Vibrio cholerae: Measuring Natural Transformation Frequency

Samit S. Watve1, Eryn E. Bernardy1, Brian K. Hammer1

1 School of Biology, Georgia Institute of Technology, Atlanta
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 6A.4
DOI:  10.1002/9780471729259.mc06a04s35
Online Posting Date:  November, 2014
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Abstract

Many bacteria can become naturally competent to take up extracellular DNA across their outer and inner membranes by a dedicated competence apparatus. Whereas some studies show that the DNA delivered to the cytoplasm may be used for genome repair or for nutrition, it can also be recombined onto the chromosome by homologous recombination: a process called natural transformation. Along with conjugation and transduction, natural transformation represents a mechanism for horizontal transfer of genetic material, e.g., antibiotic resistance genes, which can confer new beneficial characteristics onto the recipient bacteria. Described here are protocols for quantifying the frequency of transformation for the human pathogen Vibrio cholerae, one of several Vibrio species recently shown to be capable of natural transformation. © 2014 by John Wiley & Sons, Inc.

Keywords: natural transformation; competence; Vibrio cholerae; chitin; TfoX

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Chitin‐Induced Transformation Assay
  • Support Protocol 1: Preparing Extracellular DNA
  • Support Protocol 2: Preparing Autoclaved Crab Shell Fragments
  • Alternate Protocol 1: Transformation Assay with Preinduction by Chitin Exposure
  • Alternate Protocol 2: Chitin‐Independent Transformation Assay with a Constitutively Active Allele
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Chitin‐Induced Transformation Assay

  Materials
  • V. cholerae frozen stock (Martinez et al., )
  • Luria‐Bertani broth (LB; appendix 4A)
  • Artificial sea water (ASW) medium (17 g/liter Instant Ocean, cat. no. SS15‐10)
  • eDNA containing an antibiotic resistance marker (AbR; protocol 2)
  • Autoclaved crab shell fragments ( protocol 3)
  • LB agar plates with and without appropriate antibiotics ( appendix 4A)
  • 24‐ml glass culture tubes with tops (Fisher Scientific, cat. no. S00182), sterile
  • 30° and 37°C incubators
  • Test tube shaker
  • Spectrophotometer with 600‐nm filter
  • 12‐well sterile, flat‐bottom, standard tissue culture–treated microtiter plates with lids (Corning Life Sciences DL, cat. no. 353043)
  • Forceps, sterile

Support Protocol 1: Preparing Extracellular DNA

  Materials
  • PCR kit (e.g., Qiagen Taq PCR core kit)
  • PCR primers (see Fig. )
  • Additional reagents and equipment for carrying out PCR (Kramer and Coen, ), agarose gel electrophoresis (Voytas, ), DNA quantification in small volumes (Desjardins and Conklin, ), and sequencing of DNA (Slatko, et al., )

Support Protocol 2: Preparing Autoclaved Crab Shell Fragments

  Materials
  • Boiled blue crab (Callinectes sapidus)
  • Sterile water

Alternate Protocol 1: Transformation Assay with Preinduction by Chitin Exposure

  Materials (see protocol 1Basic Protocol) 

Alternate Protocol 2: Chitin‐Independent Transformation Assay with a Constitutively Active Allele

  Additional Materials (also see protocol 1Basic Protocol)
  • V. cholerae strain EA305 (Antonova et al., 2012), a derivative of C6706str2 (see unit 6.1), carrying a chromosomally‐encoded, constitutive tfoX allele
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Figures

Videos

Literature Cited

Literature Cited
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  Antonova, E.S., Bernardy, E.E., and Hammer, B.K. 2012. Natural competence in Vibrio cholerae is controlled by a nucleoside scavenging response that requires CytR‐dependent anti‐activation. Mol. Microbiol. 86:1215‐1231.
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Key References
  Meibom et al., 2005. See above.
  The first demonstration that Vibrio cholerae is naturally competent for DNA uptake when incubated in the presence of chitin.
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