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J. Biol. Chem., Vol. 276, Issue 47, 43618-43626, November 23, 2001

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Phosphorylation of the Response Regulator CheV Is Required for Adaptation to Attractants during Bacillus subtilis Chemotaxis^*

Ece Karatan, Michael M. Saulmon, Michael W. Bunn, and George W. Ordal

From the Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received for publication, May 30, 2001, and in revised form, September 11, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the Gram-positive soil bacterium Bacillus subtilis, the chemoreceptors are coupled to the central two-component kinase CheA via two proteins, CheW and CheV. CheV is a two-domain protein with an N-terminal CheW-like domain and a C-terminal two-component receiver domain. In this study, we show that CheV is phosphorylated in vitro on a conserved aspartate in the presence of phosphorylated CheA (CheA-P). This reaction is slower compared with the phospho-transfer reaction between CheA-P and one other response regulator of the system, CheB. CheV-P is also highly stable in comparison with CheB-P. Both of these properties are more pronounced in the full-length protein compared with a truncated form composed only of the receiver domain, that is, deletion of the CheW-like domain results in increase in the rate of the phospho-transfer reaction and decrease in stability of the phosphorylated protein. Phosphorylation of CheV is required for adaptation to the addition of the chemoattractant asparagine. In tethered-cell assays, strains expressing an unphosphorylatable point mutant of cheV or a truncated mutant lacking the entire receiver domain are severely impaired in adaptation to the addition of asparagine. Both of these strains, however, show near normal counterclockwise biases, suggesting that in the absence of the attractant the chemoreceptors are efficiently coupled to CheA kinase by the mutant CheV proteins. Inability of the CheW-like domain of CheV to support complete adaptation to the addition of asparagine also suggests that unlike CheW, this domain by itself may lead to the formation of signaling complexes that stay overactive in the presence of the attractant. A possible structural basis for this feature is discussed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemotaxis is based on a two-component signal transduction network that allows bacteria to navigate within their environment and move toward more favorable conditions. Environmental cues are detected by transmembrane receptors called methyl-accepting chemotaxis proteins (MCPs),¹ which are methyl-esterified on conserved glutamate residues within their cytoplasmic domains by the methyltransferase CheR (1-4). The receptors are coupled via the adapter protein CheW to the two-component histidine kinase CheA (5, 6). In Bacillus subtilis, the autophosphorylating activity of CheA is up-regulated by binding of attractant molecules to the MCPs (7). This event is thought to increase the phosphoryl group flux to two response regulators, CheB and CheY. CheB is activated upon phosphorylation and hydrolyzes the methyl groups on the MCPs (8-11). The main response regulator of the system, CheY, upon phosphoryl group transfer from CheA-P binds the switch complex at the base of each flagellum (12, 13). Binding of CheY-P to the flagellar switch brings about an increase in the probability of counterclockwise (CCW) rotation of the flagella, resulting in smooth swimming (13, 14). In B. subtilis the default clockwise (CW) rotation of the flagella in the absence of CheY-P binding is associated with tumbling (14). Tumbling randomly reorients the bacteria in space, enabling them to swim in another direction in response to subsequent environmental signals.

Under steady state conditions, the wild-type B. subtilis has an average bias (defined as the percentage of time the flagella rotate CCW) of about 55%. A net increase in the attractant concentrations in the environment causes a transient increase in bias followed by an almost immediate return to the prestimulus level despite the continued presence of the attractant. This adaptation process allows bacteria to reset their systems to the new level of attractant concentration in the environment so that they can further respond to the net changes with respect to this new level. In Escherichia coli, adaptation involves changes in receptor methylation (15, 16). High levels of methylation are associated with increased CheA activity; the purpose of net methylation changes is to cause adaptation to the stimulus caused by ligand binding to the receptor (17).

In B. subtilis, adaptation is more complex. McpB, the sole receptor for the attractant asparagine, is demethylated not only upon asparagine addition but also upon asparagine removal (18). These demethylation events appear to target different glutamate residues; relative methylation states of the various glutamates have been correlated with adaptation (19). For example if one of the sites of methylation, residue 637, is mutated to aspartate and thus prevented from becoming methylated, whereas another site of methylation, residue 630, is unchanged, then the bacteria cannot adapt to the addition of asparagine (19). The same phenotype is also seen in null mutants lacking another chemotaxis protein, CheC (20). Thus, both selective methylation changes and CheC have been implicated in the mechanism of adaptation to the addition of attractants in B. subtilis.

In this study, we have investigated the existence of another adaptation system involving CheV, the third response regulator of the B. subtilis chemotaxis network. CheV is a two-domain protein with an N-terminal CheW-like domain and a C-terminal two-component receiver domain (21). Earlier experiments showed that cheW and cheV null mutants had wild-type biases and were able to respond and adapt to large stepwise increase of the attractant azetidine-2-carboxylic acid (22). A cheWcheV null mutant, however, had a very low bias and was unable to respond to the addition of attractant (22). In capillary assays, both cheW and cheV single mutants were impaired in their ability to migrate up attractant gradients (22). Together, these results led to the conclusion that CheW and CheV may be partially redundant in coupling the receptors to CheA; however, they are both necessary for efficient chemotaxis (22). In the present study we demonstrate that CheV is phosphorylated as a result of phosphoryl group transfer from CheA-P and that the purpose of this phosphorylation is to facilitate adaptation to attractants.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Bacterial Strains and Plasmids Used in This Study-- All bacterial strains used in this study are listed in Table I, and all plasmids used are listed in Table II. All plasmids were propagated in E. coli strain TG-1 (Amersham Pharmacia Biotech).

Chemicals, Solutions, and Growth Media-- L-[methyl-³H]Methionine (80-85 Ci/mmol) and [-³²P]ATP (>5000 Ci/mmol) were obtained from Amersham Pharmacia Biotech. All other chemicals were obtained from Sigma. Growth media-tryptone, tryptose blood agar base, and yeast extract were from Difco. Luria-Bertani (LB) medium is 1% tryptone, 0.5% yeast extract, and 1% NaCl. LBON (LB medium without NaCl) is 1% tryptone and 0.5% yeast extract.

Construction of thrC Integration Plasmids-- Both mutant alleles of the cheV gene were created by PCR mutagenesis using pKF13 as template (21, 23). To create pK33 (pSK::cheV_D235A), a set of primers, one of which was coded for the mutation, was used to PCR the entire plasmid. The PCR product was ligated, digested with DpnI (Life Technologies, Inc.) to remove the parent plasmid, and transformed into Tg1. Several transformants were selected, and the presence of the mutation was confirmed by sequencing. A 1.4-kilobase EcoRI-BamHI fragment was excised from pK33 and cloned into the thrC integration plasmid pDG1664 to create pK34 (24). To create pK35 (pSK::cheV_1-168), which encodes a truncated form of CheV containing the N-terminal CheW-like domain, PCR primers were constructed as follows: the reverse primer annealed starting at the codon encoding Phe¹⁶⁸, and the forward primer annealed at the stop codon. Ligation of the PCR product created a truncated form of the cheV gene with the entire C-terminal domain deleted. This construct was digested with DpnI and transformed into Tg1. Several transformants with the correct size insert were picked, and the junctions were sequenced. A 1.0-kilobase EcoRI-BamHI fragment was excised from pK35 and cloned into pDG1664 to make pK53. Both mutants were constructed such that they contained the native promoter, native ribosome binding site, the start codon (TTG), and the terminator of the cheV gene. Pfu polymerase (Stratagene) was used for all PCR reactions.

Construction of Chromosomal Integrations of the cheV Mutants-- pK34 and pK53 were transformed into OI2737 (cheW::cat) as well as OI3061 (cheW::cat, cheV::kan). The transformants were scored for macrolide/lincosamide antibiotic (MLS) resistance and spectinomycin sensitivity. Western blot analysis done on these strains as well as on OI2737 and OI1085 showed that the expression levels of CheV were identical in all strains.

Construction of cheV, cheV_164-303, and cheA Expression Plasmids-- A T7 expression system was used to overexpress the CheV proteins (25). In order not to add extra amino acids to the start of the protein, a NdeI site was used as the upstream cloning site. Because the cheV gene has an internal NdeI site, pKF13 and pK33 were first used as templates for PCR mutagenesis to delete the NdeI site by a silent mutation, creating pK39 and pK41, respectively. The mutations were confirmed by sequencing. PK39 and pK41 were in turn used as templates for PCR to add a NdeI site to the start of the gene, a 6xhistidine tag, and a BamHI site at the end of the cheV gene. These constructs were cloned into pT7-7 vectors to create pK44 (cheV) and pK42 (cheV_D235A). The 6xhistidine-tagged wild-type cheV gene (cheV_his) was also integrated into the thrC locus in a cheWcheV double null mutant background, creating strain OI3463, to test whether the tag interfered with function. cheV_his rescued the nonchemotactic phenotype of the cheWcheV double mutant in capillary assays, indicating that the tag did not interfere with function.

To create the vector expressing the C-terminal receiver domain of cheV, a 455-base pair fragment was amplified using pKF13 as template. This fragment was cloned into the BamHI and NotI sites of pUSH1, creating pK52 (26).

Purification of CheV and CheV_D235A-- pK44 and pK42 were transformed into strain OI3378 (27). 1L cultures were grown to A₆₀₀ 0.7 at 37 °C in LBON medium, induced by adding solid NaCl to a final concentration of 0.3 M, and grown for an additional 3 h. Purification was carried out using metal affinity chromatography according to the manufacturer's instructions (28). The matrix used for the metal affinity purification (Talon) was from CLONTECH. Briefly, the cell pellet was resuspended in extraction buffer (20 mM Tris-HCl, 150 mM NaCl), and cells were sonicated 3 times for 30 s each with a 1-min pause in between at 34% amplitude. The extract was clarified by centrifugation at 12,000 × g for 20 min followed by an additional centrifugation at 50,000 × g for 20 min. The supernatant was incubated with the Talon beads for 20 min at room temperature. The beads were pelleted by centrifugation by 2 min at 700 × g. The supernatant was aspirated, and the beads were washed twice with wash buffer (extraction buffer with 5 mM imidazole). After the second wash the beads were loaded onto a column, and the protein was eluted in elution buffer (extraction buffer with 50 mM imidazole).

Purification of CheV_164-303-- pK52 was transformed into OI3229. The resulting strain was used to overexpress the C-terminal domain of cheV. Cultures were grown to an A₆₀₀ of 0.7 at 37 °C, induced by adding 1 mM isopropyl-1-thio--D-galactopyranoside, and grown for an additional 3 h. The pUSH1 expression plasmid adds a 6xhistidine tag to the N terminus of the proteins; therefore, CheV_164-303 was also purified using metal affinity chromatography as described above with the exception of using a step gradient of imidazole during elution.

Purification of CheA-- pLG104 was transformed into OI3378 creating OI3454. A 5-liter culture was grown to an A₆₀₀ of 0.7 at 37 °C in LBON, induced by adding solid NaCl to a final concentration of 0.3 M, and grown for an additional 3 h. Cell extracts were prepared by centrifugation and clarified as described previously. Saturated ammonium sulfate solution was added to 32% saturation; solution was mixed gently at 4 °C for 1 h and then centrifuged at 27,000 × g for 30 min. Saturated ammonium sulfate solution was added to the supernatant to 55% saturation, the solution was mixed gently at 4 °C for 1 h, and the precipitate, which contained most of the CheA, was collected by centrifugation at 27,000 × g for 30 min. The precipitate was dissolved in 20 ml of Tris-HCL buffer (20 mM Tris-HCl, pH 7.5) and dialyzed overnight against 4 liters of buffer containing 20 mM Tris-HCl, 20% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, pH 7.5. 250-µl samples were loaded onto a MemSep 1000 anion exchange cartridge (Millipore) driven by an HPLC pump (Waters, model 510). The column was first washed with 250 mM NaCl for 10 min and then developed with a gradient of 250-500 mM NaCl in the above described buffer delivered over 25 min at a flow rate of 1 ml/min. 1-ml fractions were collected, and those containing CheA were identified by Coomassie staining following SDS-polyacrylamide gel electrophoresis. These fractions were pooled and concentrated using a 50-ml Amicon concentration unit with a 10,000 molecular weight cut-off membrane (Amicon).

Purification of CheB-- An 8-liter culture of OI3637 was grown to an A₆₀₀ of 0.7 at 37 °C in LBON, induced by adding solid NaCl to a final concentration of 0.3 M, and grown for an additional 4 h. Cells were harvested, washed in 20 mM Tris-HCl, pH 8.5, at 10 ml/g wet weight and resuspended at 3 ml/g wet weight in Tris-HCl buffer with 0.05 mM -mercaptoethanol and 0.5 mM phenylmethylsulfonyl fluoride. Cells were lysed by sonication and the suspension clarified by centrifugation for 15 min at 4000 × g followed by a 1-h, 95,000 × g centrifugation. CheB was fractionated by 50 and 65% ammonium sulfate cuts. The 65% fraction was resuspended at 75 mg/ml in 20 mM Tris-HCl, pH 8.0, 0.05 -mercaptoethanol, 5 mM EDTA and 0.5 mM phenylmethylsulfonyl fluoride and dialyzed overnight against the same buffer. The dialyzed solution was passed over a 2.0 × 10.0-cm Macro-prep DEAE (Bio-Rad) column equilibrated in the same buffer. The flow-through was pooled and fractionated over a Protein Pak 300SW gel filtration column (Waters). CheB-containing fractions were pooled and concentrated.

All of the proteins were dialyzed against TKMD buffer (50 mM Tris-HCl, 5 mM MgCl₂, 50 mM KCl, 0.2 mM dithiothreitol, and 10% glycerol, pH 8.0). The purity of the final protein preparations, assessed by Coomassie staining, was judged to be about 85-95%. Protein concentrations were estimated by measuring absorbance at 280 nm using the following extinction coefficients calculated using the method of Gill and von Hippel (29): for CheV and CheV_D235A, E₂₈₀ = 17210 M¹ cm¹; for CheA, E₂₈₀ = 23380 M¹ cm¹; and for CheV_164-303, E₂₈₀ = 5120 M¹ cm¹. Concentration of CheB was determined using the Bradford assay.

In Vitro Phosphorylation Assays-- For single time point phosphorylation reactions, CheA was incubated alone or with wild-type or mutant CheV proteins in TKMD buffer with 20 µM ATP containing 10 µCi of [-³²P]ATP. The final concentration of each of the proteins was 10 µM. For time course phosphorylation assays, CheA was autophosphorylated in TKMD buffer with 20 µM ATP containing 50 µCi of [-³²P]ATP for 1 h at room temperature. CheA-P was separated from unincorporated nucleotides by passing the incubation mixture through G-30 microspin columns (Bio-Rad) twice (7). Purified CheA-P was mixed with each of the response regulators (final concentration of CheA-P was 0.44 µM), and at the given times, 10-µl aliquots were drawn and stopped by adding an equal amount stop buffer (2× SDS buffer containing 100 mM EDTA). For CheV-P decay experiments, CheA-P was mixed with a 200-fold excess of either CheV or CheV_164-303. All phospho-transfer reactions were carried out at room temperature unless indicated otherwise. Reaction mixtures were separated by SDS-polyacrylamide gel electrophoresis (30), washed twice for 15 min with phosphate-buffered saline, dried, and exposed to x-ray film. Autoradiographs were scanned using a scanning densitometer (Precision Digital Images (PDI), model 420e). Quantifications of the scans were done using Quantity One software from PDI. Apparent half-time (t_1/2) for CheA-P dephosphorylation is defined by amount of time it takes for CheA-P decrease to 50% of its original value. These values were calculated using computer-generated fits of the data to exponential curves with R values of 0.96.

Tethered-cell Assay-- Tethered-cell assays were performed as described previously (18) using Hobson Tracker, bacterial edition (Hobson Tracking Systems Ltd., Sheffield, UK). Asparagine was used as an attractant at 506 µM, which corresponds to 90% receptor occupancy for asparagine. Prestimulus bias was calculated by averaging data points before the attractant was added. Post-addition bias was calculated by averaging data points at the steady state that the cells reached in the presence of asparagine.

Calculation of Mean Event Duration Times-- The Hobson Tracker system generates raw event text files that sequentially lists the rotational direction and duration of each distinct CCW or CW event. Using a program written with the scripting component of the MatLab software package (The Mathworks, Natick, MA) the events duration times generated by individual cells of a given strain were pooled together and averaged to obtain a mean event duration value. The start and end points of the addition and the post-addition response duration times were determined on a cell by cell basis. The addition phase extends from the point at which the cell responds to the addition of asparagine to the point at which it has reached a stable CCW bias.

Sequence Analysis-- All sequence analyses, including sequence alignments with ClustalW (31, 32) and similarity searches with BLAST (33), were done using the Biology Workbench.²

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Phosphoryl Group Transfer between CheA and CheV-- To test whether CheV is phosphorylated as a result of phosphoryl group transfer from CheA-P, purified proteins were incubated in vitro in the presence of [-³²P]ATP as a substrate for CheA. The appearance of a radiolabeled band corresponding to the molecular weight of CheV indicated that CheV was phosphorylated, as expected (Fig. 1, lane 2). A sequence alignment of the C-terminal response regulator domain of CheV with CheY proteins from various organisms predicted that aspartate 235 is the phospho-acceptor residue on CheV (21). To determine whether this residue is the site of phosphorylation, this aspartate was mutated to alanine. The resulting mutant protein CheV_D235A did not become phosphorylated when incubated with CheA and [-³²P]ATP in vitro (Fig. 1, lane 3). This result suggested that aspartate 235 is the phospho-acceptor residue and that it is the only site of phosphorylation on the CheV protein. To test whether the N-terminal coupling domain of CheV is necessary for the phosphorylation reaction, a truncated mutant of CheV was constructed, which lacked the predicted coupling domain (21). This truncated mutant, CheV_164-303, did become phosphorylated in vitro when incubated with CheA and [-³²P]ATP (Fig. 1, lane 4). This result showed that the C-terminal response regulator domain of CheV is sufficient for the phospho-transfer reaction between CheA-P and CheV. To ensure that CheV was phosphorylated as a result of phosphoryl group transfer from CheA-P and not from [-³²P]ATP, CheV and CheV_164-303 were each incubated with [-³²P]ATP in the absence of CheA. As expected, neither the wild-type nor the truncated CheV protein was able to use [-³²P]ATP as a phosphoryl group donor (Fig. 1, lanes 5 and 6).

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Phospho-transfer from CheA to wild-type and mutant CheV proteins. An assay was performed as described under "Materials and Methods." Reactions contained each of the proteins as indicated at 10 µM, 20 µM ATP, and 10 µCi of [32P]ATP in a final volume of 50 µl in TKMD buffer. Reaction time was 1 min. Reactions were separated by electrophoresis on a 15% SDS-polyacrylamide gel and subjected to autoradiography. The presence (+) or absence () of the reactants is indicated below the lanes. Molecular mass markers (Bio-Rad) are indicated in kilodaltons.

Rates of Phosphoryl Group Transfer Reactions-- The rates of dephosphorylation of CheA-P in the presence of CheV or CheV_164-303 were compared using purified CheA-P as a phosphoryl group donor. Twenty-fold concentrations of CheV and CheV_164-303 relative to CheA-P were used. Least square fittings of the time course for dephosphorylation of CheA-P to single exponentials revealed pseudo first-order rate constants of 0.032 s¹ and 0.16 s¹ for CheV and CheV_164-303, respectively (Fig. 2). These values were used to calculate the apparent half-time (t_1/2) of dephosphorylation of CheA-P by CheV and CheV_164-303 as 21.6 and 4.2 s, respectively. These results suggested that the presence of the N-terminal coupling domain might decrease the rate of the phosphoryl group transfer reaction between CheA-P and the receiver domain of CheV.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Time course of phospho-transfer reactions between CheA-P and CheV or CheV164-303. Reactions contained 0.44 µM CheA-P and 8.8 µM CheV or CheV164-303. 10-µl aliquots were withdrawn every 10 s, and reactions were stopped. For the zero time point, an amount of unreacted CheA-P similar to that withdrawn in the aliquots was mixed with stop buffer. Reactions were separated by electrophoresis on 10 and 15% SDS-polyacrylamide gels for CheV and CheV164-303, respectively. Each time point is an average of two independent experiments. CheA-P decay in the presence of CheV (circles) and CheV164-303 (squares).

These rates were compared with that between CheA-P and CheB. When a 10-fold concentration of CheB relative to CheA-P was used, the phospho-transfer reaction was too rapid to follow at room temperature (Fig. 3, lanes 1 and 2). The entire label had been transferred to and hydrolyzed from CheB within the first time point of 10 s. Reducing the temperature to 4 °C resulted in the appearance of a labeled band corresponding to the molecular weight of CheB (Fig. 3, lanes 3 and 4). These results showed the phosphoryl group transfer from CheA-P to CheB is much faster than to either the wild-type or the truncated CheV and that CheB-P is highly unstable.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Phospho-transfer between CheA-P and CheB. 0.44 µM CheA-P and 4.4 µM CheB were mixed. 10-µl aliquots were withdrawn at the indicated times, and reactions were stopped by mixing with stop buffer. Reaction mixtures were separated by electrophoresis on a 10% SDS-polyacrylamide gel. The autoradiogram shows phospho-transfer reactions at either 25 °C (lanes 1 and 2) or 4 °C (lanes 3 and 4). Lanes 1 and 3 are CheA-P only, and lanes 2 and 4 are 10-s time points after mixing.

Stabilities of CheV-P and CheV_164-303-P-- To investigate the relative stabilities of CheV-P and CheV_164-303-P, 200-fold concentrations of CheV and CheV_164-303 relative to CheA-P were used in in vitro phosphorylation reactions. Under these conditions all of the label was transferred from CheA-P to CheV in 60 s , and after 4 min of incubation some CheV-P still remained (Fig. 4A). By contrast, CheV_164-303-P was highly unstable, and all of the CheV_164-303-P had decayed by 60 s (Fig. 4B). This result showed that the presence of the N-terminal coupling domain stabilizes CheV-P.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   CheV-P and CheV164-303-P stability. 0.44 µM CheA-P was mixed with 88 µM CheV or CheV164-303. 10-µl aliquots were withdrawn at the indicated intervals for 4 min, and reactions were stopped by mixing with stop buffer. Zero time point is as shown in Fig. 3, lane 1. Reaction mixtures were separated by 10 and 15% SDS-polyacrylamide gel electrophoresis for CheV and CheV164-303, respectively. A, autoradiogram showing decay of CheV-P. B, autoradiogram showing decay of CheV164-303-P (no CheV164-303-P remained by the third time point; data not shown).

Effect of the cheV_D235A Mutant on Chemotactic Behavior-- The receiver domains of most two-domain response regulators control the activity of their effector domains in a phosphorylation-dependent manner (34). In most cases, phosphorylation of the receiver domain results in the activation of the response regulator (34). To determine whether the coupling activity of CheV is also regulated by phosphorylation, the cheV_D235A allele was integrated into the thrC locus of a cheWcheV double null mutant, creating strain OI3447. The cheWcheV double null mutant (OI3061) has a prestimulus CCW bias of less than 5% and is unable to respond to addition of attractants (21). Therefore, if the unphosphorylatable point mutant were still functional as a coupling protein, any increase in the prestimulus bias or response to the addition of attractants resulting from the cheV_D235A allele would be easily detectable in this background. If, however, the unphosphorylated receiver domain of CheV inhibits its coupling domain, then the prestimulus CCW bias of this strain would remain at about 5% and no response to the stimulus would be detected. The chemotactic behavior of strain OI3447 was assessed by tethered-cell assay in which the probability of CCW rotation of the flagella is determined under steady state conditions and upon addition and removal of attractants, and a characteristic behavioral profile is obtained for each strain. As shown in Fig. 5A, strain OI3447 had a prestimulus bias of about 60% and was able to respond and partially adapt to addition of the chemoattractant asparagine. This result indicated that CheV_D235A is able to effectively couple the MCPs to CheA kinase, which suggests that the unphosphorylated receiver domain of CheV does not inhibit its coupling domain.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Behavioral analysis of various strains in response to addition and removal of asparagine. Tethered-cell assay was performed as described under "Materials and Methods." The downward and upward arrows represent the addition and removal of asparagine, respectively. A, OI3446 (cheV::kan, thrC::cheVD235A) (thick line) and OI3447 (cheW::cat, cheV::kan, thrC::cheVD235A) (thin line). B, OI3450 (cheV::kan, thrcC::cheV1-168) (thick line) and OI3452 (cheW::cat, cheV::kan, thrC::cheV1-168) (thin line). C, OI3059 (cheV::kan) (thick line), OI1085 (wild-type) (medium weight line), and OI2737 (cheW::cat) (thin line).

Although CheV_D235A was able to support normal coupling in the absence of stimuli, as well as normal response to the addition of asparagine, it was unable to support efficient adaptation. Strain OI3447 partially adapted to the addition of asparagine; however, the post-addition CCW bias remained high until the attractant was removed (Fig. 5A). To determine the extent of this impairment, we calculated the prestimulus and the post-addition CCW biases as well as the ratio of the prestimulus bias to the post-addition bias. A ratio of close to 1 indicates that adaptation to the addition of asparagine is complete. As seen in Table III, the wild-type strain (OI1085) had a prestimulus to post-addition CCW bias ratio of close to 1, whereas strain OI3447 had a ratio of 0.77, indicating that the adaptation to the addition of asparagine is not complete.

To ensure that this behavior resulted from the mutant CheV protein rather than the absence of CheW, a cheW null mutant was tested for its response to addition of asparagine. As seen in Fig. 5C, as well as Table III, the cheW null mutant was able to adapt completely to the addition of asparagine with a prestimulus to post-addition CCW bias ratio of 1.04. Therefore, the wild-type CheV, which is the only coupler protein in the cheW mutant, is able to support complete adaptation to the addition of asparagine.

To determine whether the presence of CheW reversed or enhanced this behavior, the cheV_D235A allele was integrated into the thrC locus in a cheV null mutant, creating strain OI3446. Although this mutant strain exhibited improved adaptation to the addition of attractant with a prestimulus to post-addition CCW bias ratio of 0.84, the effect of the mutant CheV protein was still observable (Fig. 5A, Table III). These results suggest that phosphorylation of CheV is necessary for complete adaptation to the addition of chemoattractant asparagine.

Effect of the cheV_1-168 Mutant on Chemotactic Behavior-- To assess coupling and adaptation in a cheV mutant lacking a receiver domain, a truncation mutant of CheV was constructed by deleting the entire C-terminal receiver domain (CheV_1-168). This mutant allele was integrated into the thrC locus of the cheWcheV null mutant, creating strain OI3452. In tethered-cell assays, strain OI3452 had a prestimulus CCW bias of ~65% and was also able to respond and partially adapt to the addition of asparagine. Thus, the truncated CheV protein coupled in the absence of stimulus; however, it failed to support normal adaptation to stimulus, similar to the CheV_D235A mutant (Fig. 5B). The prestimulus to post-addition CCW bias ratio in this strain was 0.72 (Fig. 5B, Table III).

To assess whether CheW itself was able to support complete adaptation to the addition of asparagine, the chemotactic behavior of a cheV null mutant, in which the only coupling protein is CheW, was analyzed. Fig. 5C shows that this mutant strain was able to support normal adaptation to the addition of asparagine, with a prestimulus to post-addition CCW bias ratio of 1.05. Therefore, the CheW protein, unlike the CheW-like domain of CheV, can support normal behavioral response to the addition of asparagine. The difference in the behavior of these two strains suggested that the CheW-like domain of CheV is not functionally equivalent to CheW.

To determine whether the effect of CheV_1-168 on adaptation was observable in the presence of CheW, the cheV_1-168 allele was integrated into the thrC locus in a cheV null mutant, creating strain OI3450. The effect of the truncated mutant of CheV was still observable in this background, although strain OI3450 was less impaired in adaptation in comparison with strain OI3452 (Fig. 5B, Table III).

Effect of the Mutant CheV Proteins on the Mean Duration of the CCW Rotational Events-- To further investigate the basis of the impairment in adaptation caused by the mutant CheV proteins, we analyzed several parameters of the behavioral response. Specifically, we compared the mean duration times of the CCW and CW rotational events characteristic of each of the strains during the prestimulus, addition, and post-addition phases of the behavioral profile (Table IV). During the prestimulus phase, all strains had mean CCW event duration times of between 0.87 and 1.43 s and mean CW event duration times of between 0.52 and 0.9 s. During the addition phase, mean duration times of the CCW events increased more than 100% in all of the strains without any significant changes in the mean duration of the CW events (Table IV). These parameters did not reveal any differences between the strains that could account for the differences in the behavioral profiles. However, a comparison between the mean duration times of the CCW events in the post-addition and the prestimulus phases did show differences that correlated with the behavioral profiles. The post-addition mean CCW event duration times were increased in strains OI3447 and OI3452 to 135 and 150%, respectively, of their prestimulus values (Table IV). Because these strains were severely impaired in adaptation to the addition of asparagine and had increased post-addition CCW biases, the elevated mean CCW duration values appear to correlate with these responses. Strains OI3446 and OI3450 exhibited 26 and 22% increases, respectively, in the mean CCW event duration times. The smaller increase in the mean CCW event duration times also appeared to correlate with the lower extent of impairment in adaptation seen in these strains. There were no significant changes between the prestimulus and post-addition mean CCW event duration times in the wild-type, cheW, and cheV single null mutants, which also correlated with the complete adaptation to the addition of asparagine seen in these strains. Although there were significant changes in the mean CCW event duration times that correlated with the increased CCW biases, the mean duration times of the CW events showed little change (Table IV). We conclude that the increase in the probability of the CCW flagellar rotation appears to result from increases in CCW event duration times as opposed to decreases in CW event duration times.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study we provide evidence that CheV, in addition to CheB and CheY, is a target for the phosphoryl group flux through CheA during Bacillus subtilis chemotaxis. Phosphorylation of CheV appears to occur on a conserved aspartate (Asp²³⁵), which is the expected phospho-acceptor residue, judged from sequence alignments with receiver domains of other response regulators (21). The N-terminal portion of CheV does not appear to be necessary for the phosphorylation reaction. The ability of the C-terminal receiver domain of CheV to fold into a unit capable of phospho-transfer suggests that it is a functional and separate domain.

The phospho-transfer reaction between CheA-P and CheV is uncharacteristically slow compared with that which occurs between E. coli CheA-P and CheY. Under conditions similar to the ones used in this study, the phosphoryl group transfer between E. coli CheA-P and CheY reached completion within 50 ms, which is several orders of magnitude faster than was observed between B. subtilis CheA-P and CheV (35). In addition to the slow phosphoryl group transfer rate to CheV, the phosphorylated form of CheV is very stable. Interestingly, the CheW-like domain both reduces the rate of phospho-transfer between CheA-P and CheV and enhances the stability of the phosphorylated form of CheV. In this aspsect CheV resembles FixJ, the response regulator that controls nitrogen fixation in Sinorhizobium meliloti. The transcriptional activator domain of FixJ has been shown to reduce the rate of phosphorylation of the receiver domain (36).

By contrast, the phosphoryl group transfer kinetics observed using CheA-P and CheB, as well as the stability of CheB-P, were similar to those observed in E. coli. E. coli CheB-P has a half-life of less than 1 s (37). Our experiments show that B. subtilis CheB-P is also highly unstable. In fact, the phosphoryl group transfer rate, as well as the CheB-P decay rate, was too high to follow at room temperature at a 1:10 ratio of CheA-P to CheB. This result is consistent with the half-life of CheB-P being on the order of hundreds of milliseconds. Because B. subtilis cheB can complement an E. coli cheB null mutant, similar kinetics might have been expected (38).

Inability to phosphorylate CheV appears to affect various phases of the chemotactic response differently. In the absence of an attractant, the unphosphorylatable and the truncated mutants of the CheV protein were both capable of maintaining near normal CCW flagellar biases, suggesting that under steady state conditions phosphorylation of CheV may not be necessary for its coupling function. During the excitation phase initiated by the addition of asparagine, these mutants were capable also of mediating normal excitation responses, suggesting that the attractant signal can be communicated efficiently to CheA kinase by either the unphosphorylatable or the truncated form of CheV. In the post-addition phase, however, both of these mutant proteins caused the same impairment in adaptation, indicated by the inability to return to prestimulus biases following excitation. This high bias appeared to result from a sustained increase in the mean duration of the CCW events in the presence of the attractant. Because the degree of CCW flagellar rotation is directly correlated to the amount of CheY-P production, there is presumably increased CheY-P production in these strains because of increased CheA activity. These results suggest that inability to phosphorylate CheV upon binding of an attractant to the chemoreceptors could result in overactive signaling complexes, which are unable to down-regulate CheA effectively. We presume that the slow phospho-transfer kinetics between CheA-P and CheV as well as the increased stability of CheV-P may have developed to allow enough time for the excitatory signal caused by attractant binding to cause a significant period of smooth swimming. Were the kinetics of phosphorylation much faster, there might be a danger of reducing the CheA activity too quickly, thus preventing a sufficient signal from being generated.

It is also interesting that in the tethered-cell assays, cheV_D235A and cheV_1-168 were epistatic over cheW. The mutants failed to adapt completely even with CheW present, although CheW did mute the phenotype somewhat. Such a result might be expected if CheV and CheW were associated with different sets of receptors. In this case, those receptors linked to the mutant CheV proteins would be impaired in adaptation even though other receptors, linked to CheW, would undergo normal adaptation, possibly resulting in the intermediate phenotype observed in strains OI3446 and OI3450. It is also noteworthy that all of the mutant strains that were analyzed had slightly elevated prestimulus biases, suggesting the cells need wild-type copies of both of the coupling proteins to maintain normal prestimulus biases. The reasons for this requirement are unknown.

Response regulators are usually inactive in their unphosphorylated forms. A variety of mechanisms have been elucidated by which phosphorylation activates the response regulator. In some cases, exemplified by the methylesterase CheB, in which the unphosphorylated receiver domain blocks the effector domain, phosphorylation relieves this inhibition, allowing the effector domain to carry out its function (39). Unphosphorylatable point mutants of these response regulators are inactive, whereas removing the receiver domains leads to constitutively active proteins. In other cases, exemplified by NtrC, the transcriptional activator for nitrogen-regulated promoters in E. coli, dimerization of the receiver domain is inhibited by the effector domain in the unphosphorylated protein (40). Phosphorylation relieves this inhibition, allowing dimerization and subsequent oligomerization of the protein, which allows transcriptional activation (40, 41). Rendering these proteins unphosphorylatable, as well as removing the receiver domains, lead to loss of activity (40). A combination of these two mechanisms has been suggested for FixJ, the transcriptional activator that controls nitrogen fixation in S. meliloti (36). In this case, phosphorylation appears to relieve inhibition of the receiver domain on the effector domain, which leads to transcriptional activation; and phosphorylation also allows dimerization of the receiver domain, which leads to an increase in affinity and specificity to the target sequence (36). There are also a few cases in which the response regulator is active in its unphosphorylated form. DegU, the transcriptional activator necessary for the induction of degradative enzyme synthesis and natural competence pathways in B. subtilis, acts as a molecular switch between these two pathways (42). In its phosphorylated form it activates the degradative enzyme pathway, and in its unphosphorylated form it activates the natural competence pathway (42). Another example is SSK1, which is involved in osmoregulation in Saccharomyces cerevisiae (43). Under high osmolarity conditions, the unphosphorylated receiver domain of SSK1 binds a MAPKKK (mitogen-activated protein kinase kinase kinase) SSK2, leading to activation of a downstream mitogen-activated protein kinase pathway (44). Phosphorylation acts as a negative regulator of SSK1 (44). CheV appears to be different from either of these proteins in that although it is partially active in its unphosphorylated form, phosphorylation does appear to be necessary for its correct function. In fact, because both the truncated and the unphosphorylatable forms of CheV are overactive, phosphorylation may be necessary to deactivate or turn down the coupling function of the effector domain. Therefore, CheV could be an example of a new class of response regulators in which the receiver domains inhibit their effector domains upon phosphorylation.

The possibility remains that the receiver domain of CheV simply constitutes a phosphate sink, which aids adaptation by draining phosphoryl groups away from CheA-P. Such a mechanism has been implicated in S. meliloti in which CheY2-P is responsible for changing the swimming behavior of the bacteria, whereas CheY1 is thought to act as a phosphate sink, which helps bring about adaptation (45, 46). We believe that slow phosphorylation kinetics of CheV and stability of CheV-P makes it a poor candidate for such a role. Furthermore, the fact that the stability of CheV-P depends in part on the presence of the coupling domain suggests that the two domains might be interacting and that the phosphorylated form of the receiver domain might be stabilized as a result of these interactions. This result further supports the notion that there is communication between the two domains. That is, upon phosphorylation, the receiver domain could feedback onto the effector domain, perturbing its coupling interactions with the receptor and/or CheA and leading to down-regulation of the CheA activity.

The CheW-like domain of CheV shares ~33% identity with B. subtilis CheW. Despite this high similarity, this domain appears to be functionally different from CheW because unlike CheW it cannot support normal adaptation to the addition of asparagine. To gain insight into the basis of this functional difference, we performed a multiple sequence alignment using CheW, CheV_1-168, and the receptor/CheW binding regulatory domain of CheA, which was recently reported to contain sequence similarity to CheW (Fig. 6) (47). In the previously published sequence alignment between CheA regulatory domains and CheW proteins from E. coli and Thermatoga maritima, several regions of high similarity were reported (47). The region of most extensive similarity contained the invariant residues Val, Arg, Gly, and Pro and formed an exposed hydrophobic surface in the crystal structure of CheA that was suggested to be a possible interface between CheA and CheW (47). Our sequence alignment also found that these residues (marked with arrows in Fig. 6) are completely conserved in CheW and CheA. In CheV, only the valine is replaced by a methionine, which is a conservative substitution. Therefore, this region is unlikely to account for the functional differences seen between CheW of B. subtilis and CheW-like domain of CheV. Another region of similarity contained two invariant residues, a valine-aspartate pair that form the kink between the two consecutive -barrels that make up the regulatory domain in the crystal structure of CheA. These residues are conserved in B. subtilis CheW and CheA as well. In CheV, however, only the valine position is conserved, whereas the aspartate is replaced by glycine, a nonconservative substitution with respect to both charge and size (Fig. 6). Furthermore, sequence comparisons of CheW, CheA, and CheV proteins from seven bacterial species revealed that the aspartate position is not conserved in most of the CheV proteins, whereas both the aspartate and the valine are conserved in the CheA and CheW proteins of these species (Table V) (48-53). Therefore, the lack of conservation of this residue appears to be a characteristic of CheV proteins. Assuming that the structure of CheW and the CheW-like domain of CheV are similar to that of the regulatory domain of CheA, the aspartate to glycine replacement at this position in B. subtilis CheV could conceivably contribute to the structural differences between the CheW-like domains of CheV and CheW.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Multiple sequence alignment of CheW, CheV1-168, and CheA531-671 from B. subtilis. Invariant residues are shaded black, highly conserved residues are shaded gray, and weakly conserved residues are boxed. The invariant residues that are believed to be at the CheW/CheA interface are marked with arrows, and the "VD" pair is marked with asterisks below the sequence.

CheW of E. coli has been shown to bind the monomeric cytoplasmic fragments of chemoreceptors corresponding to the tight turn between the two -helical extensions, which form antiparallel coiled coils (54). It has been suggested that one possible role of CheW could be to fix the angle of this turn to control the proper placement of the cytoplasmic helices of a monomer with respect to each other (55). Although this type of binding has not been shown between chemoreceptors and CheW from B. subtilis, because of the high similarity between the signaling domains of the MCPs from the two organisms, such a mechanism is conceivable in B. subtilis as well. It is also likely that the CheW-like domain of CheV also binds this region; however, the binding could be slightly different because of a possible difference in positioning of the putative -barrels of CheV with respect to each other. These different interactions with the receptor could cause the functional differences between CheW and the CheW-like domain of CheV.

	ACKNOWLEDGEMENTS

We thank Dr. John Kirby and Dr. Ted Zerucha for helpful comments on the manuscript and Joe Tin and Tim Niewold for help with tethering the bacteria and observing the effect of stimuli on them.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM54365 (to G. W. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 217-333-9098; Fax: 217-333-8868; E-mail: g-ordal@uiuc.edu.

Published, JBC Papers in Press, September 11, 2001, DOI 10.1074/jbc.M104955200

² Found on the Web at workbench.sdsc.edu.

	ABBREVIATIONS

The abbreviations used are: MCP, methyl-accepting chemotaxis protein; CheA, autophosphorylating kinase; CheA-P, phosphorylated CheA; CCW, counterclockwise; CW, clockwise; PCR, polymerase chain reaction.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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