Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Sep;82(17):5850–5854. doi: 10.1073/pnas.82.17.5850

Molecular cloning of staphylococcal enterotoxin B gene in Escherichia coli and Staphylococcus aureus.

D M Ranelli, C L Jones, M B Johns, G J Mussey, S A Khan
PMCID: PMC390651  PMID: 3898073

Abstract

We have cloned the Staphylococcus aureus entB gene in Escherichia coli, using pBR322 as the vector plasmid; however, no detectable staphylococcal enterotoxin B (SEB) was produced by the E. coli clones. When the entB gene was placed downstream from the strong lambda phage promoter, PR, SEB was synthesized at readily detectable levels in E. coli. Interestingly, mature SEB was almost exclusively present in the cytoplasmic fraction. The SEB precursor was found associated with the cell membrane. The entB gene was introduced back into S. aureus, and the clones were shown to produce SEB. The entB gene has been located to a 2.1-kilobase-pair region. Maxam-Gilbert sequencing of a part of the entB gene yielded a DNA sequence that corresponds to the known amino acid sequence of SEB. Southern hybridization experiments showed that the entB gene was present on identical restriction fragments in the chromosomes of SEB-producer strains. The entB gene is absent from SEB-nonproducer strains.

Full text

PDF
5850

Images in this article

5851Image
on p.5851
5852Image
on p.5852
5852Image
on p.5852
5853Image
on p.5853
5853Image
on p.5853
5853Image
on p.5853

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Betley M. J., Löfdahl S., Kreiswirth B. N., Bergdoll M. S., Novick R. P. Staphylococcal enterotoxin A gene is associated with a variable genetic element. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5179–5183. doi: 10.1073/pnas.81.16.5179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Chang S., Cohen S. N. High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet. 1979 Jan 5;168(1):111–115. doi: 10.1007/BF00267940. [DOI] [PubMed] [Google Scholar]
  5. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Czop J. K., Bergdoll M. S. Staphylococcal enterotoxin synthesis during the exponential, transitional, and stationary growth phases. Infect Immun. 1974 Feb;9(2):229–235. doi: 10.1128/iai.9.2.229-235.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dalidowicz J. E., Silverman S. J., Schantz E. J., Stefanye D., Spero L. Chemical and biological properties of reduced and alkylated staphylococcal enterotoxin B. Biochemistry. 1966 Jul;5(7):2375–2381. doi: 10.1021/bi00871a029. [DOI] [PubMed] [Google Scholar]
  8. Dornbusch K., Hallander H. O., Löfquist F. Extrachromosomal control of methicillin resistance and toxin production in Staphylococcus aureus. J Bacteriol. 1969 May;98(2):351–358. doi: 10.1128/jb.98.2.351-358.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dyer D. W., Iandolo J. J. Plasmid-chromosomal transition of genes important in staphylococcal enterotoxin B expression. Infect Immun. 1981 Aug;33(2):450–458. doi: 10.1128/iai.33.2.450-458.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Emr S. D., Hall M. N., Silhavy T. J. A mechanism of protein localization: the signal hypothesis and bacteria. J Cell Biol. 1980 Sep;86(3):701–711. doi: 10.1083/jcb.86.3.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Frei E., Levy A., Gowland P., Noll M. Efficient transfer of small DNA fragments from polyacrylamide gels to diazo or nitrocellulose paper and hybridization. Methods Enzymol. 1983;100:309–326. doi: 10.1016/0076-6879(83)00064-6. [DOI] [PubMed] [Google Scholar]
  12. Grunstein M., Wallis J. Colony hybridization. Methods Enzymol. 1979;68:379–389. doi: 10.1016/0076-6879(79)68027-8. [DOI] [PubMed] [Google Scholar]
  13. Hanahan D., Meselson M. Plasmid screening at high colony density. Methods Enzymol. 1983;100:333–342. doi: 10.1016/0076-6879(83)00066-x. [DOI] [PubMed] [Google Scholar]
  14. Horinouchi S., Weisblum B. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol. 1982 May;150(2):815–825. doi: 10.1128/jb.150.2.815-825.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Horinouchi S., Weisblum B. Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies. J Bacteriol. 1982 May;150(2):804–814. doi: 10.1128/jb.150.2.804-814.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Huang I. Y., Bergdoll M. S. The primary structure of staphylococcal enterotoxin B. 3. The cyanogen bromide peptides of reduced and aminoethylated enterotoxin B, and the complete amino acid sequence. J Biol Chem. 1970 Jul 25;245(14):3518–3525. [PubMed] [Google Scholar]
  17. Jarvis A. W., Lawrence R. C. Production of extracellular enzymes and enterotoxins A, B, and C by Staphylococcus aureus. Infect Immun. 1971 Aug;4(2):110–115. doi: 10.1128/iai.4.2.110-115.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kehoe M., Duncan J., Foster T., Fairweather N., Dougan G. Cloning, expression, and mapping of the Staphylococcus aureus alpha-hemolysin determinant in Escherichia coli K-12. Infect Immun. 1983 Sep;41(3):1105–1111. doi: 10.1128/iai.41.3.1105-1111.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Khan S. A., Novick R. P. Structural analysis of plasmid pSN2 in Staphylococcus aureus: no involvement in enterotoxin B production. J Bacteriol. 1982 Feb;149(2):642–649. doi: 10.1128/jb.149.2.642-649.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kreiswirth B. N., Löfdahl S., Betley M. J., O'Reilly M., Schlievert P. M., Bergdoll M. S., Novick R. P. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature. 1983 Oct 20;305(5936):709–712. doi: 10.1038/305709a0. [DOI] [PubMed] [Google Scholar]
  21. Löfdahl S., Guss B., Uhlén M., Philipson L., Lindberg M. Gene for staphylococcal protein A. Proc Natl Acad Sci U S A. 1983 Feb;80(3):697–701. doi: 10.1073/pnas.80.3.697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Maniatis T., Jeffrey A., Kleid D. G. Nucleotide sequence of the rightward operator of phage lambda. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1184–1188. doi: 10.1073/pnas.72.3.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  24. Middlebrook J. L., Spero L., Argos P. The secondary structure of staphylococcal enterotoxins A, B and C. Biochim Biophys Acta. 1980 Feb 27;621(2):233–240. doi: 10.1016/0005-2795(80)90175-0. [DOI] [PubMed] [Google Scholar]
  25. Morrison D. A. Transformation and preservation of competent bacterial cells by freezing. Methods Enzymol. 1979;68:326–331. doi: 10.1016/0076-6879(79)68023-0. [DOI] [PubMed] [Google Scholar]
  26. Murray N. E., Bruce S. A., Murray K. Molecular cloning of the DNA ligase gene from bacteriophage T4. II. Amplification and preparation of the gene product. J Mol Biol. 1979 Aug 15;132(3):493–505. doi: 10.1016/0022-2836(79)90271-7. [DOI] [PubMed] [Google Scholar]
  27. Oliver D. B., Beckwith J. E. coli mutant pleiotropically defective in the export of secreted proteins. Cell. 1981 Sep;25(3):765–772. doi: 10.1016/0092-8674(81)90184-7. [DOI] [PubMed] [Google Scholar]
  28. Pattee P. A., Neveln D. S. Transformation analysis of three linkage groups in Staphylococcus aureus. J Bacteriol. 1975 Oct;124(1):201–211. doi: 10.1128/jb.124.1.201-211.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pearson G. D., Mekalanos J. J. Molecular cloning of Vibrio cholerae enterotoxin genes in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1982 May;79(9):2976–2980. doi: 10.1073/pnas.79.9.2976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. SILVERMAN S. J. SEROLOGICAL ASSAY OF CULTURE FILTRATES FOR STAPHYLOCOCCUS ENTEROTOXIN. J Bacteriol. 1963 Apr;85:955–956. doi: 10.1128/jb.85.4.955-956.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sako T., Sawaki S., Sakurai T., Ito S., Yoshizawa Y., Kondo I. Cloning and expression of the staphylokinase gene of Staphylococcus aureus in Escherichia coli. Mol Gen Genet. 1983;190(2):271–277. doi: 10.1007/BF00330650. [DOI] [PubMed] [Google Scholar]
  32. Schantz E. J., Roessler W. G., Wagman J., Spero L., Dunnery D. A., Bergdoll M. S. Purification of staphylococcal enterotoxin B. Biochemistry. 1965 Jun;4(6):1011–1016. doi: 10.1021/bi00882a005. [DOI] [PubMed] [Google Scholar]
  33. Sero L., Metzger J. F., Warren J. R., Griffin B. Y. Biological activity and complementation of the two peptides of staphylococcal enterotoxin B formed by limited tryptic hydrolysis. J Biol Chem. 1975 Jul 10;250(13):5026–5032. [PubMed] [Google Scholar]
  34. Shafer W. M., Iandolo J. J. Chromosomal locus for staphylococcal enterotoxin B. Infect Immun. 1978 Apr;20(1):273–278. doi: 10.1128/iai.20.1.273-278.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shafer W. M., Iandolo J. J. Genetics of staphylococcal enterotoxin B in methicillin-resistant isolates of Staphylococcus aureus. Infect Immun. 1979 Sep;25(3):902–911. doi: 10.1128/iai.25.3.902-911.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shafer W. M., Iandolo J. J. Transduction of staphylococcal enterotoxin B synthesis: establishment of the toxin gene in a recombination-deficient mutant. Infect Immun. 1980 Jan;27(1):280–282. doi: 10.1128/iai.27.1.280-282.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shalita Z., Hertman I., Sarid S. Isolation and characterization of a plasmid involved with enterotoxin B production in Staphylococcus aureus. J Bacteriol. 1977 Jan;129(1):317–325. doi: 10.1128/jb.129.1.317-325.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  39. Studier F. W. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J Mol Biol. 1973 Sep 15;79(2):237–248. doi: 10.1016/0022-2836(73)90003-x. [DOI] [PubMed] [Google Scholar]
  40. Tanaka T., Weisblum B. Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. J Bacteriol. 1975 Jan;121(1):354–362. doi: 10.1128/jb.121.1.354-362.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tweten R. K., Iandolo J. J. Purification and partial characterization of a putative precursor to staphylococcal enterotoxin B. Infect Immun. 1981 Dec;34(3):900–907. doi: 10.1128/iai.34.3.900-907.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Vogelstein B., Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. doi: 10.1073/pnas.76.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES