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Biotechnology Letters

, Volume 41, Issue 2, pp 283–292 | Cite as

Thermophilin 109 is a naturally produced broad spectrum bacteriocin encoded within the blp gene cluster of Streptococcus thermophilus

  • John A. RenyeJr.Email author
  • George A. Somkuti
  • Dennis H. Steinberg
Original Research Paper
  • 179 Downloads

Abstract

Objectives

To demonstrate that S. thermophilus ST109 produces an antimicrobial peptide encoded within the bacteriocin-like peptide (blp) gene cluster, and to determine its broad spectrum activity against potential human pathogens.

Results

Analysis of the cell free supernatant (CFS) revealed that antimicrobial activity was associated with the presence of a heat-stable peptide of approximately 5–6 kDa; and activity was lost after protease treatment or exposure to α-amylase. Deletion of blpC, which encodes a quorum sensing induction peptide, resulted in a loss of antimicrobial activity showing that thermophilin 109 was encoded within the blp gene cluster of ST109. Sequencing of the ST109 blp gene cluster showed 90% and 99% identity to clusters previously characterized in S. thermophilus strains LMD-9 and ST106, both of which are unable to naturally produce their bacteriocins. Real-time qPCR showed that blpC and blpD were expressed approximately 24 and 100-fold higher in ST109 as compared to strain LMD-9; and broad spectrum activity was demonstrated against lactobacilli, enterococci and the human pathogen Streptococcus pyogenes.

Conclusions

The high level of similarity observed between the ST109 and ST106 blp gene clusters at the nucleic acid level suggests bacteriocin expression may be regulated by factors encoded elsewhere on the chromosome. Activity against E. faecalis and S. pyogenes suggest S. thermophilus ST109 could be used for food safety and probiotic applications.

Keywords

Streptococcus thermophilus Bacteriocin Antimicrobial activity Lactic acid bacteria Quorum sensing 

Notes

Acknowledgements

We would like to thank J. Hernandez, Temple University, for technical assistance in determining the antimicrobial spectra of thermophilin 109; and D. Needleman, USDA-ARS, for technical assistance for sequencing the ST109 blp gene cluster.

Supporting information

Supplementary Fig. 1—Genotype confirmation of the S. thermophilus ST109 blpC knock out mutant (ST109CKO). (A) PCR confirming the presence of both the erythromycin (erm; lane 1) and kanamycin (kan; lane 2) resistance genes on plasmid pSTKOC. (B) PCR analysis of strain ST109CKO, where both the erm (lane 1) and blpC (lane 3) were absent; and the kan gene was present (lane 2). blpC was successfully amplified from the parent ST109 culture (lane 4) to confirm proper function of the primer set. M: 1kb ladder.

Supplementary Table 1—Real-time PCR of blp components in S. thermophilus strains ST109, ST109CKO and LMD-9.

Supplementary material

10529_2018_2637_MOESM1_ESM.docx (3.6 mb)
Supplementary material 1 (DOCX 3654 KB)

References

  1. Aslam M, Shahid M, Rehman FU, Naveed NH, Batool AI, Sharif S, Asia A (2011) Purification and characterization of bacteriocin isolated from Streptococcus thermophilus. Afr J Microbiol Res 5:2642–2648CrossRefGoogle Scholar
  2. Begley M, Cotter PD, Hill C, Ross RP (2009) Identification of a novel two-peptide lantibiotic, lichenicidin, following rational genome mining for LanM proteins. Appl Environ Microbiol 75:5451–5460CrossRefGoogle Scholar
  3. Blomqvist T, Steinmoen H, Havarstein LS (2006) Pheromone-induced expression of recombinant proteins in Streptococcus thermophilus. Arch Microbiol 186:465–473CrossRefGoogle Scholar
  4. Burianek LL, Yousef AE (2000) Solvent extraction of bacteriocins from liquid cultures. Lett Appl Microbiol 31:193–197CrossRefGoogle Scholar
  5. Cotter PD, Hill C, Ross RP (2005) Bacteriocins: developing innate immunity for food. Nat Rev Microbiol 3:777–788CrossRefGoogle Scholar
  6. de Saizieu A, Gardes C, Flint N, Wagner C, Kamber M, Mitchell TJ, Keck W, Amrein KE, Lange R (2000) Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide. J Bacteriol 182:4696–4703CrossRefGoogle Scholar
  7. Di Pierro F, Colombo M, Giuliani MG, Danza ML, Basile I, Bollani T, Conti AM, Zanvit A, Rottoli AS (2016) Effects of administration of Streptococcus salivarius K12 on the occurrence of streptococcal pharyngo-tonsillitis, scarlet fever and acute otitis media in 3 year old children. Eur Rev Med Pharmacol Sci 20:4601–4606Google Scholar
  8. Fontaine L, Hols P (2008) The inhibitory spectrum of thermophilin 9 from Streptococcus thermophilus LMD-9 depends on the production of multiple peptides and the activity of BlpG(St), a thiol-disulfide oxidase. Appl Environ Microbiol 74:1102–1110CrossRefGoogle Scholar
  9. Fontaine L, Boutry C, Guedon E, Guillot A, Ibrahim M, Grossiord B, Hols P (2007) Quorum-sensing regulation of the production of Blp bacteriocins in Streptococcus thermophilus. J Bacteriol 189:7195–7205CrossRefGoogle Scholar
  10. Fontaine L, Goffin P, Dubout H, Delplace B, Baulard A, Lecat-Guillet N, Chambellon E, Gardan R, Hols P (2013) Mechanism of competence activation by the ComRS signalling system in streptococci. Mol Microbiol 87:1113–1132CrossRefGoogle Scholar
  11. Gilbreth SE, Somkuti GA (2005) Thermophilin 110: a bacteriocin of Streptococcus thermophilus ST110. Curr Microbiol 51:175–182CrossRefGoogle Scholar
  12. Giraffa G (2002) Enterococci from foods. FEMS Microbiol Rev 26:163–171CrossRefGoogle Scholar
  13. Hegarty J, Guinane CM, Ross RP, Hill C, Cotter PD (2016) Bacteriocin production: a relatively unharnessed probiotic trait? F1000Res 5:2587Google Scholar
  14. Hols P, Hancy F, Fontaine L, Grossiord B, Prozzi D, Leblond-Bourget N, Decaris B, Bolotin A, Delorme C, Dusko Ehrlich S, Guedon E, Monnet V, Renault P, Kleerebezem M (2005) New insights in the molecular biology and physiology of Streptococcus thermophilus revealed by comparative genomics. FEMS Microbiol Rev 29:435–463Google Scholar
  15. Hyink O, Wescombe PA, Upton M, Ragland N, Burton JP, Tagg JR (2007) Salivaricin A2 and the novel lantibiotic salivaricin B are encoded at adjacent loci on a 190-kilobase transmissible megaplasmid in the oral probiotic strain Streptococcus salivarius K12. Appl Environ Microbiol 73:1107–1113CrossRefGoogle Scholar
  16. Ivanova I, Miteva V, Stefanova T, Pantev A, Budakov I, Danova S, Moncheva P, Nikolova I, Dousset X, Boyaval P (1998) Characterization of a bacteriocin produced by Streptococcus thermophilus 81. Int J Food Microbiol 42:147–158CrossRefGoogle Scholar
  17. Kabuki T, Uenishi H, Watanabe M, Seto Y, Nakajima H (2007) Characterization of a bacteriocin, Thermophilin 1277, produced by Streptococcus thermophilus SBT1277. J Appl Microbiol 102:971–980Google Scholar
  18. Khalil R (2009) Evidence for probiotic potential of a capsular-producing Streptococcus thermophilus CHCC 3534 strain. Pol J Microbiol 58:49–55Google Scholar
  19. Kjos M, Borrero J, Opsata M, Birri DJ, Holo H, Cintas LM, Snipen L, Hernandez PE, Nes IF, Diep DB (2011) Target recognition, resistance, immunity and genome mining of class II bacteriocins from Gram-positive bacteria. Microbiol 157:3256–3267CrossRefGoogle Scholar
  20. Marciset O, Jeronimus-Stratingh MC, Mollet B, Poolman B (1997) Thermophilin 13, a nontypical antilisterial poration complex bacteriocin that functions without a receptor. J Biol Chem 272:14277–14284CrossRefGoogle Scholar
  21. Mathot AG, Beliard E, Thuault D (2003) Streptococcus thermophilus 580 produces a bacteriocin potentially suitable for inhibition of Clostridium tyrobutyricum in hard cheese. J Dairy Sci 86:3068–3074CrossRefGoogle Scholar
  22. Renye JA Jr, Somkuti GA (2017) Bacteriocin with novel activity. U.S. Patent 9,598,471 Issued 21 March 2017Google Scholar
  23. Renye JA Jr, Somkuti GA (2013) BlpC-regulated bacteriocin production in Streptococcus thermophilus. Biotechnol Lett 35:407–412CrossRefGoogle Scholar
  24. Renye JA Jr, Somkuti GA, Garabal JI, Steinberg DH (2016) Bacteriocin production by Streptococcus thermophilus in complex growth media. Biotechnol Lett 38:1947–1954CrossRefGoogle Scholar
  25. Rossi F, Marzotto M, Cremonese S, Rizzotti L, Torriani S (2013) Diversity of Streptococcus thermophilus in bacteriocin production; inhibitory spectrum and occurrence of thermophilin genes. Food Microbiol 35:27–33CrossRefGoogle Scholar
  26. Shatalin KY, Neyfakh AA (2005) Efficient gene inactivation in Bacillus anthracis. FEMS Microbiol Lett 245:315–319CrossRefGoogle Scholar
  27. Somkuti GA, Renye JA Jr (2015) Effect of a BlpC-based quorum-sensing induction peptide on bacteriocin production in Streptococcus thermophilus. J Food Res 4:88–96CrossRefGoogle Scholar
  28. Somkuti GA, Steinberg DH (1986) Distribution and analysis of plasmids in Streptococcus thermophilus. J Ind Microbiol 1:157–163CrossRefGoogle Scholar
  29. Somkuti GS, Steinberg DH (1988) Genetic transformation of Streptococcus thermophilus by electroporation. Biochimie 70:579–585CrossRefGoogle Scholar
  30. Villani F, Pepe O, Mauriello G, Salzano G, Moschetti G, Coppola S (1995) Antilisterial activity of thermophilin 347, a bacteriocin produced by Streptococcus thermophilus. Int J Food Microbiol 25:179–190CrossRefGoogle Scholar
  31. Ward DJ, Somkuti GA (1995) Characterization of a bacteriocin produced by Streptococcus thermophilus ST134. Appl Microbiol Biotechnol 43:330–335CrossRefGoogle Scholar
  32. Wescombe PA, Upton M, Dierksen KP, Ragland NL, Sivabalan S, Wirawan RE, Inglis MA, Moore CJ, Walker GV, Chilcott CN, Jenkinson HF, Tagg JR (2006) Production of the lantibiotic salivaricin A and its variants by oral streptococci and use of a specific induction assay to detect their presence in human saliva. Appl Environ Microbiol 72:1459–1466CrossRefGoogle Scholar

Copyright information

© This is a U.S. government work and its text is not subject to copyright protection in the United States; however, its text may be subject to foreign copyright protection 2018

Authors and Affiliations

  1. 1.Dairy and Functional Foods Research Unit, Agricultural Research ServiceUSDAWyndmoorUSA

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