Skip to main content
SpringerLink
Log in

A food-grade delivery system for Lactococcus lactis and evaluation of inducible gene expression

  • Original Paper
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The genetic improvement of Lactococcus lactis is a matter of biotechnological interest in the food industry and in the pharmaceutical and medical fields. However, to construct a food-grade delivery system, both the presence of antibiotic markers or plasmid sequences should be avoided and the maintenance and expression of the cloned gene should be guaranteed. The objective of this work was to produce crossover mutants of L. lactis with a reporter gene under the control of an inducible promoter in order to evaluate the level of gene expression. We utilized a nuclease gene of Staphylococcus aureus as a reporter gene, P nisA as the nisin-inducible promoter, a non-essential gene involved in histidine biosynthesis of L. lactis as the site for homologous recombination, and pRV300 as a suicide vector for the genomic integration in L. lactis NZ9000. Single- and double-crossover mutants were identified by genotype and phenotype. Relative to episomal transformants of L. lactis, the level of expression of the heterologous protein after nisin induction was similar in the crossover mutants, suggesting that a single copy of the heterologous gene can be used to produce the protein of interest.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2A, B
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Asseldonk M van, Rutten G, Oteman M, Siezen RJ, Vos WM de, Simons G (1990) Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subs. lactis MG1363. Gene 95:155–160

    Google Scholar 

  • Asseldonk M van, Vos WM de, Simons G (1993) Functional analysis of the Lactococcus lactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologous α-amylase. Mol Gen Genet 240:428–434

    Google Scholar 

  • Bron PA, Benchimol MG, Lambert J, Palumbo E, Deghorain M, Delcour J, Vos WM de, Kleerebezem M Hols P (2002) Use of the alr gene as a food-grade selection marker in lactic acid bacteria. Appl Environ Microbiol 68:5663–5670

    Google Scholar 

  • Cuatrecasas P, Fuchs S, Anfisen CB (1967) Catalytic properties and specificity of the extracellular nuclease of Staphylococcus aureus. J Biol Chem 242:1541–1547

    Google Scholar 

  • Davis A, Moore IB, Parker DS, Taniuchi H (1977) Nuclease B: a possible precursor of nuclease A, an extracellular nuclease of Staphylococcus aureus. J Biol Chem 252:6544–6553

    Google Scholar 

  • Delorme C, Ehrlich SD, Renault P(1992) Histidine biosynthesis genes in Lactococcus lactis subsp. lactis. J Bacteriol 174:6571–6579

    Google Scholar 

  • Dickley F, Nilsson D, Hansen EB, Johansen E (1995) Isolation of Lactococcus lactis nonsense suppressors and construction of a food-grade cloning vector. Mol Microbiol 15:839–847

    Google Scholar 

  • Djordjevic GM, Klaenhammer TR (1998) Inducible gene expression systems in Lactococcus lactis. Mol Biotechnol 9:127–139

    Google Scholar 

  • Eichenbaum Z, Federle MJ, Marra D, Vos WM de, Kuipers OP, Kleerebezem M, Scott JR (1998) Use of the lactococcal nisA promoter to regulate gene expression in gram-positive bacteria: comparison of induction level and promoter strength. Appl Environ Microbiol 64:2763–2769

    Google Scholar 

  • Emond E, Lavallee R, Drolet G, Moineau S, LaPointe G (2001) Molecular characterization of a theta replication plasmid and its use for development of a two-component food-grade cloning system for Lactococcus lactis. Appl Environ Microbiol 67:1700–1709

    Google Scholar 

  • Enouf V, Langella P, Commissaire J, Cohen J, Corthier G (2001) Bovine rotavirus nonstructural protein 4 produced by Lactococcus lactis is antigenic and immunogenic. Appl Environ Microbiol 67:1423–1428

    Google Scholar 

  • Federal Register (1988) Nisin preparation: affirmation of GRAS status as a direct human food ingredient. Fed Regist 54:11247–11251

    Google Scholar 

  • Gory L, Montel MC, Zagorec M (2001) Use of green fluorescent protein to monitor Lactobacillus sakei in fermented meat products. FEMS Microbiol Lett 194:127–133

    Google Scholar 

  • Henrich B, Klein JR, Weber B, Delorme C, Renault P, Wegmann U (2002) Food-grade delivery system for controlled gene expression in Lactococcus lactis. Appl Environ Microbiol 68:5429–5436

    Google Scholar 

  • Kleerebezem M, Beerthuyzen MM, Vaughan EE, Vos WM de, Kuipers OP (1997) Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp. Appl Environ Microbiol 63:4581–4584

    Google Scholar 

  • Kovacevic S, Veal LE, Hsiung HM, Miller JR (1985) Secretion of staphylococcal nuclease by Bacillus subtilis. J Bacteriol 162:521–528

    Google Scholar 

  • Kuipers OP, Beerthuyzen MM, Ruyter PG de, Luesink EJ, Vos WM de (1995) Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction. J Biol Chem 270:27299–27304

    Google Scholar 

  • Kuipers OP, Ruyter PG de, Kleerebezem M, Vos WM de (1997) Controlled overproduction of proteins by lactic acid bacteria. Trends Biotechnol 15:135–140

    Google Scholar 

  • Langella P, Le Loir Y (1999) Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system. Braz J Med Biol Res 32:191–198

    Google Scholar 

  • Langella P, Le Loir Y, Ehrlich SD, Gruss A (1993) Efficient plasmid mobilization by pIP501 in Lactococcus lactis subsp. lactis. J Bacteriol 175:5806–5813

    Google Scholar 

  • Le Loir Y, Gruss A, Ehrlich SD, Langella P (1994) Direct screening of recombinants in Gram-positive bacteria using the secreted staphylococcal nuclease as a reporter. J Bacteriol 176:5135–5139

    Google Scholar 

  • Le Loir Y, Gruss A, Ehrlich SD, Langella P (1998) A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis. J Bacteriol 180:1895–1903

    Google Scholar 

  • Le Loir Y, Nouaille S, Ribeiro L, Comissaire J, Cortheier G, Gilbert S, Chatel J, L’Haridon R, Gruss A, Langella P (2001) Sécrétion de protéines d’intérêt thérapeutique chez Lactococcus lactis. Lait 81:217–226

    Google Scholar 

  • Leenhouts K, Buist G, Bolhuis A, Berge A ten, Kiel J, Mierau I, Dabrowska M, Venema G, Kok J (1996) A general system for generating unlabelled gene replacements in bacterial chromosomes. Mol Gen Genet 253:217–224

    Google Scholar 

  • Maguin E, Prevost H, Ehrlich SD, Gruss A (1996) Efficient insertional mutagenesis in lactococci and other Gram-positive bacteria. J Bacteriol 178:931–935

    Google Scholar 

  • Meer JR van der, Polman J, Beerthuyzen MM, Siezen RJ, Kuipers OP, Vos WM de (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 175:2578–2588

    Google Scholar 

  • Piard JC, Hautefort I, Fischetti VA, Ehrlich SD, Fons M, Gruss A (1997) Cell-wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria. J Bacteriol 179:3068–3072

    Google Scholar 

  • Platteeuw C, Alen-Boerrigter I van, Shalkwijk S van, Vos WM de (1996) Food-grade cloning and expression system for Lactococcus lactis. Appl Environ Microbiol 62:1008–1013

    Google Scholar 

  • Poquet I, Ehrlich SD, Gruss A (1997) An export-specific reporter designed for gram-positive bacteria: application to Lactococcus lactis. J Bacteriol 180:1904–1912

    Google Scholar 

  • Ravn P, Arnay J, Madsen MS, Vrang A, Israelsen H (2003) Optimization of signal peptide SP310 for heterologous protein production in Lactococcus lactis. Microbiology 149:2193–2201

    Google Scholar 

  • Ribeiro LA, Azevedo V, Le Loir Y, Oliveira SC, Dieye Y, Piard JC, Gruss A, Langella P (2002) Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grade live vaccines against brucellosis. Appl Environ Microbiol 68:910–916

    Google Scholar 

  • Rooijen RJ van, Gasson MJ, Vos WM de (1992) Characterization of the Lactococcus lactis lactose operon promoter: contribution of flanking sequences and LacR repressor to promoter activity. J Bacteriol 174:2273–2280

    Google Scholar 

  • Ruiter PG de, Kuipers OP, Vos WM de (1996) Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 62:3662–3667

    Google Scholar 

  • Sambrook J, Fritish EF, Maniatis T (2001) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

  • Shortle D (1983) A genetic system for analysis of staphylococcal nuclease. Gene 22:181–189

    Google Scholar 

  • Simon D, Chopin A (1988) Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochemie 70:559–566

    Google Scholar 

  • Soresen KI, Larsen R, Kibenich A, Junge MP, Johansen E (2000) A food-grade cloning system for industrial strains of Lactococcus lactis. Appl Environ Microbiol 66:1253–1258

    Google Scholar 

  • Steidler L, Robinson K, Chamberlain L, Schofield KM, Remaut E, Le Page RW, Wells JM (1998) Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen and cytokine. Infect Immun 66:3183–3189

    Google Scholar 

  • Takala TM, Saris PE (2002) A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI. Appl Microbiol Biotechnol 59:467–471

    Google Scholar 

  • Vos WM de (1999a) Safe and sustainable systems for food-grade fermentations by genetically modified lactic acid bacteria. Int Dairy J 9:3–10

    Google Scholar 

  • Vos WM de (1999b) Gene expression systems for lactic acid bacteria. Curr Opin Microbiol 2:289–295

    Google Scholar 

  • Vos WM de, Simons GFM (1994) Gene cloning and expression in lactococci. In: Gasson MJ, Vos WM de (eds) Genetics and biotechnology of lactic acid bacteria. Chapman and Hall, London, pp 52–105

  • Vossen JM van der, Lelie D van der, Venema G (1987) Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. Appl Environ Microbiol 53:2452–2457

    Google Scholar 

Download references

Acknowledgements

This work was supported by the Institut National de la Recherche Agronomique, France, CNPq, Brazil and the Universidade Católica de Brasília, Brazil.

Author information

Authors and Affiliations

  1. Programa de Pós-graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, SGAN 916, 70790-160, Brasília, DF, Brazil

    A. Simões-Barbosa, H. Abreu & A. Silva Neto

  2. Unité de Recherches Laitières et de Génétique Appliquée, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352, Jouy en Josas Cedex, France

    A. Gruss & P. Langella

Authors
  1. A. Simões-Barbosa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H. Abreu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Silva Neto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Gruss
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. P. Langella
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A. Simões-Barbosa.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Simões-Barbosa, A., Abreu, H., Silva Neto, A. et al. A food-grade delivery system for Lactococcus lactis and evaluation of inducible gene expression. Appl Microbiol Biotechnol 65, 61–67 (2004). https://doi.org/10.1007/s00253-004-1555-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-004-1555-0

Keywords

Search

Navigation