Advertisement

Molecular and General Genetics MGG

, Volume 243, Issue 4, pp 409–416 | Cite as

The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene

  • Martine Crasnier
  • Valérie Dumay
  • Antoine Danchin
Original Paper

Abstract

In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIAGlc, a component of the phosphotransferase system for glucose transport. In strains deficient in EnzymeIIAGlc, CAMP levels are very low. Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIAGlc. In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIAGlc were obtained. The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end. Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIAGlc. In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished. This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose. Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain.

Key words

Adenylate cyclase Phosphotransferase system cAMP synthesis Glucose effects Deletion 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Aiba H, Mori K, Tanaka M, Ooi T, Roy A, Danchin A (1984) The complete nucleotide sequence of the adenylate cyclase gene of Escherichia coli. Nucleic Acids Res 12:9427–9440PubMedGoogle Scholar
  2. Albertini AM, Hofer M, Calos MP, Miller JH (1982) On the formation of spontaneous deletions: The importance of short sequence homologies in the generation of large deletions. Cell 29:319–328PubMedCrossRefGoogle Scholar
  3. Auerswald EA, Ludwig G, Schaller H (1980) Structural analysis of Tn5. Cold Spring Harbor Symp Quant Biol 45:107–113Google Scholar
  4. Botsford JL, Harman JG (1992) Cyclic AMP in prokaryotes. Microbiol Rev 56:100–122PubMedGoogle Scholar
  5. Close TJ, Zaitlin D, Kado C (1984) Design and development of amplifiable broad-host-range cloning vectors: analysis of the vir region of Agrobacterium tumefaciens plasmid pTIC58. Plasmid 12:111–118PubMedCrossRefGoogle Scholar
  6. Crasnier M, Danchin A (1990) Characterization of Escherichia coli adenylate cyclase mutants with modified regulation. J Gen Microbiol 136:1825–1831PubMedGoogle Scholar
  7. Crenon I, Ullmann A (1984) The role of cAMP excretion in the regulation of enzyme synthesis in Escherichia coli. FEMS Lett 22:47–51CrossRefGoogle Scholar
  8. Danchin A (1993) Phylogeny of adenylyl cyclases. Adv Second Messenger Phosphoprotein Res 27:109–162PubMedGoogle Scholar
  9. Den Blaauwen JL, Postma PW (1985) Regulation of cyclic AMP synthesis by Enzyme IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system in crp strains of Salmonella typhimurium. J Bacteriol 164:477–478Google Scholar
  10. Epstein W, Rothman-Denes LB, Hesse J (1975) Adenosine 3′:5′-cyclic monophosphate as mediator of catabolite repression in Escherichia coli. Proc Natl Acad Sci USA 72:2300–2304PubMedCrossRefGoogle Scholar
  11. Feucht BU, Saier MH (1980) Fine control of adenylate cyclase by the phosphoenolpyruvate:sugar phosphotransferase systems in Escherichia coli and Salmonella typhimurium. J Bacteriol 141:603–610PubMedGoogle Scholar
  12. Fink GR, Klopotowski R, Ames BN (1967) Histidine regulatory mutants in Salmonella typhimurium. IV. A positive selection for polar histidine mutants. J Mol Biol 30:81–95PubMedCrossRefGoogle Scholar
  13. Guidi-Rontani C, Danchin A, Ullmann A (1981) Isolation and characterization of an Escherichia coli mutant affected in the regulation of adenylate cyclase. J Bacteriol 148:753–761PubMedGoogle Scholar
  14. Holland MM, Leib TK, Gerlt JA (1988) Isolation and characterization of a small catalytic domain released from the adenylate cyclase from Escherichia coli by digestion with trypsin. J Biol Chem 263:14661–14668PubMedGoogle Scholar
  15. Joseph E, Danchin A, Ullmann A (1981) Regulation of galactose operon expression: glucose effects and role of cyclic adenosine 3′,5′-monophosphate. J Bacteriol 146:149–154PubMedGoogle Scholar
  16. Kolb A, Busby S, Buc H, Garges S, Adhya S (1993) Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem 62:749–795PubMedCrossRefGoogle Scholar
  17. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophase T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  18. Leib TK, Gerlt JA (1983) Evidence for a small catalytic domain in the adenylate cyclase from Salmonella typhimurium. J Biol Chem 258:12982–12987PubMedGoogle Scholar
  19. Levitt M (1978) Conformational preferences of amino acids in globular proteins. Biochemistry 17:4277–4285PubMedCrossRefGoogle Scholar
  20. Levy S, Zeng GQ, Danchin A (1990) cAMP synthesis in strains bearing well characterized deletions in the central pts genes of Escherichia coli. Gene 86:27–33PubMedCrossRefGoogle Scholar
  21. Matin A, Matin MK (1982) Cellular levels, excretion, and syntehsis rates of cyclic AMP in Escherichia coli grown in continuous culture. J Bacteriol 149:801–807PubMedGoogle Scholar
  22. Miller JF (1992) A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  23. Minton NP (1984) Improved plasmid vectors for the isolation of translational lac gene fusions. Gene 31:269–273PubMedCrossRefGoogle Scholar
  24. Monod J (1945) Sur la nature du phénomène de diauxie. Ann Inst Pasteur Microbiol 72:868–878Google Scholar
  25. Mori K, Aiba H (1985) Evidence for negative control of cya transcription by cAMP and cAMP receptor protein in intact Escherichia coli cells. J Biol Chem 260:14838–14843PubMedGoogle Scholar
  26. Pardee AB, Jacob F, Monod J (1959) The genetic control and cytoplasmic expression of inducibility in the synthesis of β-galacctosidase of Escherichia coli. J Mol Biol 1:165–178CrossRefGoogle Scholar
  27. Postma PW, Schuitema A, Kwa C (1981) Regulation of methyl-β-galactoside permease activity in pts and crr mutants of Salmonella typhimurium. Mol Gen Genet 181:448–453PubMedCrossRefGoogle Scholar
  28. Postma PW, Lengeler JW, Jacobson GR (1993) Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. Microbiol Rev 57:543–594PubMedGoogle Scholar
  29. Rambach A, Hogness DS (1977) Translation of Drosophila melanogaster sequences in Escherichia coli. Proc Natl Acad Sci USA 74:5041–5045PubMedCrossRefGoogle Scholar
  30. Reddy P, Peterkofsky A, McKenney K (1985) Translational efficiency of the Escherichia coli adenylate cyclase gene: mutating the UUG initiation codon to GUG or AUG results in increased gene expression. Proc Natl Acad Sci USA 82:5656–5660PubMedCrossRefGoogle Scholar
  31. Reddy P, Peterkofsky A, McKenney K (1989) Hyperexpression and purification of Escherichia coli adenylate cyclase using a vector designed for expression of lethal gene products. Nucleic Acids Res 17:10473–10488PubMedGoogle Scholar
  32. Roy A, Danchin A (1982) The cya locus of Escherichia coli K12: organization and gene products. Mol Gen Genet 188:465–471PubMedCrossRefGoogle Scholar
  33. Roy A, Danchin A, Joseph E, Ullmann A (1983) Two functional domains in adenylate cyclase of Escherichia coli. J Mol Biol 165:197–202PubMedGoogle Scholar
  34. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467PubMedCrossRefGoogle Scholar
  35. Ullmann A, Contesse G, Crepin M, Gros F, Monod J (1969) Cyclic AMP and catabolite repression in Escherichia coli. In: Rall TW, Rodbell M, Condliffe P (eds) The role of adenyl cyclase and cyclic 3′-5′AMP in biological systems. National Institutes of Health, Bethesda, Md, pp 215–231Google Scholar
  36. Wang JYJ, Clegg DO, Koshland Jr DE (1981) Molecular cloning and amplification of the adenylate cyclase gene. Proc Natl Acad Sci USA 78:4684–4688PubMedCrossRefGoogle Scholar
  37. Yang JK, Epstein W (1983) Purification and characterization of adenylate cyclase from Escherichia coli K12. J Biol Chem 258:3750–3758PubMedGoogle Scholar
  38. Zubay G, Schwartz D, Beckwith J (1970) Mechanism of activation of catabolite-sensitive genes: a positive control system. Proc Natl Acad Sci USA 66:104–110PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Martine Crasnier
    • 1
  • Valérie Dumay
    • 1
  • Antoine Danchin
    • 1
  1. 1.Unité de Régulation de l'Expression Génétique (Centre National de la Recherche Scientifique Unité Associée 1129)Institut PasteurParis Cedex 15France

Personalised recommendations