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.
Similar content being viewed by others
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
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
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
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
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
Butler AR, Bate N, Cundliffe E (1999) Impact of thioesterase activity on tylosin biosynthesis in Streptomyces fradiae. Chem Biol 6:287–292
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
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
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
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
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
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
Donadio S, Staver MJ, McAlpine JB, Swanson SJ, Katz L (1991) Modular organization of genes required for complex polyketide biosynthesis. Science 252:675–679
Dove SL, Joung JK, Hochschild A (1997) Activation of prokaryotic transcription through arbitrary protein-protein contacts. Nature 386:627–630
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
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
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
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
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
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
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
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
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
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
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by W. Goebel
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00438-004-1088-3