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Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator

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Summary

Several versatile, multi-copy, promoter-probe plasmid vectors have been constructed that replicate in a wide range of Streptomyces species. Transcriptional activity is detected by the expression of a promoter-less aminoglycoside phosphotransferase gene (neo) derived from the transposon Tn5; expression of this gene confers kanamycin and neomycin resistance on Streptomyces lividans. An efficient transcriptional terminator from E. coli phage fd has been inserted upstream of the neo coding region to prevent significant transcriptional read-through from vector promoters. A translational stop codon situated downstream from the site(s) used for cloning and preceding and in frame with the ATG start codon of the neo gene ensures the detection of transcriptional, rather than translational, fusions. Relative promoter strengths can be determined by gradient plate assays of kanamycin resistance, by measuring the amount of aminoglycoside phosphotransferase produced or by estimating neo mRNA synthesised. The high copy number of the vectors facilitates the rapid isolation and characterisation of promoter-active fragments and convenient restriction sites are available for DNA sequencing and S1 mapping of cloned inserts. Some derivatives contain a poly-linker that facilitates the insertion, excision and analysis of cloned fragments and which enhances the use of these plasmids as general cloning vectors.

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References

  • Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller H (1982) Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene 19:327–336

    Google Scholar 

  • Bernan V, Filpula D, Herber W, Bibb MJ, Katz E (1985) The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product. Gene 33:101–110

    Google Scholar 

  • Bibb MJ, Cohen SN (1982) Gene expression in Streptomyces: construction and application of promoter-probe plasmid vectors in Streptomyces lividans. Mol Gen. Genet. 187:265–277

    Google Scholar 

  • Bibb MJ, Bibb MJ, Ward JM, Cohen SN (1985a) Nucleotide sequences encoding and promoting expression of three antibiotic resistance genes indigenous to Streptomyces. Mol. Gen. Genet. 199:26–36

    Google Scholar 

  • Bibb MJ, Janssen GR, Ward JM (1985b) Cloning and analysis of the promoter region of the erythromycin resistance (ermE) gene of Streptomyces erythraeus. Gene 38:215–226

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513–1523

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254

    Google Scholar 

  • Bryson V, Szybalski W (1952) Microbial selection. Science 116:45–51

    Google Scholar 

  • Buttner MJ, Brown NL (1985) RNA polymerase-DNA interactions in Streptomyces: in vitro studies of a S. lividans plasmid promoter with S. coelicolor RNA polymerase. J. Mol. Biol. 185:177–188

    Google Scholar 

  • Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138:179–207

    Google Scholar 

  • Chater KF, Bruton CJ (1985) Resistance, regulatory and production genes for the antibiotic methylenomycin are clustered. EMBO J. 4:1893–1897

    Google Scholar 

  • Chater KF, Hopwood DA, Kieser T, Thompson CJ (1982) Gene cloning in Streptomyces. Curr. Topics Microbiol. Immunol. 97:69–95

    Google Scholar 

  • Chater KF, King AA, Rodicio RM, Bruton CJ, Fisher SH, Piret JM, Smith CP, Foster SG (1985) The cloning and analysis of Streptomyces DNA in ϕC31-derived vectors. In: G. Hegemon and CL. Hershberger (eds) Microbiology 1985. ASM, Washington, pp 421–426

    Google Scholar 

  • Cohen SN, Chang ACY, Hsu L (1972) Non-chromosomal antibiotic resistance in bacteria.: VII. Genetic transformation of E. coli by R-factor DNA. Proc. Natl. Acad. Sci. USA 69:2110–2114

    Google Scholar 

  • Cohen SN, Chang ACY, Boyer HW, Helling RB (1973) Construction of biologically functional bacterial plasmids in vivo. Proc. Natl. Acad. Sci. USA 70:3240–3244

    Google Scholar 

  • Favaloro J, Triesman R, Kamen R (1980) Transcription maps of polyoma virus-specific RNA: Analysis by two-dimensional nuclease S1 gel mapping. Methods Enzymol 65:718–749

    Google Scholar 

  • Foster SG (1983) A search for transposons active on Streptomyces DNA. Ph.D Thesis, University of East Anglia, Norwich

  • Gentz R, Langner A, Chang ACY, Cohen SN, Bujard H (1981) Cloning and analysis of strong promoters is made possible by the downstream placement of a RNA termination signal. Proc. Natl. Acad. Sci. USA 78:4936–4940

    Google Scholar 

  • Hopwood DA (1967) Genetic analysis and genome structure in Streptomyces coelicolor. Bacteriol. Rev. 31:373–403

    Google Scholar 

  • Hopwood DA, Bibb MJ, Chater KF, Kieser T, Bruton CJ, Kieser HM, Lydiate DJ, Smith CP, Ward JM, Schrempf H (1985) Genetic manipulation of Streptomyces: a laboratory manual. John Innes Foundation. Norwich

    Google Scholar 

  • Hopwood DA, Chater KF, Dowding JE, Vivian A (1973) Recent advances in Streptomyces coelicolor genetics. Bacteriol. Rev. 37:371–405

    Google Scholar 

  • Hopwood DA, Wright HM, Bibb MJ, Cohen SN (1977) Genetic recombination through protoplast fusion in Streptomyces. Nature 268:171–173

    Google Scholar 

  • Hopwood DA, Kieser T, Wright HM, Bibb MJ (1983) Plasmids, recombination and chromosome mapping in Streptomyces lividans 66. J. Gen. Microbiol. 129:2257–2269

    Google Scholar 

  • Horinouchi S, Beppu T (1985) Construction and application of a promoter-probe plasmid that allows chromogenic identification in Streptomyces lividans. J. Bacteriol. 185:406–412

    Google Scholar 

  • Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res. 9:2989–2998

    Google Scholar 

  • Janssen GR, Bibb MJ, Smith CP, Ward JM, Kieser T, Bibb MJ (1985) Isolation and analysis of Streptomyces promoters. In: G. Hegemon and CL. Hershberger (eds) Microbiology 1985. ASM, Washington, pp 392–396

    Google Scholar 

  • Jaurin B, Cohen SN (1984) Streptomyces lividans RNA polymerase recognizes and uses escherichia coli transcriptional signals. Gene 28:83–91

    Google Scholar 

  • Katz E, Thompson CJ, Hopwood DA (1983) Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. J. Gen. Microbiol. 129:2703–2714

    Google Scholar 

  • Kieser T (1984a) Factors affecting the isolation of ccc DNA from Streptomyces lividans and Escherichia coli. Plasmid 12:19–36

    Google Scholar 

  • Kieser T (1984b) DNAGEL: a computer program for determining DNA fragment sizes using a small computer equipped with a graphics tablet. Nucleic Acids Res. 12:679–688

    Google Scholar 

  • Kieser T, Hopwood DA, Wright HM, Thomson CJ (1982) pIJ101, a multi-copy broad host range Streptomyces plasmid: Functional analysis and development of DNA cloning vectors. Mol. Gen. Genet. 185:223–238

    Google Scholar 

  • Kirby KS, Fox-Carter E, Guest M (1967) Isolation of deoxyribonucleic acid and ribonucleic acid from bacteria. Biochem. J. 104:258–262

    Google Scholar 

  • Lennox ES (1955) Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190–206

    Google Scholar 

  • Lydiate DJ, Malpartida F, Hopwood DA (1985) The Streptomyces plasmid SCP2*: its functional analysis and development into useful cloning vectors. Gene 35:223–235

    Google Scholar 

  • Malpartida F, Hopwood DA (1984) Molecular cloning of the whole biosynthetic pathway of a Streptomyces antibiotic and its expression in a heterologous host. Nature 309:462–464

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning — a laboratory manual. Cold Spring Harbor, New York

  • Maxam AM, Gilbert W (1980) Sequencing end-labelled DNA with base specific chemical cleavages. Methods Enzymol 65:449–560

    Google Scholar 

  • Norrander J, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106

    Google Scholar 

  • Piret JM, Chater KF (1985) Phage-mediated cloning of bldA, a region involved in Streptomyces coelicolor morphological development, and its analysis by genetic complementation. J. Bacteriol. 163:965–972

    Google Scholar 

  • Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Ann. Rev. Genet. 13:319–353

    Google Scholar 

  • Ruther U, Koenen M, Otto K, Müller-Hill B (1981) pUR222, a vector for cloning and rapid chemical sequencing of DNA. Nucleic Acids Res 9:4087–4098

    Google Scholar 

  • Sanger F, Coulson AR (1978) The use of thin acrylamide gels for DNA sequenicng. FEBS Lett. 87:107–110

    Google Scholar 

  • Sanger F, Coulson AR, Barrell BG, Smith AJH, Roe BA (1980) Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J. Mol. Biol. 143:161–178

    Google Scholar 

  • Schottel JL, Bibb MJ, Cohen SN (1981) Cloning and expression in Streptomyces lividans of antibiotic resistance genes derived from Escherichia coli. J. Bacteriol. 146:360–368

    Google Scholar 

  • Seno ET, Bruton CJ, Chater KF (1984) The glycerol utilization operon of Streptomyces coelicolor: genetic mapping of gyl mutations and the analysis of cloned gyl DNA. Mol. Gen. Genet. 193:119–128

    Google Scholar 

  • Shapira SK, Chou J, Richaud FV, Casadaban MJ (1983) New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase. Gene 25:71–82

    Google Scholar 

  • Smith CP (1986) Molecular biology of the glycerol utilization operon of Streptomyces coelicolor A3(2). Ph.D Thesis University of East Anglia, Norwich

  • Thompson CJ, Kieser T, Ward JM, Hopwood DA (1982) Physical analysis of antibiotic resistance genes from Streptomyces and their use in vector construction. Gene 20:51–62

    Google Scholar 

  • Timmis KF, Cabello F, Cohen SN (1978) Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6. Mol. Gen. Genet. 162:121–137

    Google Scholar 

  • Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-drived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268

    Google Scholar 

  • Westpheling J, Ranes M, Losick R (1985) RNA polymerase heterogeneity in Streptomyces coelicolor. Nature 313:22–27

    Google Scholar 

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Authors and Affiliations

  1. John Innes Institute, Colney Lane, NR4 7UH, Norwich, England

    Judith M. Ward, Gary R. Janssen, Tobias Kieser, Maureen J. Bibb, Mark J. Buttner & Mervyn J. Bibb

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  1. Judith M. Ward
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  2. Gary R. Janssen
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  3. Tobias Kieser
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  4. Maureen J. Bibb
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  5. Mark J. Buttner
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  6. Mervyn J. Bibb
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Communicated by H. Saedler

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Ward, J.M., Janssen, G.R., Kieser, T. et al. Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator. Mol Gen Genet 203, 468–478 (1986). https://doi.org/10.1007/BF00422072

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  • DOI: https://doi.org/10.1007/BF00422072

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