1887

Abstract

The genetic determinants for production and immunity to the two-component lantibiotic lacticin 3147 are encoded by a 12·6 kb region of the plasmid pMRC01. This region contains ten genes arranged in two divergent clusters; these include the structural genes and a number of genes whose products show significant similarity to proteins involved in the biosynthesis of other lantibiotics. Using a strategy of deletion and mutational analysis, the effect of disruption of a number of these genes was investigated. Inactivation of either of the structural genes, or , resulted in mutants that were incapable of producing active lacticin 3147; however, the combination of the cell-free supernatant from both mutants resulted in a restoration of bacteriocin activity, confirming that processing and export of the structural peptides can occur independently. An unusual feature of the lacticin 3147 gene cluster is the presence of two homologues, whose gene products are proposed to be involved in the dehydration and thioether-forming reactions which result in lanthionine bridge formation. Mutants created in the and genes were also incapable of lantibiotic production, confirming an essential role for these enzymes in the lacticin 3147 biosynthetic pathway and supporting the assertion that these proteins are modification enzymes. Interestingly, addition of purified LtnA1, but not purified LtnA2, to the cell-free supernatant of the mutant restored bacteriocin activity; in contrast, only purified LtnA2 could complement the cell-free supernatant of the mutant. Creation of a number of double mutants supported these findings, and confirmed that LtnM1 is required to produce mature LtnA1, while LtnM2 is required to produce mature LtnA2.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-146-9-2147
2000-09-01
2019-12-14
Loading full text...

Full text loading...

/deliver/fulltext/micro/146/9/1462147a.html?itemId=/content/journal/micro/10.1099/00221287-146-9-2147&mimeType=html&fmt=ahah

References

  1. Anderson, D. G. & McKay, L. L. ( 1983; ). Simple and rapid methods for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol 46, 549-552.
    [Google Scholar]
  2. Augustin, J., Rosenstein, R., Wieland, B., Schneider, U., Schnell, N., Engelke, G., Entian, K.-D. & Götz, F. ( 1992; ). Genetic analysis of biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis. Eur J Biochem 204, 1149-1154.[CrossRef]
    [Google Scholar]
  3. Berridge, N. J., Newton, G. C. F. & Abraham, E. P. ( 1952; ). Purification and nature of the antibiotic nisin. Biochem J 52, 529-535.
    [Google Scholar]
  4. Dougherty, B. A., Hill, C., Weidman, J. F., Richardson, D. R., Venter, J. C. & Ross, R. P. ( 1998; ). Sequence and analysis of the 60 kb conjugative, bacteriocin-producing plasmid pMRC01 from Lactococcus lactis DPC3147. Mol Microbiol 29, 1029-1038.[CrossRef]
    [Google Scholar]
  5. Engelke, G., Gutowski-Eckel, Z., Hammelmann, M. & Entian, K.-D. ( 1992; ). Biosynthesis of the lantibiotic nisin: genomic organization and membrane localization of the NisB protein. Appl Environ Microbiol 58, 3730-3743.
    [Google Scholar]
  6. Gasson, M. J. ( 1983; ). Plasmid complements of Streptococcus lactis NCDO712 and other lactic streptococci after protoplast curing. J Bacteriol 154, 1-9.
    [Google Scholar]
  7. Gilmore, M. S., Segarra, R. A., Booth, M. C., Bogie, C. P., Hall, L. R. & Clewell, D. B. ( 1994; ). Genetic structure of the Enterococcus faecalis plasmid pAD1-encoded cytolytic toxin system and its relationship to lantibiotic determinants. J Bacteriol 176, 7335-7344.
    [Google Scholar]
  8. Gutowski-Eckel, Z., Klein, C., Siegers, K., Bohm, K., Hammelmann, M. & Entian, K.-D. ( 1994; ). Growth phase-dependent regulation and membrane localisation of SpaB, a protein involved in biosynthesis of the lantibiotic subtilin. Appl Environ Microbiol 60, 1-11.
    [Google Scholar]
  9. Hayes, F., Daly, C. & Fitzgerald, G. F. ( 1990; ). Identification of the minimal replicon of Lactococcus lactis subsp. lactis UC317 plasmid pCI305. Appl Environ Microbiol 56, 202-209.
    [Google Scholar]
  10. Ingram, L. C. ( 1969; ). Synthesis of the antibiotic nisin: formation of lanthionine and β-methyllanthionine. Biochim Biophys Acta 184, 216-219.[CrossRef]
    [Google Scholar]
  11. Jack, R. W., Tagg, J. R. & Ray, B. ( 1995; ). Bacteriocins of gram-positive bacteria. Microbiol Rev 59, 171-200.
    [Google Scholar]
  12. Jung, G. ( 1991; ). Lantibiotics: a survey. In Nisin and Novel Lantibiotics , pp. 320-332. Edited by G. Jung & H.-G. Sahl. Leiden, The Netherlands: ESCOM.
  13. Karakas Sen, A., Narbad, A., Horn, N., Dodd, H. M., Parr, A. J., Colquhoun, I. & Gasson, M. J. ( 1999; ). Post-translational modification of nisin: the involvement of NisB in the dehydration process. Eur J Biochem 261, 524-532.[CrossRef]
    [Google Scholar]
  14. Klein, C., Kaletta, C., Schnell, N. & Entian, K.-D. ( 1992; ). Analysis of genes involved in biosynthesis of the lantibiotic subtilin. Appl Environ Microbiol 58, 132-142.
    [Google Scholar]
  15. Kupke, T., Kempter, C., Gnau, V., Jung, G. & Götz, F. ( 1994; ). Mass spectroscopic analysis of a novel enzymatic reaction: oxidative decarboxylation of the lantibiotic precursor peptide EpiA catalyzed by the flavoprotein EpiD. J Biol Chem 269, 5653-5659.
    [Google Scholar]
  16. Leenhouts, K. J., Buist, G., Bolhuis, A., ten Berge, A., Kiel, J., Mierau, I., Dabrowska, M., Venema, G. & Kok, J. ( 1996; ). A gene system for generating unlabeled gene replacements in bacterial chromosomes. Mol Gen Genet 253, 217-224.[CrossRef]
    [Google Scholar]
  17. McAuliffe, O., Ryan, M. P., Ross, R. P., Hill, C., Breeuwer, P. & Abee, T. ( 1998; ). Lacticin 3147, a broad-spectrum bacteriocin which selectively dissipates the membrane potential. Appl Environ Microbiol 64, 439-445.
    [Google Scholar]
  18. McAuliffe, O., Hill, C. & Ross, R. P. ( 1999; ). Inhibition of Listeria monocytogenes in cottage cheese manufactured with a lacticin-3147 producing starter culture. J Appl Microbiol 86, 251-256.[CrossRef]
    [Google Scholar]
  19. McAuliffe, O., Hill, C. & Ross, R. P. ( 2000; ). Identification and overexpression of ltnI a novel gene, which confers immunity to the two-component lantibiotic lacticin 3147. Microbiology 146, 129-138.
    [Google Scholar]
  20. Meyer, C., Bierbaum, G., Heidrich, C., Reis, M., Süling, J., Iglesias-Wind, M. I., Kempter, C., Molitor, E. & Sahl, H.-G. ( 1995; ). Nucleotide sequence of the lantibiotic Pep5 biosynthetic gene cluster and functional analysis of PepP and PepC: evidence for a role of PepC in thioether formation. Eur J Biochem 232, 478-489.[CrossRef]
    [Google Scholar]
  21. Navaratna, M. A., Sahl, H.-G. & Tagg, J. R. ( 1999; ). Identification of genes encoding two-component lantibiotic production in Staphylococcus aureus strain C55 and other phage group II S. aureus strains and demonstration of an association with the exfoliative toxin B gene. Infect Immun 67, 4268-4271.
    [Google Scholar]
  22. Parente, E. & Hill, C. ( 1992; ). A comparison of factors affecting the production of two bacteriocins from lactic acid bacteria. J Appl Bacteriol 73, 290-298.[CrossRef]
    [Google Scholar]
  23. Rince, A., Dufour, A., LePogam, S., Thuault, D., Bourgeois, C. M. & LePennec, J. P. ( 1994; ). Cloning, expression and nucleotide sequence of genes involved in production of lactococcin DR, a bacteriocin from Lactococcus lactis subsp. lactis. Appl Environ Microbiol 60, 1652-1657.
    [Google Scholar]
  24. Ryan, M. P., Rea, M. C., Hill, C. & Ross, R. P. ( 1996; ). An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147. Appl Environ Microbiol 62, 612-619.
    [Google Scholar]
  25. Ryan, M. P., Jack, R. W., Josten, M., Sahl, H.-G., Jung, G., Ross, R. P. & Hill, C. ( 1999; ). Extensive post-translational modification, including serine to d-alanine conversion, in the two-component lantibiotic, lacticin 3147. J Biol Chem 274, 37544-37550.[CrossRef]
    [Google Scholar]
  26. Sahl, H.-G., Jack, R. W. & Bierbaum, G. ( 1995; ). Biosynthesis and biological activities of lantibiotics with unique post-translational modifications. Eur J Biochem 230, 827-853.[CrossRef]
    [Google Scholar]
  27. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  28. Schnell, N., Entian, K.-D., 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, 276-278.[CrossRef]
    [Google Scholar]
  29. Siegers, K., Heinzmann, S. & Entian, K.-D. ( 1996; ). Biosynthesis of lantibiotic nisin: post-translational modification of its prepeptide occurs at a multimeric membrane-associated lanthionine synthetase complex. J Biol Chem 271, 12294-12301.[CrossRef]
    [Google Scholar]
  30. Siezen, R. J., Kuipers, O. P. & de Vos, W. M. ( 1996; ). Comparison of lantibiotic gene clusters and encoded proteins. Antonie Leeuwenhoek 69, 171-184.[CrossRef]
    [Google Scholar]
  31. Skaugen, M., Nissen-Meyer, J., Jung, G., Stevanovic, S., Sletten, K., Abildgaard, C. I. M. & Nes, I. F. ( 1994; ). In vivo conversion of l-serine to d-alanine in a ribosomally synthesized polypeptide. J Biol Chem 269, 27183-27185.
    [Google Scholar]
  32. Skaugen, M., Abildgaard, C. I. M. & Nes, I. F. ( 1997; ). Organization and expression of a gene cluster involved in the biosynthesis of the lantibiotic lactocin S. Mol Gen Genet 253, 674-686.[CrossRef]
    [Google Scholar]
  33. de Vos, W. M., Kuipers, O. P., van der Meer, J. R. & Siezen, R. J. ( 1995; ). Maturation pathway of nisin and other lantibiotics: post-translationally modified antimicrobial peptides exported by gram-positive bacteria. Mol Microbiol 17, 427-437.[CrossRef]
    [Google Scholar]
  34. Wells, J. M., Wilson, P. W. & Page, R. W. F. ( 1993; ). Improved cloning vectors and transformation procedure for Lactococcus lactis. J Appl Bacteriol 74, 629-636.[CrossRef]
    [Google Scholar]
  35. Yanisch-Perron, C., Vieira, J. & Messing, J. ( 1985; ). Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mp18 and pUC19 vectors. Gene 33, 103-119.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-146-9-2147
Loading
/content/journal/micro/10.1099/00221287-146-9-2147
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error