Genetic Manipulation of Clostridium difficile

Laurent Bouillaut1, Shonna M. McBride1, Joseph A. Sorg2

1 Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, 2 Department of Biology, Texas A & M University, College Station, Texas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 9A.2
DOI:  10.1002/9780471729259.mc09a02s20
Online Posting Date:  February, 2011
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Abstract

Clostridium difficile is a Gram‐positive, spore forming, anaerobic, intestinal bacterium and is the most common cause of antibiotic‐associated colitis. For many years this organism was considered genetically intractable, but in the past 10 years, multiple methods have been developed or adapted for genetic manipulation of C. difficile. This unit describes the molecular techniques used for genetic modification of this organism, including methods for gene disruption, complementation, plasmid introduction and integration, and cross‐species conjugations. Curr. Protoc. Microbiol. 20:9A.2.1‐9A.2.17. © 2011 by John Wiley & Sons, Inc.

Keywords: Clostridium difficile; firmicute; anaerobic chamber; plasmid; genomic DNA; RNA; transposon; conjugation; group II intron; mutagenesis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Extraction of C. difficile Genomic DNA
  • Alternate Protocol 1: Quick Genomic DNA Extraction from C. difficile for PCR
  • Basic Protocol 2: Extraction of C. difficile RNA
  • Basic Protocol 3: Transfer of Tn916 from a Bacillus subtilis Donor Strain to a C. difficile Recipient Strain
  • Basic Protocol 4: Transfer of Plasmid DNA from an E. coli Donor to C. difficile Recipient
  • Basic Protocol 5: Gene Disruption in C. difficile Using Group II Intron Targeting
  • Basic Protocol 6: Site‐Directed Mutagenesis Using Unstable Plasmids
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Extraction of C. difficile Genomic DNA

  Materials
  • Liquid BHIS for C. difficile growth (see recipe)
  • Viable C. difficile on a petri plate (unit 9.1)
  • TE buffer ( appendix 2A)
  • Genomic DNA solution (see recipe)
  • Fresh lysozyme solution (see recipe)
  • 20% Sarkosyl (see recipe)
  • 10 mg/ml RNase A (see recipe)
  • 10 mg/ml proteinase K (see recipe)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (unit 2.1)
  • Chloroform
  • 3 M sodium acetate (see recipe)
  • 75% and 95% ethanol
  • 37°C anaerobic chamber
  • Refrigerated centrifuge
  • 37°C water bath or heating block

Alternate Protocol 1: Quick Genomic DNA Extraction from C. difficile for PCR

  • 1× PCR buffer
  • Sterile inoculating loops or pipet tips
  • Microcentrifuge tubes
  • 58°C and 90°C water baths or heating blocks

Basic Protocol 2: Extraction of C. difficile RNA

  Materials
  • Viable C. difficile grown on a petri plate (unit 9.1)
  • Liquid medium for C. difficile growth (i.e., BHIS or other suitable medium; see reciperecipes)
  • 1:1 (v/v) acetone/ethanol mix
  • TE buffer ( appendix 2A)
  • TRIzol (Invitrogen)
  • Chloroform
  • Cold isopropanol
  • Diethylpyrocarbonate‐treated water (DEPC water)
  • 75% ethanol in DEPC water
  • 37°C anaerobic chamber
  • Refrigerated centrifuge
  • Filter tips
  • 2‐ml screw‐cap tubes (with gaskets)
  • 0.1‐mm glass beads
  • Cell disrupter
  • Spectrophotometer

Basic Protocol 3: Transfer of Tn916 from a Bacillus subtilis Donor Strain to a C. difficile Recipient Strain

  Materials
  • DNA of interest
  • pSMB47 plasmid DNA (Manganelli et al., ) or a plasmid that contains homology to Tn916 to allow homologous recombination into the Tn916 tetracycline gene
  • B. subtilis donor that carries Tn916 on the chromosome, e.g., BS49 (Mullany et al., )
  • Agar plate with medium containing erythromycin
  • Petri plate containing viable C. difficile colonies (unit 9.1)
  • Reduced BHIS broth medium (see recipe)
  • Non‐reduced BHIS broth medium (see recipe)
  • Erythromycin (see recipe)
  • Tetracycline (see recipe)
  • Reduced BHIS(KNO 3) Petri plates (see recipe)
  • Reduced BHIS(E) Petri plates (see recipe)
  • Reduced BHIS(ECC) Petri plates (see recipe)
  • 15‐ml culture tubes
  • Anaerobic chamber at 37°C
  • Aerobic chamber at 37°C
  • Additional reagents and equipment for PCR

Basic Protocol 4: Transfer of Plasmid DNA from an E. coli Donor to C. difficile Recipient

  Materials
  • DNA of interest
  • E. coli/C. difficile shuttle plasmid, e.g., thiamphenicol‐resistant shuttle plasmid, pJIR1456
  • E. coli strain carrying a broad host range plasmid RP4 derivative, e.g., E. coli HB101 pRK24
  • LB medium ( appendix 4A)
  • Petri plate containing C. difficile colonies (see unit 9.1)
  • BHIS medium (see recipe)
  • Appropriate antibiotics (e.g., Amp, Cm)
  • Reduced BHIS agar Petri plates (see recipe)
  • Reduced BHIS(TCC) Petri plates (see recipe)
  • 15‐ml culture tubes
  • 37°C anaerobic chamber
  • Sterile inoculating loops

Basic Protocol 5: Gene Disruption in C. difficile Using Group II Intron Targeting

  Materials
  • Primers: IBS, EBS2, EBS1d, and EBS universal primer (see recipe)
  • Intron PCR template DNA (Sigma) or pMTL20IT1 and pMTL20IT2
  • High‐fidelity Taq polymerase
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (unit 2.1)
  • Cold 75% and 95% ethanol
  • TE buffer ( appendix 2A)
  • HindIII and BsrGI restriction enzymes (New England Biolabs)
  • pMTL007 plasmid DNA (Heap et al., )
  • Reduced BHIS(TCC) plates (see recipe)
  • Petri plate containing viable C. difficile (unit 9.1)
  • Reduced BHIS(E) plates (see recipe)
  • 37°C anaerobic chamber
  • Sterile inoculating loops

Basic Protocol 6: Site‐Directed Mutagenesis Using Unstable Plasmids

  Materials
  • 400‐ to 1000‐bp DNA fragment, internal to the target gene
  • E. coli strain carrying an RP4 plasmid derivative (see protocol 5)
  • BHIS Petri plate containing viable C. difficile JIR8094 colonies (unit 9.1)
  • Reduced BHIS medium (see recipe)
  • BHIS(T) plate containing viable C. difficile JIR8094 pJIR1456 colonies (see protocol 4 for introduction of plasmid DNA)
  • Reduced BHIS(TCC) Petri plates (see recipe)
  • Reduced BHIS Petri plate (see recipe)
  • 15‐ml culture tubes
  • 37°C anaerobic chamber
  • Sterile inoculating loops
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Literature Cited

Literature Cited
   Bartlett, J.G., Onderdonk, A.B., Cisneros, R.L., and Kasper, D.L. 1977. Clindamycin‐associated colitis due to a toxin‐producing species of Clostridium in hamsters. J. Infect. Dis. 136:701‐705.
   Bartlett, J.G., Moon, N., Chang, T.W., Taylor, N., and Onderdonk, A.B. 1978. Role of Clostridium difficile in antibiotic‐associated pseudomembranous colitis. Gastroenterology 75:778‐782.
   Burns, D.A., Heap, J.T., and Minton, N.P. 2010. SleC is essential for germination of Clostridium difficile spores in nutrient‐rich medium supplemented with the bile salt taurocholate. J. Bacteriol. 192:657‐664.
   Dineen, S.S., Villapakkam, A.C., Nordman, J.T., and Sonenshein, A.L. 2007. Repression of Clostridium difficile toxin gene expression by CodY. Mol. Microbiol. 66:206‐219.
   Dupuy, B. and Sonenshein, A.L. 1998. Regulated transcription of Clostridium difficile toxin genes. Mol. Microbiol. 27:107‐120.
   Emerson, J.E., Reynolds, C.B., Fagan, R.P., Shaw, H.A., Goulding, D., and Fairweather, N.F. 2009. A novel genetic switch controls phase variable expression of CwpV, a Clostridium difficile cell wall protein. Mol. Microbiol. 74:541‐556.
   Fekety, R. and Shah, A.B. 1993. Diagnosis and treatment of Clostridium difficile colitis. JAMA 269:71‐75.
   Glaser, P., Danchin, A., Kunst, F., Zuber, P., and Nakano, M.M. 1995. Identification and isolation of a gene required for nitrate assimilation and anaerobic growth of Bacillus subtilis. J. Bacteriol. 177:1112‐1115.
   Hall, I.C. and O'Toole, E. 1935. Intestinal flora in newborn infants with a description of a new pathogenic anaerobe, Bacillus difficilis. Am. J. Dis. Child 49:390‐402.
   Heap, J.T., Pennington, O.J., Cartman, S.T., Carter, G.P., and Minton, N.P. 2007. The ClosTron: A universal gene knock‐out system for the genus Clostridium. J. Microbiol. Methods 70:452‐464.
   Heap, J.T., Pennington, O.J., Cartman, S.T., and Minton, N.P. 2009. A modular system for Clostridium shuttle plasmids. J. Microbiol. Methods 78:79‐85.
   Heap, J.T., Kuehne, S.A., Ehsaan, M., Cartman, S.T., Cooksley, C.M., Scott, J.C., and Minton, N.P. 2010. The ClosTron: Mutagenesis in Clostridium refined and streamlined. J. Microbiol. Methods 80:49‐55.
   Hummel, R.P., Altemeier, W.A., and Hill, E.O. 1964. Iatrogenic staphylococcal enterocolitis. Ann. Surg. 160:551‐560.
   Just, I., Selzer, J., Wilm, M., von Eichel‐Streiber, C., Mann, M., and Aktories, K. 1995. Glucosylation of Rho proteins by Clostridium difficile toxin B. Nature 375:500‐503.
   Karlstrom, O., Fryklund, B., Tullus, K., and Burman, L.G. 1998. A prospective nationwide study of Clostridium difficile‐associated diarrhea in Sweden. The Swedish C. difficile Study Group. Clin. Infect. Dis. 26:141‐145.
   Khan, M.Y. and Hall, W.H. 1966. Staphylococcal enterocolitis—Treatment with oral vancomycin. Ann. Intern. Med. 65:1‐8.
   Kirby, J.M., Ahern, H., Roberts, A.K., Kumar, V., Freeman, Z., Acharya, K.R., and Shone, C.C. 2009. Cwp84, a surface‐associated cysteine protease, plays a role in the maturation of the surface layer of Clostridium difficile. J. Biol. Chem. 284:34666‐34673.
   Lyras, D. and Rood, J.I. 1998. Conjugative transfer of RP4‐oriT shuttle vectors from Escherichia coli to Clostridium perfringens. Plasmid 39:160‐164.
   Lyras, D., O'Connor, J.R., Howarth, P.M., Sambol, S.P., Carter, G.P., Phumoonna, T., Poon, R., Adams, V., Vedantam, G., Johnson, S., Gerding, D.N., and Rood, J.I. 2009. Toxin B is essential for virulence of Clostridium difficile. Nature 458:1176‐1179.
   Manganelli, R., Provvedi, R., Berneri, C., Oggioni, M.R., and Pozzi, G. 1998. Insertion vectors for construction of recombinant conjugative transposons in Bacillus subtilis and Enterococcus faecalis. FEMS Microbiol. Lett. 168:259‐268.
   McDonald, L.C., Killgore, G.E., Thompson, A., Owens, R.C. Jr., Kazakova, S.V., Sambol, S.P., Johnson, S., and Gerding, D.N. 2005. An epidemic, toxin gene‐variant strain of Clostridium difficile. N. Engl. J. Med. 353:2433‐2441.
   McFarland, L.V., Mulligan, M.E., Kwok, R.Y., and Stamm, W.E. 1989. Nosocomial acquisition of Clostridium difficile infection. N. Engl. J. Med. 320:204‐210.
   Mullany, P., Wilks, M., and Tabaqchali, S. 1991. Transfer of Tn916 and Tn916 delta E into Clostridium difficile: Demonstration of a hot‐spot for these elements in the C. difficile genome. FEMS Microbiol. Lett. 63:191‐194.
   O'Brien, J.A., Lahue, B.J., Caro, J.J., and Davidson, D.M. 2007. The emerging infectious challenge of Clostridium difficile–associated disease in Massachusetts hospitals: Clinical and economic consequences. Infect. Control Hosp. Epidemiol. 28:1219‐1227.
   O'Connor, J.R., Lyras, D., Farrow, K.A., Adams, V., Powell, D.R., Hinds, J., Cheung, J.K., and Rood, J.I. 2006. Construction and analysis of chromosomal Clostridium difficile mutants. Mol. Microbiol. 61:1335‐1351.
   Perelle, S., Gibert, M., Bourlioux, P., Corthier, G., and Popoff, M.R. 1997. Production of a complete binary toxin (actin‐specific ADP‐ribosyltransferase) by Clostridium difficile CD196. Infect. Immun. 65:1402‐1407.
   Redelings, M.D., Sorvillo, F., and Mascola, L. 2007. Increase in Clostridium difficile–related mortality rates, United States, 1999‐2004. Emerg. Infect. Dis. 13:1417‐1419.
   Schwan, C., Stecher, B., Tzivelekidis, T., van Ham, M., Rohde, M., Hardt, W.D., Wehland, J., and Aktories, K. 2009. Clostridium difficile toxin CDT induces formation of microtubule‐based protrusions and increases adherence of bacteria. PLoS Pathog. 5:e1000626.
   Showsh, S.A. and Andrews, R.E. Jr. 1992. Tetracycline enhances Tn916‐mediated conjugal transfer. Plasmid 28:213‐224.
   Tedesco, F.J., Barton, R.W., and Alpers, D.H. 1974. Clindamycin‐associated colitis. A prospective study. Ann. Intern. Med. 81:429‐433.
   Trieu‐Cuot, P., Carlier, C., Poyart‐Salmeron, C., and Courvalin, P. 1991. Shuttle vectors containing a multiple cloning site and a lacZ alpha gene for conjugal transfer of DNA from Escherichia coli to Gram‐positive bacteria. Gene 102:99‐104.
   Twine, S.M., Reid, C.W., Aubry, A., McMullin, D.R., Fulton, K.M., Austin, J., and Logan, S.M. 2009. Motility and flagellar glycosylation in Clostridium difficile. J. Bacteriol. 191:7050‐7062.
   Underwood, S., Guan, S., Vijayasubhash, V., Baines, S.D., Graham, L., Lewis, R.J., Wilcox, M.H., and Stephenson, K. 2009. Characterization of the sporulation initiation pathway of Clostridium difficile and its role in toxin production. J. Bacteriol. 191:7296‐7305.
   Viscidi, R., Willey, S., and Bartlett, J.G. 1981. Isolation rates and toxigenic potential of Clostridium difficile isolates from various patient populations. Gastroenterology 81:5‐9.
   Voth, D.E. and Ballard, J.D. 2005. Clostridium difficile toxins: Mechanism of action and role in disease. Clin. Microbiol. Rev. 18:247‐263.
   Warny, M., Pepin, J., Fang, A., Killgore, G., Thompson, A., Brazier, J., Frost, E., and McDonald, L.C. 2005. Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet 366:1079‐1084.
   Wren, B.W. and Tabaqchali, S. 1987. Restriction endonuclease DNA analysis of Clostridium difficile. J. Clin. Microbiol. 25:2402‐2404.
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