Adaptation in bacterial chemotaxis: CheB-dependent modification permits additional methylations of sensory transducer proteins

Sensory transduction in E. coli consists of two phases, excitation and adaptation, both of which involve the methyl-accepting chemotaxis proteins (MCPs). These molecules relay transmembrane signals and are reversibly methylated during adaptation of E. coli to environmental stimuli. Each MCP contains multiple sites of methylation, and we identified six of these sites in MCPI. Recently, a second covalent modification of MCPs has been identified, which is not methylation. This modification, designated CheB-dependent modification, is stimulated by repellents and causes a net increase in the negative charge of MCPI and MCPII by one or two charges. We demonstrate that one CheB modification occurs on the methyl-accepting methionine-and lysine-containing tryptic peptide in MCPI and MCPII, and the second CheB modification is on an arginine-containing tryptic peptide. The CheB modification allows three additional methyl groups to be incorporated into the methyl-accepting methionine-lysine peptide, while not actually creating all of these methylation sites. The two CheB modifications occur sequentially. A possible mechanism by which CheB modification permits additional methylations and the role of CheB modification in bacterial chemotaxis are discussed.     

The methyl-accepting chemotaxis proteins of Escherichia coli. Identification of the multiple methylation sites on methyl-accepting chemotaxis protein I

The methyl-accepting chemotaxis proteins (MCPs) are integral membrane proteins that undergo reversible methylation during adaptation of bacterial cells to environmental attractants and repellents. The numerous methylated forms of each MCP are seen as a pattern of multiple bands on polyacrylamide gels. We have characterized the methylation sites in MCPI by analyzing methyl-accepting tryptic peptides. At least two different tryptic peptides accept methyl esters; one methyl-accepting peptide contains methionine and lysine and may be methylated a maximum of four times. The second methyl-accepting tryptic peptide contains arginine and may be methylated twice. Base-catalyzed demethylations of tryptic peptides and analysis of the charge differences between the different methylated forms of MCPI show that MCPI molecules may be methylated a total of six times. The two methyl esters on the methyl-accepting arginine peptide appear to be preferentially methylated in most of the forms of MCPI in attractant-stimulated cells. The ability to acquire six methylations on MCPI allows the bacterial cells to adapt to a broad range of attractant and repellent concentrations.     

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.     

Multiple electrophoretic forms of methyl-accepting chemotaxis proteins generated by stimulus-elicited methylation in Escherichia coli.

The tsr and tar genetic loci of Escherichia coli determine the presence in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of methyl-accepting chemotaxis proteins (MCPs) I and II, respectively, each of which consists of a distinct group of multiple bands. Synthesis of the tsr and tar products was directed in ultraviolet-irradiated bacteria by lambda transducing phages. The addition of appropriate chemotactic stimuli to these cells resulted in the appearance of additional, faster migrating electrophoretic forms of the Tsr and Tar polypeptides which disappeared upon removal of the stimulus. The stimulus-elicited forms comigrated with component bands of the corresponding MCPs. These results indicate that methylation itself caused shifts in electrophoretic mobility and hence led to the observed MCP band patterns. The number of Tsr species suggested that there were at least three methylated sites on the Tsr polypeptide. The conclusion that methylation generates multiplicity was supported by the results of experiments in which the tsr product was synthesized in mutant bacteria defective in specific chemotaxis functions concerned with methylation or demethylation of MCPs. Thus, the presence of a cheX defect blocked the stimulus-elicited appearance of faster migrating forms of the tsr product; conversely, the presence of a cheB defect resulted in a pronounced shift toward these forms in the absence of a chemotactic stimulus.     

Ligand-specific activation of Escherichia coli chemoreceptor transmethylation

Adaptation in the chemosensory pathways of bacteria like Escherichia coli is mediated by the enzyme-catalyzed methylation (and demethylation) of glutamate residues in the signaling domains of methyl-accepting chemotaxis proteins (MCPs). MCPs can be methylated in trans, where the methyltransferase (CheR) molecule catalyzing methyl group transfer is tethered to the C terminus of a neighboring receptor. Here, it was shown that E. coli cells exhibited adaptation to attractant stimuli mediated through either engineered or naturally occurring MCPs that were unable to tether CheR as long as another MCP capable of tethering CheR was also present, e.g., either the full-length aspartate or serine receptor (Tar or Tsr). Methylation of isolated membrane samples in which engineered tethering and substrate receptors were coexpressed demonstrated that the truncated substrate receptors (trTsr) were efficiently methylated in the presence of tethering receptors (Tar with methylation sites blocked) relative to samples in which none of the MCPs had tethering sites. The effects of ligand binding on methylation were investigated, and an increase in rate was produced only with serine (the ligand specific for the substrate receptor trTsr); no significant change in rate was produced by aspartate (the ligand specific for the tethering receptor Tar). Although the overall efficiency of methylation was lower, receptor-specific effects were also observed in trTar- and trTsr-containing samples, where neither Tar nor Tsr possessed the CheR binding site at the C terminus. Altogether, the results are consistent with a ligand-induced conformational change that is limited to the methylated receptor dimer and does not spread to adjacent receptor dimers.     

Increased methyl esterification of membrane proteins in aged red-blood cells. Preferential esterification of ankyrin and band-4.1 cytoskeletal proteins

The enzymatic carboxyl methyl esterification of erythrocyte membrane proteins has been investigated in three different age-related fractions of human erythrocytes. When erythrocytes of different mean age, separated by density gradient centrifugation, were incubated under physiological conditions (pH 7.4, 37 degrees C) in the presence of L-[methyl-3H]methionine, the precursor in vivo of the methyl donor S-adenosylmethionine, a fourfold increase in membrane-protein carboxyl methylation was observed in the oldest cells compared with the youngest ones. The identification of methylated species, based on comigration of radioactivity with proteins stained with Coomassie blue, analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, shows, in all cell fractions, a pattern similar to that reported for unfractionated erythrocytes. However in the membrane of the oldest erythrocytes the increase in methylation of the cytoskeletal proteins, bands 2.1 and 4.1, appears to be significantly more marked compared with that observed in the other methylated polypeptides. Furthermore the turnover rate of incorporated [3H]methyl groups in the membrane proteins of the oldest cells markedly increases during cell ageing. Particularly in band 4.1 the age-related increase in methyl esterification is accompanied by a significant reduction of the half-life of methyl esters. The activity of cytoplasmic protein methylase II does not change during cell ageing, while the isolated ghosts from erythrocytes of different age show an age-related increased ability to act as methyl-accepting substrates, when incubated in presence of purified protein methylase II and methyl-labelled S-adenosylmethionine, therefore the relevance of membrane structure in determining membrane protein methylation levels can be postulated. Finally the possible correlation of this posttranslational protein modification with erythrocyte ageing is discussed.     

Studies on bacterial chemotaxis. II. Effect of cheB and cheZ mutations on the methylation of methyl-accepting chemotaxis protein of Escherichia coli.

Radioactive proteins from chemotactic mutants of Escherichia coli with continuous tumbling phenotype (cheB and cheZ) and their otherwise isogenic parent were compared by two-dimensional gel electrophoresis. The system was capable of separating non-methylated methyl-accepting chemotaxis protein (MCP) from its methylated equivalent. The analysis of proteins from the envelope fraction of the bacteria showed that the cheB mutants contained a larger portion of methylated MCP than did the parent. However, the change of MCP methylation level was small, if any, in cheZ strains. The results suggest that the product of cheB gene and the product of cheZ gene are not functional complementary. The product of cheB gene functions in controlling the level of methylation at the stationary state of the organisms. In addition to known MCP species, a new MCP of about 43,000 daltons was found. This MCP appears to be involved in transducing signals of some sugars.     

Transmembrane signaling by bacterial chemoreceptors: E. coli transducers with locked signal output

Methyl-accepting chemotaxis proteins (MCPs) function as transmembrane signalers in bacteria. We isolated and characterized mutants of the E. coli Tsr protein that produce output signals in the absence of overt stimuli and that are refractory to sensory adaptation. The properties of these "locked" transducers indicate that MCP molecules are capable of generating signals that actively augment clockwise and counter-clockwise rotation of the flagellar motors. Transitions between MCP signaling states can be influenced by amino acid replacements in many parts of the molecule, including the methylation sites, at least one of the two membrane-spanning segments, and a linker region connecting the receptor and signaling domains. These findings suggest that transmembrane signaling may involve direct propagation of conformational changes between the periplasmic and cytoplasmic portions of the MCP molecule.     

Methylation of erythrocyte membrane proteins at extracellular and intracellular D-aspartyl sites in vitro. Saturation of intracellular sites in vivo

A cytosolic protein carboxyl methyltransferase (S-adenosyl-L-methionine:protein O-methyltransferase, E.C. 2.1.1.24) purified from human erythrocytes catalyzes the methylation of erythrocyte membrane proteins in vitro using S-adenosyl-L-[methyl-3H]methionine as the methyl group donor. The principal methyl-accepting proteins have been identified by sodium dodecyl sulfate-gel electrophoresis at pH 2.4 and fluorography as the anion transport protein (band 3), ankyrin (band 2.1), and integral membrane proteins with molecular weights of 45,000, 28,000, and 21,000. Many of the methylation sites associated with intrinsic membrane proteins may reside in their extracellular portions, since these same proteins are methylated when intact cells are used as the substrate. The maximal number of methyl groups transferred in these experiments is approximately 30 pmol/mg of membrane protein, a value which represents less than one methyl group/50 polypeptide chains of any methyl-accepting species. The number of methylation sites associated with the membranes is increased, but not to stoichiometric levels, by prior demethylation of the membranes. The additional sites are associated primarily with bands 2.1 and 4.1, the principal methyl acceptors in vivo, suggesting that most methylation sites are fully modified in vivo. Extracellular methylation sites are not increased by demethylation of membranes. The aspartic acid beta-methyl ester which can be isolated from carboxypeptidase Y digests of [3H]methylated membranes is in the unusual D-stereoconfiguration. Similar results have been obtained with [3H]methylated membranes isolated from intact cells (McFadden, P.N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2460-2464). It is proposed that the methyltransferase recognizes D-aspartyl residues in proteins and is involved with the metabolism of damaged proteins in vivo.     

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.     

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.     

Methylation of chemotaxis-specific proteins in Escherichia coli cells permeable to S-adenosylmethionine

Using a modification of the EGTA treatment of Oishi and Smith [Oishi, M., & Smith, C. L. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3569], Escherichia coli cells have been made permeable to S-adenosylmethionine and other related molecules in order to facilitate the study of methylation in chemotaxis. The permeable cells are nonmotile but respond to chemotactic stimuli by reversible methylation of their methyl-accepting chemotactic proteins (MCP I and MCP II) in a manner similar to that of untreated, motile cells. Addition of S-adenosyl-L-[methyl-3H]methionine to the permeable cells specifically labels two proteins, MCP I and MCP II. Methylation of these MCP's is dependent on the presence of wild-type gene products of flaI, flaA, cheB, cheX, tsr, and tar. The extent of methylation of the MCP's is affected by the presence of attractants or repellents: addition of attractant increases the steady-state level of methylation; addition of repellent causes rapid demethylation to a new steady-state level. Methylation is inhibited by the addition of the transmethylase inhibitors A9145C and Sinefungin, which are S-adenosylmethionine analogues, and by S-adenosylhomocysteine.     

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.     

Chemotaxis of Pseudomonas aeruginosa: involvement of methylation.

The involvement of a protein methyl transfer system in the chemotaxis of Pseudomonas aeruginosa was investigated. When a methionine auxotroph of P. aeruginosa was starved for methionine, chemotaxis toward serine, measured by a quantitative capillary assay, was reduced 80%, whereas background motility was unaffected or increased. When unstarved bacteria were labeled with L-[methyl-3H]methionine, a labeled species of 73,000 molecular weight which was methylated in response to stimulation by L-serine was identified. Under appropriate electrophoretic conditions, the 73,000 molecular weight species was resolved into two bands, both of which responded to stimulation by L-serine, L-arginine, and alpha-aminoisobutyrate (AIB) with an increased incorporation of methyl label. Arginine, which elicited the strongest chemotactic response in the capillary assay, also stimulated the greatest methylation response. Methylation of the 73,000 molecular weight species reached a maximum 10 min after stimulation by AIB and returned to the unstimulated level upon removal of the AIB. In vitro labeling of cell extracts with S-adenosyl[methyl-3H]methionine indicated that the 73,000 molecular weight species are methylated by an S-adenosylmethionine-mediated reaction. These results indicate that chemotaxis of P. aeruginosa toward amino acids is mediated by dynamic methylation and demethylation of methyl-accepting chemotaxis proteins analogous to those of the enteric bacteria.     

Methylation sites in Escherichia coli ribosomal RNA: localization and identification of four new sites of methylation in 23S rRNA.

Four previously undetermined sites of methylation are mapped in Escherichia coli 23S rRNA employing a novel combination of methods. First, using a double-isotope approach, the total number of methyl groups in 23S rRNA was determined to be 14.9 +/- 1.6. Second, hybridization of methyl-labeled rRNA to complementary DNA restriction fragments and PAGE analysis were used to purify RNA-DNA heteroduplexes and to quantify methyl groups within specific 23S rRNA fragments. Third, the methylated nucleosides in these fragments were identified and quantified using HPLC, confirming the presence of 14 methylation sites in 23S rRNA, four more than had been previously identified. In contrast, a similar set of analyses conducted on 16S rRNA gave evidence for 10 sites of methylation, at all approximate locations consistent with published 16S methylated nucleoside identities and locations. Selected regions of the 23S rRNA molecule containing previously unidentified methylated nucleosides were released by site-directed cleavage with ribonuclease H and isolated by PAGE. Sites of methylation within the RNA fragments were determined by classical oligonucleotide analyses. The four newly identified methylation sites in 23S rRNA are m2G-1835, m5C-1962, m6A-2503, and m2G at one of positions 2445-2447. Together with previously described sites of modification, these new sites form a group that is clustered in a current model for the three-dimensional organization of the 23S rRNA in the 50S ribosomal subunit, at a locus congruent with nucleotides previously implicated in ribosomal function.     

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.     

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.     

Methyl-accepting taxis proteins in Halobacterium halobium.

Methyl-accepting taxis proteins were identified and characterized in Halobacterium halobium, an archaebacterial species that is both chemotactic and phototactic. The data suggest direct involvement of methylation and demethylation in mechanisms of both chemotaxis and phototaxis and identify adaptation as the sensory process in which those reactions are likely to be involved. Analysis by electrophoresis and fluorography revealed methyl-accepting species, of apparent Mr between 90,000 and 135,000, that exhibited characteristics of sensory components. Those methyl-3H-labeled species were absent in a mutant blocked in taxis. Methylation of specific bands increased after positive chemostimuli and decreased after negative stimuli. Other methyl-3H-labeled bands, from 17 to 29 kd, exhibited features of biosynthetic intermediates, not of sensory components. Assay of rates of demethylation by measuring release of volatile forms of radiolabeled methyl groups revealed transient changes following chemo- or photostimuli that persisted for periods roughly equivalent to adaptation times. Negative chemostimuli induced increased rates of demethylation, as expected from fluorographic analysis, but positive chemostimuli also resulted in an increase. Photostimuli of either sign were followed by increases in rates of demethylation of shorter duration and lesser magnitude than chemostimuli-induced increases, a relationship that corresponded to differences in adaptation time.     

In vitro methylation of bovine papillomavirus alters its ability to transform mouse cells.

Bovine papillomavirus (BPV) was methylated in vitro at either the 29 HpaII sites, the 27 HhaI sites, or both. Methylation of the HpaII sites reduced transformation by the virus two- to sixfold, while methylation at HhaI sites increased transformation two- to fourfold. DNA methylated at both HpaII and HhaI sites did not differ detectably from unmethylated DNA in its efficiency of transformation. These results indicate that specific methylation sites, rather than the absolute level of methylated cytosine residues, are important in determining the effects on transformation and that the negative effects of methylation at some sites can be compensated for by methylation at other sites. BPV molecules in cells transformed by methylated BPV DNA contained little or no methylation, indicating that the pattern of methylation was not faithfully retained in these extrachromosomally replicating molecules. Methylation at the HpaII sites (but not the HhaI sites) in the cloned BPV plasmid or in pBR322 also inhibited transformation of the plasmids into Escherichia coli HB101 cells.     

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.