tested.
Loss of CheW1or CheW3affects chemoreceptor
assembly at the cell poles
In E. coli, CheW is essential for the assembly of chemore-
ceptor arrays at the cell poles (Maddock and Shapiro,
1993; Sourjik and Berg, 2000; Studdert and Parkinson,
2005). Our previous studies showed that B. burgdorferi
MCPs also form arrays at the cell poles (Xu et al., 2011). To
determine whether the B. burgdorferi cheW mutants are
defective in chemoreceptor assembly, the cellular location
of the MCPs in the three mutants was determined by IFA
using an antibody targeted specifically against B. burgdor-
feri MCP3(Xu et al., 2011).As expected, bright fluorescent
loci were observed at both poles in wild-type cells (Fig. 5A).
A similar pattern was observed in DW2cells (Fig. 5C), but
not in DW3cells, in which the fluorescence was diffused
(Fig. 5D). Although fluorescent loci were still evident at the
poles of DW1mutant cells, the fluorescence signals were
considerably reduced, and even absent in many cells
(Fig. 5B). The IFA results suggest that CheW1and CheW3,
but not CheW2, are involved in the assembly and localiza-
tion of the chemoreceptor arrays.
Cryo-ET was conducted to determine the cellular loca-
tions and ultrastructures of the chemoreceptor arrays in the
three cheW mutants more precisely. Chemoreceptor
arrays could be readily recognized as prominent ‘basal
plate’-like structures (Zhang et al., 2004; Briegel et al.,
2009; 2012; Xu et al., 2011; Liu et al., 2012) at the poles of
wild-type (Fig. 6A) and DW2cells (Fig. 6B).The arrays had
an average length of 159 86 nm (n=19 cells, Table 2).
No chemoreceptor arrays were observed in any of the DW3
cells examined (0 out of 25 cells, Fig. 6C). However, the
Fig. 5. Localization of B. burgdorferi
chemoreceptor arrays using IFA. The wild
type (A), the DW1(B), DW2(C) and DW3(D)
mutant cells were fixed with methanol, stained
with anti-MCP3antibody, and counterstained
with anti-rat Texas red antibody as previously
described (Li et al., 2010; Xu et al., 2011).
The micrographs were taken under DIC light
microcopy or fluorescence microscopy with a
tetramethylrhodamine isothiocyanate (TRITC)
emission filter, and the resultant images were
merged. Arrows point to the location of the
chemoreceptor arrays within cells. This figure
is available in colour online at
Fig. 6. Detection of B. burgdorferi chemoreceptor arrays by
cryo-ET. The cryo-ET analysis was carried out as previously
described (Xu et al., 2011). Six strains were included: (A) B31A, (B)
DW2, (C) and (D) DW3and its complemented strain DW3+, and (E)
and (F) DW1and its complemented strain DW1+. Arrows point to
chemoreceptor arrays. OM, outer membrane; CM, cytoplasmic
membrane; A/W, the basal plate composed of CheA and CheW.
Borrelia burgdorferi
© 2012 Blackwell Publishing Ltd, Molecular Microbiology,85, 782–794
arrays could be readily detected in its complemented strain
DW3+(12 out of 30 cells, Fig. 6D). With the DW1mutant, the
arrays were still evident in a small portion of the cells (4 out
31 cells, Fig. 6E), but their sizes were substantially
reduced (average length of 75 7 nm, n=4 cells) com-
pared with those of the wild type or the complemented
DW1+strain (Fig. 6F, 152 58 nm, n=10 cells). The
cryo-ET results are consistent with the IFA data and thus
further confirm that both CheW1and CheW3are involved in
assembly of the chemoreceptor arrays, whereas CheW2is
CheW1and CheW3interact with CheA2, whereas CheW2
binds CheA1
In E. coli, the ternary complex of MCP–CheW–CheA is the
core structural unit in the signalling pathway of chemotaxis
(Wadhams and Armitage, 2004; Hazelbauer et al., 2008).
B. burgdorferi has two CheA homologues, CheA1and
CheA2. Identifying the interactions between the two CheAs
and the three CheWs will help us understand the complex-
ity of chemotaxis signalling pathways in B. burgdorferi.
Co-IP experiments were carried out to reveal the interac-
tions between the two CheAs and the three CheWs. For the
co-IP assays, either CheA1antibody (aCheA1) or CheA2
antibody (aCheA2) was first co-incubated with whole-cell
lysates of the B31A wild type and a previously constructed
double cheA1cheA2mutant (designated as DA1A2and used
as a negative control) (Li et al., 2002). The co-precipitated
products were then probed with aCheW1,aCheW2or
aCheW3respectively.As shown in Fig. 7, CheW1(Fig. 7A)
and CheW3(Fig. 7C) were detected in the samples precipi-
tated by aCheA2(left panel, Fig. 7) but not by aCheA1(right
panel, Fig. 7), whereas CheW2was detected in the
samples precipitated by aCheA1(right panel, Fig. 7B) but
not by aCheA2(left panel, Fig. 7B), suggesting that both
CheW1and CheW3interact with CheA2, whereas CheW2
binds CheA1. To confirm that CheW1and CheW3interact
with CheA2,aCheW1and aCheW3were used in the co-IP
assays, and the co-IP samples were probed with aCheA2.
As expected, CheA2was detected in the co-precipitated
products from the wild type but not from the DW1and DW3
mutants (Fig. 7D). Collectively, the results of the co-IP
assays show that CheW1and CheW3interact with CheA2
but not with CheA1, whereas CheW2interacts with CheA1
but not with CheA2.
As a coupling protein, CheWEc has four known activities:
binding to CheA, binding to MCPs, promoting formation of
MCP–CheW–CheA ternary complexes and chemorecep-
tor arrays, and enabling MCPs to modulate CheA autoki-
nase activity (Liu and Parkinson, 1989; Gegner et al.,
1992; Cardozo et al., 2010). In this report, a comprehen-
sive approach has been applied to investigate the roles of
the products of the three cheW genes in B. burgdorferi. The
results indicate that CheW1and CheW3play a similar role
as the CheW of E. coli, because the DW1and DW3mutants
showed an altered swimming behaviour (Table 1 and Vid-
eos S3 and S4) and failed to respond to attractant stimuli
(Fig. 4D and F). Also, the IFAand cryo-ET studies showed
that these two mutants are unable to assemble intact
chemoreceptor arrays at the cell poles of B. burgdorferi
(Figs 5 and 6). In contrast to DW1and DW3, the DW2mutant
behaved like the wild type with respect to chemotactic
response to attractants (Fig. 4E), swimming behaviour
(Table 1), and chemoreceptor assembly (Figs 5C and 6B).
Collectively, these results indicate that CheW1and CheW3
are essential for the chemotaxis of B. burgdorferi, whereas
CheW2is dispensable for the chemotaxis under the tested
Table 2. Impact of CheWs on B. burgdorferi chemoreceptor
length (nm)
B31A 30 19 159 86
DW131 4 75 7
DW230 12 130 30
DW325 0 NA
DW1+20 10 152 58
DW3+30 12 155 77
Fig. 7. Detecting the interactions between two CheAs and three
CheWs of B. burgdorferi by co-IP.
A–C. Pull-down of the CheWs using aCheA1(right panel) or
aCheA2(left panel). Precipitated proteins were probed with
aCheW1(A), aCheW2(B) or aCheW3(C). A previously constructed
cheA1A2double-deletion mutant (DA1A2)ofB. burgdorferi (Li et al.,
2002) was used as a negative control for the co-IP.
D. Pull-down of CheA2using aCheW1(left panel) and aCheW3
(right panel). Precipitated proteins were probed with aCheA2.
Extracts from the DW1or DW3mutants were used as negative
controls for the co-IP.
K. Zhang
et al
© 2012 Blackwell Publishing Ltd, Molecular Microbiology,85, 782–794
in vitro conditions. Consistent with this proposition, the
homology modelling analysis predicts that CheW2shares
the least structural similarity to CheWEc and CheWTm
(Fig. 2). It is noteworthy to point out that CheW2has a long
loop insertion near the binding interface of CheW and
CheA (Fig. 2B). This insertion may disrupt the local envi-
ronment of the CheA binding surface and consequently
prevent CheW2from interacting effectively with CheA2,a
histidine kinase that is essential for chemotaxis of B. burg-
dorferi (Li et al., 2002; Sze et al., 2012).
IFA and cryo-ET assays demonstrate that CheW3plays
a more important role than CheW1in the assembly of
chemoreceptor arrays at the cell poles of B. burgdorferi.
The IFA results showed that the polar-localized chemore-
ceptor arrays were completely disrupted in DW3cells
(Fig. 5D and Fig. S5), nor did cryo-ET analyses find any
array-like structures in the mutant (Fig. 6C, Table 2). The
observed phenotype of the DW3mutant is very similar to
that of an E. coli cheW mutant (Maddock and Shapiro,
1993; Sourjik and Berg, 2000; Zhang et al., 2004). Unlike
the situation in DW3, IFAstill detected weak polar localized
signals in DW1cells (Fig. 5B), and arrays could still be
observed by cryo-ET in a small portion of the DW1cells
(Fig. 6E). The average length of the chemoreceptor
arrays observed in the DW1cells was approximately
twofold less than those in the wild type and its comple-
mented strain, DW1+(Table 2). Recent cryo-ET studies of
E. coli MCPs show that the basal plates of the arrays
consist primarily of CheA and CheW (Briegel et al., 2009;
2012; Liu et al., 2012). Thus, it is conceivable that CheW1
contributes to the stability of the basal plates but is not
essential for their formation. Approximately one half of the
DW1cells swim smoothly, and the other half still reverse
but with a lower frequency than wild type (Video S4 and
Table 1). The observed heterogeneous phenotype of the
DW1mutant is not due to genetic heterogeneity because
when the mutation was recloned, the same mixed pheno-
type of the original DW1mutant was observed. Moreover,
genetic complementation totally restored the wild-type
phenotype (Video S4A and Table 1).
In E. coli, CheW tethers CheA to the MCPs and affects
the activity of CheA, which in turn controls the level of
CheY-P. The inactivation of cheW completely blocks pro-
duction of CheY-P. Thus, the flagellar motors are locked in
CCW rotation, and a cheW mutant constantly runs (Gegner
et al., 1992; Wadhams and Armitage, 2004; Hazelbauer
et al., 2008). Our previous studies show that, of the two
CheAs and three CheYs of B. burgdorferi, only CheA2and
CheY3are involved in chemotaxis (Li et al., 2002; Motaleb
et al., 2011b). The cheA2and cheY3mutants are smooth
swimming and non-chemotactic, suggesting that CheY3-P
directly controls the rotation of flagellar motors. Because
the chemoreceptor arrays in the DW1cells are only partially
disrupted (Figs 5B and 6E), it is possible that CheW1plays
an auxiliary role in coupling CheA2to the MCPs. The
decrease in CheY3-P associated with the reduced coupling
of CheA2results in a baseline concentration that spans the
threshold required to elicit reversals.
The results presented here raise the possibility that
B. burgdorferi may have two different chemosensory path-
ways (Charon and Goldstein, 2002; Li et al., 2002;
Motaleb et al., 2011b).Among the multiple homologues of
cheA,cheW and cheY, only cheA2,cheY3,cheW1and
cheW3are essential for chemotaxis in vitro. Other than
cheW1, all of the essential che genes are located within the
flaA operon (cheA2, cheW3, cheX and cheY3), whereas
most of the che genes that are dispensable for chemotaxis
reside within an operon that contains cheA1,cheY2and
cheW2(Fraser et al., 1997; Ge and Charon, 1997; Charon
and Goldstein, 2002; Li et al., 2002). Co-IP assays dem-
onstrated that CheW1and CheW3interact with CheA2,
whereas CheW2binds CheA1. Thus, we favour the idea
that B. burgdorferi has two chemosensory pathways:
CheW1/CheW3-CheA2-CheY3form the pathway that is
essential for chemotaxis under the conditions usually used
in vitro, and CheW2-CheA1-CheY2and/or CheY1form
another pathway that may be used only under other con-
ditions that have yet to be duplicated in the laboratory.
Why might B. burgdorferi have two chemosensory path-
ways? In nature, B. burgdorferi is maintained via an
enzootic cycle comprising both mammalian hosts and an
Ixodes tick vector (for recent reviews, see Steere et al.,
2004; Rosa et al., 2005; Samuels, 2011; Radolf et al.,
2012). The enzootic cycle begins with the feeding by an
uninfected tick on an infected vertebrate. After the feeding,
the spirochaetes remain in the tick gut throughout the
moulting process. At the time that the infected tick takes a
blood meal on a mammal, the spirochaetes begin to mul-
tiply and migrate from the tick gut to the salivary glands,
from which they are transmitted to a new host, thereby
completing the enzootic cycle. To adapt to different hosts
and complete its enzootic cycle, B. burgdorferi may need
one chemosensory pathway, perhaps represented by
CheW1/CheW3-CheA2-CheY3, for chemotaxis in mamma-
lian hosts. The pathway involving CheW2-CheA1-CheY2
and/or CheY1may be activated in the tick vector and/or
during the transmission from tick to mammal. Our recent
study of the role of cheA2in the enzootic cycle of B. burg-
dorferi (Sze et al., 2012) is consistent with the proposal that
the CheW1/CheW3-CheA2-CheY3pathway is important in
the mammalian host. Inactivation of cheA2decreased the
ability of B. burgdorferi to establish infection in mice, but
not in ticks. The true function of the CheW2-CheA1-CheY2
and/or CheY1pathway remains obscure. It could be
involved in chemotaxis in the tick vector, perhaps in migra-
tion of the spirochaetes to the salivary glands, or it may
function in a signal transduction pathway that regulates
gene expression of B. burgdorferi.
Borrelia burgdorferi
© 2012 Blackwell Publishing Ltd, Molecular Microbiology,85, 782–794
The incorporation of two coupling proteins (CheW1and
CheW3) into one chemosensory pathway is different from
the situation in other bacteria that have more than one
homologue of CheW, such as Vibrio cholera and Rhodo-
bacter sphaeroides (for recent review, see Butler and
Camilli, 2005; Rao et al., 2008; Alexander et al., 2010;
Porter et al., 2011). In these organisms, either only one
CheW homologue functions as a key coupling protein
essential for chemotaxis, or CheW homologues are func-
tionally redundant. For instance, V. cholera has three
CheW homologues, but only CheW-1 is required for
chemotaxis (Butler et al., 2006). Among the four CheW
homologues of R. sphaeroides, CheW2is essential for
chemotaxis and chemoreceptor clustering; and deletions
of other three cheWs either have no impact on chemot-
axis or only conditionally affect chemotactic responses
and chemoreceptor localization (Hamblin et al., 1997a;
1997b; Martin et al., 2001). It is intriguing to think that the
requirement for two CheW proteins in B. burgdorferi may
have to do with the extra task of co-ordinating flagellar
reversals at the two ends of an elongated cell body.
Experimental procedures
Bacterial strains and growth conditions
High-passage, avirulent B. burgdorferi sensu stricto strain
B31A (wild type) (Bono et al., 2000) and its derivative
mutants were grown in BSK-II liquid medium or on semi-solid
agar plates at 34°C in a humidified incubator in the presence
of 3.4% CO2, as previously documented (Li et al., 2002). The
E. coli strains were grown in LB medium at 37°C with appro-
priate antibiotics.
Construction of cheW mutants
The cheW1,cheW2and cheW3genes were inactivated by
allelic exchange mutagenesis as illustrated in Fig. S6. To
construct the vector for inactivation of cheW1(gene locus
bb0312; gene length, 531 bp), a 120 bp HindIII fragment was
deleted and replaced by a kanamycin-resistance cassette
(aphI) (Elias et al., 2003). To construct the vector for inactiva-
tion of cheW2(bb0565; gene length, 543 bp), the aphI cassette
was directly inserted into an EcoRV restriction cut site within
the gene. To construct the vector for inactivation of cheW3
(bb0670; gene length, 1401 bp), the entire open reading frame
(orf) was deleted and replaced with a promoterless streptomy-
cin resistance marker (aadA1), as recently described (Frank
et al., 2003; Motaleb et al., 2011a). The resultant constructs
were designated as W1::aphI,W2::aphI and W3::aadA1
(Fig. S6) respectively. The PCR primers for constructing these
vectors are listed in Table S1. To knock out the cheW genes,
these vectors were first linearized and then separately trans-
formed into B31A competent cells via electroporation as
previously reported (Samuels, 1995). Transformants were
selected on BSK-II agar plates containing 350 mgml
mycin (for W1::aphI and W2::aphI)or50mgml
(for W3::aadA1).
Constructing genetic complementation vectors
To construct the vector for the complementation of the cheW3
mutant, the entire cheW3gene and its native promoter (Pami)
(Yang and Li, 2009) were first amplified by PCR with two pairs
of primers (P17/P18 for Pami;P19/P20 for cheW3). The resultant
PCR products were then fused together via PCR using
primers P17/P20. The resultant PamicheW3fragment was first
cloned into the pGEM®-T Easy vector (Promega, Madison,
WI) and then subcloned into pBSV2G, a shuttle vector of
B. burgdorferi that contains a gentamicin-resistance cassette
(aacC1) (Elias et al., 2003; Rosa et al., 2005). The final con-
struct was named CheW3/pBSV2G (Fig. S6). A similar strat-
egy was used to construct a vector for complementation of
the cheW1mutant (CheW1/pBSV2G) and the vector for
complementation of the cheW3mutant (CheW3N+/pBSV2G)
with the N-terminal domain of CheW3(1–210 amino acids).
The PCR primers for constructing the complementation
vectors are listed in Table S1.
Generation of polyclonal antisera against CheW1,
CheW2or CheW3
The entire orfs (without the translation initiation ATG/GTG
codon) of cheW1,cheW2and cheW3were amplified by PCR
(the primers are listed in Table S1). The obtained PCR prod-
ucts were first cloned into the pGEM®-T Easy vector
(Promega), and then subcloned into the pQE30 expression
vector (Qiagen, Valencia, CA), which encodes an N-terminal
histidine tag. The expression of these three genes was
induced using 1 mM isopropyl-b-D-thiogalactoside (IPTG).
The recombinant proteins were purified by a nickel agarose
column and concentrated in 10 kDa molecular weight cut-off
Amicon Ultra centrifugal concentrators (Millipore, Billerica,
MA). Rats (for rCheW1and rCheW2) and rabbits (for rCheW3)
were immunized with 1–5 mg of purified recombinant pro-
teins during a 1-month period using standard methods. The
obtained polyclonal antisera were further purified using affin-
ity chromatography with the AminoLink Plus Immobilization
Kit (Thermo Scientific, Rockford, IL) and eluted as recom-
mended by the manufacturer.
Bacterial motion tracking analysis, swim plate and
capillary assays
The swimming velocity of B. burgdorferi cells was measured
using a computer-based motion tracking system. Swim plate
assays were carried out using 0.35% agarose with BSK-II
medium diluted 1:10 with Dulbecco’s phosphate-buffered
saline (DPBS, pH 7.5) without divalent cations, as previously
documented (Motaleb et al., 2000; Li et al., 2002). The plates
were incubated for 3–4 days at 34°C in the presence of 3.4%
CO2. Diameters of the swim rings that appeared on the plates
were measured and recorded in millimetres (mm). A previ-
ously constructed non-motile flaB-mutant (DflaB) (Motaleb
et al., 2000) was used as a negative control to determine the
initial inoculum size. Capillary assays were carried out as
previously documented with minor modifications (Li et al.,
2002; Bakker et al., 2007). Briefly, B. burgdorferi cells were
grown to late-logarithmic-phase (~5–7 ¥107cells ml-1) and
K. Zhang
et al
© 2012 Blackwell Publishing Ltd, Molecular Microbiology,85, 782–794
harvested by low-speed centrifugations (1800 g). The har-
vested cells were then resuspended in the motility buffer
(Bakker et al., 2007). Capillary tubes filled with either the
attractant [0.1 M N-acetyl-glucosamine (GlcNAc) dissolved in
the motility buffer] or only motility buffer (negative control)
were sealed and inserted into microcentrifuge tubes contain-
ing 200 ml of resuspended cells (7 ¥108cells ml-1). After 2 h
incubation at 34°C in a humidified chamber, the solutions
were expelled from the capillary tubes, and the spirochaete
cells were enumerated using Petroff-Hausser counting cham-
bers under a dark-field microscope. A positive chemotactic
response was defined as at least twice as many cells entering
the attractant-filled tubes as the buffer-filled tubes. For the
swim plate, motion tracking and capillary assays, results
are expressed as means standard errors of the means
(SEM). The significance of the difference between different
strains was evaluated with an unpaired Student’s t-test
(Pvalue <0.01).
Electrophoresis and immunoblot analyses
Sodium-dodecyl-sulphate polyacrylamide-gel electrophore-
sis (SDS-PAGE) and immunoblotting using the enhanced
chemiluminescent detection system were carried out as
described before (Li et al., 2010; Sze et al., 2011).
B. burgdorferi cells were grown at 34°C and harvested at
early stationary phase (approximately 108cells ml-1). The
whole-cell lysates were prepared by washing cells once in
PBS buffer (phosphate-buffered saline, pH 7.5) and then
boiling for 5 min in Laemmli sample buffer. The same amount
of cell lysates (~10–20 mg) were separated on SDS-PAGE
gels and transferred to PVDF membrane (Bio-Rad Laborato-
ries, Hercules, CA). The immunoblots were probed with spe-
cific antibodies against various proteins (CheA1, CheA2,
CheW1, CheW2and CheW3) and developed using horserad-
ish peroxidase-coupled secondary antibody with an ECL
luminol assay.
Co-IP assay
The co-IP assay was carried out as previously described
(Motaleb et al., 2004). Briefly, 200 ml of the late-logarithmic-
phase (~5–7 ¥107cells ml-1)B. burgdorferi cultures were
harvested by centrifugation and washed twice with PBS
buffer containing 5 mM MgCl2. The resultant cell pellets were
resuspended in TSEA buffer (50 mM Tris-HCl, 150 mM NaCl,
5 mM EDTA, 0.05% sodium azide, pH 7.5) containing
Nonidet P-40 (1%, v/v) and phenylmethylsulphonyl fluoride
(50 mgml
-1) and then incubated at 37°C for 1 h. After the
incubation, the obtained samples were centrifuged (1600 g
for 30 min, 25°C). The resultant cell pellets were resus-
pended in the PBS buffer and French pressed followed by
centrifugation (15 000 gfor 30 min, 25°C). Approximately
200 ml of the obtained supernatants were incubated with
50 ml of the polyclonal anti-CheAs (aCheA1and aCheA2)or
anti-CheWs (aCheW1,aCheW2and aCheW3) for 1 h at 25°C
in the presence of 1% bovine serum albumin (BSA). After the
incubation, 50 ml of protein A (Calbiochem-Behring Corpora-
tion, La Jolla, CA) was added to each sample and further
incubated at 25°C for 1 h. The immunoprecipitates and con-
trols were centrifuged at 1600 gat 25°C and washed three
times with 1 ml of TSEA buffer containing 0.05% Tween-20.
The final pellets were suspended in 100 ml of electrophoresis
sample buffer, boiled for 5 min and briefly centrifuged. For the
immunoblots, 10 ml of the supernatants was applied to each
lane of SDS-PAGE gels as described above.
IFA and cryo-ET
IFA and cryo-ET assays were carried out to determine the
cellular locations of MCPs in B31A and the three cheW
mutants as previously described (Xu et al., 2011). For the
IFA, aMCP3, a specific antibody against B. burgdorferi MCP3,
was used. For the cryo-ET analysis, freshly prepared
B. burgdorferi cultures were deposited onto a glow-
discharged holey carbon EM grid, blotted, and rapidly frozen
in liquid ethane. The frozen-hydrated specimens were
imaged at -170°C using a Polara G2 electron microscope
(FEI Company, Hillsboro, Oregon) equipped with a field emis-
sion gun and a 4 K ¥4 K CCD camera (TVIPS; GMBH,
Germany). The microscope was operated at 300 kV with a
magnification of 31 000¥. Low-dose single-axis tilt series
were collected from each bacterium at -6mm defocus with a
cumulative dose of ~100 e-2distributed over 65 images
with an angular increment of 2°, covering a range from -64°
to +64°. The tilt series images were aligned and recon-
structed using the IMOD software package (Kremer et al.,
1996). In total, cryo tomograms of B31A (30 cells), a cheW1
mutant (31 cells) and its complemented strain (20 cells), a
cheW2mutant (30 cells), a cheW3mutant (25 cells) and its
complemented strain (30 cells) were reconstructed and visu-
alized using IMOD (Kremer et al., 1996).
Homology model construction of CheW1, CheW2
and CheW3
The NMR structure of T. maritima CheW (Protein Data Bank
ID: 1K0S) (Griswold and Dahlquist, 2002) was selected as a
template for the homology modelling analysis of CheW1,
CheW2and N-terminus CheW-like domain of CheW3. Pair-
wise sequence alignment of CheW homologues was con-
ducted using CLUSTAL X. Automodel module in Modeller 9v7
(Sali and Blundell, 1993) was applied to obtain the final
refined structures. All structures were analysed and visual-
ized in PyMol (The PyMol Molecular Graphic System, Version, Schrodinger, LLC). The qualities of the models were
evaluated by PDBsum (Laskowski, 2001).
This research was supported by Public Health Service grants
(AI073354 and AI078958) to C. Li, GM072004 to C. Wolge-
muth; J. Liu was supported in part by Grants AI087946 from
the National Institute of Allergy and Infectious Diseases
(NIAID) and AU-1714 from the Welch Foundation.
Alexander, R.P., Lowenthal, A.C., Harshey, R.M., and Otte-
mann, K.M. (2010) CheV: CheW-like coupling proteins at
Borrelia burgdorferi
© 2012 Blackwell Publishing Ltd, Molecular Microbiology,85, 782–794
the core of the chemotaxis signaling network. Trends Micro-
biol 18: 494–503.
Alexandre, G., and Zhulin, I.B. (2003) Different evolutionary
constraints on chemotaxis proteins CheW and CheY
revealed by heterologous expression studies and protein
sequence analysis. J Bacteriol 185: 544–552.
Bakker, R.G., Li, C., Miller, M.R., Cunningham, C., and
Charon, N.W. (2007) Identification of specific chemoattrac-
tants and genetic complementation of a Borrelia burgdor-
feri chemotaxis mutant: flow cytometry-based capillary
tube chemotaxis assay. Appl Environ Microbiol 73: 1180–
Bilwes, A.M., Alex, L.A., Crane, B.R., and Simon, M.I. (1999)
Structure of CheA, a signal-transducing histidine kinase.
Cell 96: 131–141.
Bono, J.L., Elias, A.F., Kupko, J.D., III, Stevenson, B., Tilly,
K., and Rosa, P. (2000) Efficient targeted mutagenesis in
Borrelia burgdorferi.J Bacteriol 182: 2445–2452.
Boukhvalova, M., VanBruggen, R., and Stewart, R.C. (2002a)
CheA kinase and chemoreceptor interaction surfaces on
CheW. J Biol Chem 277: 23596–23603.
Boukhvalova, M.S., Dahlquist, F.W., and Stewart, R.C.
(2002b) CheW binding interactions with CheA and Tar.
Importance for chemotaxis signaling in Escherichia coli.
J Biol Chem 277: 22251–22259.
Briegel, A., Ortega, D.R., Tocheva, E.I., Wuichet, K., Li, Z.,
Chen, S., et al. (2009) Universal architecture of bacterial
chemoreceptor arrays. Proc Natl Acad Sci USA 106:
Briegel, A., Li, X., Bilwes, A.M., Hughes, K.T., Jensen, G.J.,
and Crane, B.R. (2012) Bacterial chemoreceptor arrays
are hexagonally packed trimers of receptor dimers net-
worked by rings of kinase and coupling proteins. Proc Natl
Acad Sci USA 109: 3766–3771.
Burgdorfer, W., Barbour, A.G., Hayes, S.F., Benach, J.L.,
Grunwaldt, E., and Davis, J.P. (1982) Lyme disease, a
tick-borne spirochetosis? Science 216: 1317–1319.
Butler, S.M., and Camilli, A. (2005) Going against the grain:
chemotaxis and infection in Vibrio cholerae.Nat Rev Micro-
biol 3: 611–620.
Butler, S.M., Nelson, E.J., Chowdhury, N., Faruque, S.M.,
Calderwood, S.B., and Camilli, A. (2006) Cholera stool
bacteria repress chemotaxis to increase infectivity. Mol
Microbiol 60: 417–426.
Cardozo, M.J., Massazza, D.A., Parkinson, J.S., and Stud-
dert, C.A. (2010) Disruption of chemoreceptor signalling
arrays by high levels of CheW, the receptor-kinase cou-
pling protein. Mol Microbiol 75: 1171–1181.
Charon, N.W., and Goldstein, S.F. (2002) Genetics of motility
and chemotaxis of a fascinating group of bacteria: the
spriochetes. Annu Rev Genet 36: 47–73.
Charon, N.W., Goldstein, S.F., Marko, M., Hsieh, C., Geb-
hardt, L.L., Motaleb, M.A., et al. (2009) The flat-ribbon con-
figuration of the periplasmic flagella of Borrelia burgdorferi
and its relationship to motility and morphology. J Bacteriol
191: 600–607.
Charon, N.W., Cockburn, A., Li, C., Liu, J., Miller, K., Miller,
M.R., et al. (2012) The unique paradigm of spirochete
motility and chemotaxis. Annu Rev Immunol (in press).
Djordjevic, S., and Stock, A.M. (1997) Crystal structure
of the chemotaxis receptor methyltransferase CheR
suggests a conserved structural motif for binding
S-adenosylmethionine. Structure 5: 545–558.
Djordjevic, S., and Stock, A.M. (1998) Chemotaxis receptor
recognition by protein methyltransferase CheR. Nat Struct
Biol 5: 446–450.
Dombrowski, C., Kan, W., Motaleb, M.A., Charon, N.W.,
Goldstein, R.E., and Wolgemuth, C.W. (2009) The elastic
basis for the shape of Borrelia burgdorferi.Biophys J 96:
Elias, A.F., Bono, J.L., Kupko, J.J., III, Stewart, P.E., Krum,
J.G., and Rosa, P.A. (2003) New antibiotic resistance cas-
settes suitable for genetic studies in Borrelia burgdorferi.
J Mol Microbiol Biotechnol 6: 29–40.
Frank, K.L., Bundle, S.F., Kresge, M.E., Eggers, C.H., and
Samuels, D.S. (2003) aadA confers streptomycin resis-
tance in Borrelia burgdorferi.J Bacteriol 185: 6723–6727.
Fraser, C.M., Casjens, S., Huang, W.M., Sutton, G.G.,
Clayton, R., Lathigra, R., et al. (1997) Genomic sequence
of a Lyme disease spirochaete, Borrelia burgdorferi.Nature
390: 580–586.
Fraser, C.M., Norris, S.J., Weinstock, C.M., White, O.,
Sutton, G.G., Dodson, R., et al. (1998) Complete genome
sequence of Treponema pallidum, the syphilis spirochete.
Science 281: 375–388.
Ge, Y., and Charon, N.W. (1997) Molecular characterization
of a flagellar/chemotaxis operon in the spirochete Borrelia
burgdorferi.FEMS Microbiol Lett 153: 425–431.
Gegner, J.A., Graham, D.R., Roth, A.F., and Dahlquist, F.W.
(1992) Assembly of an MCP receptor, CheW, and kinase
CheA complex in the bacterial chemotaxis signal transduc-
tion pathway. Cell 70: 975–982.
Goldstein, S.F., Charon, N.W., and Kreiling, J.A. (1994) Bor-
relia burgdorferi swims with a planar waveform similar to
that of eukaryotic flagella. Proc Natl Acad Sci USA 91:
Griswold, I.J., and Dahlquist, F.W. (2002) The dynamic
behavior of CheW from Thermotoga maritima in solution,
as determined by nuclear magnetic resonance: implica-
tions for potential protein-protein interaction sites. Biophys
Chem 101–102: 359–373.
Hamblin, P.A., Bourne, N.A., and Armitage, J.P. (1997a)
Characterization of the chemotaxis protein CheW from
Rhodobacter sphaeroides and its effect on the behaviour of
Escherichia coli.Mol Microbiol 24: 41–51.
Hamblin, P.A., Maguire, B.A., Grishanin, R.N., and Armitage,
J.P. (1997b) Evidence for two chemosensory pathways in
Rhodobacter sphaeroides.Mol Microbiol 26: 1083–1096.
Harman, M.W., Dunham-Ems, S.M., Caimano, M.J., Belper-
ron, A.A., Bockenstedt, L.K., Fu, H.C., et al. (2012) The
heterogeneous motility of the Lyme disease spirochete in
gelatin mimics dissemination through tissue. Proc Natl
Acad Sci USA 109: 3059–3064.
Hazelbauer, G.L., Falke, J.J., and Parkinson, J.S. (2008)
Bacterial chemoreceptors: high-performance signaling in
networked arrays. Trends Biochem Sci 33: 9–19.
Kremer, J.R., Mastronarde, D.N., and McIntosh, J.R. (1996)
Computer visualization of three-dimensional image data
using IMOD. J Struct Biol 116: 71–76.
Laskowski, R.A. (2001) PDBsum: summaries and analyses
of PDB structures. Nucleic Acids Res 29: 221–222.
Li, C., Bakker, R.G., Motaleb, M.A., Sartakova, M.L., Cabello,
K. Zhang
et al
© 2012 Blackwell Publishing Ltd, Molecular Microbiology,85, 782–794
F.C., and Charon, N.W. (2002) Asymmetrical flagellar rota-
tion in Borrelia burgdorferi nonchemotactic mutants. Proc
Natl Acad Sci USA 99: 6169–6174.
Li, C., Xu, H., Zhang, K., and Liang, F.T. (2010) Inactivation of
a putative flagellar motor switch protein FliG1 prevents
Borrelia burgdorferi from swimming in highly viscous media
and blocks its infectivity. Mol Microbiol 75: 1563–1576.
Li, Y., Hu, Y., Fu, W., Xia, B., and Jin, C. (2007) Solution
structure of the bacterial chemotaxis adaptor protein CheW
from Escherichia coli.Biochem Biophys Res Commun 360:
Liu, J., Lin, T., Botkin, D.J., McCrum, E., Winkler, H., and
Norris, S.J. (2009) Intact flagellar motor of Borrelia burg-
dorferi revealed by cryo-electron tomography: evidence for
stator ring curvature and rotor/C-ring assembly flexion.
J Bacteriol 191: 5026–5036.
Liu, J., Hu, B., Morado, D.R., Jani, S., Manson, M.D., and
Margolin, W. (2012) Molecular architecture of chemorecep-
tor arrays revealed by cryoelectron tomography of Escheri-
chia coli minicells. Proc Natl Acad Sci USA 109: E1481–
Liu, J.D., and Parkinson, J.S. (1989) Role of CheW protein in
coupling membrane receptors to the intracellular signaling
system of bacterial chemotaxis. Proc Natl Acad Sci USA
86: 8703–8707.
Liu, J.D., and Parkinson, J.S. (1991) Genetic evidence for
interaction between the CheW and Tsr proteins during
chemoreceptor signaling by Escherichia coli.J Bacteriol
173: 4941–4951.
Maddock, J.R., and Shapiro, L. (1993) Polar location of the
chemoreceptor complex in the Escherichia coli cell.
Science 259: 1717–1723.
Martin, A.C., Wadhams, G.H., and Armitage, J.P. (2001) The
roles of the multiple CheW and CheA homologues in
chemotaxis and in chemoreceptor localization in Rhodo-
bacter sphaeroides.Mol Microbiol 40: 1261–1272.
Motaleb, M.A., Corum, L., Bono, J.L., Elias, A.F., Rosa, P.,
Samuels, D.S., and Charon, N.W. (2000) Borrelia burgdor-
feri periplasmic flagella have both skeletal and motility
functions. Proc Natl Acad Sci USA 97: 10899–10904.
Motaleb, M.A., Sal, M.S., and Charon, N.W. (2004) The
decrease in FlaA observed in a flaB mutant of Borrelia
burgdorferi occurs posttranscriptionally. J Bacteriol 186:
Motaleb, M.A., Miller, M.R., Li, C., Bakker, R.G., Goldstein,
S.F., Silversmith, R.E., et al. (2005) CheX is a phosphory-
lated CheY phosphatase essential for Borrelia burgdorferi
chemotaxis. J Bacteriol 187: 7963–7969.
Motaleb, M.A., Pitzer, J.E., Sultan, S.Z., and Liu, J. (2011a) A
novel gene inactivation system reveals altered periplasmic
flagellar orientation in a Borrelia burgdorferi fliL mutant.
J Bacteriol 193: 3324–3331.
Motaleb, M.A., Sultan, S.Z., Miller, M.R., Li, C., and Charon,
N.W. (2011b) CheY3 of Borrelia burgdorferi is the key
response regulator essential for chemotaxis and forms a
long-lived phosphorylated intermediate. J Bacteriol 193:
Park, S.Y., Borbat, P.P., Gonzalez-Bonet, G., Bhatnagar, J.,
Pollard, A.M., Freed, J.H., et al. (2006) Reconstruction of
the chemotaxis receptor-kinase assembly. Nat Struct Mol
Biol 13: 400–407.
Parkinson, J.S. (1977) Behavioral genetics in bacteria. Annu
Rev Genet 11: 397–414.
Parkinson, J.S., and Houts, S.E. (1982) Isolation and behav-
ior of Escherichia coli deletion mutants lacking chemotaxis
functions. J Bacteriol 151: 106–113.
Porter, S.L., Wadhams, G.H., and Armitage, J.P. (2011)
Signal processing in complex chemotaxis pathways. Nat
Rev Microbiol 9: 153–165.
Radolf, J.D., Caimano, M.J., Stevenson, B., and Hu, L.T.
(2012) Of ticks, mice and men: understanding the dual-
host lifestyle of Lyme disease spirochaetes. Nat Rev Micro-
biol 10: 87–99.
Rao, C.V., Glekas, G.D., and Ordal, G.W. (2008) The three
adaptation systems of Bacillus subtilis chemotaxis. Trends
Microbiol 16: 480–487.
Rosa, P.A., Tilly, K., and Stewart, P.E. (2005) The burgeoning
molecular genetics of the Lyme disease spirochaete. Nat
Rev Microbiol 3: 129–143.
Sali, A., and Blundell, T.L. (1993) Comparative protein mod-
elling by satisfaction of spatial restraints. J Mol Biol 234:
Samuels, D.S. (1995) Electrotransformation of the spirochete
Borrelia burgdorferi.Methods Mol Biol 47: 253–259.
Samuels, D.S. (2011) Gene regulation in Borrelia burgdorferi.
Annu Rev Microbiol 65: 479–499.
Sarkar, M.K., Paul, K., and Blair, D. (2010) Chemotaxis sig-
naling protein CheY binds to the rotor protein FliN to control
the direction of flagellar rotation in Escherichia coli.Proc
Natl Acad Sci USA 107: 9370–9375.
Seshadri, R., Myers, G.S., Tettelin, H., Eisen, J.A., Heidel-
berg, J.F., Dodson, R.J., et al. (2004) Comparison of the
genome of the oral pathogen Treponema denticola with
other spirochete genomes. Proc Natl Acad Sci USA 101:
Shih, C.M., Chao, L.L., and Yu, C.P. (2002) Chemotactic
migration of the Lyme disease spirochete (Borrelia burg-
dorferi) to salivary gland extracts of vector ticks. Am J Trop
Med Hyg 66: 616–621.
Shiomi, D., Zhulin, I.B., Homma, M., and Kawagishi, I. (2002)
Dual recognition of the bacterial chemoreceptor by
chemotaxis-specific domains of the CheR methyltrans-
ferase. J Biol Chem 277: 42325–42333.
Sourjik, V., and Armitage, J.P. (2010) Spatial organization in
bacterial chemotaxis. EMBO J 29: 2724–2733.
Sourjik, V., and Berg, H.C. (2000) Localization of components
of the chemotaxis machinery of Escherichia coli using fluo-
rescent protein fusions. Mol Microbiol 37: 740–751.
Steere, A.C., Coburn, J., and Glickstein, L. (2004) The emer-
gence of Lyme disease. J Clin Invest 113: 1093–1101.
Studdert, C.A., and Parkinson, J.S. (2005) Insights into the
organization and dynamics of bacterial chemoreceptor
clusters through in vivo crosslinking studies. Proc Natl
Acad Sci USA 102: 15623–15628.
Sze, C.W., Morado, D.R., Liu, J., Charon, N.W., Xu, H., and
Li, C. (2011) Carbon storage regulator A (CsrA(Bb)) is a
repressor of Borrelia burgdorferi flagellin protein FlaB. Mol
Microbiol 82: 851–864.
Sze, C.W., Zhang, K., Kariu, T., Pal, U., and Li, C. (2012)
Borrelia burgdorferi needs chemotaxis to establish infec-
tion in mammals and to accomplish its enzootic cycle.
Infect Immun 80: 2485–2492.
Borrelia burgdorferi
© 2012 Blackwell Publishing Ltd, Molecular Microbiology,85, 782–794
Vu, A., Wang, X., Zhou, H., and Dahlquist, F.W. (2012) The
receptor-CheW binding interface in bacterial chemotaxis.
J Mol Biol 415: 759–767.
Wadhams, G.H., and Armitage, J.P. (2004) Making sense of
it all: bacterial chemotaxis. Nat Rev Mol Cell Biol 5: 1024–
Xu, H., Raddi, G., Liu, J., Charon, N.W., and Li, C. (2011)
Chemoreceptors and flagellar motors are subterminally
located in close proximity at the two cell poles in spiro-
chetes. J Bacteriol 193: 2652–2656.
Yang, J., Huber, G., and Wolgemuth, C.W. (2011) Forces and
torques on rotating spirochete flagella. Phys Rev Lett 107:
Yang, Y., and Li, C. (2009) Transcription and genetic analyses
of a putative N-acetylmuramyl-L-alanine amidase in Borre-
lia burgdorferi.FEMS Microbiol Lett 290: 164–173.
Zhang, P., Bos, E., Heymann, J., Gnaegi, H., Kessel, M.,
Peters, P.J., and Subramaniam, S. (2004) Direct visualiza-
tion of receptor arrays in frozen-hydrated sections and
plunge-frozen specimens of E. coli engineered to overpro-
duce the chemotaxis receptor Tsr. J Microsc 216: 76–83.
Supporting information
Additional supporting information may be found in the online
version of this article.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the
K. Zhang
et al
© 2012 Blackwell Publishing Ltd, Molecular Microbiology,85, 782–794
    • "Bb contains the most redundant set of chemotaxis-related genes found among eubacteria.18 Once inside an animal host, Bb uses chemoreceptor arrays at its cell poles to follow chemoattractant trails to reach specific host cells or tissue compartments.59,60 Sze et al61 have shown that when Bb becomes nonchemotactic to attractants, it abrogates infectivity even when the host is immunodeficient. "
    [Show abstract] [Hide abstract] ABSTRACT: Is chronic illness in patients with Lyme disease caused by persistent infection? Three decades of basic and clinical research have yet to produce a definitive answer to this question. This review describes known and suspected mechanisms by which spirochetes of the Borrelia genus evade host immune defenses and survive antibiotic challenge. Accumulating evidence indicates that Lyme disease spirochetes are adapted to persist in immune competent hosts, and that they are able to remain infective despite aggressive antibiotic challenge. Advancing understanding of the survival mechanisms of the Lyme disease spirochete carry noteworthy implications for ongoing research and clinical practice.
    Full-text · Article · Apr 2013
  • [Show abstract] [Hide abstract] ABSTRACT: Homology models of the E. coli and T. maritima chemotaxis protein CheW were constructed to assess the quality of structural predictions and their applicability in chemotaxis research: i) a model of E. coli CheW was constructed using the T. maritima CheW NMR structure as a template, and ii) a model of T. maritima CheW was constructed using the E. coli CheW NMR structure as a template. The conformational space accessible to the homology models and to the NMR structures was investigated using molecular dynamics and Monte Carlo simulations. The results show that even though static homology models of CheW may be partially structurally different from their corresponding experimentally determined structures, the conformational space they can access through their dynamic variations can be similar, for specific regions of the protein, to that of the experimental NMR structures. When CheW homology models are allowed to explore their local accessible conformational space, modeling can provide a rational path to predicting CheW interactions with the MCP and CheA proteins of the chemotaxis complex. Homology models of CheW (and potentially, of other chemotaxis proteins) should be seen as snapshots of an otherwise larger ensemble of accessible conformational space.
    Full-text · Article · Aug 2013
  • [Show abstract] [Hide abstract] ABSTRACT: Borrelia burgdorferi, the agent of Lyme disease, is maintained in nature within an enzootic cycle involving a mammalian reservoir and an Ixodes sp. tick vector. The transmission, survival and pathogenic potential of B. burgdorferi depend on the bacterium's ability to modulate its transcriptome as it transits between vector and reservoir host. Herein, we employed an amplification-microarray approach to define the B. burgdorferi transcriptomes in fed larvae, fed nymphs and in mammalian host-adapted organisms cultivated in dialysis membrane chambers. The results show clearly that spirochetes exhibit unique expression profiles during each tick stage and during cultivation within the mammal; importantly, none of these profiles resembles that exhibited by in vitro-grown organisms. Profound shifts in transcript levels were observed for genes encoding known or predicted lipoproteins as well as proteins involved in nutrient uptake, carbon utilization and lipid synthesis. Stage-specific expression patterns of chemotaxis-associated genes also were noted, suggesting that the composition and interactivities of the chemotaxis machinery components vary considerably in the feeding tick and mammal. The results as a whole make clear that environmental sensing by B. burgdorferi directly or indirectly drives an extensive and tightly integrated modulation of cell envelope constituents, chemotaxis/motility machinery, intermediary metabolism and cellular physiology. These findings provide the necessary transcriptional framework for delineating B. burgdorferi regulatory pathways throughout the enzootic cycle as well as defining the contribution(s) of individual genes to spirochete survival in nature and virulence in humans. This article is protected by copyright. All rights reserved.
    Article · Nov 2014
Show more