Applied Microbiology and Biotechnology

, Volume 103, Issue 9, pp 3819–3827 | Cite as

Bile-induced promoters for gene expression in Lactobacillus strains

  • José Alberto Martínez-Fernández
  • Daniel Bravo
  • Ángela Peirotén
  • Juan Luis Arqués
  • José María LandeteEmail author
Applied genetics and molecular biotechnology


Bioengineering of probiotics allows the improvement of their beneficial characteristics. In this work, we develop a molecular tool that would allow the activation of desirable traits in probiotics once they reach the intestine. The activity of upstream regions of bile-inducible genes of Lactobacillus casei BL23 and Lactobacillus plantarum WCFS1 was analyzed using plasmids encoding an anaerobic fluorescent protein as reporter. The promoter P16090 from Lb. casei BL23 was selected and its bile induction confirmed in Lb. casei BL23, Lb. plantarum WCFS1, and in Lactobacillus rhamnosus and Lactobacillus reuteri strains. However, the induction did not occur in Lactococcus lactis MG1363 or Bifidobacterium strains. Studies with different bile compounds revealed the importance of cholic acid in the bile induction process. Induction of fluorescence was also confirmed for transformed Lb. casei BL23 under simulated colonic conditions and in the presence of intestinal microbiota. The developed vector, pNZ:16090-aFP, constitutes a promising tool suitable for the expression of genes of interest under intestinal conditions in probiotic strains of the species Lb. casei, Lb. plantarum, Lb. rhamnosus, and Lb. reuteri.


Lactobacillus Gene expression Probiotic Promoter Bile induction 



This study was funded by RTA2013-00029-00-00 and RTA2017-00002-00-00 from the Spanish Ministry of Science, Innovation and Universities.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interest.


  1. Alcántara C, Zúñiga M (2012) Proteomic and transcriptomic analysis of the response to bile stress of Lactobacillus casei BL23. Microbiology 158:1206–1218CrossRefGoogle Scholar
  2. Aukrust T, Blom H (1992) Transformation of Lactobacillus strains used in meat and vegetable fermentations. Food Res Int 25:253–261CrossRefGoogle Scholar
  3. Bravo D, Landete JM (2017) Genetic engineering as a powerful tool to improve probiotic strains. Biotechnol Genet Eng Rev 33:173–189CrossRefGoogle Scholar
  4. Bron PA, Marco M, Hoffer SM, Van Mullekom E, de Vos WM, Kleerebezem M (2004) Genetic characterization of the bile salt response in Lactobacillus plantarum and analysis of responsive promoters in vitro and in situ in the gastrointestinal tract. J Bacteriol 186:7829–7835CrossRefGoogle Scholar
  5. Bron PA, Molenaar D, de Vos WM, Kleerebezem M (2006) DNA micro-array-based identification of bile-responsive genes in Lactobacillus plantarum. J Appl Microbiol 100:728–738CrossRefGoogle Scholar
  6. Dashkevicz MP, Feighner SD (1989) Development of a differential medium for bile salt hydrolase-active Lactobacillus spp. Appl Environ Microbiol 55:11–16Google Scholar
  7. Gasson MJ (1983) Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol 154:1–9Google Scholar
  8. Jordan S, Hutchings MI, Mascher T (2008) Cell envelope stress response in Gram-positive bacteria. FEMS Microbiol Rev 32:107–146CrossRefGoogle Scholar
  9. Koskenniemi K, Laakso K, Koponen J, Kankainen M, Greco D, Auvinen P, Savijoki K, Nyman TA, Surakka A, Salusjärvi T, de Vos WM, Tynkkynen S, Kalkkinen N, Varmanen P (2011) Proteomics and transcriptomics characterization of bile stress response in probiotic Lactobacillus rhamnosus GG. Mol Cell Proteomics MCP 10:M110.002741CrossRefGoogle Scholar
  10. Kristoffersen SM, Ravnum S, Tourasse NJ, Økstad OA, Kolstø AB, Davies W (2007) Low concentrations of bile salts induce stress responses and reduce motility in Bacillus cereus ATCC 14579. J Bacteriol 189:5302–5313CrossRefGoogle Scholar
  11. Kuipers OP, de Ruyter PGGA, Kleerebezem M, de Vos WM (1998) Quorum sensing controlled gene expression in lactic acid bacteria. J Biotechnol 64:15–21CrossRefGoogle Scholar
  12. Lambert JM, Bongers RS, de Vos WM, Kleerebezem M (2008) Functional analysis of four bile salt hydrolase and penicillin acylase family members in Lactobacillus plantarum WCFS1. Appl Environ Biotechnol 74:4719–4726Google Scholar
  13. Landete JM (2016) Effector molecules and regulatory proteins: applications. Trends Biotechnol 34:770–780CrossRefGoogle Scholar
  14. Landete JM, Arqués J (2017) Fluorescent lactic acid bacteria and bifidobacteria as vehicles of DNA microbial biosensors. Int J Mol Sci 18(8):1728. CrossRefGoogle Scholar
  15. Landete JM, Arqués JL, Peirotén A, Langa S, Medina M (2014a) An improved method for the electrotransformation of lactic acid bacteria: a comparative survey. J Microbiol Methods 105:130–133CrossRefGoogle Scholar
  16. Landete JM, Langa S, Revilla C, Margolles A, Medina M, Arqués JL (2015) Use of anaerobic green fluorescent protein versus green fluorescent protein as reporter in lactic acid bacteria. Appl Microbiol Biotechnol 99:6865–6877CrossRefGoogle Scholar
  17. Landete JM, Peirotén A, Rodriguez E, Margolles A, Medina M, Arqués JL (2014b) Anaerobic green fluorescent protein as a marker of Bifidobacterium strains. Int J Food Microbiol 175:6–13CrossRefGoogle Scholar
  18. Linares DM, Gómez C, Renes E, Fresno JM, Tornadijo ME, Ross RP, Stanton C (2017) Lactic acid bacteria and bifidobacteria with potential to design natural biofunctional health-promoting dairy foods. Front Microbiol 8:846CrossRefGoogle Scholar
  19. Mathipa MG, Thantsha MS (2017) Probiotic engineering: towards development of robust probiotic strains with enhanced functional properties and for targeted control of enteric pathogens. Gut Pathogens 9:28CrossRefGoogle Scholar
  20. Pfeiler EA, Azcárate-Peril MA, Klaenhammer TR (2007) Characterization of a novel bile-inducible operon encoding a two-component regulatory system in Lactobacillus acidophilus. J Bacteriol 189:4624–4634CrossRefGoogle Scholar
  21. Rodríguez E, Arqués JL, Rodríguez R, Nuñez M, Medina M (2003) Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits. Lett Appl Microbiol 37(3):259–263CrossRefGoogle Scholar
  22. Rodríguez E, Arqués JL, Rodríguez R, Peiroten A, Landete JM, Medina M (2012) Antimicrobial properties of probiotic strains isolated from breast-fed infants. J Funct Foods 4(2):542–551CrossRefGoogle Scholar
  23. Ruiz L, Álvarez-Martín P, Mayo B, de los Reyes-Gavilán CG, Gueimonde M, Margolles A (2012) Controlled gene expression in bifidobacteria by use of a bile-responsive element. Appl Environ Microbiol 78:581–585CrossRefGoogle Scholar
  24. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  25. Schulze-Gahmen U, Pelaschier J, Yokota H, Kim R, Kim SH (2003) Crystal structure of a hypothetical protein, TM841 of Thermotoga maritima, reveals its function as a fatty acid-binding protein. Proteins 50:526–530CrossRefGoogle Scholar
  26. Vulevic J, Rastall RA, Gibson GR (2004) Developing a quantitative approach for determining the in vitro prebiotic potential of dietary oligosaccharides. FEMS Microbiol Lett 236:153–159CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Departamento de Tecnología de AlimentosInstituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)MadridSpain

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