Applied Microbiology and Biotechnology

, Volume 27, Issue 5–6, pp 521–527 | Cite as

Stability during fermentation of a recombinant α-amylase plasmid in Bacillus subtilis

  • Alexis Harington
  • Terence G. Watson
  • Maureen E. Louw
  • Jill E. Rodel
  • Jennifer A. Thomson
Applied Genetics and Regulation


We have studied the stability during fermentation of a hybrid plasmid carrying a Bacillus α-amylase gene in Bacillus subtilis. In the absence of antibiotic selection plasmid loss was associated largely with the post-exponential phases of growth and decline. In fermentations containing selective antibiotics, various deleted plasmids were recovered during late stationary phase, regardless of whether the host was rec+ or recE. We therefore propose that the plasmid loss observed during late growth in antibiotic-free fermentations is due to deletion events which include the origin of plasmid replication. The structure of the deleted plasmids was determined and the sequences in the vicinity of the end-points analysed. When the deleted plasmids were subjected to further fermentations in the absence of selective antibiotics, they were completely stable.


Fermentation Bacillus Stationary Phase Bacillus Subtilis Late Growth 
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  1. Aiba S, Kitai K, Imanaka T (1983) Cloning and expression of thermostable α-amylase gene from Bacillus stearothermophilus in Bacillus stearothermophilus and Bacillus subtilis. Appl Environ Microbiol 64:1059–1065Google Scholar
  2. Anderson MLM, Young BD (1985) Quantitative filter hybridisation. In: Hames BD, Higgins SJ (eds) Nucleic Acid Hybridisation, Oxford: IRL Press, pp 73–111Google Scholar
  3. Bron S, Luxen E (1985) Segregational instability of pUB110 derived recombinant plasmids in Bacillus subtilis. Plasmid 14:235–244Google Scholar
  4. Corfield VA, Reid SJ, Bodmer J, Thomson JA (1984) A modified protoplast-regeneration protocol facilitating the detection of cloned exoenzyme genes in Bacillus subtilis. Gene 30:17–22Google Scholar
  5. Corfield VA, Sugrue JA, Thomson JA (1987) Towards an understanding of hybrid plasmid instability in Bacillus subtilis. In: Thomson JA (ed) The Role of Recombinant DNA in Bacterial Fermentation, CRC Press (in Press)Google Scholar
  6. Doi RH (1984) Genetic engineering in Bacillus subtilis. Biotech Genet Eng Rev 2:121–155Google Scholar
  7. Fujii M, Takagi M, Imanaka T, Aiba S (1983) Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus and Bacillus subtilis. J Bacteriol 154:831–837Google Scholar
  8. Gandhi AP, Kjaergaard L (1975) Effect of carbon dioxide on the formation of α-amylase by Bacillus subtilis growing in continuous and batch cultures. Biotechnol Bioeng 17:1109–1118Google Scholar
  9. Hirata H, Negoro S, Okada H (1985) High production of thermostable β-galactosidase of Bacillus stearothermophilus in Bacillus subtilis. Appl Environ Microbiol 49:1547–1549Google Scholar
  10. Honjo M, Akaoka A, Nakayama A, Furutani Y (1986) Secretion of human growth hormone in Bacillus subtilis using prepropeptide coding region of B. amyloliquefaciens neutral protease gene. J Biotech 4:63–72Google Scholar
  11. Kreft J, Hughes C (1982) Cloning vectors derived from plasmids and phage of Bacillus. In: Hofschneider PH, Goebel W (eds) Curr Top Microbiol Immunol 96, New York: Springer, pp 1–17Google Scholar
  12. Lovett PS, Keggins KM (1979) Bacillus subtilis as a host for molecular cloning. In: Wu R (ed) Methods in Enzymology, New York: Academic Press 68:342–357Google Scholar
  13. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  14. Marrero R, Lovett PS (1980) Transductional selection of cloned bacteriophage ϕ105 and SPO2 deoxyribonucleic acids in B. subtilis. J Bacteriol 143:879–886Google Scholar
  15. Mason PJ, Williams JG (1985) Hybridisation in the analysis of recombinant DNA. In: Hames BD, Higgins SJ (eds) Nucleic Acid Hybridisation, Oxford: IRL Press, pp 113–137Google Scholar
  16. McCullough JE (1983) Gene cloning in Bacilli related to enhanced penicillin acylase production. Bio/Technol 1:879–882Google Scholar
  17. McKenzie T, Hoshino T, Tanaka T, Sueoka N (1986) The nucleotide sequence of pUB110: Some salient features in relation to replication and its regulation. Plasmid 15:93–103Google Scholar
  18. Meinkoth J, Wahl G (1984) Hybridization of nucleic acids immobilized on solid supports. Anal Biochem 138:267–284Google Scholar
  19. Michel B, Ehrlich SD (1986) Illegitimate recombination occurs between the replication origin of the plasmid pC194 and a progression replication fork. EMBO J 5:3691–3696Google Scholar
  20. Ortlepp SA, Ollington JF, McConnell DJ (1983) Molecular cloning in Bacillus subtilis of a Bacillus licheniformis gene encoding a thermostable alpha-amylase. Gene 23:267–276Google Scholar
  21. Palva I (1982) Molecular cloning of α-amylase gene from Bacillus amyloliquefaciens and its expression in B. subtilis. Gene 19:81–87Google Scholar
  22. Piggott RP, Rossiter A, Ortlepp SA, Pembroke JT, Ollington JF (1984) Cloning in Bacillus subtilis of an extremely thermostable α-amylase: comparison with other cloned heat stable α-amylases. Biochem Biophys Res Commun 122:175–183Google Scholar
  23. Pinches A, Louw ME, Watson TG (1985) Growth, plasmid stability and α-amylase production in batch fermentations using a recombinant Bacillus subtilis strain. Biotechnol Lett 7:621–626Google Scholar
  24. Primrose SB, Ehrlich SD (1981) Instability associated with deletion formation in a hybrid plasmid. Plasmid 6:193–201Google Scholar
  25. Shiroza T, Nakazawa K, Tashiro N, Yamane K, Yanagi K, Yamasaki M, Tamura G, Saito H, Kawade Y, Taniguchi T (1985) Synthesis and secretion of biologically active mouse interferon-β using a Bacillus subtilis α-amylase secretion vector. Gene 34:1–8Google Scholar
  26. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517Google Scholar
  27. te, Riele H, Michel B, Ehrlich SD (1986a) Are single stranded circles intermediates in plasmid replication? EMBO J 5:631–637Google Scholar
  28. te Riele H, Michel B, Ehrlich SD (1986b) Single-stranded plasmid DNA in Bacillus subtilis and Staphylococcus aureus. Proc Natl Acad Sci USA 83:2541–2545Google Scholar
  29. Vehmaanperä JO, Korhola MP (1986) Stability of the recombinant plasmid carrying the Bacillus amyloliquefaciens α-amylase gene in B. subtilis. Appl Microbiol Biotechnol 23:456–461Google Scholar
  30. Watson TG, Pinches A, Louw ME (1986) Effect of growth rate on the maintenance of a recombinant plasmid in Bacillus subtilis. Biotechnol Lett 8:687–690Google Scholar
  31. Wells JA, Ferrari E, Henner DJ, Estell DA, Chen EY (1983) Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis. Nucleic Acids Res 11:7911–7925Google Scholar
  32. Yang RC, Lis Y, Wu R (1979) Electroelution. In: Wu R (ed) Methods in enzymology, New York: Academic Press 68:176–182Google Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • Alexis Harington
    • 1
  • Terence G. Watson
    • 2
  • Maureen E. Louw
    • 2
  • Jill E. Rodel
    • 3
  • Jennifer A. Thomson
    • 4
  1. 1.CSIR Laboratory for Molecular and Cell BiologyJohannesburg
  2. 2.CSIR National Food Research InstitutePretoriaSouth Africa
  3. 3.Department of Membrane ResearchWeizmann Institute of ScienceRehovotIsrael
  4. 4.CSIR Laboratory for Molecular and Cell BiologyBraamfonteinSouth Africa

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