Regulation of toxin synthesis in Clostridium botulinum and Clostridium tetani (original) (raw)

Abstract

Botulinum and tetanus neurotoxins are structurally and functionally related proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin (BoNT) associates with non-toxic proteins (ANTPs) to form complexes of various sizes, whereas tetanus toxin (TeNT) does not form any complex. The BoNT and ANTP genes are clustered in a DNA segment called the botulinum locus, which has different genomic localization (chromosome, plasmid, phage) in the various Clostridium botulinum types and subtypes. The botulinum locus genes are organized in two polycistronic operons (ntnh-bont and ha/orfX operons) transcribed in opposite orientations. A gene called botR lying between the two operons in C. botulinum A encodes an alternative sigma factor which regulates positively the synthesis of BoNT and ANTPs at the late exponential growth phase and beginning of the stationary phase. In C. tetani, the gene located immediately upstream of tent encodes a positive regulatory protein, TetR, which is related to BotR. C. botulinum and C. tetani genomes contain several two-component systems and predicted regulatory orphan genes. In C. botulinum type A, three two-component systems have been found to positively regulate the synthesis of BoNT and ANTPs independently of BotR/A. The synthesis of neurotoxin in Clostridia seems to be under the control of complex network of regulation. 1. INTRODUCTION Among the large number of Clostridium species, which are bacteria from the environment, some of them produce potent neurotoxins and are responsible for neurological disorders in human and animals. Botulinum neurotoxins (BoNTs) inhibit the release of acetylcholine at peripheral cholinergic nerve terminals causing the flaccid paralysis of botulism, whereas tetanus neurotoxin (TeNT) blocks neurotransmitter release at synapses at the central nervous system causing the spastic paralysis of tetanus. In the last decades, numerous works have been performed to elucidate the structure and mechanism of action of the clostridial neurotoxins as well as their genetic organization and regulation. 2. CLOSTRIDIA PRODUCING NEUROTOXINS Clostridia capable of producing BoNT display heterogeneous bacteriological characters and are divided into several species and groups. The taxonomic position of the C. botulinum species was originally based on only one phenotype, the production of a BoNT, and non-toxic variant strains, although genetically related to C. botulinum, were assigned to different species such as Clostridium sporogenes and Clostridium subterminale. It appeared

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References (85)

  1. Argaman, L., Hershberg, R., Vogel, J., Bejerano, G., Wagner, E.G., Margalit, H., Altuvia, S., 2001. Novel small RNA-encoding genes in the intergenic regions of Escherichia coli. Curr Biol 11, 941-950.
  2. Artin, I., Carter, A.T., Holst, E., Lovenklev, M., Mason, D.R., Peck, M.W., Radstrom, P., 2008. Effects of carbon dioxide on neurotoxin gene expression in nonproteolytic Clostridium botulinum Type E. Appl Environ Microbiol 74, 2391-2397.
  3. Artin, I., Mason, D.R., Pin, C., Schelin, J., Peck, M.W., Holst, E., Radstrom, P., Carter, A.T., 2010. Effects of carbon dioxide on growth of proteolytic Clostridium botulinum, its ability to produce neurotoxin, and its transcriptome. Appl Environ Microbiol 76, 1168- 1172.
  4. Ba-Thein, W., Lyristis, M., Ohtani, K., Nisbet, I.T., Hayashi, H., 1996. The virR/virS locus regulates the transcription of genes encoding extracellular toxin production in Clostridium perfringens. J. Bacteriol. 178, 2514-2520.
  5. Bhandari, M., Campbell, K.D., Collins, M.D., East, A.K., 1997. Molecular characterization of the clusters of genes encoding the botulinum neurotoxin complex in Clostridium botulinum (Clostridium argentinense) type G and nonproteolytic Clostridium botulinum type B. Curr. Microbiol. 35, 207-214.
  6. Bradshaw, M., Dineen, S.S., Maks, N.D., Johnson, E.A., 2004. Regulation of neurotoxin complex expression in Clostridium botulinum strains 62A, Hall A-hyper, and NCTC2916. Anaerobe 10, 321-333.
  7. Brüggemann, H., Bäumer, S., Fricke, W.F., Wiezr, A., Liesagang, H., Decker, I., Herzberg, C., Martinez-Arias, R., Henne, A., Gottschalk, G., 2003. The genome sequence of Clostridium tetani, the causative agent of tetanus disease. Proc. Ntl. Acad. Sci. (USA) 100, 1316-1321.
  8. Carter, A.T., Paul, C.J., Mason, D.R., Twine, S.M., Alston, M.J., Logan, S.M., Austin, J.W., Peck, M.W., 2009. Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum. BMC Genomics 10, 115.
  9. Chen, Y., Indurthi, D.C., Jones, S.W., Papoutsakis, E.T., 2011. Small RNAs in the genus Clostridium. MBio 2, e00340-00310.
  10. Chen, Y., Korkeala, H., Aarnikunnas, J., Lindstrom, M., 2007. Sequencing the botulinum neurotoxin gene and related genes in Clostridium botulinum type E strains reveals orfx3 and a novel type E neurotoxin subtype. J Bacteriol 189, 8643-8650.
  11. Cheung, J.K., Awad, M.M., McGowan, S., Rood, J.I., 2009. Functional analysis of the VirSR phosphorelay from Clostridium perfringens. PLoS One 4, e5849.
  12. Cheung, J.K., Keyburn, A.L., Carter, G.P., Lanckriet, A.L., Van Immerseel, F., Moore, R.J., Rood, J.I., 2010. The VirSR two-component signal transduction system regulates NetB toxin production in Clostridium perfringens. Infect Immun 78, 3064-3072.
  13. Connan, C., Brueggemann, H., Mazuet, C., Raffestin, S., Cayet, N., Popoff, M.R., 2012. Two- Component Systems Are Involved in the Regulation of Botulinum Neurotoxin Synthesis in Clostridium botulinum Type A Strain Hall. PLoS One 7.
  14. Cooksley, C.M., Davis, I.J., Winzer, K., Chan, W.C., Peck, M.W., Minton, N.P., 2010. Regulation of neurotoxin production and sporulation by a Putative agrBD signaling system in proteolytic Clostridium botulinum. Appl Environ Microbiol 76, 4448-4460.
  15. Couesnon, A., Raffestin, S., Popoff, M.R., 2006. Expression of botulinum neurotoxins A and E, and associated non-toxin genes, during the transition phase and stability at high temperature : analysis by quantitative reverse transcription-PCR. Microbiol. 152, 759- 770.
  16. Dineen, S.S., Bradshaw, M., Karasek, C.E., Johnson, E.A., 2004. Nucleotide sequence and transcriptional analysis of the type A2 neurotoxin gene cluster in Clostridium botulinum. FEMS Microbiol. Lett. 235, 9-16.
  17. Dupuy, B., Mani, N., Katayama, S., Sonenshein, A.L., 2005. Transcription activation of a UV-inducible Clostridium perfringens bacteriocin gene by a novel sigma factor. Mol Microbiol 55, 1196-1206.
  18. Dupuy, B., Raffestin, S., Matamouros, S., Mani, N., Popoff, M.R., Sonenshein, A.L., 2006. Regulation of toxin and bacteriocin gene expression in Clostridium by interchangeable RNA polymerase sigma factors. Mol. MIcrobiol. 60, 1044-1057.
  19. Dupuy, B., Sonenshein, A.L., 1998. Regulated transcription of Clostridium difficile toxin genes. Mol. Microbiol. 27, 107-120.
  20. East, A.K., Bhandari, M., Stacey, J.M., Campbell, K.D., Collins, M.D., 1996. Organization and phylogenetic interrelationships of genes encoding components of the botulinum toxin complex in proteolytic Clostridium botulinum types A, B, and F: evidence of chimeric sequences in the gene encoding the nontoxic nonhemagglutinin component. Int. J. Syst. Bacteriol. 46, 1105-1112.
  21. Eberhard, A., 1972. Inhibition and activation of bacterial luciferase synthesis. J Bacteriol 109, 1101-1105.
  22. Eklund, M.W., Poysky, F.T., Habig, W.H., 1989. Bacteriophages and plasmids in Clostridium botulinum and Clostridium tetani and their relationship to production of toxins., in: Simpson, L.L. (Ed.), Botulinum Neurotoxin and Tetanus Toxin. Academic Press, San Diego, pp. 25-51.
  23. Federle, M.J., Bassler, B.L., 2003. Interspecies communication in bacteria. J Clin Invest 112, 1291-1299.
  24. Franciosa, G., Ferreira, J.L., Hatheway, C.L., 1994. Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR: evidence of unexpressed type B toxin genes in type A toxigenic organisms. J. Clin. Microbiol. 32, 1911-1917.
  25. Franciosa, G., Maugliani, A., Scalfaro, C., Aureli, P., 2009. Evidence that plasmid-borne botulinum neurotoxin type B genes are widespread among Clostridium botulinum serotype B strains. PLoS One 4, e4829.
  26. Galperin, M.Y., Higdon, R., Kolker, E., 2010. Interplay of heritage and habitat in the distribution of bacterial signal transduction systems. Mol Biosyst 6, 721-728.
  27. Gutierrez, P., Li, Y., Osborne, M.J., Pomerantseva, E., Liu, Q., Gehring, K., 2005. Solution structure of the carbon storage regulator protein CsrA from Escherichia coli. J Bacteriol 187, 3496-3501.
  28. Hall, J.D., McCroskey, L.M., Pincomb, B.J., Hatheway, C.L., 1985. Isolation of an organism resembling Clostridium barati which produces type F botulinal toxin from an infant with botulism. J. Clin. Microbiol. 21, 654-655.
  29. Hassan, S., Ohtani, K., Wang, R., Yuan, Y., Wang, Y., Yamaguchi, Y., Shimizu, T., 2010. Transcriptional regulation of hemO encoding heme oxygenase in Clostridium perfringens. J Microbiol 48, 96-101.
  30. Hauser, D., Gibert, M., Marvaud, J.C., Eklund, M.W., Popoff, M.R., 1995. Botulinal neurotoxin C1 complex, Clostridial neurotoxin homology and genetic transfer in Clostridium botulinum. Toxicon 33, 515-526.
  31. Helmann, J.D., 2002. The extracytoplasmic function (ECF) sigma factors. Adv. Microbial. Physiol. 46, 47-110.
  32. Henderson, I., Davis, T., Elmore, M., Minton, N., 1997. The genetic basis of toxin production in Clostridium botulinum and Clostridium tetani, in: Rood, I. (Ed.), The Clostridia: Molecular Biology and Pathogenesis. Academic Press, New York, pp. 261-294.
  33. Henderson, I., Whelan, S.M., Davis, T.O., Minton, N.P., 1996. Genetic characterization of the botulinum toxin complex of Clostridium botulinum strain NCTC2916. FEMS Microbiol. Lett. 140, 151-158.
  34. Hill, K.K., Smith, T.J., Helma, C.H., Ticknor, L.O., Foley, B.T., Svensson, R.T., Brown, J.L., Johnson, E.A., Smith, L.A., Okinaka, R.T., Jackson, P.J., Marks, J.D., 2007. Genetic diversity among Botulinum Neurotoxin-producing clostridial strains. J Bacteriol 189, 818-832.
  35. Hill, K.K., Xie, G., Foley, B.T., Smith, T.J., Munk, A.C., Bruce, D., Smith, L.A., Brettin, T.S., Detter, J.C., 2009. Recombination and insertion events involving the botulinum neurotoxin complex genes in Clostridium botulinum types A, B, E and F and Clostridium butyricum type E strains. BMC Biol 7, 66.
  36. Hirst, T.R., 1995. Biogenesis of cholera toxin and related oligomeric toxins, in: Moss, J., Iglewsski, B., Vaughan, M., Tu, A.T. (Eds.), Bacterial Toxins and Virulence Factors in Disease. Marcel Dekker, New York, pp. 123-184.
  37. Hundsberger, T., Braun, V., Weidmann, M., Leukel, P., Sauerborn, M., von Eichel-Streiber, C., 1997. Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile. Eur. J. Biochem. 244, 735-742.
  38. Hutson, R.A., Zhou, Y., Collins, M.D., Johnson, E.A., Hatheway, C.L., Sugiyama, H., 1996. Genetic characterization of Clostridium botulinum type A containing silent type B neurotoxin gene sequences. J. Biol. Chem. 271, 10786-10792.
  39. Inoue, K., Fujinaga, Y., Watanabe, T., Ohyama, T., Takeshi, K., Moriishi, K., Nakajima, H., Inoue, K., Oguma, K., 1996. Molecular composition of Clostridium botulinum type A progenitor toxins. Infect. Immun. 64, 1589-1594.
  40. Jacobson, M.J., Lin, G., Raphael, B., Andreadis, J., Johnson, E.A., 2008. Analysis of neurotoxin cluster genes in Clostridium botulinum strains producing botulinum neurotoxin serotype A subtypes. Appl Environ Microbiol 74, 2778-2786.
  41. Jovita, M.R., Collins, M.D., East, A.K., 1998. Gene organization and sequence determination of the two botulinum neurotoxin gene clusters in Clostridium botulinum. Cur. Microbiol. 36, 226-231.
  42. Karlsson, S., Burman, L.G., Akerlund, T., 1999. Suppression of toxin production in Clostridium difficile VPI10463 by amino acids. Microbiol. 145, 1683-1693.
  43. Karlsson, S., Dupuy, B., Mukherjee, K., Norin, E., Burman, L.G., Akerlund, T., 2003. Expression of Clostridium difficile toxins A and B and their sigma factor TcdD is controlled by temperature. Infect. Immun. 71, 1784-1793.
  44. Klinkert, B., Narberhaus, F., 2009. Microbial thermosensors. Cell Mol Life Sci 66, 2661- 2676.
  45. Lee, A.S., Song, K.P., 2005. LuxS/autoinducer-2 quorum sensing molecule regulates transcriptional virulence gene expression in Clostridium difficile. Biochem Biophys Res Commun 335, 659-666.
  46. Li, B., Qian, X., Sarkar, H.K., Singh, B.R., 1998. Molecular characterization of type E Clostridium botulinum and comparison to other types of Clostridium botulinum. Biochim. Biophys. Acta 1395, 21-27.
  47. Lin, W.J., Johnson, E.A., 1995. Genome analysis of Clostridium botulinum type A by pulsed- field gel electrophoresis. Appl. Environ. Microbiol. 61, 4441-4447.
  48. Lindstrom, M., 2012. Identification of a two-component signal transduction system repressing neurotoxin production in group I Clostridium botulinum type A strain ATCC3502, Toxins2012, Miami.
  49. Lindstrom, M., Dahlsten, E., Soderholm, H., Selby, K., Somervuo, P., Heap, J.T., Minton, N.P., Korkeala, H., 2012. Involvement of Two-Component System CBO0366/CBO0365 in the Cold Shock Response and Growth of Group I (Proteolytic) Clostridium botulinum ATCC 3502 at Low Temperatures. Appl Environ Microbiol 78, 5466-5470.
  50. Lövenklev, M., Artin, I., Hagberg, O., Borch, E., Holst, E., Radström, P., 2004. Quantitative interaction effects of carbon dioxide, sodium chloride, and sodium nitrite on neurotoxin gene expression in nonproteolytic Clostridium botulinum type B. Appl. Environ. Micrcobiol. 70, 2928-2934.
  51. Ma, M., Vidal, J., Saputo, J., McClane, B.A., Uzal, F., 2011. The VirS/VirR two-component system regulates the anaerobic cytotoxicity, intestinal pathogenicity, and enterotoxemic lethality of Clostridium perfringens type C isolate CN3685. MBio 2, e00338-00310.
  52. Mani, N., Lyras, D., Barroso, L., Howarth, P., Wilkins, T., Rood, J.I., Sonenshein, A.L., Dupuy, B., 2002. Environmental response and autoregulation of Clostridiium difficile TxeR, a sigma factor for toxin gene expression. J. Bacteriol. 184, 5971-5978.
  53. Marshall, K.M., Bradshaw, M., Pellet, S., Johnson, E.A., 2007. Plasmid encoded neurotoxin genes in Clostridium botulinum serotype A subtypes. Biochem Biophys Res Commun 361, 49-54.
  54. Marvaud, J.C., Eisel, U., Binz, T., Niemann, H., Popoff, M.R., 1998a. tetR is a positive regulator of the Tetanus toxin gene in Clostridium tetani and is homologous to botR. Infect. Immun. 66, 5698-5702.
  55. Marvaud, J.C., Gibert, M., Inoue, K., Fujinaga, V., Oguma, K., Popoff, M.R., 1998b. botR is a positive regulator of botulinum neurotoxin and associated non toxic protein genes in Clostridium botulinum A. Mol. Microbiol. 29, 1009-1018.
  56. McCroskey, L.M., Hatheway, C.L., Fenicia, L., Pasolini, B., Aureli, P., 1986. Characterization of an organism that produces type E botulinal toxin but which resembles Clostridium butyricum from the feces of an infant with type E botulism. J. Clin. Microbiol. 23, 201-202.
  57. McCroskey, L.M., Hatheway, C.L., Woodruff, B.A., Greenberg, J.A., Jurgenson, P., 1991. Type F botulism due to neurotoxigenic Clostridium baratii from an unknown source in an adult. J. Clin. Microbiol. 29, 2618-2620.
  58. Nakamura, S., Okado, I., Abe, T., Nishida, S., 1979. Taxonomy of Clostridium tetani and related species. J. Gen. Microbiol. 113, 29-35.
  59. Narberhaus, F., Waldminghaus, T., Chowdhury, S., 2006. RNA thermometers. FEMS Microbiol Rev 30, 3-16.
  60. Novick, R.P., Geisinger, E., 2008. Quorum sensing in staphylococci. Annu Rev Genet 42, 541-564.
  61. Ohtani, K., Hayashi, H., Shimizu, T., 2002. The luxS gene is involved in cell-cell signalling for toxin production in Clostridium perfringens. Mol. Microbiol. 44, 171-179.
  62. Ohtani, K., Hirakawa, H., Tashiro, K., Yoshizawa, S., Kuhara, S., Shimizu, T., 2009. Identification of a two-component VirR/VirS regulon in Clostridium perfringens. Anaerobe 16, 258-264.
  63. Ohtani, K., Kawsar, H.I., Okumura, K., Hayashi, H., Shimizu, T., 2003. The VirR/VirS regulatory cascade affects transcription of plasmid-encoded putative virulence genes in Clostridium perfringens strain 13. FEMS Microbiol. Lett. 222, 137-141.
  64. Okumura, K., Ohtani, K., Hayashi, H., Shimizu, T., 2008. Characterization of genes regulated directly by the VirR/VirS system in Clostridium perfringens. J Bacteriol 190, 7719-7727.
  65. Patterson-Curtis, S.I., Johnson, E.A., 1989. Regulation of neurotoxin and protease formation in Clostridium botulinum Okra B and Hall by arginine. Appl. Environ. Microbiol. 55, 1544-1548.
  66. Popoff, M.R., Marvaud, J.C., 1999. Structural and genomic features of clostridial neurotoxins, in: Alouf, J.E., Freer, J.H. (Eds.), The Comprehensive Sourcebook of Bacterial Protein Toxins, 2 ed. Academic Press, London, pp. 174-201.
  67. Porfirio, Z., Prado, S.M., Vancetto, M.D.C., Fratelli, F., Alves, E.W., Raw, I., Fernandes, B.L., Camargo, A.C.M., Lebrun, I., 1997. Specific peptides of casein pancreatic digestion enhance the production of tetanus toxin. J. Appl. Microbiol. 83, 678-684.
  68. Poulain, B., Stiles, B.G., Popoff, M.R., Molgó, J., 2006. Attack of the nervous system by clostridial toxins: Physical findings, cellular and molecular actions, in: Alouf, J.E., Popoff, M.R. (Eds.), The Sourcebook of Bacterial Protein Toxins, 3° ed. Elsevier, Academic Press, Amsterdam, pp. 348-389.
  69. Raffestin, S., Dupuy, B., Marvaud, J.C., Popoff, M.R., 2005. BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani. Mol. MIcrobiol. 55, 235-249.
  70. Raphael, B.H., Lautenschlager, M., Kalb, S.R., de Jong, L.I., Frace, M., Luquez, C., Barr, J.R., Fernandez, R.A., Maslanka, S.E., 2012. Analysis of a unique Clostridium botulinum strain from the Southern hemisphere producing a novel type E botulinum neurotoxin subtype. BMC Microbiol 12, 245.
  71. Sebaihia, M., Peck, M.W., Minton, N.P., Thomson, N.R., Holden, M.T., Mitchell, W.J., Carter, A.T., Bentley, S.D., Mason, D.R., Crossman, L., Paul, C.J., Ivens, A., Wells- Bennik, M.H., Davis, I.J., Cerdeno-Tarraga, A.M., Churcher, C., Quail, M.A., Chillingworth, T., Feltwell, T., Fraser, A., Goodhead, I., Hance, Z., Jagels, K., Larke, N., Maddison, M., Moule, S., Mungall, K., Norbertczak, H., Rabbinowitsch, E., Sanders, M., Simmonds, M., White, B., Whithead, S., Parkhill, J., 2007. Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes. Genome Res 17, 1082-1092.
  72. Shimizu, T., Yaguchi, H., Ohtani, K., Banu, S., Hayashi, H., 2002. Clostridial VirR/VirS regulon involves a regulatory RNA molecule for expression of toxins. Mol. Microbiol. 43, 257-265.
  73. Skarin, H., Segerman, B., 2011. Horizontal gene transfer of toxin genes in Clostridium botulinum: Involvement of mobile elements and plasmids. Mob Genet Elements 1, 213- 215.
  74. Smith, L.D.S., 1975. Clostridium tetani, in: Smith, L.D.S. (Ed.), The Pathogenic Anaerobic Bacteria. Charles C. Thomas Publisher, Springfield Ill, pp. 177-201.
  75. Smith, T.J., Hill, K.K., Foley, B.T., Detter, J.C., Munk, A.C., Bruce, D.C., Doggett, N.A., Smith, L.A., Marks, J.D., Xie, G., Brettin, T.S., 2007. Analysis of the Neurotoxin Complex Genes in Clostridium botulinum A1-A4 and B1 Strains: BoNT/A3, /Ba4 and /B1 Clusters Are Located within Plasmids. PLoS ONE 2, e1271.
  76. Smith, T.J., Lou, J., Geren, I.N., Forsyth, C.M., Tsai, R., Laporte, S.L., Tepp, W.H., Bradshaw, M., Johnson, E.A., Smith, L.A., Marks, J.D., 2005. Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization. Infect Immun 73, 5450-5457.
  77. Sorek, R., Kunin, V., Hugenholtz, P., 2008. CRISPR--a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat Rev Microbiol 6, 181- 186.
  78. Strom, M.S., Eklund, M.W., Poysky, F.T., 1984. Plasmids in Clostridium botulinum and related species. Appl. Environ. Microbiol. 48, 956-963.
  79. Suen, J.C., Hatheway, C.L., Steigerwalt, A.G., Brenner, D.J., 1988. Clostridium argentinense sp. nov.: a genetically homogeneous group composed of all strains of Clostridium botulinum toxin type G and some nontoxigenic strains previously identified as Clostridium subterminale or Clostridium hastiforme. Int. J. Syst. Bacteriol. 38, 375-381.
  80. Thomason, M.K., Storz, G., 2010. Bacterial antisense RNAs: how many are there, and what are they doing? Annu Rev Genet 44, 167-188.
  81. Toledo-Arana, A., Repoila, F., Cossart, P., 2007. Small noncoding RNAs controlling pathogenesis. Curr Opin Microbiol 10, 182-188.
  82. Waters, C.M., Bassler, B.L., 2005. Quorum sensing: cell-to-cell communication in bacteria. Annu Rev Cell Dev Biol 21, 319-346.
  83. Weickert, M.J., Chambliss, G.H., Sugiyama, H., 1986. Production of toxin by Clostridium botulinum type A strains cured of plasmids. Appl. Environ. Microbiol. 51, 52-56.
  84. Wilde, E., Hippe, H., Tosunoglu, N., Schallehn, G., Herwig, K., Gottschalk, G., 1989. Clostridium tetanomorphum sp. nov. , nom rev. Int. J. SysT. Bacteriol. 39, 127-134.
  85. Yamakawa, K., Karasawa, T., Ohta, T., Hayashi, H., Nakamura, S., 1998. Inhibition of enhanced toxin production by Clostridium difficile in biotin-limited conditions. J. Med. Microbiol. 47, 767-771.