Methylation Of Methyl-accepting Chemotaxis Proteins Research Articles

Characterization of the Thermotoga maritima chemotaxis methylation system that lacks pentapeptide-dependent methyltransferase CheR:MCP tethering.

Sensory adaptation in bacterial chemotaxis is mediated by covalent modifications of specific glutamate and glutamine residues within the cytoplasmic domains of methyl-accepting chemotaxis proteins (MCPs). In Escherichia coli and Salmonella enterica, efficient methylation of MCPs depends on the localization of methyltransferase CheR to MCP clusters through an interaction between the CheR beta-subdomain and a pentapeptide sequence (NWETF or NWESF) at the C-terminus of the MCP. In vitro methylation analyses utilizing S. enterica and Thermotoga maritima CheR proteins and MCPs indicate that MCP methylation in T. maritima occurs independently of a pentapeptide-binding motif. Kinetic and binding measurements demonstrate that despite efficient methylation, the interaction between T. maritima CheR and T. maritima MCPs is of relatively low affinity. Comparative protein sequence analyses of CheR beta-subdomains from organisms having MCPs that contain and/or lack pentapeptide-binding motifs identified key similarities and differences in residue conservation, suggesting the existence of two distinct classes of CheR proteins: pentapeptide-dependent and pentapeptide-independent methyltransferases. Analysis of MCP C-terminal ends showed that only approximately 10% of MCPs contain a putative C-terminal binding motif, the majority of which are restricted to the different proteobacteria classes (alpha, beta, gamma, delta). These findings suggest that tethering of CheR to MCPs is a relatively recent event in evolution and that the pentapeptide-independent methylation system is more common than the well-characterized pentapeptide-dependent methylation system.

Aerotaxis in Halobacterium salinarium is methylation-dependent

The behavioural response to a gradient of oxygen (aerotaxis) has been characterized in the archaeon, Halobacterium salinarium. When the gas surrounding a drop of H. salinarium strain S9-P culture was changed abruptly from 10% (v/v) O2 to 100% N2, the bacteria transiently increased the frequency of reversing before they adapted and resumed random swimming. When the gas was returned to 10% O2 the bacteria responded by swimming smoothly for approximately 45 s. Aerotaxis was strongest when respiration in H. salinarium was highest and when bacteriorhodopsin and halorhodopsin were not contributing to the proton motive force. Starvation for methionine of the auxotrophic H. salinarium essentially abolished the step-down aerotactic response. Methanol production from demethylation of methyl-accepting chemotaxis proteins was transiently increased in H. salinarium S9-P by a step down or step up in oxygen concentration, as observed in methylation-dependent chemotaxis in H. salinarium. The taxis-negative and methyltransferase-deficient mutant, H. salinarium strain Pho72 did not exhibit changes in methanol release in response to aerotaxis or chemotaxis stimuli. This is the first report of an aerotactic response that is dependent on methylation of methyl-accepting chemotaxis proteins. Aerotaxis in Escherichia coli and Salmonella typhimurium is independent of transducer methylation.

Effect of outer membrane permeability on chemotaxis in Escherichia coli

The relationship between outer membrane permeability and chemotaxis in Escherichia coli was studied on mutants in the major porin genes ompF and ompC. Both porins allowed passage of amino acids across the outer membrane sufficiently to be sensed by the methyl-accepting chemotaxis proteins, although OmpF was more effective than OmpC. A mutant deleted for both ompF and ompC, AW740, was almost completely nonchemotactic to amino acids in spatial assays. AW740 required greater stimulation with L-aspartate than did the wild type to achieve full methylation of methyl-accepting chemotaxis protein II. Induction of LamB protein allowed taxis to maltose but not to L-aspartate, which indicates that the maltoporin cannot rapidly pass aspartate. Salt taxis was less severely inhibited by the loss of porins than was amino acid taxis, which implies an additional mechanism of outer membrane permeability. These results show that chemotaxis can be used as a sensitive in vivo assay for outer membrane permeability to a range of compounds and imply that E. coli can regulate chemotactic sensitivity by altering the porin composition of the outer membrane.

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.

Chemotaxis in Bacillus subtilis: effects of attractants on the level of methylation of methyl-accepting chemotaxis proteins and the role of demethylation in the adaptation process.

By performing in vivo methylation experiments and using highly resolving NaDodSO4-polyacrylamide gels, we have examined the effects of amino acid attractants on the methylation profile of Bacillus subtilis MCPs. Both increases and decreases have been found to occur in the level of methylation of these proteins. By using competition experiments and Conway diffusion cells, we have found that the demethylation event is correlated with the adaptation process. Gas chromatographic analysis indicates that methanol is evolved upon demethylation of these proteins. As more attractant receptors are titrated, corresponding increases in methanol evolution result. During this period of increased rate of methanol production, bacteria swim smoothly.

An in vitro study of the methylation of methyl-accepting chemotaxis protein of Escherichia coli. Construction of the system and effect of mutant proteins on the system.

An in vitro system for the methylation of methyl-accepting chemotaxis proteins (MCP's), which have been shown to be membrane integral proteins, was constructed. The system, consisting of the membrane, the cytoplasm, and labeled S-adenosyl methionine, showed the following characteristics. 1. The methylation of MCP in the membrane required the cytoplasm. The rate of incorporation of the labeled methyl group into MCP was dependent on the amount of the cytoplasm. 2. Incorporation of the labeled methyl moiety into MCP reached a steady state, and the level of the steady state incorporation was dependent on the concentration of the cytoplasm when the concentration of the membrane protein was constant. 3. The methyl moiety which had been incorporated into MCP before the steady state could be exchanged. It was suggested that the amount of methyl group introduced into MCP was equal to that of taken from MCP. 4. The methylated MCP was demethylated faster in the presence of a methyl donor than in its absence. 5. The membranes obtained from cheX-, cheB-, and cheZ mutants were inactive in the present in vitro system even when they were mixed with the wild type cytoplasm.

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.

Studies on bacterial chemotaxis. VI. Effect of cheX mutation on the methylation of methyl-accepting chemotaxis protein of Escherichia coli.

A membrane from Escherichia coli cheX mutants failed to accept the methyl-moiety when incubated with a cytoplasm of wild type bacteria. The possibility of reduced production of methyl-accepting chemotaxis protein (MCP) as a result of the effect of cheX mutation was considered. However, analysis of sulfur labeled proteins from cheX mutants by two-dimensional gel electrophoresis showed that the proportion of non-esterified MCP increased in cheX mutants while the total amount of MCP was not seriously affected. Thus the decreased methylation of MCP in cheX mutants, as detected by the methyl-labeling experiment, is not caused by an effect of cheX mutation on the production of MCP back-bone protein but by a defect in the device for methylation. The presence of four species of MCP's whose extent of methylation was affected by cheX mutation as well as cheB mutation was also shown by this experiment.

Attractants and repellents control demethylation of methylated chemotaxis proteins in Escherichia coli.

A group of methylated proteins, the methyl-accepting chemotaxis proteins (MCP), has been shown to play a central role in bacterial chemotaxis. Both methylation and demethylation of MCP occur continuously in the absence of added stimuli; these two processes are in balance such that a basal level of methylation is maintained. Attractants cause the methylation level to increase to a new value, whereas repellents bring about a decrease in level. Therefore, attractants and repellents must somehow perturb the balance between methylation and demethylation of MCP. In this report the effect of attractants on demethylation of MCP was monitored in two ways: (i) by following the disappearance of [methyl-3H]MCP and (ii) by measuring formation of [3H]methanol, the product of MCP demethylation. Both methods showed that addition of attractants causes a transient inhibition of MCP demethylation. Repellent addition has previously been shown to stimulate MCP demethylation. It is therefore concluded that control of demethylation plays a crucial role in changing the level of methylation of MCP in response to attractants and repellents.