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. 1991 Feb;57(2):385–388. doi: 10.1128/aem.57.2.385-388.1991

Improved vector for promoter screening in lactococci.

B Bojovic 1, G Djordjevic 1, L Topisirovic 1
PMCID: PMC182721  PMID: 1901705

Abstract

Fragments of Lactococcus lactis subsp. lactis NP45 chromosomal DNA provided promoter activity in Escherichia coli when cloned into the promoter probe vector pGKV210. Only 13% of these recombinant plasmids promoted detectable cat-86 activity when transferred to L. lactis, i.e., expressed chloramphenicol resistance. In these promoter-containing versions of pGKV210, the cat-86 gene specifies chloramphenicol-inducible chloramphenicol acetyltransferase expression. This could be a limiting factor for cloning of promoters with lower activity in L. lactis. Therefore, we have constructed a new promoter probe vector, pBV5030, with the mutated version of the cat-86 gene, which is constitutively expressed when transcriptionally activated by the insertion of a promoter. We found that in L. lactis IL1403 the constitutively expressed cat-86 gene (on a pBV5030 derivative) has four times higher activity than the inducible version of the same gene (on a pGKV210 derivative) when both have the same promoter inserted upstream of the cat-86 gene. These results suggest that plasmid pBV5030 could be a more efficient vector for the cloning of promoters from lactococci.

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Selected References

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

  1. Alexieva Z., Duvall E. J., Ambulos N. P., Jr, Kim U. J., Lovett P. S. Chloramphenicol induction of cat-86 requires ribosome stalling at a specific site in the leader. Proc Natl Acad Sci U S A. 1988 May;85(9):3057–3061. doi: 10.1073/pnas.85.9.3057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ambulos N. P., Jr, Mongkolsuk S., Kaufman J. D., Lovett P. S. Chloramphenicol-induced translation of cat-86 mRNA requires two cis-acting regulatory regions. J Bacteriol. 1985 Nov;164(2):696–703. doi: 10.1128/jb.164.2.696-703.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bates E. E., Gilbert H. J. Characterization of a cryptic plasmid from Lactobacillus plantarum. Gene. 1989 Dec 21;85(1):253–258. doi: 10.1016/0378-1119(89)90491-5. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Chen J. D., Morrison D. A. Cloning of Streptococcus pneumoniae DNA fragments in Escherichia coli requires vectors protected by strong transcriptional terminators. Gene. 1987;55(2-3):179–187. doi: 10.1016/0378-1119(87)90278-2. [DOI] [PubMed] [Google Scholar]
  6. Chopin A., Chopin M. C., Moillo-Batt A., Langella P. Two plasmid-determined restriction and modification systems in Streptococcus lactis. Plasmid. 1984 May;11(3):260–263. doi: 10.1016/0147-619x(84)90033-7. [DOI] [PubMed] [Google Scholar]
  7. Duvall E. J., Williams D. M., Lovett P. S., Rudolph C., Vasantha N., Guyer M. Chloramphenicol-inducible gene expression in Bacillus subtilis. Gene. 1983 Oct;24(2-3):171–177. doi: 10.1016/0378-1119(83)90077-x. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Kozar W., Rajchert-Trzpil M., Dobrzański W. T. The effect of proflavin, ethidium bromide and an elevated temperature on the appearance of nisin-negative clones in nisin-producing strains of Streptococcus lactis. J Gen Microbiol. 1974 Aug;83(2):295–302. doi: 10.1099/00221287-83-2-295. [DOI] [PubMed] [Google Scholar]
  11. Lakshmidevi G., Davidson B. E., Hillier A. J. Molecular characterization of promoters of the Lactococcus lactis subsp. cremoris temperate bacteriophage BK5-T and identification of a phage gene implicated in the regulation of promoter activity. Appl Environ Microbiol. 1990 Apr;56(4):934–942. doi: 10.1128/aem.56.4.934-942.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mongkolsuk S., Ambulos N. P., Jr, Lovett P. S. Chloramphenicol-inducible gene expression in Bacillus subtilis is independent of the chloramphenicol acetyltransferase structural gene and its promoter. J Bacteriol. 1984 Oct;160(1):1–8. doi: 10.1128/jb.160.1.1-8.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. West C. A., Warner P. J. Plasmid profiles and transfer of plasmid-encoded antibiotic resistance in Lactobacillus plantarum. Appl Environ Microbiol. 1985 Nov;50(5):1319–1321. doi: 10.1128/aem.50.5.1319-1321.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]

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