Multiple covalent modifications of Trg, a sensory transducer of Escherichia coli

The sensory transducers of Escherichia coli are integral membrane proteins that mediate the tactic response of cells to chemical stimuli. Adaptation to environmental stimuli is correlated with methylation of the transducer proteins. Two transducer genes, tsr and tar, exhibit extensive homologies while no homology has been detected between a third transducer, trg, and those genes. The Tsr and Tar proteins have been shown to contain multiple sites for methylation as well as two sites for another modification that requires an active cheB gene product and is designated the CheB-dependent modification. In this study, covalent modifications of the Trg protein were characterized by analysis of tryptic peptides. We found that methylation occurred at several sites on the Trg protein and that the protein contained at least three sites for CheB-dependent modification, two of which were located on a tryptic peptide that contains both methionine and lysine. This tryptic peptide is analogous to the methionine- and lysine-containing methyl-accepting peptides isolated from the Tsr and Tar proteins and like those peptides may contain several methyl-accepting sites. We estimated the pKa of the group created by the CheB-dependent modification on the methionine- and lysine-containing peptide of Trg to be between pH 2.2 and 5.8. This result supports the idea that the CheB-dependent modification is an enzymatic deamidation of glutamine to glutamic acid.     

Cloning of trg, a gene for a sensory transducer in Escherichia coli.

Clones of trg, a gene which codes for a chemotactic transducer, were isolated linked to ColE1 and pBR322 vectors. Studies with the hybrid plasmids demonstrated unequivocally that trg is the structural gene for methyl-accepting chemotaxis protein III. The Trg protein was found to be structurally complex, electrophoresing as a series of seven bands on high-resolution sodium dodecyl sulfate-polyacrylamide gels. The multiplicity of bands is a function of the activity of cheR, which codes for a methyltransferase, and of cheB, which codes for a demethylase. It appears that Trg, a quantitatively minor transducer, resembles the two major transducer proteins, Tsr and Tar, in that all three are multiply methylated and also multiply modified in a second way which requires an active cheB gene. However, preliminary analysis of the Trg protein indicated that it is significantly less related structurally to the Tsr or Tar protein than those two transducers are to each other. This implies that the features of multiple methylation and cheB-dependent modification are likely to be critical for the common physiological functions in chemotactic excitation and adaptation performed by all three transducers.     

Desensitization by covalent modification of the chemoreceptor of Escherichia coli

Chemoreceptors in Escherichia coli were studied in situ in chemotactic mutants, deficient in the ability to modify the receptors, by using membrane vesicles prepared from the mutants. The affinity of the receptors for the ligands is related to the level of modification of the receptors. Unmodified serine receptor had a dissociation constant of 0.8 microM, while modified receptor had a dissociation constant that was at least 100-times higher. The results are discussed in relation to the two-state model of the chemoreceptor.     

Sites of deamidation and methylation in Tsr, a bacterial chemotaxis sensory transducer

The sensory transducer proteins in bacterial chemotaxis undergo two covalent modifications, deamidation and reversible methylation, in response to attractants and repellents. Oligonucleotide-directed mutagenesis was used to alter putative methylation and deamidation sites in one of the transducers to further define these sites and their role in chemotaxis. The mutations, in combination with peptide maps and Edman analysis, have clarified the sites of covalent modification in Tsr. Tsr contains six specific glutamates and glutamines that serve as methyl-accepting sites. An arginine-containing tryptic peptide (R1) has two sites, one at glutamate 493 and a newly located site at glutamate 502. A lysine-containing peptide (K1) has four methyl-accepting sites. Two of the lysine peptide sites are glutamates and can accept methyl groups without deamidation. The other two sites are glutamines and two methyl-accepting sites are created by two distinct deamidations. Both deamidations can occur on the same polypeptide chain. Single glutamate mutants have shown that one deamidation (at glutamine 311) proceeds rapidly, while the other deamidation (at glutamine 297) has a half-life of approximately 60 min under our experimental conditions.     

Thermosensing ability of Trg and Tap chemoreceptors in Escherichia coli.

The thermosensing ability of the Trg and Tap chemoreceptors in Escherichia coli was investigated after amplifying these receptors in a host strain lacking all four known chemoreceptors (Tar, Tsr, Trg, and Tap). Cells with an increased amount of either Trg or Tap showed mostly smooth swimming and no response to thermal stimuli. However, when the smooth-swimming bias of the cells was reduced by adding Trg- or Tap-mediated repellents, the cells showed clear changes in the swimming pattern upon temperature changes; Trg-containing cells showed tumbling at 23 degrees C but mostly smooth swimming at 32 degrees C, while Tap-containing cells showed smooth swimming at 20 degrees C but tumbling at 32 degrees C. These results indicate that although both Trg and Tap have the ability to sense thermal stimuli, Trg functions as a warm receptor, as reported previously for Tar and Tsr, while Tap functions as a cold receptor.     

Tuning the responsiveness of a sensory receptor via covalent modification.

Down-regulation or adaptation of receptors is an essential part of the chemotaxis mechanism to sense gradients. Using localized mutagenesis it is shown that the covalent modification of the receptors makes a slight change in the binding constant (factor of 2) which is far too small to explain the adaptation. The modification does, however, alter the signaling dramatically, an increasing tumbling signal being correlated with increased covalent modification. Responses in the two extreme cases, namely, completely unmodified and completely modified receptor, occur at attractant concentrations separated by 2 orders of magnitude. Amidation of the regulatory glutamate residues causes essentially the same signaling change as methylation. Thus, adaptation in chemotaxis is due to modulation of the receptor's signaling properties, not its affinity for the chemoeffector.     

Repellent response functions of the Trg and Tap chemoreceptors of Escherichia coli.

The chemoreceptors responsible for the repellent response of Escherichia coli to phenol were investigated. In the absence of all four known methyl-accepting chemoreceptors (Tar, Tsr, Trg, and Tap), cells showed no response to phenol. However, when Trg, which mediates the attractant response to ribose and galactose, was introduced via a plasmid, the cells acquired a repellent response to phenol. About 1 mM phenol induced a clear repellent response; this response was suppressed by 1 mM ribose. Thus, Trg mediates the repellent response to phenol. Mutant Trg proteins with altered sensing for ribose and galactose showed a normal response to phenol, indicating that the interaction site for phenol differs from that for the ribose- and galactose-binding proteins. Tap, which mediates the attractant response to dipeptides, mediated a weaker repellent response to phenol. Tsr, which mediates the attractant response to serine, mediated an even weaker response to phenol. Trg and Tap were also found to function as intracellular pH sensors. Upon a pH decrease, Trg mediated an attractant response, whereas Tap mediated a repellent response. These results indicate that all the receptors in E. coli have dual functions, mediating both attractant and repellent responses.     

Purification of receptor protein Trg by exploiting a property common to chemotactic transducers of Escherichia coli

The methyl-accepting chemotactic transducers of Escherichia coli were found to bind strongly to Cibacron blue-Sepharose. Among potential elutants tested, only S-adenosylmethionine at moderate concentrations and NaCl at concentrations greater than 1.5 M caused dissociation of these detergent-solubilized transmembrane proteins from the dye. Release by S-adenosylmethionine may be a generalized effect rather than the result of a specific binding site for that compound on transducers. A truncated trg gene was created that coded for the carboxyl-terminal three-fifths of the transducer, which constitutes the cytoplasmic domain common to all four transducers in E. coli. This domain bound to Cibacron blue-Sepharose and was eluted in a pattern similar to that exhibited by intact Trg, indicating that interaction with the dye occurred in this conserved domain. Adherence to Cibacron blue and elution by high salt formed the core of an efficient purification scheme, developed for Trg but applicable to all transducers in E. coli and perhaps to methyl-accepting chemotaxis proteins in other species. Determination of the amino acid sequence at the beginning of purified Trg confirmed that it contained a longer hydrophilic segment at its amino terminus than other transducers of E. coli. The initial methionine of Trg is neither cleaved nor modified, in contrast to the Tar transducer in which the amino terminus was found previously to be blocked. Circular dichroic measurements of purified Trg indicated that the secondary structural organization of the protein is predominantly alpha-helix.     

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

Chemotactic transducer proteins of Escherichia coli contain four or five methyl-accepting glutamates that are crucial for sensory adaptation and gradient sensing. Two residues arise from posttranslational deamidation of glutamines to yield methyl-accepting glutamates. We addressed the significance of this arrangement by creating two mutated trg genes: trg(5E), coding for a transducer in which all five modification sites were synthesized as glutamates, and trg(5Q), in which all five were glutamines. We found that the normal (3E,2Q) configuration was not an absolute requirement for synthesis, assembly, or stable maintenance of transducers. Both mutant proteins were methylated, although Trg(5Q) had a reduced number of methyl-accepting sites because two glutamines at adjacent residues were blocked for deamidation and thus could not become methyl-accepting glutamates. The glutamine-glutamate balance had striking effects on signaling state. Trg(5E) was in a strong counterclockwise signaling configuration, and Trg(5Q) was in a strong clockwise signaling induced by ligand binding, and alanines substituted at modification sites had an intermediate effect. Chemotactic migration by growing cells containing trg(5E) or trg(5Q) exhibited reduced effectiveness, probably reflecting perturbations of the counterclockwise/clockwise ratio caused by newly synthesized transducers not modified rapidly enough to produce a balanced signaling state during growth. These defects were evident for cells in which other transducers were not available to contribute to balanced signaling or were present at lower levels than the mutant proteins.     

A signal transducer for aerotaxis in Escherichia coli.

The newly discovered aer locus of Escherichia coli encodes a 506-residue protein with an N terminus that resembles the NifL aerosensor and a C terminus that resembles the flagellar signaling domain of methyl-accepting chemoreceptors. Deletion mutants lacking a functional Aer protein failed to congregate around air bubbles or follow oxygen gradients in soft agar plates. Membranes with overexpressed Aer protein also contained high levels of noncovalently associated flavin adenine dinucleotide (FAD). We propose that Aer is a flavoprotein that mediates positive aerotactic responses in E. coli. Aer may use its FAD prosthetic group as a cellular redox sensor to monitor environmental oxygen levels.     

Mutations specifically affecting ligand interaction of the Trg chemosensory transducer.

The Trg transducer mediates chemotactic response to galactose and ribose by interacting, respectively, with sugar-occupied galactose- and ribose-binding proteins. Adaptation is linked to methylation of specific glutamyl residues of the Trg protein. This study characterized two trg mutations that affect interaction with binding protein ligands but do not affect methylation or adaptation. The mutant phenotypes indicated that the steady-state activity of methyl-accepting sites is independent of ligand-binding activity. The mutation trg-8 changed arginine 85 to histidine, and trg-19 changed glycine 151 to aspartate. The locations of the mutational changes provided direct evidence for functioning of the amino-terminal domain of Trg in ligand recognition. Cross-inhibition of tactic sensitivity by the two Trg-linked attractants implies competition for a common site on Trg. However, the single amino acid substitution caused by trg-19 greatly reduced the response to galactose but left unperturbed the response to ribose. Thus Trg must recognize the two sugar-binding proteins at nonidentical sites, and the complementary sites on the respective binding proteins should differ. trg-8 mutants were substantially defective in the response to both galactose and ribose. An increase in cellular content of Trg-8 protein improved the response to galactose but not to ribose. It appears that Trg-8 protein is defective in the generation of the putative conformational change induced by ligand interaction. The asymmetry of the mutational defect implies that functional separation of interaction sites could persist beyond the initial stage of ligand binding.     

Importance of Multiple Methylation Sites in Escherichia coli Chemotaxis

Bacteria navigate within inhomogeneous environments by temporally comparing concentrations of chemoeffectors over the course of a few seconds and biasing their rate of reorientations accordingly, thereby drifting towards more favorable conditions. This navigation requires a short-term memory achieved through the sequential methylations and demethylations of several specific glutamate residues on the chemotaxis receptors, which progressively adjusts the receptors’ activity to track the levels of stimulation encountered by the cell with a delay. Such adaptation also tunes the receptors’ sensitivity according to the background ligand concentration, enabling the cells to respond to fractional rather than absolute concentration changes, i.e. to perform logarithmic sensing. Despite the adaptation system being principally well understood, the need for a specific number of methylation sites remains relatively unclear. Here we systematically substituted the four glutamate residues of the Tar receptor of Escherichia coli by non-methylated alanine, creating a set of 16 modified receptors with a varying number of available methylation sites and explored the effect of these substitutions on the performance of the chemotaxis system. Alanine substitutions were found to desensitize the receptors, similarly but to a lesser extent than glutamate methylation, and to affect the methylation and demethylation rates of the remaining sites in a site-specific manner. Each substitution reduces the dynamic range of chemotaxis, by one order of magnitude on average. The substitution of up to two sites could be partly compensated by the adaptation system, but the full set of methylation sites was necessary to achieve efficient logarithmic sensing.     

The methyl-accepting chemotaxis proteins of E. coli: a repellent-stimulated, covalent modification, distinct from methylation

The methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli are integral membrane proteins that have been shown to undergo reversible methylation in response to the addition of attractants. We have shown that a second, rapid modification of MCPI and MCPII occurs, which is repellent-stimulated. This modification, which is not methylation, was detected because it causes a decrease in mobility of the MCPs on 7.5% SDS-polyacrylamide gels with a high acrylamide to bisacrylamide ratio. We have designated this modification as the CheB-modification, as it is dependent on the CheB gene product. The CheB-modification causes a decrease in the isoelectric point of MCPII by one or two charge groups. The CheB-modification is not necessary for the methylation, nor does it preclude methylation of the MCPs. Both the CheB-modified form and the unmodified, unmethylated forms of the MCPs are stable to treatment with base, which results in the hydrolysis of the methylesters (demethylation) of the MCPs. The potential role of CheB-modification in chemotaxis is discussed.     

Proteolytic modification of Escherichia coli alkaline phosphatase

Proteolytic modification of the native alkaline phosphatase dimer is restricted to sites in the amino-terminal portion of the sequence. Complementing previous studies of the product of trypsin cleavage at the R-11, A-12 bond (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733; Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 260, 7557-7561) circular dichroic spectroscopy indicates that cleavage at this site results in a rearrangement of secondary structure and change in tertiary structure as monitored in the far and near UV regions, respectively. Under more vigorous reaction conditions, trypsin cleaves at the R-35, D-36 bond. The deletion of an additional 24 residues yields a species whose functional and structural properties are similar to the initial product of trypsin cleavage. Treatment of the enzyme with Protease V-8 results in cleavage at the E-9, N-10 bond. In contrast to the products of trypsin treatment, this truncated enzyme is similar to the native enzyme. These results indicate that the residues at the N-10 and R-11 positions play a unique role in maintaining the structural integrity and catalytic potency of the enzyme although this locus is distant from the enzyme active centers. These observations are discussed in terms of the three-dimensional structure of the enzyme.     

Adaptation in bacterial chemotaxis: CheB-dependent modification permits additional methylations of sensory transducer proteins

Sensory transduction in E. coli consists of two phases, excitation and adaptation, both of which involve the methyl-accepting chemotaxis proteins (MCPs). These molecules relay transmembrane signals and are reversibly methylated during adaptation of E. coli to environmental stimuli. Each MCP contains multiple sites of methylation, and we identified six of these sites in MCPI. Recently, a second covalent modification of MCPs has been identified, which is not methylation. This modification, designated CheB-dependent modification, is stimulated by repellents and causes a net increase in the negative charge of MCPI and MCPII by one or two charges. We demonstrate that one CheB modification occurs on the methyl-accepting methionine-and lysine-containing tryptic peptide in MCPI and MCPII, and the second CheB modification is on an arginine-containing tryptic peptide. The CheB modification allows three additional methyl groups to be incorporated into the methyl-accepting methionine-lysine peptide, while not actually creating all of these methylation sites. The two CheB modifications occur sequentially. A possible mechanism by which CheB modification permits additional methylations and the role of CheB modification in bacterial chemotaxis are discussed.     

Prolipoprotein modification and processing enzymes in Escherichia coli

Prolipoprotein signal peptidase, a unique endopeptidase which recognizes glycyl glyceride cysteine as a cleavage site, was characterized in an in vitro assay system using purified prolipoprotein as the substrate. This enzyme did not require phospholipids for its catalytic activity and was found to be localized in the inner cytoplasmic membrane of the Escherichia coli cell envelope. Globomycin inhibited this enzyme activity in vitro with a half-maximal inhibiting concentration of 0.76 nM. Nonionic detergent, such as Nikkol or Triton X-100, was required for the in vitro activity. The optimum pH and reaction temperature of prolipoprotein signal peptidase were pH 7.9 and 37-45 degrees C, respectively. Phosphatidylglycerol:prolipoprotein glyceryl transferase (glyceryl transferase) activity was measured using [2-3H]glycerol-labeled JE5505 cell envelope and [35S]cysteine-labeled MM18 cell envelope as the donor and acceptor of glyceryl moiety, respectively. 3H and 35S dual-labeled glyceryl cysteine was identified in the product of this enzymatic reaction. The optimal pH and reaction temperature for glyceryl transferase were pH 7.8 and 37 degrees C, respectively.