Genetic Manipulation of Porphyromonas gingivalis

Myriam Bélanger1, Paulo Rodrigues1, Ann Progulske‐Fox1

1 University of Florida, Gainesville, Florida
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 13C.2
DOI:  10.1002/9780471729259.mc13c02s05
Online Posting Date:  June, 2007
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Abstract

Porphyromonas gingivalis, an oral anaerobic bacterium, is an important etiological agent of periodontal disease and may contribute to cardiovascular disease, preterm birth, and diabetes as well. Therefore, genetic studies are of crucial importance in investigating molecular mechanisms of P. gingivalis virulence. Although molecular genetic tools have been available for many bacterial species for some time, genetic manipulations of Porphyromonas species were not developed until more recently and remain limited. In this unit, current molecular genetic approaches for mutant construction in P. gingivalis using the suicide vector pPR‐UF1 and the transposon Tn4351 are described, as are protocols for performing electroporation and conjugation. Furthermore, a technique to restore the wild‐type phenotype of the mutant by complementation using vector pT‐COW is provided. Finally, a description of a noninvasive reporter system allowing the study of gene expression and regulation in P. gingivalis completes this unit.

Keywords: Porphyromonas gingivalis; molecular genetics; mutation; transposon; vector; recombinant DNA; conjugation; Bacteroides; oral pathogens; anaerobic bacteria

     
 
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Table of Contents

  • Basic Protocol 1: Genetic Transformation of Porphyromonas gingivalis
  • Basic Protocol 2: Mutant Construction in Porphyromonas gingivalis Using Suicide Vector pPR‐UF1
  • Basic Protocol 3: Introducing DNA into Porphyromonas gingivalis by Conjugation (Agar Plate Method)
  • Alternate Protocol 1: Introducing DNA Into Porphyromonas gingivalis by Conjugation (Broth Culture Method)
  • Basic Protocol 4: Complementation of Porphyromonas gingivalis Mutants
  • Basic Protocol 5: Reporter Gene System Using Xylosidase/Arabinosidase
  • Basic Protocol 6: Mutant Construction Using Transposon Tn4351 (Broth Culture Method)
  • Alternate Protocol 2: Mutant Construction Using Transposon Tn4351 (Agar Plate Method)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Genetic Transformation of Porphyromonas gingivalis

  Materials
  • Supplemented tryptic soy broth (TSB), prereduced and prewarmed (see recipe)
  • 3‐ to 4‐day blood agar plate culture of P. gingivalis W83 (ATCC #BAA‐308)
  • Electroporation (EP) buffer (see recipe), cold
  • DNA for transforming P. gingivalis (see protocol 2)
  • Blood agar plates (see recipe) containing appropriate antibiotics (e.g., 50 µg/ml gentamicin plus 5.0 µg/ml erythromycin; see protocol 2)
  • Refrigerated centrifuge and sterile 50‐ml centrifuge tubes
  • 0.5‐ml microcentrifuge tubes
  • 0.2‐cm‐gap electroporation cuvettes, sterile and prechilled
  • Electroporation apparatus: gene pulser and pulse controller (e.g., Bio‐Rad)
  • Additional reagents and equipment for Southern blotting (unit 14.1; also see Brown, , )

Basic Protocol 2: Mutant Construction in Porphyromonas gingivalis Using Suicide Vector pPR‐UF1

  Materials
  • Supplemented tryptic soy broth (TSB), prereduced and prewarmed (see recipe)
  • 3‐ to 4‐day blood agar plate culture of P. gingivalis W83 (ATCC #BAA‐308) or other P. gingivalis strain of interest
  • Wizard Genomic DNA Purification Kit (Promega; use manufacturer's Isolation of Genomic DNA from Gram Positive and Gram Negative protocol)
  • Nuclease‐free distilled deionized H 2O
  • PCR primers designed to amplify fragments A and B of the gene of interest
  • QIAquick PCR Purification Kit (Qiagen)
  • Restriction enzymes to cut fragments (inserts) A and B of the gene of interest (Fig. )
  • LigaFast Rapid DNA Ligation System (Promega)
  • Competent E. coli cells (e.g., DH5α, Invitrogen)
  • Plasmid pPR‐UF1 (can be obtained from the author's laboratory; )
  • LB plates ( appendix 4A) containing 300 µg/ml erythromycin (add from 300 mg/ml erythromycin stock; see recipe)
  • QIAprep Spin Miniprep Kit (Qiagen)
  • 1% agarose gel (Voytas, )
  • Restriction enzyme HindIII for screening to confirm the presence of inserts in recombinant plasmids pPR‐UF1A and pPR‐UF1AB
  • Restriction enzyme AfeI, PciL, DrdI, ApaLI, AlwNI, or Eco57I (Fig. ) for linearizing the recombinant plasmid, pPR‐UF1AB
  • Electrocompetent P. gingivalis cells (see protocol 1, steps 1 to 7)
  • Blood agar plates (see recipe) containing 50 µg/ml gentamicin (add from 50 mg/ml stock; see recipe) and 5.0 µg/ml erythromycin (add from 5.0 mg/ml stock; see recipe)
  • Refrigerated centrifuge
  • DNase‐free pipet tips and tubes
  • Additional reagents and equipment for DNA purification (Moore and Dowhan, ), determining DNA concentration (Gallagher and Desjardins, ), the polymerase chain reaction (PCR; Kramer and Coen, ), restriction digestion (Bloch and Grossman, ), transformation of E. coli (Seidman et al., ), agarose gel electrophoresis (Voytas, ), transformation of P. gingivalis by electroporation ( protocol 1), and Southern blotting (unit 14.1; also see Brown, , )

Basic Protocol 3: Introducing DNA into Porphyromonas gingivalis by Conjugation (Agar Plate Method)

  Materials
  • Recipient P. gingivalis strain W83 (ATCC #BAA‐308) or other P. gingivalis strain of interest
  • Blood agar plates (see recipe) without antibiotics
  • Donor E. coli strain (S17‐1; not available commercially; can be obtained from authors' laboratory, )
  • LB plates ( appendix 4A) containing appropriate antibiotics (see Critical Parameters and Troubleshooting)
  • Supplemented tryptic soy broth (TSB), prereduced and prewarmed (see recipe)
  • Blood agar plates (see recipe) containing 150 µg/ml gentamicin and appropriate antibiotic to select for transconjugants (see Critical Parameters and Troubleshooting)
  • Blood agar plates (see recipe) containing 50 µg/ml gentamicin and appropriate antibiotic to ensure antibiotic resistance and purity of transconjugants (see Critical Parameters and Troubleshooting)

Alternate Protocol 1: Introducing DNA Into Porphyromonas gingivalis by Conjugation (Broth Culture Method)

  • LB liquid medium ( appendix 4A) without antibiotics
  • Sterile filter membrane (cellulose esters membrane; Millipore)
  • Anaerobic chamber (unit 12.1)

Basic Protocol 4: Complementation of Porphyromonas gingivalis Mutants

  Materials
  • PCR primers designed to amplify the gene of interest
  • P. gingivalis chromosomal DNA, purified (see protocol 2)
  • Restriction enzymes appropriate for cutting the insert and for cutting the plasmid pT‐COW (see Bloch and Grossman, ; also see Fig. )
  • QIAquick PCR Purification Kit (Qiagen)
  • Plasmid pT‐COW (Shoemaker et al., ; not available commercially; contact author at for more information)
  • LigaFast Rapid DNA Ligation System (Promega)
  • Electrocompetent E. coli strain S17‐1 cells (can be obtained from the author's laboratory; )
  • LB agar plates ( appendix 4A) containing 50 µg/ml carbenicillin (add from 50 mg/ml stock; see recipe) or 5.0 µg/ml tetracycline (add from 10 mg/ml stock; see recipe)
  • QIAprep Spin Miniprep Kit (Qiagen)
  • 1% agarose gel (Voytas, )
  • Additional reagents and equipment for the polymerase chain reaction (PCR; Kramer and Coen, ), DNA purification (Moore and Dowhan, ), determining DNA concentration (Gallagher and Desjardins, ), the polymerase chain reaction (PCR; Kramer and Coen, ), restriction digestion (Bloch and Grossman, ), transformation of E. coli (Seidman et al., ), agarose gel electrophoresis (Voytas, ), transformation of P. gingivalis by conjugation ( protocol 3 or protocol 4), large‐scale preparation of plasmid DNA (Heilig et al., ), and Southern blotting (unit 14.1; also see Brown, , )

Basic Protocol 5: Reporter Gene System Using Xylosidase/Arabinosidase

  Materials
  • PCR primers designed to amplify the gene of interest
  • P. gingivalis chromosomal DNA, purified (see protocol 2)
  • High‐fidelity Taq DNA polymerase
  • QIAquick PCR Purification Kit (Qiagen)
  • Restriction enzymes appropriate for cutting the insert and for cutting the plasmid pT‐COW (see Bloch and Grossman, ; also see Fig. )
  • QIAquick Gel Extraction Kit (Qiagen)
  • Plasmid pXA1 (Whitehead, )
  • Restriction enzymes EcoRI and BamHI for cutting plasmid pXA1 (also see Bloch and Grossman, )
  • Plasmid pT‐COW (Shoemaker et al., ; not available commercially; contact author at for more information)
  • 1% agarose gels (see Voytas, )
  • LigaFastTM Rapid DNA Ligation System (Promega)Nuclease‐free H 2O
  • Electrocompetent E. coli strain S17‐1 cells (can be obtained from the author's laboratory; )
  • QIAprep Miniprep Kit (Qiagen)
  • 25°C water bath
  • Additional reagents and equipment for the polymerase chain reaction (PCR; Kramer and Coen, ), DNA purification (Moore and Dowhan, ), restriction digestion (Bloch and Grossman, ), agarose gel electrophoresis (Voytas, ), transformation of E. coli (Seidman et al., ), transformation of P. gingivalis by conjugation ( protocol 3 or protocol 4), and large‐scale preparation of plasmid DNA (Heilig et al., )

Basic Protocol 6: Mutant Construction Using Transposon Tn4351 (Broth Culture Method)

  Materials
  • Supplemented tryptic soy broth (TSB), prereduced and prewarmed (see recipe)
  • 3‐ to 4‐day blood agar plate culture of P. gingivalis W83 (ATCC #BAA‐308)
  • E. coli strain HB101 containing plasmid R751::Tn4351Ω4 (not available commercially; see Shoemaker et al., ): culture overnight
  • LB liquid medium ( appendix 4A) containing 300 µg/ml erythromycin (add from 300 mg/ml stock; see recipe)
  • Blood agar plates (see recipe), antibiotic free
  • Blood agar plates (see recipe) containing 150 µg/ml gentamicin (add from 50 mg/ml stock; see recipe) and 5 µg/ml of erythromycin (add from 300 mg/ml stock; see recipe)
  • 37°C shaking incubator
  • Additional reagents and equipment for Southern blotting (unit 14.1; also see Brown, , )

Alternate Protocol 2: Mutant Construction Using Transposon Tn4351 (Agar Plate Method)

  • E. coli strain HB101 containing plasmid R751::Tn4351Ω4 (not available commercially; see Shoemaker et al., )
  • LB agar plates ( appendix 4A) containing 10 µg/ml tetracycline (add from 10 mg/ml tetracycline stock; see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Cellulose esters membrane (Millipore)
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Figures

Videos

Literature Cited

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Key Reference
   Kuramitsu, H. K. 2003. Molecular genetic analysis of the virulence of oral bacterial pathogens: An historical perspective. Crit. Rev. Oral Biol. Med. 14:331‐344.
  Describes genetic manipulations that can be performed with various bacteria, including P. gingivalis.
   Lamont, R.J. and Jenkinson, H.F. 1998. Life below the gum line: Pathogenic mechanisms of Porphyromonas gingivalis. Microbiol. Mol. Biol. Rev. 62:1244‐1263.
  Comprehensive review article describing the various virulence factors of P. gingivalis.
   Nakayama, K. 2003. Molecular genetics of Porphyromonas gingivalis: Gingipains and other virulence factors. Curr. Protein Pept. Sci. 4:389‐395.
  Review describing the introduction of molecular genetics to analysis of pathogenesis of P. gingivalis. Special emphasis is placed on proteinases.
Internet Resources
  http://www.tigr.org/tigr‐scripts/CMR2/GenomePage3.spl?database=gpg
  Access to whole genome sequence of Porphyromonas gingivalis strain W83 as well as genomic analysis tools.
   http://www.tigr.org
  The Institute for Genomic Research (TIGR) provides structural, functional and comparative analysis of genomes and gene products.
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