Methyl esterification of glutamic acid residues of methyl-accepting chemotaxis proteins in Bacillus subtilis.

The amino acid residue modified in the reversible methylation of Bacillus subtilis methyl-accepting chemotaxis proteins was identified as glutamic acid; methylation results in the formation of glutamate 5-methyl ester. Identification was made by comparing the behaviour of a 3H-labelled compound isolated from proteolytically hydrolysed methyl-accepting chemotaxis proteins labelled in vivo with that of authentic methylated amino acids by chromatographic and electrophoretic techniques. Also, the isolated compound on mild alkaline hydrolysis shows behaviour identical with that of authentic glutamate 5-methyl ester. [3H]Methanol released by mild alkaline hydrolysis was made to react with 3,5-dinitrobenzyl chloride to form [3H]methyl 3,5-dinitrobenzoate, which was identified by reverse-phase high-pressure liquid chromatography.     

Evidence for methyl group transfer between the methyl-accepting chemotaxis proteins in Bacillus subtilis.

We present evidence for methyl (as methyl or methoxy) transfer from the methyl-accepting chemotaxis proteins H1 and possibly H3 of Bacillus subtilis to the methyl-accepting chemotaxis protein H2. This methyl transfer, which has been observed in vitro (D. J. Goldman and G. W. Ordal, Biochemistry 23:2600-2606, 1984), was strongly stimulated by the chemoattractant aspartate and thus may play an important role in the sensory processing system of this organism. Although radiolabeling of H1 and H3 began at once after the addition of [3H]methionine, radiolabeling of H2 showed a lag. Furthermore, the addition of excess nonradioactive methionine caused immediate exponential delabeling of H1 and H3 while labeling of H2 continued to increase. Methylation of H2 required the chemotactic methyltransferase, probably to first methylate H1 and H3. Aspartate caused increased labeling of H2 and strongly decreased labeling of H1 and H3 after the addition of nonradioactive methionine. Without the addition of nonradioactive methionine, aspartate caused demethylation of H1 and to a lesser extent H3, with an approximately equal increase of methylation of H2.     

Influence of attractants and repellents on methyl group turnover on methyl-accepting chemotaxis proteins of Bacillus subtilis and role of CheW.

The ability of attractants and repellents to affect the turnover of methyl groups on the methyl-accepting chemotaxis proteins (MCPs) was examined for Bacillus subtilis. Attractants were found to cause an increase in the turnover of methyl groups esterified to the MCPs, while repellents caused a decrease. These reactions do not require CheW. However, a cheW null mutant exhibits enhanced turnover in unstimulated cells. Assuming that the turnover of methyl groups on the MCPs reflects a change in the activity of CheA, these results suggest that the activation of CheA via chemoeffector binding at the receptor does not require CheW.     

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.     

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.     

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.     

Purification and characterization of Bacillus subtilis methyl-accepting chemotaxis protein methyltransferase II

A Bacillus subtilis methyltransferase capable of methylating membrane-bound methyl-accepting chemotaxis proteins (MCPs) of a chemotaxis mutant was purified to homogeneity. MCPs are normally unmethylated in this strain. Results of gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the enzyme is a 30,000 molecular weight monomer. The enzyme transfers methyl groups from S-adenosylmethionine to glutamate residues of the substrates. The enzyme is activated by divalent cations and has a Km for S-adenosylmethionine of about 5 microM. It is competitively inhibited by S-adenosylhomocysteine, with a Ki of about 0.2 microM, and exhibits an in vitro assay pH optimum of 6.9. This methyltransferase is very different from another methyltransferase from B. subtilis, described previously (Ullah, A. H. J., and Ordal, G. W. (1981) Biochem. J. 199, 795-805).     

Methyl transfer in chemotaxis toward sugars by Bacillus subtilis.

Like amino acids, the sugars glucose and the nonmetabolizable 2-deoxyglucose caused a turnover of methyl groups on the methyl-accepting chemotaxis proteins. These sugars also caused methanol formation on addition. Thus, in contrast to chemotaxis in Escherichia coli, taxis to phosphotransferase sugars by Bacillus subtilis utilizes the methyl-accepting chemotaxis proteins.     

Purification and characterization of methyl-accepting chemotaxis protein methyltransferase I in Bacillus subtilis.

A methyltransferase that methylates one of the proteins involved in chemotactic adaptation to sensory stimuli in Bacillus subtilis was purified to homogeneity. The enzyme utilizes S-adenosylmethionine as donor for a methyl group that is transferred to a glutamate residue in a 69 000-mol.wt. membrane protein and also to a protein of 19 000 mol.wt. The molecular weights of the denatured enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and of the native enzyme by gel-filtration chromatography both show the protein to be a 44 000-mol.wt. monomer. Isoelectric focusing of the purified methyltransferase showed the protein to be a single species with isoelectric point pI 5.4. On the basis of a molecular weight of 44 000, the molar absorption coefficient at 262 nm of the enzyme is 10.9 x 10(4) M-1 . cm-1. The Km of the enzyme for S-adenosylmethionine is about 2 microM. The Ki for S-adenosylhomocysteine is about 0.2 microM. Ca2+ is a competitive inhibitor of methylation, with a Ki of 0.065 microM. The enzyme methylates membranes from the wild-type more efficiently than membranes isolated from a mutant strain defective in chemotaxis. The enzyme is unable to methylate Escherichia coli membranes.     

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.     

Novel methyl transfer during chemotaxis in Bacillus subtilis

If Bacillus subtilis is incubated in radioactive methionine in the absence of protein synthesis, the methyl-accepting chemotaxis proteins (MCPs) become radioactively methylated. If the bacteria are further incubated in excess nonradioactive methionine ("cold-chased") and then given the attractant aspartate, the MCPs lose about half of their radioactivity due to turnover, in which lower specific activity methyl groups from S-adenosylmethionine (AdoMet) replace higher specific activity ones. Due to the cold-chase, the specific activity of the AdoMet pool is reduced at least 2-fold. If, later, the attractant is removed, higher specific activity methyl groups return to the MCPs. Thus, there must exist an unidentified methyl carrier that can "reversibly" receive methyl groups from the MCPs. In a similar experiment, labeled cells were transferred to a flow cell and exposed to addition and removal of attractant and of repellent. All four kinds of stimuli were found to cause methanol production. Bacteria with maximally labeled MCPs were exposed to many cycles of addition and removal of attractant; the maximum amount of radioactive methanol was evolved on the third, not the first, cycle. This result suggests that there is a precursor-product relationship between methyl groups on the MCPs and on the unidentified carrier, which might be the direct source of methanol. However, since no methanol was produced when a methyltransferase mutant, whose MCPs were unmethylated, was exposed to addition and removal of attractant or repellent, the methanol must ultimately derive from methylated MCPs.     

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.     

Receptor conformational changes enhance methylesterase activity during chemotaxis by Bacillus subtilis

Addition and removal of the attractant asparagine causes methanol formation as a consequence of methylation and demethylation of conserved glutamate residues in the Bacillus subtilis chemotaxis receptor McpB C-terminal domain. We found that methanol was released on both addition and removal of asparagine even when the response regulator domain of CheB was removed (to produce CheB(141-357)). Thus, in undergoing the transition from unbound receptor to ligand-bound adapted receptor, the receptor must pass through a state of heightened susceptibility to demethylation by CheB that is independent of phosphorylation. The same result occurred when the aspartate phosphorylation site of CheB, Asp54, had been mutated to an asparagine residue, provided the enzyme was sufficiently induced. However, no methanol release was observed for an active site point mutant, cheB(S173C), in response to addition or removal of asparagine even when induced. Finally, methanol release was observed only for attractant addition in a mutant background lacking the coupling proteins, CheW and CheV, provided CheB(141-357) was present. Thus, on attractant addition, methanol must arise from a transient conformation of the receptor C-terminal domain that is an intrinsic property of the receptor; on attractant removal, however, methanol must arise from a different transient conformation, one dependent on the presence of coupling proteins.     

Effect of methionine on chemotaxis by Bacillus subtilis.

Bacillus subtilis, like Escherichia coli and Salmonella typhimurium, carries out chemotaxis by modulating the relative frequency of smooth swimming and tumbling. Like these enteric bacteria, methionine auxotrophs starved for methionine show an abnormally long-period of smooth swimming after addition of attractant. This "hypersensitive" state requires an hour of starvation for its genesis, which can be hastened by including alanine, a strong attractant, in starvation medium. Susceptibility to repellent, which causes transient tumbling when added, if anything, increases slightly by starvation for methionine. The results are interpreted by postulating the existence of a methionine-derived structure that hastens recovery of attractant-stimulated bacteria back to normal.     

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.     

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.     

Receptors for chemotaxis in Bacillus subtilis.

At least three receptors for chemotaxis toward L-amino acids in Bacillus subtilis could be found with the aid of taxis competition experiments. They are called the asparagine receptor, which detects asparagine and glutamine, the isoleucine receptor, which detects isoleucine, leucine, valine, phenylalanine, serine, threonine, cysteine, and methionine, and the alanine receptor, which detects alanine and proline. Histidine and glycine could not be assigned to one of these receptors. Cysteine and methionine were found to be general inhibitors of chemotaxis and serine was found to be a general stimulator of chemotaxis. Some structural analogues of amino acids were tested for chemotactic activity. The chemotactic activity of B. subtilis is compared with that of Escherichia coli.     

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.     

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.