Transfer of nisin gene cluster from Lactococcus lactis ATCC 11454 into the chromosome of Bacillus subtilis 168

Applied Genetics and Molecular Biotechnology

Abstract

Nisin is an antimicrobial peptide produced by certain strains of Lactococcus lactis. It is a gene-encoded peptide that contains unusual amino acid residues. These novel residues are introduced by posttranslational modification machinery and confer unique chemical and physical properties that are not attainable by regular amino acid residues. To study the modification mechanisms and to create structural analogs with superior properties, it would be advantageous to insert the nisin genes into a bacterial strain that is amenable to genetic manipulation. In this study, we report the cloning and integration of the complete and intact nisin gene cluster into the Bacillus subtilis 168 chromosome. Furthermore, we demonstrate that the nisin genes are transcriptionally active. These results should greatly facilitate the studies of the genes and proteins involved in nisin expression, as well as provide a standard system for the manipulation and expression of genes involved in other members of the lantibiotic family of antimicrobial peptides.

Keywords

Nisin Lantibiotic Nisin biosynthesis Antimicrobial peptide Genetic engineering 

Notes

Acknowledgement

This work was supported by National Institutes of Health Grant R01-AI24454-12, and by TEDCO and JIFSAN.

References

  1. Chakicherla A, Hansen JN (1995) Role of the leader and structural regions of prelantibiotic peptides as assessed by expressing nisin-subtilin chimeras in Bacillus subtilis 168, and characterization of their physical, chemical, and antimicrobial properties. J Biol Chem 270:3533–23539CrossRefGoogle Scholar
  2. Chatterjee C, Paul M, Xie L, Van Der Donk WA (2005) Biosynthesis and mode of action of lantibiotics. Chem Rev 105(2):633–684CrossRefGoogle Scholar
  3. De Vos WM, Mulders JW, Siezen RJ, Hugenholtz J, Kuipers OP (1993) Properties of nisin Z and distribution of its gene, nisZ, in Lactococcus lactis. Appl Environ Microbiol 59:213–218Google Scholar
  4. Feeney RE, Garibaldi JA, Humphreys EM (1948) Nutritional studies on subtilin formation by Bacillus subtilis. Arch Biochem Biophys 17:435–445Google Scholar
  5. Gross E, Morell JL, Craig LC (1969) Dehydroalanyllysine: identical COOH-terminal structures in the peptide antibiotics nisin and subtilin. Proc Natl Acad Sci USA 62L:952–956CrossRefGoogle Scholar
  6. Guder A, Wiedemann I, Sahl HG (2000) Posttranslationally modified bacteriocins—the lantibiotics. Biopolymers 55:62–73CrossRefGoogle Scholar
  7. Hansen JN (1993) Antibiotics synthesized by posttranslational modification. Annu Rev Microbiol 47:535–564CrossRefGoogle Scholar
  8. Jack RW, Tagg JR, Ray B (1995) Bacteriocins of gram-positive bacteria. Microbiol Rev 59:171–200Google Scholar
  9. Kluskens LD, Kuipers A, Rink R, De Boef E, Fekken S, Driessen AJ, Kuipers OP, Moll GN (2005) Post-translational modification of therapeutic peptides by NisB, the dehydratase of the lantibiotic nisin. Biochemistry 44:12827–12834CrossRefGoogle Scholar
  10. Kuipers A, De Boef E, Rink R, Fekken S, Kluskens LD, Driessen AJ, Leenhouts K, Kuipers OP, Moll G (2004) NisT, the transporter of the lantibiotic nisin, can transport fully modified, dehydrated, and unmodified prenisin and fusions of the leader peptide with non-lantibiotic peptides. J Biol Chem 279:22176–22182CrossRefGoogle Scholar
  11. Liu W, Hansen JN (1990) Some chemical and physical properties of nisin, a small-protein antibiotic produced by Lactococcus lactis. Appl Environ Microbiol 56:2551–2558Google Scholar
  12. Liu W, Hansen JN (1991) Conversion of Bacillus subtilis 168 to a subtilin producer by competence transformation. J Bacteriol 173:7387–7390Google Scholar
  13. Nishio CS, Komura S, Kurahashi K (1983) Peptide antibiotic subtilin is synthesized via precursor proteins. Biochem Biophys Res Commun 116:751–758CrossRefGoogle Scholar
  14. Paik SH, Chakicherla A, Hansen JN (1998) Identification and characterization of the structural and transporter genes for, and the chemical and biological properties of, sublancin 168, a novel lantibiotic produced by Bacillus subtilis 168. Appl Environ Microbiol 273:23134–23142Google Scholar
  15. Paul LK (1999) Ph. D. Thesis. The role of subtilin leader in the biosynthesis of subtilin in E. coli and B. subtilis 168 and the role of sublancin leader in the biosynthesis of sublancin in B. subtilis 168. University of Maryland, College Park, MDGoogle Scholar
  16. Rauch PJG, Beerthuyzen MM, De Vos WM (1991) Molecular analysis and evolution of conjugative transposons encoding nisin production and sucrose fermentation in Lactococcus lactis. In: Jung G, Sahl HG (eds) Nisin and novel lantibiotics. ESCOM, Leiden, The Netherlands, pp 243–249Google Scholar
  17. Rink R, Kuipers A, De Boef E, Leenhouts KJ, Driessen AJ, Moll GN, Kuipers OP (2005) Lantibiotic structures as guidelines for the design of peptides that can be modified by lantibiotic enzymes. Biochemistry 44:8873–8882CrossRefGoogle Scholar
  18. Rogers LA (1928) Limiting factors in the lactic acid fermentation. J Bacteriol 16:321Google Scholar
  19. Sahl HG (1994) Gene-encoded antibiotics made in bacteria. Ciba Found Symp 186:27–42Google Scholar
  20. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, USAGoogle Scholar
  21. Schnell N, Entian KD, Schneider U, Götz F, Zähner H, Kellner R, Jung G (1988) Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings. Nature 333:276–278CrossRefGoogle Scholar
  22. Siezen RJ, Kuipers OP, De Vos WM (1996) Comparison of lantibiotic gene clusters and encoded proteins. Antonie Van Leeuwenhoek 69:171–184CrossRefGoogle Scholar
  23. Stein T, Heinzmann S, Solovieva I, Entian KD (2003) Function of Lactococcus lactis nisin immunity genes nisI and nisFEG after coordinated expression in the surrogate host Bacillus subtilis. J Biol Chem 278:89–94CrossRefGoogle Scholar
  24. Van den Hooven HW, Lagerwerf FM, Heerma W, Haverkamp J, Piard JC, Hilbers CW, Siezen RJ, Kuipers OP, Rollema HS (1996) The structure of the lantibiotic lacticin 481 produced by Lactococcus lactis: location of the thioether bridges. FEBS Lett 391:317–322CrossRefGoogle Scholar
  25. Van der Meer JR, Polman J, Beerthuyzen MM, Siezen RJ, Kuipers OP, De Vos WM (1993) 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 175:2578–2588Google Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of MarylandCollege ParkUSA
  2. 2.Department of Molecular Biology and GeneticsBogazici UniversityIstanbulTurkey

Personalised recommendations