Applied Microbiology and Biotechnology

, Volume 76, Issue 6, pp 1403–1411

Sequencing and expression analysis of sakacin genes in Lactobacillus curvatus strains

Applied Genetics and Molecular Biotechnology

Abstract

In this study, we focused our investigation on two strains of Lactobacillus curvatus, L442 and LTH1174, which are able to produce bacteriocins. L. curvatus LTH1174 is widely studied for its capability to produce curvacin A, while L. curvatus L442 was isolated from traditional Greek fermented sausages and was shown to possess a strong inhibitory activity toward Listeria monocytogenes. By polymerase chain reaction, we were able to target in both strains the genes for the production of sakacin P and sakacin Q, sppA and sppQ, respectively, both encoded chromosomally. While sppA was found to be conserved when compared with other sakacin P genes, sppQ showed a deletion of about 15 nucleotides when aligned with sequences obtained from Lactobacillus sakei. This difference did not affect the activity of sakacin Q as determined by testing sensitive strains. Expression analysis highlighted that sakacin P was expressed in L. curvatus L442 but not in L. curvatus LTH1174. Curing experiments were performed on L. curvatus LTH1174 to study the effect of the megaplasmid, present in this strain. In the plasmid-cured strain, expression of the sppA gene was detected. sppQ was expressed in both plasmid-cured and wild-type L. curvatus LTH1174, although expression was higher in the plasmid-cured strain.

Keywords

Bacteriocins Expression analysis Gene sequencing Bioprotection 

References

  1. Altschul SF, Madden TL, Shaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefGoogle Scholar
  2. Anderson DG, McKay LL (1983) Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol 46:549–552Google Scholar
  3. Aymerich MT, Garriga M, Monfort JM, Nes I, Hugas M (2000) Bacteriocin producing lactobacilli in Spanish-styled fermented sausages: characterization of bacteriocins. Food Microbiol 17:33–45CrossRefGoogle Scholar
  4. Barefoot SF, Klaenhammer TR (1983) Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus. Appl Environ Microbiol 45:1808–1815Google Scholar
  5. Cocolin L, Rantsiou K, Iacumin L, Urso R, Cantoni C, Comi G (2004) Study of the ecology of fresh sausages and characterization of populations of lactic acid bacteria by molecular methods. Appl Environ Microbiol 70:1883–1894CrossRefGoogle Scholar
  6. Dunny GM, Leonard BAB (1997) Cell–cell communication in Gram-positive bacteria. Annu Rev Microbiol 51:527–564CrossRefGoogle Scholar
  7. Eijsink VGH, Axelsson L, Diep DB, Havarstein LS, Holo H, Nes IF (2002) Production of class II bacteriocins by lactic acid bacteria: an example of biological warfare and communication. Antonie van Leeuwenhoek 81:639–654CrossRefGoogle Scholar
  8. Ganzle MG, Hertel C, Hammes WP (1997) Modelling of the effect of pH, NaCl and nitrite concentrations on the antimicrobial activity of sakacin P against Listeria ivanovii DSM20750. Fleischwirtschaft Int 4:22–25Google Scholar
  9. Holck A, Axelsson L, Birkeland SE, Aukrust T, Blom H (1992) Purification and aminoacid sequence of sakacin A, a bacteriocin from Lactobacillus sakei Lb706. J Gen Microbiol 138:2715–2720Google Scholar
  10. Hugas M (1998) Bacteriocinogenic lactic acid bacteria for the biopreservation of meat and meat products. Meat Sci 49:S139–S150CrossRefGoogle Scholar
  11. Hugas M, Garriga M, Aymerich T, Monfort JM (1993) Biochemical characterization of lactobacilli from dry fermented sausages. Int J Food Microbiol 18:107–113CrossRefGoogle Scholar
  12. Huhne K, Axelsson L, Holck A, Krockel L (1996) Analysis of the sakacin P gene cluster from Lactobacillus sake Lb674 and its expression in sakacin-negative Lb. sake strains. Microbiol 142:1437–1448Google Scholar
  13. Jack RW, Tagg JR, Ray B (1995) Bacteriocins of gram-positive bacteria. Microbiol Rev 59:171–200Google Scholar
  14. Larsen AG, Vogensen FK, Josephsen J (1993) Antimicrobial activity of lactic acid bacteria isolated from sour doughs: purification and characterization of bavaricin A, a bacteriocin roduced by Lactobacillus bavaricus MI401. J Appl Bacteriol 75:113–122Google Scholar
  15. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  16. Mataragas M, Metaxopoulos J, Galiotou M, Drosinos EH (2003) Influence of pH and temperature on growth and bacteriocin production by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442. Meat Sci 64:265–271CrossRefGoogle Scholar
  17. Mathiesen G, Huehne K, Kroeckel L, Axelsson L, Eijsink VGH (2005) Characterization of a new bacteriocin operon in sakacin P-producing Lactobacillus sakei, showing strong translational coupling between the bacteriocin and immunity genes. Appl Environ Microbiol 71:3565–3574CrossRefGoogle Scholar
  18. Messens W, Verluyten J, Leroy F, De Vuyst L (2003) Modelling growth and bacteriocin production by lactobacillus curvatus LTH1174 in response to temperature and pH values used for European sausage fermentation processes. Int J Food Microbiol 81:41–52CrossRefGoogle Scholar
  19. Moretro T, Naterstad K, Wang E, Aasen IM, Chaillou S, Zagorec M, Axelsson L (2005) Sakacin P non-producing Lactobacillus sakei strains contain homologues of the sakacin P gene cluster. Res Microbiol 156:949–960CrossRefGoogle Scholar
  20. Nes IF, Eijsink VGH (1999) Regulation of group II peptide bacteriocin synthesis by quorum-sensing mechanisms. In: Dunny GM, Winans SC (eds) Cell–cell signaling in bacteria. American Society for Microbiology, Washington, DC, pp 175–192Google Scholar
  21. Quadri LEN (2002) Regulation of antimicrobial peptide production by autoinducer-mediated quorum sensing in lactic acid bacteria. Antonie van Leeuwenhoek 82:133–145CrossRefGoogle Scholar
  22. Remiger A, Ehrmann MA, Vogel RF (1996) Identification of bacteriocin-encoding genes in lactobacilli by polymerase chain reaction (PCR). Syst Appl Microbiol 19:28–34Google Scholar
  23. Schillinger U, Lücke FK (1989) Antibacterial activity of Lactobacillus sake isolated from meat. Appl Environ Microbiol 55:1901–1906Google Scholar
  24. Simon L, Fremaux C, Cenatiempo Y, Berjeaud JM (2002) Sakacin G, a new type of antilisterial bacteriocin. Appl Environ Microbiol 68:6416–6420CrossRefGoogle Scholar
  25. Sobrino OJ, Rodriguez JM, Moreira WL, Fernandez MF, Sanz B, Hernandez PE (1992) Sakacin M, a bacteriocin-like substance from Lactobacillus sakei 148. Int J Food Microbiol 16:215–225CrossRefGoogle Scholar
  26. Tichaczek PS, Nissen-Meyer J, Nes IF, Vogel RF, Hammes WP (1992) Characterization of the bacteriocins curvacin A from Lactobacillus curvatus LTH1174 and sakacin P from L. sake LTH673. Syst Appl Microbiol 15:460–468Google Scholar
  27. Tichaczek PS, Vogel RF, Hammes WP (1993) Cloning and sequencing of the curA encoding curvacin A, the bacteriocin produced by Lactobacillus curvatus LTH1174. Arch Microbiol 160:279–283CrossRefGoogle Scholar
  28. Tichaczek PS, Vogel RF, Hammes WP (1994) Cloning and sequencing of sakP encoding sakacin P, the bacteriocin produced by Lactobacillus sake LHT673. Microbiol 140:361–367CrossRefGoogle Scholar
  29. Trevors JT (1986) Plasmid curing in bacteria. FEMS Microbiol Rev 32:149–157CrossRefGoogle Scholar
  30. Urso R, Rantsiou K, Cantoni C, Comi G, Cocolin L (2006a) Sequencing and expression analysis of the sakacin P bacteriocin produced by a Lactobacillus sakei strain isolated from naturally fermented sausages. Appl Microbiol Biotechnol 71:480–485CrossRefGoogle Scholar
  31. Urso R, Rantsiou K, Cantoni C, Comi G, Cocolin L (2006b) Technological characterization of a bacteriocin-producing Lactobacillus sakei and its use in fermented sausage production. Int J Food Microbiol 110:232–239CrossRefGoogle Scholar
  32. Vandenbergh PA (1993) Lactic acid bacteria, their metabolic products and interference with microbial growth. FEMS Microbiol Rev 12:221–237CrossRefGoogle Scholar
  33. Varabioff Y (1992) Incidence of Listeria in small goods. Lett Appl Microbiol 14:167–169Google Scholar
  34. Vaughan A, O’Mahony J, Eijsink VGH, O’Connell-Motherway M, van Sinderen D (2004) Transcriptional analysis of bacteriocin production by malt isolate Lactobacillus sakei 5. FEMS Microbiol Lett 235:377–384CrossRefGoogle Scholar
  35. Verluyten J, Messens W, Vuyst L (2003) Sodium chloride reduces production of curvacin A, a bacteriocin produced by Lactobacillus curvatus strain LTH 1174, originating from fermented sausage. Appl Environ Microbiol 70:2271–2278CrossRefGoogle Scholar
  36. Verluyten J, Messens W, De Vuyst L (2004a) The curing agent sodium nitrite, used in the production of fermented sausages, is less inhibiting to the bacteriocin-producing meat starter culture Lactobacillus curvatus LTH1174 under anaerobic conditions. Appl Environ Microbiol 69:3833–3839CrossRefGoogle Scholar
  37. Verluyten J, Leroy F, De Vuyst L (2004b) Effect of different spices used in production of fermented sausages on growth of and curvacin A production by Lactobacillus curvatus LTH1174. Appl Environ Microbiol 70:4807–4813CrossRefGoogle Scholar
  38. Verluyten J, Leroy F, De Vuyst L (2004c) Influence of complex nutrient source on growth of and curvacin A production by sausage isolate Lactobacillus curvatus LTH1174. Appl Environ Microbiol 70:5081–5088CrossRefGoogle Scholar
  39. Xiraphi N, Georgalaki M, Van Driessche G, Devreese B, Van Beeumen J, Tsakalidou E, Metaxopoulos J, Drosinos EH (2005) Purification and characterization of curvaticin L442, a bacteriocin produced by Lactobacillus curvatus L442. Antonie van Leeuwenhoek 22:1–8Google Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.Dipartimento di Valorizzazione e Protezione delle Risorse Agroforestali, Facoltà di AgrariaUniversity of TurinTurinItaly

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