Extraction, Purification, and Identification of Yersiniabactin, the Siderophore of Yersinia pestis

M. Clarke Miller1, Edward DeMoll2

1 James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky, 2 Department of Biology, University of Kentucky, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 5B.3
DOI:  10.1002/9780471729259.mc05b03s23
Online Posting Date:  November, 2011
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This unit describes in detail the extraction, purification, and identification of Yersiniabactin the siderophore of Yersinia pestis. Iron is essential for bacterial growth. Although relatively abundant, access to iron is limited in nature by low solubility. This problem is exacerbated for pathogenic bacteria, which must also defeat the host organism's innate defenses, including mechanisms to sequester iron. One solution to these problems is production of water soluble, small molecules with high affinities for iron called siderophores. This protocol has been fine tuned for Yersiniabactin purification but may be easily modified for use in isolating other siderophores or similar molecules. Curr. Protoc. Microbiol. 23:5B.3.1‐5B.3.22. © 2011 by John Wiley & Sons, Inc.

Keywords: siderophore; iron; extraction; purification; Yersiniabactin; Yersinia pestis; plague

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Extraction, Purification, and Identification of Yersiniabactin
  • Support Protocol 1: Concentration of HPLC Fractions by Sep‐Pak Reversed‐Phase Cartridge
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Extraction, Purification, and Identification of Yersiniabactin

  • Yersinia pestis KIM6+ (units 5.1& 5.2)
  • PMH2 medium (see recipe)
  • Ethyl acetate
  • Ethanol
  • Methanol
  • HPLC mobile phases:
    • Buffer A: water with 10% (v/v) acetonitrile, pH 8.0 (adjusted with NH 4OH)
    • Buffer B: water with 25% (v/v) acetonitrile, pH 8.0 (adjusted with NH 4OH)
    • Buffer C: acetonitrile (HPLC‐grade)
    • Buffer D: water (HPLC‐grade or 22‐µm filtered distilled deionized)
  • Acetonitrile
  • Centrifuge (e.g., Beckman‐Coulter Avanti J‐26 equipped with J‐10 or JLA‐10.500 rotor)
  • 500‐ml centrifuge bottles (e.g., Beckmann, cat. no. 355607)
  • 0.22‐µm bottle top filter (e.g., Corning bottle‐top filter system, part no. 341098)
  • Aluminum foil
  • 3‐ and 4‐liter separatory funnels
  • Ring (large enough to support separatory funnel) and ring stand
  • Rotary evaporator with water bath
  • Test tube clamp for ring stand
  • 5‐ or 10‐ml syringes
  • Sep‐Pak C18 cartridges (Waters, cat. no. WAT051910)
  • Syringe filters (0.22‐µm)
  • Centrivap concentrator or lyophilizer
  • HPLC system with:
    • Waters 600 pump and controller
    • Waters 2998 UV‐vis photodiode array detector
    • Waters 2702 auto‐sampler with 250‐µl sample loop
    • Waters fraction collector III (optional)
  • µRPC C2/C18 ST 4.6/100 column (GE Healthcare, cat. no. 17‐5057‐01)
  • 1.5‐ml microcentrifuge tubes (optional)
  • UV‐vis (optional)

Support Protocol 1: Concentration of HPLC Fractions by Sep‐Pak Reversed‐Phase Cartridge

  • Methanol
  • HPLC‐purified Yersiniabactin fractions
  • Ring stand
  • Test tube clamp for ring stand
  • 5‐ and 10‐ml syringes
  • Sep‐Pak C18 cartridges (Waters, cat. no. WAT051910)
  • Aluminum foil
  • Centrivap concentrator or lyophilizer
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Literature Cited

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