Microbiology (2004), 150, 581–589
DOI 10.1099/mic.0.26463-0
Effect of loss of CheC and other adaptational
proteins on chemotactic behaviour in Bacillus
subtilis
Michael M. Saulmon, Ece Karatan and George W. Ordal
Department of Biochemistry, Colleges of Medicine and Liberal Arts and Sciences,
University of Illinois, Urbana, IL 61801, USA
Correspondence
George W. Ordal
g-ordal@uiuc.edu
Received 6 May 2003
Revised
25 September 2003
Accepted 7 November 2003
Bacillus subtilis has a more complex mechanism of chemotaxis than does the paradigm
organism, Escherichia coli. In order to understand better the role of the novel chemotaxis
proteins – CheC, CheD and CheV – mutants in which increasing numbers of the corresponding
genes had been deleted were studied as tethered cells and their biases and sometimes durations
of counterclockwise (CCW) and clockwise (CW) flagellar rotations in response to addition and
removal of the attractant asparagine were observed. The cheC mutant was found to have
considerably reduced switching frequency (that is, prolonged CCW and CW rotations) without a
significantly different prestimulus CCW bias, compared with wild-type. This result may indicate
that in absence of CheC the switch might be in a conformation less resembling the transition state
than in presence of CheC. Conversely, the cheB (methylesterase) mutant showed considerably
increased switching frequency without affecting CCW bias, compared with wild-type. Removal
of all known adaptation systems – the methylation, CheC and CheV systems – resulted in a
mutant (cheRBCDV ) that still retained some adaptation following the addition of attractant.
INTRODUCTION
Chemotaxis is the process by which bacteria travel to
high concentrations of attractant or low concentrations of
repellent and it occurs by biasing an otherwise random walk
(Berg & Brown, 1972). The basic mechanism in Bacillus
subtilis (reviewed by Aizawa et al., 2001) and in Escherichia
coli (reviewed by Bourret & Stock, 2002; Bren & Eisenbach,
2000; Falke & Kim, 2000; Falke & Hazelbauer, 2001; Stock
& Levit, 2000; Taylor et al., 1999) involves complexes of
transmembrane receptors (called methyl-accepting chemotaxis proteins or MCPs), CheA autokinase and CheW
coupling protein, which allows the receptors to activate the
kinase (Morrison & Parkinson, 1997). The CheA kinase
autophosphorylates at the expense of ATP with production of ADP (Garrity & Ordal, 1997; Hess et al., 1988a, b).
The response regulator, CheY, phosphorylates itself using
CheA-P as the phosphoryl donor (Hess et al., 1988b) and
is thought to bind to the switch protein FliM (Bren &
Eisenbach, 1998; Szurmant et al., 2003; Toker & Macnab,
1997; Toker et al., 1996). Since the default direction of
flagellar rotation in E. coli is counterclockwise (CCW),
CheY-P binding causes an increase in the probability of
CW rotation (Alon et al., 1998; Sourjik & Berg, 2002; Bren
& Eisenbach, 2001; Scharf et al., 1998), to produce tumbling, which is uncoordinated motion that permits the next
Abbreviations: CCW, counterclockwise; CW, clockwise; MCP, methylaccepting chemotaxis protein.
0002-6463 G 2004 SGM
smooth swim to be in a different direction (Berg & Brown,
1972). Since the default direction of flagellar rotation in
B. subtilis is clockwise (CW) (Bischoff et al., 1993), binding
of CheY-P to FliM causes smooth swimming in B. subtilis
(Bischoff et al., 1993; Szurmant et al., 2003). In the
unstimulated, prestimulus state, the flagella in B. subtilis
rotate CCW approximately 55 % of the time. Stimulation by
attractants results in an upregulation of CheA kinase and
leads to increased CheY-P levels. This results in an increase
in the CCW flagellar rotation.
Adaptation, which brings the levels of CheA-P and CheY-P
back to prestimulus levels following addition of attractant,
involves a coordinated methylation and demethylation of
different sites on the receptors in B. subtilis (Zimmer et al.,
2000) and involves only methylation of several possible
sites in E. coli/Salmonella enterica (Terwilliger et al., 1986).
CheR methyltransferase carries out the methylation in
these organisms (Burgess-Cassler et al., 1982; Springer
& Koshland, 1977) and the CheB methylesterase, the
demethylation (Goldman et al., 1984; Stock & Koshland,
1978). The B. subtilis chemotaxis mechanism is more
elaborate than that of the paradigm organism E. coli and
involves several proteins not found in E. coli – CheC, CheD
and CheV (Aizawa et al., 2001; Fredrick & Helmann, 1994;
Karatan et al., 2001; Kirby et al., 2001; Kristich & Ordal,
2002; Rosario & Ordal, 1996; Rosario et al., 1995). However,
B. subtilis lacks CheZ, which in E. coli dephosphorylates
CheY-P (Blat & Eisenbach, 1994; Lukat & Stock, 1993;
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581
M. M. Saulmon, E. Karatan and G. W. Ordal
Zhao et al., 2002). CheD deamidates certain selected
glutamine residues in the receptors, changing them to
glutamate residues (Kristich & Ordal, 2002), a reaction that
makes the receptors more amenable to stimulation by
attractant, possibly due to an effect on higher-order
complexes within receptor clusters (Kirby et al., 2001).
The other two proteins – CheC and CheV – are involved
in mechanisms of adaptation. CheV has two domains, a
CheW-type coupling domain and a CheY-type responseregulator domain (Fredrick & Helmann, 1994), which
undergoes phosphorylation to help bring about adaptation
(Karatan et al., 2001). CheC works by an unknown
mechanism. There is evidence that it can bind to the
receptor complex, including the receptors, the CheA kinase
and CheD, and it is homologous to a region within the
switch proteins FliM and FliY (Kirby et al., 2001). It has
been suggested that CheC binds to one or both of these
switch proteins (Kirby et al., 2001). Interestingly, CheC and
CheD interact (Rosario & Ordal, 1996) but the purpose is
unknown, for there are many bacteria that have cheD but
lack cheC (Kirby et al., 2001). A summary of the properties
of the chemotactic proteins of B. subtilis is presented in
Table 1 and a diagram of their proposed locations is given
in Fig. 1.
In order to gain further insight into the roles of the various
chemotaxis proteins, we created strains lacking combinations of them, subjected them to addition and removal of
attractant, and observed the effect on the time-course of
direction of flagellar rotation using tethered cells. Tethered
cells are cells that are sheared of most of their flagella and
tethered using anti-flagellar antibody by the remaining
flagellum to a glass cover slip that forms the ceiling of a
laminar-flow chamber. The stimuli of addition and removal
of attractant are administered by flowing buffer with or
without attractant past the bacteria and the resulting
changes in rotational behaviour of the bacteria are
monitored using a microscope fitted with a CCD camera
and suitable recording devices (Kirby et al., 1999).
METHODS
Growth of bacteria. Bacteria were grown and prepared for the
tethered-cell assay as described by Kirby et al. (2001) for capillary
assays except that the bacteria were grown overnight on tryptose
blood agar (TBAB) plates at 30 uC, suspended at OD600 0?014, and
grown in minimal medium supplemented with 0?02 % tryptone. The
methods of Ordal et al. (1983) were followed for transformation and
transduction.
Construction of strains. The strains studied in this article are
given in Table 2. Strain OI3627, the cheRBCD mutant, was constructed by the same procedure as described by Kirby et al. (1999).
The linkage of cheCD, as determined by Cmr, and unkU29 : : spc (due
to integration of a Spcr cassette in an unknown gene upstream of
the major fla/che operon), was 70 % by PBS1 transduction. Strain
OI3642, the cheRBCDV mutant, was created by transducing OI3627
to Knr using PBS1 grown on HB4004, which contains a null mutation in cheV (Rosario et al., 1994). Strain OI3511, the cheRBCDW
mutant, was created in two stages. First, plasmid pC11 (Hanlon
et al., 1992), which contains part of cheA as well as cheW, cheC,
cheD and part of sigD, was gutted of cheW, cheC and cheD, which
was replaced with a terminatorless cat cassette (Bischoff & Ordal,
1991) to make plasmid pK37. This plasmid was linearized and
introduced into the cheB mutant OI2715 by transformation, with
selection for Cmr, to make strain OI3512. Finally, strain OI2680
was made His+ by selection for histidine prototrophy, and the cheR
allele was introduced into OI3512 by PBS1 transduction with selection for His+. Presence of the cheR allele was confirmed by growing
PBS1 on candidates, transducing OI1085 to His+ and scoring for Cmr.
Table 1. Roles of proteins in B. subtilis chemotaxis
Protein
Role
Bias of null mutant
Comments
CheA
Autokinase
Tumbly
For excitation
CheY
Response
regulator
Coupling
Completely tumbly,
no switching
Random
Binds to flagellar switch to
cause CCW rotation
Couples receptor to CheA
CheB
Coupling,
adaptation
Methylesterase
Random (cheWcheV
is tumbly)
Random
CheR
Methyltransferase Tumbly
CheC
Adaptation
Random
CheD
Deamidation of
receptors
CheY-P
hydrolysis
Tumbly
CheW
CheV
FliY
582
Non-motile (fliYD6–15
is smooth swimming)
Reference(s)
Fuhrer & Ordal (1991); Garrity & Ordal (1997);
Zimmer et al. (2002)
Bischoff & Ordal (1991); Bischoff et al. (1993)
Fredrick & Helmann (1994); Hanlon et al. (1992);
Karatan et al. (2001); Rosario et al. (1994)
Couples receptor to CheA and Fredrick & Helmann (1994); Karatan et al. (2001);
plays role in adaptation
Rosario et al. (1994)
Demethylates receptors upon
Goldman et al. (1984); Kirby et al. (1997, 2000);
being phosphorylated
Kirsch et al. (1993b); Zimmer et al. (2000, 2002)
Methylates receptors using
Burgess-Cassler & Ordal (1982); Burgess-Cassler
S-adenosylmethionine
et al. (1982); Kirsch et al. (1993b); Rosario et al.
(1995); Zimmer et al. (2000, 2002)
Plays role in adaptation
Kirby et al., 2001); Rosario & Ordal (1996);
Rosario et al. (1995)
Deamidates receptors to
Kirby et al. (2001); Kristich & Ordal (2002);
activate them
Rosario & Ordal (1996); Rosario et al. (1995)
Dephosphorylates CheY-P
Szurmant et al. (2003)
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Microbiology 150
Behaviour of mutants of adaptational proteins
RESULTS
Mean CCW and CW event durations in cheD,
cheC and cheB mutants in the prestimulus state
Both cheC and cheB mutants have wild-type prestimulus
biases (Table 3). However, analysis of mean CCW and CW
durations of flagellar rotations in these mutants showed
gross differences. When compared to the wild-type, the
mean CCW and CW event durations in the cheC mutant
were increased by 70 % and 67 %, respectively, both of
which were statistically significant values (Table 3). Because
both values increased by a similar magnitude, the cheC
prestimulus bias was very close to the wild-type value. In
contrast, in the cheB mutant, mean CCW and CW values
were lower than that of the wild-type by 37 % and 27 %,
respectively (Table 3). Again, because both parameters
decreased equally, the cheB strain exhibited a wild-type
prestimulus bias.
Fig. 1. Cartoon depicting locations of chemotaxis protein in
B. subtilis. Receptors are portrayed as a dimer of a transmembrane protein that is predominately a-helical. Proteins in
circles or ellipses are chemotaxis proteins (the letter referring to
the corresponding protein, so that ‘A’ means ‘CheA’, etc.) that
are cytoplasmic but can be associated with the receptors or
the switch, as indicated. Switch proteins are in boxes, with
designations as follows: G, FliG; M, FliM; Y, FliY. CheC
migrates between the receptor complex (the receptor and associated proteins) and the switch complex (FliG, FliM and FliY)
(see Discussion). When attractant binds to the receptor, it activates the CheA kinase to autophosphorylate, to make CheA-P.
CheY then becomes phosphorylated, and CheY-P goes to FliM
in the switch to cause CCW rotation of the flagella. Adaptation, to restore prestimulus CheY-P levels, occurs by several
mechanisms, involving CheR (which methylates the receptors),
CheB (which demethylates them), CheV, CheC and FliY.
Besides CheY, both CheB and CheV become phosphorylated
by CheA-P. (See text for a fuller explanation of these events.)
Assay of behaviour of tethered cells and analysis of data.
The tethered-cell assay and calculation of the averaged behaviour
have been previously described by Kirby et al. (1999). A CCW bias
for an individual behavioural period of a single cell was calculated
by averaging all 4 s data points within that period. The CCW biases
for all cells of a given strain were then averaged to obtain the mean
CCW bias for that behavioural period. A Student t-test was used to
compare two CCW bias distributions, either the same behavioural
period of two different strains or two behavioural periods of the
same strain. Two distributions were considered different if the
probability of committing a type I error (ptype I, the probability of
considering two distributions different when they are in fact the
same) was less than 0?05. Otherwise, they were considered not
significantly different. Mean CCW and clockwise (CW) event durations for a given period were calculated by averaging all rotational
events that occurred within the period of all cells for a given strain.
An identical Student t-test as that described above was then used
to determine the difference between two distributions.
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Interestingly, although the wild-type adapted completely
(Fig. 2, Table 4), the mean postaddition CCW and CW
event durations were significantly longer than those of the
prestimulus period (Table 4).
Comparison of the effect of the cheD, cheC
and cheB null alleles on bias
CheD is known to interact with CheC and the MCP receptor
complex (Kirby et al., 2001; Rosario & Ordal, 1996). It was
therefore of interest to understand the effect of deleting
cheD on the event duration parameters described above. It
has long been known that the cheD strain has a very low
prestimulus CCW bias (Kirby et al., 2001; Rosario & Ordal,
1996); analysis of the mean event duration parameters
showed that this low bias was due to a large decrease in
mean CCW event duration and a small increase in the
mean CW event duration (Table 3). This phenotype was
very similar to that of the cheCD strain, indicating that the
cheD null allele is much more important than the cheC
null allele in determining bias. However, even in these
strains, the cheC allele had a qualitatively similar effect as
in wild-type: the mean CCW and CW event durations were
longer in the strain lacking cheC, the ptype I values being
1?43E–02 and 3?51E–17, respectively. Additionally, the
cheD null allele was also much more important than the
cheB null allele, at least when the cheC null allele is also
present, in that the bias was also very low (Table 3). These
results underscore the importance of CheD in setting the
prestimulus bias.
Chemotactic behaviour of the cheBcheCcheD
mutant
Behavioural responses of cheB and cheC mutants to addition and removal of asparagine have been reported (Kirby
et al., 2000, 2001). The cheB mutant could respond to high
concentrations of asparagine (504 mM, corresponding to
90 % receptor occupancy: Ordal et al., 1977). However, it
was impaired in adaptation to this stimulus as well as its
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M. M. Saulmon, E. Karatan and G. W. Ordal
Table 2. Chemotaxis mutant strains used in this study
Strain
Genotype
Relevant characteristics
Reference(s)
Ullah & Ordal (1981)
Kirsch et al. (1993b)
Kirby et al. (2000); Kirsch
et al. (1993a)
Kirsch et al. (1993a)
Rosario et al. (1995)
Rosario et al. (1995)
Kirby et al. (1999)
OI1085
OI2680
OI2715
hisC2trpF7metC1
hisC2trpF7metC1cheR3 : : cat
hisC2trpF7metC1cheB7
Wild-type for chemotaxis
Lacks CheR
Lacks CheB
OI2836
OI2934
OI3135
OI3375
hisC2trpF7metC1cheB8 : : cat
hisC2trpF7metC1 cheD1 : : cat
hisC2trpF7metC1cheC1
hisC2trpF7metC1cheB7unkU29 : : spc
(cheCcheD)501 : : cat
trpF7metC1cheR3 : : catcheB7
(cheWcheCcheD)1 : : cat
hisC2trpF7metC1cheB7unkU29 : : spc
(cheCcheD)501 : : catcheR3 : : cat
hisC2trpF7metC1cheB7unk : : spc
(cheCcheD)501 : : catcheR3 : : catcheV1 : : kan
Lacks CheB
Lacks CheD
Lacks CheC
Lacks CheB, CheC and
CheD
Lacks CheB, CheR, CheC,
CheD and CheW
Lacks CheB CheR CheC
and CheD
Lacks CheB, CheR, CheC,
CheD and CheV
OI3511
OI3627
OI3642
This work
This work
This work
Table 3. CCW bias and mean event durations for various mutant strains
Strain*
CCW
bias
ptype ID
CCW event
duration
Wild-type
cheB
cheC
cheD
cheCD
cheBCD
0?55
0?54
0?59
0?10
0?04
0?09
NA
1?02
0?64
1?73
0?22
0?25
0?29
NS
NS
8?2E–12
2?0E–02
9?9E–04
ptype ID
CW event
duration
NA
0?75
0?55
1?25
1?03
1?40
1?03
1?2E–33
4?5E–15
5?5E–158
1?4E–02
2?4E–04
ptype ID
NA
1?1E–33
4?1E–20
1?2E–22
3?5E–17
3?2E–19
*Number of cells observed: wild-type, 19; cheB, 48; cheC, 28; cheD, 24; cheCD, 17; cheBCD, 48.
DAs compared to the corresponding wild-type value, except that the values for cheCD are compared with the
values for cheD and the values for cheBCD are compared with the values for cheCD. See Methods for
definition. E–33 (etc.), 610233 (etc.); NA, not applicable; NS, difference not significant.
Table 4. Comparison of wild-type steady-state period
parameter values
Period
Prestimulus
Postaddition
Postremoval
Duration or bias
Mean value*
ptype ID
CCW event duration
CW event duration
CCW bias
CCW event duration
CW event duration
CCW bias
CCW event duration
CW event duration
CCW bias
1?02
0?75
0?55
1?16
0?91
0?59
1?08
0?84
0?57
NA
NA
NA
3?42E–4
4?25E–14
NS
NS
3?71E–3
NS
*CCW and CW event durations (in s).
DAs compared to the corresponding prestimulus period value; see
Methods for definition. Nineteen cells were observed. NA, Not
applicable; NS, difference not significant.
584
response to the removal (Kirby et al., 2000; Zimmer et al.,
2002). The cheC mutant showed impaired adaptation to
the addition of 504 mM asparagine, but normal response
to its removal (Kirby et al., 2001; Rosario et al., 1995).
To determine the behavioural responses of the cheBCD
mutant, tethered-cell assays were performed with this
concentration of asparagine. Previous analysis of rotational
behaviour of the cheD and cheCD strains had indicated that
only 50 % of the cells were capable of responding to the
addition of asparagine in either of the strains (Kirby et al.,
2001). However, in the cheBCD mutant every cell analysed
showed a response to the addition of asparagine (Fig. 2).
This strain was also able to partially adapt to the addition
of asparagine.
Chemotactic behaviour generated by minimal
systems
In order to determine the minimal chemotaxis system still
capable of response and adaptation to a positive stimulus,
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Behaviour of mutants of adaptational proteins
Table 5. CCW bias and mean
cheRBCD, cheRBCDW, cheRBDCV
Fig. 2. Tethered-cell assay on wild-type (OI1085) and
cheBCD mutant (OI3375) in response to 0?5 mM asparagine.
Each line represents an averaged CCW bias: OI1085, 19
cells; OI3375, 48 cells. Up arrow, step addition of asparagine;
down arrow, step removal of asparagine. See Methods for
details on the strains and the tethering assay.
three strains were constructed: cheRBCD (Kirby et al., 1999),
cheRBCDW and cheRBCDV. The latter two strains each lack
one of the two proteins that couple the CheA autokinase
to the MCP receptor complex. A cheWV strain has a very
tumbly bias and is unresponsive to all stimuli (Karatan
et al., 2001; Rosario et al., 1994). The cheRBCD strain had
a very low prestimulus CCW bias and was unresponsive to
the addition and removal of asparagine (Fig. 3), and the
mean prestimulus CCW and CW event durations (Table 5)
were not significantly different from those of the other
strains lacking CheD (Table 5). Interestingly, further deletion
of cheW restored the responsive nature of the chemotaxis
system (Fig. 3) and significantly increased the prestimulus
CCW bias (Table 5). This increase in bias was entirely due to
an increase in the mean CCW event duration.
The cheRBCDV strain did not show a statistically significant
increase in bias; however, the response to addition and
removal of asparagine was restored in this strain as well.
Fig. 3. Tethered-cell assay on cheRBCD (OI3627), cheRBCDW
(OI3511) and cheRBCDV (OI3642) mutants. Each line represents an averaged CCW bias: OI3627, 16 cells; OI3511, 13
cells; OI3642, 14 cells. Up arrow, step addition of asparagine;
down arrow, step removal of asparagine. See Methods for
details on the strains and the tethering assay.
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event
durations
for
Strain
CCW
bias
CCW event
duration
CW event
duration
Wild-type
cheRBCD
cheRBCDW
cheRBCDV
0?55
0?10
0?27*
0?15
1?02
0?31
0?56*
0?45*
0?75
1?30
1?31
1?19*
*Statistically different from corresponding cheRBCD value. Values of
ptype I were as follows: CCW bias for cheRBCD compared with
cheRBCDW, 4?91E–03; CCW event duration for cheRBCD compared
with cheRBCDW, 2?91E–26; CCW event duration for cheRBCD
compared with cheRBCDV, 2?28E–08; CW event duration for
cheRBCD compared with cheRBCDV, 4?87E–02.
Furthermore, the ability of this strain to partially adapt to
the addition of asparagine is significant because it suggests
that there still exists some measure of post-additional adaptation, despite the lack of all three known adaptation mechanisms: CheR/CheB methylation/demethylation (Hanlon et al.,
1993; Kirby et al., 1997; Zimmer et al., 2000, 2002), the
CheC mechanism (Kirby et al., 2001; Rosario et al., 1995),
and CheV phosphorylation (Karatan et al., 2001). It is
possible that a fourth (as-yet-unidentified) adaptation
mechanism is also present in the chemotaxis system of
B. subtilis. In this connection, FliY has recently been found
to have CheY-P phosphatase activity (Szurmant et al., 2003).
DISCUSSION
Over the past 20 years much insight has been gained into
the roles of chemotaxis proteins in both B. subtilis and other
bacteria by analysing the rotational behaviour of cells that
are subjected to addition and removal of chemoeffectors.
The response profile of mutant strains reveals which phase
of the chemotactic response – prestimulus, addition, postaddition, removal or post-removal – the strains are defective in. The parameter used has been the CCW bias, that
is, the fraction of time the flagella rotate CCW. Here we
have shown that while the CCW bias is an important
parameter for analysis of chemotactic behaviour of cells,
it can sometimes be incomplete. A complementary parameter to use is the mean durations of CCW and CW events.
By using this parameter, we were able to show that cheB
and cheC mutants were altered in their prestimulus
behaviour even though they had normal prestimulus
biases and that adaptation in wild-type was complete for
bias but not for these parameters. The cheC mutant had
increased CCW and CW event durations and the cheB
mutant had decreased CCW and CW event durations. In
other words, the cheC and cheB mutants exhibited a
significant decrease and increase in the flagellar switching
frequencies, respectively. These defects suggest that the
switch complexes in these mutants are in a different
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M. M. Saulmon, E. Karatan and G. W. Ordal
conformational state compared to the wild-type strain as
well as each other.
The observation that the cheC mutant has reduced flagellar
switching frequency suggests that CheC reduces the durations of CCW and CW events. This result is consistent with
a reduction in the energy of activation for the transition
between CCW and CW directions of flagellar rotation. One
way this might occur would be by binding of CheC to
FliY or FliM (see Kirby et al., 2001) and stabilizing a
conformation of the switch proteins that is closer to the
transition state. Kirby et al. (2001) suggested that since
CheC has similarity to switch proteins, it might transiently
associate with the switch complex and since it binds to the
receptors, CheD and CheA, it might go from the switch
to the receptors as part of a mechanism of adaptation
(cheC mutants adapt poorly: Kirby et al., 2001; Rosario
et al., 1995). Our results support this hypothesis by showing that CheC does have an effect on switch properties
and it is hard to imagine a way that such an effect can occur
other than by direct binding.
Not only is it likely that CheC binds to the switch, but also
two lines of evidence are consistent with the hypothesis
that CheC is at least transiently associated with the ternary
receptor complex. (i) CheC was shown to interact with the
asparagine receptor McpB as well as CheA and CheD in a
yeast two-hybrid assay (Kirby et al., 2001). (Both CheA and
CheD interact with receptors directly: see Introduction.)
(ii) It has also been shown that receptor methylation is
affected by CheC because cheC mutants have overmethylated receptors (Rosario & Ordal, 1996). Thus, not only is
there evidence that CheC can interact at both the receptors
and at the switch – and hence might travel between them
as part of a mechanism – but also it is possible that the
affinity of CheC for the receptors could be determined by
the methylation state of the receptors.
In order to account for the effect of a null mutation in
cheB on switching frequency (much shorter durations of
CCW and CW rotations; see Table 3), two hypotheses are
apparent. One is that CheB binds directly to the switch to
affect switching frequency and the other is that in the cheB
mutant, there is more CheC available to bind to the switch.
We favour the latter hypothesis since there is no evidence
that CheB, an enzyme that demethylates receptors, binds
to the switch. By contrast, the effect of the cheB null
mutation on switching frequency can be accounted for
by assuming that overmethylated receptors have minimal
affinity for CheC (see preceding paragraph). In this hypothesis, a surplus of CheC could result in increased binding
at the switch and further lowering of the energy of activation of transition beyond that in wild-type, leading to
the increased switching frequency observed in the cheB
mutant.
This hypothesis that effects of different conditions (such as
lack of CheB methylesterase) on switching frequency are
mediated via CheC levels also may provide understanding
586
of the incomplete adaptation for durations of CW and CCW
rotation following addition of attractant (Table 4). It is
generally accepted that bacteria fully adapt to stimuli (Alon
et al., 1999; Barkai & Leibler, 1997; Macnab & Koshland,
1972), and it is true that B. subtilis adapts fully in terms
of bias (Table 4). However, it does not adapt in terms of
durations of CCW and CW rotations; in fact, both are
longer after ‘adaptation’ (Table 4). No current model of
chemotaxis accounts for this failure of adaptation. However, if we assume that the ‘adapted’ receptor complex has
increased affinity for CheC, then there would be less CheC
binding at the switch and the switching frequency would
be longer.
This line of thinking, where CheC binding to the switch
affects switching frequency, might provide insight into a
longstanding puzzle. Many archaea and some bacteria
have CheC or the closely related CheX (Kirby et al., 2001).
Some of these have polar flagella and, on negative stimuli,
undergo repeated reversals of motion, such as the spirochaete Spirochaeta aurantia (Cercignani et al., 1998;
Fosnaugh & Greenberg, 1988) and the archaeon Halobacterium salinarum (Spudich et al., 1989). It is clear that
the main output of the receptor/CheA complex is to regulate CheY-P levels in these as in all chemotactic bacteria/
archaea but how CheY-P levels can regulate switching
frequency (it primarily affects bias) has never been understood. In line with the effect of CheC on switching frequency
in B. subtilis, we propose a possible mechanism for the
increased switching frequency observed in these organisms
upon negative stimulation, namely change in CheC (or
CheX for S. aurantia) binding at the switch.
To make this argument, we first point out that that in the
case of H. salinarum, at least, CheY-P has a similar function as in E. coli and B. subtilis in that it permits the nondefault direction of flagellar rotation. In a cheY mutant of
H. salinarum, only forward swimming (chronic CW rotation of the flagella at each end of the archeon) occurs
(Rudolph & Oesterhelt, 1996). Backward swimming and
reversals between directions, however, occur in wild-type.
The increased reversals on negative stimulus might be due
to changing CheY-P levels affecting affinity of CheC (or
CheX) for the switch and possibly also to changing affinity
for CheC (or CheX) at the receptors (due to methylation
changes that occur there: Nordmann et al., 1994; Spudich
et al., 1989). As a further parallel with B. subtilis, we note
that a cheB mutant of H. salinarum shows increased
frequency of reversals, with no effect on the ratio of CW
and CCW rotation of the flagella, compared with wild-type
(Rudolph & Oesterhelt, 1996). Since in the B. subtilis cheB
mutant the ratio of CW and CCW rotation (that is, bias) of
the flagella is unchanged but switching frequency is
increased (Table 3), it is not hard to imagine that reversal
frequency in H. salinarum and B. subtilis are controlled by
the same mechanism, namely, amount of CheC bound at
the switch.
One of the hallmarks of B. subtilis chemotaxis is that
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Microbiology 150
Behaviour of mutants of adaptational proteins
multiple adaptational mechanisms appear to operate
concurrently. Besides the CheC mechanism, there is one
that involves methylation/demethylation of the receptors
(Hanlon & Ordal, 1994; Kirby et al., 1997; Zimmer et al.,
2000, 2002) and another that involves phosphorylation of
the response regulator CheV (Karatan et al., 2001). We
sought to understand how deletion of these adaptational
systems affected the unstimulated behaviour of bacteria
and their response to attractant. We were already aware of
the effect of deleting cheD and of the fact that the phenotype of the cheD and cheCD mutants was quite similar
(considerably different from the cheC mutant) (Kirby et al.,
2000). We confirmed and extended this previous observation to include strains cheBCD, cheBCD, cheRBCDW and
cheRBCDV. The low prestimulus biases that occur in these
appear to be a result of reduced durations of mean CCW
events due, probably, to receptors undeamidated by CheD
(Kristich & Ordal, 2002) being fairly inactive at stimulating
the CheA kinase. Earlier work had also shown that in both
the cheD and cheCD mutants about half the cells responded
and half did not respond to addition of asparagine (Kirby
et al., 2001). We have shown here that this responsiveness
was influenced by methylation in the sense that further
deletion of cheB made the bacteria all responsive but,
curiously, further deletion of cheR made all of them
unresponsive. Obviously, the methylation system has a
profound effect on the overall geometry and functioning
of the receptor/kinase complex.
It should be noted that the failure of the cheRBCD mutant
to respond to asparagine in the tethered-cell assay (or on
swarm plates; data not shown) is in contrast to the results
of Kirby et al. (1999), who reported that oscillations in
CCW bias occurred on addition of attractant and stopped
on its removal. Those results were reproducible; however,
the linkage between cheD and a Spcr marker in an
unidentified gene upstream of the beginning of the fla/che
operon (Slack et al., 1995) was later found to be tenfold
lower than expected for unknown reasons. The conclusion
of the paper that the adaptation was due to CheY-P feedback
at the receptors may have been premature since at that
time CheV was not recognized as an adaptational protein.
Interestingly, deletion of either gene encoding the coupling
proteins cheW or cheV (Karatan et al., 2001; Rosario et al.,
1994) to make a cheRBCDW or cheRBCDV mutant made
the bacteria again responsive to stimuli, and in the former
strain also significantly increased the mean prestimulus
CCW bias (Table 5). In this sense cheW had a moderating
effect on cheD, the only allele that we observed to have this
effect. In some way, the conformation of the receptors/
CheA complex was very stable in a refractory state when
both coupling proteins were present and became more
amenable to changing to an active state when one of
the coupling proteins was deleted. Very interestingly, the
cheRBCDV mutant lacked all known adaptation systems –
the methylation/demethylation mechanism, the CheV
mechanism and the CheC mechanism – and yet showed a
http://mic.sgmjournals.org
progressive decrease in CCW bias following the response
to the addition of attractant. Very recent experiments
have revealed that FliY, one of the switch proteins, can
catalyse dephosphorylation of CheY-P (Szurmant et al.,
2003) and in future experiments we will investigate the
behavioural phenotype of mutants unable to carry out this
dephosphorylation.
ACKNOWLEDGEMENTS
This work was supported by Public Health Service Grant RO1
GM54365 (to G. W. O.) from the National Institutes of Health. We
thank Vincent Cannistraro for making Fig. 1.
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