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. 1987 Feb;53(2):379–384. doi: 10.1128/aem.53.2.379-384.1987

Protease-deficient Bacillus subtilis host strains for production of Staphylococcal protein A.

S R Fahnestock, K E Fisher
PMCID: PMC203669  PMID: 3032102

Abstract

We constructed strains of Bacillus subtilis which produced very low levels of extracellular proteases. These strains carried insertion or deletion mutations in the subtilisin structural gene (apr) which were constructed in vitro by using the cloned gene. The methods used to construct the mutations involved the use of a plasmid vector which allowed the selection of chromosomal integrates and their subsequent excision by homologous recombination to effect replacement of the chromosomal apr gene by a derivative carrying an inactivating insert with a selectable marker (a cat gene conferring chloramphenicol resistance). The strains produced no subtilisin, no detectable extracellular metalloprotease activity, and residual extracellular serine protease levels as low as 0.5% of that of the standard strain from which they were derived. The strains proved to be superior host strains for the production of staphylococcal protein A, accumulating higher levels of intact protein than do previously available B. subtilis strains.

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Selected References

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  1. 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]
  2. Fahnestock S. R., Fisher K. E. Expression of the staphylococcal protein A gene in Bacillus subtilis by gene fusions utilizing the promoter from a Bacillus amyloliquefaciens alpha-amylase gene. J Bacteriol. 1986 Mar;165(3):796–804. doi: 10.1128/jb.165.3.796-804.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ferrari E., Henner D. J., Hoch J. A. Isolation of Bacillus subtilis genes from a charon 4A library. J Bacteriol. 1981 Apr;146(1):430–432. doi: 10.1128/jb.146.1.430-432.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gutterson N. I., Koshland D. E., Jr Replacement and amplification of bacterial genes with sequences altered in vitro. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4894–4898. doi: 10.1073/pnas.80.16.4894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Haldenwang W. G., Banner C. D., Ollington J. F., Losick R., Hoch J. A., O'Connor M. B., Sonenshein A. L. Mapping a cloned gene under sporulation control by inserttion of a drug resistance marker into the Bacillus subtilis chromosome. J Bacteriol. 1980 Apr;142(1):90–98. doi: 10.1128/jb.142.1.90-98.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hawkes R., Niday E., Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. doi: 10.1016/0003-2697(82)90677-7. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. 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]
  11. Michel J. F., Millet J. Physiological studies on early-blocked sporulation mutants of Bacillus subtilis. J Appl Bacteriol. 1970 Mar;33(1):220–227. doi: 10.1111/j.1365-2672.1970.tb05246.x. [DOI] [PubMed] [Google Scholar]
  12. Roitsch C. A., Hageman J. H. Bacillopeptidase F: two forms of a glycoprotein serine protease from Bacillus subtilis 168. J Bacteriol. 1983 Jul;155(1):145–152. doi: 10.1128/jb.155.1.145-152.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Saunders C. W., Schmidt B. J., Mirot M. S., Thompson L. D., Guyer M. S. Use of chromosomal integration in the establishment and expression of blaZ, a Staphylococcus aureus beta-lactamase gene, in Bacillus subtilis. J Bacteriol. 1984 Mar;157(3):718–726. doi: 10.1128/jb.157.3.718-726.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Scherer S., Davis R. W. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951–4955. doi: 10.1073/pnas.76.10.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Uehara H., Yamane K., Maruo B. Thermosensitive, extracellular neutral proteases in Bacillus subtilis: isolation, characterization, and genetics. J Bacteriol. 1979 Aug;139(2):583–590. doi: 10.1128/jb.139.2.583-590.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Uhlén M., Guss B., Nilsson B., Götz F., Lindberg M. Expression of the gene encoding protein A in Staphylococcus aureus and coagulase-negative staphylococci. J Bacteriol. 1984 Aug;159(2):713–719. doi: 10.1128/jb.159.2.713-719.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Vasantha N., Thompson L. D., Rhodes C., Banner C., Nagle J., Filpula D. Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein. J Bacteriol. 1984 Sep;159(3):811–819. doi: 10.1128/jb.159.3.811-819.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

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