DOI QR코드

DOI QR Code

아욱 잎에서 분리한 Bacillus velezensis MV2의 유전체 염기서열 분석과 항균활성능 연구

Complete Genome Sequence and Antimicrobial Activities of Bacillus velezensis MV2 Isolated from a Malva verticillate Leaf

  • 이현주 (부산대학교 미생물학과) ;
  • 조은혜 (부산대학교 미생물학과) ;
  • 김지혜 (부산대학교 미생물학과) ;
  • 문금옥 (부산대학교 미생물학과) ;
  • 김민지 (경북대학교 응용생명과학부) ;
  • 신재호 (경북대학교 응용생명과학부) ;
  • 차재호 (부산대학교 미생물학과)
  • Lee, Hyeonju (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Jo, Eunhye (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Kim, Jihye (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Moon, Keumok (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Kim, Min Ji (School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University) ;
  • Shin, Jae-Ho (School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University) ;
  • Cha, Jaeho (Department of Microbiology, College of Natural Sciences, Pusan National University)
  • 투고 : 2020.11.19
  • 심사 : 2020.12.07
  • 발행 : 2021.03.28

초록

본 연구에서는 국내 자생 식물인 아욱으로부터 새로운 균주를 분리 및 동정하였고 해당 미생물이 생산하는 항미생물질의 활성과 관련 생합성 유전자들을 확인하고자 하였다. 16S rRNA 유전자 서열 정보를 토대로 비교한 결과, 아욱에서 분리된 균주는 Bacillus velezensis이었으며 strain은 MV2라고 명명되었다. 유전체 염기서열 분석을 통해 전체 유전정보를 확인할 수 있었으며, 45.57% GC 함량을 가지는 4,191,702 bp 크기의 1개 컨티그(contig)가 존재하는 것으로 확인되었다. B. velezensis MV2가 정지기에 생산하는 물질 중 항균 활성이 확인된 소수성 물질 분획을 이용하여 항균 활성 스펙트럼 테스트를 진행한 결과, 그람음성균보다 그람양성균에서 더 높은 억제능이 확인되었다. 6종의 곰팡이를 이용한 항진균 활성 테스트에서는 모든 진균에 대해 강한 저해 활성을 보였으며, 특히 F. fujikuroi와 F. graminearum에 대한 항진균 활성이 매우 강하게 나타났다. 세균에 대한 항균물질의 작용 기작 분석을 통해 해당 항균물질은 균을 용해시키는 살균(bactericidal) 특성을 가진 것으로 추측할 수 있었다. B. velezensis MV2의 유전체 염기서열 정보를 통해 이차대사산물 생합성 유전자 cluster를 탐색한 결과 총 47가지 이차대사산물 생산이 예측되었으며, 기존에 밝혀져 있는 물질들과 유사도 80% 이상인 물질은 14개로 확인되었다. 앞서 확인된 내용들을 바탕으로 B. velezensis MV2에서 생성되는 항균물질은 비리보솜 펩타이드성 물질로 예상되며, 향후 항균물질의 동정과 활용 가능성에 대한 추가적인 연구가 필요할 것으로 생각되었다. 기존에 상업적으로 이용되었던 곰팡이에 대한 활성이 낮은 항균물질 생물제제들과 함께 복합기능성 미생물제로 활용하여 식품산업 및 농업에서의 이용 가능성이 있음을 보여주었다.

A bacterial strain isolated from a Malva verticillata leaf was identified as Bacillus velezensis MV2 based on the 16S rRNA sequencing results. Complete genome sequencing revealed that B. velezensis MV2 possessed a single 4,191,702-bp contig with 45.57% GC content. Generally, Bacillus spp. are known to produce diverse antimicrobial compounds including bacteriocins, polyketides, and non-ribosomal peptides. Antimicrobial compounds in the B. velezensis MV2 were extracted from culture supernatants using hydrophobic interaction chromatography. The crude extracts showed antimicrobial activity against both gram-positive bacteria and gram-negative bacteria; however, they were more effective against gram-positive bacteria. The extracts also showed antifungal activity against phytopathogenic fungi such as Fusarium fujikuroi and F. graminearum. In time-kill assays, these antimicrobial compounds showed bactericidal activity against Bacillus cereus, used as indicator strain. To predict the type of antimicrobial compounds produced by this strain, we used the antiSMASH algorithm. Forty-seven secondary metabolites were predicted to be synthesized in MV2, and among them, fourteen were identified with a similarity of 80% or more with those previously identified. Based on the antimicrobial properties, the antimicrobial compounds may be nonribosomal peptides or polyketides. These compounds possess the potential to be used as biopesticides in the food and agricultural industry as an alternative to antibiotics.

키워드

과제정보

This research was supported by PNU-RENovation (2019-2020).

참고문헌

  1. Joseph WK, Leong J, Teintze M, Schroth MN. 1980. Enhanced plant growth by siderophores produced by plant growth promoting rhizobacteria. Nature 286: 885-886. https://doi.org/10.1038/286885a0
  2. Glick B. 1995. The enhancement of plant growth by free-living bacteria. Can. J. Microbiol. 41: 109-117. https://doi.org/10.1139/m95-015
  3. Rodelas BJ, Gonzalez-Lopez, Martinez-Toledo MV, Pozo C, Salmeron V. 1999. Influence of Rhizobium/Azotobacter and Rhizobium/Azospirillum combined inoculation on mineral composition of faba bean (Vicia faba L.). Biol. Fertil Soils. 29: 165-169. https://doi.org/10.1007/s003740050540
  4. Bashan Y, Levanony H. 1990. Current status of Azospirillum inoculation technology: Azospirillum as a challenge for agriculture. Can. J. Microbiol. 36: 591-608. https://doi.org/10.1139/m90-105
  5. Chakraborty U, Purkayastha RP. 1983. Role of rhizobitoxine in protecting soybean roots from Macrophomina phaseolina infection. Can. J. Microbiol. 30: 285-289. https://doi.org/10.1139/m84-043
  6. Yuan WM, Crawford DL. 1995. Characterization of Streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots. Appl. Environ. Microbiol. 61: 3119-3128. https://doi.org/10.1128/aem.61.8.3119-3128.1995
  7. Orhan E, Esitken A, Ercisli S, Turan M, Sahin F. 2006. Effects of plant growth promoting rhizobacteria (PGPR) on yield, growth and nutrient contents in organically growing raspberry. Sci. Hortic. 111: 38-43. https://doi.org/10.1016/j.scienta.2006.09.002
  8. Maksimov IV, Abizgil'dina RR, Pusenkova LI. 2011. Plant growth promoting rhizobacteria as alternative to chemical crop protectors from pathogens (review). Appl. Biochem. Microbiol. 47: 333-345. https://doi.org/10.1134/S0003683811040090
  9. Stein T. 2005. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol. Microbiol. 56: 845-857. https://doi.org/10.1111/j.1365-2958.2005.04587.x
  10. Dunlap C, Kim SJ, Kwon SW, Rooney AP. 2016. Bacillus velezensis is not a later heterotypic synonym of Bacillus amyloliquefaciens; Bacillus methylotrophicus, Bacillus amyloliquefaciens subsp. plantarum and 'Bacillus oryzicola' are later heterotypic synonyms of Bacillus velezensis based on phylogenomics. Int. J. Syst. Evol. Microbiol. 66: 1212-1217. https://doi.org/10.1099/ijsem.0.000858
  11. Rabbee MF, Ali MS, Choi J, Hwang BS, Jeong SC, Baek KH. 2019. Bacillus velezensis: A valuable member of bioactive molecules within plant microbiomes. Molecules 24: 1-13. https://doi.org/10.3390/molecules24010001
  12. Abriouel H, Franz CM, Omar NB, Galvez A. 2011. Diversity and applications of Bacillus bacteriocins. FEMS Microbiol. Rev. 35: 201-232. https://doi.org/10.1111/j.1574-6976.2010.00244.x
  13. Cleveland J, Montville TJ, Nes IF, Chikindas ML. 2001. Bacteriocins: safe, natural antimicrobials for food preservation. Int. J. Food Microbiol. 71: 1-20. https://doi.org/10.1016/S0168-1605(01)00560-8
  14. Caulier S, Nannan C, Gillis A, Licciardi F, Bragard C, Mahillon J. 2019. Overview of the antimicrobial compounds produced by members of the Bacillus subtilis Group. Front. Microbiol. 10: 302. https://doi.org/10.3389/fmicb.2019.00302
  15. Rangarajan V, Kim GC. 2016. Towards bacterial lipopeptide products for specific applications - a review of appropriate downstream processing schemes. Process Biochem. 51: 2176-2185. https://doi.org/10.1016/j.procbio.2016.08.026
  16. Ma Z, Zhang S, Zhang S, Wu G, Shao Y, Mi Q, et al. 2020. Isolation and characterization of a new cyclic lipopeptide surfactin from a marine-derived Bacillus velezensis SH-B74. J. Antibiot. 73: 863-867. https://doi.org/10.1038/s41429-020-0347-9
  17. Kang DW, Ryu IH, Han SS. 2012. The isolation of Bacillus subtilis KYS-10 with antifungal activity against plant pathogens. Korean J. Pestic. Sci. 16: 178-186. https://doi.org/10.7585/kjps.2012.16.2.178
  18. Smith JL, Collins HP, Crump AR, Bailey VL. 2015. Management of soil biota and their processes, pp. 539-572. In Paul EA (ed.), Soil Microbiology, Ecology and Biochemistry, 4th Ed. Waltham, MA : Academic Press, Boston, USA.
  19. Blin K, Shaw S, Steinke K, Villebro R, Ziemert N, Lee SY, et al. 2019. AntiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res. 47: W81-W87. https://doi.org/10.1093/nar/gkz310
  20. Russell AD. 1998. Mechanisms of Bacterial Resistance to Antibiotics and Biocides, pp. 133-197. In Ellis GP, Luscombe DK, Oxford AW (ed.), Prog. Med. Chem., Ed. Elsevier, Amsterdam, Netherlands, Oxford, England.
  21. Russell AD. 1998. Mechanisms of bacterial resistance to antibiotics and biocides. Prog. Med. Chem. 35: 133-197. https://doi.org/10.1016/S0079-6468(08)70036-5
  22. Patel P, Huangn S, Fisher S, Pirnik D, Aklonis C, Dean L, et al. 1995. Bacillaene, a novel inhibitor of procaryotic protein synthesis produced by Bacillus subtilis. J. Antibiot. 48: 997-1003. https://doi.org/10.7164/antibiotics.48.997
  23. Muller S, Strack SN, Hoefler BC, Straight PD, Kearns DB, Kirby JR. 2014. Bacillaene and sporulation protect Bacillus subtilis from predation by Myxococcus xanthus. Appl. Environ. Microbiol. 80: 5603-5610. https://doi.org/10.1128/AEM.01621-14
  24. Gong A, Li HP, Yuan QS, Song XS, Yao W, He WJ, et al. 2015. Antagonistic mechanism of iturin A and plipastatin A from Bacillus amyloliquefaciens S76-3 from wheat spikes against Fusarium graminearum. PLoS One 10: e0116871. https://doi.org/10.1371/journal.pone.0116871
  25. Koumoutsi A, Chen XH, Henne A, Liesegang H, Hitzeroth G, Franke P, et al. 2004. Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42. J. Bacteriol. 186: 1084-1096. https://doi.org/10.1128/JB.186.4.1084-1096.2004
  26. Chen M, Wang J, Liu B, Zhu Y, Xiao R, Yang W, et al. 2020. Bio-control of tomato bacterial wilt by the new strain Bacillus velezensis FJAT-46737 and its lipopeptides. BMC Microbiol. 20: 160-172. https://doi.org/10.1186/s12866-020-01851-2
  27. Stincone P, Veras FF, Pereira JQ, Mayer FQ, Varela APM, Brandelli A. 2020. Diversity of cyclic antimicrobial lipopeptides from Bacillus P34 revealed by functional annotation and comparative genome analysis. Microbiol. Res. 238: 126515. https://doi.org/10.1016/j.micres.2020.126515