Skip to main content
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1992 Sep;58(9):3142–3149. doi: 10.1128/aem.58.9.3142-3149.1992

Characterization of transcription initiation and termination signals of the proteinase genes of Lactococcus lactis Wg2 and enhancement of proteolysis in L. lactis.

J M van der Vossen 1, J Kodde 1, A J Haandrikman 1, G Venema 1, J Kok 1
PMCID: PMC183061  PMID: 1444428

Abstract

The transcription initiation signals of the prtP and prtM genes specifying the proteolytic activity of Lactococcus lactis subsp. cremoris Wg2 were mapped by primer extension. The strength of these promoters was analyzed with promoter-screening vector pGKV410, and they appeared to be weaker than previously isolated promoters of strain Wg2. In addition, a putative transcription terminator downstream of the prtP gene was characterized by using the terminator-screening vector pGKV259. The putative terminator decreased the transcription activity of lactococcal promoter P59 by approximately 70% in both Bacillus subtilis and L. lactis. Deletion of a part of the stem-loop structure of the terminator decreased the negative effect on transcription, indicating that the structure could indeed function as a terminator of transcription. The proteolytic activity of the lactococcal host was enhanced by placing the originally oppositely oriented prt genes in tandem and replacing the relatively weak promoters upstream of the prt genes with the stronger promoter, P32, from the chromosome of L. lactis Wg2.

Full text

PDF
3142

Images in this article

Selected References

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

  1. 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]
  2. Casadaban M. J., Cohen S. N. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol. 1980 Apr;138(2):179–207. doi: 10.1016/0022-2836(80)90283-1. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Graves M. C., Rabinowitz J. C. In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for "extended" promoter elements in gram-positive organisms. J Biol Chem. 1986 Aug 25;261(24):11409–11415. [PubMed] [Google Scholar]
  7. Haandrikman A. J., Kok J., Laan H., Soemitro S., Ledeboer A. M., Konings W. N., Venema G. Identification of a gene required for maturation of an extracellular lactococcal serine proteinase. J Bacteriol. 1989 May;171(5):2789–2794. doi: 10.1128/jb.171.5.2789-2794.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kiwaki M., Ikemura H., Shimizu-Kadota M., Hirashima A. Molecular characterization of a cell wall-associated proteinase gene from Streptococcus lactis NCDO763. Mol Microbiol. 1989 Mar;3(3):359–369. doi: 10.1111/j.1365-2958.1989.tb00181.x. [DOI] [PubMed] [Google Scholar]
  9. Kok J., Hill D., Haandrikman A. J., de Reuver M. J., Laan H., Venema G. Deletion analysis of the proteinase gene of Streptococcus cremoris Wg2. Appl Environ Microbiol. 1988 Jan;54(1):239–244. doi: 10.1128/aem.54.1.239-244.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kok J., Leenhouts K. J., Haandrikman A. J., Ledeboer A. M., Venema G. Nucleotide sequence of the cell wall proteinase gene of Streptococcus cremoris Wg2. Appl Environ Microbiol. 1988 Jan;54(1):231–238. doi: 10.1128/aem.54.1.231-238.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kok J., Venema G. Genetics of proteinases of lactic acid bacteria. Biochimie. 1988 Apr;70(4):475–488. doi: 10.1016/0300-9084(88)90084-3. [DOI] [PubMed] [Google Scholar]
  12. Kok J., van Dijl J. M., van der Vossen J. M., Venema G. Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis. Appl Environ Microbiol. 1985 Jul;50(1):94–101. doi: 10.1128/aem.50.1.94-101.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Moran C. P., Jr, Lang N., LeGrice S. F., Lee G., Stephens M., Sonenshein A. L., Pero J., Losick R. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet. 1982;186(3):339–346. doi: 10.1007/BF00729452. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shaw W. V. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 1975;43:737–755. doi: 10.1016/0076-6879(75)43141-x. [DOI] [PubMed] [Google Scholar]
  20. Trieu-Cuot P., Courvalin P. Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III. Gene. 1983 Sep;23(3):331–341. doi: 10.1016/0378-1119(83)90022-7. [DOI] [PubMed] [Google Scholar]
  21. Vos P., van Asseldonk M., van Jeveren F., Siezen R., Simons G., de Vos W. M. A maturation protein is essential for production of active forms of Lactococcus lactis SK11 serine proteinase located in or secreted from the cell envelope. J Bacteriol. 1989 May;171(5):2795–2802. doi: 10.1128/jb.171.5.2795-2802.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. van de Guchte M., Kodde J., van der Vossen J. M., Kok J., Venema G. Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease. Appl Environ Microbiol. 1990 Sep;56(9):2606–2611. doi: 10.1128/aem.56.9.2606-2611.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. van der Lelie D., van der Vossen J. M., Venema G. Effect of Plasmid Incompatibility on DNA Transfer to Streptococcus cremoris. Appl Environ Microbiol. 1988 Apr;54(4):865–871. doi: 10.1128/aem.54.4.865-871.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. van der Vossen J. M., Kok J., Venema G. Construction of cloning, promoter-screening, and terminator-screening shuttle vectors for Bacillus subtilis and Streptococcus lactis. Appl Environ Microbiol. 1985 Aug;50(2):540–542. doi: 10.1128/aem.50.2.540-542.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. van der Vossen J. M., van der Lelie D., Venema G. Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. Appl Environ Microbiol. 1987 Oct;53(10):2452–2457. doi: 10.1128/aem.53.10.2452-2457.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES