Journal of Industrial Microbiology

, Volume 8, Issue 1, pp 1–12 | Cite as

Transcriptional organization and regulation of the nosiheptide resistance gene inStreptomyces actuosus

  • Yun Li
  • Donald C. Dosch
  • Robert H. Woodman
  • Heinz G. Floss
  • William R. Strohl


The nosiheptide resistance gene (nshR) and a putative regulatory gene (nshA) are found together on a 2326 bpBamHI-PstI DNA fragment isolated fromStreptomyces actuosus ATCC 25421. The putative regulatory gene,nshA, situated upstream from the nosiheptide resistance gene in the 2326 bp DNA fragment, contains apparent DNA-binding and RNA-binding domains. Interruption ofnshA in the chromosome ofS. actuosus alters nosiheptide production, suggesting thatnshA is involved in regulation of nosiheptide biosynthesis. Two transcription initiation sites were found upstream ofnshA as demonstrated by high-resolution Sl nuclease mapping. A weak transcription start site fornshR was found which initiated transcription from the first nucleotide of the open reading frame. Although a stem-loop structure with apparent termination activity was found betweennshA andnshR, readthrough of transcription betweennshA andnshR was demonstrated by S1 nuclease mapping of the 3′ terminus of thenshA transcript. Time-course S1 experiments of the three promoters (nshA-pl, nshA-p2, nshR-p) indicated highly regulated differential expression of the promoters.nshA-p2 is a strong, constitutive promoter whereasnshA-pl being regulated temporally with maximal activity at 96 h. Approximately 30% of the totalnshA-p1/p2 transcript reads through the terminator and into thenshR gene, accounting for more than half of the total steady-statenshR transcript. The implications of the regulation ofnshA andnshR gene expression, as well as the expression of two other linked genes, are presented.

Key words

Nosiheptide resistance Thiopeptide antibiotics Streptomyces Antitermination Regulation of transcription 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Anzai, H., T. Murakami, S. Imai, A. Satoh, K. Nagoaka and C. Thompsom. 1987. Transcriptional regulation of bialophos biosynthesis inStreptomyces hygroscopicus. J. Bacteriol. 169: 3482–3488.Google Scholar
  2. 2.
    Babcock, M.J. 1990. Characterization and regulation of a gene involved in sporulation ofStreptomyces griseus. Ph.D. Thesis, The Ohio State University, Columbus, OH. 148 pp.Google Scholar
  3. 3.
    Babcock, M. and K.E. Kendrick. 1990. Nucleotide sequence and transcription of DNA that restores sporulatio to bald mutants ofStreptomyces griseus. Gene 95: 57–63.Google Scholar
  4. 4.
    Banerjee, S. and J.N. Hansen. 1988. Structure and expression of a gene encoding the precursor of subtilin, a small protein antibiotic. J. Biol. Chem. 263: 9508–9514.Google Scholar
  5. 5.
    Benazet, F., M. Cartier, J. Florent, C. Godard, G. Jung, J. Lunel, D. Mancy, C. Pascal, J. Renaut, P. Tarridec, J. Theilleux, R. Tissier, M. Dubost and L. Ninet. 1980. Nosiheptide, a sulfur-containing peptide antibiotic isolated fromStreptomyces actuosus 40037. Experientia 36: 414–416.Google Scholar
  6. 6.
    Betlach, J., J. Friedman, H.W. Boyer and F. Pfeifer. 1984. Characterization of a halobacterial gene affecting bacterioopsin gene expression. Nucl. Acids Res. 12: 7949–7959.Google Scholar
  7. 7.
    Bibb, M.J., M.J. Bibb, J.M. Ward and S.N. Cohen. 1985. Nucleotide sequences encoding and promoting expression of three antibiotic resistance genes indigenous toStreptomyces. Mol. Gen. Genet. 199: 26–36.Google Scholar
  8. 8.
    Bibb, M.J. and G.R. Janssen. 1986. Unusual features of transcription and translation of antibiotic resistance genes in antibiotic-producingStreptomyces. In: Proceedings of the Fifth International Symposium on the Genetics of Industrial Microorganisms, Split, Yugoslavia (Alacevic, A., Hranueli, D. and Toman, Z., eds). pp. 309–318. Ognjen Prica Printing Works, Karlovac, Yugoslavia.Google Scholar
  9. 9.
    Bibb, M.J., G.R. Janssen and J.M. Ward. 1985. Cloning and analysis of the promoter region of the erythromycin-resistance gene (ermE) ofStreptomyces erythraeus. Gene 38: 215–226 (and erratum in Gene 41: E357–E368, 1986).Google Scholar
  10. 10.
    Billig, A. and R. Zocher. 1987. Enzymatic synthesis of cyclosporin. J. Biol. Chem. 262: 17258–17259.Google Scholar
  11. 11.
    Blanck, A. and D. Oesterhelt. 1987. The halo-opsin gene. II. Sequence, primary structure of halorhodopsin and comparison with bacteriorhodopsin. EMBO J. 6: 265–273.Google Scholar
  12. 12.
    Butler, M.J., E.J. Friend, I.S. Hunter, F.S. Kaczmarek, D.A. Sugden and M. Warren. 1989. Molecular cloning of resistance genes and architecture of a linked gene cluster involved in the biosynthesis of oxytetracycline byStreptomyces rimosus. Mol. Gen. Genet. 215: 231–238.Google Scholar
  13. 13.
    Buttner, M.J., I.M. Fearnley and M.J. Bibb. 1987. The agarase gene (dagA) ofStreptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis. Mol. Gen. Genet. 209: 101–109.Google Scholar
  14. 14.
    Chater, K.F. 1990. The improving prospects for yield increase by genetic engineering in antibiotic-producing streptomycetes. Biotechnology 8: 115–121.Google Scholar
  15. 15.
    Chater, K.F. and C.J. Bruton. 1985. Resistance, regulatory and production genes for the antibiotic methylenomycin are clustered. EMBO J. 4: 1893–1897.Google Scholar
  16. 16.
    Chater, K.F., C.J. Bruton, K.A. Plaskitt, M.J. Buttner, C. Mèndez and J.D. Helmann. 1989. The developmental fate ofS. coelicolor hyphae depends upon a gene product homologous with the motility σ factor ofB. subtilis. Cell 59: 133–143.Google Scholar
  17. 17.
    Chater, K.F. and D.A. Hopwood. 1989. Antibiotic biosynthesis inStreptomyces. In: Genetics of Bacterial Diversity, (Hopwood, D.A. and Chater, K.F., eds.). pp. 129–151. Academic Press, London.Google Scholar
  18. 18.
    Cho, H., T. Horisaka, H. Ohkishi and H. Ogawara. 1989. Cloning of biosynthesis and resistance genes of nosiheptide. In: Trends in Actinomycetology in Japan, Actinomycetologica Forum 1989. (Koyama, Y. ed.). pp. 85–86. Society for Actinomycetes, Japan.Google Scholar
  19. 19.
    Cundliffe, E. 1989. How antibiotic-producing organisms avoid suicide. Annu. Rev. Microbiol. 43: 207–233.Google Scholar
  20. 20.
    Cundliffe, E. and J. Thompson. 1981. The mode of action of nosiheptide (multhiomycin) and the mechanism of resistance in the producing organisms. J. Gen. Microbiol. 126: 185–192.Google Scholar
  21. 21.
    Dary, A., B. Simonet, J.-M. Simonet and B. Decaris. 1989. Clonage d'un gene de resistance au nosiheptide deStreptomyces actuosus. C.R. Acad. Sci. Paris. 308: 35–41.Google Scholar
  22. 22.
    Debarbouille, M., M. Arnaud, A. Fouet, A. Klier and G. Rapoport. 1990. ThesacT gene regulating thesacPA operon inBacillus subtilis shares strong homology with transcriptional antiterminators. J. Bacteriol. 172: 3966–3973.Google Scholar
  23. 23.
    Distler, J., K. Mansouri and W. Piepersberg. 1985. Streptomycin biosynthesis inStreptomyces griseus. II. Adjacent genomic location of biosynthetic genes and one of two streptomycin resistance genes. FEMS Microbiol. Lett. 30: 151–154.Google Scholar
  24. 24.
    Distler, J., A. Ebert, K. Mansouri, K. Pissowotzki, M. Stockmann and W. Piepersberg. 1987. Gene cluster for streptomycin biosynthesis inStreptomyces griseus: nucleotide sequence of three genes and analysis of transcriptional activity. Nucl. Acids Res. 15: 8041–8056.Google Scholar
  25. 25.
    Dosch, D.C., W.R. Strohl and H.G. Floss. 1988. Molecular cloning of the nosiheptide resistance gene fromStreptomyces actuosus ATCC 25421. Biochem. Biophys. Res. Commun. 156: 517–523.Google Scholar
  26. 26.
    Dunn, R., J. McCoy, M. Simsek, A. Kajumdar, S.H. Chang, U.L. RajBhandry and H.G. Khorana. 1981. The bacteriorhodopsin gene. Proc. Natl. Acad. Sci. USA 78: 6744–6748.Google Scholar
  27. 27.
    Floss, H.G. and J.M. Beale. 1989. Biosynthetic studies on antibiotics. Angew. Chem. Ind. Ed. Engl. 28: 146–177.Google Scholar
  28. 28.
    Gold, L., D. Pribnow, T. Schneider, S. Shinedling, B.S. Singer and G. Stormo. 1981. Translational initiation in prokaryotes. Annu. Rev. Microbiol. 35: 365–403.Google Scholar
  29. 29.
    Guijarro, J., R. Santamaria, A. Schauer and R. Losick. 1988. Promoter determining the timing and spatial localization of transcription of a clonedStreptomyces coelicolor gene encoding a spore-associated protein. J. Bacteriol. 170: 1895–1901.Google Scholar
  30. 30.
    Hallam, S.E., F. Malpartida and D.A. Hopwood. 1988. DNA sequence, transcription, and deduced function of a gene involved in polyketide antibiotic biosynthesis inStreptomyces coelicolor. Gene 74: 305–320.Google Scholar
  31. 31.
    Hopwood, D.A., M.J. Bibb, K.F. Chater, G.R. Janssen, F. Malpartida and P. Smith. 1986. Regulation of gene expression in antibiotic-producing streptomycetes. In: Regulation of Gene Expression, 25 Year On. Symp. Soc. Gen. Microbiol. (Booth, I.R. and Higgins, C.F. eds.). pp. 251–276. University of Cambridge Press, Cambridge, U.K.Google Scholar
  32. 32.
    Hopwood, D.A., M.J. Bibb, K.F. Chater, T. Kieser, C.J. Bruton, H.M. Kieser, D.J. Lydiate, C.P. Smith, J.M. Ward and H. Schrempf. 1985. Genetic manipulation ofStrepomyces: a laboratory manual. The John Innes Foundation, Norwich, U.K.Google Scholar
  33. 33.
    Horinouchi, S., K. Furuya, M. Nishiyama, H. Suzuki and T. Beppu. 1987. Nucleotide sequence of the streptothricin acetyltransferase gene fromStreptomyce lavendulae and its expression in heterologous hosts. J. Bacteriol. 169: 1929–1937.Google Scholar
  34. 34.
    Horinouchi, S., H. Suzuki and T. Beppu. 1986. Nucleotide sequence ofafsB, a pleiotropic gene involved in secondary metabolism inStreptomyces coelicolor A3(2) andStreptomyces lividans. J. Bacteriol. 168: 257–269.Google Scholar
  35. 35.
    Horinouchi, S., H. Suzuki, M. Nishiyama and T. Beppu. 1989. Nucleotide sequence and transcriptional analysis of theStreptomyces griseus gene (afsA) responsible for A-factor biosynthesis. J. Bacteriol. 171: 1206–1210.Google Scholar
  36. 36.
    Hoshiko, S., C. Nojiri, K. Matsunaga, K. Katsumata, E. Satoh and K. Nagaoka. 1988. Nucleotide sequence of the ribostamycin phosphotransferase gene and of its control region inStreptomyces ribosidificus. Gene 68: 285–296.Google Scholar
  37. 37.
    Houck, D.R., L.-C. Chen, P.J. Keller, J.M. Beale and H.G. Floss. 1988. Biosynthesis of the modified peptide antibiotic nosiheptide inStreptomyces actuosus. J. Am. Chem. Soc. 110: 5800–5806.Google Scholar
  38. 38.
    Hunter, I.S. and S. Baumberg. 1989. In: Microbial Products: New Approaches. Soc. Gen. Microbiol., 44th Symp. (Baumberg, S., Hunter I. and Rhodes M. eds.). pp. 121–162. Cambridge University Press, Cambridge.Google Scholar
  39. 39.
    Ishihara, H., N. Hara and T. Iwabuchi. 1989. Molecular cloning and expression inEscherichia coli of theBacillus licheniformis bacitracin synthetase 2 gene. J. Bacteriol. 171: 1705–1711.Google Scholar
  40. 40.
    Janssen, G.R. and M.J. Bibb. 1988. Tandem promoters transcribe the thiostrepton resistance gene fromStreptomyces azureus and the viomycin resistance gene fromStreptomyces vinaceus. In: Biology of actinomycetes '88. (Okami, Y., Beppu, T. and Ogawara H. eds.). pp. 374–379. Japan Scientific Societies Press, Tokyo.Google Scholar
  41. 41.
    Janssen, G.R. and M.J. Bibb. 1988. Complex and unusual patterns of transcriptional initiation precede two antibiotic resistance genes from antibiotic-producing streptomycetes. Dev. Industr. Microbiol. 29: 89–96.Google Scholar
  42. 42.
    Janssen, G.R., J.M. Ward and M.J. Bibb. 1989. Unusual transcriptional and translational features of the aminoglycoside phosphotransferase gene (aph) fromStreptomyces fradiate. Genes Devel. 3: 415–429.Google Scholar
  43. 43.
    Kaletta, C. and K.-D. Entian. 1989. Nisin, a peptide antibiotic: Cloning and sequencing of thenisA gene and posttranslational processing of its peptide product. J. Bacteriol. 171: 1597–1601.Google Scholar
  44. 44.
    Klock, G. and W. Hillen. 1986. Expression, purification and operator binding of the transposon Tn1721-encoded Tet repressor. J. Mol. Biol. 189: 633–641.Google Scholar
  45. 45.
    Kratzschmar, J., M. Krause and M.A. Marahiel. 1989. Gramicidin S biosynthesis operon containing the structural genesgrsA andgrsB has an open reading frame encoding a protein homologous to fatty acid thioesterases. J. Bacteriol. 171: 5422–5429.Google Scholar
  46. 46.
    Lawlor, E.J., H.A. Baylis and K.F. Chater. 1987. Pleiotropic morphological and antibiotic deficiencies result from mutations in a gene encoding a tRNA-like product inStreptomyces coelicolor A3(2). Genes. Devel. 1: 1305–1310.Google Scholar
  47. 47.
    Lazinski, D., E. Grzadzielska and A. Das. 1989. Sequencespecific recognition of RNA hairpins by bacteriophage anti-terminators requires a conserved arginine-rich motif. Cell 59: 207–218.Google Scholar
  48. 48.
    Li, Y., D.C. Dosch, W.R. Strohl and H.G. Floss. 1990. Nucleotide sequence and transcriptional analysis of the nosiheptide-resistance gene fromStreptomyces actuosus. Gene 91: 9–17.Google Scholar
  49. 49.
    Lopez-Cabrera, M., J.A. Perez-Gonzales, P. Heinzel, W. Piepersberg and A. Jiminez. 1989. Isolation and nucleotide sequencing of an aminocyclitol acetyltransferase gene fromStreptomyces rimosus formaparomomycinus. J. Bacteriol. 171: 321–328.Google Scholar
  50. 50.
    Maxam, A.M. and W. Gilbert. 1980. Sequencing end-labelled DNA with base specific chemical cleavages. Methods Enzymol. 65: 449–560.Google Scholar
  51. 51.
    Mittenhuber, G., R. Weckermann and M.A. Marahiel. 1989. Gene cluster containing the genes for tyrocidine synthetases 1 and 2 fromBacillus brevis: Evidence for an operon. J. Bacteriol. 171: 4881–4887.Google Scholar
  52. 52.
    Murakami, R., H. Anzai, S. Imai, A. Satoh, K. Nagaoka and C.J. Thompson. 1986. The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster. Mol. Gen. Genet. 205: 42–50.Google Scholar
  53. 53.
    Murray, M.G. 1986. Use of sodium trichloroacetate and mung bran nuclease to increase sensitivity and precision during transcript mapping. Anal. Biochem. 158: 165–170.Google Scholar
  54. 54.
    Narva, K.E. and J.S. Feitelson. 1990. Nucleotide sequence and transcriptional analysis of theredD locus ofStreptomyces coelicolor A3(2). J. Bacteriol. 172: 326–333.Google Scholar
  55. 55.
    Ochi, K. 1989. Thetsr gene-coding plasmid pIJ702 prevents thiopeptin from inhibiting ppGpp synthesis inStreptomyces lividans. FEMS Microbiol. Lett. 61: 219–224.Google Scholar
  56. 56.
    Ohnuki, T., T. Imanaka and S. Aiba. 1985. Self-cloning inStreptomyces griseus of anstr gene cluster for streptomycin biosynthesis and streptomycin resistance. J. Bacteriol. 164: 85–94.Google Scholar
  57. 57.
    Pascard, C., A. Ducruix, J. Lunel and T. Prange. 1977. Highly modified cysteine-containing antibiotics. Chemical structure and configuration of nosiheptide. J. Am. Chem. Soc. 99: 6418–6423.Google Scholar
  58. 58.
    Piepersberg, W., J. Distler, A. Ebert, P. Heinzel, K. Mansouri, G. Kayer and K. Pissowotzki. 1988. Expression of genes for streptomycin biosynthesis. In: Biology of actinomycetes '88. (Okami, Y., Beppu, T. and Ogawara H., eds.). pp. 86–91. Japan Scientific Societies Press, Tokyo.Google Scholar
  59. 59.
    Ptashne, M., K. Backman, M.Z. Humayaun, A. Jeffrey, R. Maurer and R.T. Sauer. 1976. Autoregulation and function of a repressor in bacteriophage lambda. Science 194: 156–161.Google Scholar
  60. 60.
    Pulido, D., M. Zalacain and A. Jiminez 1988. Thehyg gene promoter fromStreptomyces hygroscopicus: a novel form ofStreptomyces promoters. Biochem. Biophys. Res. Commun. 150: 270–274.Google Scholar
  61. 61.
    Schnell, N., K.-D. Entian, U. Schneider, F. Gotz, H. Zahner, R. Kellner and G. Jung. 1988. Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings. Nature (London) 333: 276–278.Google Scholar
  62. 62.
    Schnetz, K., C. Toloczyki and B. Rak. 1987. β-Glucoside [bgl] operon ofEscherichia coli K-12: Nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of twoBacillus subtilis genes. J. Bacteriol. 169: 2579–2590.Google Scholar
  63. 63.
    Sprengart, M.L., H.P. Fatscher and E. Fuchs. 1990. The initiation of translation inE. coli: apparent base pairing between the 16SrRNA and downstream sequences of the mRNA. Nucl. Acids Res. 18: 1719–1723.Google Scholar
  64. 64.
    Stanzak, R., P. Matsushima, R.H. Baltz and R.N. Rao. 1986. Cloning and expression inStreptomyces lividans of clustered erythromycin biosynthetic genes fromStreptomyces erythraeus. Biotechnology 4: 229–232.Google Scholar
  65. 65.
    Stein, D. and S.N. Cohen. 1989. A cloned regulatory gene ofStreptomyces lividans can suppress the pigment deficiency phenotype of different developmental mutants. J. Bacteriol. 171: 2258–2261.Google Scholar
  66. 66.
    Thompson, C.J., T. Kieser, J.M. Ward and D.A. Hopwood. 1982. Physical analysis of antibiotic-resistance genes fromStreptomyces and their use in vector construction. Gene 20: 51–62.Google Scholar
  67. 67.
    Thompson, J., F. Schmidt and E. Cundliffe. 1982. Site of action of a ribosomal RNA methylase conferring resistance to thiostrepton. J. Biol. Chem. 257: 7915–7917.Google Scholar
  68. 68.
    Vögtli, M. and R. Hütter. 1987. Characterization of the hydroxystreptomycin phosphotransferase gene (sph) ofStreptomyces glaucescens: nucleotide sequence and promoter analysis. Molec. Gen. Genet. 208: 195–203.Google Scholar
  69. 69.
    Ward, J.M., G.R. Janssen, T. Kieser, M.J. Bibb, M.J. Buttner and M.J. Bibb. 1986. Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors forStreptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator. Mol. Gen. Genet. 203: 468–478.Google Scholar
  70. 70.
    Woodman, R.H. 1991. Molecular and Biochemical Studies on Thiopeptide Antibiotic Biosynthetis. Ph.D. dissertation. Ohio State University, Columbus, OH.Google Scholar

Copyright information

© Society for Industrial Microbiology 1991

Authors and Affiliations

  • Yun Li
    • 1
  • Donald C. Dosch
    • 1
  • Robert H. Woodman
    • 1
  • Heinz G. Floss
    • 2
  • William R. Strohl
    • 1
  1. 1.The Ohio State UniversityColumbusUSA
  2. 2.University of WashingtonSeattleUSA
  3. 3.Department of MicrobiologyThe Ohio state UniversityColumbusUSA
  4. 4.Department of BiologyKenyon CollegeGambierUSA

Personalised recommendations