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. 1990 Apr;172(4):1846–1852. doi: 10.1128/jb.172.4.1846-1852.1990

Characterization of malT mutants that constitutively activate the maltose regulon of Escherichia coli.

B Dardonville 1, O Raibaud 1
PMCID: PMC208678  PMID: 2180908

Abstract

The expression of the maltose regulon of Escherichia coli is controlled by a transcriptional activator, the product of the malT gene, and is induced by the presence of maltose or maltodextrins in the growth medium. We isolated eight mutants with mutations in malT which lead to constitutive expression of the regulon. The nucleotide sequences of the mutated genes revealed that the eight mutations are clustered in two small regions in the first one-third of the malT gene. Two mutated MalT proteins (corresponding to a mutation in each cluster) were purified and examined for in vitro activation of the MalT-dependent malPp promoter. Whereas wild-type MalT activity was absolutely dependent upon the presence of maltotriose, even at high protein concentrations, both mutated proteins were partially active in the absence of this sugar. Indeed, while the activity of the mutated proteins was still increased by maltotriose at low protein concentrations, the proteins were fully active in the absence of maltotriose at high protein concentrations. Both proteins exhibited a fivefold-higher affinity for maltotriose than the wild-type protein did.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bedouelle H., Duplay P. Production in Escherichia coli and one-step purification of bifunctional hybrid proteins which bind maltose. Export of the Klenow polymerase into the periplasmic space. Eur J Biochem. 1988 Feb 1;171(3):541–549. doi: 10.1111/j.1432-1033.1988.tb13823.x. [DOI] [PubMed] [Google Scholar]
  2. Bukau B., Ehrmann M., Boos W. Osmoregulation of the maltose regulon in Escherichia coli. J Bacteriol. 1986 Jun;166(3):884–891. doi: 10.1128/jb.166.3.884-891.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  4. Cole S. T., Raibaud O. The nucleotide sequence of the malT gene encoding the positive regulator of the Escherichia coli maltose regulon. Gene. 1986;42(2):201–208. doi: 10.1016/0378-1119(86)90297-0. [DOI] [PubMed] [Google Scholar]
  5. Debarbouille M., Schwartz M. The use of gene fusions to study the expression of malT the positive regulator gene of the maltose regulon. J Mol Biol. 1979 Aug 15;132(3):521–534. doi: 10.1016/0022-2836(79)90273-0. [DOI] [PubMed] [Google Scholar]
  6. Duplay P., Szmelcman S., Bedouelle H., Hofnung M. Silent and functional changes in the periplasmic maltose-binding protein of Escherichia coli K12. I. Transport of maltose. J Mol Biol. 1987 Apr 20;194(4):663–673. doi: 10.1016/0022-2836(87)90243-9. [DOI] [PubMed] [Google Scholar]
  7. Débarbouillé M., Schwartz M. Mutants which make more malT product, the activator of the maltose regulon in Escherichia coli. Mol Gen Genet. 1980;178(3):589–595. doi: 10.1007/BF00337865. [DOI] [PubMed] [Google Scholar]
  8. Débarbouillé M., Shuman H. A., Silhavy T. J., Schwartz M. Dominant constitutive mutations in malT, the positive regulator gene of the maltose regulon in Escherichia coli. J Mol Biol. 1978 Sep 15;124(2):359–371. doi: 10.1016/0022-2836(78)90304-2. [DOI] [PubMed] [Google Scholar]
  9. Ehrmann M., Boos W. Identification of endogenous inducers of the mal regulon in Escherichia coli. J Bacteriol. 1987 Aug;169(8):3539–3545. doi: 10.1128/jb.169.8.3539-3545.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Englesberg E., Irr J., Power J., Lee N. Positive control of enzyme synthesis by gene C in the L-arabinose system. J Bacteriol. 1965 Oct;90(4):946–957. doi: 10.1128/jb.90.4.946-957.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Freundlieb S., Boos W. Alpha-amylase of Escherichia coli, mapping and cloning of the structural gene, malS, and identification of its product as a periplasmic protein. J Biol Chem. 1986 Feb 25;261(6):2946–2953. [PubMed] [Google Scholar]
  12. Garges S., Adhya S. Cyclic AMP-induced conformational change of cyclic AMP receptor protein (CRP): intragenic suppressors of cyclic AMP-independent CRP mutations. J Bacteriol. 1988 Apr;170(4):1417–1422. doi: 10.1128/jb.170.4.1417-1422.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  14. Harman J. G., McKenney K., Peterkofsky A. Structure-function analysis of three cAMP-independent forms of the cAMP receptor protein. J Biol Chem. 1986 Dec 15;261(35):16332–16339. [PubMed] [Google Scholar]
  15. Hatfield D., Hofnung M., Schwartz M. Genetic analysis of the maltose A region in Escherichia coli. J Bacteriol. 1969 May;98(2):559–567. doi: 10.1128/jb.98.2.559-567.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hofnung M., Schwartz M., Hatfield D. Complementation studies in the maltose-A region of the Escherichia coli K12 genetic map. J Mol Biol. 1971 Nov 14;61(3):681–694. doi: 10.1016/0022-2836(71)90072-6. [DOI] [PubMed] [Google Scholar]
  17. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  18. Michaelis S., Chapon C., D'Enfert C., Pugsley A. P., Schwartz M. Characterization and expression of the structural gene for pullulanase, a maltose-inducible secreted protein of Klebsiella pneumoniae. J Bacteriol. 1985 Nov;164(2):633–638. doi: 10.1128/jb.164.2.633-638.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mizuno T., Kato M., Jo Y. L., Mizushima S. Interaction of OmpR, a positive regulator, with the osmoregulated ompC and ompF genes of Escherichia coli. Studies with wild-type and mutant OmpR proteins. J Biol Chem. 1988 Jan 15;263(2):1008–1012. [PubMed] [Google Scholar]
  20. Nathanson N. M., Schleif R. Paucity of sites mutable to constitutivity in the araC activator gene of the L-arabinose operon of Escherichia coli. J Mol Biol. 1975 Jul 25;96(1):185–199. doi: 10.1016/0022-2836(75)90190-4. [DOI] [PubMed] [Google Scholar]
  21. Palmer T. N., Ryman B. E., Whelan W. J. The action pattern of amylomaltase from Escherichia coli. Eur J Biochem. 1976 Oct 1;69(1):105–115. doi: 10.1111/j.1432-1033.1976.tb10863.x. [DOI] [PubMed] [Google Scholar]
  22. Raibaud O., Débarbouillé M., Schwartz M. Use of deletions created in vitro to map transcriptional regulatory signals in the malA region of Escherichia coli. J Mol Biol. 1983 Jan 25;163(3):395–408. doi: 10.1016/0022-2836(83)90065-7. [DOI] [PubMed] [Google Scholar]
  23. Raibaud O., Gutierrez C., Schwartz M. Essential and nonessential sequences in malPp, a positively controlled promoter in Escherichia coli. J Bacteriol. 1985 Mar;161(3):1201–1208. doi: 10.1128/jb.161.3.1201-1208.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Raibaud O., Richet E. Maltotriose is the inducer of the maltose regulon of Escherichia coli. J Bacteriol. 1987 Jul;169(7):3059–3061. doi: 10.1128/jb.169.7.3059-3061.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Raibaud O., Schwartz M. Restriction map of the Escherichia coli malA region and identification of the malT product. J Bacteriol. 1980 Aug;143(2):761–771. doi: 10.1128/jb.143.2.761-771.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Raibaud O., Vidal-Ingigliardi D., Richet E. A complex nucleoprotein structure involved in activation of transcription of two divergent Escherichia coli promoters. J Mol Biol. 1989 Feb 5;205(3):471–485. doi: 10.1016/0022-2836(89)90218-0. [DOI] [PubMed] [Google Scholar]
  27. Reyes M., Shuman H. A. Overproduction of MalK protein prevents expression of the Escherichia coli mal regulon. J Bacteriol. 1988 Oct;170(10):4598–4602. doi: 10.1128/jb.170.10.4598-4602.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Richet E., Raibaud O. MalT, the regulatory protein of the Escherichia coli maltose system, is an ATP-dependent transcriptional activator. EMBO J. 1989 Mar;8(3):981–987. doi: 10.1002/j.1460-2075.1989.tb03461.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Richet E., Raibaud O. Purification and properties of the MalT protein, the transcription activator of the Escherichia coli maltose regulon. J Biol Chem. 1987 Sep 15;262(26):12647–12653. [PubMed] [Google Scholar]
  30. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shevell D. E., LeMotte P. K., Walker G. C. Alteration of the carboxyl-terminal domain of Ada protein influences its inducibility, specificity, and strength as a transcriptional activator. J Bacteriol. 1988 Nov;170(11):5263–5271. doi: 10.1128/jb.170.11.5263-5271.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Spratt B. G., Hedge P. J., te Heesen S., Edelman A., Broome-Smith J. K. Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9. Gene. 1986;41(2-3):337–342. doi: 10.1016/0378-1119(86)90117-4. [DOI] [PubMed] [Google Scholar]
  33. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Takano K., Nakabeppu Y., Sekiguchi M. Functional sites of the Ada regulatory protein of Escherichia coli. Analysis by amino acid substitutions. J Mol Biol. 1988 May 20;201(2):261–271. doi: 10.1016/0022-2836(88)90137-4. [DOI] [PubMed] [Google Scholar]
  35. Yamaguchi K., Tomizawa J. Establishment of Escherichia coli cells with an integrated high copy number plasmid. Mol Gen Genet. 1980;178(3):525–533. doi: 10.1007/BF00337857. [DOI] [PubMed] [Google Scholar]

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