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{at}uiuc.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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.


Abbreviations: CCW, counterclockwise; CW, clockwise; MCP, methyl-accepting chemotaxis protein


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 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; 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 response-regulator 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.


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Table 1. Roles of proteins in B. subtilis chemotaxis

 


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Fig. 1. Cartoon depicting locations of chemotaxis protein in B. subtilis. Receptors are portrayed as a dimer of a transmembrane protein that is predominately {alpha}-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.)

 
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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 °C, 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.


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Table 2. Chemotaxis mutant strains used in this study

 
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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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.


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Table 3. CCW bias and mean event durations for various mutant strains

 
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).



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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.

 

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Table 4. Comparison of wild-type steady-state period parameter values

 
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 µM, corresponding to 90 % receptor occupancy: Ordal et al., 1977). However, it was impaired in adaptation to this stimulus as well as its response to the removal (Kirby et al., 2000; Zimmer et al., 2002). The cheC mutant showed impaired adaptation to the addition of 504 µM 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, 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.



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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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Table 5. CCW bias and mean event durations for cheRBCD, cheRBCDW, cheRBDCV

 
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. 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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, post-addition, 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 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 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 non-default 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 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 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.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Aizawa, S.-I., Zhulin, I. B., Márquez-Magaña, L. & Ordal, G. W. (2001). Chemotaxis and motility. In Bacillus subtilis and its Relatives: from Genes to Cells, pp. 437–452. Edited by A. Sonenshein, R. Losick & J. Hoch. Washington, DC: American Society for Microbiology.

Alon, U., Camarena, L., Surette, M. G., Aguera y Arcas, B., Liu, Y., Leibler, S. & Stock, J. B. (1998). Response regulator output in bacterial chemotaxis. EMBO J 17, 4238–4248.[Abstract/Free Full Text]

Alon, U., Surette, M. G., Barkai, N. & Leibler, S. (1999). Robustness in bacterial chemotaxis. Nature 397, 168–171.[CrossRef][Medline]

Barkai, N. & Leibler, S. (1997). Robustness in simple biochemical networks. Nature 387, 913–917.[CrossRef][Medline]

Berg, H. C. & Brown, D. A. (1972). Chemotaxis in Escherichia coli analysed by three-dimensional tracking. Nature 239, 500–504.[Medline]

Bischoff, D. S. & Ordal, G. W. (1991). Sequence and characterization of Bacillus subtilis CheB, a homolog of Escherichia coli CheY, and its role in a different mechanism of chemotaxis. J Biol Chem 266, 12301–12305.[Abstract/Free Full Text]

Bischoff, D. S., Bourret, R. B., Kirsch, M. L. & Ordal, G. W. (1993). Purification and characterization of Bacillus subtilis CheY. Biochemistry 32, 9256–9261.[Medline]

Blat, Y. & Eisenbach, M. (1994). Phosphorylation-dependent binding of the chemotaxis signal molecule CheY to its phosphatase, CheZ. Biochemistry 33, 902–906.[Medline]

Bourret, R. B. & Stock, A. M. (2002). Molecular information processing: lessons from bacterial chemotaxis. J Biol Chem 277, 9625–9628.[Free Full Text]

Bren, A. & Eisenbach, M. (1998). The N terminus of the flagellar switch protein, FliM, is the binding domain for the chemotactic response regulator, CheY. J Mol Biol 278, 507–514.[CrossRef][Medline]

Bren, A. & Eisenbach, M. (2000). How signals are heard during bacterial chemotaxis: protein-protein interactions in sensory signal propagation. J Bacteriol 182, 6865–6873.[Free Full Text]

Bren, A. & Eisenbach, M. (2001). Changing the direction of flagellar rotation in bacteria by modulating the ratio between the rotational states of the switch protein FliM. J Mol Biol 312, 699–709.[CrossRef][Medline]

Burgess-Cassler, A. & Ordal, G. W. (1982). Functional homology of Bacillus subtilis methyltransferase II and Escherichia coli CheR protein. J Biol Chem 257, 12835–12838.[Abstract/Free Full Text]

Burgess-Cassler, A., Ullah, A. H. & Ordal, G. W. (1982). Purification and characterization of Bacillus subtilis methyl-accepting chemotaxis protein methyltransferase II. J Biol Chem 257, 8412–8417.[Abstract/Free Full Text]

Cercignani, G., Lucia, S. & Petracchi, D. (1998). Photoresponses of Halobacterium salinarum to repetitive pulse stimuli. Biophys J 75, 1466–1472.[Abstract/Free Full Text]

Falke, J. J. & Hazelbauer, G. L. (2001). Transmembrane signaling in bacterial chemoreceptors. Trends Biochem Sci 26, 257–265.[CrossRef][Medline]

Falke, J. J. & Kim, S. H. (2000). Structure of a conserved receptor domain that regulates kinase activity: the cytoplasmic domain of bacterial taxis receptors. Curr Opin Struct Biol 10, 462–469.[CrossRef][Medline]

Fosnaugh, K. & Greenberg, E. P. (1988). Motility and chemotaxis of Spirochaeta aurantia: computer-assisted motion analysis. J Bacteriol 170, 1768–1774.[Medline]

Fredrick, K. L. & Helmann, J. D. (1994). Dual chemotaxis signaling pathways in Bacillus subtilis: a sigma D-dependent gene encodes a novel protein with both CheW and CheY homologous domains. J Bacteriol 176, 2727–2735.[Abstract]

Fuhrer, D. K. & Ordal, G. W. (1991). Bacillus subtilis CheN, a homolog of CheA, the central regulator of chemotaxis in Escherichia coli. J Bacteriol 173, 7443–7448.[Medline]

Garrity, L. F. & Ordal, G. W. (1997). Activation of the CheA kinase by asparagine in Bacillus subtilis chemotaxis. Microbiology 143, 2945–2951.[Medline]

Goldman, D. J., Nettleton, D. O. & Ordal, G. W. (1984). Purification and characterization of chemotactic methylesterase from Bacillus subtilis. Biochemistry 23, 675–680.[Medline]

Hanlon, D. W. & Ordal, G. W. (1994). Cloning and characterization of genes encoding methyl-accepting chemotaxis proteins in Bacillus subtilis. J Biol Chem 269, 14038–14046.[Abstract/Free Full Text]

Hanlon, D. W., Marquez-Magana, L. M., Carpenter, P. B., Chamberlin, M. J. & Ordal, G. W. (1992). Sequence and characterization of Bacillus subtilis CheW. J Biol Chem 267, 12055–12060.[Abstract/Free Full Text]

Hanlon, D. W., Ying, C. & Ordal, G. W. (1993). Purification and reconstitution of the methyl-accepting chemotaxis proteins from Bacillus subtilis. Biochim Biophys Acta 1158, 345–351.[Medline]

Hess, J. F., Bourret, R. B. & Simon, M. I. (1988a). Histidine phosphorylation and phosphoryl group transfer in bacterial chemotaxis. Nature 336, 139–143.[CrossRef][Medline]

Hess, J. F., Oosawa, K., Kaplan, N. & Simon, M. I. (1988b). Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis. Cell 53, 79–87.[Medline]

Karatan, E., Saulmon, M. M., Bunn, M. W. & Ordal, G. W. (2001). Phosphorylation of the response regulator CheV is required for adaptation to attractants during Bacillus subtilis chemotaxis. J Biol Chem 276, 43618–43626.[Abstract/Free Full Text]

Kirby, J. R., Kristich, C. J., Feinberg, S. L. & Ordal, G. W. (1997). Methanol production during chemotaxis to amino acids in Bacillus subtilis. Mol Microbiol 24, 869–878.[CrossRef][Medline]

Kirby, J. R., Saulmon, M. M., Kristich, C. J. & Ordal, G. W. (1999). CheY-dependent methylation of the asparagine receptor, McpB, during chemotaxis in Bacillus subtilis. J Biol Chem 274, 11092–11100.[Abstract/Free Full Text]

Kirby, J. R., Niewold, T. B., Maloy, S. & Ordal, G. W. (2000). CheB is required for behavioural responses to negative stimuli during chemotaxis in Bacillus subtilis. Mol Microbiol 35, 44–57.[CrossRef][Medline]

Kirby, J. R., Kristich, C. J., Saulmon, M. M., Zimmer, M. A., Garrity, L. F., Zhulin, I. B. & Ordal, G. W. (2001). CheC is related to the family of flagellar switch proteins and acts independently from CheD to control chemotaxis in Bacillus subtilis. Mol Microbiol 42, 573–585.[CrossRef][Medline]

Kirsch, M. L., Peters, P. D., Hanlon, D. W., Kirby, J. R. & Ordal, G. W. (1993a). Chemotactic methylesterase promotes adaptation to high concentrations of attractant in Bacillus subtilis. J Biol Chem 268, 18610–18616.[Abstract/Free Full Text]

Kirsch, M. L., Zuberi, A. R., Henner, D., Peters, P. D., Yazdi, M. A. & Ordal, G. W. (1993b). Chemotactic methyltransferase promotes adaptation to repellents in Bacillus subtilis. J Biol Chem 268, 25350–25356.[Abstract/Free Full Text]

Kristich, C. J. & Ordal, G. W. (2002). Bacillus subtilis CheD is a chemoreceptor modification enzyme required for chemotaxis. J Biol Chem 277, 25356–25362.[Abstract/Free Full Text]

Lukat, G. S. & Stock, J. B. (1993). Response regulation in bacterial chemotaxis. J Cell Biochem 51, 41–46.[Medline]

Macnab, R. M. & Koshland, D. E., Jr (1972). The gradient-sensing mechanism in bacterial chemotaxis. Proc Natl Acad Sci U S A 69, 2509–2512.[Abstract]

Morrison, T. B. & Parkinson, J. S. (1997). A fragment liberated from the Escherichia coli CheA kinase that blocks stimulatory, but not inhibitory, chemoreceptor signaling. J Bacteriol 179, 5543–5550.[Abstract]

Nordmann, B., Lebert, M. R., Alam, M., Nitz, S., Kollmannsberger, H., Oesterhelt, D. & Hazelbauer, G. L. (1994). Identification of volatile forms of methyl groups released by Halobacterium salinarium. J Biol Chem 269, 16449–16454.[Abstract/Free Full Text]

Ordal, G. W., Villani, D. P. & Gibson, K. J. (1977). Amino acid chemoreceptors of Bacillus subtilis. J Bacteriol 129, 156–165.[Medline]

Ordal, G. W., Nettleton, D. O. & Hoch, J. A. (1983). Genetics of Bacillus subtilis chemotaxis: isolation and mapping of mutations and cloning of chemotaxis genes. J Bacteriol 154, 1088–1097.[Medline]

Rosario, M. M. & Ordal, G. W. (1996). CheC and CheD interact to regulate methylation of Bacillus subtilis methyl-accepting chemotaxis proteins. Mol Microbiol 21, 511–518.[Medline]

Rosario, M. M., Fredrick, K. L., Ordal, G. W. & Helmann, J. D. (1994). Chemotaxis in Bacillus subtilis requires either of two functionally redundant CheW homologs. J Bacteriol 176, 2736–2739.[Abstract]

Rosario, M. M., Kirby, J. R., Bochar, D. A. & Ordal, G. W. (1995). Chemotactic methylation and behavior in Bacillus subtilis: role of two unique proteins, CheC and CheD. Biochemistry 34, 3823–3831.[Medline]

Rudolph, J. & Oesterhelt, D. (1996). Deletion analysis of the che operon in the archaeon Halobacterium salinarium. J Mol Biol 258, 548–554.[CrossRef][Medline]

Scharf, B. E., Fahrner, K. A., Turner, L. & Berg, H. C. (1998). Control of direction of flagellar rotation in bacterial chemotaxis. Proc Natl Acad Sci U S A 95, 201–206.[Abstract/Free Full Text]

Slack, F. J., Serror, P., Joyce, E. & Sonenshein, A. L. (1995). A gene required for nutritional repression of the Bacillus subtilis dipeptide permease operon. Mol Microbiol 15, 689–702.[Medline]

Sourjik, V. & Berg, H. C. (2002). Binding of the Escherichia coli response regulator CheY to its target measured in vivo by fluorescence resonance energy transfer. Proc Natl Acad Sci U S A 99, 12669–12774.[Abstract/Free Full Text]

Springer, W. R. & Koshland, D. E., Jr (1977). Identification of a protein methyltransferase as the cheR gene product in the bacterial sensing system. Proc Natl Acad Sci U S A 74, 533–537.[Abstract]

Spudich, E. N., Takahashi, T. & Spudich, J. L. (1989). Sensory rhodopsins I and II modulate a methylation/demethylation system in Halobacterium halobium phototaxis. Proc Natl Acad Sci U S A 86, 7746–7750.[Abstract]

Stock, J. & Levit, M. (2000). Signal transduction: hair brains in bacterial chemotaxis. Curr Biol 10, R11–14.[CrossRef][Medline]

Stock, J. B. & Koshland, D. E., Jr (1978). A protein methylesterase involved in bacterial sensing. Proc Natl Acad Sci U S A 75, 3659–3663.[Abstract]

Szurmant, H., Bunn, M. W., Cannistraro, V. J. & Ordal, G. W. (2003). Bacillus subtilis hydrolyzes CheY-P at the location of its action, the flagellar switch. J Biol Chem 278, 48611–48616.[Abstract/Free Full Text]

Taylor, B. L., Zhulin, I. B. & Johnson, M. S. (1999). Aerotaxis and other energy-sensing behavior in bacteria. Annu Rev Microbiol 53, 103–128.[CrossRef][Medline]

Terwilliger, T. C., Wang, J. Y. & Koshland, D. E., Jr (1986). Kinetics of receptor modification. The multiply methylated aspartate receptors involved in bacterial chemotaxis. J Biol Chem 261, 10814–10820.[Abstract/Free Full Text]

Toker, A. S. & Macnab, R. M. (1997). Distinct regions of bacterial flagellar switch protein FliM interact with FliG, FliN and CheY. J Mol Biol 273, 623–634.[CrossRef][Medline]

Toker, A. S., Kihara, M. & Macnab, R. M. (1996). Deletion analysis of the FliM flagellar switch protein of Salmonella typhimurium. J Bacteriol 178, 7069–7079.[Abstract]

Ullah, A. H. & Ordal, G. W. (1981). Purification and characterization of methyl-accepting chemotaxis protein methyltransferase I in Bacillus subtilis. Biochem J 199, 795–805.[Medline]

Zhao, R., Collins, E. J., Bourret, R. B. & Silversmith, R. E. (2002). Structure and catalytic mechanism of the E. coli chemotaxis phosphatase CheZ. Nat Struct Biol 9, 570–575.[Medline]

Zimmer, M. A., Tiu, J., Collins, M. A. & Ordal, G. W. (2000). Selective methylation changes on the Bacillus subtilis chemotaxis receptor McpB promote adaptation. J Biol Chem 275, 24264–24272.[Abstract/Free Full Text]

Zimmer, M. A., Szurmant, H., Saulmon, M. M., Collins, M. A., Bant, J. S. & Ordal, G. W. (2002). The role of heterologous receptors in McpB-mediated signalling in Bacillus subtilis chemotaxis. Mol Microbiol 45, 555–568.[CrossRef][Medline]

Received 6 May 2003; revised 25 September 2003; accepted 7 November 2003.