Characterization of cyanobacteria isolated from the Portuguese coast: diversity and potential to produce D bioactive compounds Ângela Marisa Oliveira Brito Programa Doutoral em Biologia de Plantas Departamento de Biologia 2015 Orientador Paula Tamagnini, Professora Associada Faculdade de Ciências, Universidade do Porto Coorientador Vitor M. Vasconcelos, Professor Catedrático Faculdade de Ciências, Universidade do Porto Coorientador Marta Vaz Mendes, Investigadora Auxiliar IBMC - Instituto de Biologia Molecular e Celular Aos meus pais One should not pursue goals that are easily achieved. One must develop an instinct for what one can just barely achieve through one’s greatest efforts. Albert Einstein FCUP vii Characterization of cyanobacteria isolated from the Portuguese coast Acknowledgments The work presented here is the result of a close collaboration of several people (involved directly or indirectly) to whom I would like to express my gratitude. First, I want to acknowledge my supervisor, Prof. Paula Tamagnini, for this opportunity and for the extraordinary support, guidance and patience (especially during the thesis writing) always expressed over all these years (not only during the PhD). Thank you for sharing your knowledge, for always keeping your door open and for the encouragement throughout this work (not always an easy path). I would like to acknowledge my co-supervisor Marta Vaz Mendes, for all the help, support, and valuable teachings. Thank you for spending time to teaching and helping me with the “PKS and NRPS world”. I would also like to thank my co-supervisor Prof. Vitor Vasconcelos for the opportunity. Thank you for the valuable advices, support and availability always expressed. I want to express my gratitude to Professor William H. Gerwick at the Scripps Institution of Oceanography, University of California San Diego, for giving me the opportunity to spend some time in his lab. It was a pleasure to work and learn so much on cyanobacteria secondary metabolites. Thank you so much for welcoming me in your lab and for showing me the wonderful world of marine natural products. A huge thank you to Vitor Ramos for the precious help and advices that were crucial for the achievement of the results presented in this thesis. Thank you for your support and friendship. I am particularly grateful to all co-authors that contributed to this work. Without you this work would not be possible. Prof. Arlete Santos, as well as Rui Fernandes and Francisco Figueiredo (Histology and Electron Microscopy facility, IBMC), for all the help with the transmission electron microscopy. I would like to thank Cristina Vieira and Jorge Vieira for assistance with the phylogenetic analysis. To all present and former members of the Bioengineering and Synthetic Microbiology (BSM) Group, a big thank you for the wonderful work environment, the viii FCUP Characterization of cyanobacteria isolated from the Portuguese coast sharing of ideas, help, support, friendship, tips and laughs. Sharing my lab time with you all was a fun and constructive experience. Special thanks to Rita Mota. Thanks for all the help (not only in the work), patience, and for constantly showing me the things in a different way. Thanks for your friendship, and for being with me in all moments (good and bad ones). Everything would be harder without you! To Sara Pereira. Thank you for your help, support and friendship. Thanks for being always there. I will never forget that my “first steps” in the lab were with you! Thanks for everything! To Meritxell Notari. Thanks for your good mood and help either in the lab and at home. I think that you deserve the award for “best housemate”, because I know that it was not easy to share almost 24h per day with me. Thanks for your patience, friendship and all good moments shared. To Filipe Pinto. Thank you for your support and for being always available to help me (and anyone who asks). Thank you for helping me with images, computer problems and I could continue... (quem tem um Filipe tem tudo!). To Catarina Pacheco (thanks for the help and support over all these years), Paulo Oliveira (thanks for constructive discussions about the work and for your help with the GC), Steeve Lima, Eunice Ferreira, Carlos Flores, Marina Santos and José Pereira for the good work environment, the sharing of ideas and help. Thank you all. To the present and former members of the MCA and RCS groups. Thank you for your support and friendship. To all members of the Gerwick Laboratory, especially Evgenia Glukhov, Ozlem Demirkiran, Karin Kleigrewe and Enora Briand. Thank you for your precious help. To all members of LEGE, CIIMAR. Thank you for all the help, support and advices. To all my friends, especially Juliana Oliveira (thank you for being by my side and for your support that encouraged me to never look back) and all members of the “team”: Rita Mota, Meritxell Notari, Sara Pereira, Filipe Pinto, Ricardo Celestino (thanks for your help with the thesis, thank you for everything…except the spiders!), Joana Gaifem, Sérgio Ferreira, Nádia Gonçalves and Nelson Ferreira. Thank you for all the good moments spent together, dinners, parties, laughs. Thank you for being always there (in the good and bad times). Special thanks to Goody and Brian. Thank you for welcoming me in your house during my stay in San Diego, for your care and for all the good moments. You made everything easier; all the words won’t be enough to thank you. I would also like to acknowledge the Fundação para a Ciência e Tecnologia (FCT) for the PhD. Grant SFRH/BD/70284/2010. FCUP ix Characterization of cyanobacteria isolated from the Portuguese coast And for last, but for sure not least… Quero agradecer à minha família, em especial aos meus pais (os meus pilares), pois sem eles nada disto teria sido possível. Não há palavras que descrevam tudo o que são para mim. Muito obrigada por estarem sempre comigo e apoiarem sempre as minhas decisões (mesmo por vezes não estarem totalmente de acordo). Obrigada pelo amor, compreensão, dedicação e pelos valores transmitidos. À minha irmã Sónia (desde sempre a minha melhor amiga) e ao meu cunhado Filipe. Obrigada pelo vosso apoio incondicional, por toda ajuda e por estarem sempre comigo. Vocês foram e são fundamentais para aquilo que sou hoje. Finalmente, como não poderia deixar de ser, aos meus sobrinhos Tiago Filipe e Ana Carolina (que nasceu mesmo na fase final de escrita da tese) que são sem dúvida o melhor do Mundo, são o meu orgulho. x FCUP Characterization of cyanobacteria isolated from the Portuguese coast This work was funded by FEDER funds through the Operational Competitiveness Programme - COMPETE and by national funds through FCT - Fundação para a Ciência e a Tecnologia under the projects FCOMP-01-0124-FEDER-010600 (PTDC/MAR/102258/2008), FCOMP-01-0124-FEDER-010568 (PTDC/MAR/099642/2008), FCOMP-01-0124-FEDER- 028314 (PTDC/BIA-MIC/2889/2012) and the PhD grant SFRH/BD/70284/2010 (POPH-QREN). FCUP xi Characterization of cyanobacteria isolated from the Portuguese coast List of publications (Ao abrigo do disposto no número 2 do Artigo 8º do decreto-Lei nº 388/70) This dissertation is based on the following publications and some unpublished results: I - Brito Â., Ramos V., Seabra R., Santos A., Santos C.L., Lopo M., Ferreira S., Martins A., Mota R., Frazão B., Martins R., Vasconcelos V., Tamagnini P. 2012. Culture-dependent characterization of cyanobacterial diversity in the intertidal zones of the Portuguese coast: a polyphasic study. Systematic and Applied Microbiology 35: 110-119. II - Brito Â., Mota R., Ramos V., Lima S., Santos A., Vieira J., Vieira C.P., Vasconcelos V.M., Tamagnini P. 2015. Isolation of cyanobacterial strains from the Portuguese Atlantic coast disclosures novel diversity. (Manuscript). III - Brito Â., Gaifem J., Ramos V., Glukhov E., Dorrestein P.C., Gerwick W.H., Vasconcelos V.M., Mendes M.V., Tamagnini P. 2015. Bioprospecting Atlantic cyanobacteria for bioactive secondary metabolites reveals untapped chemodiversity. Algal Research 9: 218-226. Other publications: Pinho L.X., Azevedo J., Brito Â., Santos A., Tamagnini P., Vilar V.J.P., Vasconcelos V. M., Boaventura R.A.R. 2015. Effect of TiO2 photocatalysis on the destruction of Microcystis aeruginosa cells and degradation of cyanotoxins microcystin-LR and cylindrospermopsin. Chemical Engineering Journal 268: 144-152. Publications (Chapter II and IV) are reproduced with the permission from the copyright holders. FCUP xiii Characterization of cyanobacteria isolated from the Portuguese coast Abstract Cyanobacteria are photosynthetic prokaryotes with a wide geographical distribution and are present in a broad spectrum of environments, including extreme ones. They play an important role in the global carbon cycle as primary producers and some strains are able to fix atmospheric nitrogen, characteristic that confers them an advantage in nutrient-poor and nitrogen-depleted environments like the marine ones. Cyanobacteria are also recognized as a prolific source of secondary metabolites with biological activities. Some of these compounds are toxic to a wide array of organisms, including humans, but others exhibit promising activities with interest in biotechnological applications, namely in the pharmacological, cosmetic and agricultural fields. Despite the ecological importance of cyanobacteria and their biotechnological potential, little is known about the diversity of these organisms on the Portuguese continental coast. In this work, the diversity of cyanobacteria on the intertidal zones of the Portuguese coast was assessed. For this purpose nine beaches were sampled and 60 of the retrieved isolates were characterized using a polyphasic approach. Based on morphology, 35 morphotypes were identified being the most representative group the non-heterocystous forms, with a predominance of the filamentous Oscillatoriales/Subsection III. The 16S rRNA gene was partially sequenced for all the isolates and phylogenetic analyses were performed. For the majority of the isolates there was a broad congruence between the phenotypic and genotypic based identifications.