Genetic Manipulation of Campylobacter jejuni

Lindsay Davis1, Kathryn Young1, Victor DiRita2

1 Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, 2 Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, Michigan
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 8A.2
DOI:  10.1002/9780471729259.mc08a02s10
Online Posting Date:  August, 2008
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Abstract

Molecular manipulation has been a limiting factor in C. jejuni research for many years. Recent advances in molecular techniques adapted for C. jejuni have furthered our understanding of the organism. This unit is dedicated to common molecular tools in bacterial research specifically tailored for C. jejuni. These include colony PCR, DNA isolation, and RNA isolation. The unit also reviews techniques for genetic manipulation, such as the use of plasmids, natural transformation, electroporation, conjugation, and transposition. In addition, a reporter system, the arylsulfatase assay, can be used to study gene expression. Curr. Protoc. Microbiol. 10:8A.2.1‐8A.2.17. © 2008 by John Wiley & Sons, Inc.

Keywords: natural transformation; arylsulfatase; genomic DNA; RNA isolation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Conjugation of C. jejuni and E. coli
  • Basic Protocol 2: Electroporation of C. jejuni
  • Basic Protocol 3: Natural Transformation of C. jejuni
  • Basic Protocol 4: Transposon Mutagenesis of C. jejuni
  • Basic Protocol 5: Preparation of C. jejuni Template for Colony PCR
  • Basic Protocol 6: Isolation of C. jejuni Genomic DNA
  • Basic Protocol 7: Isolation of RNA from C. jejuni
  • Alternate Protocol 1: Two‐Step Isolation of RNA From C. jejuni Using Trizol
  • Basic Protocol 8: Arylsulfatase Assay
  • Reagents and Solution
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Conjugation of C. jejuni and E. coli

  Materials
  • DNA fragment of interest
  • C. jejuni plasmid (e.g., pRY108 or pECO101; also see Table 8.2.1)
  • Donor strain: E. coli DH5α [pRK212.1]
  • LB agar plates with appropriate selective antibiotics ( appendix 4A)
  • Recipient strain: C. jejuni (unit 8.1)
  • Mueller Hinton (MH) agar plates, with and without appropriate selective antibiotics (BD Biosciences)
  • LB liquid medium with appropriate selective antibiotics ( appendix 4A)
  • Mueller Hinton (MH) broth without antibiotics (BD Biosciences)
  • Additional reagents and equipment for cloning of DNA (Struhl, ), introduction of plasmid DNA into E. coli (Seidman et al., ), growth of E. coli on solid medium (Elbing and Brent, ) and in liquid medium (Elbing and Brent, ), and growth of C. jejuni (unit 8.1)

Basic Protocol 2: Electroporation of C. jejuni

  Materials
  • C. jejuni frozen stock (unit 8.1)
  • Mueller Hinton (MH) agar plates with appropriate selective antibiotics (BD Biosciences)
  • Mueller Hinton (MH) broth without antibiotics (BD Biosciences)
  • Wash buffer: 15% (v/v) glycerol/9% (w/v) sucrose
  • DNA of interest, to be electroporated into C. jejuni (15 µg DNA/reaction)
  • SOC medium (see recipe)
  • Nitrocellulose membrane (0.025‐µm pore size VSWP (Millipore, cat. no. VSWP04700)
  • Electroporation cuvettes (BioRad Gene Pulser Cuvettes, 0.2‐cm electrode; cat. no. 165‐2006)
  • Electroporator (BioRad E. coli Pulser)
  • Additional reagents and equipment for streaking bacteria ( appendix 4A) and growing C. jejuni (unit 8.1)

Basic Protocol 3: Natural Transformation of C. jejuni

  Materials
  • C. jejuni frozen stock (unit 8.1)
  • Mueller Hinton (MH) agar plates with and without appropriate selective antibiotics (BD Biosciences)
  • Mueller Hinton (MH) broth without antibiotics (BD Biosciences)
  • DNA of interest, to be transformed into C. jejuni (C. jejuni genomic DNA prepared as in protocol 6)
  • Additional reagents and equipment for streaking bacteria ( appendix 4A) and growing C. jejuni (unit 8.1)

Basic Protocol 4: Transposon Mutagenesis of C. jejuni

  Materials
  • 5× salt buffer (see recipe)10 mg/ml bovine serum albumin (BSA)
  • 100 mM dithiothreitol (DTT)
  • Donor DNA (containing transposon)
  • Recipient DNA (containing target of mutagenesis or total C. jejuni genomic DNA prepared as in protocol 6)
  • Transposase: (available from Dr. David Lampe (Lampe et al., )
  • TE buffer, pH 8.0 ( appendix 2A)
  • dNTP mix: 1.25 mM each dNTP ( appendix 2A)
  • T4 DNA polymerase and T4 DNA polymerase buffer (Invitrogen)
  • T4 ligase and T4 ligase buffer (Invitrogen)
  • 30° and 70°C water baths and 11° and 16°C recirculating water baths (or thermal cycler)
  • Nitrocellulose membrane (0.025 µm pore size VSWP (Millipore, cat. no. VSWP04700)
  • Additional reagents and equipment for phenol:chloroform extraction and ethanol precipitation of DNA (Moore and Dowhan, )

Basic Protocol 5: Preparation of C. jejuni Template for Colony PCR

  Materials
  • Proteinase K
  • K buffer (see recipe)
  • 100°C water bath
  • Additional reagents and equipment for PCR (Kramer and Coen, )

Basic Protocol 6: Isolation of C. jejuni Genomic DNA

  Materials
  • C. jejuni frozen stock (unit 8.1)
  • Mueller Hinton (MH) agar plates (BD Biosciences) with 10 µg/ml trimethoprim (TMP)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 10 mM Tris·Cl, pH 8.0 ( appendix 2A)/0.1 mM EDTA
  • 10 mM Tris·Cl, pH 8.0 ( appendix 2A)/0.1 mM EDTA containing 1 mg/ml lysozyme
  • Proteinase K
  • 20% (w/v) SDS ( appendix 2A)
  • Buffered phenol ( appendix 2A)
  • 24:1 chloroform:isoamyl alcohol
  • 7.5 M ammonium acetate
  • Isopropanol
  • 70% (v/v) ethanol
  • 1 mM Tris·Cl, pH 7.5 ( appendix 2A)/0.1 mM EDTA
  • Filter paper (Whatman)
  • Additional reagents and equipment for growing C. jejuni (unit 8.1) and phenol/chloroform extraction of DNA (Moore and Dowhan, )

Basic Protocol 7: Isolation of RNA from C. jejuni

  Materials
  • C. jejuni frozen stock (unit 8.1)
  • Mueller Hinton (MH) agar plates (BD Biosciences) with 10 µg/ml trimethoprim (TMP)
  • Mueller Hinton (MH) broth (BD Biosciences) with 10 µg/ml trimethoprim (TMP)
  • RNA Protect Bacteria Reagent (Qiagen)
  • TE buffer, pH 8.0 ( appendix 2A) containing 1 mg/ml lysozyme
  • RNeasy kit (Qiagen) including:
    • Buffer RLT
    • Buffer RW1
    • Buffer RPE
    • RNA Mini Spin columns
    • 2‐ml collection tubes
    • RNase‐free H 2O
  • 2‐mercaptoethanol (2‐ME)
  • 100% ethanol, ice cold
  • 10× DNase buffer
  • 10 U/µl DNase (Invitrogen)
  • Buffered phenol ( appendix 2A)
  • Chloroform
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 70% (v/v) ethanol
  • 75‐cm2 tissue culture flask
  • 15‐ml conical centrifuge tubes
  • Centrifuge
  • Spectrophotometer
  • Additional reagents and equipment for growing C. jejuni (unit 8.1)

Alternate Protocol 1: Two‐Step Isolation of RNA From C. jejuni Using Trizol

  • 10× stop Solution: 5% (v/v) buffered phenol ( appendix 2A) in 100% ethanol
  • Liquid N 2
  • TE buffer, pH 8.0, containing 0.4 mg/ml lysozyme
  • Trizol reagent (Invitrogen)
  • Buffer RDD from RNeasy Kit (Qiagen)

Basic Protocol 8: Arylsulfatase Assay

  Materials
  • p‐Nitrophenol (Sigma, cat. no 104‐8)
  • Arylsulfatase buffer 1 (AB1): 0.1 M Tris·Cl, pH 7.2 ( appendix 2A)
  • C. jejuni (unit 8.1)
  • Mueller Hinton (MH) agar plates with antibiotics (BD Biosciences)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Arylsulfatase buffer 2 (AB2): 2 mM tyramine in 0.1 M Tris·Cl, pH 7.2
  • Arylsulfatase buffer 3 (AB3): 20 mM nitrophenyl sulfate (potassium 4‐nitrophenyl sulfate; Sigma, cat. no. N3877) in 0.1 M Tris·Cl, pH 7.2 ( appendix 2A)
  • 0.2 N NaOH
  • Spectrophotometer
  • Additional reagents and equipment for growing C. jejuni (unit 8.1)
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Figures

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Literature Cited

Literature Cited
   Elbing, K. and Brent, R. 2002a. Growth on solid media. Curr. Protoc. Mol. Biol. 59:1.3.1‐1.3.6.
   Elbing, K. and Brent, R. 2002b. Growth in liquid media. Curr. Protoc. Mol. Biol. 59:1.2.1‐1.2.2.
   Gaynor, E.C., Wells, D.H., MacKichan, J.K., and Falkow, S. 2005. The Campylobacter jejuni stringent response controls specific stress survival and virulence‐associated phenotypes. Mol. Microbiol. 56:8‐27.
   Golden, N.J., Camilli, A., and Acheson, D.W. 2000. Random transposon mutagenesis of Campylobacter jejuni. Infect. Immun. 68:5450‐5453.
   Hendrixson, D.R., Akerley, B.J., and DiRita, V.J. 2001. Transposon mutagenesis of Campylobacter jejuni identifies a bipartite energy taxis system required for motility. Mol. Microbiol. 40:214‐224.
   Hendrixson, D.R. and DiRita, V.J. 2003. Transcription of sigma54‐dependent but not sigma28‐dependent flagellar genes in Campylobacter jejuni is associated with formation of the flagellar secretory apparatus. Mol. Microbiol. 50:687‐702.
   Hendrixson, D.R. and DiRita, V.J. 2004. Identification of Campylobacter jejuni genes involved in commensal colonization of the chick grastrointestinal tract. Mol. Microbiol. 52:471‐484.
   Kakuda, T. and DiRita, V.J. 2006. Cj1496c encodes a Campylobacter jejuni glycoprotein that influences invasion of human epithelial cells and colonization of the chick gastrointestinal tract. Infect. Immun. 74:4715‐4723.
   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.
   Lampe, D.J., Akerley, B.J., Rubin, E.J., Mekalanos, J.J., and Robertson, H.M. 1999. Hyperactive transposase mutants of the Himar1 mariner transposon. Proc. Natl. Acad. Sci. U.S.A. 96:11428‐11433.
   Miller, J.F., Dower, W.J., and Tompkins, L.S. 1988. High‐voltage electroporation of bacteria: Genetic transformation of Campylobacter jejuni with plasmid DNA. Proc. Natl. Acad. Sci. U.S.A. 85:856‐860.
   Moore, D. and Dowhan, D. 2002. Purification and concentration of DNA from aqueous solutions. Curr. Protoc. Mol. Biol. 59:2.1.1‐2.1.10.
   Rubin, E.J., Akerley, B.J., Novik, V.N., Lampe, D.J., Husson, R.N., and Mekalanos, J.J. 1999. In vivo transposition of mariner‐based elements in enteric bacteria and Mycobacteria. Proc. Natl. Acad. Sci. U.S.A. 96:1645‐1650.
   Seidman, C.E., Struhl, K., Sheen, J., and Jessen, T. 1997. Introduction of plasmid DNA into cells. Curr. Protoc. Mol. Biol. 37:1.8.1‐1.8.10.
   Struhl, K. 2000. Subcloning of DNA fragments. Curr. Protoc. Mol. Biol. 13:3.16.1‐3.16.11.
   Wang, Y. and Taylor, D.E. 1990. Natural transformation in Campylobacter species. J. Bacteriol. 172:949‐955.
   Wiesner, R.S., Hendrixson, D.R., and DiRita, V.J. 2003. Natural transformation of Campylobacter jejuni requires components of a type II secretion system. J. Bacteriol. 185:5408‐5418.
   Yao, R., Alm, R.A., Trust, T.J., and Guerry, P. 1993. Construction of a new Campylobacter cloning vector and a new mutational cat cassette. Gene 130:127‐130.
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