생태와환경 (Korean Journal of Ecology and Environment)

  • 제50권1호
  • /
  • Pages.137-147
  • /
  • 2017
  • /
  • 2288-1115(pISSN)
  • /
  • 2288-1123(eISSN)
한국수생태학회 (The Korean Society of Limnology)
권호 표지
DOI QR코드

DOI QR Code

낙동강에서 분리된 Aphanizomenon flos-aquae (Cyanophyceae) 균주의 목표 유전자를 이용한 잠재적 독소 생성능 및 계통학적 분석

Analysis of Potential Toxigenicity and Phylogeny using Target Genes in Aphanizomenon flos-aquae (Cyanophyceae) strains isolated from the Nakdong River

  • 류희성 (대구대학교 생물교육전공) ;
  • 안성민 (한국해양과학기술원) ;
  • 임창건 (대구대학교 생명과학과) ;
  • 신라영 (대구대학교 생물교육전공) ;
  • 박종근 (대구대학교 지구과학교육전공) ;
  • 이정호 (대구대학교 생물교육전공)
  • Ryu, Hui-Seong (Department of Biology Education, Daegu University) ;
  • An, Sung-Min (Korean Institute of Ocean Science & Technology) ;
  • Lim, Chang-Kun (Department of Biological Science, Daegu University) ;
  • Shin, Ra-Young (Department of Biology Education, Daegu University) ;
  • Park, Jong-Guen (Department of Earth Science Education, Daegu University) ;
  • Lee, Jung-Ho (Department of Biology Education, Daegu University)
  • 투고 : 2017.02.15
  • 심사 : 2017.03.27
  • 발행 : 2017.03.31
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

독소 생성 분류군의 정의는 분리균주에 의해서 동정되고, 단일배양에 의한 독소 생성 여부 및 유전적 검토가 확인된 분류군을 의미한다. 이러한 관점에서 Aphanizomenon flos-aquae의 독소 생성능은 세계적으로 아직 논쟁의 여지가 있다. 본 연구는 낙동강에서 분리한 Aphanizomenon flos-aquae (DGUC001, DGUC003)을 대상으로 16S rRNA 염기서열을 이용하여 계통학적 위치를 확인하고, 남세균독소인 saxitoxin (STX)과 cylindrospermopsin (CYN)의 잠재적 생성능력을 유전자 수준에서 검토하였다. 연구에 사용된 균주는 2016년 8월과 2016년 10월에 낙동강 본류구간의 하천수에서 분리되었다. 계통학적 분석에는 16S rRNA가 사용되었으며, 독소 생성 유전자는 CYN과 STX 생합성에 관여하는 cyrA, cyrJ, sxtA, sxtI 유전자가 선택되었다. 분리된 균주 DGUC001과 DGUC003은 육안으로 관찰 가능한 크기의 다발(fascicles)을 형성하였으며, 세포사(trichome)가 병렬 형태로 나열되고, 세포사의 양쪽 끝에 위치한 말단 세포(terminal cell)가 거의 투명하거나 긴 끈 형태의 세포질을 가지고 있었다. 또한, 두 개의 균주는 98.4%의 유전적 유사도를 나타내어 동일종으로 판단되었고, 유전자 은행에서 선별한 Cluster I의 Aph. flos-aquae strains과도 계통수에서 66~82%의 bootstrap value의 지지도로 단일 cluster에 포함되었다. 확보된 두 개 균주의 유전자 정보는 유전자은행 NCBI에 등록되었으며, KY327795, KY327796의 Accession no.를 부여받았다. 한편, 세포독소 CYN의 생합성에 관여하는 유전자 cyrA와 cyrJ는 두 개 균주 모두에서 확인되지 않았다. STX의 생합성을 담당하는 유전자 중 sxtA 유전자는 두 개의 균주에서 확보되었으며, 독소생합성 과정의 분자생물학적 지표 역할을 하는 sxtI 유전자는 발견되지 않았다. 따라서 낙동강 현장시료에서 분리된 두 개의 균주는 형태학적 및 계통분류학적으로 동일종인 Aphanizomenon flos-aquae Ralfs ex Bornet et Flahault 1888로 동정되었으며, 두 개의 균주는 CYN과 STX의 잠재적인 독소 비생성 균주로 확인되었다. 이 결과를 통하여 Aph. flos-aquae가 독소 생성 분류군으로 분류되는 것에 대한 보다 면밀한 검토가 필요할 것으로 판단되었다.

The identity of toxin producers remains only hypothesis unless there were identified by strain isolation and analytical confirmation of both the cyanotoxin production and the genetic identity of the monoculture. The purposes of this study were to identify a morphologic and phylogenetic classification in Aphanizomenon flos-aquae strains isolated from the Nakdong River and to investigate the potential ability of the strains to produce toxins such as saxitoxin and cylindrospermopsin using target genes. The 16S rRNA and sxtA, sxtI, cyrA, cyrJ genes were analyzed on two strains (DGUC001, DGUC003) isolated from the Nakdong River. Morphological features of the strains were observed a shape of aggregated trichomes in parallel fascicles which can reach up to macroscopic size and a hyaline terminal cell without aerotope. In addition, the 16S rRNA phylogenetic analyses showed that the strains were identified as the same species with high genetic similarity of 98.4% and grouped within a monospecific andsupported cluster I of Aphanizomenon flos-aquae selected from GenBank of the NCBI. The cyrA and cyrJ genes encoding for the cylindrospermopsin-biosynthesis were not detected in the present study. The sxtA gene was in detected both the two strains, whereas the sxtI gene which had been suggested as a suitable molecular marker to detect saxitoxin-producing cyanobacteria was not found both the strains. Thus, the two strains isolated from Nakdong River were identified as the same species of Aphanizomenon flos-aquae Ralfs ex Bornet et Flahault 1888, the two strains were confirmed as potential non-producing strains of the saxitoxin and cylindrospermopsin.

키워드

참고문헌

  1. Alam, M., Y. Shimizu, M. Ikawa and J.J. Sasner. 1978. Reinvestigation of toxins from blue-green-alga, Aphanizomenon flos-aquae, by a high-performance chromatographic method. Journal of Environmental Science and Health, Part A. 13(7): 493-499. https://doi.org/10.1080/10934527809374828
  2. Ballot, A., J. Fastner, M. Lentz and C. Wiedner. 2010a. First report of anatoxin-a-producing cyanobacterium Aphanizomenon issatschenkoi in Northeastern Germany. Toxicon 56(6): 964-971. https://doi.org/10.1016/j.toxicon.2010.06.021
  3. Ballot, A., J. Fastner and C. Wiedner. 2010b. Paralytic shellfish poisoning toxin-producing cyanobacterium Aphanizomenon gracile in Northest Germany. Applied and Environmental Microbiology 76(4): 1173-1180. https://doi.org/10.1128/AEM.02285-09
  4. Brient, L., M. Lengronne, M. Bormans and J. Fastner. 2009. First occurrence of cylindrospermopsin in freshwater in France. Environmental Toxicology 24(4): 415-420. https://doi.org/10.1002/tox.20439
  5. Campo, E., R. Agha, S. Cires, A. Quesada and R. El-Shehawy. 2013. First TaqManassay to identify and quantify the cylindrospermopsin-producing cyanobacterium Aphanizomenon ovalisporum in water. Advances in Microbiology 3(5): 430-437. https://doi.org/10.4236/aim.2013.35058
  6. Carmichael, W.W., C. Drapeau, D.M. Anderson. 2000. Harvesting of Aphanizomenon flos-aquae Ralfsex Born & Flah. var. flos-aquae (cyanobacteria) from Klamath Lake for human dietary use. Journal of Applied Phycology 2(6): 585-595.
  7. Casero, M.C., A. Ballot, R. Agha, A. Quesada and S. Cires. 2014. Characterization of saxitoxin production and release and phylogeny of sxt genes in paralytic shellfish poisoning toxin-producion Aphanizomenon gracile. Harmful Algae 37: 28-37. https://doi.org/10.1016/j.hal.2014.05.006
  8. Cires, S. and A. Ballot. 2016. A review of the phylogeny, ecology and toxin production of bloom-forming Aphanizomenon spp. and related species within the Nostocales (Cyanobacteria). Harmful Algae 52: 21-43.
  9. Cires, S., L. Wormer, A. Ballot, R. Agha, C. Wiedner, D. Velazquez, M.C. Casero and A. Quesada. 2014. Phylogeography of cylindrospermopsin and paralytic shellfish toxin-producing Nostocales cyanobacteria from Mediterranean Europe (Spain). Applied and Environmental Microbiology 80: 1359-1370. https://doi.org/10.1128/AEM.03002-13
  10. Edwards, U.T.R., H. Blocker, M. Emde and E.C. Bottger. 1989. Isolation and direct complete nucleotide determination of entire genes. characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Research 17: 7843-7853. https://doi.org/10.1093/nar/17.19.7843
  11. Gentile, J.H. and T.E. Maloney. 1969. Toxicity and environmental requirements of a strain of Aphanizomenon flos-aquae (L.) Ralfs. Can. Journal of Microbiology 15(2): 165-173. https://doi.org/10.1139/m69-028
  12. Kim, M., H.S. Oh, S.C. Park and J. Chun. 2014. Toward a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. International Journal of Systematic and Evolutionary Microbiology 64(Pt 2): 346-351. https://doi.org/10.1099/ijs.0.059774-0
  13. KokoCinski, M., J. Mankiewicz-Boczek, T. Jurczak, L. Spoof, J. Meriluoto, E. Rejmonczyk, H. Hautala, M. Vehniainen, J. Pawelczyk and J. Soininen. 2013. Aphanizomenon gracile (Nostocales), a cylindrospermopsin-producing cyanobacterium in Polish lakes. Environmental Science and Pollution Research 20: 5243-5264. https://doi.org/10.1007/s11356-012-1426-7
  14. Komarek, J. 2013. Cyanoprokaryota 3.Teil/3rd part: Heterocytous Genera. pp. 1-1131. In: B. Bubel, G. Gärtner, L. Krienitz and M. Schagerl (eds.), SuBwasser flora von Mutteleuropa, 19/3. Springer Spektrum.
  15. Komarek, J. and J. Komarkova. 2006. Diversity of Aphanizomenon- like cyanobacteria. Czech Phycology, Olomouc 6: 1-32.
  16. Ledreux, A., S. Thomazeau, A. Catherine, C. Duval, C. Yepremian, A. Marie and C. Bernard. 2010. Evidence for saxitoxins production by the cyanobacterium Aphanizomenon gracile in a French recreational water body. Harmful Algae 10: 88-97. https://doi.org/10.1016/j.hal.2010.07.004
  17. Lepere, C., A. Wilmotte and B. Meyer. 2000. Molecular diversity of Microcystis strains (cyanophyceae, chroococcales) based on 16S rDNA sequences. Systematics and Geography of Plants 70: 275-283. https://doi.org/10.2307/3668646
  18. Li, R., W.W. Carmichael and P. Pereira. 2003. Morphological and 16S rRNA gene evidence for reclassification of the paralytic shellfish toxin producing Aphanizomenon flosaquae LMECYA31 as Aphanizomenon issatschenkoi (cyanophyceae). Journal of Phycology 39(4): 814-818. https://doi.org/10.1046/j.1529-8817.2003.02199.x
  19. Li, R., W.W. Carmichael, Y. Liu and M.W. Watanabe. 2000. Taxonomic re-evaluation of Aphanizomenon flos-aquae NH-5 based on morphology and 16S rRNA gene sequences. Hydrobiologia 438: 99-105. https://doi.org/10.1023/A:1004166029866
  20. Mahmood, N.A. and W.W. Carmichael. 1986. Paralytic shellfish poisons produced by the freshwater cyanobacterium Aphanizomenon flos-aquae NH-5. Toxicon 24(2): 175-186. https://doi.org/10.1016/0041-0101(86)90120-0
  21. Mazmouz, R., F. Chapuis-Hugon, S. Mann, V. Pichon, A. Mejean and O. Ploux. 2010. Biosynthesis of cylindrospermopsin and 7-epicylindrospermopsin in Oscillatoria sp. strain PCC 6506: Identification of the cyr gene cluster and toxin analysis. Applied and Environmental Microbiology 76(15): 4943-4949. https://doi.org/10.1128/AEM.00717-10
  22. McDonald, K.E. and J.T. Lehman. 2013. Dynamics of Aphanizomenon and Microcystis (cyanobacteria) during experimental manipulation of an urban impoundment. lake reservoir management 29(2): 272-276.
  23. Mihali, T.K., R. Kellmann and B.A. Neilan. 2009. Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5. BMC Biochemistry 10(1): 8. https://doi.org/10.1186/1471-2091-10-8
  24. Mihali, T.K., R. Kellmann, J. Muenchhoff, K.D. Barrow and B.A. Neilan. 2008. Characterization of the gene cluster responsible for cylindrospermopsin biosynthesis. Applied and Environmental Microbiology 74: 716-722. https://doi.org/10.1128/AEM.01988-07
  25. Moustafa, A., J.E. Loram, J.D. Hackett, D.M. Anderson, F.G. Plumley and D. Bhattacharya. 2009. Origin of saxitoxin biosynthetic genes in cyanobacteria. PLoS ONE 4(6): e5758. https://doi.org/10.1371/journal.pone.0005758
  26. Nubel, U., F. Garciapichel and G. Muyzer. 1997. PCR primers Ti amplify 16S rRNA genes from cyanobacteria. Applied and Environmental Microbiology 63(8): 3327-3332.
  27. Park, H.K., R.Y. Shin, H.J. Lee, K.L. Lee and S.U. Cheon. 2015. Spatio-temporal characteristics of cyanobacterial communities in the middle-downstream of Nakdong River and Lake Dukdong. Journal of Korean Society on Water Environment 31(3): 286-294. (in Korean) https://doi.org/10.15681/KSWE.2015.31.3.286
  28. Presel, K., G. Wessel, J. Fastner and I. Chorus. 2009. Response of cylindrospermopsin production and release in Aphanizomenon flos-aquae (cyanobacteria) to varying light and temperature conditions. Harmful Algae 8: 645-650. https://doi.org/10.1016/j.hal.2008.10.009
  29. Preussel, K., A. Stuken, C. Wiedner, I. Chorus and J. Fastner. 2006. First report on cylindrospermopsinproducing Aphanizomenon flos-aquae (Cyanobacteria) isolated from two German lakes. Toxicon 47(2): 156-162. https://doi.org/10.1016/j.toxicon.2005.10.013
  30. Preussel, K., I. Chorus and J. Fastner. 2014. Nitrogen limitation promotes accumulation and suppresses release of cylindrospermopsins in cells of Aphanizomenon sp. Toxins 6(10): 2932-2947. https://doi.org/10.3390/toxins6102932
  31. Rajaniemi, P., P. Hrouzek, K. Kaštovska, R. Willame, A. Rantala, L. Hoffmann, J. Komarek and K. Sivonen. 2005a. Phylogenetic and morphological evaluation of the genera Anabaena, Aphanizomenon, Trichormus and Nostoc (nostocales, cyanobacteria). International Journal of Systematic and Evolutionary Microbiology 55: 11-26. https://doi.org/10.1099/ijs.0.63276-0
  32. Rajaniemi, P., J. Komarek, R. Willame, P. Hrouzek, K. Kastovska, L. Hoffmann and K. Sivonen. 2005b. Taxonomic consequences from the combined molecular and phenotype evaluaion of selected Anabaena and Aphanizomenon strains. Algolocial Studies 117: 371-391. https://doi.org/10.1127/1864-1318/2005/0117-0371
  33. Rapala, J., K. Sivonen, R. Luukkainen and S.I. Niemela. 1993. Anatoxin-a concentration in Anabaena and Aphanizomenon under different environmental conditions and comparison of growth by toxic and non toxic Anabaena strains a laboratory study. Journal of Applied Phycology 5(6): 581-591. https://doi.org/10.1007/BF02184637
  34. Ryu, H.S., H.K. Park, H.J. Lee, R.Y. Shin and S.U. Cheon. 2016. Occurrence and succession pattern of cyanobacteria in the upper region of the Nakdong River: Factors influencing Aphanizomenon bloom. Journal of Korean Society on Water Environment 32(1): 52-59. (in Korean) https://doi.org/10.15681/KSWE.2016.32.1.52
  35. Sabour, B., M. Loudiki, B. Oudra, V. Vasconcelos, S. Oubraim and B. Fawzi. 2005. Dynamics and toxicity of Anabaena aphanizomenoides (cyanobacteria) water-blooms in the shallow Brackish OuedMellah lake (Morocco). Aquatic Ecosystem Health and Management 8(1): 95-104. https://doi.org/10.1080/14634980590914944
  36. Shirai, M., K. Matumaru, A. Ohotake, Y. Takamura, T. Aida and M. Nakano. 1989. Development of a solid medium for growth and isolation of axenic Microcystis strains (cyanobacteria). Applied and Envieonmental Microbiology 55(10): 2569-2571.
  37. Stuken, A. and K.S. Jakobsen. 2010. The cylindrospermopsingene cluster of Aphanizomenon sp. strain 10E6: Organization and recombination. Microbiology 156: 2438-2451. https://doi.org/10.1099/mic.0.036988-0
  38. Stuken, A., R.J. Campbell, A. Quesada, A. Sukenik, P.K. Dadheech and C. Wiedner. 2009. Genetic and morphologic characterization of four putative cylindrospermopsin producing species of the cyanobacterial genera Anabaena and Aphanizomenon. Journal of Plankton Research 31(5): 465-480. https://doi.org/10.1093/plankt/fbp011
  39. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei and S. Kumar. 2011. MEGA 5: Molecular evolution genetics analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution 28(10): 2731-2739. https://doi.org/10.1093/molbev/msr121
  40. Wilmotte, A., C. Demonceae, A. Goffart, J.H. Hecq, V. Demoilin and A.C. Crossley. 2002. Molecular and pigment studies of the picophytoplankton in a region of the Southern Ocean (42-54 degrees S. 141-144 degrees E) in March 1998. Deep-Sea Research Part II: Topical Studies in Oceanography 49(16): 3351-3363. https://doi.org/10.1016/S0967-0645(02)00087-5
  41. Yu, J.J., H.J. Lee, K.L. Lee, H.S. Lyu, J.H. Hwang, L.Y. Shin and S.U. Chen. 2014. Relations between distribution of the dominant phytoplankton species and water temperature in the Nakdong River, Korea. Korean Journal of Ecology and Environment 47(4): 247-257. (in Korean) https://doi.org/10.11614/KSL.2014.47.4.247
  42. Zhang, D., S.Y. Liu, J. Zhang, C.X. Hu, D.H. Li and Y.D. Liu. 2015. Antioxidative reponses in zebrafish liver exposed to sublethal doses Aphanizomenon flos-aquae DC-1 aphantoxins. Ecotoxicology and Environmental Safety 113: 425-432. https://doi.org/10.1016/j.ecoenv.2014.12.029