Molecular and General Genetics MGG

, Volume 248, Issue 5, pp 610–620 | Cite as

Identification ofStreptomyces violaceoruber Tü22 genes involved in the biosynthesis of granaticin

  • Andreas Bechthold
  • Jae Kyung Sohng
  • Todd M. Smith
  • Xin Chu
  • Heinz G. Floss
Original Paper


A 50 kb region of DNA fromStreptomyces violaceoruber Tü22, containing genes encoding proteins involved in the biosynthesis of granaticin, was isolated. The DNA sequence of a 7.3 kb fragment from this region, located approximately 10 kb from the genes that encode the polyketide synthetase responsible for formation of the benzoisochromane quinone skeleton, revealed five open reading frames (ORF1-ORF5). The deduced amino acid sequence of GraE, encoded by ORF2, shows 60.8% identity (75.2% similarity) to a dTDP-glucose dehydratase (StrE) fromStreptomyces griseus. Cultures ofEscherichia coli containing plasmids with ORF2, on a 2.1 kbBamHI fragment, were able to catalyze the formation of dTDP-4-keto-6-deoxy-d-glucose from dTDP-glucose at 5 times the rate of control cultures, confirming that ORF2 encodes a dTDP-glucose dehydratase. The amino acid sequence encoded by ORF3 (GraD) is 51.4% identical (69.9% similar) to that of StrD, a dTDP-glucose synthase fromStreptomyces griseus. The amino acid sequence encoded by ORF4 shares similarities with proteins that confer resistance to tetracycline and methylenomycin, and is suggested to be involved in transporting granaticin out of the cells by an active efflux mechanism.


Amino Acid Sequence Tetracycline Quinone Deduce Amino Acid Sequence Control Culture 
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  1. Bibb MJ, Findlay PR, Johnson MW (1984) The relationship between base composition and codon usage in bacterial genes and its use in the simple and reliable identification of protein codon sequences. Gene 30:157–166CrossRefPubMedGoogle Scholar
  2. Bierman M, Logan R, O'Brien K, Seno ET, Rao RN, Schoner BE (1992) Plasmid cloning vectors for the conjugal transfer of DNA fromEscherichia coli toStreptomyces spp. Gene 116:43–49CrossRefPubMedGoogle Scholar
  3. Caballero JL, Malpartida FA, Hopwood DA (1991) Transcriptional organization and regulation of an antibiotic export complex in the producingStreptomyces culture. Mol Gen Genet 228:372–380CrossRefPubMedGoogle Scholar
  4. Calcutt MJ, Schmidt FJ (1991) Bleomycin biosynthesis: characterization of the resistance region of the producer streptomycete. International Symposium on Biology of Actinomycetes, University of Wisconsin, Madison wis USAGoogle Scholar
  5. Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395PubMedGoogle Scholar
  6. Fernández-Moreno MA, Caballero JL, Hopwood DA, Malpartida F (1991) Theact cluster contains regulatory and antibiotic export genes, direct targets for translational control by thebldA tRNA gene ofStreptomyces. Cell 66:769–780CrossRefPubMedGoogle Scholar
  7. Griesebach H (1978) Biosynthesis of sugar components of antibiotic substances. Adv Carbohyd Chem Biochem 35:81–126CrossRefGoogle Scholar
  8. Guilfoile PG, Hutchinson CR (1991) A bacterial analog of themdr gene of mammalian tumor cells in present inStreptomyces peuceticus, the producer of daunorubicin and doxorubicin. Proc Natl Acad Sci USA 88:8553–8557PubMedGoogle Scholar
  9. Guilfoile PG, Hutchinson CR (1992) Sequence and transcriptional analysis of theStreptomyces glaucescens tcmAR tetracenomycin C resistance and repressor gene loci. J Bacteriol 174:3651–3658PubMedGoogle Scholar
  10. He X-G, Chang C-C, C-J. C, Vederas JC, McInnes AG, Walter JA, Floss HG (1986) Further studies on the biosynthesis of granaticin. Z Naturforsch 41c:215–221Google Scholar
  11. Hopwood DA, Bibb MJ, Chater KF, Kieser T, Bruton C, Kieser HM, Lydiate DJ, Smith CP, Ward JM (1985) Genetic manipulation ofStreptomyces: a laboratory manual. John Innes Foundation, Norwich, UKGoogle Scholar
  12. Hui-Zahn Z, Schmidt H, Piepersberg W (1992) Molecular cloning and characterization of two genes fromStreptomyces lincolnensis 78-11. Mol Microbiol 6:3147–3157Google Scholar
  13. Jenkins G, Cundliffe E (1991) Cloning and characterization of two genes fromStreptomyces lividans that confer resistance to lincomycin and macrolide antibiotics. Gene 108:55–62CrossRefPubMedGoogle Scholar
  14. Khosla C, McDaniel R, Ebert-Khosla S, Torres R, Sherman DH, Bibb MJ, Hopwood DA (1993) Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins. J Bacteriol 175:2197–2204PubMedGoogle Scholar
  15. Malpartida F, Hopwood DA (1986) Physical and genetic characterization of the gene cluster for the antibiotic actinorhodin inStreptomyces coelicolor A3 (2). Mol Gen Genet 205:66–73PubMedGoogle Scholar
  16. Mansouri K, Piepersberg W (1991) Genetics of streptomycin production inStreptomyces griseus: nucleotide sequence of five genes,strFGHIK, including a phosphatase gene. Mol Gen Genet 228:459–469CrossRefPubMedGoogle Scholar
  17. Martin JF, Liras P (1989) Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annu Rev Microbiol 43:173–206CrossRefPubMedGoogle Scholar
  18. Matsudaira PT (1993). A practical guide to protein and peptide purification for microsequencing. Academic Press, San DiegoGoogle Scholar
  19. Neal RJ, Chater KF (1987) Nucleotide sequence analysis reveals similarities between proteins conferring methylenomycin A resistance inStreptomyces and tetracycline resistance in eubacteria. Gene 8:229–241CrossRefGoogle Scholar
  20. Normark S, Bergström S, Edlund T, Grundström T, Jaurin B, Lindberg FP, Olsson O (1983) Overlapping genes. Annu Rev Genet 58:229–241Google Scholar
  21. Pearson WR (1990) Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol 183:63–98PubMedCrossRefGoogle Scholar
  22. Pissowotzki K, Mansouri K, Piepersberg W (1991) Genetics of streptomycin production inStreptomyces griseus: molecular structure and putative function of genesstrELMB2N. Mol Gen Genet 231:113–123CrossRefPubMedGoogle Scholar
  23. Reynes LP, Calmels T, Drocourt D, Tibay G (1988) Cloning, expression inEscherichia coli and nucleotide sequence of a tetracycline resistance gene fromStreptomyces rimosus. J Gen Microbiol 134:585–598PubMedGoogle Scholar
  24. Riordan JR, Ling V (1985) Genetic and biochemical characterization of multidrug resistance. Pharmac Ther 28:51–75CrossRefGoogle Scholar
  25. Rouch DA, Cram DS, DiBeradino D, Littlejohn TG, Skurray RA (1990) Effluxmediated antiseptic resistance geneqacA fromStaphylococcus aureus: common ancestry with tetracycline- and sugar-transport proteins. Mol Microbiol 4:2051–2062PubMedGoogle Scholar
  26. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  27. Schoner B, Geistlich M, Rosteck Jr P, Rao RN, Seno E, Reynolds P, Cox C, Burgett S, Hershberger C (1982) Sequence similarity between macrolide resistance determinants and ATP-binding transport proteins. Gene 115:93–96CrossRefGoogle Scholar
  28. Sheridan RP, Chopra I (1991) Origin of tetracycline efflux proteins: conclusions from nucleotide sequence analysis. Mol Microbiol 5:895–900PubMedGoogle Scholar
  29. Sherman DH, Malpartida F, Bibb MJ, Kieser HM, Bibb MJ, Hopwood DA (1989) Structure and deduced function of the granaticin-producing polyketide synthase gene cluster ofStreptomyces violaceoruber Tü22. EMBO J 8:2717–2725PubMedGoogle Scholar
  30. Sherman DH, Kim E-S, Bibb MJ, Hopwood DA (1992) Functional replacement of genes for individual polyketide synthase components inStreptomyces coelicolor A3(2) by heterologous genes from a different polyketide pathway. J Bacteriol 174: 6184–6190PubMedGoogle Scholar
  31. Snipes CE, Brillinger G-U, Sellers L, Mascaro L, Floss HG (1977) Sterochemistry of the dTDP-glucose oxidoreductase reaction. J Biol Chem 252:8113–8117PubMedGoogle Scholar
  32. Snipes CE, Chang C-J, Floss HG (1979) Biosynthesis of the antibiotic granaticin. J Amer Chem Soc 101:701–706CrossRefGoogle Scholar
  33. Stockmann M, Piepersberg W (1992) Gene probes for the detection of 6-deoxyhexose metabolism in secondary metabolite-producingStreptomycetes. FEMS Microbiol Lett 90:185–190Google Scholar
  34. Thorson JS, Lo SF, Liu H-W (1993b) Biosynthesis of 3,6-dideoxyhexoses: new mechanistic reflections upon 2,6-dideoxy, 4,6-dideoxy and amino sugar construction. J Am Chem Soc 115:6993–6994CrossRefGoogle Scholar
  35. Thorson JS, Lo SF, Liu H-W, Hutchinson CR (1993a) Molecular basis of 3,6-dideoxyhexose biosynthesis: elucidation of CDP-ascarylose biosynthetic genes and their relationship to other 3,6-dideoxyhexose pathways. J Am Chem Soc 115: 5827–5828CrossRefGoogle Scholar
  36. Vara JA, Hutchinson CR (1988) Purification of thymidine-diphospho-d-glucose 4,6-dehydratase from an erythromycin-producing strain ofSaccharopolyspora erythrea by high resolution liquid chromatography. J Biol Chem 263:14992–14995PubMedGoogle Scholar
  37. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Andreas Bechthold
    • 1
  • Jae Kyung Sohng
    • 1
  • Todd M. Smith
    • 2
  • Xin Chu
    • 1
  • Heinz G. Floss
    • 1
    • 2
  1. 1.Department of ChemistryUniversity of WashingtonSeattleUSA
  2. 2.Department of Medicinal ChemistryUniversity of WashingtonSeattleUSA

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