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Cloning, production and expression of the bacteriocin enterocin A produced by Enterococcus faecium PLBC21 in Lactococcus lactis

  • Applied Genetics and Molecular Biotechnology
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Abstract

Replacement of the leader sequence of enterocin A (EntA), a bacteriocin produced by Enterococcus faecium PLBC21, by the signal peptide of enterocin P (EntP), a sec-dependent bacteriocin produced by E. faecium P13, permitted production of EntA in Lactococcus lactis. Chimeras encoding the EntP signal peptide (SPentP) fused to mature EntA (entA), with or without its immunity gene (entiA), were cloned into the expression vector pMG36c to generate the recombinant plasmids, pMPA15 (SPentP:entA) and pMPA10i (SPentP:entA + entiA). Transformation of competent L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris NZ9000 with the recombinant plasmids permitted production of EntA by the transformed cells, and the co-production of nisin A and EntA by the L. lactis subsp. lactis DPC5598 transformants. Mature EntA fused to SPEntP is the minimum requirement for synthesis, processing and secretion of biologically active EntA in L. lactis. The production of EntA by most recombinant L. lactis hosts was larger than in the E. faecium control strains. All L. lactis derivatives showed antimicrobial activity against Listeria spp., and L. lactis (pMPA15) displayed the highest antilisterial effect.

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References

  • Beaulieu L, Groleau D, Míguez CB, Jetté JF, Aomari H, Subirade M (2005) Production of pediocin PA-1 in the methylotrophic yeast Pichia pastoris reveals unexpected inhibition of its biological activity due to the presence of collagen-like material. Protein Expr Purif 43:111–125

    Article  CAS  Google Scholar 

  • Biet F, Berjeaud JM, Worobo RW, Cenatempo Y, Fremaux C (1998) Heterologous expression of the bacteriocin mesenterocin Y105 using the dedicated transport system and the general secretory pathway. Microbiology 144:2845–2854

    Article  CAS  Google Scholar 

  • Casaus P, Nilsen T, Cintas LM, Nes IF, Hernández PE, Holo H (1997) Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A. Microbiology 143:2287–2294

    Article  CAS  Google Scholar 

  • Cintas LM (1995) Ph.D. thesis. Universidad Autónoma de Madrid, Spain

  • Cintas LM, Casaus P, Håvarstein LS, Hernández PE, Nes IF (1997) Biochemical and genetic characterization of EntP, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum. Appl Environ Microbiol 63:4321–4330

    Article  CAS  Google Scholar 

  • Cintas LM, Casaus P, Herranz C, Håvarstein LS, Holo H, Hernández PE, Nes IF (2000) Biochemical and genetic evidence that Enterococcus faecium L50 produces enterocins L50A and L50B, the sec-dependent enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q. J Bacteriol 182:6806–6814

    Article  CAS  Google Scholar 

  • Cintas LM, Casaus P, Herranz C, Nes IF, Hernández PE (2001) Review: bacteriocins of lactic acid bacteria. Food Sci Technol Int 7:281–305

    Article  CAS  Google Scholar 

  • Cotter PD, Hill C, Ross RP (2005) Bacteriocins: developing innate immunity for food. Nat Rev Microbiol 3:777–788

    Article  CAS  Google Scholar 

  • Chopin MC, Chopin A, Moillo-Bott A, Langella P (1984) Two plasmid-determined restriction and modification systems in Streptococcus lactis. Plasmid 11:260–263

    Article  CAS  Google Scholar 

  • Ennahar S, Aoude-Werner D, Sorokine O, van Dorsselaer A, Bringel F, Hubert JC, Hassemann C (1996) Production of pediocin AcH by Lactobacillus plantarum WHE 92 isolated from cheese. Appl Environ Microbiol 62:4381–4387

    Article  CAS  Google Scholar 

  • Franz CMAP, Worobo RW, Quadri LEN, Schillinger U, Holzapfel WH, Vederas JC, Stiles ME (1999) Atypical genetic locus associated with constitutive production of enterocin B by Enterococcus faecium BFE 900. Appl Environ Microbiol 65:2170–2178

    Article  CAS  Google Scholar 

  • Franz CMAP, Stiles ME, Schleifer KH, Holzapfel WH (2003) Enterococci in foods - a conundrum for food safety. Int J Food Microbiol 88:105–122

    Article  CAS  Google Scholar 

  • Freitas DA, Leclerc S, Miyoshi A, Oliveira SC, Sommer PSM, Rodrigues L, Correa A, Gautier M, Langella P, Azevedo VA, Le Loir Y (2005) Secretion of Streptomyces tendae antifungal protein 1 by Lactococcus lactis. Braz J Med Biol Res 38:1585–1592

    Article  CAS  Google Scholar 

  • Gasson MJ (1983) Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol 154:1–9

    Article  CAS  Google Scholar 

  • Guinane CM, Cotter PD, Hill C, Ross RP (2005) Microbial solutions to microbial problems; lactococcal bacteriocins for the control of undesirable biota in food. J Appl Microbiol 98:1316–1325

    Article  CAS  Google Scholar 

  • Gutiérrez J, Criado R, Citti R, Martín M, Herranz C, Fernández MF, Cintas LM, Hernández PE (2004) Performance and applications of polyclonal anti-peptide antibodies with specificity for the enterococcal bacteriocin enterocin P. J Agric Food Chem 52:2247–2255

    Article  CAS  Google Scholar 

  • Gutiérrez J, Criado R, Citti R, Martín M, Herranz C, Nes IF, Cintas LM, Hernández PE (2005a) Cloning, production and functional expression of enterocin P, a sec-dependent bacteriocin produced by Enterococcus faecium P13, in Escherichia coli. Int J Food Microbiol 103:239–250

    Article  CAS  Google Scholar 

  • Gutiérrez J, Criado R, Martín M, Herranz C, Cintas LM, Hernández PE (2005b) Production of enterocin P, an antilisterial pediocin-like bacteriocin from Enterococcus faecium P13, in Pichia Pastoris. Antimicrob Agents Chemother 49:3004–3008

    Article  CAS  Google Scholar 

  • Gutiérrez J, Larsen R, Cintas LM, Kok J, Hernández PE (2006) High-level heterologous production and functional expression of the sec-dependent enterocin P from Enterococcus faecium P13 in Lactococcus lactis. Appl Microbiol Biotechnol 72:41–51

    Article  CAS  Google Scholar 

  • Herranz C, Driessen AJM (2005) Sec-mediated secretion of enterocin P by Lactococcus lactis. Appl Environ Microbiol 71:1959–1963

    Article  CAS  Google Scholar 

  • Holo H, Nes IF (1989) High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol 55:3119–3123

    Article  CAS  Google Scholar 

  • Horn N, Martínez MI, Martínez JM, Hernández PE, Gasson MJ, Rodríguez JM, Dodd HM (1998) Production of pediocin PA-1 by Lactococcus lactis using the lactococcin A secretory apparatus. Appl Environ Microbiol 64:818–823

    Article  CAS  Google Scholar 

  • Ingham AB, Sproat KW, Tizard MLV, Moore RJ (2005) A versatile system for the expression of nonmodified bacteriocins in Escherichia coli. J Appl Microbiol 98:676–683

    Article  CAS  Google Scholar 

  • Klocke M, Mundt K, Idler F, Jung S, Bachausen JE (2005) Heterologous expression of enterocin A, a bacteriocin from Enterococcus faecium, fused to a cellulose-binding domain in Escherichia coli results in a functional protein with inhibitory activity against Listeria. Appl Microbiol Biotechnol 67:532–538

    Article  CAS  Google Scholar 

  • Kuipers OP, de Ruyter PGGA, Kleerebezem M, de Vos WM (1998) Quorum sensing-controlled gene expression in lactic acid bacteria. J Biotechnol 64:15–21

    Article  CAS  Google Scholar 

  • Martín M, Gutiérrez J, Criado R, Herranz C, Cintas LM, Hernández PE (2006) Genes encoding bacteriocins and their expression, and potential virulence factors of enterococci isolated from wood pigeons (Columba palumbus). J Food Prot 69:520–531

    Article  Google Scholar 

  • Martínez JM, Kok J, Sanders JW, Hernández PE (2000) Heterologous co-production of enterocin A and pediocin PA-1 by Lactococcus lactis: detection by specific peptide-directed antibodies. Appl Environ Microbiol 66:3543–3549

    Article  Google Scholar 

  • Moon GS, Pyun YR, Park MS, Ji GE, Kim WJ (2005) Secretion of recombinant pediocin PA-1 by Bifidobacterium longum, using the signal sequence for bifidobacterial a-amylase. Appl Environ Microbiol 71:5630–5632

    Article  CAS  Google Scholar 

  • Nilsen T, Nes IF, Holo H (1998) An exported inducer regulates bacteriocin production in Enterococcus faecium CTC492. J Bacteriol 180:1848–1854

    Article  CAS  Google Scholar 

  • O’Keefee T, Hill C, Ross RP (1999) Characterization and heterologous expression of the genes encoding enterocin A production, immunity and regulation in Enterococcus faecium DPC1146. Appl Environ Microbiol 65:1506–1515

    Article  Google Scholar 

  • Rodríguez JM, Cintas LM, Casaus P, Horn N, Dodd HM, Hernández PE, Gasson MJ (1995) Isolation of nisin-producing Lactococcus lactis strain from dry fermented sausages. J Appl Bacteriol 78:109–115

    Article  Google Scholar 

  • Schoeman H, Vivier MA, du Toit M, Dicks LMT, Pretorius IS (1999) The development of bactericidal yeast strains expressing the Pediococcus acidilactici pediocin gene (pedA) in Saccharomyces cerevisae. Yeast 15:647–656

    Article  CAS  Google Scholar 

  • Trotter M, Ross RP, Fitzgerald GF, Coffey A (2002) Lactococcus lactis DPC5598, a plasmid-free derivative of a commercial starter, provides a valuable alternative host for culture improvement studies. J Appl Microbiol 93:134–143

    Article  CAS  Google Scholar 

  • Van de Guchte M, van der Vossen JMBM, Kok J, Venema G (1989) Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis. Appl Environ Microbiol 55:224–228

    Article  Google Scholar 

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Acknowledgements

The authors wish to express their gratitude to Prof. R.P. Ross (Teagasc Dairy Products Research Centre, Moorepark, Fermoy, Cork, Ireland) for providing the strain L. lactis DPC5598 and to Prof. Jan Kok (Department of Genetics, University of Groningen, The Netherlands) for the supply of pMG36c. This work was partially supported by grants AGL2003-01508 and AGL2006-01042 from the Ministerio de Educación y Ciencia (MEC) and by grant S-0505/AGR/0265 from the Comunidad de Madrid (CAM), Spain. MM and RC hold a fellowship from the Ministerio de Educación, Cultura y Deporte (MECD), whereas JG is recipient of a fellowship from the Ministerio de Ciencia y Tecnología (MCYT), Spain.

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Author notes

  1. Jorge Gutiérrez

    Present address: School of Food Science and Technology, Dublin Institute of Technology, Dublin, Ireland

Authors and Affiliations

  1. Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040, Madrid, Spain

    María Martín, Jorge Gutiérrez, Raquel Criado, Carmen Herranz, Luis M. Cintas & Pablo E. Hernández

Authors
  1. María Martín
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  2. Jorge Gutiérrez
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  3. Raquel Criado
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  4. Carmen Herranz
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  5. Luis M. Cintas
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  6. Pablo E. Hernández
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Corresponding author

Correspondence to Pablo E. Hernández.

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Martín, M., Gutiérrez, J., Criado, R. et al. Cloning, production and expression of the bacteriocin enterocin A produced by Enterococcus faecium PLBC21 in Lactococcus lactis . Appl Microbiol Biotechnol 76, 667–675 (2007). https://doi.org/10.1007/s00253-007-1044-3

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