Site-specific methylation in Bacillus subtilis chemotaxis: effect of covalent modifications to the chemotaxis receptor McpB.
ABSTRACT The Bacillus subtilis chemotaxis pathway employs a receptor methylation system that functions differently from the one in the canonical Escherichia coli pathway. Previously, we hypothesized that B. subtilis employs a site-specific methylation system for adaptation where methyl groups are added and removed at different sites. This study investigated how covalent modifications to the adaptation region of the chemotaxis receptor McpB altered its apparent affinity for its cognate ligand, asparagine, and also its ability to activate the CheA kinase. This receptor has three closely spaced adaptation sites located at residues Gln371, Glu630 and Glu637. We found that amidation, a putative methylation mimic, of site 371 increased the receptor's apparent affinity for asparagine and its ability to activate the CheA kinase. Conversely, amidation of sites 630 and 637 reduced the receptor's ability to activate the kinase but did not affect the apparent affinity for asparagine, suggesting that activity and sensitivity are independently controlled in B. subtilis. We also examined how electrostatic interactions may underlie this behaviour, using homology models. These findings further our understanding of the site-specific methylation system in B. subtilis by demonstrating how the modification of specific sites can have varying effects on receptor function.
- SourceAvailable from: George Winford Ordal[show abstract] [hide abstract]
ABSTRACT: The Bacillus subtilis gene encoding CheB, which is homologous to Escherichia coli CheY, the regulator of flagellar rotation, has been cloned and sequenced. It has been verified, using a phage T7 expression system, by showing that a small protein, the same size as E. coli CheY, is actually made from this DNA. Despite the fact that the two proteins are 36% identical, with many highly conserved residues, they appear to play different roles. Unlike CheY null mutants, which swim smoothly, CheB null mutants tumble incessantly. However, a CheB point mutant swims smoothly, even in the presence of a plasmid bearing cheB, which restores the null mutants to wild type. Expression of CheB in wild type B. subtilis makes the cells exhibit more tumbling. Since both absence of CheB and presence of high levels of CheB cause tumbling, CheB appears to be required, in certain circumstances, for both smooth swimming and tumbling. Expression in wild type E. coli makes the cells smooth swimmers and strongly inhibits chemotaxis.Journal of Biological Chemistry 08/1991; 266(19):12301-5. · 4.65 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: As an important model for transmembrane signaling, methyl-accepting chemotaxis proteins (MCPs) have been extensively studied by using genetic, biochemical, and structural techniques. However, details of the molecular mechanism of signaling are still not well understood. The availability of genomic information for hundreds of species enables the identification of features in protein sequences that are conserved over long evolutionary distances and thus are critically important for function. We carried out a large-scale comparative genomic analysis of the MCP signaling and adaptation domain family and identified features that appear to be critical for receptor structure and function. Based on domain length and sequence conservation, we identified seven major MCP classes and three distinct structural regions within the cytoplasmic domain: signaling, methylation, and flexible bundle subdomains. The flexible bundle subdomain, not previously recognized in MCPs, is a conserved element that appears to be important for signal transduction. Remarkably, the N- and C-terminal helical arms of the cytoplasmic domain maintain symmetry in length and register despite dramatic variation, from 24 to 64 7-aa heptads in overall domain length. Loss of symmetry is observed in some MCPs, where it is concomitant with specific changes in the sensory module. Each major MCP class has a distinct pattern of predicted methylation sites that is well supported by experimental data. Our findings indicate that signaling and adaptation functions within the MCP cytoplasmic domain are tightly coupled, and that their coevolution has contributed to the significant diversity in chemotaxis mechanisms among different organisms.Proceedings of the National Academy of Sciences 03/2007; 104(8):2885-90. · 9.74 Impact Factor
Site-specific methylation in Bacillus subtilis
chemotaxis: effect of covalent modifications to the
chemotaxis receptor McpB
George D. Glekas,1Joseph R. Cates,1Theodore M. Cohen,1
Christopher V. Rao2and George W. Ordal1
George W. Ordal
Christopher V. Rao
Received 16 August 2010
Revised17 September 2010
Accepted 22 September 2010
1Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
2Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-
Champaign, Urbana, IL 61801, USA
The Bacillus subtilis chemotaxis pathway employs a receptor methylation system that functions
differently from the one in the canonical Escherichia coli pathway. Previously, we hypothesized
that B. subtilis employs a site-specific methylation system for adaptation where methyl groups are
added and removed at different sites. This study investigated how covalent modifications to the
adaptation region of the chemotaxis receptor McpB altered its apparent affinity for its cognate
ligand, asparagine, and also its ability to activate the CheA kinase. This receptor has three closely
spaced adaptation sites located at residues Gln371, Glu630 and Glu637. We found that
amidation, a putative methylation mimic, of site 371 increased the receptor’s apparent affinity for
asparagine and its ability to activate the CheA kinase. Conversely, amidation of sites 630 and 637
reduced the receptor’s ability to activate the kinase but did not affect the apparent affinity for
asparagine, suggesting that activity and sensitivity are independently controlled in B. subtilis. We
also examined how electrostatic interactions may underlie this behaviour, using homology models.
These findings further our understanding of the site-specific methylation system in B. subtilis by
demonstrating how the modification of specific sites can have varying effects on receptor function.
Peritrichiously flagellated bacteria can swim towards
attractants and away from repellents by altering the
frequency of smooth runs and reorientating tumbles
through a process known as chemotaxis (Rao & Ordal,
2009). At the core of this navigational system is a
mechanism for gradient sensing. Bacteria detect gradients
by comparing their presently sensed chemical environment
with that sensed in the recent past (Berg & Purcell, 1977;
Macnab & Koshland, 1972). If the bacterium is travelling in
a favourable direction, up a gradient of attractant or down
one of repellent, then it will tend to continue along its
current trajectory by decreasing the frequency of tumbles
(Berg & Brown, 1972). A critical element of this gradient-
sensing mechanism is sensory adaptation: the increase in
bias (tendency for counter-clockwise rotation of the
flagella) caused by addition of attractant does not persist
and, despite continued presence of the attractant, the bias
returns to its pre-stimulus value. In fact, the bias is
proportional to the rate of change in the average number of
ligand-bound receptors and not their absolute levels (Segall
et al., 1986).
In the soil bacterium Bacillus subtilis, the chemotaxis
pathway employs a modified two-component system (Rao
& Ordal, 2009). Briefly, the transmembrane chemotaxis
receptors form complexes with a histidine kinase, CheA,
and two coupling proteins, CheW and CheV. Upon
addition of attractant, the receptors change their con-
formation to cause increased levels of phosphorylated
CheA kinase (CheAp). The receptors are able to commu-
nicate with the flagellar motors as the phosphoryl group on
the kinase is transferred to the soluble response regulator,
CheY. Phosphorylated CheY (CheYp) binds to the flagellar
motors and increases the likelihood of counter-clockwise
rotation and runs (Bischoff & Ordal, 1991; Bischoff et al.,
1993; Garrity & Ordal, 1997).
B. subtilis employs three parallel systems for sensory
adaptation (Rao et al., 2008). One of these involves
phosphorylation of CheV, which has both a CheW domain
and a CheY domain, by the kinase (Karatan et al., 2001).
Phosphorylated CheV is believed to inhibit the coupling
between the kinase and the chemotaxis receptors (Rao
et al., 2008). Another involves CheC, whose binding of
CheD is enhanced by CheYp. When CheYp concentrations
are high, CheC recruits CheD away from the receptors to
make them less active (Muff & Ordal, 2007).
The third system involves receptor methylation, the focus
of this study. In the methylation adaptation system, two
Microbiology (2011), 157, 56–65
56044685G2011 SGMPrinted in Great Britain
enzymes, CheR and CheB, add and remove methyl groups,
respectively, at conserved glutamate residues on the
receptors in response to changes in attractant binding
(Burgess-Cassler et al., 1982; Goldman et al., 1984). When
the receptors bind attractant, they are rapidly demethylated
and then slowly remethylated, with the remethylation
phase persisting until the degree of receptor methylation
returns to its pre-stimulus levels (Kirby et al., 1999).
Irrespective of the ambient concentration of attractant, the
net level of methylation in B. subtilis is roughly constant at
steady state (Kirby et al., 1999). Interestingly, this remethy-
lation step takes many times longer than the behavioural
adaptation period. The same two-step process also occurs
when the attractant is removed: rapid demethylation
followed by slow remethylation.
Based on these observations, we previously hypothesized
that receptor methylation is site-specific in B. subtilis, so
that during adaptation to changes in attractant or repellent
concentration, methyl groups are added and removed at
these different sets of residues (Zimmer et al., 2000).
Specifically, we hypothesized that the methylation of some
residues activates the kinase whereas the methylation of
others inhibits the kinase. In this study, we tested this
hypothesis by exploring how covalent modifications to the
three adaptation sites of the canonical B. subtilis receptor
McpB altered the apparent affinity for its cognate ligand,
asparagine, and affected kinase activity. Consistent with
this model, we found that the modification of Glu371
increased McpB’s apparent affinity for asparagine and also,
in most cases, increased the activity of the receptor in
stimulating the kinase. Modifications of Glu630 and
Glu637, on the other hand, decreased the activity of the
receptor in stimulating the kinase. Furthermore, we also
examined how electrostatic interactions may underlie this
Bacterial strains and plasmids. All B. subtilis strains are derived
from the chemotactic strain (Che+) OI1085 (Ullah & Ordal, 1981).
All cloning and plasmid propagation were performed in Escherichia
coli strain TG1 (GE Healthcare Life Sciences).
The B. subtilis strains were created by using long PCR mutagenesis on
the pAIN750 plasmid, which contains the full-length mcpB receptor
gene under control of its native promoter, as previously described
(Zimmer et al., 2000, 2002). These mutated receptors were then
integrated back into the amyE locus of the D10mcp cheB cheR strain
OI3635 (Hou et al., 2000; Kristich & Ordal, 2002), which lacks all ten
B. subtilis chemoreceptors along with CheR and CheB. Expression
levels of McpB were confirmed using Western blots, and found to be
similar to wild-type for all mutant alleles tested.
Sensitivity capillary assay. Sensitivity capillary assays were
performed as previously described (Mesibov et al., 1973; Ordal
et al., 1977). In these assays, each capillary contained 3.16-fold more
attractant than the concentration found in the corresponding pond.
The experiments were performed over a range of concentrations
spanning eight orders of magnitude. The number of bacteria that
enter the capillary is proportional to the difference in the number of
receptors titrated with attractant at the two end points – the capillary
concentration and the pond concentration. The numbers of bacteria
per capillary were plotted as a function of the geometric mean of the
concentrations of attractant in the capillary and in the pond. The
centre of symmetry of the peak in this corresponding graph is the
concentration at which chemotaxis is most potent and, hence,
provides an apparent dissociation constant for the receptors (Mesibov
et al., 1973).
Cells were grown overnight at 30 uC on TBAB [tryptose blood agar:
1% (w/v) tryptone, 0.3% (w/v) beef extract, 0.5% (w/v) NaCl, 1.5%
agar] plates. The cells were then scraped from the plate and
resuspended to OD600 0.014 in 40 ml capillary assay minimal
medium (50 mM K3PO4, pH 7.0, 1.2 mM MgCl2, 0.14 mM CaCl2,
1 mM (NH4)2SO4, 0.01 mM MnCl2, 20 mM sorbitol and 0.02%
tryptone, supplemented with 50 mg ml21
methionine and tryptophan). The cultures were grown to OD6000.4
at 37 uC with shaking, after which a 50 ml aliquot of a 5% (v/v)
glycerol/0.5 M sodium lactate solution was added, and the cells were
incubated for an additional 15 min. They were then washed three
times with chemotaxis buffer and diluted to OD6000.001. The cells
were supplemented with the appropriate asparagine concentration
and aliquoted into 0.3 ml ponds on a temperature-controlled plate at
37 uC. Closed-end capillary tubes filled with the corresponding
amount of asparagine were inserted into the pond. After 30 min, the
cells in the capillaries were harvested and transferred to 0.5 ml top
agar [1% (w/v) tryptone, 0.8% (w/v) NaCl, 0.8% (w/v) agar, 0.5 mM
EDTA] and plated onto TB [1% (w/v) tryptone, 0.5% (w/v) NaCl]
agar plates. These plates were incubated at 37 uC for 16 h, at which
point colonies were counted to derive the data. Experiments were
performed in triplicate and on two different days to ensure
reproducibility. The error for the dissociation constant (Kd) estimates
is less than 5%, as determined by five separate experiments
performed on the wild-type strain OI1085.
each of histidine,
Tethered cell assay. The tethered cell assay was performed as
described previously (Block et al., 1983; Kirby et al., 1999; Saulmon
et al., 2004). Bacterial cells were adhered by their flagella to a
microscope coverslip by an anti-flagellin antibody, and the rotational
direction, clockwise (CW) or counter-clockwise (CCW), was tracked
for a period of 5 min. Data averaged over a population of at least 16
cells resulted in a probability, or bias, of CCW rotation (smooth swim).
The cells were grown using the same protocol as in the sensitivity
capillary assay. Once the cells were harvested, the flagella were sheared
in a Waring blender by two 15 s bursts. The cells were then pelleted
and resuspended in fresh chemotaxis buffer [10 mM K3PO4, pH 7.0,
0.14 mM CaCl2, 0.3 mM (NH4)2SO4, 0.1 mM EDTA, 5 mM sodium
lactate, 0.05% (v/v) glycerol]. They were then applied to a round
coverslip previously incubated with anti-flagellin antibodies for
20 min. The coverslip was fixed to a flow cell, and rotation of the
cells was observed with a phase-contrast microscope. Chemotaxis
buffer supplemented with asparagine was continuously flowed past
the cells. Hobson Tracker software (Hobson Tracking Systems) was
employed to track the rotational direction of individual cells, and
Matlab (The Mathworks) was used to process the resultant data.
Structural modelling and electrostatic analysis. Structural
models of the McpB cytoplasmic domain were generated using
SPDBV software and the SWISS-MODEL homology server (Arnold
etal., 2006; Kieferetal., 2009).The closelyrelatedThermatogamaritima
TM1143 receptor crystal structure served as the template (Park et al.,
2006). The cytoplasmic domains of McpB and TM1143 share 27%
identity, and the root mean square deviation (RMSD) of the final McpB
model when compared with the TM1143 crystal structure was 0.063 A˚
(0.0063 nm). The solvent-accessible electrostatic surface potential of
each model was analysed using APBS (Baker et al., 2001). The potentials
were calculated for a protein core dielectric of 2 and a solvent dielectric
Adaptation mechanism of a B. subtilis chemoreceptor
constant of 80. The isopotential contours are shown for an electrostatic
energy of±8 kT/e.
Behavioural effect of glutamine substitutions
McpB, the sole receptor for chemotaxis towards asparagine
(Hanlon & Ordal, 1994), has three adaptation sites located
at residues Gln371, Glu630 and Glu637 (Zimmer et al.,
2000), confirmed using tandem mass spectroscopy (G.
Glekas, unpublished). A structural model of the cytoplasmic
domain of McpB, based on the crystal structure of the
closely related T. maritima chemoreceptor
(Alexander & Zhulin, 2007; Park et al., 2006), shows the
spatial orientation of the three adaptation sites as
constituting a triad (Fig. 1). By contrast, the methylation
sites in the E. coli Tar receptor are arranged in a line (Fig. 1).
To determine how the covalent modification of these three
residues affects the sensitivity and activity of the associated
kinase, we tested the effect of substituting glutamines and
glutamates in all possible combinations at the three
adaptationsites ofthecanonical McpB receptor.
Substituting glutamates with glutamines has long been
used to study the effects of methylation in E. coli
chemotaxis (Dunten & Koshland, 1991). In particular,
the amine replaces the negatively charged carboxylate
anion, mimicking the effect of carboxyl methylation.
While the chemotaxis pathways in B. subtilis and E. coli differ
in many ways (Szurmant & Ordal, 2004), we reasoned that
the mimicking effect of carboxyl methylation by amidation
would be conserved in B. subtilis. Our justification is as
follows. The structure of the adaptation region, the coiled-
coil region of the receptor where the adaptation sites are
located, is similar in E. coli and B. subtilis (Alexander &
Zhulin, 2007; Park et al., 2006). The adaptation sites in B.
subtilis and E. coli also share the same heptadic motif
(Zimmer et al., 2000). Moreover, CheB and CheR from B.
subtilis functionally complement the corresponding E. coli
null mutant (Kirsch et al., 1993a, b), indicating that the
recognition sequence and local structure of the receptors is
the same, as are the governing reactions for reversible
methylation. Finally, the evidence to date strongly indicates
located predominantly at the cell poles (Briegel et al., 2009).
Since the cytoplasmic parts of the receptors in B. subtilis and
T. maritima are very similar (Alexander & Zhulin, 2007),
they would probably be organized in similar arrays.
In E. coli the amidation, and presumably methylation, of the
negatively charged glutamates has previously been shown to
neutralize repulsive electrostatic interactions between neigh-
bouring receptors (Starrett & Falke, 2005). Given that the
structure of the adaptation region and higher-order
organization of the receptors appear to be the same in both
E. coli and B. subtilis, carboxyl methylation probably
neutralizes the repulsive effects of the negatively charged
glutamates in the B. subtilis receptors as well. While we lack
definitive evidence, all lines of evidence suggest that
amidation will also mimic methylation in B. subtilis.
To test the effect of covalent modifications of the adaptation
sites the mutated McpB receptors were expressed in a strain
lacking the CheR methyltransferase, the CheB methylesterase,
the wild-type McpB receptor and the other nine chemotaxis
receptors. While CheD, a chemotaxis protein present in B.
subtilis but not E. coli, is also capable of deamidating specific
glutamine residues on McpB (Kristich & Ordal, 2002), we
have previously found that it did not deamidate residue
Gln371 (Kristich, 2002), which presumably gets deamidated
by CheB. In addition, we found that CheD does not
deamidate engineered glutamines at the adaptation sites
(data not shown). To determine the in vivo effect of these
modifications, we carried out two types of experiments, a
sensitivity capillary assay and a tethered cell assay.
Sensitivity capillary assay to assess ‘apparent Kd’
In the sensitivity capillary assay, the traditional capillary
assay is altered such that a wide range of attractant
Fig. 1. Structural depictions of the B. subtilis McpB and E. coli Tar
cytoplasmic domains. The homology model shown for McpB is
based on the crystal structure of the cytoplasmic domain of the T.
maritima TM1143 chemoreceptor (Park et al., 2006). The
adaptation sites, Gln371, Glu630 and Glu637, form a closely
spaced, interacting triad on McpB. In comparison, the E. coli Tar
cytoplasmic domain shows a different orientation of adaptation
sites, all in a line and spaced further apart. Shown is the
methylation domain from the structural model for the full receptor
(Kim et al., 1999). For easier visualization, the adaptation sites
(shown in red) are only highlighted on one face of the receptor.
G. D. Glekas and others
concentrations is spanned by 3.16-fold gradients, where the
capillary concentration is 3.16 times that in the pond
(bacterial suspension). Bacteria will swim up into the
capillary, and the number that do so is proportional to the
number of receptors titrated at the upper concentration
minus the number titrated at the lower concentration.
From a graph of the results, the ‘apparent Kd’ can be
determined (Mesibov et al., 1973). This value is the
concentration where chemotaxis is maximal, namely the
concentration at which the receptors are half bound with
attractant and half free. The numerical value reflects not
only the intrinsic Kdof the receptor for the attractant but
also constraints on receptor conformation and signalling
caused by the binding of other chemotaxis proteins,
including CheA, CheD, CheW and CheV.
One great value of the experiments performed here is that
they were carried out on a static methylation system in
which various combinations of glutamines and glutamates
at each of the three methylation sites were assessed without
the possibility of their being altered during the course of
the experiment. However, since chemotaxis requires both
excitation and adaptation, this experimental set-up would
not be possible in many bacteria. But, in B. subtilis, there
are two other functional adaptation systems besides the
methylation system to facilitate taxis.
When sensitivity capillary assays were carried out on the
various mutants, it was discovered that the ‘apparent Kd’
(as defined above) was 10-fold to 20-fold higher (less
apparent affinity) when there was a glutamate at site 371
than when a glutamine was at this site (results shown in
Fig. 2 and summarized in Table 1). However, the presence
of a glutamine or glutamate at sites 630 or 637 did not
much affect the ‘apparent Kd’. These results show that the
modification state of residue 371 affects McpB’s sensitivity
There were two instances, however, when both sites 630
and 637 were encoded as glutamines, that chemotaxis was
too feeble for the ‘apparent Kd’ to be measured, regardless
of the allelic state of site 371 (371E/630Q/637Q and 371Q/
630Q/637Q). Interestingly, as outlined below, these are the
states where kinase activity would be expected to be the
Measuring kinase activity using the tethered cell
We also measured the counter-clockwise rotational bias of
the flagellar motors using the tethered cell assay (Block
et al., 1983; Kirby et al., 1999; Saulmon et al., 2004). In this
assay, the cells are tethered to a glass coverslip using an
anti-flagellin antibody and their rotation is recorded using
video microscopy at varying concentrations of asparagine.
The average counter-clockwise rotational bias of the
motors reflects the concentration of CheYp and thus
provides an indirect measure of CheA kinase activity. It
should be noted that these experiments were performed in
the presence of asparagine, since in the absence of
attractant, cheRcheB cells expressing only McpB rotate
almost exclusively clockwise (Zimmer et al., 2002).
When we looked for a general pattern among the data
(Table 2), the results could best be classified based on the
amidation state of site 371, the very site that affects the
‘apparent Kd’ of the receptor. When site 371 was encoded
Fig. 2. Sensitivity assay results. (a) Comparison of the Gln371/
Glu630/Glu637 (QEE) mutant (thin line, &) and the Glu371/
Glu630/Glu637 (EEE) strain (thicker line, $). (b) Comparison of
the Gln371/Gln630/Glu637 (QQE) mutant (thin line, &) and the
Glu371/Gln630/Glu637 (EQE) mutant (thicker line, $). (c)
Comparison of the Gln371/Glu630/Gln637 (QEQ) mutant (thin
line, &) and the Glu371/Glu630/Gln637 (EEQ) mutant (thicker
Adaptation mechanism of a B. subtilis chemoreceptor
as a glutamate, the amidation of sites 630 and 637
decreased the bias at the two asparagine concentrations
tested (25 and 130 mM). Moreover, the effect was additive;
receptors with both sites 630 and 637 modified (371E/
630Q/637Q) had a lower bias relative to the unmodified
receptor (371E/630E/637E) than those with just one site
modified (371E/630Q/637E and 371E/630E/637Q). When
considering the two sites individually, however, we did not
observe any significant difference between them. When site
371 was encoded as a glutamine, it was found that both
sites 630 and 637 needed to be amidated in order to reduce
the bias at the two concentrations tested (371Q/630E/637E
versus 371Q/630Q/637Q). Individually, the modification
state of these two sites did not lead to a significant
reduction in the bias (371Q/630E/637E versus 371Q/630Q/
637E and 371Q/630E/637Q). In summary, these results
indicate that amidation of sites 630 and 637 reduces the
ability of the receptor complex to stimulate the kinase.
By contrast, amidation of site 371 mostly increased, rather
than decreased, kinase activity (Table 2). In addition, we
observed a much higher relative bias at 130 mM asparagine
than at 25 mM in 371E strains as compared with 371Q
strains. Presumably, most of the receptors are already
titrated at 25 mM asparagine in the 371Q strains due to
their lower ‘apparent Kd’. The only exception to this rule is
the case of 371Q/630E/637E, where the kinase is less active
than the 371E/630E/637E case at 130 mM asparagine, but
not at 25 mM. We cannot explain why amidation of site
371 decreases kinase activity at 130 mM asparagine,
although we suspect that it is likely to be due to a
confluence of competing effects. In particular, while the
amidation of site 371 in general increases kinase activity it
may also reduce electrostatic interactions as described
below, leading in some cases to lower kinase activity.
Electrostatic surface potential of the receptor
Researchers at the Falke laboratory have previously
proposed that electrostatic interactions along the dimer
subunit interface of the adaptation region alter the stability
and conformational dynamics of the E. coli aspartate
receptor (Starrett & Falke, 2005; Swain et al., 2009).
According to their model, electrostatic repulsions between
negatively charged glutamates repress the kinase by
destabilizing helical packing within the adaptation region,
having the effect of stabilizing the receptor where it
interacts with the kinase and CheW. Likewise, neutraliza-
tion of these negatively charged glutamates by amidation,
and presumably methylation, activates the kinase by
stabilizing the helical packing within the methylation
region, having the effect of destabilizing the receptor
where it interacts with the kinase and CheW.
Electrostatic interactions potentially explain some of the in
vivo data regarding McpB. To explore this hypothesis in
more detail, we compared the electrostatic surface potential
of the solvent-exposed methylation region using homology
models of McpB (Fig. 3). The examination of surface
potential and isopotential patterns can reveal insights into
the biochemical data (Honig & Nicholls, 1995). Similar to
what has been found in both E. coli and T. maritima (Park
et al., 2006; Starrett & Falke, 2005), there is a distinct
surface of negative potential that contains the adaptation
sites in the cytoplasmic region of McpB. Comparison of the
electrostatic surface potential for the adaptation region in
different modification states indicates that the potential is
primarily determined by sites 630 and 637. Specifically, the
surface potential for the 371E/630E/637E cytoplasmic
domain, which corresponds to the receptor that showed
Table 1. Apparent Kdvalues of mcpB mutants
Kdvalues were determined using the capillary sensitivity assay. In the
experiments, a capillary containing asparagine is inserted into a
suspension of bacteria containing 3.16-fold less asparagine. By
counting the number of bacteria that accumulate in the capillary as
a function of concentration, one can determine the Kdby determining
the concentration at which accumulation is greatest. In the
experiments involving the EQQ and QQQ mutants, no accumulation
was observed at any concentration (denoted by ND). The raw data for
the apparent Kdare shown in Fig. 2.
Table 2. Rotational bias of McpB mutants
The rotational bias of different McpB mutants at two concentrations
of asparagine was determined using the tethered cell assay. In these
experiments, the cells were adhered to the glass coverslip by their
flagella using an anti-flagellin antibody. The direction of cell rotation
was recorded for approximately 5 min. Results are the mean±SEM of
at least 16 cells.
Receptor modificationCounter-clockwise bias (%)
371630637 130 mM25 mM
G. D. Glekas and others
the highest counter-clockwise bias in the tethered cell assay,
shows a surface of negative potential that contains sites 630
and 637 but, interestingly, not site 371. When these two
sites are changed to glutamines (371E/630Q/637Q), which
corresponds to the receptor that showed the lowest bias in
the tethered cell assay, the region of negative surface
potential is greatly reduced. Based on the work of Falke and
coworkers (Starrett & Falke, 2005), we hypothesize
specifically that negatively charged glutamates at sites 630
and 637 destabilize the helical packing of the receptors.
Amidation, and presumably methylation, of these two sites
can then neutralize the negative charges and stabilize the
helical packing of the receptors (Fig. 3).
In the case of site 371, we found that it had a much weaker
effect on the surface potential than sites 630 and 637 (Fig.
3). This suggests that this site may affect receptor function
by some other, still unknown mechanism.
Receptor methylation plays an integral role in bacterial
chemotaxis. With the notable exception of Helicobacter
pylori (Pittman et al., 2001), all flagellated bacteria appear
to employ some form of receptor methylation for
chemotaxis (Alexander & Zhulin, 2007; Wuichet et al.,
2007), although the mechanistic details can vary signifi-
cantly across species. Despite its prevalence, receptor
methylation has only been extensively studied so far in E.
coli. Little is known about this mechanism in other species
of bacteria. In this work, we explored how covalent
modification of the adaptation sites affects chemotaxis
signalling in B. subtilis. The canonical asparagine receptor,
McpB, has three adaptation sites, located at residues 371,
630 and 637 (Zimmer et al., 2000). We found that the
amidation of site 371 increased the sensitivity (i.e. binding
affinity) of the receptor to asparagine whereas the
corresponding changes to sites 630 and 637 had no
substantive effect. We also found that the amidation of
the negatively charged glutamates at sites 630 and 637
decreased kinase activity whereas the amidation of the
glutamate at site 371, with a single exception, increased
kinase activity (Table 2). Finally, the electrostatic surface
potential of the cytoplasmic domain of McpB showed that
negative charges at the adaptation sites might further
explain the effect of covalent modifications on chemotactic
The B. subtilis strains used in this study all contain CheD,
which functions in the CheC/D/Y adaptation system by
interacting with CheC when CheYp levels are high,
effectively recruiting CheD away from the receptors. Past
experiments have shown that mutants lacking cheD activate
CheA kinase poorly (Kirby et al., 2001). It is possible that
the glutamate and glutamine substitutions at the three
methylation sites may change the receptor’s affinity for
CheD, thereby influencing CheA kinase activity. Thus, the
net kinase activity in the cell could reflect not only the
direct change in the receptor caused by covalent modifica-
tion of the adaptation sites but also the secondary effect of
changing the receptor’s affinity for CheD. However,
preliminary experiments indicate that if there are such
changes in affinity, they are slight.
As mentioned in Results, the reason that we were able to
use chemotaxis assays to study receptor methylation is that
there are two other adaptation systems in B. subtilis, the
CheC/D/Y and CheV systems. These systems are redund-
ant, with two sufficient for chemotaxis, albeit at reduced
efficiency (Rao et al., 2008). In our experiments, the
methylation system was inactivated. Nonetheless, the cells
were still able to migrate up gradients of attractant
although with varying efficiencies based on the modifica-
tion state (Fig. 2). Moreover, the cells were incapable of
perfect adaptation (Table 2).
Role of electostatic interactions
As outlined in Results, the presence of negative charges
(glutamates) at sites 630 and 637 greatly increases the
surface negative potential of the adaptation region (Fig. 3).
Based on a model proposed by the Falke laboratory for the
Fig. 3. Electrostatic potentials and isopotential contours of the
solvent-accessible surface of McpB show a change in surface
potential upon the neutralization of a negatively charged glutamic
acid residue. The adaptation sites are shown as black circles.
Clockwise from the top, site 371, site 630 and site 637, with the
corresponding residue in that position labelled above.
Adaptation mechanism of a B. subtilis chemoreceptor
E. coli receptors, we hypothesized that these electrostatic
interactions probably affect the intra- and inter-subunit
packing of the B. subtilis receptors. However, the receptor–
kinase complexes in B. subtilis and E. coli have reciprocal
polarity. Attractant binding increases kinase activity in B.
subtilis whereas it inhibits it in E. coli. This reciprocal
polarity would potentially suggest that the E. coli model
cannot be fully applied to B. subtilis. However, the
differences in kinase activation most likely are due to
how the two sets of receptors interact with the kinase and
not to the receptors themselves. Evidence comes from
dynamic receptor localization studies, where the binding of
attractant has been shown to disrupt receptor packing in
both B. subtilis and E. coli (Lamanna et al., 2005). The
packing was restored once the cells adapted to the
attractant, presumably due in part to methylation and
the concomitant neutralization of repulsive electrostatic
interactions. Moreover, these results demonstrate that
despite the differences in polarity, the same changes in
receptor packing are observed in the two species of
bacteria. Also, they demonstrate that these same changes
lead alternatively to kinase activation in B. subtilis and
kinase inhibition in E. coli, providing further evidence that
the differences are due to how the receptors interact with
the kinase and not the adaptation region, which directly
affects packing. Finally, these patterns of reorganization are
also consistent with our model where repulsive interactions
between negatively charged glutamates at sites 630 and 637
destabilize the receptor and lead to increased kinase
Dissimilarity between B. subtilis and E. coli is also intrinsic
to the receptors themselves. In E. coli, substitutions of
glutamines or glutamates at all methylatable positions both
increase the kinase activity and the ‘apparent Kd’ of the
receptor (Li & Weis, 2000; Sourjik & Berg, 2002a). In B.
subtilis, however, we found that the modification state of
sites 630 and 637 affects only the kinase activity but not the
‘apparent Kd’. Only the amidation state of site 371 affects
the ‘apparent Kd’. Interestingly, we found in our structural
modelling that the amidation state of site 371 does not
affect the surface potential as greatly as amidation states of
sites 630 and 637.
Implication of activation and sensitivity being
decoupled from one another
As mentioned earlier, activity and sensitivity are not
directly correlated with one another in McpB. In particular,
there are modifications with high activity and high
apparent affinity (371Q/630E/637E) and others with low
activity and low apparent affinity (371E/630Q/637E and
371E/630E/637Q). Likewise, there are modifications with
high activity and low apparent affinity (371E/630E/637E)
and perhaps even one with low activity and high apparent
affinity (371Q/630Q/637Q). In E. coli, on the other hand,
there is a direct, inverse correlation between the two
(Sourjik & Berg, 2002b, 2004). In the models commonly
used to explain receptor activity in E. coli, the receptor
complex is assumed to exist in one of two states: (1) a high-
affinity, low-activity state and (2) a low-affinity, high-
activity state (Keymer et al., 2006; Mello & Tu, 2005; Rao
et al., 2004). The equilibrium partitioning between these
two states is determined by the concentration of chemoat-
tractant and degree of methylation/amidation. Our data for
McpB, however, imply that the mechanism for receptor
activation cannot be described by a simple two-state model
but instead requires a more complicated model involving
additional conformational states. While we still lack the
requisite biochemical data to construct such a quantitative
model, our results nonetheless suggest that B. subtilis is able
to independently tune these two factors.
In particular, why do modifications to site 371 affect the
‘apparent Kd’ whereas ones to site 630 and 637 do not? While
the actual mechanism is still unknown, we note that the
associated mechanism of attractant binding is different in
E. coli and B. subtilis. In E. coli, attractants bind across the
dimer interface and induce a piston-like movement in the
descending helix, the one emerging from the transmembrane
attractant binds within an individual monomer and induces
a rotation between the helices (Glekas et al., 2010; Szurmant
modifications to it may affect the ‘information flow’ towards
both the sensing domain and the kinase, located at the turn
(‘bottom’) of the receptor (Fig. 4). By contrast, sites 630 and
637 are on the ascending helix. Modifications to these sites
may affectjust the information flow solely towards the kinase
the three sitesappear toform a closely spaced triad.The close
proximity of the sites suggests that they may be affected by
electrostatic interactions between them. It also suggests that
modification of each site may affect not only the conforma-
tion of each monomer of the cytoplasmic domain separately
but also the interface of the two monomers that form the
tightly wound dimer. Finally, comparing a sequence align-
ment of other B. subtilis receptors such as McpA and McpC,
we find that the triad of putative methylation sites, and
presumably mode of action, is conserved in other B. subtilis
receptors (Le Moual & Koshland, 1996).
Comparison with previous work
Previously it was argued that the modification, amidation
or methylation, of site 630 increased kinase activity whereas
the modification of site 637 decreased it. These results were
obtained from experiments where aspartate substitutions at
each of the three sites and in combinations were examined
using the tethered cell assay (Zimmer et al., 2000). This
approach was based on previous work from the Koshland
lab (Shapiro & Koshland, 1994), in which glutamate/
glutamine residues were substituted with aspartate residues
G. D. Glekas and others
where a ‘permanent’ negative charge would be at the sites
that could not be neutralized by methylation. Based on
these previous studies, we anticipated that there would be
some difference between sites 630 and 637 in the
experiments reported in Tables 1 or 2, but evidently there
is none. One possible explanation for this discrepancy is
that previously employed aspartate-for-glutamate/gluta-
mine substitutions, which are shorter by one methylene
group, may alter the conformation of the receptor in some
unnatural way. Considering the close proximity of the
three adaptation sites, it does seem plausible that moving
the negative charge from its position in a glutamate to its
position in an aspartate (the distance of a methylene group
or 1.33 A˚) could have unnatural effects.
Model for site-specific methylation during taxis in
a concentration gradient of attractant
Based on the results of this work, we propose the following
model for site-specific methylation. In the absence of
attractant, we expect that site 371 is either amidated (i.e.
glutamine) or methylated and sites 630 and 637 are
unmethylated (i.e. glutamates). Such a modification state
would be optimal in the sense that the kinase is maximally
active and the ‘apparent Kd’ lowest. Thus, the bacteria
would be able to detect a concentration gradient of
attractant beginning at quite low concentrations. As the
bacterium swam up the gradient, site 371 would gradually
be deamidated when it is a glutamine or demethylated
when a methyl-glutamate. This would have the effect of
reducing kinase activity due to higher ambient concentra-
tions of asparagine as part of the adaptation process and
also increasing the ‘apparent Kd’, enabling the bacterium to
optimally sense gradients at higher concentrations of
attractant. Similarly, we expect that sites 630 and 637
would gradually become more methylated (for which
amidation was used in this study as a mimic). This would
have the effect of further reducing kinase activity as part of
the adaptation process. Based on the relative timing of the
demethylation and methylation steps (Kirby et al., 1999),
we expect that changes at site 371 would occur more
rapidly than those at sites 630 and 637. The reason why
these two processes occur on different timescales, however,
is still unknown.
In summary, we have found that amidation of site 371
increases McpB’s apparent affinity for asparagine and also,
in most cases, increases kinase activity. In addition, we
found that amidation of sites 630 and 637 decreases kinase
activity but does not affect the apparent affinity. These
findings further our understanding of the site-specific
methylation system in B. subtilis by demonstrating how the
modification of specific sites can have varying effects on
This work was supported by NIH grant GM054365 to G.W.O and
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The cartoon shows that site 371 is in close proximity to the HAMP
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Edited by: J. G. Shaw
Adaptation mechanism of a B. subtilis chemoreceptor