DOI QR코드

DOI QR Code

Identification and Characterization of a Bacteriocin from the Newly Isolated Bacillus subtilis HD15 with Inhibitory Effects against Bacillus cereus

  • Sung Wook Hong (Technology Innovation Research Division, World Institute of Kimchi) ;
  • Jong-Hui Kim (National Institute of Animal Science, Rural Development Administration) ;
  • Hyun A Cha (National Institute of Animal Science, Rural Development Administration) ;
  • Kun Sub Chung (Division of Biological Science and Technology, Yonsei University) ;
  • Hyo Ju Bae (National Institute of Animal Science, Rural Development Administration) ;
  • Won Seo Park (National Institute of Animal Science, Rural Development Administration) ;
  • Jun-Sang Ham (National Institute of Animal Science, Rural Development Administration) ;
  • Beom-Young Park (National Institute of Animal Science, Rural Development Administration) ;
  • Mi-Hwa Oh (National Institute of Animal Science, Rural Development Administration)
  • 투고 : 2022.08.05
  • 심사 : 2022.10.11
  • 발행 : 2022.11.28

초록

Natural antimicrobial substances are needed as alternatives to synthetic antimicrobials to protect against foodborne pathogens. In this study, a bacteriocin-producing bacterium, Bacillus subtilis HD15, was isolated from doenjang, a traditional Korean fermented soybean paste. We sequenced the complete genome of B. subtilis HD15. This genome size was 4,173,431 bp with a G + C content of of 43.58%, 4,305 genes, and 4,222 protein-coding genes with predicted functions, including a subtilosin A gene cluster. The bacteriocin was purified by ammonium sulfate precipitation, Diethylaminoethanol-Sepharose chromatography, and Sephacryl gel filtration, with 12.4-fold purification and 26.2% yield, respectively. The purified protein had a molecular weight of 3.6 kDa. The N-terminal amino acid sequence showed the highest similarity to Bacillus subtilis 168 subtilosin A (78%) but only 68% similarity to B. tequilensis subtilosin proteins, indicating that the antimicrobial substance isolated from B. subtilis HD15 is a novel bacteriocin related to subtilosin A. The purified protein from B. subtilis HD15 exhibited high antimicrobial activity against Listeria monocytogenes and Bacillus cereus. It showed stable activity in the range 0-70℃ and pH 2-10 and was completely inhibited by protease, proteinase K, and pronase E treatment, suggesting that it is a proteinaceous substance. These findings support the potential industrial applications of the novel bacteriocin purified from B. subtilis HD15.

키워드

과제정보

This work was carried out with the support of "Cooperative Research Program for Agricultural Science & Technology Development (Project No. PJ009221012014)" Rural Development Administration and was financially supported by the Ministry of Trade, Industry and Energy (MOTIE) and Korea Institute for Advancement of Technology (KIAT) through the National Innovation Cluster R&D program(P0015309), Republic of Korea.

참고문헌

  1. Singh A, Poshtiban S, Evoy S. 2013. Recent advances in bacteriophage based biosensors for food-borne pathogen detection. Sensors 13: 1763-1786. https://doi.org/10.3390/s130201763
  2. Oliver SP, Jayarao BM, Almeida RA. 2005. Foodborne pathogens in milk and the dairy farm environment: food safety and public health implications. Foodborne Pathog. Dis. 2: 115-129. https://doi.org/10.1089/fpd.2005.2.115
  3. Maragkoudakis PA, Mountzouris KC, Psyrras D, Cremonese S, Fischer J, Cantor MD, et al. 2009. Functional properties of novel protective lactic acid bacteria and application in raw chicken meat against Listeria monocytogenes and Salmonella enteritidis. Int. J. Food Microbiol. 130: 219-226. https://doi.org/10.1016/j.ijfoodmicro.2009.01.027
  4. Rasko DA, Altherr MR, Han CS, Ravel J. 2005. Genomics of the Bacillus cereus group of organisms. FEMS Microbiol. Rev. 29: 303-329.
  5. McLauchlin J. 1997. The identification of Listeria species. Int. J. Food Microbiol. 381: 77-81. https://doi.org/10.1016/S0168-1605(97)00086-X
  6. Cowan MM. 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12: 564-582 https://doi.org/10.1128/CMR.12.4.564
  7. Kim YS, Shin DH. 2003. Researches on the volatile antimicrobial compounds from edible plants and their food application. Korean J. Food Sci. Technol. 35: 159-165.
  8. Vendrell D, Balcazar JL, Ruiz-Zarzuela I, de Blas I, Girones O, Muzquiz JL. 2006. Lactococcus garvieae in fish: a review. Compar. Immunol. Microbiol. Infect. Dis. 29: 177-198. https://doi.org/10.1016/j.cimid.2006.06.003
  9. Hurst A. 1981. Nisin. Adv. Appl. Microbiol. 27: 85-123. https://doi.org/10.1016/S0065-2164(08)70342-3
  10. Motta AS, Flores FS, Souto AA, Brandelli A. 2008. Antibacterial activity of a bacteriocin-like substance produced by Bacillus sp. P34 that targets the bacterial cell envelope. Antonie Van Leeuwenhoek 93: 275-284. https://doi.org/10.1007/s10482-007-9202-2
  11. Sirtori LR, Cladera-Olivera F, Lorenzini DM, Tsai SM, Brandelli A. 2006. Purification and partial characterization of an antimicrobial peptide produced by Bacillus sp. strain P45, a bacterium from the Amazon basin fish Piaractus mesopotamicus. J. Gen. Appl. Microbiol. 52: 357-363. https://doi.org/10.2323/jgam.52.357
  12. Abriouel H, Franz CM, Omar NB, Galvez A. 2011. Diversity and applications of Bacillus bacteriocins. FEMS Microbiol. Rev. 5: 201-232. https://doi.org/10.1111/j.1574-6976.2010.00244.x
  13. Perez RH, Perez MTM, Elegado FB. 2015. Bacteriocins from lactic acid bacteria: A review of biosynthesis, mode of action, fermentative production, uses, and prospects. Int. J. Philippine Sci. Technol. 8: 61-67. https://doi.org/10.18191/2015-08-2-027
  14. Serpil U, Kati H. 2013. Purification and characterization of the bacteriocin Thuricin Bn1 produced by Bacillus thuringiensis subsp. kurstaki Bn1 isolated from a hazelnut pest. J. Microbiol. Biotechnol. 23: 167-176. https://doi.org/10.4014/jmb.1209.09056
  15. Le Marrec C, Hyronimus B, Bressollier P, Verneuil B, Urdaci MC. 2000. Biochemical and genetic characterization of coagulin, a new antilisterial bacteriocin in the pediocin family of bacteriocins, produced by Bacillus coagulans I4. Appl. Environ. Microbiol. 66: 5213-5220. https://doi.org/10.1128/AEM.66.12.5213-5220.2000
  16. Lee H, Churey JJ, Worobo RW. 2009. Biosynthesis and transcriptional analysis of thurincin H, a tandem repeated bacteriocin genetic locus, produced by Bacillus thuringiensis SF361. FEMS Microbiol. Lett. 299: 205-213. https://doi.org/10.1111/j.1574-6968.2009.01749.x
  17. Pattnaik P, Kaushik JK, Grover S, Batish VK. 2001. Purification and characterization of a bacteriocin-like compound (lichenin) produced anaerobically by Bacillus licheniformis isolated from water buffalo. J. Appl. Microbiol. l91: 636-645.
  18. Yang EJ, Chang HC. 2007. Characterization of bacteriocin-like substances produced by Bacillus subtilis MJP1. Korean J. Microbiol. Biotechnol. 35: 339-346.
  19. Xie J, Zhang R, Shang C, Guo Y. 2009. Isolation and characterization of a bacteriocin produced by an isolated Bacillus subtilis LFB112 that exhibits antimicrobial activity against domestic animal pathogens. Afr. J. Biotechnol. 8: 5611-5619.
  20. Tagg J, Mcgiven AR. 1971. Assay system for bacteriocin. Appl. Environ. Microbiol. 21: 943-948. https://doi.org/10.1128/am.21.5.943-943.1971
  21. Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, et al. 2013. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat. Methods 10: 563-569. https://doi.org/10.1038/nmeth.2474
  22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265-275. https://doi.org/10.1016/S0021-9258(19)52451-6
  23. Bhunia AK, Johnson MC, Ray B. 1987. Direct detection of an antimicrobial peptide of Pediococcus acidilactici in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Ind. Microbiol. 2: 319-322. https://doi.org/10.1007/BF01569434
  24. Edman P, Begg G. 1967. A protein sequenator. Eur. J. Biochem. 1: 80-91. https://doi.org/10.1111/j.1432-1033.1967.tb00047.x
  25. Khochamit N, Siripornadulsil S, Sukon P, Siripornadulsil W. 2015. Antibacterial activity and genotypic-phenotypic characteristics of bacteriocin-producing Bacillus subtilis KKU213: potential as a probiotic strain. Microbiol. Res. 170: 36-50. https://doi.org/10.1016/j.micres.2014.09.004
  26. Rood JI, Cole ST. 1991. Molecular genetics and pathogenesis of Clostridium perfringens. Microbiol. Rev. 55: 621-648. https://doi.org/10.1128/mr.55.4.621-648.1991
  27. Regamey A, Karamata D. 1998. The N-acetylmuramoyl-L-alanine amidase encoded by the Bacillus subtilis 168 prophage SPβ. Microbiology 144: 885-893. https://doi.org/10.1099/00221287-144-4-885
  28. Stein T. 2008. Whole-cell matrix-assisted laser desorption/ ionization mass spectrometry for rapid identification of bacteriocin/ lantibiotic-producing bacteria. Rapid Commun. Mass Spectrom. 22: 1146-1152. https://doi.org/10.1002/rcm.3481
  29. Stein T, Borchert S, Conrad B, Feesche J, Hofemeister B, Hofemeister J, et al. 2002. Two different lantibiotic like peptides originate from the ericin gene cluster of Bacillus subtilis A1/3. J. Bacteriol. 184: 1703-1711. https://doi.org/10.1128/JB.184.6.1703-1711.2002
  30. Marx R, Stein T, Entian KD, Glaser SJ. 2001. Structure of the Bacillus subtilis peptide antibiotic subtilosin A determined by 1H-NMR and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. J. Protein Chem. 20: 501-506. https://doi.org/10.1023/A:1012562631268
  31. Paik SH, Chakicherla A, Hansen JN. 1998. Identification and characterization of the structural and transporter genes for, and the chemical and biological properties of sublancin 168, a novel lantibiotic produced by Bacillus subtilis 168. J. Biol. Chem. 273: 23134-23142. https://doi.org/10.1074/jbc.273.36.23134
  32. Stein T, Dusterhus S, Stroh A, Entian KD. 2004. Subtilosin production by two Bacillus subtilis subspecies and variance of the sbo-alb cluster. Appl. Environ. Microbiol. 70: 2349-2353. https://doi.org/10.1128/AEM.70.4.2349-2353.2004
  33. Sutyak KE, Wirawan RE, Aroutcheva AA, Chikindas ML. 2008. Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product?derived Bacillus amyloliquefaciens. J. Appl. Microbiol. 104: 1067-1074. https://doi.org/10.1111/j.1365-2672.2007.03626.x
  34. Rani RP, Anandharaj M, Hema S, Deepika R, Ravindran AD. 2016. Purification of antilisterial Peptide (subtilosin A) from Novel Bacillus tequilensis FR9 and demonstrate their pathogen invasion protection ability using human carcinoma cell line. Front. Microbiol. 7: 1910.
  35. Kwon GH, Lee HA, Kim JH. 2010. A bacteriocin of 5-kDa in size secreted by Bacillus subtilis 168. Korean J. Microbiol. Biotechnol. 38: 163-167.
  36. Kindoli S, Lee HA, Kim JH. 2012. Properties of a bacteriocin from Bacillus subtilis H27 isolated from Cheonggukjang. J. Microbiol. Biotechnol. 21: 1745-1751.
  37. Jack RW, Tagg JR, Ray B. 1995. Bacteriocin of gram-positive bacteria. Microbiol. Rev. 59: 171-200. https://doi.org/10.1128/mr.59.2.171-200.1995
  38. Lee NK, Yeo IC, Park JW, Kang BS, Hahm YT. 2010. Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus. J. Biosci. Bioeng. 110: 298-303. https://doi.org/10.1016/j.jbiosc.2010.03.002
  39. Kamoun F, Mejdoub H, Aouissaoui H, Reinbolt J, Hammani A, Jaoua S. 2005. Purification, amino acid sequence and characterization of Bacthuricin F4, a new bacteriocin produced by Bacillus thuringiensis. J. Appl. Microbiol. 98: 881-888. https://doi.org/10.1111/j.1365-2672.2004.02513.x
  40. Chen L, Gu Q, Li P, Li Y, Song D, Yang J. 2018. Purification and Characterization of Plantaricin ZJ316, a Novel Bacteriocin against Listeria monocytogenes from Lactobacillus plantarum ZJ316. J. Food Protect. 81: 1929-1935.   https://doi.org/10.4315/0362-028X.JFP-18-306