1887

Abstract

In the chemotaxis of , receptor methylation is the key process of adaptation. The methyltransferase CheR binds to the carboxy-terminal NWETF sequence of major chemoreceptors. The substitution of Ala for Trp of this sequence (W550A) of the aspartate chemoreceptor (Tar) abolishes its CheR-binding ability. In this study, six independent intragenic suppressors of the mutation were isolated. They were divided into two classes. Tar carrying the class I suppressors (G278A-L488M, T334A, G278A, G278C and A398T) showed signal biases toward tumbling, corresponding to increased activities of the receptor-associated histidine kinase CheA. These suppressors further reduced the unstimulated methylation level of Tar-W550A, but allowed slight but significant stimulation of methylation by aspartate. Some other CheA-activating mutations were also found to serve as class I suppressors. These results suggest that the class I suppressors compensate for the signal bias of Tar-W550A caused by its low methylation level and that the NWETF sequence is required primarily to maintain an appropriate level of methylation by increasing the local concentration of CheR around the receptor. The class II suppressor was a mutation in the termination codon (Op554W) resulting in the addition of 11 residues containing an xWxxF motif. This revertant Tar supported chemotaxis and was methylated almost as effectively as wild-type Tar. This effect was reversed by introducing a mutation in the xWxxF motif. These results reinforce the importance of the xWxxF motif and suggest that the motif does not have to be located at the extreme carboxy terminus.

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2002-10-01
2020-01-28
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References

  1. Alon U, Surette M. G, Barkai N., Leibler S. 1999; Robustness in bacterial chemotaxis. Nature397:168–171
    [Google Scholar]
  2. Anand G. S, Goudreau P. N., Stock A. M. 1998; Activation of methyltransferase CheB: evidence of a dual role for the regulatory domain. Biochemistry37:14038–14047
    [Google Scholar]
  3. Barak R., Eisenbach M. 1992; Correlation between phosphorylation of the chemotaxis protein CheY and its activity at the flagellar motor. Biochemistry31:1821–1826
    [Google Scholar]
  4. Barkai N., Leibler S. 1997; Robustness in simple biochemical networks. Nature387:913–917
    [Google Scholar]
  5. Barnakov A. N, Barnakova L. A., Hazelbauer G. L. 1999; Efficient adaptational demethylation of chemoreceptors requires the same enzyme-docking site as efficient methylation. Proc Natl Acad Sci USA96:10667–10672
    [Google Scholar]
  6. Barnakov A. N, Barnakova L. A., Hazelbauer G. L. 2001; Location of the receptor-interaction site on CheB, the methylesterase response regulator of bacterial chemotaxis. J Biol Chem276:32984–32989
    [Google Scholar]
  7. Bass R. B., Falke J. J. 1998; Detection of a conserved α-helix in the kinase-docking region of the aspartate receptor by cysteine and disulfide scanning. J Biol Chem273:25006–25014
    [Google Scholar]
  8. Bass R. B., Falke J. J. 1999; The aspartate receptor cytoplasmic domain: in situ chemical analysis of structure, mechanism and dynamics. Structure7:829–840
    [Google Scholar]
  9. Blair D. F. 1995; How bacteria sense and swim. Annu Rev Microbiol49:489–522
    [Google Scholar]
  10. Borkovich K. A, Kaplan N, Hess J. F., Simon M. I. 1989; Transmembrane signal transduction in bacterial chemotaxis involves ligand-dependent activation of phosphate group transfer. Proc Natl Acad Sci USA86:1208–1212
    [Google Scholar]
  11. Borkovich K. A, Alex L. A., Simon M. I. 1992; Attenuation of sensory receptor signaling by covalent modification. Proc Natl Acad Sci USA89:6756–6760
    [Google Scholar]
  12. Boyd A., Simon M. I. 1980; Multiple electrophoretic forms of methyl-accepting chemotaxis proteins generated by stimulus-elicited methylation in Escherichia coli . J Bacteriol143:809–815
    [Google Scholar]
  13. Bulter S. L., Falke J. J. 1998; Cysteine and disulfide scanning reveals two amphiphilic helices in the linker region of the aspartate chemoreceptor. Biochemistry37:10746–10756
    [Google Scholar]
  14. Chelsky D., Dahlquist F. W. 1980; Structural studies of methyl-accepting chemotaxis proteins of Escherichia coli : evidence for multiple methylation sites. Proc Natl Acad Sci USA77:2434–2438
    [Google Scholar]
  15. Danielson M. A, Bass R. B., Falke J. J. 1997; Cysteine and disulfide scanning reveals a regulatory α-helix in the cytoplasmic domain of the aspartate receptor. J Biol Chem272:32878–32888
    [Google Scholar]
  16. DeFranco A. L., Koshland D. E. Jr. 1980; Multiple methylation in processing of sensory signals during bacterial chemotaxis. Proc Natl Acad Sci USA77:2429–2433
    [Google Scholar]
  17. Djordjevic S., Stock A. M. 1998; Chemotaxis receptor recognition by protein methyltransferase CheR. Nature Struct Biol5:446–450
    [Google Scholar]
  18. Engström P., Hazelbauer G. L. 1980; Multiple methylation of methyl-accepting chemotaxis proteins during adaptation of E. coli to chemical stimuli. Cell20:165–171
    [Google Scholar]
  19. Falke J. J, Bass R. B, Butler S. L, Chervitz S. A., Danielson M. A. 1997; The two component signaling pathway of bacterial chemotaxis: a molecular view of signal transduction by receptors, kinases, and adaptation enzymes. Annu Rev Cell Dev Biol13:457–512
    [Google Scholar]
  20. Feng X, Baumgartner J. W., Hazelbauer G. L. 1997; High- and low-abundance chemoreceptors in Escherichia coli : differential activities associated with closely related cytoplasmic domains. J Bacteriol179:6714–6720
    [Google Scholar]
  21. Feng X, Lilly A. A., Hazelbauer G. L. 1999; Enhanced function conferred on low abundance chemoreceptor Trg by a methyltransferase-docking site. J Bacteriol181:3164–3171
    [Google Scholar]
  22. Gegner J. A, Graham D. R, Roth A. F., Dahlquist F. W. 1992; Assembly of an MCP, CheW, and kinase CheA complex in the bacterial chemotaxis signal transduction pathway. Cell70:975–982
    [Google Scholar]
  23. Hazelbauer G. L., Engström P. 1980; Parallel pathways for transduction of chemotactic signals in Escherichia coli . Nature283:98–100
    [Google Scholar]
  24. Hess J. F, Oosawa K, Kaplan N., Simon M. I. 1988; Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis. Cell53:79–87
    [Google Scholar]
  25. Iwama T, Homma M., Kawagishi I. 1997; Uncoupling of ligand-binding affinity of the bacterial serine chemoreceptor from methylation- and temperature-modulated signaling states. J Biol Chem272:13810–13815
    [Google Scholar]
  26. Kim K. K, Yokota H., Kim S.-H. 1999; Four-helical-bundle structure of the cytoplasmic domain of a serine chemotaxis receptor. Nature400:787–792
    [Google Scholar]
  27. Kondoh H, Ball C. B., Adler J. 1979; Identification of a methyl-accepting chemotaxis protein for the ribose and galactose chemoreceptors of Escherichia coli . Proc Natl Acad Sci USA76:260–264
    [Google Scholar]
  28. Landt O, Grunert H. P., Hahn U. 1990; A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene96:125–128
    [Google Scholar]
  29. Le Moual H., Koshland D. E. Jr. 1997; Methylation of the Escherichia coli chemotaxis receptors: intra- and interdimer mechanisms. Biochemistry36:13441–13448
    [Google Scholar]
  30. Li J, Li G., Weis R. M. 1997; The serine receptor from Escherichia coli is methylated through an inter-dimer process. Biochemistry36:11851–11857
    [Google Scholar]
  31. Liu Y, Levit M, Lurz R, Surette M. G., Stock J. B. 1997; Receptor-mediated protein kinase activation and the mechanism of transmembrane signaling in bacterial chemotaxis. EMBO J16:7231–7240
    [Google Scholar]
  32. Lupas A., Stock J. 1989; Phosphorylation of an N-terminal regulatory domain activates the CheB methylesterase in bacterial chemotaxis. J Biol Chem264:17337–17342
    [Google Scholar]
  33. Lybarger S. R., Maddock J. R. 2000; Differences in the polar clustering of the high- and low-abundance chemoreceptors of Escherichia coli . Proc Natl Acad Sci USA97:8057–8062
    [Google Scholar]
  34. Maddock J. R., Shapiro L. 1993; Polar location of the chemoreceptor complex in the Escherichia coli cell. Science259:1717–1723
    [Google Scholar]
  35. Manson M. D. 1992; Bacterial motility and chemotaxis. Adv Microb Physiol33:277–346
    [Google Scholar]
  36. Milligan D. L., Koshland D. E. Jr. 1988; Site-directed cross linking: establishing the dimeric structure of the aspartate receptor of bacterial chemotaxis. J Biol Chem263:6268–6275
    [Google Scholar]
  37. Nishiyama S, Nara T, Imae Y, Homma M., Kawagishi I. 1997; Thermosensing properties of mutant aspartate receptors having methyl-accepting sites substituted multiply or singly with alanine. J Bacteriol179:6573–6580
    [Google Scholar]
  38. Nishiyama S, Umemura T, Nara T, Homma M., Kawagishi I. 1999; Conversion of a bacterial warm sensor to a cold sensor by methylation of a single residue in the presence of an attractant. Mol Microbiol32:357–365
    [Google Scholar]
  39. Okumura H, Nishiyama S, Sasaki A, Homma M., Kawagishi I. 1998; Chemotactic adaptation is altered by changes in the C-terminal sequence conserved among the major methyl accepting chemoreceptors. J Bacteriol180:1862–1868
    [Google Scholar]
  40. Oosawa K., Simon M. I. 1986; Analysis of mutations in the transmembrane region of aspartate chemoreceptor in Escherichia coli . Proc Natl Acad Sci USA83:6930–6934
    [Google Scholar]
  41. Parkinson J. S. 1993; Signal transduction schemes of bacteria. Cell73:857–871
    [Google Scholar]
  42. Schuster S. C, Swanson R. V, Alex L. A, Bourret R. B., Simon M. I. 1993; Assembly and function of a quaternary signal transduction complex monitored by surface plasmon resonance. Nature365:343–347
    [Google Scholar]
  43. Shiomi D, Okumura H, Homma M., Kawagishi I. 2000; The aspartate chemoreceptor Tar is effectively methylated by binding to the methyltransferase mainly through hydrophobic interaction. Mol Microbiol36:132–140
    [Google Scholar]
  44. Shiomi D, Zhulin I. B, Homma M., Kawagishi I. 2002; Dual recognition of the bacterial chemoreceptor by chemotaxis-specific domains of the CheR methyltransferase. J Biol Chem in press
    [Google Scholar]
  45. Simms S. A, Stock A. M., Stock J. B. 1987; Purification and characterization of S -adenosylmethionine: glutamyl methyltransferase that modifies membrane chemoreceptor proteins in bacteria. J Biol Chem262:8537–8543
    [Google Scholar]
  46. Springer M. S, Goy M. F., Adler J. 1977; Sensory transduction in Escherichia coli : two complementary pathways of information processing that involve methylated proteins. Proc Natl Acad Sci USA74:3312–3316
    [Google Scholar]
  47. Springer W. R., Koshland D. E. Jr. 1977; Identification of a protein methyltransferase as the cheR gene product in the bacterial sensing system. Proc Natl Acad Sci USA74:533–537
    [Google Scholar]
  48. Stock A. M, Wylie D. C, Mottonen J. M, Lupas A. M, Ninfa E. G, Ninfa A. J, Schutt C. E., Stock J. B. 1988; Phospho-proteins involved in bacterial signal transduction. Cold Spring Harbor Symp Quant Biol53:49–57
    [Google Scholar]
  49. Stock J. B., Surette M. G. others 1996; Chemotaxis. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, 2nd edn. pp1103–1129 Edited by Neidhardt F. C.. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  50. Tatsuno I, Homma M, Oosawa K., Kawagishi I. 1996; Signaling by the Escherichia coli aspartate chemoreceptor Tar with a single cytoplasmic domain per dimer. Science274:423–425
    [Google Scholar]
  51. Trammell M. A., Falke J. J. 1999; Identification of a site critical for kinase regulation on the central processing unit (CPU) helix of the aspartate receptor. Biochemistry38:329–336
    [Google Scholar]
  52. Umemura T, Tatsuno I, Shibasaki M, Homma M., Kawagishi I. 1998; Intersubunit interaction between transmembrane helices of the bacterial aspartate chemoreceptor homodimer. J Biol Chem273:30110–30115
    [Google Scholar]
  53. Wang E. A., Koshland D. E. Jr. 1980; Receptor structure in the bacterial sensing system. Proc Natl Acad Sci USA77:7157–7161
    [Google Scholar]
  54. Weerasuriya S, Schneider B. M., Manson M. D. 1998; Chimeric chemoreceptors in Escherichia coli : signaling and methylation properties of Tar-Tap and Tap-Tar hybrids. J Bacteriol180:914–920
    [Google Scholar]
  55. Welch M, Oosawa K, Aizawa S.-I., Eisenbach M. 1993; Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria. Proc Natl Acad Sci USA90:8787–8791
    [Google Scholar]
  56. Wolfe A. J., Berg H. C. 1989; Migration of bacteria in semisolid agar. Proc Natl Acad Sci USA86:6973–6977
    [Google Scholar]
  57. Wolfe A. J, Conley M. P, Kramer T. J., Berg H. C. 1987; Reconstitution of signaling in bacterial chemotaxis. J Bacteriol169:1878–1885
    [Google Scholar]
  58. Wu J, Li J, Li G, Long D. G., Weis R. M. 1996; The receptor binding site for the methyltransferase of bacterial chemotaxis is distinct from the sites of methylation. Biochemistry35:4984–4993
    [Google Scholar]
  59. Wylie D, Stock A. M, Wong C.-Y., Stock J. 1988; Sensory transduction in bacterial chemotaxis involves phosphotransfer between Che proteins. Biochem Biophys Res Commun151:891–896
    [Google Scholar]
  60. Yamamoto K, Macnab R. M., Imae Y. 1990; Repellent response functions of the Trg and Tap chemoreceptors of Escherichia coli . J Bacteriol172:383–388
    [Google Scholar]
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