Skip to main content
SpringerLink
Log in

Complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in Clostridium botulinum type B strain 111 isolated from an infant patient in Japan

  • Original Paper
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

Botulinum neurotoxins (BoNTs) are highly potent toxins that are produced by Clostridium botulinum. We determined the complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in C. botulinum type B strain 111 in order to obtain an insight into the toxigenicity and evolution of the bont gene in C. botulinum. Group I C. botulinum type B strain 111 was isolated from the first case of infant botulism in Japan in 1995. In previous studies, botulinum neurotoxin subtype B2 (BoNT/B2) produced by strain 111 exhibited different antigenic properties from those of authentic BoNT/B1 produced by strain Okra. We have recently shown that the isolates of strain 111 that lost toxigenicity were cured of the plasmid containing the bont/B2 gene. In the present study, the plasmid (named pCB111) was circular 265,575 bp double-stranded DNA and contained 332 predicted open reading frames (ORFs). 85 gene products of these ORFs could be functionally assigned on the basis of sequence homology to known proteins. The bont/B2 complex genes were located on pCB111 and some gene products may be involved in the conjugative plasmid transfer and horizontal transfer of bont genes. pCB111 was similar to previously identified plasmids containing bont/B1, /B5, or/A3 complex genes in other group I C. botulinum strains. It was suggested that these plasmids had been derived from a common ancestor and had played important roles for the bont gene transfer between C. botulinum.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Alouf JE, Freer JH (1999) The comprehensive sourcebook of bacterial protein toxins. In: Popoff MR, Marvaud JC (eds) Structural and genomic features of clostridial neurotoxins, 2nd edn. Academic Press, San Diego, pp 174–201

    Google Scholar 

  • Arnon SS, Schechter R, Inglesby TV, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, Fine AD, Hauer J, Layton M, Lillibridge S, Osterholm MT, O’Toole T, Parker G, Perl TM, Russell PK, Swerdlow DL, Tonat K (2001) Botulinum toxin as a biological weapon: medical and public health management. J Am Med Assoc 285(8):1059–1070

    Article  CAS  Google Scholar 

  • Barash JR, Arnon SS (2013) A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins. J Infect Dis 209(2):183–191. doi:10.1093/infdis/jit449

    Article  PubMed  Google Scholar 

  • Cornet F, Mortier I, Patte J, Louarn JM (1994) Plasmid pSC101 harbors a recombination site, psi, which is able to resolve plasmid multimers and to substitute for the analogous chromosomal Escherichia coli site dif. J Bacteriol 176(11):3188–3195

    CAS  PubMed Central  PubMed  Google Scholar 

  • Das B, Martinez E, Midonet C, Barre FX (2013) Integrative mobile elements exploiting Xer recombination. Trends Microbiol 21(1):23–30. doi:10.1016/j.tim.2012.10.003

    Article  CAS  PubMed  Google Scholar 

  • Dover N, Barash JR, Hill KK, Xie G, Arnon SS (2013) Molecular characterization of a novel botulinum neurotoxin type h gene. J Infect Dis 209(2):192–202. doi:10.1093/infdis/jit450

    Article  PubMed  Google Scholar 

  • Edmond BJ, Guerra FA, Blake J, Hempler S (1977) Case of infant botulism in Texas. Tex Med 73(10):85–88

    CAS  PubMed  Google Scholar 

  • Efron B, Halloran E, Holmes S (1996) Bootstrap confidence levels for phylogenetic trees. Proc Natl Acad Sci USA 93(23):13429–13434

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Eklund MW, Poysky FT, Reed SM, Smith CA (1971) Bacteriophage and the toxigenicity of Clostridium botulinum type C. Science 172(3982):480–482

    Article  CAS  PubMed  Google Scholar 

  • Fox CK, Keet CA, Strober JB (2005) Recent advances in infant botulism. Pediatr Neurol 32(3):149–154

    Article  PubMed  Google Scholar 

  • Fujinaga Y, Matsumura T, Jin Y, Takegahara Y, Sugawara Y (2009) A novel function of botulinum toxin-associated proteins: HA proteins disrupt intestinal epithelial barrier to increase toxin absorption. Toxicon 54(5):583–586. doi:10.1016/j.toxicon.2008.11.014

    Article  CAS  PubMed  Google Scholar 

  • Hatheway CL (1990) Toxigenic clostridia. Clin Microbiol Rev 3(1):66–98

    CAS  PubMed Central  PubMed  Google Scholar 

  • Hielm S, Bjorkroth J, Hyytia E, Korkeala H (1998) Genomic analysis of Clostridium botulinum group II by pulsed-field gel electrophoresis. Appl Environ Microbiol 64(2):703–708

    CAS  PubMed Central  PubMed  Google Scholar 

  • Hill KK, Smith TJ (2013) Genetic diversity within clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes. Curr Top Microbiol Immunol 364:1–20. doi:10.1007/978-3-642-33570-9_1

    PubMed  Google Scholar 

  • Hill KK, Xie G, Foley BT, Smith TJ, Munk AC, Bruce D, Smith LA, Brettin TS, Detter JC (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. doi:10.1186/1741-7007-7-66

    Article  PubMed Central  PubMed  Google Scholar 

  • Huber KE, Waldor MK (2002) Filamentous phage integration requires the host recombinases XerC and XerD. Nature 417(6889):656–659

    Article  CAS  PubMed  Google Scholar 

  • Ihara H, Kohda T, Morimoto F, Tsukamoto K, Karasawa T, Nakamura S, Mukamoto M, Kozaki S (2003) Sequence of the gene for Clostridium botulinum type B neurotoxin associated with infant botulism, expression of the C-terminal half of heavy chain and its binding activity. Biochim Biophys Acta 1625(1):19–26

    Article  CAS  PubMed  Google Scholar 

  • Kakinuma H, Maruyama H, Takahashi H, Yamakawa K, Nakamura S (1996) The first case of type B infant botulism in Japan. Acta Paediatr Jpn 38(5):541–543

    Article  CAS  PubMed  Google Scholar 

  • Kohda T, Ihara H, Seto Y, Tsutsuki H, Mukamoto M, Kozaki S (2007) Differential contribution of the residues in C-terminal half of the heavy chain of botulinum neurotoxin type B to its binding to the ganglioside GT1b and the synaptotagmin 2/GT1b complex. Microb Pathog 42(2–3):72–79

    Article  CAS  PubMed  Google Scholar 

  • Kozaki S, Kamata Y, Nishiki T, Kakinuma H, Maruyama H, Takahashi H, Karasawa T, Yamakawa K, Nakamura S (1998) Characterization of Clostridium botulinum type B neurotoxin associated with infant botulism in japan. Infect Immun 66(10):4811–4816

    CAS  PubMed Central  PubMed  Google Scholar 

  • Lamanna C, Glassman HN (1947) The isolation of type B Botulinum Toxin. J Bacteriol 54(5):575–584

    CAS  PubMed Central  PubMed  Google Scholar 

  • Mahillon J, Chandler M (1998) Insertion sequences. Microbiol Mol Biol Rev 62(3):725–774

    CAS  PubMed Central  PubMed  Google Scholar 

  • Matsumura T, Jin Y, Kabumoto Y, Takegahara Y, Oguma K, Lencer WI, Fujinaga Y (2008) The HA proteins of botulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption. Cell Microbiol 10(2):355–364

    CAS  PubMed  Google Scholar 

  • Montecucco C, Schiavo G (1995) Structure and function of tetanus and botulinum neurotoxins. Q Rev Biophys 28(4):423–472

    Article  CAS  PubMed  Google Scholar 

  • Nakayama K, Takashima K, Ishihara H, Shinomiya T, Kageyama M, Kanaya S, Ohnishi M, Murata T, Mori H, Hayashi T (2000) The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage. Mol Microbiol 38(2):213–231

    Article  CAS  PubMed  Google Scholar 

  • Ohtsubo Y, Ikeda-Ohtsubo W, Nagata Y, Tsuda M (2008) GenomeMatcher: a graphical user interface for DNA sequence comparison. BMC Bioinform 9:376. doi:10.1186/1471-2105-9-376

    Article  Google Scholar 

  • Peck MW (2009) Biology and genomic analysis of Clostridium botulinum. Adv Microb Physi 55(183–265):320. doi:10.1016/S0065-2911(09)05503-9

    Google Scholar 

  • Raffestin S, Marvaud JC, Cerrato R, Dupuy B, Popoff MR (2004) Organization and regulation of the neurotoxin genes in Clostridium botulinum and Clostridium tetani. Anaerobe 10(2):93–100

    Article  CAS  PubMed  Google Scholar 

  • Sakaguchi Y, Hayashi T, Kurokawa K, Nakayama K, Oshima K, Fujinaga Y, Ohnishi M, Ohtsubo E, Hattori M, Oguma K (2005) The genome sequence of Clostridium botulinum type C neurotoxin-converting phage and the molecular mechanisms of unstable lysogeny. Proc Natl Acad Sci USA 102(48):17472–17477

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sebaihia M, Peck MW, Minton NP, Thomson NR, Holden MT, Mitchell WJ, Carter AT, Bentley SD, Mason DR, Crossman L, Paul CJ, Ivens A, Wells-Bennik MH, Davis IJ, Cerdeno-Tarraga AM, Churcher C, Quail MA, 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(7):1082–1092

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Smith TJ, Hill KK, Foley BT, Detter JC, Munk AC, Bruce DC, Doggett NA, Smith LA, Marks JD, Xie G, Brettin TS (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(12):e1271

    Article  PubMed Central  PubMed  Google Scholar 

  • Sugiyama H (1980) Clostridium botulinum neurotoxin. Microbiol Rev 44(3):419–448

    CAS  PubMed Central  PubMed  Google Scholar 

  • Umeda K, Seto Y, Kohda T, Mukamoto M, Kozaki S (2009) Genetic characterization of Clostridium botulinum associated with type B infant botulism in Japan. J Clin Microbiol 47(9):2720–2728. doi:10.1128/JCM.00077-09

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Umeda K, Seto Y, Kohda T, Mukamoto M, Kozaki S (2012) Stability of toxigenicity in proteolytic Clostridium botulinum type B upon serial passage. Microbiol Immunol 56(5):338–341. doi:10.1111/j.1348-0421.2012.00441.x

    Article  CAS  PubMed  Google Scholar 

  • Umeda K, Wada T, Kohda T, Kozaki S (2013) Multi-locus variable number tandem repeat analysis for Clostridium botulinum type B isolates in Japan: comparison with other isolates and genotyping methods. Infect Genet Evol 16:298–304. doi:10.1016/j.meegid.2013.02.022

    Article  CAS  PubMed  Google Scholar 

  • Yamaichi Y, Niki H (2000) Active segregation by the Bacillus subtilis partitioning system in Escherichia coli. Proc Natl Acad Sci USA 97(26):14656–14661

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yamakawa K, Karasawa T, Kakinuma H, Maruyama H, Takahashi H, Nakamura S (1997) Emergence of Clostridium botulinum type B-like nontoxigenic organisms in a patient with type B infant botulism. J Clin Microbiol 35(8):2163–2164

    CAS  PubMed Central  PubMed  Google Scholar 

  • Ye Q, Roh Y, Carroll SL, Blair B, Zhou J, Zhang CL, Fields MW (2004) Alkaline anaerobic respiration: isolation and characterization of a novel alkaliphilic and metal-reducing bacterium. Appl Environ Microbiol 70(9):5595–5602. doi:10.1128/AEM.70.9.5595-5602.2004

    Article  PubMed Central  PubMed  Google Scholar 

  • Zielenkiewicz U, Ceglowski P (2001) Mechanisms of plasmid stable maintenance with special focus on plasmid addiction systems. Acta Biochim Pol 48(4):1003–1023

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Orai Kita, Izumisano, Osaka, 598-8531, Japan

    Koji Hosomi, Tomoko Kohda, Shunji Kozaki & Masafumi Mukamoto

  2. Interdisciplinary Research Organization, University of Miyazaki, Miyazaki, Japan

    Yoshihiko Sakaguchi

  3. Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan

    Kazuyoshi Gotoh, Daisuke Motooka, Shota Nakamura & Tetsuya Iida

  4. Department of Microbiology, Osaka City Institute of Public Health and Environmental Sciences, Osaka, Japan

    Kaoru Umeda

Authors
  1. Koji Hosomi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yoshihiko Sakaguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tomoko Kohda
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kazuyoshi Gotoh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Daisuke Motooka
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shota Nakamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kaoru Umeda
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tetsuya Iida
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Shunji Kozaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Masafumi Mukamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Masafumi Mukamoto.

Additional information

Communicated by S. Hohmann.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PPTX 799 kb)

Supplementary material 2 (TXT 43 kb)

Supplementary material 3 (XLSX 39 kb)

Supplementary Fig. 1 PFGE of undigested DNA and Southern blot hybridization.

The PFGE patterns of undigested DNA (a) and Southern blot hybridization detection of the bont/B genes (b) of strain 111 (lane 1) and a non-toxigenic derivative (lane 2) are shown. Lane M, bacteriophage lambda ladder.

Supplementary Fig. 2 Circular plasmid map of pCB111.

The plasmid map shows the location of the neurotoxin complex genes (in red) and transposases (in green) within pCB111 and the predicted sites of gene transcription. Data are described from the outermost to the innermost circle. Circles 1 and 2 show gray bars to indicate the predicted coding sequences on the plus strand (circle 1) and minus strand (circle 2). Circle 3 shows the G + C content. Circle 4 shows the GC skew (G-C/G + C).

Supplementary Fig. 3 Phylogenetic tree based on mobile element enzyme amino acid sequences.

CB111_321 (a) and CB111_322 (b) were aligned to highly homologous proteins (transposase, recombinase, or integrase) identified within Genbank by BLAST. CB111_321 and CB111_322 are indicated in red, the top ten matches are indicated in blue. The numbers on each branch indicate bootstrap values (>950) for the cluster supported by that branch.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hosomi, K., Sakaguchi, Y., Kohda, T. et al. Complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in Clostridium botulinum type B strain 111 isolated from an infant patient in Japan. Mol Genet Genomics 289, 1267–1274 (2014). https://doi.org/10.1007/s00438-014-0887-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-014-0887-4

Keywords

Search

Navigation