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. 1996 Jun;178(12):3434–3439. doi: 10.1128/jb.178.12.3434-3439.1996

Functional analysis of promoters in the nisin gene cluster of Lactococcus lactis.

P G de Ruyter 1, O P Kuipers 1, M M Beerthuyzen 1, I van Alen-Boerrigter 1, W M de Vos 1
PMCID: PMC178110  PMID: 8655538

Abstract

The promoters in the nisin gene cluster nisABTCIPRKFEG of Lactococcus lactis were characterized by primer extension and transcriptional fusions to the Escherichia coli promoterless beta-glucuronidase gene (gusA). Three promoters preceding the nisA, nisR, and nisF genes, which all give rise to gusA expression in the nisin-producing strain L. lactis NZ9700, were identified. The transcriptional autoregulation of nisA by signal transduction involving the sensor histidine kinase NisK and the response regulator NisR has been demonstrated previously (0. P. Kuipers, M. M. Beerthuyzen, P. G. G. A. de Ruyter, E. J. Luesink, and W. M. de Vos, J. Biol. Chem. 270: 27299-27304, 1995), and therefore the possible nisin-dependent expression of gusA under control of the nisR and nisF promoters was also investigated. The nisR promoter was shown to direct nisin-independent gusA expression in L. lactis MG 1363, which is a nisin-transposon- and plasmid-free strain. L. lactis NZ9800, which does not produce nisin because of a deletion in the nisA gene, containing the nisF-gusA fusion plasmid, gave rise to beta-glucuronidase production only after induction by nisin. A similar regulation was found in L. lactis NZ3900, which contains a single copy of the nisR and nisK genes but no other genes of the nisin gene cluster. In contrast, when the nisK gene was disrupted, no beta-glucuronidase activity directed by the nisF promoter could be detected even after induction with nisin. These results show that, like the nisA promoter, the nisF promoter is nisin inducible. The nisF and nisA promoter sequences have significant similarities and contain a conserved region that could be important for transcriptional control.

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Selected References

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  1. Buchman G. W., Banerjee S., Hansen J. N. Structure, expression, and evolution of a gene encoding the precursor of nisin, a small protein antibiotic. J Biol Chem. 1988 Nov 5;263(31):16260–16266. [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. Dodd H. M., Horn N., Gasson M. J. Analysis of the genetic determinant for production of the peptide antibiotic nisin. J Gen Microbiol. 1990 Mar;136(3):555–566. doi: 10.1099/00221287-136-3-555. [DOI] [PubMed] [Google Scholar]
  4. Engelke G., Gutowski-Eckel Z., Hammelmann M., Entian K. D. Biosynthesis of the lantibiotic nisin: genomic organization and membrane localization of the NisB protein. Appl Environ Microbiol. 1992 Nov;58(11):3730–3743. doi: 10.1128/aem.58.11.3730-3743.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Engelke G., Gutowski-Eckel Z., Kiesau P., Siegers K., Hammelmann M., Entian K. D. Regulation of nisin biosynthesis and immunity in Lactococcus lactis 6F3. Appl Environ Microbiol. 1994 Mar;60(3):814–825. doi: 10.1128/aem.60.3.814-825.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Horn N., Swindell S., Dodd H., Gasson M. Nisin biosynthesis genes are encoded by a novel conjugative transposon. Mol Gen Genet. 1991 Aug;228(1-2):129–135. doi: 10.1007/BF00282457. [DOI] [PubMed] [Google Scholar]
  8. Immonen T., Ye S., Ra R., Qiao M., Paulin L., Saris P. E. The codon usage of the nisZ operon in Lactococcus lactis N8 suggests a non-lactococcal origin of the conjugative nisin-sucrose transposon. DNA Seq. 1995;5(4):203–218. doi: 10.3109/10425179509030968. [DOI] [PubMed] [Google Scholar]
  9. Jack R. W., Tagg J. R., Ray B. Bacteriocins of gram-positive bacteria. Microbiol Rev. 1995 Jun;59(2):171–200. doi: 10.1128/mr.59.2.171-200.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kaletta C., Entian K. D. Nisin, a peptide antibiotic: cloning and sequencing of the nisA gene and posttranslational processing of its peptide product. J Bacteriol. 1989 Mar;171(3):1597–1601. doi: 10.1128/jb.171.3.1597-1601.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Klein C., Kaletta C., Entian K. D. Biosynthesis of the lantibiotic subtilin is regulated by a histidine kinase/response regulator system. Appl Environ Microbiol. 1993 Jan;59(1):296–303. doi: 10.1128/aem.59.1.296-303.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kuipers O. P., Beerthuyzen M. M., Siezen R. J., De Vos W. M. Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis. Requirement of expression of the nisA and nisI genes for development of immunity. Eur J Biochem. 1993 Aug 15;216(1):281–291. doi: 10.1111/j.1432-1033.1993.tb18143.x. [DOI] [PubMed] [Google Scholar]
  13. Kuipers O. P., Beerthuyzen M. M., de Ruyter P. G., Luesink E. J., de Vos W. M. Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction. J Biol Chem. 1995 Nov 10;270(45):27299–27304. doi: 10.1074/jbc.270.45.27299. [DOI] [PubMed] [Google Scholar]
  14. Mulders J. W., Boerrigter I. J., Rollema H. S., Siezen R. J., de Vos W. M. Identification and characterization of the lantibiotic nisin Z, a natural nisin variant. Eur J Biochem. 1991 Nov 1;201(3):581–584. doi: 10.1111/j.1432-1033.1991.tb16317.x. [DOI] [PubMed] [Google Scholar]
  15. Parkinson J. S., Kofoid E. C. Communication modules in bacterial signaling proteins. Annu Rev Genet. 1992;26:71–112. doi: 10.1146/annurev.ge.26.120192.000443. [DOI] [PubMed] [Google Scholar]
  16. Platteeuw C., Simons G., de Vos W. M. Use of the Escherichia coli beta-glucuronidase (gusA) gene as a reporter gene for analyzing promoters in lactic acid bacteria. Appl Environ Microbiol. 1994 Feb;60(2):587–593. doi: 10.1128/aem.60.2.587-593.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rauch P. J., De Vos W. M. Characterization of the novel nisin-sucrose conjugative transposon Tn5276 and its insertion in Lactococcus lactis. J Bacteriol. 1992 Feb;174(4):1280–1287. doi: 10.1128/jb.174.4.1280-1287.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Runar Ra S., Saris P. E. Characterization of prokaryotic mRNAs by RT-PCR. Biotechniques. 1995 May;18(5):792–795. [PubMed] [Google Scholar]
  19. 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]
  20. Siegers K., Entian K. D. Genes involved in immunity to the lantibiotic nisin produced by Lactococcus lactis 6F3. Appl Environ Microbiol. 1995 Mar;61(3):1082–1089. doi: 10.1128/aem.61.3.1082-1089.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Simon D., Chopin A. Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochimie. 1988 Apr;70(4):559–566. doi: 10.1016/0300-9084(88)90093-4. [DOI] [PubMed] [Google Scholar]
  22. Stock J. B., Stock A. M., Mottonen J. M. Signal transduction in bacteria. Nature. 1990 Mar 29;344(6265):395–400. doi: 10.1038/344395a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Wanner B. L. Is cross regulation by phosphorylation of two-component response regulator proteins important in bacteria? J Bacteriol. 1992 Apr;174(7):2053–2058. doi: 10.1128/jb.174.7.2053-2058.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wells J. M., Wilson P. W., Le Page R. W. Improved cloning vectors and transformation procedure for Lactococcus lactis. J Appl Bacteriol. 1993 Jun;74(6):629–636. doi: 10.1111/j.1365-2672.1993.tb05195.x. [DOI] [PubMed] [Google Scholar]
  26. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  27. de Vos W. M., Kuipers O. P., van der Meer J. R., Siezen R. J. Maturation pathway of nisin and other lantibiotics: post-translationally modified antimicrobial peptides exported by gram-positive bacteria. Mol Microbiol. 1995 Aug;17(3):427–437. doi: 10.1111/j.1365-2958.1995.mmi_17030427.x. [DOI] [PubMed] [Google Scholar]
  28. van Alen-Boerrigter I. J., Baankreis R., de Vos W. M. Characterization and overexpression of the Lactococcus lactis pepN gene and localization of its product, aminopeptidase N. Appl Environ Microbiol. 1991 Sep;57(9):2555–2561. doi: 10.1128/aem.57.9.2555-2561.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. van Rooijen R. J., Gasson M. J., de Vos W. M. Characterization of the Lactococcus lactis lactose operon promoter: contribution of flanking sequences and LacR repressor to promoter activity. J Bacteriol. 1992 Apr;174(7):2273–2280. doi: 10.1128/jb.174.7.2273-2280.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. van der Meer J. R., Polman J., Beerthuyzen M. M., Siezen R. J., Kuipers O. P., De Vos W. M. Characterization of the Lactococcus lactis nisin A operon genes nisP, encoding a subtilisin-like serine protease involved in precursor processing, and nisR, encoding a regulatory protein involved in nisin biosynthesis. J Bacteriol. 1993 May;175(9):2578–2588. doi: 10.1128/jb.175.9.2578-2588.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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