Skip to main content
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
. 1982 Sep;79(18):5582–5586. doi: 10.1073/pnas.79.18.5582

Secretion of Escherichia coli beta-lactamase from Bacillus subtilis by the aid of alpha-amylase signal sequence.

I Palva, M Sarvas, P Lehtovaara, M Sibakov, L Kääriäinen
PMCID: PMC346948  PMID: 6182566

Abstract

We describe a secretion vector system for introducing foreign genes into Bacillus subtilis. We constructed secretion vectors from the plasmid pUB110 and the promoter and signal sequence region of the alpha-amylase gene from Bacillus amyloliquefaciens. Foreign structural genes can be inserted into the various vectors after the signal sequence region of the alpha-amylase gene. Demonstrating secretion of a foreign gene product from Bacillus, we here report that the Escherichia coli beta-lactamase gene, devoid of its own signal sequence coding region, can be expressed in B. subtilis by the aid of the secretion vectors so that greater than 95% of the enzyme activity is secreted to the growth medium. Efficient secretion of beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) is observed if the complete signal sequence coding region of the alpha-amylase gene precedes the beta-lactamase structural gene. However, an incomplete alpha-amylase signal peptide lacking the six carboxy-terminal amino acid residues does not promote secretion of the fused beta-lactamase, which remains unprocessed and cell-associated.

Full text

PDF
5585

Images in this article

Selected References

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

  1. Anagnostopoulos C., Spizizen J. REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1961 May;81(5):741–746. doi: 10.1128/jb.81.5.741-746.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Ehrlich S. D. DNA cloning in Bacillus subtilis. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1433–1436. doi: 10.1073/pnas.75.3.1433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Goldfarb D. S., Doi R. H., Rodriguez R. L. Expression of Tn9-derived chloramphenicol resistance in Bacillus subtilis. Nature. 1981 Sep 24;293(5830):309–311. doi: 10.1038/293309a0. [DOI] [PubMed] [Google Scholar]
  6. Gryczan T. J., Contente S., Dubnau D. Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J Bacteriol. 1978 Apr;134(1):318–329. doi: 10.1128/jb.134.1.318-329.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hardy K., Stahl S., Küpper H. Production in B. subtilis of hepatitis B core antigen and a major antigen of foot and mouth disease virus. Nature. 1981 Oct 8;293(5832):481–483. doi: 10.1038/293481a0. [DOI] [PubMed] [Google Scholar]
  8. Jeppesen P. G. Separation and isolation of DNA fragments using linear polyacrylamide gradient gel electrophoresis. Methods Enzymol. 1980;65(1):305–319. doi: 10.1016/s0076-6879(80)65041-1. [DOI] [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. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. McLaughlin J. R., Murray C. L., Rabinowitz J. C. Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene. J Biol Chem. 1981 Nov 10;256(21):11283–11291. [PubMed] [Google Scholar]
  13. O'Callaghan C. H., Morris A., Kirby S. M., Shingler A. H. Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother. 1972 Apr;1(4):283–288. doi: 10.1128/aac.1.4.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Palva I., Pettersson R. F., Kalkkinen N., Lehtovaara P., Sarvas M., Söderlund H., Takkinen K., Käriäinen L. Nucleotide sequence of the promoter and NH2-terminal signal peptide region of the alpha-amylase gene from Bacillus amyloliquefaciens. Gene. 1981 Oct;15(1):43–51. doi: 10.1016/0378-1119(81)90103-7. [DOI] [PubMed] [Google Scholar]
  15. Sutcliffe J. G. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3737–3741. doi: 10.1073/pnas.75.8.3737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Talmadge K., Kaufman J., Gilbert W. Bacteria mature preproinsulin to proinsulin. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3988–3992. doi: 10.1073/pnas.77.7.3988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Talmadge K., Stahl S., Gilbert W. Eukaryotic signal sequence transports insulin antigen in Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3369–3373. doi: 10.1073/pnas.77.6.3369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Uhlin B. E., Molin S., Gustafsson P., Nordström K. Plasmids with temperature-dependent copy number for amplification of cloned genes and their products. Gene. 1979 Jun;6(2):91–106. doi: 10.1016/0378-1119(79)90065-9. [DOI] [PubMed] [Google Scholar]
  20. Williams D. M., Schoner R. G., Duvall E. J., Preis L. H., Lovett P. S. Expression of Escherichia coli trp genes and the mouse dihydrofolate reductase gene cloned in Bacillus subtilis. Gene. 1981 Dec;16(1-3):199–206. doi: 10.1016/0378-1119(81)90076-7. [DOI] [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