Genetic Manipulation of Streptomyces Species

S. Eric Nybo1, Micah D. Shepherd1, Mary A. Bosserman1, Jürgen Rohr1

1 College of Pharmacy, University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10E.3
DOI:  10.1002/9780471729259.mc10e03s19
Online Posting Date:  November, 2010
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Abstract

This unit includes general protocols for the genetic manipulation of Streptomyces species, including genomic DNA isolation, genomic library preparation, intergeneric conjugation of Streptomyces with E. coli, generation and transformation of Streptomyces protoplasts, electroporation of Streptomyces mycelia, and colony PCR. Curr. Protoc. Microbiol. 19:10E.3.1‐10E.3.26. © 2010 by John Wiley & Sons, Inc.

Keywords: Streptomyces; protoplast; conjugation; genomic DNA isolation; transformation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Genomic DNA Isolation from Streptomycetes
  • Basic Protocol 2: Genomic Library Preparation
  • Basic Protocol 3: Intergeneric Conjugation Between Streptomyces coelicolor CH999 or Streptomyces lividans TK 64 and E. coli
  • Alternate Protocol 1: Intergeneric Conjugation Between S. globisporus 1912, S. kanamyceticus, and S. cyanogenus S136, and E. coli
  • Alternate Protocol 2: Intergeneric Conjugation Between Streptomyces verticillus and Escherichia coli
  • Basic Protocol 4: Generation and Transformation of Streptomyces Protoplasts
  • Basic Protocol 5: Electroporation of Streptomyces Mycelia
  • Basic Protocol 6: Preparation of Samples for Colony PCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Genomic DNA Isolation from Streptomycetes

  Materials
  • Tryptic Soy Broth (TSB) or appropriate growth medium (see unit 10.1)
  • Mycelia or spore stock of Streptomyces (see unit 10.1)
  • Lysis buffer (see recipe)
  • 50 mg/ml lysozyme in 0.01 M Tris⋅Cl, pH 8.0 (see appendix 2A for Tris buffer)
  • 10% SDS ( appendix 2A)
  • 0.5 M EDTA, pH 8.0 ( appendix 2A)
  • 20 mg/ml pronase E (Sigma‐Aldrich, cat. no. P‐6911)
  • 5 M potassium acetate, pH adjusted to 7.5 with 2 M acetic acid (store in aliquots at –20°C)
  • 1:1 (v/v) phenol:chloroform
  • Chloroform
  • RNase solution (see recipe)
  • 100% ethanol and 70% ethanol
  • TE buffer (see recipe)
  • 250‐ml baffled Erlenmeyer flasks
  • 28°C shaking incubator
  • 50‐ml screw top vials (Fisher, cat. no. 05‐539‐13)
  • Centrifuge
  • Microscope slides
  • 72°C water bath
  • Blunted Pasteur pipets (see recipe)
  • Large‐bore (with ends cut off) and smaller‐bore 1000‐µl pipet tips

Basic Protocol 2: Genomic Library Preparation

  Materials
  • BfuCI restriction endonuclease with 10× NEB Buffer 4 (New England Biolabs)
  • Genomic DNA ( protocol 1)
  • DNA loading buffer (see recipe)
  • 0.8% agarose gel (Voytas, )
  • 1:1 (v/v) phenol:chloroform
  • TE buffer ( appendix 2A)
  • Chloroform
  • 100% and 70% ethanol
  • ∼0.4 µg/µl purified pOJ446 (The John Innes Centre; http://www.jic.ac.uk/)
  • HpaI restriction endonuclease (New England Biolabs)
  • DNA miniprep kit (available from various suppliers; optional)
  • 10 U/µl calf intestinal alkaline phosphatase (CIP; New England Biolabs)
  • 10× NEB Buffer 2 (New England Biolabs)
  • BamHI restriction endonuclease with 10× NEB Buffer 3 (New England Biolabs)
  • 400 U/µl T4 DNA ligase and 10× buffer (New England Biolabs)
  • Gigapack III XL (Agilent Technologies), or similar kit
  • LB agar plates with appropriate antibiotics ( appendix 4A)
  • LB liquid medium ( appendix 4A)
  • 50% (v/v) glycerol
  • 50°C drying oven
  • 1.5 ml microcentrifuge tubes
  • 16°C water bath
  • 50‐ml screw‐cap vials (Fisher, cat. no. 05‐539‐13)
  • Sterile spreader or wire loop
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )

Basic Protocol 3: Intergeneric Conjugation Between Streptomyces coelicolor CH999 or Streptomyces lividans TK 64 and E. coli

  Materials
  • E. coli ET12567/pUZ8002 competent cells (ATCC BAA‐525; plasmid pUX8002 is obtained from The John Innes Centre; http://www.jic.ac.uk/)
  • LB liquid medium ( appendix 4A) containing 25 µg/ml chloramphenicol (add from 25 mg/ml stock in ethanol) and 25 µg/ml kanamycin (add from 25 mg/ml stock in sterile double‐distilled H 2O)
  • LB agar plates ( appendix 4A) containing 25 µg/ml chloramphenicol (add from 25 mg/ml stock in ethanol) and 25 µg/ml kanamycin (add from 25 mg/ml stock in sterile double‐distilled H 2O)
  • Plasmid containing oriT (for this example, pOJ446; The John Innes Centre, http://www.jic.ac.uk/; see protocol 2)
  • Antibiotic to select for plasmid with oriT (antibiotic no. 3; for this example, apramycin; prepare 50 mg/ml stock in sterile H 2O)
  • LB liquid medium ( appendix 4A) without antibiotics
  • Streptomyces recipient strain (Streptomyces lividans TK 64 or Streptomyces coelicolor CH999; The John Innes Centre, http://www.jic.ac.uk/), well‐sporulated, growing on oatmeal agar (see recipe) plates
  • 2× YT broth (see recipe)
  • Sterile mannitol soya flour agar plates (MS agar; see recipe)
  • Nalidixic acid: dissolve 50 mg nalidixic acid (Sigma‐Aldrich) in 1 ml 0.15 M NaOH
  • R2YE medium (see recipe) supplemented with antibiotic no. 3
  • 37°C incubator with orbital shaker
  • 15‐ml culture tubes
  • Centrifuge
  • Sterile toothpicks
  • 50°C water bath
  • 28°C incubator
  • Sterile spreaders
  • Additional reagents and equipment for preparing competent E. coli (Sambrook and Russell, )

Alternate Protocol 1: Intergeneric Conjugation Between S. globisporus 1912, S. kanamyceticus, and S. cyanogenus S136, and E. coli

  Materials
  • E. coli ET12567/pUB307 competent cells (ATCC BAA‐525; plasmid pUB307 is obtained from The John Innes Centre; http://www.jic.ac.uk/)
  • LB liquid medium ( appendix 4A) containing 50 µg/ml chloramphenicol (add from 25 mg/ml stock in ethanol) and 50 µg/ml kanamycin (add from 25 mg/ml stock in sterile double‐distilled H 2O)
  • LB agar plates ( appendix 4A) containing 50 µg/ml each of kanamycin (add from 25 mg/ml stock in sterile double‐distilled H 2O), chloramphenicol (add from 25 mg/ml stock in ethanol), and apramycin (add from 50 mg/ml stock in sterile double‐distilled H 2O)
  • pSET152 (Amr, ori pUC18, int‐attP, φC31, oriT) harboring the desired genes for manipulation (Bierman et al., ; obtained from The John Innes Centre; pSET152 has restriction sites to create XbaI, BamHI, EcoRV, and EcoRI restriction fragments)
  • LB liquid medium ( appendix 4A) containing 50 µg/ml each of kanamycin (add from 25 mg/ml stock in sterile double‐distilled H 2O), chloramphenicol (add from 25 mg/ml stock in ethanol), and apramycin (add from 50 mg/ml stock in sterile double‐distilled H 2O)
  • LB liquid medium ( appendix 4A) without antibiotics
  • Oatmeal agar plates (see recipe) of S. globisporus 1912 (Luzhetskii et al., ; can be obtained from authors), S. kanamyceticus (ATCC 12853), or S. cyanogenus S136 (DSM 5087; http://www.dsm.com) grown to confluence over 3 to 5 days (well sporulated)
  • Tryptic Soy Broth (TSB; unit 10.1)
  • Oatmeal agar plates (see recipe)
  • 50 mg/ml nalidixic acid in 0.15 M NaOH
  • 50 mg/ml apramycin in sterile H 2O
  • Southern blot kit (optional; Roche) for analysis of transformants including:
    • Roche nylon membranes (cat. no. 11‐699‐075‐001 (85 mm) or cat. no. 11‐699‐083‐001 (132 mm)
    • Anti‐digoxigenin‐AP conjugate, Fab fragments (cat. no. 11‐093‐274‐910)
    • NBT/BCIP (cat. no. 11‐681‐451‐001 (8 ml)
    • DIG Easy Hyb granules [cat. no. 11‐796‐895‐001 (6× 100 ml)]
    • Blocking reagent [cat. no. 11‐096‐176‐001 (50 g)]
    • Hybridization bags [cat. no. 11‐666‐649‐001 (50 bags)]
    • DIG DNA labeling kit [cat. no. 11‐175‐033‐910 (40 labeling reactions)]
  • 37°, 29°, and 28°C incubators with orbital shakers
  • 15‐ml culture tubes
  • Centrifuge
  • Sterile toothpicks
  • 1.5‐ml microcentrifuge tubes
  • 50°C water bath
  • 250‐ml baffled Erlenmeyer flask with ventilated screw cap
  • Sterile spreaders
  • Additional reagents and equipment for preparing competent E. coli (Sambrook and Russell, )

Alternate Protocol 2: Intergeneric Conjugation Between Streptomyces verticillus and Escherichia coli

  Materials
  • E. coli S17‐1 competent cells (ATCC 47055)
  • LB liquid medium ( appendix 4A)
  • pSET152 or pRT801 (Bierman et al., ; available from The John Innes Centre)
  • LB agar plates ( appendix 4A) supplemented with 50 µg/ml apramycin (add from 50 mg/ml stock in sterile water)
  • LB liquid medium supplemented with 50 µg/ml apramycin (add from 50 mg/ml stock in sterile water)
  • Streptomyces verticillus ATCC15003 spores from a well‐sporulated ISP4 plate (see recipe) grown for 10 days
  • Tryptic Soy Broth (TSB; unit 10.1) supplemented with 10.0% (w/v) sucrose and 0.4% (w/v) glycine (add 10 g sucrose and 0.4 g glycine to 100 ml TSB, then autoclave)
  • 50 mg/ml nalidixic acid in 0.15 M NaOH
  • 50 mg/ml apramycin in sterile double‐distilled H 2O
  • ISP4 agar plates (see recipe) supplemented with 20 ml/liter of 1 M 1MgCl 2⋅6 H 2O (20 mM final), 1 g/liter of tryptone (0.1% w/v final ), and 500 mg/liter yeast extract (0.5% w/v final)
  • Southern blot kit (optional; Roche) for analysis of transformants including:
    • Roche nylon membranes (cat. no. 11‐699‐075‐001 (85 mm) or cat. no. 11‐699‐083‐001 (132 mm)
    • Anti‐digoxigenin‐AP conjugate, Fab fragments (cat. no. 11‐093‐274‐910)
    • NBT/BCIP (cat. no. 11‐681‐451‐001 (8 ml)
    • DIG Easy Hyb granules [cat. no. 11‐796‐895‐001 (6× 100 ml)]
    • Blocking reagent [cat. no. 11‐096‐176‐001 (50 g)]
    • Hybridization bags [cat. no. 11‐666‐649‐001 (50 bags)]
    • DIG DNA labeling kit [cat. no. 11‐175‐033‐910 (40 labeling reactions)]
  • 37° and 30° incubator with orbital shaker
  • Spectrophotometer
  • 15‐ml culture tubes
  • Sterile 100‐ml baffled Erlenmeyer flask
  • Sterile toothpicks
  • 1.5‐ml microcentrifuge tubes
  • 50°C water bath

Basic Protocol 4: Generation and Transformation of Streptomyces Protoplasts

  Materials
  • Yeast extract malt extract (YEME) medium (see recipe)
  • 2.5 M MgCl 2⋅6H 2O
  • 20% (w/v) glycine
  • R2YE plate of S. lividans TK 64 or S. coelicolor CH999 grown for 3 to 4 days
  • YEME medium (see recipe)
  • 10.3% (w/v) sucrose: dissolve 103 g sucrose per liter H 2O
  • Lysozyme (powder)
  • P Buffer (see recipe)
  • DNA sample
  • E. coli ET12567/pUZ8002 for passaging of DNA through non‐methylating host for S. coelicolor CH999, and/or generic E. coli strain for routine cloning
  • T Buffer (see recipe)
  • Sterile R2YE plates (see recipe)
  • Appropriate antibiotic for selection
  • Baffled 250‐ml Erlenmeyer flasks with ventilated screw caps
  • 28°C incubator with orbital shaker
  • Sterile 50‐ml centrifuge tubes with cotton filter plugs
  • Centrifuge
  • 15‐ml centrifuge tubes
  • Sterile spreaders

Basic Protocol 5: Electroporation of Streptomyces Mycelia

  Materials
  • Two plates of Streptomyces lividans TK 64 spores from fresh R2YE plate (suspended in 15% glycerol)
  • CRM liquid medium (see recipe)
  • 10.3% (w/v) sucrose: dissolve 103 g sucrose per liter H 2O
  • 15% (v/v) glycerol, ice cold
  • Lysozyme
  • Electroporation solution (see recipe)
  • Miniprep of plasmid DNA to be electroporated
  • Selective medium for plasmid
  • Sterile toothpicks
  • 50‐ml centrifuge tubes
  • 50°C water bath
  • Centrifuge
  • Electroporator (capable of administering a 2‐kV pulse)
  • Sterilized 2‐mm‐gapped electroporation cuvettes
  • 1.5‐ml microcentrifuge tubes
  • 28°C incubator with orbital shaker

Basic Protocol 6: Preparation of Samples for Colony PCR

  Materials
  • Agar plate containing Streptomyces mycelia (see unit 10.1, protocol 2)
  • PCR‐grade H 2O
  • Sterile toothpicks
  • 1.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for PCR (Kramer and Coen, )
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Figures

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Literature Cited

Literature Cited
   Bibb, M., Schottel, J.L., and Cohen, S.N. 1980. A DNA cloning system for interspecies gene‐transfer in antibiotic‐producing Streptomyces. Nature 284:526‐531.
   Bierman, M., Logan, R., O'Brien, K., Seno, E.T., Rao, R.N., and Schoner, B.E. 1992. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116:43‐49.
   Evans, G.A., Lewis, K., and Rothenberg, B.E. 1989. High efficiency vectors for cosmid microcloning and genomic analysis. Gene 79:9‐20.
   Galm, U., Wang, L., Wendt‐Pienkowski, E., Yang, R., Liu, W., Tao, M., Coughlin, J.M., and Shen, B. 2008. In vivo manipulation of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 revealing new insights into its biosynthetic pathway. J. Biol. Chem. 283:28236‐28245.
   Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F., and Hopwood, D.A. 2000. Practical Streptomyces Genetics. John Innes Foundation, Norwich, U.K.
   Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 56:15.1.1‐15.1.14.
   Luzhetskii, A.N., Ostash, B.E., and Fedorenko, V.A. 2001. Interspecies conjugation of Escherichia coli–Streptomyces globisporus 1912 using integrative plasmid pSET152 and its derivatives. Genetika 37:1340‐1347.
   Luzhetskii, A., Fedoryshyn, M., Gromyko, O., Ostash, B., Rebets, Y., Bechthold, A., and Fedorenko, V. 2006. IncP plasmids are most effective in mediating conjugation between Escherichia coli and streptomycetes. Genetika 42:595‐601.
   Mazodier, P., Petter, R., and Thompson, C. 1989. Intergeneric conjugation between Escherichia coli and Streptomyces species. J. Bacteriol. 171:3583‐3585.
   Okanishi, M., Suzuki, K., and Umezawa, H. 1974. Formation and reversion of Streptomycete protoplasts—Cultural condition and morphological study. J. Gen. Microbiol. 80:389‐400.
   Pigac, J. and Schrempf, H. 1995. A simple and rapid method of transformation of Streptomyces rimosus R6 and other Streptomycetes by electroporation. Appl. Environ. Microb. 61:352‐356.
   Rao, R.N., Richardson, M.A., and Kuhstoss, S. 1987. Cosmid shuttle vectors for cloning and analysis of Streptomyces DNA. Methods Enzymol. 153:166‐198.
   Sambrook, J. and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Vol. 1, pp. 1.117‐1.118. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
   Simon, R., Priefer, U., and Puhler, A. 1983. A broad host range mobilization system for in vivo genetic engineering—Transposon mutagenesis in Gram‐negative bacteria. Bio‐Technol. 1:784‐791.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
   Wahl, G.M., Lewis, K.A., Ruiz, J.C., Rothenberg, B., Zhao, J., and Evans, G.A. 1987. Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer. Proc. Natl. Acad. Sci. U.S.A. 84:2160‐2164.
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