The roles of the multiple CheW and CheA homologues in chemotaxis and in chemoreceptor localization in Rhodobacter sphaeroides

Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in two major operons and other, unlinked, loci. These include cheA1 and cheW1 (che Op1) and cheA2, cheW2 and cheW3 (che Op2). We have deleted each of these cheA and cheW homologues in-frame and examined the chemosensory behaviour of these strains on swarm plates and in tethered cell assays. In addition, we have examined the effect of these deletions on the polar localization of the chemoreceptor McpG. In E. coli, deletion of either cheA or cheW results in a non-chemotactic phenotype, and these strains also show no receptor clustering. Here, we demonstrate that CheW2 and CheA2 are required for the normal localization of McpG and for normal chemotactic responses under both aerobic and photoheterotrophic conditions. Under aerobic conditions, deletion of cheW3 has no significant effect on McpG localization and only has an effect on chemotaxis to shallow gradients in swarm plates. Under photoheterotrophic conditions, however, CheW3 is required for McpG localization and also for chemotaxis both on swarm plates and in the tethered cell assay. These phenotypes are not a direct result of delocalization of McpG, as this chemoreceptor does not mediate chemotaxis to any of the compounds tested and can therefore be considered a marker for general methyl-accepting chemotaxis protein (MCP) clustering. Thus, there is a correlation between the normal localization of McpG (and presumably other chemoreceptors) and chemotaxis. We propose a model in which the multiple different MCPs in R. sphaeroides are contained within a polar chemoreceptor cluster. Deletion of cheW2 and cheA2 under both aerobic and photoheterotrophic conditions, and cheW3 under photoheterotrophic conditions, disrupts the cluster and hence reduces chemotaxis to any compound sensed by these MCPs.     

Evidence for an intermediate methyl-acceptor for chemotaxis in Bacillus subtilis

Bacillus subtilis responds to chemotactic attractants by demethylating certain membrane-bound proteins, termed methyl-accepting chemotaxis proteins (MCPs) and by augmenting the evolution of methanol. We propose that the methanol comes from a methylated intermediate rather than directly from the MCPs themselves. First, repellent blocks attractant-induced smooth swimming and methanol formation, but not MCP demethylation. Second, prior treatment of cells with much attractant to reduce radiolabeling of MCPs and increase that of the putative intermediate caused increased, rather than decreased, production of methanol upon addition and then removal of the repellent. Third, such cells also produced much, rather than little, methanol upon addition of less attractant than during the pretreatment. We speculate that unmethylated intermediate causes tumbling; attractant causes its methylation and hence absence of tumbling (smooth swimming). Its demethylation during the period of smooth swimming affords adaptation.     

Chemotaxis of Escherichia coli to pyrimidines: a new role for the signal transducer tap

Escherichia coli exhibits chemotactic responses to sugars, amino acids, and dipeptides, and the responses are mediated by methyl-accepting chemotaxis proteins (MCPs). Using capillary assays, we demonstrated that Escherichia coli RP437 is attracted to the pyrimidines thymine and uracil and the response was constitutively expressed under all tested growth conditions. All MCP mutants lacking the MCP Tap protein showed no response to pyrimidines, suggesting that Tap, which is known to mediate dipeptide chemotaxis, is required for pyrimidine chemotaxis. In order to confirm the role of Tap in pyrimidine chemotaxis, we constructed chimeric chemoreceptors (Tapsr and Tsrap), in which the periplasmic and cytoplasmic domains of Tap and Tsr were switched. When Tapsr and Tsrap were individually expressed in an E. coli strain lacking all four native MCPs, Tapsr mediated chemotaxis toward pyrimidines and dipeptides, but Tsrap did not complement the chemotaxis defect. The addition of the C-terminal 19 amino acids from Tsr to the C terminus of Tsrap resulted in a functional chemoreceptor that mediated chemotaxis to serine but not pyrimidines or dipeptides. These results indicate that the periplasmic domain of Tap is responsible for detecting pyrimidines and the Tsr signaling domain confers on Tapsr the ability to mediate efficient chemotaxis. A mutant lacking dipeptide binding protein (DBP) was wild type for pyrimidine taxis, indicating that DBP, which is the primary chemoreceptor for dipeptides, is not responsible for detecting pyrimidines. It is not yet known whether Tap detects pyrimidines directly or via an additional chemoreceptor protein.     

The conserved cytoplasmic module of the transmembrane chemoreceptor McpC mediates carbohydrate chemotaxis in Bacillus subtilis

Escherichia coli cells use two distinct sensory circuits during chemotaxis towards carbohydrates. One circuit requires the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and is independent of any specific chemoreceptor, whereas the other uses a chemoreceptor-dependent sensory mechanism analogous to that used during chemotaxis towards amino acids. Work on the carbohydrate chemotaxis sensory circuit of Bacillus subtilis reported in this article indicates that the B. subtilis circuit is different from either of those used by E. coli. Our chemotactic analysis of B. subtilis strains expressing various chimeric chemoreceptors indicates that the cytoplasmic, C-terminal module of the chemoreceptor McpC acts as a sensory-input element during carbohydrate chemotaxis. Our results also indicate that PTS-mediated carbohydrate transport, but not carbohydrate metabolism, is required for production of a chemotactic signal. We propose a model in which PTS-transport-induced chemotactic signals are transmitted to the C-terminal module of McpC for control of chemotaxis towards PTS carbohydrates.     

Methylation segments are not required for chemotactic signalling by cytoplasmic fragments of Tsr, the methyl-accepting serine chemoreceptor of Escherichia coli

The serine chemoreceptor Tsr and other methyl-accepting chemotaxis proteins (MCPs) control the swimming behaviour of Escherichia coli by generating signals that influence the direction of flagellar rotation. MCPs produce clockwise (CW) signals by stimulating the autophosphorylation activity of CheA, a cytoplasmic histidine kinase, and counter-clockwise signals by inhibiting CheA. CheW couples CheA to chemoreceptor control by promoting formation of MCP/CheW/CheA ternary complexes. To identify MCP structural determinants essential for CheA stimulation, we inserted fragments of the tsr coding region into an inducible expression vector and used a swimming contest called 'pseudotaxis' to select for transformant cells carrying CW-signalling plasmids. The shortest active fragment we found, Tsr (350–470), stimulated CheA in a CheW-dependent manner, as full-length Tsr molecules do. It spans a highly conserved 'core' (370–420) that probably specifies the CheA and CheW contact sites and other determinants needed for stimulatory control of CheA. Tsr (350–470) also carries portions of the left and right arms flanking the core, which probably play roles in regulating MCP signalling state. However, this Tsr fragment lacks all of the methylation sites characteristic of MCP molecules, indicating that methylation segments are not essential for generating receptor output signals.     

Lateral density of receptor arrays in the membrane plane influences sensitivity of the E. coli chemotaxis response

In chemotactic bacteria, transmembrane chemoreceptors, CheA and CheW form the core signalling complex of the chemotaxis sensory apparatus. These complexes are organized in extended arrays in the cytoplasmic membrane that allow bacteria to respond to changes in concentration of extracellular ligands via a cooperative, allosteric response that leads to substantial amplification of the signal induced by ligand binding. Here, we have combined cryo-electron tomographic studies of the 3D spatial architecture of chemoreceptor arrays in intact E. coli cells with computational modelling to develop a predictive model for the cooperativity and sensitivity of the chemotaxis response. The predictions were tested experimentally using fluorescence resonance energy transfer (FRET) microscopy. Our results demonstrate that changes in lateral packing densities of the partially ordered, spatially extended chemoreceptor arrays can modulate the bacterial chemotaxis response, and that information about the molecular organization of the arrays derived by cryo-electron tomography of intact cells can be translated into testable, predictive computational models of the chemotaxis response.     

Stabilization of polar localization of a chemoreceptor via its covalent modifications and its communication with a different chemoreceptor

In the chemotaxis of Escherichia coli, polar clustering of the chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW is thought to be involved in signal amplification and adaptation. However, the mechanism that leads to the polar localization of the receptor is still largely unknown. In this study, we examined the effect of receptor covalent modification on the polar localization of the aspartate chemoreceptor Tar fused to green fluorescent protein (GFP). Amidation (and presumably methylation) of Tar-GFP enhanced its own polar localization, although the effect was small. The slight but significant effect of amidation on receptor localization was reinforced by the fact that localization of a noncatalytic mutant version of GFP-CheR that targets to the C-terminal pentapeptide sequence of Tar was similarly facilitated by receptor amidation. Polar localization of the demethylated version of Tar-GFP was also enhanced by increasing levels of the serine chemoreceptor Tsr. The effect of covalent modification on receptor localization by itself may be too small to account for chemotactic adaptation, but receptor modification is suggested to contribute to the molecular assembly of the chemoreceptor/histidine kinase array at a cell pole, presumably by stabilizing the receptor dimer-to-dimer interaction.     

The Range of Attractant Concentrations for Bacterial Chemotaxis and the Threshold and Size of Response over This Range : Weber law and related phenomena

Attractant was added to a suspension of bacteria (the background concentration of attractant) and then these bacteria were exposed to a yet higher concentration of attractant in a capillary. Chemotaxis was measured by determining how many bacteria accumulated in the capillary. The response range for chemotaxis lies between the threshold concentration and the saturating concentration. The breadth of this range is different for attractants detected by different chemoreceptors. Attractants detected by the same chemoreceptor can have their response ranges in widely different places. Over the center of the response range (on a logarithmic scale), bacteria give similar sized responses to similar fractional increases of concentration, i.e. they respond to ratios of attractant concentration, but the response peaks at the center of the range. The size of the response is different for attractants detected by different chemoreceptors. For a detectable response, a smaller increase in attractant concentration is needed for attractants detected by some chemoreceptors than for attractants detected by others. Although the data are inadequate, it appears that the Weber law may be observed over a wide range of concentrations for some attractants but not for others. In the Appendix we aim to explain some of these results in terms of the interaction of an attractant with its chemoreceptor according to the law of mass action.     

Direct sensing and signal transduction during bacterial chemotaxis toward aromatic compounds in Comamonas testosteroni

Micro‐organisms sense and chemotactically respond to aromatic compounds. Although the existence of chemoreceptors that bind to aromatic attractants and subsequently trigger chemotaxis have long been speculated, such a chemoreceptor has not been demonstrated. In this report, we demonstrated that the chemoreceptor MCP2901 from Comamonas testosteroni CNB‐1 binds to aromatic compounds and initiates downstream chemotactic signaling in addition to its ability to trigger chemotaxis via citrate binding. The function of gene MCP2901 was investigated by genetic deletion from CNB‐1 and genetic complementation of the methyl‐accepting chemotaxis protein (MCP)‐null mutant CNB‐1Δ20. Results showed that the expression of MCP2901 in the MCP‐null mutant restored chemotaxis toward nine tested aromatic compounds and nine carboxylic acids. Isothermal titration calorimetry (ITC) analyses demonstrated that the ligand‐binding domain of MCP2901 (MCP2901LBD) bound to citrate, and weakly to gentisate and 4‐hydroxybenzoate. Additionally, ITC assays indicated that MCP2901LBD bound strongly to 2,6‐dihydroxybenzoate and 2‐hydroxybenzoate, which are isomers of gentisate and 4‐hydroxybenzoate respectively that are not metabolized by CNB‐1. Agarose‐in‐plug and capillary assays showed that these two molecules serve as chemoattractants for CNB‐1. Through constructing membrane‐like MCP2901‐inserted Nanodiscs and phosphorelay activity assays, we demonstrated that 2,6‐dihydroxybenzoate and 2‐hydroxybenzoate altered kinase activity of CheA. This is the first evidence of an MCP binding to an aromatic molecule and triggering signal transduction for bacterial chemotaxis.     

Activated chemoreceptor arrays remain intact and hexagonally packed

Bacterial chemoreceptors cluster into exquisitively sensitive, tunable, highly ordered, polar arrays. While these arrays serve as paradigms of cell signalling in general, it remains unclear what conformational changes transduce signals from the periplasmic tips, where attractants and repellents bind, to the cytoplasmic signalling domains. Conflicting reports support and contest the hypothesis that activation causes large changes in the packing arrangement of the arrays, up to and including their complete disassembly. Using electron cryotomography, here we show that in Caulobacter crescentus, chemoreceptor arrays in cells grown in different media and immediately after exposure to the attractant galactose all exhibit the same 12 nm hexagonal packing arrangement, array size and other structural parameters. ΔcheB and ΔcheR mutants mimicking attractant- or repellent-bound states prior to adaptation also show the same lattice structure. We conclude that signal transduction and amplification must be accomplished through only small, nanoscale conformational changes.     

Demethylation of methyl-accepting chemotaxis proteins in Escherichia coli induced by the repellents glycerol and ethylene glycol.

The addition of glycerol or ethylene glycol caused not only severe tumbling but also a drastic decrease in the methylation level of methyl-accepting chemotaxis proteins (MCPs) in Escherichia coli. Experiments with various mutants having defects in their MCPs showed that the demethylation occurred in all three kinds of MCPs, MCPI, II, and III. The addition of an attractant to the glycerol- or ethylene glycol-treated cells resulted in a distinct increase in the methylation level of the relevant MCP, indicating that glycerol and ethylene glycol do not directly damage the methylation-demethylation system in the cell. The time courses of adaptation and MCP demethylation upon addition of these repellents were consistent with each other. Furthermore, both the response time and the extent of MCP demethylation were increased in parallel with increasing concentrations of glycerol or ethylene glycol. These results indicate that the adaptation to these repellents is performed by the demethylation of MCPs. Thus, glycerol and ethylene glycol are novel repellents, which utilize not just one but all three kinds of MCPs for both information processing and adaptation.     

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.     

Chemoattractants elicit methylation of specific polypeptides in Spirochaeta aurantia

On the basis of this investigation, chemotaxis in Spirochaeta aurantia correlates with methylation of specific polypeptides which are presumed to be analogous to the methyl-accepting chemotaxis proteins (MCPs) in bacteria such as Escherichia coli. The polypeptides exhibited apparent molecular weights in the range of 55,000 to 65,000. Generally, two major presumptive MCP bands and three minor bands were observed on sodium dodecyl sulfate-polyacrylamide gels. Upon addition of D-glucose to S. aurantia cells, methylation of the presumptive MCPs increased for 10 to 12 min to a level greater than 4 times the level of methylation in the absence of D-glucose. Removal of D-glucose resulted in a decrease in methylation of the presumptive MCPs to a level similar to that in unstimulated cells. All attractants tested, including a non-metabolizable attractant (alpha-methyl-D-glucoside) stimulated methylation of the presumptive MCPs (from 1.7 to 4.3 times the level of methylation in unstimulated cells). D-Mannitol, a metabolizable sugar which is not an attractant for S. aurantia, did not stimulate methylation. Stimulation of methylation by D-galactose occurred in cells induced for D-galactose taxis but not in uninduced cells. These data are indicative of an evolutionary relationship between the chemotaxis systems of spirochetes and of flagellated bacteria.     

Designed potent multivalent chemoattractants for Escherichia coli.

Bacterial chemotactic responses are initiated when certain small molecules (i.e., carbohydrates, amino acids) interact with bacterial chemoreceptors. Although bacterial chemotaxis has been the subject of intense investigations, few have explored the influence of attractant structure on signal generation and chemotaxis. Previously, we found that polymers bearing multiple copies of galactose interact with the chemoreceptor Trg via the periplasmic binding protein glucose/galactose binding protein (GGBP). These synthetic multivalent ligands were potent agonists of Escherichia coli chemotaxis. Here, we report on the development of a second generation of multivalent attractants that possess increased chemotactic activities. Strikingly, the new ligands can alter bacterial behavior at concentrations 10-fold lower than those required with the original displays; thus, they are some of the most potent synthetic chemoattractants known. The potency depends on the number of galactose moieties attached to the oligomer backbone and the length of the linker tethering these carbohydrates. Our investigations reveal the plasticity of GGBP; it can bind and mediate responses to several carbohydrates and carbohydrate derivatives. These attributes of GGBP may underlie the ability of bacteria to sense a variety of ligands with relatively few receptors. Our results provide insight into the design and development of compounds that can modulate bacterial chemotaxis and pathogenicity.     

Ensifer alkalisoli YIC4027趋化受体基因的比较及相关蛋白序列分析

[目的] 田菁共生根瘤菌Ensifer alkalisoli YIC4027是从宿主植物田菁的根瘤中分离出来的一株新型高效的固氮菌。本研究对E.alkalisoli的趋化受体基因与其他研究透彻的物种进行比较以及相关蛋白分析。[方法] 利用NCBI的BLAST对E.alkalisoli趋化受体基因进行序列相似性搜索。以Pfam数据库为基础,用HMMR3对甲基化趋化受体蛋白（MCP）进行比较分析。[结果] E.alkalisoli有2个趋化基因簇,共有13个MCP,含有不同的信号传感结构。此外,这些MCPs的胞质结构域除了一个是由40个七肽重复序列组成,其余都是由36个七肽重复序列组成。[结论] 尽管E.alkalisoli的趋化受体与已被广泛研究的物种的趋化受体具有较高的相似性,但仍显示出其特性。通过基因的比对以及相关蛋白的分析,我们能够更好地理解E.alkalisoli是如何通过趋化系统来响应外界变化的。     

Analysis of chimeric chemoreceptors in Bacillus subtilis reveals a role for CheD in the function of the McpC HAMP domain

Motile prokaryotes use a sensory circuit for control of the motility apparatus in which ligand-responsive chemoreceptors regulate phosphoryl flux through a modified two-component signal transduction system. The chemoreceptors exhibit a modular architecture, comprising an N-terminal sensory module, a C-terminal output module, and a HAMP domain that connects the N- and C-terminal modules and transmits sensory information between them via an unknown mechanism. The sensory circuits mediated by two chemoreceptors of Bacillus subtilis have been studied in detail. McpB is known to regulate chemotaxis towards the attractant asparagine in a CheD-independent manner, whereas McpC requires CheD to regulate chemotaxis towards the attractant proline. Although CheD is a phylogenetically widespread chemotaxis protein, there exists only a limited understanding of its function. We have constructed chimeras between McpB and McpC to probe the role of CheD in facilitating sensory transduction by McpC. We found that McpC can be converted to a CheD-independent receptor by the replacement of one-half of its HAMP domain with the corresponding sequence from McpB, suggesting that McpC HAMP domain function is complex and may require intermolecular interactions with the CheD protein. When considered in combination with the previous observation that CheD catalyzes covalent modification of the C-terminal modules of B. subtilis receptors, these results suggest that CheD may interact with chemoreceptors at multiple, functionally distinct sites.     

CheC and CheD interact to regulate methylation of Bacillus subtilis methyl-accepting chemotaxis proteins

In this study, we have demonstrated that two unique proteins in Bacillus subtilis chemotaxis, CheC and CheD, interact. We have shown this interaction both by using the yeast two-hybrid system and by precipitation of in vitro translated products using glutathione-S-transferase fusions and glutathione agarose beads. We have also shown that CheC inhibits B. subtilis CheR-mediated methylation of B. subtilis methyl-accepting chemotaxis proteins (MCPs) but not of Escherichia coli MCPs. It was previously reported that cheC mutants tend to swim smoothly and do not adapt to addition of attractant; cheD mutants have very poorly methylated MCPs and are very tumbiy, similar to cheA mutants. We hypothesize that CheC exerts its effect on MCP methylation in B. subtilis by controlling the binding of CheD to the MCPs. In absence of CheD, the MCPs are poor substrates for CheR and appear to tie up, rather than activate, CheA. The regulation of CheD by CheC may be part of a unique adaptation system for chemotaxis in B. subtilis, whereby high levels of CheY-P brought about by attractant addition would allow CheC to interact with CheD and consequently leave the MCPs, reducing CheA activity and hence the levels of CheY-P.     

Elucidation of a PTS-carbohydrate chemotactic signal pathway in Escherichia coli using a time-resolved behavioral assay

Chemotaxis of Escherichia coli toward phosphotransferase systems (PTSs)-carbohydrates requires phosphoenolpyruvate-dependent PTSs as well as the chemotaxis response regulator CheY and its kinase, CheA. Responses initiated by flash photorelease of a PTS substrates D-glucose and its nonmetabolizable analog methyl alpha-D-glucopyranoside were measured with 33-ms time resolution using computer-assisted motion analysis. This, together with chemotactic mutants, has allowed us to map out and characterize the PTS chemotactic signal pathway. The responses were absent in mutants lacking the general PTS enzymes EI or HPr, elevated in PTS transport mutants, retarded in mutants lacking CheZ, a catalyst of CheY autodephosphorylation, and severely reduced in mutants with impaired methyl-accepting chemotaxis protein (MCP) signaling activity. Response kinetics were comparable to those triggered by MCP attractant ligands over most of the response range, the most rapid being 11.7 +/- 3.1 s-1. The response threshold was <10 nM for glucose. Responses to methyl alpha-D-glucopyranoside had a higher threshold, commensurate with a lower PTS affinity, but were otherwise kinetically indistinguishable. These facts provide evidence for a single pathway in which the PTS chemotactic signal is relayed rapidly to MCP-CheW-CheA signaling complexes that effect subsequent amplification and slower CheY dephosphorylation. The high sensitivity indicates that this signal is generated by transport-induced dephosphorylation of the PTS rather than phosphoenolpyruvate consumption.     

BasT, a membrane-bound transducer protein for amino acid detection in Halobacterium salinarum

Halophilic archaea, such as eubacteria, use methyl-accepting chemotaxis proteins (MCPs) to sense their environment. We show here that BasT is a halobacterial transducer protein (Htp) responsible for chemotaxis towards five attractant amino acids. The C-terminus of the protein exhibits the highly conserved regions that are diagnostic for MCPs: the signalling domain for communication with the histidine kinase and the methylation sites that interact with the methylation/demethylation enzymes for adaptation. Hydropathy analysis predicts an enterobacterial-type transducer protein topology for BasT, with an extracellular putative ligand-binding domain flanked by two transmembrane helices and a cytoplasmic domain. BasT-inactivated mutant cells are missing a membrane protein radiolabelled with L-[methyl-3H]-methionine in wild-type cells, confirming that BasT is methylatable and membrane bound. Behavioural analysis of the basT mutant cells by capillary and chemical-in-plug assays demonstrates complete loss of chemotactic responses towards five (leucine, isoleucine, valine, methionine and cysteine) of the six attractant amino acids for Halobacterium salinarum, whereas they still respond to arginine. The volatile methyl group production assays also corroborate these findings and confirm that BasT signalling induces methyl group turnover. Our data identify BasT as the chemotaxis transducer protein for the branched chain amino acids leucine, isoleucine and valine as well as for methionine and cysteine. Thus, BasT and the arginine sensor Car cover the entire spectrum of chemotactic responses towards attractant amino acids in H. salinarum.     

Bacillus subtilis CheD is a chemoreceptor modification enzyme required for chemotaxis

The chemotaxis machinery of Bacillus subtilis is similar to that of the well characterized system of Escherichia coli. However, B. subtilis contains several chemotaxis genes not found in the E. coli genome, such as cheC and cheD, indicating that the B. subtilis chemotactic system is more complex. In B. subtilis, CheD is required for chemotaxis; the cheD mutant displays a tumbly phenotype, has abnormally methylated chemoreceptors, and responds poorly to most chemical stimuli. Homologs of B. subtilis CheD have been found in chemotaxis-like operons of a large number of bacteria and archaea, suggesting that CheD plays an important role in chemotactic sensory transduction for many organisms. However, the molecular function of CheD has remained unknown. In this study, we show that CheD catalyzes amide hydrolysis of specific glutaminyl side chains of the B. subtilis chemoreceptor McpA. In addition, we present evidence that CheD deamidates other B. subtilis chemoreceptors including McpB and McpC. Previously, deamidation of B. subtilis receptors was thought to be catalyzed by the CheB methylesterase, as is the case for E. coli receptors. Because cheD mutant cells do not respond to most chemoattractants, we conclude that deamidation by CheD is required for B. subtilis chemoreceptors to effectively transduce signals to the CheA kinase.