Abstract
The genus Clostridium is a diverse assemblage of Gram positive, anaerobic, endospore-forming bacteria. Whilst certain species have achieved notoriety as important animal and human pathogens (e.g. Clostridium difficile, Clostridium botulinum, Clostridium tetani, and Clostridium perfringens), the vast majority of the genus are entirely benign, and are able to undertake all manner of useful biotransformations. Prominent amongst them are those species able to produce the biofuels, butanol and ethanol from biomass-derived residues, such as Clostridium acetobutylicum, Clostridium beijerinkii, Clostridium thermocellum, and Clostridium phytofermentans. The prominence of the genus in disease and biotechnology has led to the need for more effective means of genetic modification. The historical absence of methods based on conventional strategies for “knock-in” and “knock-out” in Clostridium has led to the adoption of recombination-independent procedures, typified by ClosTron technology. The ClosTron uses a retargeted group II intron and a retro-transposition-activated marker to selectively insert DNA into defined sites within the genome, to bring about gene inactivation and/or cargo DNA delivery. The procedure is extremely efficient, rapid, and requires minimal effort by the operator.
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References
Dürre P. (2005) Handbook on Clostridia, CRC Press, New York. 2005.
Onderdonk A.B., and Allen S.D. (1994) Clostridium. Manual of clinical microbiology. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, editors. 6 th ed. Washington, DC: ASM Press; p. 1210.
Dürre P. (2008) Fermentative butanol production: bulk chemical and biofuel. Ann. N. Y. Acad. Sci. 1125, 353–362.
Demain A.L., Newcomb M., and Wu J.H.D. (2005) Cellulase, clostridia, and ethanol. Microbiol. Mol. Biol. Rev. 69, 124–154.
Minton N.P. (2003) Nature Rev. Microbiol. 1, 237–242.
Johnson E.A. (2005) Clostridium botulinum neurotoxins – applications in medicine and potential agents of bioterrorism. Clin. Microbiol. News. 27, 147–151.
Hurst L.C., Badalamente M.A., Hentz V.R., Hotchkiss R.N., Kaplan T.T.D., Meals R.A., Smith T.M., and Rodzvilla J. (2009) Injectable collagenase Clostridium histolyticum for Dupuytren’s contracture. N Engl J Med 361, 968–979.
Heap J.T., Cartman S.T., Pennington O.J., Cooksley C.M., Scott J.C., Blount B., Burns D., and Minton N.P. (2008) Development of genetic knock-out systems for clostridia. In: Bruggermann, H, Gottschalk, G. (Eds), Clostridia: Molecular biology in the post-genomic era. Caister Academic Press. Norfolk, UK, pp. 179–198.
Karberg M., Guo H., Zhong J., Coon R., Perutka J., and Lambowitz A.M. (2001) Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nature Biotechnol. 19, 1162–7.
Zhong J., Karberg M., and Lambowitz A.M. (2003) Targeted and random bacterial gene disruption using a group II intron (targetron) vector containing a retrotransposition-activated selectable marker. Nucleic Acids Res. 31, 1656–64.
Mohr G., Smith D., Belfort M., and Lambowitz A.M. (2000) Rules for DNA target-site recognition by a lactococcal group II intron enable retargeting of the intron to specific DNA sequences. Genes Dev. 14, 559–73.
Perutka J., Wang W., Goerlitz D., and Lambowitz A.M. (2004) Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes. J Mol Biol. 336, 421–39.
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, 452–64.
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.
Heap T.J., Pennington O.J., Cartman S.T., and Minton N.P. (2009) “A modular system for Clostridium shuttle plasmids. J. Microbiol. Methods 78, 79–85.
Bradshaw M., Marshall K.M., Heap J.T., Tepp W.H., Minton N.P., and Johnson E.A. (2010) Construction of a Nontoxigenic Clostridium botulinum Strain for Food Challenge Studies Appl. Environ. Microbiol. 76, 387–93.
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, 657–64.
Cooksley C.M., Davis I.J., Winzer K., Chan W.C., Peck M.W., and Minton N.P. (2010) Regulation of neurotoxin production and sporulation by a putative agrBD signaling system in proteolytic Clostridium botulinum. Appl. Environ. Microbiol. 76, 4448–60.
Camiade E., Peltier J., Bourgeois I., Couture-Tosi E., Courtin P., Antunes A., Chapot-Chartier M.P., Dupuy B., and Pons J.L. (2010) Characterization of Acp, a peptidoglycan hydrolase of Clostridium perfringens with N-acetylglucosaminidase activity that is implicated in cell separation and stress-induced autolysis. J Bacteriol. 192, 2373–84.
Dong H., Zhang Y., Dai Z., and Li Y. (2010) Engineering clostridium strain to accept unmethylated DNA. PLoS One. 5, e9038.
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.
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.
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.
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.
Warrens A.N., Jones M.D., and Lechler R.I. (1997) Splicing by overlap extension by PCR using asymmetric amplification: an improved technique for the generation of hybrid proteins of immunological interest. Gene. 186, 29–35.
Purdy D., O’Keeffe T.A., Elmore M., Herbert M., McLeod A., Bokori-Brown M., Ostrowski A., and Minton N.P. (2002) Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier. Mol Microbiol. 46, 439–452.
Williams D.R., Young D.I., and Young M. (1990) Conjugative plasmid transfer from Escherichia coli to Clostridium acetobutylicum. J. Gen. Microbiol. 136, 819–26.
Theys J., Pennington O.P, Dubois L., Anlezark G., Vaughan T., Mengesha A., Landuyt W., Anné J., Burke P.J., Dûrre P., Wouters B.G., Minton N.P., and Lambin P. (2006) Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo. British J. Cancer 95, 1212–9.
Davis T.O., Henderson I., Brehm J.K., and Minton N.P. (2000) Development of a transformation and gene reporter system for group II, non-proteolytic Clostridium botulinum type B strains. J. Mol. Microbiol. Biotechnol. 2, 59–69.
Mauchline M.L., Davis T.O., and Minton N.P. (1999) Clostridia. In: Manual of Industrial Microbiology and Biotechnology, Demain AL, Davies JE (eds), ASM Press, pp. 475–492.
Mermelstein L.D., and Papoutsakis E.T. (1993) In vivo methylation in Escherichia coli by the Bacillus subtilis phage ϕ3tI methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 59, 1077–1081.
Hussain H.A., Roberts A.P., and Mullany P. (2005) Generation of an Em-sensitive derivative of Clostridium difficile strain 630 (630Deltaerm) and demonstration that the conjugative transposon Tn916DeltaE enters the genome of this strain at multiple sites. J. Med. Microbiol. 54, 137–141.
Cartman S.T., Heap J.T., Kuehne S.A., Cockayne A., and Minton N.P. (2010) The Emergence of ‘Hypervirulence’ in Clostridium difficile. Int. J. Med. Microbiol. 300, 387–395.
Kuehne S.A., Cartman S.T., Heap J.T., Kelly M.L., Cockayne A., and Minton N.P. (2010) The role of toxin A and toxin B in Clostridium difficile infection. Nature 467, 711–713.
Cooksley C.M., Davis I.J., Winzer K., Cockayne A., Chan W.C., Peck M.W., and Minton N.P. (2010) A putative agrBD signalling system regulates toxin production and sporulation in proteolytic Clostridium botulinum. J Bacteriol 76, 4448–4460.
Acknowledgments
The authors acknowledge the financial support of UK Medical Research Council (G0601176), the European Union (HEALTH-F3-2008-223585), the UK Biotechnology and Biological Sciences Research Council (BB/E021271/1, BB/D001498/1, BB/F003390/1, and BB/G016224/1), SysMO (Systems Biology of Microorganisms) and Morvus Technologies Ltd.
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Kuehne, S.A., Heap, J.T., Cooksley, C.M., Cartman, S.T., Minton, N.P. (2011). ClosTron-Mediated Engineering of Clostridium . In: Williams, J. (eds) Strain Engineering. Methods in Molecular Biology, vol 765. Humana Press. https://doi.org/10.1007/978-1-61779-197-0_23
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DOI: https://doi.org/10.1007/978-1-61779-197-0_23
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