The methyl-accepting chemotaxis proteins of Escherichia coli. Identification of the multiple methylation sites on methyl-accepting chemotaxis protein I

The methyl-accepting chemotaxis proteins (MCPs) are integral membrane proteins that undergo reversible methylation during adaptation of bacterial cells to environmental attractants and repellents. The numerous methylated forms of each MCP are seen as a pattern of multiple bands on polyacrylamide gels. We have characterized the methylation sites in MCPI by analyzing methyl-accepting tryptic peptides. At least two different tryptic peptides accept methyl esters; one methyl-accepting peptide contains methionine and lysine and may be methylated a maximum of four times. The second methyl-accepting tryptic peptide contains arginine and may be methylated twice. Base-catalyzed demethylations of tryptic peptides and analysis of the charge differences between the different methylated forms of MCPI show that MCPI molecules may be methylated a total of six times. The two methyl esters on the methyl-accepting arginine peptide appear to be preferentially methylated in most of the forms of MCPI in attractant-stimulated cells. The ability to acquire six methylations on MCPI allows the bacterial cells to adapt to a broad range of attractant and repellent concentrations.     

Glycerol and ethylene glycol: members of a new class of repellents of Escherichia coli chemotaxis

By using the chemical-in-plug method, we found that glycerol and ethylene glycol caused negative chemotaxis in wild-type cells of Escherichia coli; the threshold concentration was about 10(-3) M for both chemicals. As with other known repellents, the addition of glycerol or ethylene glycol induced a brief tumble response in wild-type cells but not in generally nonchemotactic mutants. Experiments with mutants defective in various methyl-accepting chemotaxis proteins (MCPs) revealed that the presence of any one of three kinds of MCPs (MCP I, MCP II, or MCP III) was necessary to give a tumble response to these repellents. Consistently, it was found that the methylation-demethylation system of MCPs was involved in the adaptation of the cells to these repellents. The effect of glycerol or ethylene glycol was not enhanced by lowering the pH of the medium, and glycerol did not alter the membrane potential of the cells. All of these results suggest that glycerol and ethylene glycol are members of a new class of repellents which produce a tumble response in the cells by perturbing the MCPs in the membrane.     

Multiple electrophoretic forms of methyl-accepting chemotaxis proteins generated by stimulus-elicited methylation in Escherichia coli.

The tsr and tar genetic loci of Escherichia coli determine the presence in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of methyl-accepting chemotaxis proteins (MCPs) I and II, respectively, each of which consists of a distinct group of multiple bands. Synthesis of the tsr and tar products was directed in ultraviolet-irradiated bacteria by lambda transducing phages. The addition of appropriate chemotactic stimuli to these cells resulted in the appearance of additional, faster migrating electrophoretic forms of the Tsr and Tar polypeptides which disappeared upon removal of the stimulus. The stimulus-elicited forms comigrated with component bands of the corresponding MCPs. These results indicate that methylation itself caused shifts in electrophoretic mobility and hence led to the observed MCP band patterns. The number of Tsr species suggested that there were at least three methylated sites on the Tsr polypeptide. The conclusion that methylation generates multiplicity was supported by the results of experiments in which the tsr product was synthesized in mutant bacteria defective in specific chemotaxis functions concerned with methylation or demethylation of MCPs. Thus, the presence of a cheX defect blocked the stimulus-elicited appearance of faster migrating forms of the tsr product; conversely, the presence of a cheB defect resulted in a pronounced shift toward these forms in the absence of a chemotactic stimulus.     

Genetics of methyl-accepting chemotaxis proteins in Escherichia coli: organization of the tar region.

The tar locus of Escherichia coli specifies one of the major species of methyl-accepting proteins involved in the chemotactic behavior of this organism. The physical and genetic organization of the tar region was investigated with a series of specialized lambda transducing phages and plasmid clones. The tar gene was mapped at the promoter-proximal end of an operon containing five other chemotaxis-related loci. Four of those genes (cheR, cheB, cheY and cheZ) are required for all chemotactic responses; consequently, polar mutations in the tar gene resulted in a generally nonchemotactic phenotype. The fifth gene, tap, was mapped between the tar and cheR loci and specified the production of a 65-kilodalton methyl-accepting protein. Unlike the tar locus, which is required for chemotaxis to aspartate and maltose, mutants lacking only the tap function had no obvious defects in chemotactic ability. Genetic and physical maps of the tar-tap region were constructed with Mu d1 (Apr lac) insertion mutations, whose polar properties conferred a phenotype suitable for deletion mapping studies. Restriction endonuclease analyses of phage and plasmid clones indicated that all of the genetic coding capacity in the tar region is now accounted for.     

Genetics of methyl-accepting chemotaxis proteins in Escherichia coli: null phenotypes of the tar and tap genes.

The tar and tap genes are located adjacent to one another in an operon of chemotaxis-related functions. They encode methyl-accepting chemotaxis proteins implicated in tactic responses to aspartate and maltose stimuli. The functional roles of these two gene products were investigated by isolating and characterizing nonpolar, single-gene deletion mutants at each locus. Deletions were obtained by selecting for loss or a defective Mu d1 prophage inserted in either the tar or tap gene. The extent of the tar deletions was determined by genetic mapping with Southern hybridization. Representative deletion mutants were surveyed for chemotactic responses on semisolid agar and by temporal stimulation in a tethered cell assay to assess flagellar rotational responses to chemoeffector compounds. The tar deletion strains exhibited complete loss of aspartate and maltose responses, whereas the tap deletion strains displayed a wild-type phenotype under all conditions tested. These findings indicate that the tap function is unable to promote chemotactic responses to aspartate and maltose, and its role in chemotaxis remains unclear.     

Proteins antigenically related to methyl-accepting chemotaxis proteins of Escherichia coli detected in a wide range of bacterial species.

The four methyl-accepting chemotaxis proteins of Escherichia coli, often called transducers, are transmembrane receptor proteins that exhibit substantial identity among the sequences of their cytoplasmic domains. Thus, antiserum raised to one of these proteins recognizes the others and might be expected to recognize related proteins in other bacteria. We used antiserum raised to the transducer Trg in immunoblot experiments to probe a wide range of bacterial species for the presence of antigenically related proteins. Such proteins were detected in over 20 different species, representing 6 of the 11 eubacterial phyla defined by analysis of rRNA sequences as well as one archaebacterial group. Species containing proteins antigenically related to the transducers of E. coli included members of all four subdivisions of the phylum in which E. coli is placed, members of four of the six subdivisions of spirochetes, and two gliding bacteria. These observations provide substantial support for the notion that methyl-accepting taxis proteins are widely distributed among the diversity of bacterial species.     

Genetics of methyl-accepting chemotaxis proteins in Escherichia coli: cheD mutations affect the structure and function of the Tsr transducer

The tsr gene specifies a methyl-accepting membrane protein involved in chemotaxis to serine and several repellent compounds. We have characterized a special class of tsr mutations designated cheD which alter the signaling properties of the Tsr transducer. Unlike tsr null mutants, cheD strains are generally nonchemotactic, dominant in complementation tests, and exhibit a pronounced counterclockwise bias in flagellar rotation. Several lines of evidence showed that cheD mutations were alleles of the tsr gene. First, cheD mutations were mapped into the same deletion segments as conventional tsr mutations. Second, restriction site analysis of the transducing phage deletions used to construct the genetic map demonstrated that the endpoints of the deletion segments fell within the tsr coding sequence. Third, a number of the cheD mutants synthesized Tsr proteins with slight changes in electrophoretic mobility, consistent with alterations in Tsr primary structure. These mutant proteins were able to undergo posttranslational deamidation and methylation reactions in the same manner as wild-type Tsr protein; however, the steady-state level of Tsr methylation in cheD strains was very high. The methylation state of the Tar protein, another species of methyl-accepting protein in Escherichia coli, was also higher than normal in cheD strains, suggesting that the aberrant Tsr transducer in cheD mutants has a generalized effect on the sensory adaptation system of the cell. These properties are consistent with the notion that the Tsr protein of cheD mutants is locked in an excitatory signaling mode that both activates the sensory adaptation system and drowns out chemotactic signals generated by other transducer species. Further study of cheD mutations thus promises to reveal valuable information about the functional architecture of the Tsr protein and how this transducer controls flagellar behavior.     

Biosynthesis of ethylene glycol in Escherichia coli

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose?→?d-xylonate?→?2-dehydro-3-deoxy-d-pentonate?→?glycoaldehyde?→?EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose?→?d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g?L^?1 of EG from 40.0 g?L^?1 of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde?→?glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.     

Influence of attractants and repellents on methyl group turnover on methyl-accepting chemotaxis proteins of Bacillus subtilis and role of CheW.

The ability of attractants and repellents to affect the turnover of methyl groups on the methyl-accepting chemotaxis proteins (MCPs) was examined for Bacillus subtilis. Attractants were found to cause an increase in the turnover of methyl groups esterified to the MCPs, while repellents caused a decrease. These reactions do not require CheW. However, a cheW null mutant exhibits enhanced turnover in unstimulated cells. Assuming that the turnover of methyl groups on the MCPs reflects a change in the activity of CheA, these results suggest that the activation of CheA via chemoeffector binding at the receptor does not require CheW.     

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. II. Effect of cheB and cheZ mutations on the methylation of methyl-accepting chemotaxis protein of Escherichia coli.

Radioactive proteins from chemotactic mutants of Escherichia coli with continuous tumbling phenotype (cheB and cheZ) and their otherwise isogenic parent were compared by two-dimensional gel electrophoresis. The system was capable of separating non-methylated methyl-accepting chemotaxis protein (MCP) from its methylated equivalent. The analysis of proteins from the envelope fraction of the bacteria showed that the cheB mutants contained a larger portion of methylated MCP than did the parent. However, the change of MCP methylation level was small, if any, in cheZ strains. The results suggest that the product of cheB gene and the product of cheZ gene are not functional complementary. The product of cheB gene functions in controlling the level of methylation at the stationary state of the organisms. In addition to known MCP species, a new MCP of about 43,000 daltons was found. This MCP appears to be involved in transducing signals of some sugars.     

Localization of proteins controlling motility and chemotaxis in Escherichia coli.

Flagellar proteins controlling motility and chemotaxis in Escherichia coli were selectively labeled in vivo with [35S]methionine. This distribution of these proteins in subcellular fractions was examined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography. The motA, motB, cheM, and cheD gene products were found to be confined exclusively to the inner cytoplasmic membrane fraction, whereas the cheY, cheW, and cheA (66,000 daltons) polypeptides appeared only in the soluble cytoplasmic fraction. The cheB, cheX, cheZ, and cheA (76,000 daltons) proteins, however, were distributed in both the cytoplasm and the inner membrane fractions. The hag gene product (flagellin) was the only flagellar protein examined that copurified with the outer lipopolysaccharide membrane. Differences in the intracellular locations of the che and mot gene prodcuts presumably reflect the functional attributes of these components.     

Chemotactic transducer proteins of Escherichia coli exhibit homology with methyl-accepting proteins from distantly related bacteria.

Transducers are transmembrane, methyl-accepting proteins central to the chemotactic systems of the enteric bacteria Escherichia coli and Salmonella typhimurium. Methyl-accepting proteins have been reported in a number of species in addition to these enteric bacteria. Those species include Bacillus subtilis and Spirochaeta aurantia, representatives of groups that diverged from ancestral enteric bacteria and from each other very early in bacterial evolution. An antiserum that reacts with all transducers of E. coli precipitated specifically methyl-accepting proteins from B. subtilis and S. aurantia, indicating that these proteins share antigenic determinants with transducers of E. coli. In addition, analysis of tryptic peptides by high-pressure liquid chromatography revealed similarities in the regions of methyl-accepting sites for proteins from all three species. These observations imply that structural features have been preserved in the three species from transducers contained in a common ancestor of eubacteria. It is thus reasonable to predict that other flagellated, chemotactic bacteria will be found to contain methyl-accepting proteins homologous to transducers of enteric bacteria.     

Experimental evolution of a metabolic pathway for ethylene glycol utilization by Escherichia coli.

Spontaneous mutants of Escherichia coli able to grow on ethylene glycol as a sole source of carbon and energy were obtained from mutants that could grow on propylene glycol. Attempts to obtain ethylene glycol-utilizing mutants from wild-type E. coli were unsuccessful. The two major characteristics of the ethylene glycol-utilizing mutants were (i) increased activities of propanediol oxidoreductase, an enzyme present in the parental strain (a propylene glycol-positive strain), which also converted ethylene glycol into glycolaldehyde; and (ii) constitutive synthesis of high activities of glycolaldehyde dehydrogenase, which converted glycolaldehyde to glycolate. Glycolate was metabolized via the glycolate pathway, which was present in the wild-type cells; this was indicated by the induction in ethylene glycol-grown cells of glycolate oxidase, the first enzyme in the pathway. Glycolaldehyde dehydrogenase was partially characterized as an enzyme of this new metabolic pathway in E. coli, and glycolate was identified as the product of the reaction. This enzyme used NAD and NADP as coenzymes, although the NADP-dependent activity was about 10 times lower than the NAD-dependent activity. Uptake of [14C]ethylene glycol was dependent on the presence of the enzymes capable of metabolism of ethylene glycol. Glycolaldehyde and glycolate were identified as intermediate metabolites in the pathway.     

Methanol formation in vivo from methylated chemotaxis proteins in Escherichia coli.

Chemotactically wild type Escherichia coli were incubated with L-[methyl-3H]methionine to label the methyl groups of their methyl-accepting chemotaxis proteins. Cells were then treated to specifically demethylate these proteins. We have identified the end product of this demethylation as [3H]methanol in the cell-free medium from treated cells.     

Multiple forms of methyl-accepting chemotaxis proteins distinguished by a factor in addition to multiple methylation.

Methyl-accepting chemotaxis proteins are central to both the excitation and adaptation phases of chemotactic behavior. Using null mutations in the genes coding for the two major methyl-accepting proteins (tsr and tar), we identified the gene products among the membrane proteins of Escherichia coli visualized on one- and two-dimensional gels. On two-dimensional gels, both the tsr and the tar proteins appeared as a group of multiple spots arranged in two to four diagonal arrays. The multiplicity of forms could not be completely explained by the previously documented heterogeneity of the methylated proteins resulting from different numbers of methylated glutamyl residues per polypeptide chain. We suggest that there is at least one other way besides extent of methylation in which the polypeptides of a methylated protein can differ.     

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.     

Evidence for methyl group transfer between the methyl-accepting chemotaxis proteins in Bacillus subtilis.

We present evidence for methyl (as methyl or methoxy) transfer from the methyl-accepting chemotaxis proteins H1 and possibly H3 of Bacillus subtilis to the methyl-accepting chemotaxis protein H2. This methyl transfer, which has been observed in vitro (D. J. Goldman and G. W. Ordal, Biochemistry 23:2600-2606, 1984), was strongly stimulated by the chemoattractant aspartate and thus may play an important role in the sensory processing system of this organism. Although radiolabeling of H1 and H3 began at once after the addition of [3H]methionine, radiolabeling of H2 showed a lag. Furthermore, the addition of excess nonradioactive methionine caused immediate exponential delabeling of H1 and H3 while labeling of H2 continued to increase. Methylation of H2 required the chemotactic methyltransferase, probably to first methylate H1 and H3. Aspartate caused increased labeling of H2 and strongly decreased labeling of H1 and H3 after the addition of nonradioactive methionine. Without the addition of nonradioactive methionine, aspartate caused demethylation of H1 and to a lesser extent H3, with an approximately equal increase of methylation of H2.     

Identification of the polyamine induced proteins in Escherichia coli.

The polyamine (PA)-induced proteins were identified by SDS-PAGE, and by two dimensional gel analysis in Escherichia coli strains. A large number of the PA-induced proteins were acidic. The molecular weights of the most highly induced proteins were 40 and 82 kDa proteins in the wild type and PA-auxotrophic mutant, respectively. Although a part of the PA-induced proteins were induced both in the wild type and PA auxotrophic mutant, most of them seem to be induced either in the wild type or mutant. These features may provide a foundation for evaluating the role of the PA-induced proteins relative to the physiology and environmental stress of Escherichia coli.