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. 1985 Jul;50(1):94–101. doi: 10.1128/aem.50.1.94-101.1985

Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis.

J Kok, J M van Dijl, J M van der Vossen, G Venema
PMCID: PMC238578  PMID: 2992377

Abstract

Previously, curing experiments suggested that plasmid pWV05 (17.5 megadaltons [Md]) of Streptococcus cremoris Wg2 specifies proteolytic activity. A restriction enzyme map of pWV05 was constructed, the entire plasmid was subcloned in Escherichia coli with plasmids pBR329 and pACYC184. A 4.3-Md HindIII fragment could not be cloned in an uninterrupted way in E. coli but could be cloned in two parts. Both fragments showed homology with the 9-Md proteinase plasmid of S. cremoris HP. The 4.3-Md HindIII fragment was successfully cloned in Bacillus subtilis on plasmid pGKV2 (3.1 Md). Crossed immunoelectrophoresis of extracts of B. subtilis carrying the recombinant plasmid (pGKV500; 7.4 Md) showed that the fragment specifies two proteins of the proteolytic system of S. cremoris Wg2. PGKV500 was introduced in a proteinase-deficient Streptococcus lactis strain via protoplast transformation. Both proteins were also present in cell-free extracts of S. lactis(pGKV500). In S. lactis, pGKV500 enables the cells to grow normally in milk with rapid acid production, indicating that the 4.3-Md HindIII fragment of plasmid pWV05 specifies the proteolytic activity of S. cremoris Wg2.

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Selected References

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  1. Blin N., von Gabain A., Bujard H. Isolation of large molecular weight DNA from agarose gels for further digestion by restriction enzymes. FEBS Lett. 1975 Apr 15;53(1):84–86. doi: 10.1016/0014-5793(75)80688-0. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Brown J. H., Howe P. E. Transparent Milk as a Bacteriological Medium. J Bacteriol. 1922 Sep;7(5):511–514. doi: 10.1128/jb.7.5.511-514.1922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Covarrubias L., Bolivar F. Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication. Gene. 1982 Jan;17(1):79–89. doi: 10.1016/0378-1119(82)90103-2. [DOI] [PubMed] [Google Scholar]
  7. Davies F. L., Gasson M. J. Reviews of the progress of dairy science: genetics of lactic acid bacteria. J Dairy Res. 1981 Jun;48(2):363–376. doi: 10.1017/s0022029900021798. [DOI] [PubMed] [Google Scholar]
  8. Elferink M. G., Hellingwerf K. J., Michels P. A., Seÿen H. G., Konings W. N. Immunochemical analysis of membrane vesicles and chromatophoresis of Rhodopseudomonas sphaeroides by crossed immunoelectrophoresis. FEBS Lett. 1979 Nov 15;107(2):300–307. doi: 10.1016/0014-5793(79)80395-6. [DOI] [PubMed] [Google Scholar]
  9. Gasson M. J., Davies F. L. High-frequency conjugation associated with Streptococcus lactis donor cell aggregation. J Bacteriol. 1980 Sep;143(3):1260–1264. doi: 10.1128/jb.143.3.1260-1264.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gasson M. J. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol. 1983 Apr;154(1):1–9. doi: 10.1128/jb.154.1.1-9.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hugenholtz J., Exterkate F., Konings W. N. The Proteolytic Systems of Streptococcus cremoris: an Immunological Analysis. Appl Environ Microbiol. 1984 Dec;48(6):1105–1110. doi: 10.1128/aem.48.6.1105-1110.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Kondo J. K., McKay L. L. Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning. Appl Environ Microbiol. 1984 Aug;48(2):252–259. doi: 10.1128/aem.48.2.252-259.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kondo J. K., McKay L. L. Transformation of Streptococcus lactis Protoplasts by Plasmid DNA. Appl Environ Microbiol. 1982 May;43(5):1213–1215. doi: 10.1128/aem.43.5.1213-1215.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kuhl S. A., Larsen L. D., McKay L. L. Plasmid Profiles of Lactose-Negative and Proteinase-Deficient Mutants of Streptococcus lactis C10, ML(3), and M18. Appl Environ Microbiol. 1979 Jun;37(6):1193–1195. doi: 10.1128/aem.37.6.1193-1195.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Larsen L. D., McKay L. L. Isolation and characterization of plasmid DNA in Streptococcus cremoris. Appl Environ Microbiol. 1978 Dec;36(6):944–952. doi: 10.1128/aem.36.6.944-952.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Law B. A., Kolstad J. Proteolytic systems in lactic acid bacteria. Antonie Van Leeuwenhoek. 1983 Sep;49(3):225–245. doi: 10.1007/BF00399500. [DOI] [PubMed] [Google Scholar]
  19. Leblanc D. J., Lee L. N. Rapid screening procedure for detection of plasmids in streptococci. J Bacteriol. 1979 Dec;140(3):1112–1115. doi: 10.1128/jb.140.3.1112-1115.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. McKay L. L., Baldwin K. A. Plasmid distribution and evidence for a proteinase plasmid in Streptococcus lactis C2-1. Appl Microbiol. 1975 Apr;29(4):546–548. doi: 10.1128/am.29.4.546-548.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McKay L. L., Baldwin K. A. Simultaneous loss of proteinase- and lactose-utilizing enzyme activities in Streptococcus lactis and reversal of loss by transduction. Appl Microbiol. 1974 Sep;28(3):342–346. doi: 10.1128/am.28.3.342-346.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ostroff G. R., Pène J. J. Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis: isolation of a spontaneous mutant of Bacillus subtilis with enhanced transformability for Escherichia coli-propagated chimeric plasmid DNA. J Bacteriol. 1983 Nov;156(2):934–936. doi: 10.1128/jb.156.2.934-936.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Otto R., de Vos W. M., Gavrieli J. Plasmid DNA in Streptococcus cremoris Wg2: Influence of pH on Selection in Chemostats of a Variant Lacking a Protease Plasmid. Appl Environ Microbiol. 1982 Jun;43(6):1272–1277. doi: 10.1128/aem.43.6.1272-1277.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  26. Rottländer E., Trautner T. A. Genetic and transfection studies with B, subtilis phage SP 50. I. Phage mutants with restricted growth on B. subtilis strain 168. Mol Gen Genet. 1970;108(1):47–60. doi: 10.1007/BF00343184. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Terzaghi B. E., Sandine W. E. Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol. 1975 Jun;29(6):807–813. doi: 10.1128/am.29.6.807-813.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Thomas T. D., Jarvis B. D., Skipper N. A. Localization of proteinase(s) near the cell surface of Streptococcus lactis. J Bacteriol. 1974 May;118(2):329–333. doi: 10.1128/jb.118.2.329-333.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]

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