Structural studies of methyl-accepting chemotaxis proteins of Escherichia coli: evidence for multiple methylation sites.

Abstract

Two-dimensional analysis of tryptic peptides from [35S]methionine-1abeled methyl-accepting chemotaxis proteins, MCP I and MCP II, demonstrates a high degree of homology between the two proteins. After the methylation sites were labeled with S-adenosyl-L-methyl-3H]methionine, peptides of three distinct migrations in each protein were found to carry a methyl group. These multiple methylations appear to be responsible in part for the observed multiple banding patterns on sodium dodecyl sulfate/polyacrylamide slab gels.Motile bacteria are able to sense, respond to, and adapt to changes in their environment. Escherichia coli, for example, will migrate toward higher concentrations of certain chemicals, typically nutrients. They also migrate away from repellent stimuli, such as acetate and cobalt ions (1). Study of this behavioral response, called chemotaxis, is revealing the mechanism, on a relatively simple level, by which sensory reception and transduction take place. Stimuli are initially detected by a number of specific receptors on the cell surface, some of which have been identified. Information about receptor occupancy is then transmitted to the chemotaxis gene products via a set of methyl-accepting chemotaxis proteins (MCPs) (2). These inner membrane proteins are products of the tsr (MCP I), tar (MCP II), and trg (MCP III) genes, and each is specific for mediating signals from a different set of stimuli (3, 4). The MCPs are reversibly methylated by a methyltransferase (5, 6) and the methyl donor S-adenosyl-L-methionine (AdoMet). The cells maintain a basal level of methylation in the absence of stimuli. When exposed to an attractant, the level of methylation of the corresponding MCP rises to a new level related to the external concentration of that stimulus. Similarly, a repellent results in the demethylation of the corresponding MCP (7). Although these molecules are methylated and demethylated in response to a different set of attractants and repellents, they share many common features. All E. coli MCPs require the cheX gene product for methylation and the cheB gene product for demethylation (5,8,9). Each appears on high-resolution NaDodSO4/polyacrylamide slab gels as a set of closely spaced protein bands in the 56,000-to 66,000-dalton range with MCP I migrating at the high end of this range and MCP III at the low end (3, 7).We have used trypsin digestion in order to determine the number of methylation sites in MCP I and MCP II. We present evidence that suggests that both MCP I and II have at least three methylation sites and that the variations in number of methyl groups per protein are correlated with the multiple banding patterns. In were used to exclusively label either MCP I or MCP II, respectively. Isolation and Trypsin Digestion of MCPs. Cells that had been labeled were dissolved in NaDodSO4 dissociation buffer and loaded onto NaDodSO4/polyacrylamide slab gels (6% acrylamide, 0.8 mm X 2.5 cm) according to Laemmli (13) in order to resolve the multiple MCP bands. Immediately after electrophores...

Effects of glutamines and glutamates at sites of covalent modification of a methyl-accepting transducer

“…The existence of multiple sites of methylation on transducer proteins was originally detected because methylation and demethylation have substantial effects on the mobility of the proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (3,5,7,8). The addition of methyl groups increases electrophoretic mobility and demethylation decreases it, causing shifts in electrophoretic position that correspond to changes in apparent molecular weight of 500 to 1,500.…”

Relationships between C4 dicarboxylic acid transport and chemotaxis in Rhizobium meliloti

“…In vivo methylation and methanol release assays gave the expected results for E. coli (5,21) but failed to show a significant increase in methylation in R. meliloti cells exposed to any of the attractants tested. The low level of protein methylation in R. meliloti may have one or more explanations.…”

“…The methanol release-diffusion assay described by Chelsky and Dahlguist (5) was also used to detect methyl esterification of proteins in R. meliloti in parallel with the in vivo methylation experiments described above. A three-to fourfold increase in release of labeled methanol was observed for E. coli cells exposed to 5 mM aspartate (Fig.…”

Intersubunit Interaction between Transmembrane Helices of the Bacterial Aspartate Chemoreceptor Homodimer

“…7). Multiple methylation of a chemoreceptor by CheR causes stepwise increases in its mobility in SDS-polyacrylamide gel electrophoresis (33)(34)(35)(36). All of the mutant receptors were detected in whole cell lysates, although their amounts and levels of methylation varied substantially.…”