Genetic Manipulation of Treponema denticola

Howard K. Kuramitsu1, Bo Chi2, Akihiko Ikegami3

1 State University of New York at Buffalo, Buffalo, New York, 2 U.S. Food and Drug Administration, Bethesda, Maryland, 3 Case Western Reserve University, Cleveland, Ohio
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 12B.2
DOI:  10.1002/9780471729259.mc12b02s00
Online Posting Date:  July, 2005
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Abstract

The oral anaerobic spirochete, Treponema denticola, has been implicated in the etiology of human periodontal diseases; however, the molecular basis for the virulence of these organisms is still unclear. Potential pathogenic factors expressed by T. denticola have recently begun to be identified through the development of gene transfer approaches in this organism following electroporetic transformation. Several antibiotic resistance markers have been developed for use in the construction of monospecific mutants in these organisms. In addition, these antibiotic resistance cassettes have been more recently utilized to construct shuttle plasmids for complementation analysis of the mutants. These plasmids were also used to express heterologous spirochete genes in T. denticola. The transformation of other spirochetes such as T. phagedenis with these plasmids further suggests that it should be possible to develop similar gene transfer systems in other cultivable treponemes.

Keywords: Spirochaetales; Treponema denticola; electroporation; gene silencing; plasmids; genetic transformation; heterologous gene expression

     
 
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Table of Contents

  • Basic Protocol 1: Transformation of T. denticola with Plasmid DNA
  • Support Protocol 1: Preparation of Electrocompetent T. denticola for Plasmid Transformation
  • Basic Protocol 2: Electrotransformation of T. denticola for Alleleic‐Exchange Mutagenesis
  • Support Protocol 2: Production of Electrocompetent T. denticola for Use in Alleleic Exchange Mutagenesis
  • Support Protocol 3: Generation of a Targeted Gene‐Deletion Construct
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Transformation of T. denticola with Plasmid DNA

  Materials
  • Electrocompetent T. denticola, ATCC #35405 or ATCC #33520 (see protocol 2)
  • Appropriate shuttle plasmid: pKMR4PE (Chi et al., ), pKMCou (Chi et al., ), or pBFC (Sliviesnki‐Gebhardt et al., )
  • TYGVS medium (unit 12.1), with and without appropriate antibiotic (Sigma) for growth: erythromycin at 40 µg/ml, chloramphenicol at 10 µg/ml, or coumermycin A1 at 10 µg/ml
  • TYGVS medium (unit 12.1) containing 0.8% (w/v) SeaPlaque agarose (VWR) and appropriate antibiotic (Sigma): erythromycin at 40 µg/ml (for pKMR4PE), chloramphenicol at 10 µg/ml (for pBFC), or coumermycin A1 at 10 µg/ml (for pKMCou)
  • Gene Pulser (Bio‐Rad)
  • 0.1‐cm‐gap electroporation cuvettes (Bio‐Rad)
  • 15‐ml plastic screw cap test tubes (VWR)
  • Anaerobic chamber (unit 12.1)
  • 100 × 15–mm petri dishes
  • Sterile inoculation loops or sterile Pasteur pipets
  • Additional reagents and equipment for cultivation of T. denticola (unit 12.1)

Support Protocol 1: Preparation of Electrocompetent T. denticola for Plasmid Transformation

  Materials
  • T. denticola, ATCC #35405 or ATCC #33520
  • TYGVS medium (unit 12.1)
  • 10% (v/v) glycerol, ice‐cold
  • Anaerobic chamber
  • 50°C water bath
  • Sorvall RC5 or equivalent refrigerated centrifuge
  • Additional reagents and equipment for cultivation of T. denticola (unit 12.1)

Basic Protocol 2: Electrotransformation of T. denticola for Alleleic‐Exchange Mutagenesis

  Materials
  • Electrocompetent T. denticola, ATCC #35405 or ATCC #33520 (see protocol 4)
  • Plasmid pVA2198 (Fletcher et al., ; contact H. Fletcher, Loma Linda University, Loma Linda, Calif.)
  • T. denticola (ATCC #35405 or ATCC #33520)
  • TYGVS medium (unit 12.1) with and without 40 µg/ml erythromycin (Sigma)
  • TYGVS medium (unit 12.1) containing 0.8% (w/v) SeaPlaque agarose (VWR) and 40 µg/ml erythromycin (Sigma)
  • Gene Pulser (Bio‐Rad)
  • 0.1‐cm‐gap electroporation cuvettes (Bio‐Rad)
  • 15‐ml plastic screw cap test tubes (VWR)
  • Anaerobic chamber (unit 12.1)
  • 100 × 15–mm petri dishes
  • Sterile inoculation loops or sterile Pasteur pipets
  • Additional reagents and equipment for cultivation of T. denticola (unit 12.1)

Support Protocol 2: Production of Electrocompetent T. denticola for Use in Alleleic Exchange Mutagenesis

  Materials
  • T. denticola, ATCC #35405 or ATCC #33520
  • TYGVS medium (unit 12.1)
  • 10% (v/v) glycerol, ice‐cold
  • Anaerobic chamber
  • 50°C water bath
  • Sorvall RC5 or equivalent refrigerated centrifuge
  • Additional reagents and equipment for cultivation of T. denticola (unit 12.1)
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Figures

Videos

Literature Cited

Literature Cited
   Cheng, S.‐L., Siboo, R., Quee, T.C., Johnson, J.L., Mayberry, W.R., and Chan, E.C.S. 1985. Comparative study of six random oral spirochete isolates. J. Periodontal Res. 20:602‐612.
   Chi, B., Chauhan, S., and Kuramitsu, H. 1999. Development of a system for expressing heterologous genes in the oral spirochete Treponema denticola and its use in expression of the Treponema pallidum flaA gene. Infect. Immun. 67:3653‐3656.
   Chi, B., Limberger, R.J., and Kuramitsu, H.K. 2002. Complementation of a Treponema denticola flgE mutant with a novel coumermycin A1‐resistant T. denticola shuttle vector system. Infect. Immun. 70:2233‐2237.
   Fenno, J.C., Wong, G.W., Hannam, P.M., and McBride, B.C. 1998. Mutagenesis of outer membrane virulence determinants of the spirochete Treponema denticola. FEMS Microbiol. Lett 163:209‐215.
   Fletcher, H.M., Schenkein, H.A., Morgan, R.M., Bailey, K.A., Berry, C.R., and Macrina, F.L. 1995. Virulence of a Porphyromonas gingivalis W83 mutant defective in the prtH gene. Infect. Immun. 63:1521‐1528.
   Ikegami, A., Honma, K., Sharma, A., and Kuramitsu, H.K. 2004. Multiple functions of the leucine‐rich repeat protein LrrA of Treponema denticola. Infect. Immun. 72:4619‐4627.
   Ishihara, K., Kuramitsu, H.K., Miura, T., and Okuda, K. 1998. Dentilisin activity affects the organization of the outer sheath of Treponema denticola. J. Bacteriol. 180:3837‐3844.
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   Slivienski‐Gebhardt, L.L., Izard, J., Samsonoff, W.A., and Limberger, R.J. 2004. Development of a novel chloramphenicol resistance expression plasmid used for genetic complementation of a fliG deletion mutant in Treponema denticola. Infect. Immun. 72:5493‐5497.
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