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Volume 150, Issue 8

Genetic and functional analysis of the family of genes in Free

Abstract

CytK is a pore-forming toxin of that has been linked to a case of necrotic enteritis. PCR products of the expected size were generated with primers in 13 of 29 strains. Six strains were PCR-positive for the related gene , which encodes haemolysin II, a protein that is 37 % identical to the original CytK. Five of the strains were positive for both genes. The DNA sequences of putative genes from three positive strains were determined, and the deduced amino acid sequences were 89 % identical to that of the original CytK. The authors have designated this new variant , and refer to the original as . The CytK-2 proteins from these three strains were isolated, and their identity was verified by N-terminal sequencing. analysis using the gene sequences revealed very high homology with two sequences in the genomes of strains ATCC 14579 and ATCC 10987. The differences between CytK-1 and the CytK-2 proteins were clustered to certain regions of the proteins. The isolated CytK-2 proteins were haemolytic and toxic towards human intestinal Caco-2 cells and Vero cells, although their toxicity was about 20 % of that of CytK-1. Both native and recombinant CytK-2 proteins from 1230-88 were able to form pores in planar lipid bilayers, but the majority of the channels observed were of lower conductance than those created by CytK-1. It is likely that CytK-2 toxins contribute to the enterotoxicity of several strains of , although not all of the CytK-2 toxins may be as harmful as the CytK-1 originally isolated.

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  • Accepted:
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  • Published Online:
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2004-08-01
2024-04-16
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References

  1. Agaisse H., Gominet M., Økstad O. A., Kolstø A.-B., Lereclus D. 1999; PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis. Mol Microbiol 32:1043–1053 [CrossRef]
     [Google Scholar]
  2. Altschul S. F., Madden T. L., Schaffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. 1997; Gapped BLAST and PSI-BLAST: a new generation of protein database programs. Nucleic Acids Res 25:3389–3402 [CrossRef]
     [Google Scholar]
  3. Baida G., Budarina Z. I., Kuzmin N. P., Solonin A. S. 1999; Complete nucleotide sequence and molecular characterization of hemolysin II gene from Bacillus cereus. FEMS Microbiol Lett 180:7–14 [CrossRef]
     [Google Scholar]
  4. Beecher D. J., Wong A. C. L. 1994; Improved purification and characterization of hemolysin BL, a hemolytic dermonecrotic vascular permeability factor from Bacillus cereus. Infect Immun 62:980–986
     [Google Scholar]
  5. Beecher D. J., Olsen T. W., Somers E. B., Wong A. C. L. 2000; Evidence for contribution of tripartite hemolysin BL, phosphatidylcholine-preferring phospholipase C, and collagenase to virulence of Bacillus cereus endophthalmitis. Infect Immun 68:5269–5276 [CrossRef]
     [Google Scholar]
  6. Blum H., Beier H., Gross H. J. 1987; Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93–99 [CrossRef]
     [Google Scholar]
  7. Budarina Z. I., Sinev M. A., Mayorov S. G., Tomashevski A. Y., Shmelev I. V., Kuzmin N. P. 1994; Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus. Arch Microbiol 161:252–257
     [Google Scholar]
  8. Chattopadhyay K., Banerjee K. K. 2003; Unfolding of Vibrio cholerae hemolysin induces oligomerization of the toxin monomer. J Biol Chem 278:38470–38475 [CrossRef]
     [Google Scholar]
  9. Coolbaugh J. C., Williams R. P. 1978; Production and characterization of two hemolysins of Bacillus cereus. Can J Microbiol 24:1289–1295 [CrossRef]
     [Google Scholar]
  10. Fedhila S., Gohar M., Slamti L., Nel P., Lereclus D. 2003; The Bacillus thuringiensis PlcR-regulated gene inhA2 is necessary, but not sufficient, for virulence. J Bacteriol 185:2820–2825 [CrossRef]
     [Google Scholar]
  11. Fossum K. 1963; Separation of hemolysin and egg yolk turbidity factor in cell-free extracts of Bacillus cereus. Acta Pathol Microbiol Scand 59:400–406
     [Google Scholar]
  12. Gohar M., Økstad O. A., Gilois N., Sanchis V., Kolstø A.-B., Lereclus D. 2002; Two-dimensional electrophoresis analysis of the extracellular proteome of Bacillus cereus reveals the importance of the PlcR regulon. Proteomics 2:784–791 [CrossRef]
     [Google Scholar]
  13. Granum P. E. 2001; Bacillus cereus. In Food Microbiology. Fundamentals and Frontiers, 2nd edn. pp 373–381 Edited by Doyle M., Beuchat L., Montville T. Washington, DC: American Society for Microbiology;
     [Google Scholar]
  14. Granum P. E., Lund T. 1997; Bacillus cereus enterotoxins. FEMS Microbiol Lett 157:223–228 [CrossRef]
     [Google Scholar]
  15. Granum P. E., Brynestad S., Kramer J. M. 1993; Analysis of enterotoxin production by Bacillus cereus from dairy products, food poisoning incidents and non-gastrointestinal infections. Int J Food Microbiol 17:269–279 [CrossRef]
     [Google Scholar]
  16. Hardy S. P., Lund T., Granum P. E. 2001; CytK toxin of Bacillus cereus forms pores in planar lipid bilayers and is cytotoxic to intestinal epithelia. FEMS Microbiol Lett 197:47–51 [CrossRef]
     [Google Scholar]
  17. Kok J., van der Vossen J. M., Venema G. 1984; Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Appl Environ Microbiol 48:726–731
     [Google Scholar]
  18. Lund T., Granum P. E. 1996; Characterization of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak. FEMS Microbiol Lett 141:151–156 [CrossRef]
     [Google Scholar]
  19. Lund T., Granum P. E. 1997; Comparison of biological effect of the two different enterotoxin complexes isolated from three different strains of Bacillus cereus. Microbiology 143:3329–3336 [CrossRef]
     [Google Scholar]
  20. Lund T., De Buyser M.-L., Granum P. E. 2000; A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Mol Microbiol 38:254–261 [CrossRef]
     [Google Scholar]
  21. Pendleton I. R., Bernheimer A. W., Grushoff P. 1973; Purification and partial characterization of hemolysins from Bacillus thuringiensis. J Invertebr Pathol 21:131–135 [CrossRef]
     [Google Scholar]
  22. Pinto M., Robine-Leon S., Appay M.-D.8 other authors 1983; Enterocyte-like differentiation and polarization of human colon carcinoma cell line Caco-2 in culture. Biol Cell 47:323–330
     [Google Scholar]
  23. Pospiech A., Neumann B. 1995; A versatile quick-prep of genomic DNA from gram-positive bacteria. Trends Genet 11:217–218 [CrossRef]
     [Google Scholar]
  24. Prevost G. 1999; The bi-component staphylococcal leukocidins and γ-haemolysins (toxins. In The Comprehensive Sourcebook of Bacterial Protein Toxins pp 402–418 Edited by Freer J. H., Alouf J. E. London, UK: Academic Press;
     [Google Scholar]
  25. Sandvig K., Olsnes S. 1982; Entry of the toxic proteins abrin, modeccin, ricin and diphtheria toxin into cells. J Biol Chem 257:7495–7503
     [Google Scholar]
  26. Sinev M. A., Budarina Z. I., Gavrilenko I. V., Tomashevski A. Y., Kuzmin N. P. 1993; Evidence of the existence of hemolysin II from Bacillus cereus: cloning the genetic determinant of hemolysin II. Mol Biol 27:753–760 (English translation of Molekulyarnaya Biologiya 27, 1218–1229
    [Google Scholar]
  27. Smith T. F., Waterman M. S. 1981; Identification of common molecular subsequences. J Mol Biol 147:195–197 [CrossRef]
     [Google Scholar]
  28. Song L., Hobaugh M. R., Shustak C., Cheley S., Bayley H., Gouaux J. E. 1996; Structure of staphylococcal α-hemolysin, a heptameric transmembrane pore. Science 274:1859–1866 [CrossRef]
     [Google Scholar]
  29. Steinthorsdottir V., Halldorsson H., Andresson O. S. 2000; Clostridium perfringens β-toxin forms multimeric transmembrane pores in human endothelial cells. Microb Pathog 28:45–50 [CrossRef]
     [Google Scholar]
  30. Stenfors L. P., Granum P. E. 2001; Psychrotolerant species from the Bacillus cereus group are not necessarilyB. weihenstephanensis. FEMS Microbiol Lett 197:223–228 [CrossRef]
     [Google Scholar]
  31. van de Guchte M., van der Vossen J. M., Kok J., Venema G. 1989; Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis. Appl Environ Microbiol 55:224–228
     [Google Scholar]
  32. Williams A. J. 1995; The measurement of the function of ion channels reconstituted into artifical membranes. In Ion Channels: a Practical Approach pp 43–67 Edited by Ashley R. H. Oxford: Oxford University Press;
     [Google Scholar]
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Genetic and functional analysis of the cytK family of genes in Bacillus cereus
Microbiology 150, 2689 (2004); https://doi.org/10.1099/mic.0.26975-0
/content/journal/micro/10.1099/mic.0.26975-0
/content/journal/micro/10.1099/mic.0.26975-0
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