The three adaptation systems of Bacillus subtilis chemotaxis

Adaptation has a crucial role in the gradient-sensing mechanism that underlies bacterial chemotaxis. The Escherichia coli chemotaxis pathway uses a single adaptation system involving reversible receptor methylation. In Bacillus subtilis, the chemotaxis pathway seems to use three adaptation systems. One involves reversible receptor methylation, although quite differently than in E. coli. The other two involve CheC, CheD and CheV, which are chemotaxis proteins not found in E. coli. Remarkably, no one system is absolutely required for adaptation or is independently capable of generating adaptation. In this review, we discuss these three novel adaptation systems in B. subtilis and propose a model for their integration.     

Rapid attractant-induced changes in methylation of methyl-accepting chemotaxis proteins in Bacillus subtilis

In Bacillus subtilis, addition of chemotactic attractant causes an immediate change in distribution of methyl groups on methyl-accepting chemotaxis proteins (MCPs), whereas in Escherichia coli, it causes changes that occur throughout the adaptation period. Thus, methylation changes in B. subtilis are probably related to excitation, not adaptation. If labeled cells are exposed to excess nonradioactive methionine, then attractant causes immediate 50% delabeling of the MCPs, suggesting that a flux of methyl groups through the MCPs occurs. Methanol is given off at a high rate during the adaptation period and probably reflects demethylation of some substance to bring about adaptation. The fact that many radioactive methyl groups are lost immediately from the MCPs but only slowly arise as methanol is consistent with the hypothesis that they are transferred from the MCPs to a carrier from which methanol arises. Demethylation of this carrier may cause adaptation.     

CheC is related to the family of flagellar switch proteins and acts independently from CheD to control chemotaxis in Bacillus subtilis

Chemotaxis by Bacillus subtilis requires the inter-acting chemotaxis proteins CheC and CheD. In this study, we show that CheD is absolutely required for a behavioural response to proline mediated by McpC but is not required for the response to asparagine mediated by McpB. We also show that CheC is not required for the excitation response to asparagine stimulation but is required for adaptation while asparagine remains complexed with the McpB chemoreceptor. CheC displayed an interaction with the histidine kinase CheA as well as with McpB in the yeast two-hybrid assay, suggesting that the mechanism by which CheC affects adaptation may result from an interaction with the receptor-CheA complex. Furthermore, CheC was found to be related to the family of flagellar switch proteins comprising FliM and FliY but is not present in many proteobacterial genomes in which CheD homologues exist. The distinct physiological roles for CheC and CheD during B. subtilis chemotaxis and the observation that CheD is present in bacterial genomes that lack CheC indicate that these proteins can function independently and may define unique pathways during chemotactic signal transduction. We speculate that CheC interacts with flagellar switch components and dissociates upon CheY-P binding and subsequently interacts with the receptor complex to facilitate adaptation.     

In vivo and in vitro chemotactic methylation in Bacillus subtilis

Two doublets of Bacillus subtilis membrane proteins with molecular weights of 69,000 and 71,000 and of 30,000 and 30,800, were labeled by C3H3 transfer in the absence of protein synthesis. In addition, there was intense methylation of several low-molecular-weight substances. Both doublets were missing in a chemotaxis mutant. The equivalent proteins in Escherichia coli and Salmonella typhimurium are believed to be the methyl-accepting chemotaxis proteins. The higher-molecular-weight doublet bands were increased in degree of methylation upon addition of attractant to the bacteria. A methyltransferase from B. subtilis that methylates the wild-type membrane significantly better than the mutant membrane, using S-adenosylmethionine, has been partly purified. The methylated product was alkali labile and is probably a gamma-glutamyl methyl ester, as in E. coli and S. typhimurium. Ca2+ ion inhibited the methyltransferase, with a Ki of about 80 nM. Analysis of the in vitro methylation product showed labeling of the 69,000-dalton methyl-accepting chemotaxis protein and a low-molecular-weight protein, using wild-type membrane. Labeling of the low-molecular-weight protein but not of the 69,000 dalton protein was observed when the mutant membrane was used. The chemotaxis mutant tumbled much longer than the wild type when diluted away from attractant.     

Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein.

The nucleotide sequence of the Bacillus subtilis fliM gene has been determined. This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium. Expression of this gene in Che+ cells of E. coli and B. subtilis interferes with normal chemotaxis. The nature of the chemotaxis defect is dependent upon the host used. In B. subtilis, overproduction of FliM generates mostly nonmotile cells. Those cells that are motile switch less frequently. Expression of B. subtilis FliM in E. coli also generates nonmotile cells. However, those cells that are motile have a tumble bias. The B. subtilis fliM gene cannot complement an E. coli fliM mutant. A frameshift mutation was constructed in the fliM gene, and the mutation was transferred onto the B. subtilis chromosome. The mutant has a Fla- phenotype. This phenotype is consistent with the hypothesis that the FliM protein encodes a component of the flagellar switch in B. subtilis. Additional characterization of the fliM mutant suggests that the hag and mot loci are not expressed. These loci are regulated by the SigD form of RNA polymerase. We also did not observe any methyl-accepting chemotaxis proteins in an in vivo methylation experiment. The expression of these proteins is also dependent upon SigD. It is possible that a functional basal body-hook complex may be required for the expression of SigD-regulated chemotaxis and motility genes.     

Role of methylation in aerotaxis in Bacillus subtilis.

Taxis to oxygen (aerotaxis) in Bacillus subtilis was characterized in a capillary assay and in a temporal assay in which the concentration of oxygen in a flow chamber was changed abruptly. A strong aerophilic response was present, but there was no aerophobic response to high concentrations of oxygen. Adaptation to a step increase in oxygen concentration was impaired when B. subtilis cells were depleted of methionine to prevent methylation of the methyl-accepting chemotaxis proteins. There was a transient increase in methanol release when wild-type B. subtilis, but not a cheR mutant that was deficient in methyltransferase activity, was stimulated by a step increase or a step decrease in oxygen concentration. The methanol released was quantitatively correlated with demethylation of methyl-accepting chemotaxis proteins. This indicated that methylation is involved in aerotaxis in B. subtilis in contrast to aerotaxis in Escherichia coli and Salmonella typhimurium, which is methylation independent.     

In vitro methylation and demethylation of methyl-accepting chemotaxis proteins in Bacillus subtilis

Bacillus subtilis responds to attractants by demethylating a group of integral membrane proteins referred to as methyl-accepting chemotaxis proteins (MCPs). We have studied the methylation and demethylation of these proteins in an in vitro system, consisting of membrane vesicles, and purified methyltransferase and methylesterase. The chemoattractant aspartate was found to inhibit methylation and stimulate demethylation of MCPs. Escherichia coli radiolabeled membranes in the presence of B. subtilis enzyme do not respond to aspartate by an increase demethylation rate. We also report that B. subtilis MCPs are multiply methylated, demethylation resulting in slower migrating proteins on sodium dodecyl sulfate-polyacrylamide gels.     

CheY-dependent methylation of the asparagine receptor, McpB, during chemotaxis in Bacillus subtilis

For the Gram-positive organism Bacillus subtilis, chemotaxis to the attractant asparagine is mediated by the chemoreceptor McpB. In this study, we show that rapid net demethylation of B. subtilis McpB results in the immediate production of methanol, presumably due to the action of CheB. We also show that net demethylation of McpB occurs upon both addition and removal of asparagine. After each demethylation event, McpB is remethylated to nearly prestimulus levels. Both remethylation events are attributable to CheR using S-adenosylmethionine as a substrate. Therefore, no methyl transfer to an intermediate carrier need be postulated to occur during chemotaxis in B. subtilis as was previously suggested. Furthermore, we show that the remethylation of asparagine-bound McpB requires the response regulator, CheY-P, suggesting that CheY-P acts in a feedback mechanism to facilitate adaptation to positive stimuli during chemotaxis in B. subtilis. This hypothesis is supported by two observations: a cheRBCD mutant is capable of transient excitation and subsequent oscillations that bring the flagellar rotational bias below the prestimulus value in the tethered cell assay, and the cheRBCD mutant is capable of swarming in a Tryptone swarm plate.     

Methanol production during chemotaxis to amino acids in Bacillus subtilis

The 20 common amino acids act as attractants during chemotaxis by the Gram-positive organism Bacillus subtilis . In this study, we report that all amino acids induce B. subtilis to produce methanol both upon addition and removal of the chemoeffector. Asparagine-induced methanol production is specific to the McpB receptor and aspartate-induced methanol production correlates with receptor occupancy. These findings suggest that addition and removal of all amino acids cause demethylation of specific receptors which results in methanol production. We also demonstrate that certain attractants cause greater production of methanol after multiple stimulations. CheC and CheD, while affecting the levels of receptor methylation, are not absolutely required for either methylation or demethylation. In contrast, CheY is necessary for methanol formation upon removal of attractant but not upon addition of attractant. We conclude that methanol formation due to negative stimuli indicates the existence of a unique adaptational mechanism in B. subtilis involving the response regulator, CheY.     

Reconstitution of the bacterial chemotaxis signal transduction system from purified components.

In bacterial chemotaxis, transmembrane receptor proteins detect attractants and repellents in the medium and send intracellular signals that control motility. The cytoplasmic proteins that transduce information from the receptors to the flagellar motor have previously been purified and many of their enzymatic activities have been identified. Here we report the reconstitution of the complete signal transduction system from purified components. The protein kinase, CheA, plays a central role in both the initial excitation response to stimuli as well as subsequent events associated with adaptation. This kinase provides phosphoryl groups to two acceptor proteins, CheY, which interacts with the flagellar motor, and CheB, which demethylates the receptors. The purified aspartate receptor, Tar, reconstituted into phospholipid vesicles, acts in conjunction with an auxiliary protein, CheW, to stimulate the rate of kinase autophosphorylation greater than 10-fold. This stimulation is inhibited by aspartate. The activity of the kinase is increased by increased levels of receptor methylation. This effect provides a mechanism that explains how changes in receptor methylation mediate adaptive responses to attractant and repellant stimuli.     

CheB is required for behavioural responses to negative stimuli during chemotaxis in Bacillus subtilis

The methyl-accepting chemotaxis protein, McpB, is the sole receptor mediating asparagine chemotaxis in Bacillus subtilis. In this study, we show that wild-type B. subtilis cells contain approximately 2,000 copies of McpB per cell, that these receptors are localized polarly, and that titration of only a few receptors is sufficient to generate a detectable behavioural response. In contrast to the wild type, a cheB mutant was incapable of tumbling in response to decreasing concentrations of asparagine, but the cheB mutant was able to accumulate to low concentrations of asparagine in the capillary assay, as observed previously in response to azetidine-2-carboxylate. Furthermore, net demethylation of McpB is logarithmically dependent on asparagine concentration, with half-maximal demethylation of McpB occurring when only 3% of the receptors are titrated. Because the corresponding methanol production is exponentially dependent on attractant concentration, net methylation changes and increased turnover of methyl groups must occur on McpB at high concentrations of asparagine. Together, the data support the hypothesis that methylation changes occur on asparagine-bound McpB to enhance the dynamic range of the receptor complex and to enable the cell to respond to a negative stimulus, such as removal of asparagine.     

Transposon Tn917lacZ mutagenesis of Bacillus subtilis: identification of two new loci required for motility and chemotaxis.

We have used Tn917lacZ to mutagenize the Bacillus subtilis chromosome and have isolated mutants that are defective in chemotaxis and motility. Mapping of the transposon inserts identified two new loci. Mutations in one of these loci generated mutants that had paralyzed flagella. Accordingly, we designate this a mot locus. The other locus is closely linked to the first and encodes proteins specifying chemotaxis functions. This locus is designated the cheX locus. Both the mot and cheX loci map close to ptsI. An additional transposon insert that maps in the hag locus was obtained. The pattern of beta-galactosidase expression from some of the transposons suggested that the mot locus is regulated by sigD, a minor sigma factor of B. subtilis. The cheX locus appeared to be under the control of vegetative sigA. Four transposon inserts were mapped to a previously characterized che locus near spcB. These mutants did not produce flagellin and were defective in the methylation of the methyl-accepting chemotaxis proteins. This locus probably encodes proteins required for flagellum biosynthesis and other proteins that are required for the methylation response.     

Cellular stoichiometry of the chemotaxis proteins in Bacillus subtilis

The chemoreceptor-CheA kinase-CheW coupling protein complex, with ancillary associated proteins, is at the heart of chemotactic signal transduction in bacteria. The goal of this work was to determine the cellular stoichiometry of the chemotaxis signaling proteins in Bacillus subtilis. Quantitative immunoblotting was used to determine the total number of chemotaxis proteins in a single cell of B. subtilis. Significantly higher levels of chemoreceptors and much lower levels of CheA kinase were measured in B. subtilis than in Escherichia coli. The resulting cellular ratio of chemoreceptor dimers per CheA dimer in B. subtilis is roughly 23.0 ± 4.5 compared to 3.4 ± 0.8 receptor dimers per CheA dimer observed in E. coli, but the ratios of the coupling protein CheW to the CheA dimer are nearly identical in the two organisms. The ratios of CheB to CheR in B. subtilis are also very similar, although the overall levels of modification enzymes are higher. When the potential binding partners of CheD are deleted, the levels of CheD drop significantly. This finding suggests that B. subtilis selectively degrades excess chemotaxis proteins to maintain optimum ratios. Finally, the two cytoplasmic receptors were observed to localize among the other receptors at the cell poles and appear to participate in the chemoreceptor complex. These results suggest that there are many novel features of B. subtilis chemotaxis compared with the mechanism in E. coli, but they are built on a common core.     

Independence and interdependence of Dif and Frz chemosensory pathways in Myxococcus xanthus chemotaxis

Dif and Frz, two Myxococcus xanthus chemosensory pathways, are required in phosphatidylethanolamine (PE) chemotaxis for excitation and adaptation respectively. DifA and FrzCD, the homologues of methyl-accepting chemoreceptors in the two pathways, were examined for methylation in the context of chemotaxis and inter-pathway interactions. Evidence indicates that DifA may not undergo methylation, but signals transmitting through DifA do modulate FrzCD methylation. Results also revealed that M. xanthus possesses Dif-dependent and Dif-independent PE-sensing mechanisms. Previous studies showed that FrzCD methylation is decreased by negative chemostimuli but increased by attractants such as PE. Results here demonstrate that the Dif-dependent sensory mechanism suppresses the increase in FrzCD methylation in attractant response and elevates FrzCD methylation upon negative stimulation. In other words, FrzCD methylation is governed by opposing forces from Dif-dependent and Dif-independent sensing mechanisms. We propose that the Dif-independent but Frz-dependent PE sensing leads to increases in FrzCD methylation and subsequent adaptation, while the Dif-dependent PE signalling suppresses or diminishes the increase in FrzCD methylation to decelerate or delay adaptation. We contend that these antagonistic interactions are crucial for effective chemotaxis in this gliding bacterium to ensure that adaptation does not occur too quickly relative to the slow speed of M. xanthus movement.     

Identification of methylation sites and effects of phototaxis stimuli on transducer methylation in Halobacterium salinarum.

The two transducers in the phototaxis system of the archaeon Halobacterium salinarum, HtrI and HtrII, are methyl-accepting proteins homologous to the chemotaxis transducers in eubacteria. Consensus sequences predict three glutamate pairs containing potential methylation sites in HtrI and one in HtrII. Mutagenic substitution of an alanine pair for one of these, Glu265-Glu266, in HtrI and for the homologous Glu513-Glu514 in HtrII eliminated methylation of these two transducers, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autofluorography. Photostimulation of the repellent receptor sensory rhodopsin II (SRII) induced reversible demethylation of HtrII, while no detectable change in the extent of methylation of HtrI was observed in response to stimulation of its cognate sensory rhodopsin, the attractant receptor SRI. Cells containing HtrI or HtrII with all consensus sites replaced by alanine still exhibited phototaxis responses and behavioral adaptation, and methanol release assays showed that methyl group turnover was still induced in response to photostimulation of SRI or SRII. By pulse-chase experiments with in vivo L-[methyl-(3)H]methionine-labeled cells, we found that repetitive photostimulation of SRI complexed with wild-type (or nonmethylatable) HtrI induced methyl group turnover in transducers other than HtrI to the same extent as in wild-type HtrI. Both attractant and repellent stimuli cause a transient increase in the turnover rate of methyl groups in wild-type H. salinarum cells. This result is unlike that obtained with Escherichia coli, in which attractant stimuli decrease and repellent stimuli increase turnover rate, and is similar to that obtained with Bacillus subtilis, which also shows turnover rate increases regardless of the nature of the stimulus. We found that a CheY deletion mutant of H. salinarum exhibited the E. coli-like asymmetric pattern, as has recently also been observed in B. subtilis. Further, we demonstrate that the CheY-dependent feedback effect does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell.     

The Importance of the Interaction of CheD with CheC and the Chemoreceptors Compared to Its Enzymatic Activity during Chemotaxis in Bacillus subtilis

Bacillus subtilis use three systems for adaptation during chemotaxis. One of these systems involves two interacting proteins, CheC and CheD. CheD binds to the receptors and increases their ability to activate the CheA kinase. CheD also binds CheC, and the strength of this interaction is increased by phosphorylated CheY. CheC is believed to control the binding of CheD to the receptors in response to the levels of phosphorylated CheY. In addition to their role in adaptation, CheC and CheD also have separate enzymatic functions. CheC is a CheY phosphatase and CheD is a receptor deamidase. Previously, we demonstrated that CheC’s phosphatase activity plays a minor role in chemotaxis whereas its ability to bind CheD plays a major one. In the present study, we demonstrate that CheD’s deamidase activity also plays a minor role in chemotaxis whereas its ability to bind CheC plays a major one. In addition, we quantified the interaction between CheC and CheD using surface plasmon resonance. These results suggest that the most important features of CheC and CheD are not their enzymatic activities but rather their roles in adaptation.     

Multiple methylation of methyl-accepting chemotaxis proteins during adaptation of E. coli to chemical stimuli

In bacterial chemotaxis, adaptation is correlated with methylation or demethylation of methyl-accepting chemotaxis proteins (MCPs). Each protein migrates as a characteristic set of multiple bands in sodium dodecylsulfate polyacrylamide gel electrophoresis. The changes in MCP methylation that accompany adaptation are not the same for all bands of a set. Adaptation to a type II repellent stimulus results in an overall decrease in MCP II methylation, but also in an increase in the amount of radioactive methyl groups in the upper band of the set. We demonstrate that this increase is not due to new methylation, but rather to reduced electrophoretic mobility of previously methylated molecules that have lost some but not all of their methyl groups. We suggest that the pattern of multiple bands is a direct reflection of multiple sites for methylation on MCP molecules, and that the distribution of radiolabel among the bands is determined by the total extent of methylation. The patterns of methylated peptides produced by limited proteolysis of different MCP bands imply that methylation of the multiple sites on a molecule may occur in a specific order.     

Selective methylation changes on the Bacillus subtilis chemotaxis receptor McpB promote adaptation

The Bacillus subtilis McpB is a class III chemotaxis receptor, from which methanol is released in response to all stimuli. McpB has four putative methylation sites based upon the Escherichia coli consensus sequence. To explore the nature of methanol release from a class III receptor, all combinations of putative methylation sites Gln(371), Gln(595), Glu(630), and Glu(637) were substituted with aspartate, a conservative substitution that effectively eliminates methylation. McpB((Q371D,E630D,E637D)) in a Delta(mcpA mcpB tlpA tlpB)101::cat mcpC4::erm background failed to release methanol in response to either the addition or removal of the McpB-mediated attractant asparagine. In the same background, McpB((E630D,E637D)) produced methanol only upon asparagine addition, whereas McpB((Q371D,E630D)) produced methanol only upon asparagine removal. Thus methanol release from McpB was selective. Mutants unable to methylate site 637 but able to methylate site 630 had high prestimulus biases and were incapable of adapting to asparagine addition. Mutants unable to methylate site 630 but able to methylate site 637 had low prestimulus biases and were impaired in adaptation to asparagine removal. We propose that selective methylation of these two sites represents a method of adaptation novel from E. coli and present a model in which a charged residue rests between them. The placement of this charge would allow for opposing electrostatic effects (and hence opposing receptor conformational changes). We propose that CheC, a protein not found in enteric systems, has a role in regulating this selective methylation.     

Discrimination between different methylation states of chemotaxis receptor Tar by receptor methyltransferase CheR

Bacterial chemotaxis receptors are posttranslationally modified by carboxyl methylation of specific glutamate residues within their cytoplasmic domains. This highly regulated, reversible modification counterbalances the signaling effects of ligand binding and contributes to adaptation. On the basis of the crystal structure of the gamma-glutamyl methyltransferase CheR, we have postulated that positively charged residues in helix alpha2 in the N-terminal domain of the enzyme may be complementary to the negatively charged methylation region of the methyltransferase substrates, the bacterial chemotaxis receptors. Several altered CheR proteins, in which positively charged arginine or lysine residues were substituted with alanines, were constructed and assayed for their methylation activities toward wild-type receptor and a series of receptor variants containing different glutamates available for methylation. One of the CheR mutant proteins (Arg53Ala) showed significantly lower activity toward all receptor constructs, suggesting that Arg53 may play a general role in catalysis of methyl transfer. The rest of the mutant proteins exhibited different patterns of relative methylation rates toward different receptor substrates, indicating specificity, probably through interaction of CheR with the receptor at sites distal to the specific site of methylation. The findings imply complementarity between positively charged residues of the alpha2 helix of CheR and the negatively charged glutamates of the receptor. It is likely that this complementarity is involved in discriminating different methylation states of the receptors.     

The role of heterologous receptors in McpB-mediated signalling in Bacillus subtilis chemotaxis

Asparagine chemotaxis in Bacillus subtilis appears to involve two partially redundant adaptation mechanisms: a receptor methylation-independent process that operates at low attractant concentrations and a receptor methylation-dependent process that is required for optimal responses to high concentrations. In order to elucidate these processes, chemotactic responses were assessed for strains expressing methylation-defective mutations in the asparagine receptor, McpB, in which all 10 putative receptors (10del), five receptors (5del) or only the native copy of mcpB were deleted. This was done in both the presence and the absence of the methylesterase CheB. We found that: (i) only responses to high concentrations of asparagine were impaired; (ii) the presence of all heterologous receptors fully compensated for this defect, whereas responses progressively worsened as more receptors were taken away; (iii) methyl-group turnover occurred on heterologous receptors after the addition of asparagine, and these methylation changes were required for the restoration of normal swimming behaviour; (iv) in the absence of the methyleste-rase, the presence of heterologous receptors in some cases caused impaired chemotaxis; and (v) either a certain threshold number of receptors must be present to promote basal CheA activity, or one or more of the receptors missing in the 10del background (but present in the 5del background) is required for establishing basal CheA activity. Taken together, these findings suggest that many or all chemoreceptors work as an ensemble that constitutes a robust chemotaxis system. We propose that the ability of non-McpB receptors to compensate for the methylation-defective McpB mutations involves lateral transmission of the adapted conformational change across the ensemble.