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. 1987 Jul;169(7):3321–3328. doi: 10.1128/jb.169.7.3321-3328.1987

Construction and use of signal sequence selection vectors in Escherichia coli and Bacillus subtilis.

H Smith, S Bron, J Van Ee, G Venema
PMCID: PMC212386  PMID: 3110136

Abstract

To study the diversity and efficiency of signal peptides for secreted proteins in gram-positive bacteria, two plasmid vectors were constructed which were used to probe for export signal-coding regions in Bacillus subtilis. The vectors contained genes coding for extracellular proteins (the alpha-amylase gene from Bacillus licheniformis and the beta-lactamase gene from Escherichia coli) which lacked a functional signal sequence. By shotgun cloning of restriction fragments from B. subtilis chromosomal DNA, a great variety of different export-coding regions were selected. These regions were functional both in B. subtilis and in E. coli. In a number of cases where protein export had been restored, intracellular precursor proteins of increased size could be detected, which upon translocation across the cellular membrane were processed to mature products. The high frequency with which export signal-coding regions were obtained suggests that, in addition to natural signal sequences, many randomly cloned sequences can function as export signal.

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Selected References

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

  1. Bron S., Venema G. Ultraviolet inactivation and excision-repair in Bacillus subtilis. I. Construction and characterization of a transformable eightfold auxotrophic strain and two ultraviolet-sensitive derivatives. Mutat Res. 1972 May;15(1):1–10. doi: 10.1016/0027-5107(72)90086-3. [DOI] [PubMed] [Google Scholar]
  2. Ghrayeb J., Kimura H., Takahara M., Hsiung H., Masui Y., Inouye M. Secretion cloning vectors in Escherichia coli. EMBO J. 1984 Oct;3(10):2437–2442. doi: 10.1002/j.1460-2075.1984.tb02151.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Hoffman C. S., Wright A. Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5107–5111. doi: 10.1073/pnas.82.15.5107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kadonaga J. T., Gautier A. E., Straus D. R., Charles A. D., Edge M. D., Knowles J. R. The role of the beta-lactamase signal sequence in the secretion of proteins by Escherichia coli. J Biol Chem. 1984 Feb 25;259(4):2149–2154. [PubMed] [Google Scholar]
  6. Kaiser C. A., Preuss D., Grisafi P., Botstein D. Many random sequences functionally replace the secretion signal sequence of yeast invertase. Science. 1987 Jan 16;235(4786):312–317. doi: 10.1126/science.3541205. [DOI] [PubMed] [Google Scholar]
  7. Kawamura F., Doi R. H. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J Bacteriol. 1984 Oct;160(1):442–444. doi: 10.1128/jb.160.1.442-444.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kok J., van der Vossen J. M., Venema G. Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Appl Environ Microbiol. 1984 Oct;48(4):726–731. doi: 10.1128/aem.48.4.726-731.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  11. Mézes P. S., Wang W., Yeh E. C., Lampen J. O. Construction of penP delta 1, Bacillus licheniformis 749/C beta-lactamase lacking site for lipoprotein modification. Expression in Escherichia coli and Bacillus subtilis. J Biol Chem. 1983 Sep 25;258(18):11211–11218. [PubMed] [Google Scholar]
  12. Nakamura K., Furusato T., Shiroza T., Yamane K. Stable hyper-production of Escherichia coli beta-lactamase by Bacillus subtilis grown on a 0.5 M succinate-medium using a B. subtilis alpha-amylase secretion vector. Biochem Biophys Res Commun. 1985 Apr 30;128(2):601–606. doi: 10.1016/0006-291x(85)90088-9. [DOI] [PubMed] [Google Scholar]
  13. Ohmura K., Shiroza T., Nakamura K., Nakayama A., Yamane K., Yoda K., Yamasaki M., Tamura G. A Bacillus subtilis secretion vector system derived from the B. subtilis alpha-amylase promoter and signal sequence region, and secretion of Escherichia coli beta-lactamase by the vector system. J Biochem. 1984 Jan;95(1):87–93. doi: 10.1093/oxfordjournals.jbchem.a134607. [DOI] [PubMed] [Google Scholar]
  14. Palva I., Sarvas M., Lehtovaara P., Sibakov M., Käriäinen L. Secretion of Escherichia coli beta-lactamase from Bacillus subtilis by the aid of alpha-amylase signal sequence. Proc Natl Acad Sci U S A. 1982 Sep;79(18):5582–5586. doi: 10.1073/pnas.79.18.5582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Priest F. G. Extracellular enzyme synthesis in the genus Bacillus. Bacteriol Rev. 1977 Sep;41(3):711–753. doi: 10.1128/br.41.3.711-753.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Roggenkamp R., Dargatz H., Hollenberg C. P. Precursor of beta-lactamase is enzymatically inactive. Accumulation of the preprotein in Saccharomyces cerevisiae. J Biol Chem. 1985 Feb 10;260(3):1508–1512. [PubMed] [Google Scholar]
  17. Silhavy T. J., Benson S. A., Emr S. D. Mechanisms of protein localization. Microbiol Rev. 1983 Sep;47(3):313–344. doi: 10.1128/mr.47.3.313-344.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Stahl M. L., Ferrari E. Replacement of the Bacillus subtilis subtilisin structural gene with an In vitro-derived deletion mutation. J Bacteriol. 1984 May;158(2):411–418. doi: 10.1128/jb.158.2.411-418.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tai P. C., Zyk N., Citri N. In situ detection of beta-lactamase activity in sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem. 1985 Jan;144(1):199–203. doi: 10.1016/0003-2697(85)90105-8. [DOI] [PubMed] [Google Scholar]
  20. Ulmanen I., Lundström K., Lehtovaara P., Sarvas M., Ruohonen M., Palva I. Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector. J Bacteriol. 1985 Apr;162(1):176–182. doi: 10.1128/jb.162.1.176-182.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Uozumi T., Ozaki A., Beppu T., Arima K. New cryptic plasmid of Bacillus subtilis and restriction analysis of other plasmids found by general screening. J Bacteriol. 1980 Apr;142(1):315–318. doi: 10.1128/jb.142.1.315-318.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Vasantha N., Thompson L. D. Secretion of a heterologous protein from Bacillus subtilis with the aid of protease signal sequences. J Bacteriol. 1986 Mar;165(3):837–842. doi: 10.1128/jb.165.3.837-842.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Watson M. E. Compilation of published signal sequences. Nucleic Acids Res. 1984 Jul 11;12(13):5145–5164. doi: 10.1093/nar/12.13.5145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Williams D. M., Duvall E. J., Lovett P. S. Cloning restriction fragments that promote expression of a gene in Bacillus subtilis. J Bacteriol. 1981 Jun;146(3):1162–1165. doi: 10.1128/jb.146.3.1162-1165.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yang M. Y., Ferrari E., Henner D. J. Cloning of the neutral protease gene of Bacillus subtilis and the use of the cloned gene to create an in vitro-derived deletion mutation. J Bacteriol. 1984 Oct;160(1):15–21. doi: 10.1128/jb.160.1.15-21.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yuuki T., Nomura T., Tezuka H., Tsuboi A., Yamagata H., Tsukagoshi N., Udaka S. Complete nucleotide sequence of a gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying alpha-amylases deduced from the DNA sequences. J Biochem. 1985 Nov;98(5):1147–1156. doi: 10.1093/oxfordjournals.jbchem.a135381. [DOI] [PubMed] [Google Scholar]

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