Genetic Manipulation of Acinetobacter baumannii

Anna C. Jacobs1, Mitchell G. Thompson1, Michael Gebhardt2, Brendan W. Corey1, Suleyman Yildirim1, Howard A. Shuman2, Daniel V. Zurawski1

1 Walter Reed Army Institute of Research, Department of Wound Infections, Silver Spring, Maryland, 2 Department of Microbiology, University of Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 6G.2
DOI:  10.1002/9780471729259.mc06g02s35
Online Posting Date:  November, 2014
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Abstract

Acinetobacter baumannii is a Gram‐negative nosocomial pathogen of clinical importance. A lack of genetic tools has hindered the research of this organism in the past; however, recently, various methods have been designed, modified, and optimized to facilitate the genetic manipulation of A. baumannii. This unit describes some of the recent genetic advances and new recombinant tools developed for this pathogen, including standard transformation and conjugation techniques specifically developed for the bacteria. As the need to understand the basic biology of A. baumannii increases with the prospect of developing new therapeutics, the use of the basic genetic methods herein can provide the critical first step to identify genes required for infection. © 2014 by John Wiley & Sons, Inc.

Keywords: Acinetobacter baumannii; mutagenesis; conjugation; transformation; electroporation; plasmid

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: DNA Transfer via Conjugation
  • Basic Protocol 2: Bacterial Transformation via Electroporation
  • Support Protocol 1: Making Electrocompetent A. baumannii Cells
  • Basic Protocol 3: Natural Bacterial Transformation via Motility
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: DNA Transfer via Conjugation

  Materials
  • A. baumannii recipient strain grown on agar plate
  • Donor strain grown on an agar plate
  • E. coli strain containing helper plasmid grown on agar plate
  • Lysogeny broth (LB; see recipe)
  • LB agar plates (see recipe)
  • LB agar plates containing selective antibiotic
  • Pipets and sterile tips
  • 14‐ml sterile plastic round‐bottom tubes
  • 37°C shaking incubator
  • 1.5‐ml microcentrifuge tubes, sterile
  • Microcentrifuge
  • 37°C stationary incubator
  • Sterile inoculation needles
  • L‐shaped plate spreader or sterile glass beads

Basic Protocol 2: Bacterial Transformation via Electroporation

  Materials
  • Electrocompetent A. baumannii cells (see the protocol 3Support Protocol)
  • SOC medium (see recipe)
  • LB agar plates containing selective antibiotic (see recipe)
  • DNA
  • Pipets and sterile tips
  • 1.5‐ml microcentrifuge tubes
  • 1‐mm electroporation cuvettes
  • Electroporator
  • 14‐ml sterile plastic round‐bottom tubes
  • 37°C shaking incubator
  • L‐shaped plate spreader or sterile glass beads
  • 37°C stationary incubator

Support Protocol 1: Making Electrocompetent A. baumannii Cells

  Materials
  • Overnight culture of A. baumannii strain(s) (see unit 6.1)
  • LB (see recipe)
  • 10% glycerol (see recipe), sterile
  • 500‐ml glass Erlenmeyer flask
  • Pipet and sterile tips
  • Pipetman and serological pipets
  • 37°C shaking incubator
  • 50‐ml conical tubes
  • Tabletop centrifuge

Basic Protocol 3: Natural Bacterial Transformation via Motility

  Materials
  • A. baumannii strain grown on agar plate
  • Phosphate‐buffered saline (PBS; see recipe)
  • DNA for transformation
  • Motility agar plates (see recipe)
  • LB agar plates containing selective antibiotic
  • Pipets and sterile tips
  • 1.5‐ml microcentrifuge tubes
  • 37°C incubator
  • L‐shaped plate spreader or sterile glass beads
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Figures

Videos

Literature Cited

Literature Cited
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