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Interaction studies on proteins encoded by the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis

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Abstract

Polyketide synthases (PKSs) of Mycobacterium tuberculosis are increasingly being seen as producers of virulence factors that are important for pathogenesis by the bacterium. Thus, the phenolphthiocerol synthase PKS cluster of M. tuberculosis is responsible, in part, for the synthesis of a virulence determinant called phthiocerol dimycocerosate (PDIM). Here, we provide evidence that the PpsE protein, which is part of that cluster, interacts with the type II thioesterase TesA of M. tuberculosis. The interaction was demonstrated by employing a two-hybrid system, and confirmed using a GST (glutathione S-transferase) pull-down’ assay after both proteins had been purified to homogeneity. Based on the present findings, a revised model for the processing of polyketides during the synthesis of PDIM is presented.

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References

  • Azad AK, Sirakova TD, Rogers LM, Kolattukudy PE (1996) Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis BCG produces a mutant that lacks mycosides. Proc Natl Acad Sci USA 93:4787–4792

    Article  CAS  PubMed  Google Scholar 

  • Azad AK, Sirakova TD, Fernandes ND, Kolattukudy PE (1997) Gene knockout reveals a novel gene cluster for the synthesis of a class of cell wall lipids unique to pathogenic mycobacteria. J Biol Chem 272:16741–16745

    Article  CAS  PubMed  Google Scholar 

  • Barberis A, Pearlberg J, Simkovich N, Farrell S, Reinagel P, Bamdad C, Sigal G, Ptashne M (1995) Contact with a component of the polymerase II holoenzyme suffices for gene activation. Cell 81:359–368

    Article  CAS  PubMed  Google Scholar 

  • Black PN, DiRusso CC, Metzger AK, Heimert TL (1992) Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. J Biol Chem 267:25513–25520

    CAS  PubMed  Google Scholar 

  • Black PN, Zhang Q, Weimar JD, DiRusso CC (1997) Mutational analysis of a fatty acyl-coenzyme A synthetase signature motif identifies seven amino acid residues that modulate fatty acid substrate specificity. J Biol Chem 272:4896–4903

    Article  CAS  PubMed  Google Scholar 

  • Butler AR, Bate N, Cundliffe E (1999) Impact of thioesterase activity on tylosin biosynthesis in Streptomyces fradiae. Chem Biol 6:287–292

    Article  CAS  PubMed  Google Scholar 

  • Caffrey P, Green B, Packman LC, Rawlings BJ, Staunton J, Leadlay PF (1991) An acyl-carrier-protein-thioesterase domain from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea. High-level production, purification and characterisation in Escherichia coli. Eur J Biochem 195:823–830

    CAS  PubMed  Google Scholar 

  • Camacho LR, Ensergueix D, Perez E, Gicquel B, Guilhot C (1999) Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis. Mol Microbiol 34:257–267

    Article  CAS  PubMed  Google Scholar 

  • Camacho LR, Constant P, Raynaud C, Laneelle MA, Triccas JA, Gicquel B, Daffe M, Guilhot C (2001) Analysis of the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis. Evidence that this lipid is involved in the cell wall permeability barrier. J Biol Chem 276:19845–19854

    Article  CAS  PubMed  Google Scholar 

  • Cho H, Cronan JE Jr (1993) Escherichia coli thioesterase I, molecular cloning and sequencing of the structural gene and identification as a periplasmic enzyme. Biol Chem 268:9238–9245

    CAS  Google Scholar 

  • Cortes J, Haydock SF, Roberts GA, Bevitt DJ, Leadlay PF (1990) An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea. Nature 348:176–178

    Article  CAS  PubMed  Google Scholar 

  • Cox JS, Chen B, McNeil M, Jacobs WR Jr (1999) Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice. Nature 4:79–83

    Google Scholar 

  • Donadio S, Staver MJ, McAlpine JB, Swanson SJ, Katz L (1991) Modular organization of genes required for complex polyketide biosynthesis. Science 252:675–679

    CAS  PubMed  Google Scholar 

  • Dove SL, Joung JK, Hochschild A (1997) Activation of prokaryotic transcription through arbitrary protein-protein contacts. Nature 386:627–630

    Article  CAS  PubMed  Google Scholar 

  • Fernandes ND, Kolattukudy PE (1997) Methylmalonyl coenzyme A selectivity of cloned and expressed acyltransferase and beta-ketoacyl synthase domains of mycocerosic acid synthase from Mycobacterium bovis BCG. J Bacteriol 179:7538–7543

    CAS  PubMed  Google Scholar 

  • Fitzmaurice AM, Kolattukudy PE (1998) An acyl-CoA synthase (acoas) gene adjacent to the mycocerosic acid synthase (mas) locus is necessary for mycocerosyl lipid synthesis in Mycobacterium tuberculosis var. bovis BCG. J Biol Chem 273:8033–8039

    Article  CAS  PubMed  Google Scholar 

  • Heathcote ML, Staunton J, Leadlay PF (2001) Role of type II thioesterases: evidence for removal of short acyl chains produced by aberrant decarboxylation of chain extender units. Chem Biol 8:207–220

    Article  CAS  PubMed  Google Scholar 

  • Kennedy J, Auclair K, Kendrew SG, Park C, Vederas JC, Hutchinson CR (1999) Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis. Science 284:1368–1372

    Article  CAS  PubMed  Google Scholar 

  • Kotowska M, Pawlik K, Butler AR, Cundliffe E, Takano E, Kuczek K (2002) Type II thioesterase from Streptomyces coelicolor A3(2). Microbiology 148:1777–1783

    CAS  PubMed  Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

  • Sanger F, Nicklen S, Coulson AR (1997) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Google Scholar 

  • Schwecke T, Aparicio JF, Molnar I, Konig A, Khaw LE, Haydock SF, Oliynyk M, Caffrey P, Cortes J, Lester JB, Bohm GA, Staunton J, Leadlay PF (1995) The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin. Proc Natl Acad Sci USA 92:7839–7843

    CAS  PubMed  Google Scholar 

  • Sirakova TD, Fitzmaurice AM, Kolattukudy PE (2002) Regulation of expression of mas and fadD28, two genes involved in production of dimycocerosyl phthiocerol, a virulence factor of Mycobacterium tuberculosis. J Bacteriol 184:6796–6802

    Article  CAS  PubMed  Google Scholar 

  • Staunton J, Caffrey P, Aparicio JF, Roberts GA, Bethell SS, Leadlay PF (1996) Evidence for a double-helical structure for modular polyketide synthases. Nat Struct Biol 3:188–192

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors wish to thank RGP lab members for helpful discussions. This work was funded by internal grants of ICGEB, New Delhi. Alka Rao wishes to thank CSIR (New Delhi) for financial support

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Correspondence to A. Ranganathan.

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Communicated by W. Goebel

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Rao, A., Ranganathan, A. Interaction studies on proteins encoded by the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis. Mol Genet Genomics 272, 571–579 (2004). https://doi.org/10.1007/s00438-004-1088-3

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  • DOI: https://doi.org/10.1007/s00438-004-1088-3

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