Phylogenetic Analysis of Frequent Recombinant Region in Micropeptin Biosynthetic Gene Cluster among the Genus <i>Microcystis</i>

The gene cluster for the biosynthetic of a nonribosomal peptide, cyanopeptolins and micropeptin (MCN), was identified in Microcystis strains and halogenated MCN-producing Microcystis were found to possess the halogenase gene, mcnD, between nonribosomal peptide synthetase genes, mcnC and mcnE. A comparative sequence analysis of the mcn gene cluster between halogenated and non-halogenated MCN-producing strains revealed mosaic sequence traces from mcnD in the non-coding region between mcnC and mcnE in the latter strains. A phylogenetic analysis based on a 170-bp non-coding region including the mcnD traces suggests that the recombination events occurred in a particular region of the Microcystis’ mcn gene. This study provides novel insight into the ecological patterning of widespread Microcystis species.

• Cyanobacteria
• <i>Microcystis</i>
• Micropeptin Biosynthetic Gene
• Phylogenetic Analysis

Acknowledgements

We thank Dr. Takashi Kitano (Ibaraki University College of Engineering) for critical comments about evolutionary divergence and valuable discussions. We thank Dr. Akito Nishizawa of the laboratory of M.S. for technical assistance.

References

1. Mur, L.R., Skulberg, O.M. and Utkilen, H. (1999) Cyanobacateria in the Environment, p. 25-54. In: Chorus, I. and Bartram, J., Eds., Toxic Cyanobacteria in Water, WHO, E and FN Spon, London.

2. van Gremberghe, I., Leliaert, F., Mergeay, J., Vanormelingen, P., van der Gucht, K., Debeer, A.-E., Lacerot, G., de Meester, L. and Vyverman, W. (2011) Lack of Phylogeographic Structure in the Freshwater Cyanobacterium Microcystis aeruginosa Suggests Global Dispersal. PLoS One, 6, e19561. http://dx.doi.org/10.1371/journal.pone.0019561

3. Cohan, F.M. (2006) Toward a Conceptual and Operation Union of Bacterial Systematics, Ecology, and Evolution. Philosophical Transactions of the Royal Society B, 361, 1985-1996. http://dx.doi.org/10.1098/rstb.2006.1918

4. Ward, D.M., Cohan, F.M., Bhaya, D., Heidelberg, J.F., Kühl, M. and Grossman, A. (2008) Genomics, Environmental Genomics and the Issue of Microbial Species. Heredity, 100, 207-219. http://dx.doi.org/10.1038/sj.hdy.6801011

5. Fraser, C., Alm, E.J., Polz, M.F., Spratt, B.G. and Hanage, W.P. (2009) The Bacterial Species Challenge: Making Sense of Genetic and Ecological Diversity. Science, 323, 741-746. http://dx.doi.org/10.1126/science.1159388

6. Kaneko, T., Nakajima, N., Okamoto, S., Suzuki, I., Tanabe, Y., Tamaoki, M., Nakamura, Y., Kasai, F., Watanabe, A., Kawashima, K., Kishida, Y., Ono, A., Shimizu, Y., Takahashi, C., Minami, C., Fujishiro, T., Kohara, M., Katoh, M., Nakazaki, N., Nakayama, S., Yamada, M., Tabata, S. and Watanabe, M.M. (2007) Complete Genomic Structure of the Bloom-Forming Toxic Cyanobacterium Microcystis aeruginosa NIES-843. DNA Research, 14, 247-256. http://dx.doi.org/10.1093/dnares/dsm026

7. Frangeul, L., Quillardet, P., Castets, A.M., Humbert, J.F., Matthijs, H.C., Cortez, D., Tolonen, A., Zhang, C.C., Gribaldo, S., Kehr, J.C., Zilliges, Y., Ziemert, N., Becker, S., Talla, E., Latifi, A., Billault, A., Lepelletier, A., Dittmann, E., Bouchier, C. and Tandeau de Marsac, N. (2008) Highly Plastic Genome of Microcystis aeruginosa PCC 7806, a Ubiquitous Toxic Freshwater Cyanobacterium. BMC Genomics, 9, 274. http://dx.doi.org/10.1186/1471-2164-9-274

8. Nishizawa, T., Asayama, M., Fujii, K., Harada, K.-I. and Shirai, M. (1999) Genetic Analysis of the Peptide Synthetase Genes for a Cyclic Heptapeptide Microcystin in Microcystis spp. Journal of Biochemistry, 126, 520-529. http://dx.doi.org/10.1093/oxfordjournals.jbchem.a022481

9. Nishizawa, T., Ueda, A., Asayama, M., Fujii, K., Harada, K.-I., Ochi, K. and Shirai, M. (2000) Polyketide Synthase Gene Coupled to the Peptide Synthetase Module Involved in the Biosynthesis of the Cyclic Heptapeptide Microcystin. Journal of Biochemistry, 127, 779-789. http://dx.doi.org/10.1093/oxfordjournals.jbchem.a022670

10. Nishizawa, T., Nishizawa, A., Asayama, M., Harada, K.-I. and Shirai, M.(2007) Diversity within the Microcystin Biosynthetic Gene Clusters among the Genus Microcystis. Microbes and Environments, 22, 380-390. http://dx.doi.org/10.1264/jsme2.22.380

11. Noguchi, T., Shinohara, A., Nishizawa, A., Asayama, M., Nakano, T., Hasegawa, M., Harada, K.-I., Nishizawa, T. and Shirai, M. (2009) Genetic Analysis of the Microcystin Biosynthesis Gene Cluster in Microcystis Strains from Four Bodies of Eutrophic Water in Japan. Journal of General and Applied Microbiology, 55, 111-123.http://dx.doi.org/10.2323/jgam.55.111

12. Tooming-Klunderud, A., Mikalsen, B., Kristensen, T. and Jakobsen, K.S. (2008) The Mosaic Structure of the mcyABC Operon in Microcystis. Microbiology, 154, 1886-1899. http://dx.doi.org/10.1099/mic.0.2007/015875-0

13. Nishizawa, T., Ueda, A., Nakano, T., Nishizawa, A., Miura, T., Asayama, M., Fujii, K., Harada, K.-I. and Shirai, M. (2011) Characterization of the Locus of Genes Encoding Enzymes Producing Heptadepsipeptide Micropeptin in the Unicellular Cyanobacterium Microcystis. Journal of Biochemistry, 149, 475-485. http://dx.doi.org/10.1093/jb/mvq150

14. Cadel-Six, S., Dauga, C., Castets, A.M., Rippka, R., Bouchier, C., Tandeau de Marsac, N. and Welker, M. (2008) Halogenase Genes in Nonribosomal Peptide Synthetase Gene Clusters of Microcystis (Cyanobacteria): Sporadic Distribution and Evolution. Molecular Biology and Evolution, 25, 2031-2041. http://dx.doi.org/10.1093/molbev/msn150

15. Matsui, M., Yoshimura, T., Wakabayashi, Y., Imamura, S., Tanaka, K., Takahashi, H., Asayama, M. and Shrai, M. (2007) Interference Expression at Levels of the Transcript and Protein among Group 1, 2, and 3 Sigma Factor Genes in a Cyanobacterium. Microbes and Environments, 22, 32-43. http://dx.doi.org/10.1264/jsme2.22.32

16. Harada, K.-I., Nakano, T., Fujii, K. and Shirai, M. (2004) Comprehensive Analysis System Using Liquid Chromatography-Mass Spectrometry for the Biosynthetic Study of Peptides Produced by Cyanobacteria. Journal of Chromatography A, 1033, 107-113. http://dx.doi.org/10.1016/j.chroma.2004.01.006

17. Ishida, K., Matsuda, H., Murakami, M. and Yamaguchi, K. (1996) The Absolute Sterochemistry of Micropeptin 90. Tetrahedron Letters, 37, 9225-9226. http://dx.doi.org/10.1016/S0040-4039(96)02189-2

18. von Elert, E., Oberer, L., Merkel, P., Huhn, T. and Blom, J.F. (2005) Cyanopeptolin 954, a Chlorine-Containing Chymotrypsin Inhibitor of Microcystis aeruginosa NIVA Cya 43. Journal of Natural Products, 68, 1324-1327.http://dx.doi.org/10.1021/np050079r

19. Martin, C., Oberer, L., Ino, T., K&ouml;nig, W.A., Busch, M. and Weckesser, J. (1993) Cyanopeptolins, New Depsipeptides from the Cyanobacterium Microcystis sp. PCC 7806. Journal of Antibiotics, 46, 1550-1556.

20. Bister, B., Keller, S., Baumann, H.I., Nicholson, G., Weist, S., Jung, G., Süssmuth, R.D. and Jüttner, F. (2004) Cyanopeptolin 963A, a Chymotrypsin Inhibitor of Microcystis PCC 7806. Journal of Natural Products, 67, 1755-1757.http://dx.doi.org/10.1021/np049828f

21. Harada, K.-I., Mayumi, T., Shimada, T., Suzuki, M., Kondo, F. and Watanabe, M.F. (1993) Occurrence of Our Depsipeptides, Aeruginopeptins, Together with Microcystins from Toxic Cyanobacteria. Tetrahedron Letters, 34, 6091-6094. http://dx.doi.org/10.1016/S0040-4039(00)61736-7

22. Welker, M., Brunke, M., Preussel, K., Lippert, I. and von D&ouml;hren, H. (2004) Diversity and Distribution of Microcystis (Cyanobacteria) Oligopeptide Chemotypes from Natural Communities Studied by Single-Colony Mass Spectrometry. Microbiology, 150, 1785-1796. http://dx.doi.org/10.1099/mic.0.26947-0

23. Weckesser, J., Martin, C. and Jakobi, C. (1996) Cyanopeptolins, Depsipeptides from Cyanobacteria. Systematic and Applied Microbiology, 19, 133-138. http://dx.doi.org/10.1016/S0723-2020(96)80038-5

24. Murakami, M., Kodani, S., Ishida, K., Matsuda, H. and Yamaguchi, K. (1997) Micropeptin 103, a Chymotrypsin Inhibitor from the Cyanobacterium Microcystis viridis (NIES-103). Tetrahedron Letters, 38, 3035-3038. http://dx.doi.org/10.1016/S0040-4039(97)00528-5

25. Tooming-Klunderud, A., Rohrlack, T., Shalchian-Tabrizi, K., Kristensen, T. and Jakobsen, K.S. (2007) Structural Analysis of a Non-Ribosomal Halogenated Cyclic Peptide and Its Putative Operon form Microcystis: Implications for Evolution of Cyanopeptolins. Microbiology, 153, 1382-1393. http://dx.doi.org/10.1099/mic.0.2006/001123-0

26. Rouhiainen, L., Vakkilainen, T., Siemer, S., Buikema, W., Haselkorn, R. and Sivonen, K. (2000) Genes Encoding Synthetases of Cyclic Depsipeptides, Anabaenopeptilides, in Anabaena Strain 90. Molecular Microbiology, 37, 156-167. http://dx.doi.org/10.1046/j.1365-2958.2000.01982.x

27. Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment through Sequence Weighting, Position Specific Gap Penalties and Weight Matrix Choice. Nucleic Acids Research, 22, 4673-4680. http://dx.doi.org/10.1093/nar/22.22.4673

28. Tamura, K., Nei, M. and Kumar, S. (2004) Prospects for Inferring Very Large Phylogenies by Using the Neighbor-Joining Method. Proceedings of the National Academy of Sciences of the United States of America, 101, 11030-11035.http://dx.doi.org/10.1073/pnas.0404206101

29. Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0. Molecular Biology and Evolution, 24, 1596-1599. http://dx.doi.org/10.1093/molbev/msm092

30. Huson, D.H. and Bryant, D. (2006) Application of Phylogenetic Networks in Evolutionary Studies. Molecular Biology and Evolution, 23, 254-267. http://dx.doi.org/10.1093/molbev/msj030