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. 1989 Feb;57(2):367–376. doi: 10.1128/iai.57.2.367-376.1989

Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens.

R W Titball 1, S E Hunter 1, K L Martin 1, B C Morris 1, A D Shuttleworth 1, T Rubidge 1, D W Anderson 1, D C Kelly 1
PMCID: PMC313106  PMID: 2536355

Abstract

A fragment of DNA containing the gene coding for the phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into Escherichia coli. The cloned DNA appeared to code only for the alpha-toxin and contained both the coding region and its associated gene promoter. The nucleotide sequence of the cloned DNA was determined, and an open reading frame was identified which encoded a protein with a molecular weight of 42,528. By comparison of the gene sequence with the N-terminal amino acid sequence of the protein, a 28-amino-acid signal sequence was identified. The gene promoter showed considerable homology with the E. coli sigma 55 consensus promoter sequences, and this may explain why the gene was expressed by E. coli. The cloned gene product appeared to be virtually identical to the native protein. A 77-amino-acid stretch that was close to the N terminus of the alpha-toxin showed considerable homology with similarly located regions of the Bacillus cereus phosphatidylcholine, preferring phospholipase C and weaker homology with the phospholipase C from Pseudomonas aeruginosa.

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Selected References

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  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Chen K. C., Chen J. S., Johnson J. L. Structural features of multiple nifH-like sequences and very biased codon usage in nitrogenase genes of Clostridium pasteurianum. J Bacteriol. 1986 Apr;166(1):162–172. doi: 10.1128/jb.166.1.162-172.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Eloy C., Fardel G., Flandrois J. P. Fast protein liquid chromatography for the isolation of Clostridium perfringens type A alpha-toxin. J Chromatogr. 1985 Mar 8;321(1):235–239. doi: 10.1016/s0021-9673(01)90440-6. [DOI] [PubMed] [Google Scholar]
  4. Garnier T., Cole S. T. Characterization of a bacteriocinogenic plasmid from Clostridium perfringens and molecular genetic analysis of the bacteriocin-encoding gene. J Bacteriol. 1986 Dec;168(3):1189–1196. doi: 10.1128/jb.168.3.1189-1196.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Godson G. N., Vapnek D. A simple method of preparing large amounts of phiX174 RF 1 supercoiled DNA. Biochim Biophys Acta. 1973 Apr 11;299(4):516–520. doi: 10.1016/0005-2787(73)90223-2. [DOI] [PubMed] [Google Scholar]
  6. Götz F., Popp F., Korn E., Schleifer K. H. Complete nucleotide sequence of the lipase gene from Staphylococcus hyicus cloned in Staphylococcus carnosus. Nucleic Acids Res. 1985 Aug 26;13(16):5895–5906. doi: 10.1093/nar/13.16.5895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  9. Hopp T. P., Woods K. R. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3824–3828. doi: 10.1073/pnas.78.6.3824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Johansen T., Holm T., Guddal P. H., Sletten K., Haugli F. B., Little C. Cloning and sequencing of the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus. Gene. 1988 May 30;65(2):293–304. doi: 10.1016/0378-1119(88)90466-0. [DOI] [PubMed] [Google Scholar]
  11. Kameyama S., Sato H., Murata R. The role of alpha-toxin of Clostridium perfringens in experimental gas gangrene in guinea pigs. Jpn J Med Sci Biol. 1972 Jun;25(3):200–200. [PubMed] [Google Scholar]
  12. Krug E. L., Kent C. Phospholipase C from Clostridium perfringens: preparation and characterization of homogeneous enzyme. Arch Biochem Biophys. 1984 Jun;231(2):400–410. doi: 10.1016/0003-9861(84)90403-x. [DOI] [PubMed] [Google Scholar]
  13. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Little C., Otnåss A. B. The metal ion dependence of phospholipase C from Bacillus cereus. Biochim Biophys Acta. 1975 Jun 24;391(2):326–333. doi: 10.1016/0005-2744(75)90256-9. [DOI] [PubMed] [Google Scholar]
  16. Macfarlane M. G., Knight B. C. The biochemistry of bacterial toxins: The lecithinase activity of Cl. welchii toxins. Biochem J. 1941 Sep;35(8-9):884–902. doi: 10.1042/bj0350884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Minton N. P., Atkinson T., Sherwood R. F. Molecular cloning of the Pseudomonas carboxypeptidase G2 gene and its expression in Escherichia coli and Pseudomonas putida. J Bacteriol. 1983 Dec;156(3):1222–1227. doi: 10.1128/jb.156.3.1222-1227.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mitsu K., Mitsui N., Hase J. Clostridium perfringens exotoxins. I. Purification and properties of -toxin. Jpn J Exp Med. 1973 Apr;43(2):65–80. [PubMed] [Google Scholar]
  19. Moran C. P., Jr, Lang N., Banner C. D., Haldenwang W. G., Losick R. Promoter for a developmentally regulated gene in Bacillus subtilis. Cell. 1981 Sep;25(3):783–791. doi: 10.1016/0092-8674(81)90186-0. [DOI] [PubMed] [Google Scholar]
  20. Möllby R., Nord C. E., Wadström T. Biological activities contaminating preparations of phospholipase C ( -toxin) from Clostridium perfringens. Toxicon. 1973 Feb;11(2):139–147. doi: 10.1016/0041-0101(73)90075-5. [DOI] [PubMed] [Google Scholar]
  21. Möllby R., Thelestam M., Wadström T. Effect of Clostridium perfringens phospholipase C(alpha-toxin) on the human diploid fibroblast membrane. J Membr Biol. 1974;16(4):313–330. doi: 10.1007/BF01872421. [DOI] [PubMed] [Google Scholar]
  22. Nelson H. C., Finch J. T., Luisi B. F., Klug A. The structure of an oligo(dA).oligo(dT) tract and its biological implications. Nature. 1987 Nov 19;330(6145):221–226. doi: 10.1038/330221a0. [DOI] [PubMed] [Google Scholar]
  23. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  24. Otnaess A. B., Little C., Sletten K., Wallin R., Johnsen S., Flengsrud R., Prydz H. Some characteristics of phospholipase C from Bacillus cereus. Eur J Biochem. 1977 Oct 3;79(2):459–468. doi: 10.1111/j.1432-1033.1977.tb11828.x. [DOI] [PubMed] [Google Scholar]
  25. Pritchard A. E., Vasil M. L. Nucleotide sequence and expression of a phosphate-regulated gene encoding a secreted hemolysin of Pseudomonas aeruginosa. J Bacteriol. 1986 Jul;167(1):291–298. doi: 10.1128/jb.167.1.291-298.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  27. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  28. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Smith L. D. Virulence factors of Clostridium perfringens. Rev Infect Dis. 1979 Mar-Apr;1(2):254–262. doi: 10.1093/clinids/1.2.254. [DOI] [PubMed] [Google Scholar]
  30. Smyth C. J., Arbuthnott J. P. Properties of Clostridium perfringens (welchii) type-A alpha-toxin (phospholipase C) purified by electrofocusing. J Med Microbiol. 1974 Feb;7(1):41–66. doi: 10.1099/00222615-7-1-41. [DOI] [PubMed] [Google Scholar]
  31. Smyth C. J., Freer J. H., Arbuthnott J. P. Interaction of Clostridium perfringens theta-haemolysin, a contaminant of commercial phospholipase C, with erythrocyte ghost membranes and lipid dispersions. A morphological study. Biochim Biophys Acta. 1975 Apr 8;382(4):479–493. doi: 10.1016/0005-2736(75)90216-3. [DOI] [PubMed] [Google Scholar]
  32. Takahashi T., Sugahara T., Ohsaka A. Purification of Clostridium perfringens phospholipase C (alpha-toxin) by affinity chromatography on agarose-linked egg-yolk lipoprotein. Biochim Biophys Acta. 1974 May 10;351(1):155–171. doi: 10.1016/0005-2795(74)90074-9. [DOI] [PubMed] [Google Scholar]
  33. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  34. Turnell W., Sarra R., Glover I. D., Baum J. O., Caspi D., Baltz M. L., Pepys M. B. Secondary structure prediction of human SAA1. Presumptive identification of calcium and lipid binding sites. Mol Biol Med. 1986 Oct;3(5):387–407. [PubMed] [Google Scholar]
  35. Yamakawa Y., Ohsaka A. Purification and some properties of phospholipase C (alpha-toxin) of Clostridium perfringens. J Biochem. 1977 Jan;81(1):115–126. doi: 10.1093/oxfordjournals.jbchem.a131426. [DOI] [PubMed] [Google Scholar]
  36. van Dam-Mieras M. C., Slotboom A. J., Pieterson W. A., de Haas G. H. The interaction of phospholipase A2 with micellar interfaces. The role of the N-terminal region. Biochemistry. 1975 Dec 16;14(25):5387–5394. doi: 10.1021/bi00696a001. [DOI] [PubMed] [Google Scholar]
  37. von Heijne G. How signal sequences maintain cleavage specificity. J Mol Biol. 1984 Feb 25;173(2):243–251. doi: 10.1016/0022-2836(84)90192-x. [DOI] [PubMed] [Google Scholar]

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