SummarySensory 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 b-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 b-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: pentapeptidedependent and pentapeptide-independent methyltransferases. Analysis of MCP C-terminal ends showed that only~10% of MCPs contain a putative C-terminal binding motif, the majority of which are restricted to the different proteobacteria classes (a, b, g, d). 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.

Adaptation in bacterial chemotaxis involves reversible methylation of specific glutamate residues within the cytoplasmic domains of methyl-accepting chemotaxis proteins. The specific sites of methylation in Salmonella enterica and Escherichia coli chemoreceptors, identified 2 decades ago, established a consensus sequence for methylation by methyltransferase CheR. Here we report the in vitro methylation of chemoreceptors from Thermotoga maritima, a hyperthermophile that has served as a useful source of chemotaxis proteins for structural analysis. Sites of methylation have been identified by liquid chromatography-mass spectrometry/ mass spectrometry. Fifteen sites of methylation were identified within the cytoplasmic domains of four different T. maritima chemoreceptors. The results establish a consensus sequence for chemoreceptor methylation sites in T. maritima that is distinct from the previously identified consensus sequence for E. coli and S. enterica. These findings suggest that consensus sequences for posttranslational modifications in one organism may not be directly extrapolated to analogous modifications in other bacteria.

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Bacterial chemotaxis receptors are posttranslationally modified by carboxyl methylation of specific glutamate residues within their cytoplasmic domains. This highly regulated, reversible modification counterbalances the signaling effects of ligand binding and contributes to adaptation. Based on the crystal structure of the γ-glutamyl-methyltransferase CheR, we have postulated that positively charged residues in helix α2 in the N-terminal domain of the enzyme may be complementary to the negatively charged methylation region of the methyltransferase substrates, the bacterial chemotaxis receptors. Several altered CheR proteins, in which positively charged arginine or lysine residues were substituted with alanines, were constructed and assayed for their methylation activities toward wild-type receptor and a series of receptor variants containing different glutamates available for methylation. One of the CheR mutant proteins (Arg53Ala) showed significantly lower activity toward all receptor constructs, suggesting that Arg53 may play a general role in catalysis of methyl transfer. The rest of the mutant proteins exhibited different patterns of relative methylation rates toward different receptor substrates, indicating specificity, probably through interaction of CheR with the receptor at sites distal to the specific site of methylation. The findings imply complementarity between positively charged residues of the α2 helix of CheR and the negatively charged glutamates of the receptor. It is likely that this complementarity is involved in discriminating different methylation states of the receptors.In enteric bacteria, chemotaxis is mediated by several homologous transmembrane receptors that sense changes in concentrations of attractant and repellent molecules (reviewed in refs. (1-4)). Ligand binding to the periplasmic domains of the receptors generates conformational signals that are transmitted to the cytoplasmic domains that regulate an associated twocomponent phosphotransfer signal transduction system. Alterations in ligand concentration evoke a cellular response involving a change in direction of flagellar rotation. Following a transient response, cells return to pre-stimulus behavior, a process known as adaptation. Adaptation is in part mediated by reversible covalent modification of the chemoreceptors. Changes in receptor ligand concentration are accompanied by changes in the level of methylation at several specific glutamate residues in the cytoplasmic domains of the † A