Divergence Analysis of 16S rRNA and rpoB Gene Sequences Revealed from the Harmful Cyanobacterium Microcystis aeruginosa

유해 남조세균 Microcystis aeruginosa의 16S rRNA 및 rpoB 유전자 염기서열 변이 분석

  • Ki, Jang-Seu (Department of Green Life Science, Sangmyung University)
  • 기장서 (상명대학교 그린생명과학과)
  • Received : 2010.07.28
  • Accepted : 2010.09.14
  • Published : 2010.09.30

Abstract

Microcystis (Cyanobacteria, Chroococcales) is one of the green tide-causing organisms in freshwaters, and some species produce microcystin that is hepatotoxin. In the aspects of freshwater quality controls and health concerns, therefore it is necessary to manage the harmful organisms. In the present study, RNA polymerase beta subunit (rpoB) gene sequences of Microcystis were determined and characterized in order to use a potential marker for the molecular detections of the species. Microcystis rpoB showed high divergences of DNA similarity and genetic distances when compared with those of 16S rRNA, and the molecular differences were statistically significant (Student t-test, p<0.05). Parsimony analyses showed the rpoB gene evolves more than 2-fold faster than 16S rRNA. In addition, phylogeny of the rpoB gene separated each M. aeruginosa strain more clearly compared with a 16S rRNA tree. This study found that the order Chroococcales, including Microcystis, has approximately two rRNA operons and single copy of the rpoB gene in their chromosomes. These results suggest that the rpoB gene is a useful marker for the molecular phylogenetics and the detection of Microcystis.

남조세균 Microcystis (Cyanobacteria, Chroococcales)는 담수 녹조원인 생물의 하나로써 일부 종은 microcystin이라는 간 독소를 분비한다. 따라서 담수 수질관리 및 보건위생 측면에서 이들에 대한 관리가 필요하다. 본 연구는 Microcystis 분자 검출을 위한 신규 마커로 RNA polymerase beta subunit (rpoB) 유전자 염기서열을 분석하여 이들의 분자적 특성을 규명하였다. Microcystis rpoB 유전자는 16S rRNA보다 염기 유사도와 유전거리에서 큰 변이가 있는 것으로 조사되었으며, 통계적으로 유의한 차이를 보였다(Student t-test, p<0.05). Parsimony 분석을 통해 rpoB 유전자가 16S rRNA 유전자보다 2배 이상 빠르게 진화하는 것으로 파악되었다. 또한 rpoB 유전자 phylogeny 분석에서 16S rRNA tree 보다 M. aeruginosa 균주를 명확하게 구분해 주었다. Microcystis가 속하는 Chroococcales 목은 염색체 안에 2개 정도의 rRNA 오페론이 있고 rpoB 유전자는 1개 있는 것으로 조사되었다. 본 연구결과는 rpoB 유전자가 Microcystis의 분자계통분류 및 분자검출 마커로 유용하다는 것을 제시해 준다.

Keywords

References

  1. Al-Thukair, A.A., R.M. Abed, and L. Mohamed. 2007. Microbial community of cyanobacteria mats in the intertidal zone of oil-polluted coast of Saudi Arabia. Mar. Pollut. Bull. 54, 173-179. https://doi.org/10.1016/j.marpolbul.2006.08.043
  2. Bartram, J. and R. Ballance. 1996. Water quality monitoring, a practical guide to the design and implementation of freshwater quality studies and monitoring programmes. E & FN SPON, London.
  3. Castiglioni, B., E. Rizzi, A. Frosini, K. Sivonen, P. Rajaniemi, A. Rantala, M.A. Mugnai, and et al. 2004. Development of a universal microarray based on the ligation detection reaction and 16S rrna gene polymorphism to target diversity of cyanobacteria. Appl. Environ. Microbiol. 70, 7161-7172. https://doi.org/10.1128/AEM.70.12.7161-7172.2004
  4. Cho, H.M. 2005. Emergency measures on algal problems in water resource of the Han River. Seoul Development Institute. Seoul.
  5. Cho, J.E., S.W. Bang, and M.S. Han. 2004. Development of oligonucleotide primers for the detection of harmful Microcystis in water. Bull. Environ. Contam. Toxicol. 72, 655-662.
  6. Dahllof, I., H. Baillie, and S. Kjelleberg. 2000. rpoB-based microbial community analysis avoids limitations inherent in 16S rRNA gene intraspecies heterogeneity. Appl. Environ. Microbiol. 66, 3376-3380. https://doi.org/10.1128/AEM.66.8.3376-3380.2000
  7. Doers, M.P. and D.L. Parker. 1988. Properties of Microcystis aeruginosa and M. flosaquae (Cyanophyta) in culture: taxonomic implications. J. Phycol. 24, 502-508.
  8. Fegatella, F., J. Lim, S. Kjelleberg, and R. Cavicchioli. 1998. Implications of rRNA operon copy number and ribosome content in the marine oligotrophic ultramicrobacterium Sphingomonas sp. strain RB2256. Appl. Environ. Microbiol. 64, 4433-4438.
  9. Fischer, W.J., B.C. Hitzfeld, F. Tencalla, J.E. Eriksson, A. Mikhailov, and D.R. Dietrich. 2000. Microcystin-LR toxicodynamics, induced pathology, and immunohistochemical localization in livers of blue-green algae exposed rainbow trout (Oncorhynchus mykiss). Toxicol. Sci. 54, 365-373. https://doi.org/10.1093/toxsci/54.2.365
  10. Frangeul, L., P. Quillardet, A.M. Castets, H.F. Humbert, H.C. Matthijs, D. Cortez, A. Tolonen, and et al. 2008. Highly plastic genome of Microcystis aeruginosa PCC 7806, a ubiquitous toxic freshwater cyanobacterium. BMC Genomics 9, 274. https://doi.org/10.1186/1471-2164-9-274
  11. Gilroy, D.J., K.W. Kauffman, R.A. Hall, X. Huang, and F.S. Chu. 2000. Assessing potential health risks from microcystin toxins in blue-green algae dietary supplements. Environ. Health Perspect. 108, 435-439. https://doi.org/10.1289/ehp.00108435
  12. Ha, J.H., T. Hidaka, and H. Tsunos. 2009. Quantification of toxic Microcystis and evaluation of its dominance ratio in blooms using real-time PCR. Environ. Sci. Technol. 43, 812-818. https://doi.org/10.1021/es801265f
  13. Harmsen, D. and H. Karch. 2004. 16S rDNA for diagnosing pathogens: a living tree. American Soc. for Microbiol. News 70, 19-24.
  14. Henriksen, P. 1996. Microcystin profiles and contents in Danish populations of cyanobacteria/ blue-green algae as determined by HPLC. Phycologia 35, 102-110. https://doi.org/10.2216/i0031-8884-35-6S-102.1
  15. Jaiswal, P., P.K. Singh, and R. Prasanna. 2008. Cyanobacterial bioactive molecules-an overview of their toxic properties. Can. J. Microbiol. 54, 701-717. https://doi.org/10.1139/W08-034
  16. Jang, M.H., J.M. Jung, J.D. Yoon, Y.J. Lee, and K. Ha. 2007. Changes in microcystin production in Microcystis aeruginosa exposed to different concentrations of filtered water from phytoplanktivorous and omnivorous fish. Korean J. Limnol. 40, 294-302.
  17. Kaneko, T., N. Nakajima, S. Okamoto, I. Suzuki, Y. Tanabe, M. Tamaoki, Y. Nakamura, and et al. 2007. Complete genomic structure of the bloom-forming toxic cyanobacterium Microcystis aeruginosa NIES-843. DNA Res. 14, 247-256. https://doi.org/10.1093/dnares/dsm026
  18. Ki, J.S., W. Zhang, and P.Y. Qian. 2009. Discovery of marine Bacillus species by 16S rRNA and rpoB comparisons and their usefulness for species identification. J. Microbiol. Methods 77, 48-57. https://doi.org/10.1016/j.mimet.2009.01.003
  19. Ki, J.S., R. Zhang, W. Zhang, Y.L. Huang, and P.Y. Qian. 2009. Analysis of RNA polymerase beta subunit (rpoB) gene sequences for the discriminative power of marine Vibrio species. Microb. Ecol. 58, 679-691. https://doi.org/10.1007/s00248-009-9519-7
  20. Ki, J.S. 2009. Heterogeneity analysis of the 16S rRNA gene sequences of the genus Vibrio. Korean J. Microbiol. 45, 430-434.
  21. Kim, B.C., E.K. Kim, D.J. Pyo, H.D. Park, and W.M. Heo. 1995. Toxic cyanobacterial blooms in Korean lakes. J. KSWQ Sep. 11, 231-237.
  22. Kim, W.H., A.R. Choi, and J.A. Lee. 1999. The occurrence of microcystins in the Naktong River. Algae 14, 319-322.
  23. Komarek, J. 1991. A review of water-bloom forming Microcystis species, with regard to population from Japan. Arch. Hydrobiol. Suppl. 92, 115-127.
  24. Lee, H.K., J.H. Kim, S.A. Yoo, T.S. Ahn, C.K. Kim, and D.H. Lee. 2003. Primer evaluation for the detection of toxigenic Microcystis by PCR. Korean J. Microbiol. 39, 166-174.
  25. Lee, J.W., H.W. Yu, and I.S. Kim. 2004. Application of quantum-dot nanocrystals for cyanobacterial toxin-microcystin detection. J. Kor. Soc. Water Qual. 23, 705-711.
  26. Lee, K.L., W.H. Jheong, J.M. Kim, Y.S. Kim, H.J. Choi, and H.S. Kim. 2008. Seasonal variations of cyanobacterial toxins (mirocystins) in Yeongchun Reservoir. Korean J. Limnol. 41, 264-274.
  27. Lyra, C., S. Suomalainen, M. Gugger, C. Vezie, P. Sundman, L. Paulin, and K. Sivonen. 2001. Molecular characterization of planktic cyanobacteria of Anabaena, Aphanizomenon, Microcystis and Planktothrix genera. Int. J. Syst. Evol. Microbiol. 51, 513-526. https://doi.org/10.1099/00207713-51-2-513
  28. Ministry of Environment. 2000. Environmental white paper, Seoul.
  29. Nakamura, Y., T. Kaneko, S. Sato, M. Ikeuchi, H. Katoh, S. Sasamoto, A. Watanabe, and et al. 2002. Complete genome structure of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. DNA Res. 9, 123-130. https://doi.org/10.1093/dnares/9.4.123
  30. Neilan, B.A., D. Jacobs, and A.E. Goodman. 1995. Genetic diversity and phylogeny of toxic cyanobacteria determined by DNA polymorphisms within the phycocyanin locus. Appl. Environ. Microbiol. 61, 3875-3883.
  31. Neilan, B.A., D. Jacobs, T. Del Dot, L.L. Blackall, P.R. Hawkins, P.T. Cox, and A.E. Goodman. 1997. rRNA sequences and evolutionary relationships among toxic and nontoxic cyanobacteria of the genus Microcystis. Int. J. Syst. Bacteriol. 47, 693-697. https://doi.org/10.1099/00207713-47-3-693
  32. Nishizawa, T., M. Asayama, K. Fujii, K. Harada, and M. Shirai. 1999. Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp. J. Biochem. 126, 520-526. https://doi.org/10.1093/oxfordjournals.jbchem.a022481
  33. Otsuka, S., S. Suda, S. Shibata, H. Oyaizu, S. Matsumoto, and M.M. Watanabe. 2001. A proposal for the unification of five species of the cyanobacterial genus Microcystis Kutzing ex Lemmermann 1907 under the rules of the Bacteriological Code. Int. J. Syst. Evol. Microbiol. 51, 873-879. https://doi.org/10.1099/00207713-51-3-873
  34. Richert, K., E. Brambilla, and Stackebrandt. 2007. The phylogenetic significance of peptidoglycan types: Molecular analysis of the genera Microbacterium and Aureobacterium based upon sequence comparison of gyrB, rpoB, recA and ppk and 16S rRNA genes. Syst. Appl. Microbiol. 30, 102-108. https://doi.org/10.1016/j.syapm.2006.04.001
  35. Rudi, K., O.M. Skulberg, F. Larsen, and K.S. Jakobsen. 1997. Strain characterization and classification of oxyphotobacteria in clone cultures on the basis of 16S rRNA sequences from the variable regions V6, V7, and V8. Appl. Environ. Microbiol. 63, 2593-2599.
  36. Stanier, R.Y., R. Kunisawa, M. Mandel, and G. Cohen-Bazire. 1971. Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol. Rev. 35, 171-205.
  37. Suh, M.Y., B.H. Kim, and M.S. Han. 2005. Distribution of cyanotoxin microcystin-LR in Han River system and ecological park in Seoul and Kyunggi districts. Korean J. Limnol. 38, 237-248.
  38. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596-1599. https://doi.org/10.1093/molbev/msm092
  39. Thompson, J.D., D.G. Higgins, and T.J. Gibbson. 1997. Clustal X: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673.
  40. Tillett, D., D.L. Parker, and B.A. Neilan. 2001. Detection of toxigenicity by a probe for the microcystin synthetase A gene (mcyA) of the cyanobacteiral genus Microcystis: comparison of toxicities with 16S rRNA and phycocyanin operon (phycocyanin intergenic spacer) phylogenies. Appl. Environ. Microbiol. 67, 2810-2818. https://doi.org/10.1128/AEM.67.6.2810-2818.2001
  41. Watanabae, M.F. and S. Oishi. 1985. Effects of environmental factors on toxicity of a cyanobacterium (Microcystis aeruginosa) under culture conditions. Appl. Environ. Microbiol. 49, 1342-1344.
  42. Welsh, E.A., M. Liberton, J. Stockel, T. Loh, T. Elvitigala, C. Wang, A. Wollam, and et al. 2008. The genome of Cyanothece 51142, a unicellular diazotrophic cyanobacterium important in the marine nitrogen cycle. Proc. Natl. Acad. Sci. USA 105, 15094-15099. https://doi.org/10.1073/pnas.0805418105