Purification and characterization of chemotactic methylesterase from Bacillus subtilis

Goldman, Daniel; Nettleton, David O.; Ordal, George

doi:10.1021/bi00299a014

Cited by 34 publications

(19 citation statements)

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“…subtilis employs three adaptation systems to sense chemical gradients (35). The methylation system consists of CheR, a methyltransferase, and CheB, a methylesterase, enzymes that, respectively, add and remove methyl groups at specific glutamate residues on the MCPs (4,12,13). A second adaptation system, the CheC-CheD-CheY system, consists of three proteins.…”

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Cellular Stoichiometry of the Chemotaxis Proteins in Bacillus subtilis

et al. 2011

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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.Motile organisms, such as the soil-based bacterium Bacillus subtilis, have the ability to sense their chemical environment and move to more favorable conditions through a process known as chemotaxis. The chemotactic pathway in B. subtilis involves 10 different chemoreceptors and eight soluble proteins (for a review, see reference 43). The core of the chemotaxis system contains the histidine kinase CheA and the coupling protein CheW (7, 15). These proteins interact with the typically membrane-bound chemoreceptors, or MCPs (methyl-accepting chemotaxis proteins), which can sense various ligands in the extracellular environment (16,33). Once attractant binds to the receptor, CheA autophosphorylates and then transfers its phosphoryl group to CheY, the response regulator (2, 8). Phosphorylated CheY (CheYp) binds to the flagellar motor and changes the direction of its rotation, which leads to a change in the swimming behavior of the bacterium.B. subtilis employs three adaptation systems to sense chemical gradients (35). The methylation system consists of CheR, a methyltransferase, and CheB, a methylesterase, enzymes that, respectively, add and remove methyl groups at specific glutamate residues on the MCPs (4, 12, 13). A second adaptation system, the CheC-CheD-CheY system, consists of three proteins. CheC has been shown to weakly dephosphorylate CheYp, and its activity is enhanced in the presence of CheD (32,38,42). CheD, apart from interacting with CheC, can also interact with the chemoreceptors and deamidate specific glutamine residues on the MCPs, me...

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Cellular Stoichiometry of the Chemotaxis Proteins in Bacillus subtilis

et al. 2011

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“…The phosphorylated form of CheA transfers a phosphoryl group to CheY to produce CheY-P (2, 3), which then interacts with switch proteins to cause CCW 1 rotation of the flagella, resulting in smooth swimming behavior (3). CheA-P also donates phosphoryl groups to CheB, 2 which thereby becomes activated to demethylate the MCPs and produce methanol (4,5). Methylation of the MCPs is known to occur on glutamate side chains (6) through the action of CheR, the chemotactic methyltransferase, which utilizes AdoMet as a substrate (7).…”

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CheY-dependent Methylation of the Asparagine Receptor, McpB, during Chemotaxis in Bacillus subtilis

Kirby

Saulmon

Kristich

et al. 1999

Journal of Biological Chemistry

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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.Chemotaxis is the process by which bacteria sense their chemical environment and migrate toward more favorable conditions. In Bacillus subtilis, chemotaxis toward the attractant asparagine has been shown to be mediated by the methylaccepting chemotaxis protein McpB (1). When asparagine is added to membranes containing McpB in vitro, the rate of autophosphorylation of the CheA autokinase increases (2). The phosphorylated form of CheA transfers a phosphoryl group to CheY to produce CheY-P (2, 3), which then interacts with switch proteins to cause CCW 1 rotation of the flagella, resulting in smooth swimming behavior (3). CheA-P also donates phosphoryl groups to CheB, 2 which thereby becomes activated to demethylate the MCPs and produce methanol (4, 5). Methylation of the MCPs is known to occur on glutamate side chains (6) through the action of CheR, the chemotactic methyltransferase, which utilizes AdoMet as a substrate (7).The B. subtilis chemotactic machinery also includes CheW, CheC, CheD, and CheV. CheW and CheV are thought to couple CheA activity to the MCPs (8 -11). CheC inhibits methylation of the MCPs by an unknown mechanism but does not interfere with the methylesterase, CheB (12, 13). CheD is required to produce a normal prestimulus bias, normal methylation, and azetidine-2-COOH-induced activation of CheA in vivo (12). How these proteins interact to regulate the chemotactic response in B. subtilis remains unknown.The chemotaxis system in Escherichia coli has been well characterized and has served as a paradigm for our studies (for reviews, see Refs. 14 -17). The E. coli system includes homologs of the MCPs, CheA, CheB, CheR, CheW, and CheY. The E. coli system also includes CheZ, which facilitates dephosphorylation of CheY-P (18 -21), but does not include homologs to CheC, CheD, or...

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“…B. subtilis methylesterase was purified by the procedure of Goldman et al (7). E. coli crude extract containing methylesterase activity was prepared by the following procedure.…”

Section: Methodsmentioning

confidence: 99%

“…B. subtilis methyl-3H-MCPs were prepared from OI1085 membranes as previously described (7). E. coli methyl-3H-MCPs were prepared by combining 1 ml of the membrane fraction from an enzyme preparation, The entire contents of the outer well were heated to 100°C for 5 min, and 25 ,ul was applied to a sodium dodecyl sulfate-polyacrylamide gel (10% acrylamide, 0.125% bisacrylamide) and electrophoresed as previously described (14).…”

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confidence: 99%

Functional homology of chemotactic methylesterases from Bacillus subtilis and Escherichia coli

Nettleton

Ordal

1989

J Bacteriol

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The methylesterase enzyme from Bacilus subtilis was compared with that from Escherichia coli. Both enzymes were able to demethylate methyl-accepting chemotaxis proteins (MCPs) from the other organism and were similarly affected by variations in glycerol, magnesium ion, or pH. When attractants were added to a mixture of B. subtlis MCPs and E. coli methylesterase, the rate of demethylation was enhanced. Conversely, when attractants were added to a mixture of E. coli MCPs and B. subtlis methylesterase, the rate of demethylation was diminished. These effects are what would be expected if, in these in vitro systems, the MCPs determined the rate of demethylation. These data suggest that, although the enzymes are from evolutionarily divergent organisms and are different in size, they have considerable functional homology.Chemotaxis is the process by which motile cells migrate toward higher concentrations of attractant and away from higher concentrations of repellent. This process has been observed in evolutionarily divergent organisms (5,13,19). In bacteria, chemotaxis has been linked to the posttranslational modification of certain membrane proteins called methylaccepting chemotaxis proteins (MCPs) (9, 10). These MCPs are methyl esterified on specific glutamic acid residues (1,3,11,27). The number of methyl esters on an MCP changes in response to chemoeffectors (9,10,12,22,26).Because the divergent organisms Escherichia coli and Bacillus subtilis both use this type of methylation-demethylation system, many comparisons have been made of the similarities and differences between the chemotactic processes of the two organisms. Similarities and differences in the chemoeffectors sensed have been reported (17,18,25), as have variations in the mechanisms of response and adaptation to similar chemoeffectors (2,8,9). MCPs from the two organisms have been found to have similar sites of methylation (3) as well as structural similarities (16). In addition, the chemotactic methyltransferase enzymes from the two organisms have been found to be functionally homologous (3). In this paper, we present evidence for the functional homology of the methylesterase enzymes of E. coli and B. subtilis. MATERIALS AND METHODS Chemicals

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Purification and characterization of chemotactic methylesterase from Bacillus subtilis

Cited by 34 publications

References 24 publications

Cellular Stoichiometry of the Chemotaxis Proteins in Bacillus subtilis

Cellular Stoichiometry of the Chemotaxis Proteins in Bacillus subtilis

CheY-dependent Methylation of the Asparagine Receptor, McpB, during Chemotaxis in Bacillus subtilis

Functional homology of chemotactic methylesterases from Bacillus subtilis and Escherichia coli

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