Cyanobacterial Bioactive Compounds: Biosynthesis, Evolution, Structure and Bioactivity

Cyanobacterial bioactive compounds: biosynthesis, evolution, structure and bioactivity

Tânia Keiko Shishido Joutsen

Division of Microbiology and Biotechnology Department of Food and Environmental Sciences Faculty of Agriculture and Forestry University of Helsinki, Finland

and

Integrative Life Science Doctoral Program University of Helsinki, Finland

ACADEMIC DISSERTATION

To be presented, with permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public examination in auditorium B2 (A109), at Viikki B- Building, Latokartanonkaari 7, on May 22nd 2015, at 12 o’clock noon.

Helsinki 2015

Supervisors Professor Kaarina Sivonen Department of Food and Environmental Sciences University of Helsinki, Finland

Docent David P. Fewer Department of Food and Environmental Sciences University of Helsinki, Finland

Docent Jouni Jokela Department of Food and Environmental Sciences University of Helsinki, Finland

Reviewers Professor Elke Dittmann Institute of Biochemistry and Biology University of Potsdam, Germany

Professor Pia Vuorela Pharmaceutical Biology, Faculty of Pharmacy University of Helsinki, Finland

Thesis committee Professor Mirja Salkinoja-Salonen Department of Food and Environmental Sciences University of Helsinki, Finland

Docent Päivi Tammela Centre for drug research, Faculty of Pharmacy University of Helsinki, Finland

Opponent Professor Vitor Vasconcelos Interdisciplinary Centre of Marine and Environmental Research University of Porto, Portugal

Custos Professor Kaarina Sivonen Department of Food and Environmental Sciences University of Helsinki, Finland

Cover layout by Anita Tienhaara and Cover image: Mare Balticum, Anabaena sp. XSPORK2A and chemical structures of microcystin, anabaenolysin, hassallidin, cyclodextrin, scytophycin (from SciFinder and courtesy by Jouni Jokela).

ISSN 2342-3161 (Print) and ISSN 2342-317X (Online) ISBN 978-951-51-1113-5 (pbk.) and ISBN 978-951-51-1114-2 (PDF) Hansaprint Helsinki 2015

Table of Contents

List of publications and author’s contribution ...... 4 Abbreviations ...... 5 Abstract ...... 6 Resumo ...... 7 Tiivistelmä ...... 8

1. Introduction ...... 9 Cyanobacteria ...... 9 Natural products biosynthesis ...... 9 Genetic diversity of cyanobacterial nonribosomal peptide and polyketide gene clusters ...... 11 Evolution of the genes involved in the synthesis of nonribosomal peptides and polyketides ... 17 Activity of cyanobacterial nonribosomal peptides and polyketides ...... 18 Drug discovery and development process ...... 32 2. Study aims ...... 34 3. Summary of material and methods ...... 35 4. Summary of results and discussion ...... 36 Anabaenolysin, hassallidin and microcystin gene clusters (I, II, III) ...... 36 Gene evolution involved in microcystin and anabaenolysin synthesis (I, III) ...... 39 Diversity of cyanobacterial strains producing the studied natural products (I, II, III, IV) ...... 41 Activity of the cyanobacterial natural products analyzed in this study (II, III, IV) ...... 43 5. Final remarks and future considerations ...... 45 6. Acknowledgements ...... 47 7. References ...... 48

Supplementary Table S1: Strains used in the present doctoral thesis...... 65

List of publications

This thesis is based on the following publications and manuscripts:

I Shishido TK, Kaasalainen U, Fewer DP, Rouhiainen L, Jokela J, Wahlsten M, Fiore MF, Yunes JS, Rikkinen J, Sivonen K. 2013. Convergent evolution of [D- Leucine1] microcystin-LR in taxonomically disparate cyanobacteria. BMC Evolutionary Biology. 13:86.

II Vestola J, Shishido TK, Jokela J, Fewer DP, Aitio O, Permi P, Wahlsten M, Wang H, Rouhiainen L, Sivonen K. 2014. Hassallidins, antifungal glycolipopeptides, are widespread among cyanobacteria and are the end-product of a nonribosomal pathway. Proceedings of the National Academy of Science U S A. 111(18):E1909- 17.

III Shishido TK, Jokela J, Kumer C-T, Fewer DP, Wahlsten M, Wang H, Rouhiainen L, Rizzi E, Debellis G, Permi P, Sivonen K. Biosynthesis of a unique antifungal synergy between the anabaenolysin lipopeptides and cyclodextrins from Anabaena (Cyanobacteria). Submitted manuscript.

IV Shishido TK, Humisto A, Jokela J, Liu L, Wahlsten M, Tamrakar A, Fewer DP, Permi P, Andreote APD, Fiore MF, Sivonen K. Antifungal compounds from cyanobacteria. Marine Drugs. 13(4):2124-2140.

The author’s contribution

I Tânia Keiko Shishido Joutsen designed the study, executed most of the experimental work, analysis and data interpretation, and wrote the article.

II Tânia Keiko Shishido Joutsen participated in the design of the study, carried out part of the research, analysis and data interpretation, and collaborated with the other authors in writing the article.

III Tânia Keiko Shishido Joutsen designed the study, carried out the research and data analysis, and wrote the article.

IV Tânia Keiko Shishido Joutsen designed the study, carried out the research and data analysis, and wrote the article.

Abbreviations

A Adenylation domain Aa Amino acid abl Anabaenolysin gene cluster Acc Acyl-CoA carboxylase AccB Biotin carboxyl carrier protein ACP Acyl carrier protein domain AT Acyl-transferase domain bp Base pairs C Condensation domain CoA Coenzyme A DH Dehydratase domain E Epimerization domain EMA European Medicines Agency ER Enoyl reductase domain FAALs Fatty acyl-AMP ligases FACLs Acyl-CoA-synthesizing fatty acyl-CoA ligases FADS Fatty acid desaturase FDA Food and Drug Administration has Hassallidin gene cluster HMM Hidden Markov model IC50 Half maximum inhibitory concentration or Concentration at 50% inhibition kb Kilo base pairs KR Ketoreductase domain KS Ketosynthase domain Leu or L Leucine Mb Mega base pairs mcy Microcystin gene cluster McyA Protein encoded by the gene mcyA McyB Protein encoded by the gene mcyB McyC Protein encoded by the gene mcyC Met Methionine MIC Minimum inhibitory concentration MT Methyltransferase domain NCBI National Center for Biotechnology Information NRPS Nonribosomal peptides synthetases OX Monooxygenase domain PCP Peptidyl carrier protein domain PKS Polyketide synthases Q Glutamine R Arginine T Threonine TE Thioesterase domain WHO World Health Organization X Unknown amino acid Y Tyrosine

Abstract

Cyanobacteria have a long evolutionary history, dating back to 3500 million years ago. They are an ancient lineage of photosynthetic bacteria that contribute to global nitrogen and carbon cycles. Cyanobacteria can be found in diverse environments, from aquatic to terrestrial systems, with specimens detected and isolated from geothermal, hypersaline, polar and desert regions. Cyanobacteria are infamous for the production of toxins such as microcystin, cylindrospermopsin, saxitoxin and anatoxin-a. However, many different types of cyanobacterial compounds with e.g. antibacterial, antifungal, anticancer, antiviral and antiprotozoal activity have also been found. This study aimed at investigating the evolution, biosynthesis, chemical variety and antifungal activity of cyanobacterial compounds. The results indicate that distantly related cyanobacteria converged on the ability to produce a rare variant of microcystin. Microcystins are commonly produced by cyanobacteria during blooms, but have recently also been found in benthic and lichen- associated cyanobacteria. A benthic, a lichen-associated cyanobacterium and two planktonic strains were shown to produce [D-Leu1] microcystin-LR. Bioinformatic analyses indicated that different evolutionary events, i.e. point mutations and gene conversion, were involved in this convergent evolution. Over 400 cyanobacterial strains were screened for the production of antifungal compounds. Genome mining allowed the discovery of the biosynthetic genes involved in the synthesis of the antifungal compounds hassallidin and anabaenolysin. Anabaena sp. SYKE748A produced more than 40 glycolipopeptide hassallidins in addition to the two main variants. Hassallidins were also identified from Aphanizomenon, Cylindrospermopsis, Nostoc and Tolypothrix species. The lipopeptides anabaenolysins were detected only in Anabaena strains. New variants of anabaenolysins C and D were chemically characterized. The antifungal activity of hassallidin D and anabaenolysin B were investigated through disc diffusion and microdilution bioassays. Synergistic antifungal activity was surprisingly observed through the production of anabaenolysin and cyclodextrins by Anabaena strains. The macrolide scytophycin was identified from Anabaena strains in this study, the first report of scytophycin from this genus. In addition, Nostoc and Scytonema strains from benthic habitats in the Finnish coastal area in the Baltic Sea were found to produce scytophycins. Unidentified antifungal compounds from the strains Fischerella sp. CENA 298, Scytonema hofmanni PCC 7110 and Nostoc sp. N107.3 were detected in the present study. Further chemical characterizations of these compounds are needed. Cyanobacteria are a prolific source for bioactive compounds, which could be toxic or potentially new drug leads. In this study, we show evidence of cyanobacterial biosynthetic genes and their evolution. We also detected new variants of the cyanobacterial compounds and their bioactivity. Furthermore, this study showed the potential of utilizing cyanobacteria for drug discovery.

Resumo

Cianobactérias possuem uma longa história evolutiva datada em 3500 milhões de anos. Elas pertencem a uma antiga linhagem de bactérias fotossintéticas que contribuem para os ciclos globais de nitrogênio e carbono. As cianobactérias podem ser encontradas em diversos ambientes, de sistemas aquáticos a terrestres, com espécimes detectados e isolados de regiões geotérmicas, supersalinas, polares e desérticas. As cianobactérias são famosas pela produção de toxinas como microcistina, cilindrospermopsina, saxitoxina e anatoxina-a. Por outro lado, muitos compostos produzidos por essas linhagens apresentam atividades antibacterianas, antifúngicas, anticancerígenas, antivirais e antiprotozoáreas. Neste sentido, este estudo teve por objetivo investigar a evolução, a biossíntese, a variação química e a atividade antifúngica de substâncias bioativas produzidas por cianobactérias. Os resultados indicam uma convergência na habilidade de cianobactérias, remotamente relacionadas, produzirem uma variante rara de microcistina. Microcistinas são geralmente produzidas por cianobactérias durante proliferações excessivas de células nos meios aquáticos, conhecidas como blooms. Porém, recentemente, microcistinas foram detectadas em linhagens bentônicas e associadas aos líquens. [D-Leu1] microcystin-LR foi encontrada em extratos celulares de uma linhagem bentônica, uma associada aos líquens e duas planctônicas. Análises bioinformáticas indicaram que eventos evolucionários diferentes, como, por exemplo, mutações pontuais e conversão de genes, estão envolvidos nessa convergência evolutiva. Mais de 400 linhagens de cianobactérias foram testadas para a produção de compostos antifúngicos. A análise de genomas (genome mining) permitiu a descoberta dos genes envolvidos na síntese dos compostos antifúngicos hassalidina e anabaenolisina. Anabaena sp. SYKE748A produziu mais de 40 variantes do glicolipopeptídeo hassalidina, além das duas principais variantes. A produção de hassalidinas também foi detectada em linhagens dos gêneros Aphanizomenon, Cylindrospermopsis, Nostoc e Tolypothrix. O lipopeptídeo anabaenolisina foi detectado somente em linhagens do gênero Anabaena. As novas variantes de anabaenolisina C e D foram caracterizadas quimicamente. As atividades antifúngicas da hassalidina D e da anabaenolisina B foram investigadas através de ensaios de disco-difusão e microdiluição. Um aumento da atividade antifúngica foi observado com a ação conjunta de anabaenolisinas e ciclodextrinas produzidas por linhagens do gênero Anabaena. O primeiro relato de produção do macrolídeo scitoficina por linhagens de Anabaena foi descrito neste estudo. Além disso, linhagens dos gêneros Nostoc e Scytonema, isoladas de ambientes bentônicos da costa finlandesa no Mar Báltico, foram descritas como produtoras de scitoficina. No mesmo estudo, foram descritos compostos não-identificados que apresentaram atividades antifúngicas, produzidos pelas linhagens Fischerella sp. CENA 298, Scytonema hofmanni PCC 7110 e Nostoc sp. N107.3. As caracterizações químicas destas substâncias bioativas precisarão ser realizadas no futuro. Cianobactérias são uma fonte promissora de compostos bioativos que podem ser toxinas ou potencialmente novos medicamentos (drug leads). No presente estudo, mostramos evidências dos genes envolvidos nas sínteses, na evolução, na detecção de novas variantes e na bioatividade de compostos produzidos por cianobactérias. Além disso, esse estudo mostra o potencial uso de cianobactérias na descoberta de medicamentos.

Tiivistelmä

Syanobakteerien evolutiivinen historia kattaa kolme ja puoli miljardia vuotta maapallon olemassaolosta. Nämä fotosynteettiset bakteerit ovat vuosituhansien ajan edesauttaneet elämää toimimalla typen ja hiilen kierrättäjinä luonnossa. Syanobakteereja esiintyy mitä moninaisimmissa elinympäristöissä niin vesi- kuin maaekosysteemeissä. Joitain lajeja on havaittu ja eristetty elämän kannalta ääriolosuhteista – suolajärvistä, napaseuduilta, aavikoilta ja maankuoren geotermisen lämmön synnyttämistä kuumista lähteistä. Syanobakteerit ovat pahamaineisia tuottamiensa toksiinien johdosta. Tällaisia ovat esimerkiksi mikrokystiini, sylindrospermopsiini, saksitoksiini ja anatoksiini-a. Syanobakteerien on havaittu tuottavan muitakin yhdisteitä kuin toksiineja. Nämä yhdisteet tappavat ja estävät esimerkiksi bakteerien, sienien, syöpäsolujen, viruksien ja alkueläimien lisääntymistä ja toimintaa. Tämän työn tavoite on tutkia syanobakteerien tuottamien yhdisteiden evoluutiota, biosynteesiä, kemiallista vaihtelua ja antifungaalista aktiivisuutta. Tulokset viittaavat siihen, että kaukaista sukua toisilleen olevat syanobakteerit ovat itsenäisesti, toisista erillään kehittäneet kyvyn tuottaa samaa mikrokystiinin harvinaista muotoa. Veden pintakerroksien planktiset syanobakteerit tuottavat mikrokystiiniä tavallisesti massaesiintymien aikana. Mikrokystiinejä on löydetty myös jäkälien symbioottisista ja vesistöjen pohjalla elävistä benttisistä syanobakteereista. Yhden, jäkälän kanssa symbionttisen, yhden benttisen sekä kahden planktisen syanobakteerikannan osoitettiin tuottavan [D-Leu1] mikrokystiini-LR:ää. Bioinformatiikka analyysit ilmaisivat, että syanobakteerien erilaisissa elinolosuhteissa syntynyt kyky tuottaa mikrokystiiniä on seurausta evolutionaarisista muutoksista esimerkiksi pistemutaation ja muun geenien muuntumisen kautta. Antifungaalisia yhdisteitä tuottavien syanobakteerien löytämiseksi tutkittiin yli 400 kantaa. Geenien analysointi paljasti biosynteesigeenit, jotka ovat vastuussa antifungaalisten yhdisteiden hassalidiinin ja anabaenolysiinin tuotannosta. Kahden päävariantin lisäksi Anabaena sp. SYKE748A tuotti yli 40 glykolipopeptidi hassallidiiniä. Hassalidiinejä tunnistettiin myös Aphanizomenon, Cylindrospermopsis, Nostoc ja Tolypothrix -lajeista. Lipopeptidi anabaenolysiinejä löydettiin vain Anabaena -kannoista. Kemiallisesti kuvattiin uudet anabaenolysiinit C ja D. Hassallidiini D:n ja anabaenolysiini B:n antifungaalista aktiivisuutta tutkittiin kiekkodiffuusio- ja mikrolaimennusbiotestimenetelmillä. Yllättäen Anabaena-kantojen havaittiin tuottavan samanaikaisesti sekä anabaenolysiiniä ja syklodekstriiniä ja jälkimmäisen havaittiin lisäävän anabaenolysiinin antifungaalista aktiivisuutta synergistisesti. Tutkimuksessa tunnistettiin ensimmäisen kerran Anabaena -suvun kannoista makroliidi skytofysiiniä. Lisäksi Itämerellä, Suomen rannikon benttisten Nostoc ja Scytonema -kantojen havaittiin tuottavan skytofysiiniä. Tutkimuksessa löydettiin myös tuntemattomia antifungaalisia yhdisteitä Fischerella sp. CENA 298, Scytonema hofmanni PCC 7110 ja Nostoc sp. N107.3 kannoista. Näiden yhdisteiden tarkempi tunnistaminen vaatii lisätutkimuksia. Syanobakteereista on löydetty runsaasti bioaktiivisia yhdisteitä, jotka ovat myrkyllisiä, mutta myös yhdisteitä joita voidaan mahdollisesti käyttää lääkkeiden lähteinä. Tässä tutkimuksessa tuotettiin uutta tietoa syanobakteerien biosynteesigeeneistä ja niiden evoluutiosta. Tutkimuksessa tuli esiin myös uusia syanobakteerien tuottamia yhdisteitä ja bioaktiivisuuksia. Lisäksi tutkimuksesta käy ilmi, kuinka syanobakteereja on mahdollista käyttää hyväksi uusien lääkkeiden löytämisessä.

1. Introduction

Cyanobacteria

Cyanobacteria are the only microorganisms capable of performing oxygenic photosynthesis. They can live in different types of environments, from aquatic (marine, brackish and freshwater) to terrestrial (rock, soil and lichen-association) habitats, including hypersaline, deserts, polar and hot springs (Whitton and Potts 2000, Stal 2012). Some of them have specialized cells, heterocysts, capable of fixing nitrogen, and resisting cells known as akinetes. However, some cyanobacteria, e.g. most of the oceanic nitrogen- fixing cyanobacteria, can fix nitrogen without requiring the ability to form heterocysts (Díez et al. 2008, Tripp et al. 2010, Stal 2012). Gas vacuoles help planktonic cyanobacteria regulate their position in the water column through buoyancy. Hormogonia are motile filaments that can improve cyanobacterial dispersal and colonization (Meeks et al. 2002). Cyanobacteria are well known for their capability to produce bioactive compounds, an important feature that might improve their survival in the environment. More than 600 peptides or other molecules have been discovered in organisms from different cyanobacteria genera (Welker and von Döhren 2006, Jones et al. 2010).

Natural products biosynthesis

Cyanobacterial natural products can be classified as peptides, polyketides, alkaloids, fatty acids, terpenes and UV-absorbing compounds (Micalleff et al. 2014). The synthesis of the cyanobacterial peptides occurs through ribosomal or nonribosomal pathways. Nonribosomal peptides and polyketides are synthesized by multifunctional modular enzymes known as nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS), respectively. These enzymes are formed by modules that incorporate each unit in the growing peptide (Figure 1). The elongation module of an NRPS is minimally composed by three domains: adenylation (A), peptidyl carrier protein (PCP) and condensation (C) (Marahiel and Essen 2009). The adenylation domain selects and activates the amino acid (Aa) that will be incorporated in the peptide (Finking and Marahiel 2004, Marahiel and Essen 2009). This amino acid is transferred to the peptidyl carrier protein and the condensation domain forms the peptide bond between two amino acids attached to the adjacent peptidyl carrier protein domain (Finking and Marahiel 2004, Marahiel and Essen 2009). An initiation module contains adenylation and peptidyl carrier protein domains (Finking and Marahiel 2004). The thioesterase domain (TE) catalyzes the possible formation of a macrocycle and the release of the compound (Marahiel and Essen 2009). Tailoring domains might appear and they increase the diversity of possible modifications of the peptide. Among other tailoring domains, it is important to highlight the epimerization domain (E) that changes the L- to D-amino acids, methyltransferase (MT) that are responsible for the methylation of amino acid residues, and monooxygenase domains (OX) that incorporate one hydroxyl group to the substrate (Finking and Marahiel 2004, Marahiel and Essen 2009). Glycosyltransferases are enzymes that add sugar units to free hydroxyl groups present in the peptide (Finking and Marahiel 2004, Welker and von Döhren 2006). In a similar way, PKS-type I modules need a minimum of three domains: acyl- transferase (AT), acyl carrier protein (ACP) and ketosynthase (KS) (Figure 1, Staunton

9 and Weissman 2001). The acyl-transferase selects one of these different units to be incorporated in the peptide malonyl-, methyl-, ethyl-, propylmalonyl-CoA (Coenzyme A) (Katz 1997). In a biosynthetic mechanism analagous to NRPS, the malonyl-CoA unit is transferred to the acyl carrier protein and subsequently the ketosynthase domain allows the formation of a bond between two units through Claisen-condensation. At the end of the elongation steps through all the PKS domains, the thioesterase domain (TE) cleaves and macrocyclizes the product. Optional domains can occur such as keto reductase (KR) (formation of hydroxyl groups), dehydratase (DH) (formation of unsaturated bonds) and enoyl reductase (ER) (reduction to saturated bonds) domains (Figure 1, Hopwood 1997, Katz 1997). Hybrid systems containing both NRPS and PKS domains are commonly involved in the synthesis of several compounds, such as microcystin from cyanobacteria (Wang et al. 2014).

Figure 1. Nonribosomal biosynthetic enzymes. A. Nonribosomal peptide synthetases (NRPS). B. Polyketide synthases (PKS). Domains showed in the figure: A- adenylation; PCP- peptidyl carrier protein; C- condensation; MT- methyltransferase; E- epimerization; AT- acyl-transferase; ACP- acyl carrier protein; KS- ketosynthase; KR- ketoreductase; DH- dehydratase; ER- enoyl reductase (Modified from Strieker et al. 2010 and Kehr et al. 2011).

Some nonribosomal peptides and polyketides have a fatty acid portion in their chemical structure. Fatty acid synthesis can occur in two different pathways: type I formed by large multifunctional proteins and type II formed by discrete proteins acting together (Campbell and Cronan 2001). The latter is more commonly found in bacteria and can be found as highly conserved protein sequences (Fab, fatty acid biosynthesis and Acc, acyl-CoA carboxylase) or the less conserved enoyl reductases (ER) (Campbell and Cronan 2001). Other proteins surrounding the previous ones are also involved in the fatty acid biosynthesis, such as acyl carrier protein (ACP) and biotin carboxyl carrier protein (AccB) (Campbell and Cronan 2001).

The free fatty acids found in the cell can be activated to their corresponding coenzyme A derivatives by fatty acyl-CoA ligases (FACLs) to be further used in fatty acid transport, protein acylation, energy generation, phospholipid biosynthesis (Arora et al. 2009) and secondary metabolism (Miao et al. 2005). Fatty acyl-AMP ligases (FAALs) can form the fatty acyladenylates, which are the intermediary units for the coenzyme A derivatives (Trivedi et al. 2004, Arora et al. 2009). These activated fatty acyladenylates can be transferred to the acyl carrier protein to be used in the synthesis of lipidic compounds (Trivedi et al. 2004, Arora et al. 2009). The fatty acid chain present in the secondary metabolites can also be formed by PKSs as in curacin A (Chang et al. 2004) or be incorporated by a starter condensation domain from NRPS that acts as an acceptor of a β-hydroxyl fatty acid (Rausch et al. 2007, Kraas et al. 2010, Qian et al. 2012). Several biosynthetic gene clusters have been described to contain these starter condensation domains, such as surfactin (Arima et al. 1968, Rausch et al. 2007), lichenysin (Horowitz and Griffin 1991), fengycin (Tosato et al. 1997), arthrofactin (Morikawa et al. 1993), polymyxin (Choi et al. 2009) and pelgipeptin (Qian et al. 2012). These different mechanisms can be used by cyanobacteria to synthesize lipopeptides. PuwC in the puwainaphycins biosynthetic gene cluster is a FAAL from Cylindrospermum alatosporum (Mareš et al. 2014). In the curacin A biosynthesis, CurG to CurM are polyketide modules that condensate seven malonyl-CoAs (Chang et al. 2004). Starter condensation domains have been described to be putatively involved in the synthesis of aeruginosin (Fewer et al. 2013), cyanopeptolin (Tooming-Klunderud et al. 2007) and nostopeptolide (Hoffmann et al. 2003). In addition to the possible incorporation of approximately 300 proteinogenic and nonproteinogenic substrates, different modification can be acquired with accessory domains present in the gene cluster that contributes to the high diversity of synthesizable compounds.

Genetic diversity of cyanobacterial nonribosomal peptide and polyketide gene clusters

The nonribosomal pathways from cyanobacteria described to date are often large (between 11 to 63 kb) and most commonly consist of hybrid PKS/NRPS systems (Table 1). Most known cyanobacterial gene clusters have been described for freshwater strains. The possibility of human poisoning due to water comsumption or recreation has focused the efforts to further study cyanobacterial toxins. The biosynthetic pathways of different cyanotoxins have been determined, such as microcystin, nodularins, cylindrospermopsins, anatoxin-a, and saxitoxins (Table 1, Dittmann et al. 2012). Marine filamentous cyanobacteria are a prolific source of drug leads, as almost 800 compounds have been described (Jones et al. 2010). Modifications increasing the diversity of cyanobacterial compounds can be obtained due to chlorination (anabaenopeptilide, curacin A, jamaicamides, hectochlorin and barbamide), bromination (jamaicamide A), methylation by methyltransferases in NRPS gene clusters and by S-adenosyl methionine (SAM)- mediated methylation or HMGCoA-like β-branching mechanisms in PKS gene clusters (curacin A, barbamide and jamaicamide) and iodination (incorporation of iodine in the cryptophycin) (Rouhiainen et al. 2000, Jones et al. 2010, Kehr et al. 2011). With the advance of next-generation sequencing methods, more genomes of cyanobacteria have been sequenced and are now available for analysis in the National

Center for Biotechnology Information (NCBI) database. The analysis of 991 organisms from the three domains of life showed high NRPS and PKS frequencies in Cyanobacteria, Proteobacteria, Actinobacteria, Firmicutes and Ascomycota (Wang et al. 2014). Another study indicates that 70% of 126 cyanobacterial genomes encoded NRPS or PKS gene clusters (Shih et al. 2013). From these 88 genomes, only 20% of the gene clusters were already known, with the other 80% (91 belonging to 19 cluster families) still needing further characterization (Calteau et al. 2014). Strains containing most of the NRPS or PKS gene clusters represented the following morphotypes: baeocystous (unicellular cyanobacteria that reproduce using multiple fissions in three planes), heterocystous (presence of specialized cell for nitrogen fixation) and ramified (filamentous cells that might divide to create branches) (Shih et al. 2013). Baeocystous strains are an under- exploited interesting source of natural products. The smallest cyanobacterial genome described to date has 1.44 mega base pairs (Mb) (marine cyanobacterium UCYN-A) and the largest has 12.07 Mb (Scytonema hofmanni PCC 7110) (Micallef et al. 2014). Genome mining will be essential for linking the metabolite to the biosynthetic gene cluster and in the near future it is likely to increase the knowledge concerning this topic.

Table 1. Gene clusters of nonribosomal peptides and alkaloids assigned for cyanobacteria. Modified from Jones et al. 2010, Méjean and Ploux 2013, and Micallef et al. 2014. ■ – Marine strains; ▲- Freshwater strains; ● - terrestrial; ♦ - brackish water; □ - rice field; ○ – root section of Macrozamia sp. ♣ - Unknown Compound Activity Cyanobacteria Biosynthetic Cluster size Accession Reference pathway number system Aeruginosins Protease PKS/NRPS 34 kb Aeruginoside 126 inhibitor ▲ Planktothrix agardhii NIVA-CYA 126/8 (aer) AM071396 Ishida et al. 2007 Aeruginosin 686 ▲ Microcystis aeruginosa PCC 7806 AM778955 Frangeul et al. 2008, Ishida et al. 2009 Aeruginosin A ▲ Planktothrix rubescensi NIVA-CYA 98 AM990465 Rounge et al. 2009 Aeruginosin 98 ▲ Microcystis aeruginosa NIES-98 FJ609416 Ishida et al. 2009 Aeruginosin 102 ▲ Microcystis aeruginosa NIES-843 AP009552 Ishida et al. 2009 ▲ Microcystis aeruginosa PCC 9432 CAIH00000000 Humbert et al. 2013* ▲ Microcystis aeruginosa PCC 9443 CAIH00000000 Humbert et al. 2013* ▲ Microcystis aeruginosa PCC 9807 CAIM00000000 Humbert et al. 2013* ▲ Microcystis aeruginosa PCC 9809 CAIO00000000 Humbert et al. 2013* ▲ Microcystis aeruginosa TAIHU 98 ANKQ00000000 Yang et al. 2013 ♦ Nodularia spumigena CCY 9414 CM001793 Fewer et a. 2013 Ambiguine Antibacterial, ♣ Fischerella ambigua UTEX1903 Alkaloid (amb) 42 kb KF664586 Hillwig et al. 2013 antifungal ♣ Fischerella ambigua UTEX1903 Alkaloid (amb) 51 kb JK742065 Micallef et al. 2014 Anabaenopeptins Protease NRPS Anabaenopeptin A,B,F, inhibitor ▲ Planktothrix rubescensi NIVA-CYA 98 (ana) 24 kb AM990463 Rounge et al. 2009 Oscillamid Y Anabaenopeptin A, B, C ▲ Anabaena sp. 90 (apt) 32 kb GU174493 Rouhiainen et al. 2010 Nodulapeptin B, C ♦ Nodularia spumigena CCY 9414 (apt) 26 kb EAW45420 Rouhiainen et al. 2010 Anabaenopeptin NZ 857 ● Nostoc punctiforme PCC 73102 (apt) 26 kb ACC81026 Rouhiainen et al. 2010 Anabaenopeptin 908, ▲ Planktothrix agardhii NIVA-CYA 126/8 (apn) 24 kb EF672686 Christiansen et al. 2011 915 Anabaenopeptin ▲ Microcystis aeruginosa PCC 9432 (apt) 24 kb CAIH0000000 Humbert et al. 2013* Anabaenopeptin ▲ Microcystis aeruginosa PCC 9701 (apt) 24 kb CAIQ01000001 Humbert et al. 2013* Anatoxin-a/ Neurotoxin ♣ Oscillatoria sp. PCC 6506 PKS/NRPS 29 kb FJ477836 Méjean et al. 2009 Homoanatoxin-a ▲ Anabaena sp. 37 (ana) JF803645 Rantala-Ylinen et al. 2011 ▲ Oscillatoria formosa PCC 6407 ALVI00000000 Calteau et al. 2014 ● Cylindrospermum sp. PCC 7417 CP003642-45 Calteau et al. 2014

Compound Activity Cyanobacteria Biosynthetic Cluster Accession Reference pathway size number system Apratoxins Cytotoxin ■ Lyngbya bouillonii PKS/NRPS 58 kb Grindberg et al. 2011 (apr) Barbamide Molluscidal ■ Lyngbya majuscula 3L PKS/NRPS 26 kb AF516145 Chang et al. 2002 (bar) Cryptophycin/ Cytotoxic ● Nostoc sp. ATCC 53789 PKS/NRPS 40 kb EF159954 Magarvey et al. 2006 arenastatins (crp) Curacin A Cytotoxin, ■ Lyngbya majuscula 3L PKS/NRPS 63 kb AY652953 Chang et al. 2004 antiproliferative (cur) Cyanopeptolins Protease NRPS 30 kb Anabaenopeptolides inhibitor ▲ Anabaena sp. 90 (apd) 28 kb AJ269505 Rouhiainen et al. 2000 Cyanopeptolin 984 ▲ Microcystis cf. wesenbergii NIVA CYA (mcn) DQ075244 Tooming-Klunderud et 172/5 al. 2007 Cyanopeptolin 1138 ▲ Planktothrix agardhii NIVA CYA 116 (oci) 30 kb DQ837301 Rounge et al. 2007 Oscillapeptin C, D, E ▲ Plankthotrix agardhii NIES-205 (oci) 32 kb EU109504 Rounge et al. 2007 Cyanopeptolin ▲ Microcystis aeruginosa NIES 843 (mcn) AP009552 Kaneko et al. 2007 Cyanopeptolin ▲ Microcystis aeruginosa PCC 7806 (mcn) AM778843- Frangeul et al. 2008 AM778958 Oscillapeptin G ▲ Planktothrix rubescensi NIVA CYA 98 (oci) 35 kb AM990463 Rounge et al. 2009 Micropeptin ▲ Microcystis aeruginosa K-139 (mcn) AB481215 Nishizawa et al. 2011 Cyanopeptolin ▲ Microcystis aeruginosa PCC 7941 (mcn) CAIK00000000 Humbert et al. 2013* Cyanopeptolin ▲ Microcystis aeruginosa PCC 9432 (mcn) CAIH00000000 Humbert et al. 2013* Cyanopeptolin ▲ Microcystis aeruginosa PCC 9701 (mcn) CAIQ01000001 Humbert et al. 2013* Cyanopeptolin ▲ Microcystis aeruginosa PCC 9717 (mcn) CAII00000000 Humbert et al. 2013* Cyanopeptolin ▲ Microcystis aeruginosa PCC 9807 (mcn) CAIM00000000 Humbert et al. 2013* Cyanopeptolin ▲ Microcystis aeruginosa PCC 9808 (mcn) CAIN00000000 Humbert et al. 2013* Cyanopeptolin ▲ Microcystis aeruginosa PCC 9809 (mcn) CAIO00000000 Humbert et al. 2013* Cylindrospermopsins Hepatotoxin ▲ Cylindrospermopsis raciborskii AWT 205 PKS/NRPS 43 kb EU140798 Mihali et al. 2008 ▲ Aphanizomenon sp. 10E6 (cyr) CQ385961 Stüken and Jacobsen 2010 ♣ Oscillatoria sp. PCC 6506 FJ418586 Mazmouz et al. 2010 ▲ Cylindrospermopsis raciborskii CS-505 ACYA01000027 Stucken et al. 2010 ▲ Aphanizomenon ovalisporum ILC146 Kaplan et al. 2012 ▲ Raphidiopsis curvata CHAB 1150 CHAB1150 Jiang et al. 2012 ▲ Cylindrospermopsis raciborskii CS-506 SRR1042336 Sinha et al. 2014

Compound Activity Cyanobacteria Biosynthetic Cluster Accession Reference pathway size number system Hapalindole Cytotoxic, ♣ Fischerella sp. ATCC 43239 Alkaloid (hpi) 40 kb KJ742064 Micallef et al. 2014 antibacterial ♣ Fischerella sp. PCC 9339 (hpi) 45 kb JGI IMG/ER: Micallef et al. 2014 2516653082 Hassallidin Antifungal ▲ Anabaena sp. 90 NRPS 62 kb ANA_C11023- Wang et al. 2012 C13074 Hectochlorin Cytotoxin, ■ Lyngbya majuscula JHB PKS/NRPS 38 kb AY974560 Ramaswamy et al. antifungal (hct) 2007 Jamaicamide Neurotoxin ■ Lyngbya majuscula JHB PKS/NRPS 57 kb AY522504 Edwards et al. 2004 (jam) Lyngbyatoxin Dermatotoxin ■ Lyngbya majuscula NRPS with 11 kb AY588942 Edwards and Gerwick modification 2004 (ltx) Microcystins Hepatotoxin ▲ Microcystis aeruginosa PCC 7806 PKS/NRPS 55 kb AF183408 Tillett et al. 2000 ▲ Planktothrix agardhii CYA 126/8 (mcy) AJ441056 Christiansen et al. 2003 ▲ Anabaena sp. 90 AJ536156 Rouhiainen et al. 2004 ▲ Microcystis aeruginosa NIES 843 AP009552 Kaneko et al. 2007 ▲ Planktothrix sp. NIVA-CYA 98 AM990462 Rounge et al. 2009 ♣ Fischerella sp. PCC 9339 ALVS00000000 Shih et al. 2013 ▲ Microcystis aeruginosa SPC 777 ASZQ00000000 Fiore et al. 2013 ▲ Microcystis aeruginosa PCC 7941 CAIK00000000 Humbert et al. 2013* ▲ Microcystis aeruginosa PCC 9807 CAIM00000000 Humbert et al. 2013* ▲ Microcystis aeruginosa PCC 9809 CAIO00000000 Humbert et al. 2013* ▲ Nostoc sp. 152 57 kb KC699835 Fewer et al. 2013 Microginins ▲ Planktothrix rubescensi NIVA CYA 98 PKS/NRPS 23 kb AM990464 Rounge et al. 2009 ▲ Microcystis aeruginosa PCC 7941 (mic) 21 kb CAIK00000000 Humbert et al. 2013* ▲ Microcystis aeruginosa PCC 9701 CAIQ01000001 Humbert et al. 2013* Nodularins Hepatotoxin ♦ Nodularia spumigena NSOR 10 PKS/NRPS 48 kb AY210783 Moffitt and Neilan (nda) 2004 ♦ Nodularia spumigena CCY 9414 48 kb AOFE00000000 Voß et al. 2013 Nostocyclopeptides Protease ● Nostoc sp. ATCC 53789 NRPS (ncp) 33 kb AY167420 Becker et al. 2004 inhibitor Nostopeptolides Antitoxins and ● Nostoc sp. GSV224 PKS/NRPS 40 kb AF204805 Hoffmann et al. 2003 hormogonium- (nos) Liu et al. 2014a repressing Liaimer et al. 2015

Compound Activity Cyanobacteria Biosynthetic Cluster Accession Reference pathway size number system Nostophycins Cytotoxic ▲ Nostoc sp. 152 PKS/NRPS 45 kb JF430079 Fewer et al. 2011 (npn) Puwainaphycins Cytotoxic ● Cylindrospermum alatosporum CCALA 988 PKS/NRPS 57 kb KM078884 Mareš et al. 2014 (puw) Saxitoxins Neurotoxin ▲ Cylindrospermopsis raciborskii T3 PKS/amino acid 35 kb DQ787200 Kellmann et al. 2008 ▲ Anabaena circinalis AWQC131C (sxt) DQ787201 Mihali et al. 2009 ▲ Aphanizomenon sp. NH5 EU603710 Mihali et al. 2009 ▲ Raphidiopsis brookii D9 ACYB00000000 Stucken et al. 2010 ▲ Lyngbya wollei Murray et al. 2011 Scytonemin Sunscreen ○ Nostoc sp. ATCC 29133 indole-alkaloid 28 kb Soule et al. 2007 Spumigin Protease ♦ Nodularia spumigena CCY 9414 NRPS/PKS 21 kb AAVW00000000 Fewer et al. 2009 inhibitor Welwitindolinone Cytotoxic ♣ Hapalosiphon welwitschii UTEX B1830 Alkaloid (wel) 36 kb KF811479 Hillwig et al. 2014 ● Westiella intricata UH HT-29-1 Alkaloid (wel) 59 kb KJ767018 Micallef et al. 2014 ● Hapalosiphon welwitschii UH IC-52-3 Alkaloid (wel) 56 kb KJ767017 Micallef et al. 2014 ♣ Fischerella sp. PCC 9431 Alkaloid (wel) 57 kb JGI IMG/ER: Micallef et al. 2014 2512875027 □ Fischerella muscicola SAG 1427-1 Alkaloid (wel) 25 kb JGI IMG/ER: Micallef et al. 2014 2548876995 *Gene clusters included in the table were analyzed data from strains producing the compound (Humbert et al. 2013).

Evolution of the genes involved in the synthesis of nonribosomal peptides and polyketides

The presence of NRPS and PKS are energetically expensive for the microorganisms hosting these pathways. One example is the 15 000 amino acids present in the cyclosporin synthase to produce an undecapeptide (Amoutzias et al. 2008). This expensive synthesis system must be important for microorganisms to adapt or survive in the environment. However, it is believed that as soon as the synthesis of these nonribosomal peptides is not needed, the organism could perhaps lose the ability to produce it. It is therefore expected that the NRPS and PKS gene content would be modified based on their ecology (Amoutzias et al. 2008). Microcystin synthetase gene clusters are an example of this theory, in which the gene cluster is vertically transferred, rather than through horizontal gene transfer as earlier hypothesized (Rantala et al. 2004). The ability to produce microcystin has been lost by some genera of cyanobacteria and explains why we occasionally find the genes. There is additionally an indication that the biosynthetic genes involved in the synthesis of nodularin (a chemically related compound) have been derived from the microcystin gene cluster (Rantala et al. 2004, Moffitt and Neilan 2004). Nevertheless, a recent broad comparative study in the cyanobacterial phylum level indicates that most of the NRPS/PKS gene clusters are not vertically inherited or obtained through horizontal gene transfer (Calteau et al. 2014). Most of the gene clusters analyzed showed a more complex evolutionary history. Studies have shown that recombination and point mutations in the domains responsible for the selection and activation of subunits (adenylation and acyl-transferase domains) increases the diversity of the peptides produced (Jenke-Kodama et al. 2006, Fewer et al. 2007). The incorporation of a diverse number of substrates in NRPS and rapid evolution resulting in high diversity of peptide synthesis might be the advantages of keeping these energetically costly modular NRPS and PKS systems (Amoutzias et al. 2008). Nonetheless, the selective value of these NRPS and PKS systems for cyanobacteria remains unclear. The recombination events occuring in the adenylation domain gene sequences result in incongruent phylogeny compared to the evolutionary history of the organism harbouring the biosynthetic pathway. However, the analysis based on the alignment of amino acid sequences helps predict the amino acid substrate to be activated and incorporated in the growing peptide by the adenylation domain. The prediction of the activated amino acid is based on the identification of ten amino acid residues (binding pocket) based in the position of core motifs (Stachelhaus et al. 1999) or 34 residues at 8 Å distant from the substrate (Rausch et al. 2005, Jenke-Kodama and Dittmann 2009). It was also shown that two point mutations in the binding pocket were sufficient to change the adenylation domain specificity from phenylalanine to leucine (Stachelhaus et al. 1999). A higher diversity of peptides could be obtained by mutations and gene conversions of the adenylation domains, allowing e.g. for a change in the peptide activity. In contrast to adenylation domains, phylogenetic studies based on condensation domain sequences show congruence in the phylogenetic trees (Rausch et al. 2007). These authors additionally provided profiles of domain sets (hidden Markov model – HMM) to help in the classification of an unknown condensation domain. The known condensation domains L are divided into the following subtypes: CL (forms the peptide bond between two D adjacents L-amino acids), CL (forms the peptide bond between one L-amino acid and an oligopeptide with a D-amino acid at the end, and is usually present after an epimerization domain in the gene cluster), cyclization (condensation and cyclization of the oligopeptide),

17 starter condensation (acylates with a fatty acid the first incorporated amino acid) and dual epimerization (responsible for epimerization and condensation) (Rausch et al. 2007). Phylogenetic analysis combined with functional data and bioinformatics can also be used to predict whether an acyl-transferase domain in PKS will recognize malonyl-CoA or methylmalonyl-CoA (Amoutzias et al. 2008). Different programmes and databases available for NRPS and PKS analyses are useful. However, caution should be excercized concerning possible unfunctional domains and systems not following the colinearity rule (Jenke-Kodama and Dittmann 2009). Studies on the evolutionary history and the function of these NRPSs and PKSs increase the chance for successful modifications of biosynthetic pathways and help genome mining link products to their genes.

Activity of cyanobacterial nonribosomal peptides and polyketides

Cyanobacteria are present in diverse environments and are able to produce natural products. Strains living in fresh- and brackish water in tropical regions as well as others living in meltwater ponds in the Antarctic were found to produce microcystin (Hitzfeld et al. 2000, Welker and von Döhren 2006, van Apeldoorn et al. 2007). While a broad global distribution of cyanobacterial strains can be seen, it is common to find microcystin- producing and non-producing cyanobacteria growth in the same bloom sample (Vezie et al. 1998, Janse et al. 2004). Microcystin has been largely studied due to toxic effects in humans and animals. It is a cyclic heptapeptide produced by a hybrid system of NRPS and PKS (Tillett et al. 2000). Microcystin inhibits the protein phosphatases 1 and 2A in the hepatocytes, which may result in haemorrhaging or liver failure (van Apeldoorn et al. 2007, Campos and Vasconcelos 2010). Although microcystin can affect its predator Daphnia (Rohrlack et al. 1999), no clear evidence was found that this intoxication could change zooplankton dynamics (Martins and Vasconcelos 2009). A variety of cyanobacterial natural products have been reported to have allelopathic activity, inhibiting other cyanobacteria, micro- and macro-algae and angiosperms (Leão et al. 2009). Nostocyclamide is produced by the freshwater strain Nostoc sp. 31 and inhibits cyanobacteria and algae (Todorova et al. 1995). Portoamides A and B, from a freshwater Oscillatoria sp. differ by one O-methyl group, showed a synergistic allelopathic effect inhibiting the micro-algae Chorella vulgaris (Leão et al. 2010). Other possible ecologic roles for the cyanobacterial compounds are resource competition (e.g. metal chelators), quorum sensing or intracellular physiology (Utkilen and Gjølme 1995, Kaebernick et al. 2000, Dittmann et al. 2001, Leão et al. 2012, Neilan et al. 2013, Pereira and Giani 2014). However, the function of these molecules for the cyanobacteria is still unknown but might provide an advantage in the ecosystems such as defense, allelopathy or fitness enhancement (Schatz et al. 2007, Sivonen 2009, Méjean and Ploux 2013, Meissner et al. 2014). While this issue is not uncovered, natural products produced by cyanobacteria are recognized as toxins or potential drug leads. Microcystins, nodularins, cylindrospermopsins, anatoxin-a and saxitoxins are infamous for their hepato- or neurotoxicity to human cells. Cyanobacteria present in water used for human consumption or recreation should be monitored due to the potential synthesis of one or more of these cyanotoxins. However, other compounds produced by cyanobacteria present

18 pharmaceutical interest for their anticancer, antiviral, antimicrobial and protease inhibition activity (Singh et al. 2011). Promising anticancer agents from cyanobacteria are e.g. cryptophycin from Nostoc sp., curacin A from Lyngbya majuscuala, dolastatin from Symploca sp. and Lyngbya sp., apratoxin A from Lyngbya sp., tolyporphin from Tolypothrix nodosa and somocystinamide from Lyngbya majuscula (Singh et al. 2011). Although the clinical trials of cryptophycin and dolastatin were discontinued, their analogues cryptophycin 249 and 309 and ILX-651 (synthadotin) still represent a hope for new trials (Singh et al. 2011). Some interesting compounds with bioactivity include anabaenolysins A and B, which are lipopeptides found in benthic Anabaena sp. (Jokela et al. 2012), and potent cytotoxins able to permeate mammalian cells that contain cholesterol (Oftedal et al. 2012). Scytophycins are macrolide polyketides produced by Scytonema (Carmeli et al. 1990a), Tolypothrix (Ishibashi et al. 1986), Cylindrospermum (Jung et al. 1991) and Nostoc (Tomsickova et al. 2014) strains that present strong antifungal and anticancer activities (Ishibashi et al. 1986, Yeung and Patterson 2002). In 2014 The World Health Organization (WHO) published its first report on the surveillance of antimicrobial resistance. This warning comes during a time when we face a globally distributed increase in the resistence to commonly used antimicrobial compounds combined with a lack of new approved antimicrobial drugs. Only three classes of antifungal compounds (azoles, echinocandins and polyenes) are available to treat serious infections caused by Candida spp., and problems such as high toxicity, increased resistence, high costs and absence of oral formulation deepens this difficult situation (WHO 2014). Some cyanobacterial antifungal compounds are listed in Table 2. There are a variety of compound classes with antifungal activity, but several of these discovered compounds are peptides and polyketides (Table 2).

Table 2. Examples of antifungal compounds from cyanobacteria (Modified from Abed et al. 2008 and Chlipala et al. 2011). Chemical class Compound family Strains of cyanobacteria Reference Alkaloid Ambiguine isonitriles Fischerella ambigua, Raveh and Carmeli 2007, Fischerella sp. Smitka et al. 1992 Carazostatin Hyella caespitose Burja et al. 2001 Fischerellin Fischerella muscicola Dahms et al. 2006 Fischerindoles Fischerella muscicola Park et al. 1992 Hapalindole Hapalosiphon fontinalis Burja et al. 2001 Norharmane Nodularia harveyana Volk and Furkert 2006 Tjipanazoles Tolypothrix tjipanasensis Bonjouklian et al. 1991, Falch et al. 1995 Welwitindolinones Hapalosiphon welwitschii, Stratmann et al. 1994a Westiella intricate

Aromatic Ambigols A-C Fischerella ambigua Wright et al. 2005, Falch et al. 1993, 1995 Cyanobacterin Scytonema hofmannii Lee and Gleason 1994, Pignatello et al. 1983 Cyclic peptide AK-3 Synechocystis sp. Yoon and Lee 2009 Laxaphycins A and B Anabaena laxa Bonnard et al. 1997, Lyngbya majuscula Frankmölle et al. 1992a,b Nostocyclamide Nostoc sp. Abed et al. 2008 Tolybyssidins A and B Tolypothrix byssoidea Jaki et al. 2001

Chemical class Compound family Strains of cyanobacteria Reference Lipopeptide Balticidins Anabaena cylindrica Bui et al. 2014 Calophycin Calothrix fusca Moon et al. 1992 Hassallidin Hassallia sp. Neuhof et al. 2005, 2006a Lobocyclamides Mat containing Lyngbya MacMillan et al. 2002 confervoides Nostofungicidine Nostoc commune Kajiyama et al. 1998 Scyzotrin A Schizothrix sp. Pergament and Carmeli 1994 Scytonemin Scytonema sp. Helms et al. 1988

Nucleoside Toyocamycin Tolypothrix tenuis Banker and Carmeli 1998, Stewart et al. 1988 Oligosaccharide Nostodione Nostoc commune Bhadury and Wright 2004

Phenolic 4,4´-dihydroxybiphenyl Nostoc insulare Volk and Furkert 2006

Polyketide Cryptophycin Nostoc sp. Schwartz et al. 1990 Mirabilene isonitriles A-F Scytonema hofmannii Carmeli et al. 1990b Scytophycins Scytonema Burja et al. 2001 pseudohofmanni Tolytoxin Tolypothrix conglutinata Carmeli et al. 1990a var. Colorata

Volatile alkane Tetradecane, heptadecane Spirulina platensis Ozdemir et al. 2004

Curiously, the analysis of a marine compounds database (MarinLit) from cyanobacteria showed that 40% of the cyanobacterial natural products were lipopeptides (Burja et al. 2001). Among these 170 cyclic and linear lipopeptides, 85% presented some bioactivity: cytotoxic, antiviral, anticancer, antifungal, antibacterial, enzyme inhibitor or other (Burja et al. 2001). Some examples of the lipopeptides are presented in Table 3. These compounds present different lengths of the fatty acid chain and different activities. The majority of the compounds are synthesized by marine Lyngbya spp. It is interesting to note the huge variation in chemical structures of the different compounds (Table 3). The pharmaceutical interest in the lipopeptides is due to their capability to pass the blood- tissue and blood-brain barrier because of their interaction with cell liposomes and membranes (Burja et al. 2001). Glycolipopeptides are lipopeptides containing sugar chains, such as hassallidins A and B, which are antifungal glycosylated lipopeptides isolated from Hassallia sp. extracts (Table 3, Neuhof et al. 2005, 2006a). The mechanism of action of hassallidin A is to affect the integrity of Candida albicans plasma membrane (Neuhof et al. 2006b). Suomilides are glycolipopeptides produced by Nodularia spumigena HKVV (Fujii et al. 1997) and belong to the aeruginosin family of protease inhibitors (Schindler et al. 2008). Other previously described compounds containing a sugar chain in the structure include bacteriopanepolyols (terpenoid), malyngamide J (glycolipid), lyngbyaloside (polyketide), malyngamides-type alkaloids and tolyporphins (porphinoid) (Tidgewell et al. 2010). Known cyanobacterial compounds composed of only carbohydrates are the iminotetrasaccharide from Anabaena sp. that have moderate inhibitory activity of β-D- glucuronidase, and the cyclodextrins from Tolypothrix byssoidea that blocks the activity of other compounds produced by this strain (Tidgewell et al. 2010). Screenings, such as the one performed in this present doctoral thesis, are urgently needed to detect new or synthetically modified bioactive compounds, and cyanobacteria

20 have proved to be a prolific source for different and complex compounds. The discovery of new chemical structures that present bioactivity is the first, and very important, step of a long process until the drug is available for marketing.

Table 3. Examples of lipopeptides produced by cyanobacteria, their bioactivity and first strains described as producers. Compound Chemical structure Activity Producer Reference Aeruginosin Protease Microcystis aeruginosa Murakami et al. 1995 inhibitor NIES-98

Anabaenolysin Cytotoxin Anabaena sp. Jokela et al. 2012 Oftedal et al. 2012

Antillatoxin Ichthyotoxic Lyngbya majuscula Orjala et al. 1995

Apratoxin D Anticancer Lyngbya majuscula and Gutiérrez et al. 2008 Lyngbya sordida

Compound Chemical structure Activity Producer Reference Balticidins Antifungal Anabaena cylindrica Bui et al. 2014

Barbamide Molluscicidal Lyngbya majuscula Orjala and Gerwick 1996

Carmabins Cytotoxic, Lyngbya majuscula Hooper et al. 1998 antimalarial McPhail et al. 2007 activity

Curacin A Antimitotic, Lyngbya majuscula Gerwick et al. 1994 brine shrimp toxin

Compound Chemical structure Activity Producer Reference Dragonamide Anticancer Lyngbya Gunasekera et al. 2008 polychroa

Hapalosin Mediated Hapalosiphon Stratmann et al. 1994b multidrug welwitschii resistance (MDR)- reversing activity Hassallidin Antifungal Hassallia sp. Neuhof et al. 2005

Hoiamide Promotes Lyngbya majuscula Pereira et al. 2009 neuronal and Phormidium gracile growth and plasticity

Compound Chemical structure Activity Producer Reference Hormothamnin Antimicrobial Hormothamnion Gerwick et al. 1992 enteromorphoides

Jamaicamides Neurotoxic, Lyngbya majuscula Edwards et al. 2004 ichthyotoxic

Kalkitoxin Neurotoxic Lyngbya majuscula Wu et al. 2000 LePage et al. 2005

Largazole Anticancer Symploca sp. Taori et al. 2008

Compound Chemical structure Activity Producer Reference Laxaphycins A and B Antifungal, Anabaena laxa Frankmölle et al. 1992a,b cytotoxic Lyngbya majuscula Bonnard et al. 1997 *synergism

Lobocyclamides Modest Lyngbya confervoides MacMillan et al. 2002 antifungal activity *synergism

Lyngbyabellin Cytotoxic Lyngbya majuscula Luesch et al. 2000 Yokokawa et al. 2002

Compound Chemical structure Activity Producer Reference Malyngamide H Ichthyotoxic Lyngbya majuscula Orjala et al. 1995

Malyngamide 2 Anti- Lyngbya sordida Malloy et al. 2011 inflammatory and cytotoxic activity

Microcolin Citotoxic and Lyngbya majuscula Koehn et al. 1992 immunosuppre Mattern et al. 1996 ssive Zhang et al. 1997

Microginin Protease Microcystis aeruginosa Ishida et al. 2000 inhibitor

Minutissamides Anticancer Anabaena sp. Kang et al. 2011, 2012

Compound Chemical structure Activity Producer Reference Mitsoamide Cytotoxicity Geitlerinema sp. Adrianasolo et al. 2007 and toxic to brine shrimp

Nostofungicidine Antifungal Nostoc commune Kajiyama et al. 1998

Nostopeptolide antitoxins Nostoc sp. Golakoti et al. 2000 (nodularins Liu et al. 2014a and Liaimer et al. 2015 microcystins) and hormogonium- repressing

Compound Chemical structure Activity Producer Reference Nostosin Protease Nostoc sp. Liu et al. 2014b inhibitor

Parguerene Unknown Moorea producens Mevers et al. 2013

Precarriebowmide Unknown Moorea producens Mevers et al. 2013

Puwainaphycins Cytotoxic Anabaena sp. Moore et al. 1989a, Cylindrospermum Hrouzek et al. 2012 alatosporum

Compound Chemical structure Activity Producer Reference Somocystinamide Anticancer Lyngbya Wrasido et al. 2008 majuscula

Spiroidesin Inhibits Anabaena spidoides Kaya et al. 2002 Microcystis aeruginosa

Suomilide Protease Nodularia spumigena Fujii et al. 1997 inhibitor Schindler et al. 2008

Compound Chemical structure Activity Producer Reference Taveuniamides Toxic to brine Lyngbya Williamson et al. 2004 shrimp majuscula and Schizothrix sp.

Drug discovery and development process

There is a constant need for more efficient and effective drugs to combat diverse diseases or clinical conditions and improve the quality of life for mankind. The process of drug discovery and development until the final approval of a product for marketing can take more than 12 years at a cost of over $ 1 billion (Gad 2005, Hughes et al. 2011). The drug discovery process begins with screening for bioactive compounds, their mechanism of action and the optimization of the compound’s activity and efficacy (Figure 2). After the selection of the best candidates, the compounds are tested on animal models during the preclinical trials to evaluate if the drug is safe to be tested on humans. The next step is to test the drug on humans during three-phased clinical trials (Figure 2). The aims of the clinical trials are to evaluate the safety and effectiveness of the compound. The results obtained will be reviewed by different agencies that will decide if the drug can be commercialized (Figure 2). The Food and Drug Administration (FDA) agency in the United States and the European Medicines Agency (EMA) of the European Union are examples of regulatory agencies that approve new drugs. The large-scale manufacture of a compound will be evaluated during the FDA review and might be a reason for any delay in the approval or denial of the drug’s commercialization. The FDA will continue to follow the adverse effects of a drug after its approval (post-marketing surveillance programme).

Figure 2. Drug discovery and development process. The average time spent on each phase is indicated below the rectangle. An asterisk (*) indicates the part of the process in which this doctoral thesis is involved.

Nearly half of the new drugs introduced in the marked during the period of 1981–2002 were natural products or semi-synthetic and synthetic compounds based on natural products (Koehn and Carter 2005). The interest of the research for new drug leads from natural products relies on the high chemical diversity and novelty found in these sources (Spainhour 2005). However, a decrease in pharmaceutical company investments in the discovery of natural products, especially related to infectious disease therapy, has been observed in past years (Koehn and Carter 2005). WHO has contradictingly warned of an increase in antimicrobial resistance indicating an urgent need for finding new antimicrobial drugs (WHO 2014). Advances obtained in synthetic chemistry (for the synthesis of complex natural products), modelling (of compounds and their targets to improve their activities) and combinatorial synthesis (to increase the diversity of compounds to be tested) (Koehn and Carter 2005) improve the success of finding new drugs based on natural products. The FDA and EMA have applied new facilitating measures in 2012 for antibiotic development that are leading to an increase in the research

32 of antimicrobial peptides by small companies (Fox 2013). This action is refuelling antimicrobial research and showing a new hope for the future.

2. Study aims

Genes involved in the synthesis of cyanobacterial bioactive peptides and their evolutionary origins were investigated, while the chemical characterizations of the compounds related to the biosynthetic genes were analyzed and their bioactivities were tested.

The specific aims of this doctoral thesis were: - to study the evolutionary history of a rare microcystin hepatotoxin based on mcy biosynthetic genes (I) - to describe the hassallidin gene cluster and the chemical variants produced by different cyanobacteria (II) - to detect cyanobacteria producing the cytotoxin anabaenolysin, to analyze its biological activity and to describe the gene cluster involved in anabaenolysin synthesis (III) - to screen cyanobacteria for the production of antifungal compounds (IV)

3. Summary of material and methods

The cyanobacterial strains used in the articles (I, II, III and IV) included in this doctoral thesis are listed in Supplementary Table S1. The methods used in each study are explained in detail in the articles. The different methods used in each article are indicated in Table 4.

Table 4. Methods used in this thesis.

Method Described in Cultivation of cyanobacterial strains I, II, III, IV

Molecular biology analysis DNA extraction I, II, III, IV PCR amplification I, II, III, IV Cloning I, II, III, IV Sequencing I, II, III, IV Site-directed mutation I Heterologous expression I

Biochemistry ATP-pyrophosphate exchange assay I

Bioinformatics Phylogenetic analyses I, II, III, IV Recombination test I Identification of gene cluster II, III Substrate prediction of adenylation domains I, II, III

Chemical analysis LC-MS I, II, III, IV Purification of compounds I, II, III, IV Stable isotope labelling (15N, 34S) I Amino acid analysis I, II, III NMR II, III, IV

Bioactivity Preparation of crude extracts II, III, IV Disk diffusion assay II, III, IV Microdilution assay II, III, IV

4. Summary of results and discussion

Anabaenolysin, hassallidin and microcystin gene clusters (I, II, III)

Cyanobacteria can produce different bioactive compounds through ribosomal or nonribosomal pathways. Figure 3 shows the genes and enzymes involved in the synthesis of three bioactive compounds investigated in this study. The present study described two new nonribosomal gene clusters involved in the synthesis of anabaenolysins and hassallidins (Figure 3A and B). The 23–24.3 kilo base pairs (kb) anabaenolysin gene cluster (abl) was located in the genomes of Anabaena spp. XSPORK2A, XPORK13A and XPORK15F, and predicted to be involved in the synthesis of anabaenolysin. The partial genomes of three Anabaena strains producing anabaenolysin B or C were obtained to unveil the biosynthetic genes of these compounds. The abl gene cluster consists of a hybrid NRPS and PKS, with additional enzymes such as the FADS (fatty acid desaturase) and a hypothetical protein. The NRPSs (AblA, AblD and AblE) contain three adenylation domains, which are predicted to activate and incorporate one aspartic acid and two glycines to anabaenolysin B. Anabaena sp. XPORK13A produces anabaenolysin C and the adenylation domain in the NRPS AblD activates and incorporates a glutamine instead of glycine. The FADS (AblC) might act in the creation of double bonds in the fatty acid chain and the PKS (AblB) in the addition of one malonyl-CoA unit and a hydroxyl group after the modified aspartic acid. Enzymes involved in the synthesis of the fatty acid chain and its incorporation in the anabaenolysin were not detected close to the putative gene cluster. However, two (XPORK13A and XPORK15F) or three (XSPORK2A) FAALs were detected elsewhere in the three genomes analyzed (III). NRPSs and tailoring enzymes constitute the 59 kb biosynthetic pathway of hassallidin (has) (II). Nine amino acids are activated and incorporated in the hassalliding by nine NRPS modules. Enzymes were found to be involved in the incorporation of fatty acid (acyl-protein synthetase, ligase and acyl carrier protein), sugars (glycosyltransferases), the acetylation of a sugar (acetyltransferase) and in the synthesis of the fatty acid chain (3- oxoacyl-acyl-carrier-protein). Interestingly, this gene cluster was found to be responsible for the synthesis of more than 40 different variants of hassallidins in Anabaena sp. SYKE 748A. The genome of Anabaena sp. 90 contained a putative hassallidin gene cluster (Wang et al. 2012). However, the deletion of 526 bp in the condensation domain of hasV turned into an inactive gene cluster, and this strain is no longer a producer of hassallidin (Wang et al. 2012).

Figure 3. Biosynthetic gene clusters analyzed in this study. A. Anabaenolysin gene cluster (abl) from Anabaena sp. XSPORK2A. B. Hassallidin gene cluster (has) from Anabaena sp. SYKE748A. C. Microcystin gene cluster (mcy) from Microcystis aeruginosa PCC 7806 (Modified from Tillett et al. 2000). The microcystin gene focused on study I is highlighted in pink. Amino acids or subunits that could be incorporated in the peptide are indicated under each module.

Microcystins are synthesized by a hybrid NRPS and PKS system first described in Microcystis aeruginosa PCC 7806 (Figure 3C, Tillett et al. 2000). The microcystin biosynthetic genes of Anabaena, Plankthotrix and Nostoc have been described before (Christiansen et al. 2003, Rouhiainen et al. 2004, Fewer et al. 2013). The present study focused on the analysis of microcystin genes, specifically the region of the McyA responsible for the incorporation of the amino acid in position one of the microcystin (I). The knowledge of the biosynthetic genes involved in toxin synthesis e.g. microcystin, improved the molecular methods for the detection of potential toxin producers in environmental samples (Dittmann et al. 2012). Knowledge of the enzymes involved in the synthesis of nonribosomal peptides additionally collaborates with further modifications in this system to improve production or modify the final product. The number of genome sequences from cyanobacteria available in databases, e.g. NCBI, is increasing. Currently there is an interest to link the information in the genome with the compounds produced by each strain. This study has succeeded in presenting two new biosynthetic gene clusters involved in the synthesis of two antifungal compounds.

Gene evolution involved in microcystin and anabaenolysin synthesis (I, III)

Cyanobacteria are ancient microorganisms and the selective forces leading to the evolutionary history of the NRPS and PKS genes are still not fully understood. Microcystin is a hepatotoxin produced by diverse cyanobacteria. They can be widely detected in strains from fresh- or brackish water, and cyanobacteria in lichen symbiosis (Sivonen and Jones 1999; Sivonen 2009, Lopes and Vasconcelos 2011, Kaasalainen et al. 2012). It was previously hypothesized that microcystin biosynthetic genes were horizontally transferred, and were later found to be more ancient in cyanobacteria (Rantala et al. 2004, Dittmann et al. 2012). Some cyanobacteria may have lost the mcy genes, while others may have experienced different selective forces (e.g. recombination) that allowed a huge diversity of microcystins to be synthesized by different strains. In the present study, the production of a rare variant of the hepatotoxin microcystin was found to be the result of convergent evolution in three distantly related cyanobacteria (I). The phylogenetic and recombination analyses indicated that the filamentous strains Nostoc sp. UK89IIa and Phormidium sp. CENA270 suffered gene conversion, while the unicellular strains Microcystis spp. NPLJ-4 and RST 9501 had point mutations in the mcyA gene (Figure 4 and I). Phylogenetic analysis revealed that these gene conversions and point mutations resulted in the synthesis of the same [D-Leu1]microcystin-LR variant by the studied strains (I). The evolutionary history based on genes involved in the microcystin synthesis (mcy) indicated recombination in the adenylation domain gene regions, but not in the other domains (condensation, peptidyl carrier protein and epimerization domains) (I). Recombination and positive selection in the adenylation domains of McyB and McyC has been related to the high variability of amino acids found in position 2 of the microcystins

(Mikalsen et al. 2003, Kurmayer et al. 2005, Fewer et al. 2007, Tooming-Klunderud et al. 2008, Christiansen et al. 2008). In this study (I), we show that McyA is also under recombination, which resulted in a rare variation in position 1 of the microcystin. The study of the enzymes involved in the synthesis of NRPSs and PKSs might contribute to further engineering and heterologous expression of new or known compounds. The results in study (I) indicates that the replacement of the entire adenylation domain, rather than using point mutations, could be a more efficient strategy for obtaining more specificity in the modification of NRPSs.

Figure 4. Independent evolutionary history of the McyA2 adenylation domain. Maximum- likelihood tree based on amino acid sequences inferred using MEGA 5. Bootstrap values above 50 per cent from 1000 respectively neighbour-joining, maximum parsimony and maximum-likelihood bootstrap replicates are given at the nodes. The studied strains are marked in bold and indicated with *. (From article I).

The gene cluster involved in the synthesis of the lipopeptide anabaenolysin is a hybrid of NRPS and PKS (III). A phylogenetic tree was obtained using the condensation domain sequences belonging to the anabaenolysin gene cluster. AblD and AblE grouped with other condensation domains that form the peptide bonds between two L- amino acids L ( CL) (III). However, AblA and other condensation domains present in hybrid NRPS/PKS

systems were grouped together, and have in common an aminotransferase and/or a monooxigenase preceding them (III). Interestingly, most of the 452 gene clusters from cyanobacteria recently analyzed presented a more complex evolutionary history than the vertical or horizontal gene transfer (Calteau et al. 2014). Different evolutionary mechanisms might be involved in the final gene cluster (Dittmann et al. 2012, Calteau et al. 2014). The evolutionary history of the different enzymes involved in nonribosomal biosynthesis helps to understand their organization and function in the unknown natural products gene clusters. A better understanding of highly recombinant regions in these genes might help lead to the rational modification of these systems according to an interest in producing more specific or variable peptide types.

Diversity of cyanobacterial strains producing the studied natural products (I, II, III, IV)

Natural products produced by cyanobacteria can be divided into different classes such as peptides, alkaloids, macrolides, fatty acids and indoles (Burja et al. 2001). Table 5 summarizes the compounds detected in this study to be produced by each genus of cyanobacteria.

Table 5. Compounds detected in different genera of cyanobacterial crude extracts.

Genera Compounds Study(s)

] ]

Leu

Hassallidin

Unidentified

Scytophycin

Cyclodextrin

Anabaenolysin Microcystin Anabaena + + + + II,III,IV Aphanizomenon + II Cylindrospermopsis + II Microcystis + I Fischerella + IV Phormidium + I Nostoc + + + + I,II,IV Scytonema + + IV Tolypothrix + II

Microcystis spp. from brackish water, a Nostoc sp. in lichen-association and a Phormidium sp. from freshwater were found to produce the same rare variant [D- Leu1]microcystin-LR (I). The Microcystis strains were found to also produce [Met1]microcystin-LR (I). Although Microcystis sp. RST 9501 produced higher amounts of [Met1]microcystin-LR, the strain Microcystis aeruginosa NPLJ-4 could produce more variants but in trace amounts, such as [Val1]microcystin-LR and [Phe1]microcystin-LR.

Most of the microcystins detected in previous studies have an alanine in position one (Sivonen and Jones 1999). This study (I) was the first description of a microcystin containing methionine in this position. Leucine (Leu or L) (Matthiensen et al. 2000, Park et al. 2001, Schripsema and Dagnino 2002, Albuquerque et al. 2007, Kaasalainen et al. 2012), glycine (Wood et al. 2008) and serine (Sivonen et al. 1992, Oksanen et al. 2004) have previously been reported. Interestingly, methionine (Met) is not commonly found in secondary metabolites (Caboche et al. 2010), and this could be due to the highly active sulfhydryl group. Microcystins have mainly been detected in planktonic strains, where the most common producers are Microcystis, Planktothrix and Anabaena (Sivonen 2009). However, benthic and terrestrial specimens are also able to produce microcystins (Izaguirre et al. 2007, Sivonen 2009, Kaasalainen et al. 2012). Cyanobacterial strains isolated from aquatic (fresh- and brackish water) and terrestrial (lichen-association and rocks) were described to produce hassallidins (II, IV). The Anabaena sp. SYKE 748A was found to produce a huge number of hassallidin variants (II). The hassallidins detected in this study varied regarding the size of the fatty acid chain, the amino acid present in position ten (glutamine (Q), tyrosine (Y) or threonine (T)), the sugars and their amounts present in the structure and the acetylation present in the hexose (Table 6).

Table 6. The structural variation of hassallidins in strains of cyanobacteria from Anabaena, Nostoc, Aphanizomenon, Tolypothrix and Cylindrospermopsis genera. Variation in aglyconic lipopeptides (A-M) can be explained by the hydrocarbon chain length (Cn = 14 -18, U = unknown) of the di-OH-fatty acid in position 1 and by amino acids in position 10 (Aa10; Q = glutamine, Y = tyrosine, X = unknown amino acid). Monosaccharides M1, M2 and M3 of hassallidins are N- acetylhexosamine (▲), pentose (▼), deoxyhexose (♦) and hexose (●), which contains 0–4 acetyl (Ac) groups. Monosaccharide positions have been deduced based on the identified positions in hassallidins A, B and D. Black squares indicate presence (Modified from article II). Aglyconic lipopeptide Monosaccharides

Code*: A B C D E F G H I J K L M M1 M2 M3 (Hex)

Cn: 14 15 16 18 14 15 16 18 18 U U U U 0 - 4 = no of Ac

Genus Aa10 : Q Q Q Q Y Y Y Y T X X X X 0 1 2 3 4

Anabaena ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ▲ ▼ ● ♦ ▼ ♦ ● ● ● ● ●

Nostoc ■ ■ ■ ▲ ● ▼ ♦ ●

Aphanizomenon ■ ▼ ▼ ● ●

Tolypothrix ■ ● ▼ ♦ ●

Cylindrospermopsis ■ ■ ▼ ● *A-M: 1220, 1234, 1248, 1276, 1255, 1269, 1283, 1311, 1249, 1232, 1241, 1282, 1298 m/z.

Hassallidins A and B have first been described in Hassallia sp. (Neuhof et al. 2005, 2006a). The present study was able to detect the production of hassallidins by different cyanobacteria, including bloom-forming strains such as Anabaena and Cylindrospermosis (II and IV). Aphanizomenon, Nostoc and Tolypothrix are also among the hassallidin producers (Table 5). Twenty-two isolated Anabaena strains producing hassalidins were detected (II and IV). Balticidins, compounds very similar to hassallidins, were recently detected in Anabaena cylindrica Bio33 isolated from the Baltic Sea (Bui et al. 2014). In

addition to the cyclic forms, hassallidins and balticidins (Bui et al. 2014) can also be detected in linear forms in the extracts, which indicate that the rings of these compounds are unstable. Anabaenolysins were detected only in Anabaena species (III). Most of the Anabaena strains producing anabaenolysins are grouped together in the phylogenetic tree, except Anabaena sp. XPORK15F (Figure 3 in III). All the anabaenolysin producers were isolated from brackish water in the Finnish coastal area. Anabaenolysins were first reported a few years ago in two benthic strains isolated from the Baltic Sea (Jokela et al. 2012). New variants of anabaenolysin C and D have been discovered in the present study (III). These new variants have a glutamine instead of a glycine in position three of the anabaenolysin. The two new variants differ from each other by one double bond in the fatty acid (III). Anabaena strains are also known to produce other lipopeptides such as hassallidins (Neuhof et al. 2005, 2006a and studies II and IV), puwainaphycins (Moore et al. 1989a, Hrouzek et al. 2012), laxaphycins (Frankmölle et al. 1992a,b), spiroidesin (Kaya et al. 2002), minutissamides (Kang et al. 2011, 2012) and balticidins (Bui et al. 2014). Anabaena strains producing anabaenolysins were also found to produce di- and tri- acetylated α-, β- and/or γ-cyclodextrins (III). These cyclodextrins have six, seven or eight hexoses that are decorated with two to three acetyl groups. Interestingly, cyclodextrins are cyclic oligosaccharides that can form complexes and are used to improve the solubility of hydrophobic compounds (Nardello-Rataj and Leclercq 2014). Cyclodextrins have a conical shape and commonly have six (α-), seven (β-) or eight (γ-) glucose units (Nardello-Rataj and Leclercq 2014). Cyclodextrins are used in the pharmaceutical industry for drug delivery, the encapsulation of biocides and in food and cosmetics products (Szejtli 1998, Astray et al. 2009, Ruiz et al. 2014, Nardello-Rataj and Leclercq 2014). Scytophycin is a macrolide, which contains a macrocyclic lactone ring. It has been discovered in Scytonema spp. (Ishibashi et al. 1986, Carmeli et al. 1990a) and later detected in Nostoc sp. (Tomsickova et al. 2013). In this study, we found new and known scytophycin variants being produced from Anabaena, Nostoc and Scytonema (IV). This is the first report of Anabaena strains producing scytophycins. Several compounds have been described as structurally related to scytophycins, e.g. tolytoxin (Carmeli et al. 1990a, Ishibashi et al. 1986), swinholides, aplyronine, sphinxolides/reidispongiolides and ulapualides/kabiramide/halichondramides/mycalolides (Yeung and Paterson 2002). Interestingly, these related compounds were detected in cyanobacteria, sea hare, sponges and their symbionts, nudibranches and coral (see discussion in IV). Three cyanobacterial strains were detected to show antifungal activity in their crude methanol extract: Fischerella sp. CENA 298, Scytonema hofmanni PCC 7110 and Nostoc sp. N107.3 (IV). Further analyses are necessary to identify their bioactive compound.

Activity of cyanobacterial natural products analyzed in this study (II, III, IV)

This study detected known and unidentified antifungal compounds produced by cyanobacteria. Hassallidin D produced by Anabaena sp. SYKE748A had a minimum inhibitory concentration (MIC) of ≤ 2.8 µg/mL (1.5 µM) against Candida albicans (II). Interestingly, the linear form of hassallidin D had an MIC of 36 µg/mL (20 µM) (II). The linear hassallidin was less potent than the cyclic version, but nevertheless it was still active against Candida albicans. Compounds might usually lose practically all of their activity when linearized (Choi et al. 1993, Herfindal et al. 2011).

Isolated anabaenolysin B from Anabaena sp. XSPORK27C did not show strong inhibition to Candida albicans (MIC of 1.47 mg ml-1 and a half maximum inhibitory -1 concentration or concentration at 50% inhibition (IC50) of 1.15 mg ml ; 2.1 mM). However, when combined to a mixture of di- and tri-acetylated α-, β- and γ-cyclodextrins produced by anabaenolysins producers, the antifungal activity was improved (IV). Cyclodextrins are known to form complexes with diverse compounds to improve their solubility. Fatty acid synthesis can be stimulated in the presence of cyclodextrins in Mycobacterium phlei (Machida et al. 1973). This complex cyclodextrin-fatty acid would avoid a high number of free fatty acids and stimulate the cell to continue its production (Machida et al. 1973). A complex formed of cyclodextrin and tolypophycins produced by the cyanobacterium Tolypothrix byssoidea blocked the antifungal activity (Entzeroth et al. 1986). However, in this study we described an increase in antifungal activity when cyclodextrins and anabaenolysin are combined (III). Similarly, γ-cyclodextrin and amphotericin B showed an up to 60% increase in antifungal activity (Ruiz et al. 2014). Cyclodextrins have been used for diverse purposes and recent studies describe cyanobacterial hepatotoxins microcystins and nodularins to be able to strongly bind γ- cyclodextrins, which could be used as a strategy to remove these toxins from the environment (Chen et al. 2011). Scytophycins are potent antifungal and cytotoxic compounds (Moore et al. 1989b, Carmeli et al. 1993). The 7-OMe-scytophycin-B derivative isolated from Anabaena sp. -1 HAN21/1 presented an MIC/IC50 of 0.33/0.16 mg ml (0.40/0.19 mM) for Candida albicans HAMBI 484 and 0.67/0.29 mg ml-1 (0.80/0.23 mM) for Candida guilliermondi HAMBI 257 (IV). Interestingly, a photocatalytic conversion of the 7-OMe-scytophycin-B might have affected antifungal activity (IV). Previous studies showed that Scytonema ocellatum increased the synthesis of tolytoxin (6-hydroxy-7-O-methyl-scytophycin B) in the presence of the fungal cell wall polyssacaride chitin (Patterson and Bolis 1997). Unidentified antifungal compounds were found in this study to be produced by Fischerella sp. CENA 298, Scytonema hofmanni PCC 7110 and Nostoc sp. N107.3 (IV). Preliminary results indicate that the antifungal compound of Nostoc sp. N107.3 does not have nitrogen and is purely polyketidic. Further analyses are needed to identify the chemical structure of these compounds. Some information is known concerning the cytotoxicity of the antifungal compounds analyzed in this study. Hassallidin B presented anticancer activity against human acute T cell leukemia (Jurkat ATCC-TIB-152; IC50 of 0.2 µg/ml) and murine aneuploid fibrosarcoma (L 929; at concentration of 1.0 µg/ml) (Neuhof et al. 2006c). Anabaenolysin lyses cells in a cholesterol-dependent manner and showed a higher haemolythic activity than the digitonin from plants (Oftedal et al. 2012). Scytophycins are potent cytotoxins against several human cancer cell lines, e.g. KB human epidermoid carcinoma (1 ng/ml) (Moore et al. 1989b, Yeung and Paterson 2002). Studies indicate that scytophycin affects the actin cytoskeleton at 100- to 1000-fold potency compared to the fungal compound cytochalasin (inhibits the polymerization of G-actin and depolymerization of F-actin) (Yeung and Paterson 2002). However, more studies concerning the bioavailability of these compounds, their action on biofilms and microbial selectivity are needed for further applications by the pharmaceutical industry. The fungal strains used in the present study were opportunistic fungi and additional tests performed with pathogenic fungal strains would provide further information of the antifungal activity of the new chemical variants studied here. Cyanobacteria are known to be a prolific source for bioactive compounds (Abed et al. 2008, Singh et al. 2011). Remarkably, we detected different strains from diverse environments producing known and yet to be discovered antifungal compounds.

5. Final remarks and future considerations

The analysis of 418 cyanobacteria isolates from diverse environments was accomplished in this study. Two new biosynthetic gene clusters were described (II and III). Analysis of the nonribosomal and nonribosomal/polyketide hybrid gene clusters aid in understanding the function of multimodular enzymatic systems producing hassallidin and anabaenolysin, respectively. The increased number of cyanobacterial genomes available in the database recently shows the huge information source for finding new biosynthetic gene clusters involved in the synthesis of natural products. However, interdisciplinary work in chemistry, biology and bioinformatics are necessary to uncover the unknown gene clusters. The connection between biosynthetic genes and their products is important for providing new information for further synthesis and biotechnological studies. The evolutionary analysis improved our knowledge concerning the well-known microcystin biosynthetic gene cluster (I) and helped in the analysis of the newly discovered anabaenolysin gene cluster (III). The analysis, based on the evolution of genes involved in the synthesis of natural products, indicates highly variable genetic regions in these biosynthetic machineries. The heterologous expression of more diverse or more specific peptides could be obtained with modifications in these highly variable genetic regions. The results obtained in this doctoral study indicate that the replacement of the entire adenylation domain could result in the synthesis of more specific products (I). This allows the use of available resources for the synthesis of one or a few chemical variants and for facilitating target compound isolation. Although cyanobacteria only require a medium based on minerals and light for growth, they are slow-growing organisms. Heterologous expression of cyanobacterial compounds aids in obtaining a high yield and quicker synthesis of the target compounds. Understanding the evolutionary history of these genes might additionally help explain why cyanobacteria are still able to produce the respective natural products. The chemical analysis included in this study improved our knowledge concerning different nonribosomal peptides and polyketides synthesized by the isolated cyanobacterial strains. The hepatotoxin microcystins are commonly detected in planktonic strains. A benthic and a lichen-associated cyanobacteria were found to produce microcystins (I), improving the information of cyanobacteria from different environments able to synthesize this well studied toxin. The new [Met1]microcystin was detected in planktonic Microcystis spp. cell extracts (I), increasing the number of microcystin variants known to the date. Hassallidin, anabaenolysin synergistically acting together with cyclodextrins, scytophycin and unidentified compounds presented antifungal activities (II, III, IV). Anabaena sp. 90 was a natural mutant of hassallidin due to a deletion that occurred some time after cultivation in the culture collection. Contrastingly, although some of the cyanobacterial strains have been isolated and cultivated for almost 30 years, they are still able to produce antifungal hassallidin (II). Interestingly, over 40 variants of hassallidin were detected in the Anabaena sp. SYKE748A cell extract, additionally with information of other new genera detected as hassallidin producers such as Aphanizomenon, Cylindrospermopsis, Nostoc and Tolypothrix (II). Hassallidins have shown some level of cytotoxicity and the synthesis of this compound by bloom-forming strains, e.g. Cylindrospermopsis, indicates the importance to deeply study the possible role of hassallidins in human and animal poisoning cases and further to improve the monitoring of water used for drinking and recreation.

Cyclodextrins were discovered over 100 years ago and are nowadays widely used by the pharmaceutical, food and chemical industries. Cyclodextrins have the ability to solubilize hydrophobic compounds and can be used to improve solubility and the delivery of drugs. The present study indicates that this method is also applied by cyanobacteria that showed an increase in antifungal activity by anabaenolysins combined with cyclodextrins (III). Due to an increase in antifungal resistence and the need for new antifungal drugs, a search for new bioactive compounds is currently needed. Three cyanobacterial strains were found to produce unidentified compounds that open the possility of finding new antifungal drugs (IV). Unfortunately, there are disadvantages in studying cyanobacteria for drug discovery such as the difficulties faced in genetic manipulation, solving the complex structures of the compounds produced and the slow growth of these organisms compared to some bacterial strains. However, with current advances in the study of chemical biology and biotechnology, there is a higher chance of success in the synthesis of cyanobacterial compounds for a broad range of uses. The fact that cyanobacteria have lived on Earth for 3500 million years in a daily combat against parasitic fungi or in competition with fungi in the natural environments, make them a great source for antifungal drug discovery. To overcome the problems faced by cyanobacterial drug research, more studies are necessary in this field.

6. Acknowledgements

I would like to thank the financial support from the Academy of Finland, the Graduate Program in Biotechnology and Molecular Biology, currently the Integrative Life Science Doctoral Program, São Paulo Research Foundation (2009/13455-0), Centre for International Mobility (TM-09-6506) and Finnish Cultural Foundation. This work was carried out in the Division of Microbiology and Biotechnology, Department of Food and Environmental Sciences, Faculty of Agriculture and Forestry, University of Helsinki. I am deeply grateful to my supervisors Professor Kaarina Sivonen, Docent David Fewer and Docent Jouni Jokela for their great guidance, support and mentoring during these five years in Finland. I want to express my gratitude to Professor Kaarina Sivonen for the opportunity to be part of the Cyanobacteria group in the University of Helsinki, for all the learning and teaching opportunities and the freedom that allowed me to fly with my own wings. I am deeply grateful to Docent David Fewer for the discussions and supervisions over these years and for his friendship, and to Docent Jouni Jokela for all the support and teaching about the chemistry world. I would like to thank Professor Elke Dittmann and Professor Pia Vuorela for reviewing and improving my doctoral thesis. I am thankful to Professor Mirja Salkinoja-Salonen and Docent Päivi Tammela for the important comments and suggestions during my thesis committee meetings. My sincere thanks go to Matti Wahlsten for all the support, teaching and discussions about science and Lyudmila Saari for all the help with our cyanobacteria. I wish to thank my co-authors for the fruitful collaboration that resulted in the four articles included in this doctoral thesis: Kaarina Sivonen, David P. Fewer, Jouni Jokela, Matti Wahlsten, Leo Rouhiainen, Perttu Permi, Hao Wang, Marli de Fátima Fiore, Ulla Kaasalainen, Jouko Rikkinen, João Sarkis Yunes, Johanna Vestola, Olli Aitio, Clara- Theresia Kumer, Ermano Rizzi, Gianluca Debellis, Liwei Liu, Anu Humisto, Anisha Tamrakar, and Ana P. D. Andreote. I would like to thank the support and help from the technicians and employees from the Division of Microbiology and Biotechnology. Many thanks to the Cyanobacterial group, alumni and present members, for all the experiences exchanged and for the good times in conferences, meetings, lunches and coffees. I wish to thank all my colleagues and friends for the nice time spent during these five years. Obrigada à minha querida família no Brasil que durante esses anos me apoiou e incentivou a correr atrás dos meus objetivos. Haluaisin kiittää myös perhettäni Suomessa kaikesta saamastani tuesta ja kaikista yhdessä vietetyistä hienoista hetkistä. I would like to thank my Finnish family for all the support and special moments shared. And last but not least, countless thanks to my beloved husband Janne for all his support, help, care, patience, love, et cetera.

7. References

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Supplementary Table S1. Strains used in the present doctoral thesis are indicated in gray or black depending on which study they have been included from. Strains detected producing the studied cyanobacterial compound(s) are highlighted in black. Study

Year of 16S rRNA

II hassallidinII I microcystin I isolation sequences antifungalIV Genera Strain City/State or State anabaenolysin III Anabaena Anabaena sp. 0TU33S16 Lake Tuusulanjärvi, Finland 2000 II Anabaena sp. 0TU43S7 Lake Tuusulanjärvi, Finland 2000 II Anabaena sp. 0TU43S8 Lake Tuusulanjärvi, Finland 2000 II Anabaena sp. 1TU23S3 Lake Tuusulanjärvi, Finland 2001 II Anabaena sp. 1TU26S10 Lake Tuusulanjärvi, Finland 2001 II Anabaena sp. 1TU28S8 Lake Tuusulanjärvi, Finland 2001 II Anabaena sp. 1TU28S13 Lake Tuusulanjärvi, Finland 2001 II Anabaena sp. 1TU32S11 Lake Tuusulanjärvi, Finland 2001 II Anabaena sp. 1TU33S8 Lake Tuusulanjärvi, Finland 2001 AJ6630432 II Anabaena sp. 1TU33S10 Lake Tuusulanjärvi, Finland 2001 II Anabaena sp. 1TU35S12 Lake Tuusulanjärvi, Finland 2001 AJ630423 II III Anabaena sp. 1TU36S8 Lake Tuusulanjärvi, Finland 2001 II Anabaena sp. 1TU36S9 Lake Tuusulanjärvi, Finland 2001 II Anabaena sp. 1TU39S17 Lake Tuusulanjärvi, Finland 2001 II Anabaena sp. 1TU44S9 Lake Tuusulanjärvi, Finland 2001 II Anabaena sp. 1TU44S16 Lake Tuusulanjärvi, Finland 2001 II Anabaena sp. 14 Lake Sääskjärvi, Finland 1985 II Anabaena sp. 37 Lake Sääskjörvi, Finland 1985 III IV Anabaena sp. 54 Lake Sääskjärvi, Finland 1986 II Anabaena sp. 66 Lake Kiikkara, Finland 1986 II Anabaena sp. 66B Lake Kiikkara, Finland 1986 II Anabaena sp. 79 Lake Sääskjärvi, Finland 1986 II Anabaena sp. 82 Lake Karpjärvi, Finland 1986 II Anabaena sp. 86 Lake Villikalanjärvi, Finland 1986 II Anabaena sp. 90 Lake Vesijärvi, Finland 1986 AJ133156 I II Anabaena sp. 90 M3 (apdA- Knock out strain of Anabaena sp. 1999 II mutant) 90 (Rouhiainen et al. 2000) Anabaena sp. 130 Lake Säyhteenjärvi, Finland 1986 II Anabaena sp. 141 Lake Vesijärvi, Finland 1986 II Anabaena sp. 191A2 Lake Rautjärvi, Finland 1986 II Anabaena sp. 202A1 Lake Vesijärvi, Finland 1987 II Anabaena sp. 202A1/35 Lake Vesijärvi, Finland 1987 II Anabaena sp. 202A2 Lake Vesijärvi, Finland 1987 II Anabaena sp. 258 Lake Tuusulanjärvi, Finland 1990 II Anabaena sp. 288 Lake Littoistenjärvi, Finland 1990 II Anabaena sp. 293 Lake Tuusulanjärvi, Finland 1992 II Anabaena sp. 299A Lake Vesijärvi, Finland 1992 II Anabaena sp. 299B Lake Vesijärvi, Finland 1992 II Anabaena sp. 301 The Baltic Sea, Gulf of Finland 1992 II Anabaena sp. 318 Helsinki coast, “39A”, BS 1998 III planktonic Anabaena sp. 326 Lake Lohjanjärvi, Finland 1998 II

Anabaena sp. 335/1 River Juupajoki, Finland 1999 II Anabaena sp. AnapI-Bos00 Unknown unknown II Anabaena sp. Anapl-Nech00 Nechranice reservoir, North-West 2000 II Bohemia, Czech Republic Anabaena sp. ANASP-URB Urbensky pond, Czech Republic unknown II Anabaena sp. Anaspi-Vrb00 Vrbenský pond, South-Bohemia, 2000 II Czech Republic Anabaena variabilis ATCC 29413 Mississippi, USA Freshwater 1964 NR_074300 IV Anabaena sp. BECID19 The Baltic Sea, Pikku-Kallahti, 2001 EF583857 II III Finland Anabaena sp. BECID 20 The Baltic Sea, Pikku-Kallahti, 2001 II III Finland, epiphytic Anabaena Helsinki, Vuosaari, Gulf of AJ630426 III BECID 22 oscillaroides Finland, epiphytic 2001 Anabaena sp. BECID 23 The Baltic Sea, Kallahti, Finland, 2001 II III epiphytic Baltic Sea, Helsinki, Finland; III Anabaena sp. BECID 30 epilithic 2001 Anabaena sp. BECID 32 The Baltic Sea, Gulf of Finland, 2001 III Pikku-Kallahti, Finland Anabaena sp. BECID38 The Baltic Sea, Pikku-Kallahti, 2001 II Finland Baltic Sea, Gulf of Finland III BIR 3 Anabaena sp. planktonic 2003 Baltic Sea, Gulf of Finland III BIR 25 Anabaena sp. planktonic 2004 Anabaena sp. BIR 32 III Anabaena sp. BIR 42 III Baltic Sea, Gulf of Finland III BIR 49 Anabaena sp. planktonic 2004 Baltic Sea, Gulf of Finland III BIR 52 Anabaena sp. planktonic 2004 Anabaena sp. BIR 54 The Baltic Sea, Gulf of Finland 2004 II Anabaena sp. BIR 78 The Baltic Sea, Gulf of Finland, 2004 II III planktonic Baltic Sea, Gulf of Finland III BIR 84 Anabaena sp. planktonic 2004 Baltic Sea, Gulf of Finland III BIR 94 Anabaena sp. planktonic 2004 Baltic Sea, Gulf of Finland III BIR 162 Anabaena sp. planktonic 2004 Baltic Sea, Gulf of Finland III BIR 208 Anabaena sp. planktonic 2004 Anabaena sp. BIR 219 The Baltic Sea, Gulf of Finland 2004 II Baltic Sea, Gulf of Finland III BIR 241 Anabaena sp. planktonic 2004 Baltic Sea, Gulf of Finland III BIR 246 Anabaena sp. planktonic 2004 Baltic Sea, Gulf of Finland III BIR 247 Anabaena sp. planktonic 2004 Anabaena sp. BIR 256 The Baltic Sea, Gulf of Finland, 2004 II III planktonic Anabaena sp. BIR 257 The Baltic Sea, Gulf of Finland 2004 II Baltic Sea, Gulf of Finland III BIR 258 Anabaena sp. planktonic 2004 Anabaena sp. BIR 259 The Baltic Sea, Gulf of Finland 2004 II Baltic Sea, Gulf of Finland III BIR 260 Anabaena sp. planktonic 2004 Baltic Sea, Gulf of Finland III BIR 348 Anabaena sp. planktonic 2004 Baltic Sea, Gulf of Finland III BIR 441 Anabaena sp. planktonic 2004 Anabaena sp. BIR JV1 The Gulf of Finland IV

Rio Camarão/CE river water, 2009 IV Anabaena sp. CENA 247 Brazil Kobben, Hanko, Finland small 2012 IV Anabaena sp. HAN 7/1 stones in the water Kobben, Hanko, Finland 2012 + IV Anabaena sp. HAN 15/2 Gastropod from waterline Kobben, Hanko, Finland 2012 + IV Anabaena sp. HAN 21/1 Gastropod 10 cm under water Hanko Casino sea shore, Hanko, 2012 + IV Anabaena sp. HAN 37/1 Finland green biofilm Hiidenvesi, Kiihkelyksenselkä IV Anabaena sp. HIID D7A 1999 sediment Anabaena sp. NIES-73 Lake Kasumigaura, Japan 1978 II Anabaena sp. NIVA 269/2 Lake Frøylandsvatnet, Norway 1990 II Anabaena sp. NIVA 269/6 Lake Frøylandsvatnet, Norway 1990 II Anabaena sp. NIVA 270/1 Lake Arefjordsvatnet, Norway 1990 II Anabaena sp. NIVA-CYA Lake Edlandsvatnet, Norway 1981 II 83/1 Anabaena sp. NIVA-CYA Lake Fammestadtjønni, Norway 1990 II 267/4 Anabaena sp. PH 57 Lake Veljesø, Denmark 1993 II Anabaena sp. PH 71 Lake Furesø, Denmark 1993 II Anabaena sp. PH 118 Lake Langesø, Denmark 1993 II Anabaena sp. PH 133 Lake Arresø, Denmark 1993 AJ293110 II IV Anabaena sp. PH 189 Lake Tuel, Denmark 1993 II Anabaena sp. PH 256 Lake Knud, Denmark 1994 II Anabaena sp. PH 262 Lake Knud, Denmark 1994 II Anabaena sp. PMC 9701 Champsanglard dam, France 1997 II Anabaena sp. PMC 9702 Champsanglard dam, France 1998 II Moss Beach, California USA 1970 AJ133162 IV Anabaena sp. PCC 7108 interdidal zone Anabaena sp. PCC 7122 Pond, England 1939 AF091150 II IV Anabaena sp. PCC 9208 Soil, Galicia, Spain unknown II Anabaena sp. Schmidke Rostock, Germany unknown II JAHNKE/4 Anabaena sp. SYKE 658 Lake Enäjärvi, Finland planktonic 1999 II III Anabaena sp. SYKE 701 Lake Enäjärvi, Finland unknown II Anabaena sp. SYKE 741 Maarian allas, Finland 1999 II III Anabaena sp. SYKE 748A Lake Tuusulanjärvi, Finland 1999 KF631396 II III Anabaena sp. SYKE 763A Lake Tuusulanjärvi, Finland 1999 II Anabaena sp. SYKE 790 Halikko, Finland unknown II Anabaena sp. SYKE 816 The Baltic Sea, Gulf of Finland 1999 II Anabaena sp. SYKE844/16 Kisakallio, Finland unknown II Anabaena sp. SYKE 844A Kisakallio, Finland 1999 III Anabaena sp. SYKE 844B Lake Lohjanjärvi, Finland 1999 II Anabaena sp. SYKE 844C Kisakallio, Finland 1999 II Anabaena sp. SYKE 966 Lake Littoistenjärvi, Finland unknown II Anabaena sp. SYKE 971/6 Lake Kotojärvi, Finland 1999 + II Anabaena sp. SYKE 971/9 Lake Kotojärvi, Finland 1999 II Anabaena sp. SYKE 1228 Joutikas, Finland 1999 II Anabaena sp. TR 232 The Baltic Sea, Gulf of Finland 1993 II Anabaena sp. Hiidenvesi, Mustionselkä IV XHIID B2A 1999 sediment, Finland Anabaena sp. Hiidenvesi, Mustionselkä IV XHIID B6 1999 sediment, Finland Anabaena sp. XPORK 1C The Baltic Sea coast, Porkkala 1999 II III cape, Finland Anabaena sp. XPORK 1D The Baltic Sea coast, Porkkala 1999 II III

cape, Finland Anabaena sp. XPORK 4C The Baltic Sea coast, Porkkala 1999 III cape, Finland, epiphytic Anabaena sp. XPORK 4D The Baltic Sea coast, Porkkala 1999 III cape, Finland, epiphytic Anabaena sp. XPORK 5C The Baltic Sea coast, Porkkala 1999 II III cape, Finland Baltic Sea, Cape Porkkala benthic III XPORK 6A Anabaena sp. sediment 1999 Anabaena sp. XPORK 6B The Baltic Sea coast, Porkkala 1999 II IV cape, Finland Anabaena sp. XPORK 6C The Baltic Sea coast, Porkkala 1999 II III cape, Finland Anabaena sp. XPORK 13A The Baltic Sea coast, Porkkala 1999 II III cape, Finland Anabaena sp. XPORK 15D The Baltic Sea coast, Porkkala 1999 II III cape, Finland Anabaena sp. XPORK 15F Baltic Sea, Cape Porkkala benthic 1999 III Anabaena sp. XPORK 34A The Baltic Sea coast, Porkkala 1999 II cape, Finland Baltic Sea, Cape Porkkala III XPORK 36C Anabaena sp. periphytic 1999 Anabaena sp. XPORK 36D The Baltic Sea coast, Porkkala 1999 II III cape, Finland Baltic Sea, Cape Porkkala benthic, III Anabaena sp. XSPORK 2A gastropod 1999 Anabaena sp. XSPORK 7B The Baltic Sea coast, Porkkala 1999 KF631399 II III cape, Finland Anabaena sp. XSPORK 14D The Baltic Sea coast, Porkkala 1999 II III cape, Finland Anabaena sp. XSPORK 27C The Baltic Sea coast, Porkkala 1999 II III cape, Finland Anabaena sp. XSPORK 36B The Baltic Sea coast, Porkkala 1999 II cape, Finland

Aphanizomenon Aphanizomenon 0tu37s7 Lake Tuusulanjärvi, Finland 2000 II III issatschenkoi Aphanizomenon sp. 1TU26S2 Lake Tuusulanjärvi, Finland 2001 II III Aphanizomenon sp. 1TU29S13 Lake Tuusulanjärvi, Finland 2001 II III Aphanizomenon 1tu37s13 Lake Tuusulanjärvi, Finland 2001 II flos-aquae Aphanizomenon sp. 3 Lake Långsjön, Åland Island 1985 II III Aphanizomenon sp. 37 Lake Sääskjärvi, Finland 1985 II Aphanizomenon sp. 201 Lake Bodomjärvi, Finland 1987 II III Aphanizomenon sp. 313 Lake Tuusulanjärvi, Finland 1997 II III

Aphanizomenon sp. 326 Lake Lohjanjärvi, Finland 1998 II III Aphanizomenon sp. 335/4 Juupajoki, Kukkolahti, Finland 1999 II III Aphanizomenon Heaney/Camb Freshwater, Lough Neagh, Ireland 1986 AJ630444 II gracile 1986 140 1/1 Aphanizomenon sp. SYKE 761/II Raisio-Naantali, Finland unknown II III

Aphanocapsa Morro Branco/CE water 2009 IV Aphanocapsa sp. CENA 223 accumulated, Brazil

Aphanothece Hiidenvesi, Kiihkelyksenselkä IV Aphanothece sp. XHIID D1 1999 sediment

Calothrix Calothrix sp. 441/2 Lake Säyhteenjärvi,Finland III Hel., Matosaari, BS, Fin. III BECID 1 Calothrix sp. periphytic 2001 Baltic Sea, Gulf of Finland III BECID 4 Calothrix sp. Brackish water 2001 Baltic Sea, Gulf of Finland, III BECID 9 Calothrix sp. epilithic 2001 Calothrix sp. BECID 16 The Baltic Sea, Helsinki, Finland; 2001 II epilithic Calothrix sp. BECID 18 The Baltic Sea, Helsinki, Finland; 2001 II III sediment benthic Calothrix sp. BECID 26 Baltic Sea, Vuosaari, epilithic 2001 III Helsinki, Vuosaari, Gulf of III BECID 33 Calothrix sp. Finland, epilithic 2001 Calothrix sp. CAL336/1 Lake Enäjärvi, Finland 1999 II III Calothrix sp. Cal 336/2 Vihti, Enäjärvi Laukilanlahti IV Calothrix sp. Cal 336/3 Lake Enäjärvi, Finland Lake water 1999 II III IV Calothrix sp. CENA 283 Piauí soil, Brazil 2009 IV Kobben, Hanko, Finland Growth 2012 + IV Calothrix sp. HAN 6/3 on rock waterline Kobben, Hanko, Finland Growth 2012 + IV Calothrix sp. HAN 6/4 on rock waterline Kobben, Hanko, Finland Growth 2012 + IV Calothrix sp. HAN 8/2 on rock waterline Kobben, Hanko, Finland black 2012 + IV Calothrix sp. HAN 12/2 spots film, benthic Kobben, Hanko, Finland 2012 + IV Calothrix sp. HAN 16/2 Waterplant from shallow water Kobben, Hanko, Finland Red 2012 + IV Calothrix sp. HAN 17/1 biofilm Kobben, Hanko, Finland 2012 + IV Calothrix sp. HAN 19/2 black/green biofilm from waterline Kobben, Hanko, Finland sediments 2012 + IV Calothrix sp. HAN 20/3 10 cm under water Kobben, Hanko, Finland 2012 + IV Calothrix sp. HAN 21/3 Gastropod 10 cm under water Kobben, Hanko, Finland 2012 + IV Calothrix sp. HAN 21/4 Gastropod 10 cm under water Kobben, Hanko, Finland 2012 + IV Calothrix sp. HAN 21/5 Gastropod 10 cm under water Kobben, Hanko, Finland Black 2012 + IV Calothrix sp. HAN 22/1 biofilm Hanko Casino sea shore, Hanko, 2012 + IV Finland Green biofilm and Calothrix sp. HAN 30/2 Gastropod Hanko Casino sea shore, Hanko, 2012 + IV Finland Green biofilm and Calothrix sp. HAN 30/3 Gastropod Hanko Casino sea shore, Hanko, 2012 + IV Calothrix sp. HAN 33/1 Finland brown biofilm on the rock Hanko Casino sea shore, Hanko, 2012 + IV Calothrix sp. HAN 33/2 Finland Brown/yellow biofilm Hanko Casino sea shore, Hanko, 2012 + IV Calothrix sp. HAN 36/2 Finland Biofilm Hanko Casino sea shore, Hanko, 2012 + IV Calothrix sp. HAN 37/2 Finland green biofilm Hanko Casino sea shore, Hanko, 2012 + IV Calothrix sp. HAN 37/3 Finland Green biofilm Hanko Casino sea shore, Hanko, 2012 + IV Calothrix sp. HAN 38/2 Finland Red biofilm Calothrix sp. PCC 7102 Antofagasta, Chile sand 1958 IV Unknown herbarium specimen of AM230700 IV Calothrix sp. PCC 7103 Anacystis Montana

Calothrix sp. PCC 7507 Sphagnum bog ,Vierwaldstättersee 1972 NR_102891 II III IV Calothrix sp. PCC 7714 Pool, Aldabra Atoll, India 1972 II III Calothrix sp. PCC 7715 Thermal spring, Dax, France 1964 II III Calothrix sp. UHCC 0022 IV Calothrix sp. XP 10A The Baltic Sea coast, Porkkala 1999 II III cape, Finland, epilithic Calothrix sp. XP 11C The Baltic Sea coast, Porkkala 1999 II III cape, Finland benthic sediment Calothrix sp. XPORK 2B The Baltic Sea coast, Porkkala 1999 II cape, Finland benthic, periphytic Calothrix sp. XPORK 4B The Baltic Sea coast, Porkkala 1999 II cape, Finland Calothrix sp. XSPORK 3 The Baltic Sea coast, Porkkala 1999 II III cape, Finland Calothrix sp. XSPORK 4A The Baltic Sea coast, Porkkala 1999 II III cape, Finland epiphytic Baltic Sea, Cape Porkkala, III Calothrix sp. XSPORK 10A epilithic 1999 Calothrix sp. XSPORK 36C The Baltic Sea coast, Porkkala 1999 II III cape, Finland periphytic

Cyanobium Cyanobium sp. HIID A3A Hiidenvesi, Kirkkojärvi sediment 1999 IV

Cyanospira Morro Branco/CE water IV Cyanospira sp. CENA 215 accumulated 2009

Cylindrospermopsis Cylindrospermopsis ATC-9502 Lake Balaton, Hungary 1994 II raciborskii Cylindrospermopsis CS505/8 Freshwater, Solomon Dam, North 1996 EU552055 II IV raciborskii Queensland, Australia

Cylindrospermum Cylindrospermum PCC 7417 Greenhouse soil, Stockholm, 1972 NR_114701 II III IV sp. Sweden Cylindrospermum Hiidenvesi, Nummelanselkä IV XHIID C12 1999 sp. sediment

Fischerella Fischerella sp. CENA161 Dam Brazil 2004 EU840724 I Fischerella sp. PCC 7414 Hot spring, New Zealand unknown II III Fischerella sp. PCC 9339 Unknown unknown AB075984 IV Fischerella SAG 1427-1 Freswater, Rice field, India 1951 II III muscicola

Geitlerinema Geitlerinema sp. Morro Branco/CE water 2009 IV CENA 224 accumulated, Brazil Geitlerinema sp. Açude São Mateus/CE water used 2009 IV CENA 228 by population (lake), Brazil Geitlerinema sp. Sodrelândia/BA water in the pond, 2009 IV CENA 252 Brazil

Gloeotrichia Gloeotrichia sp. Rio Camarão/CE river water, 2009 IV CENA 250 Brazil Gloeotrichia sp. Paulista/PA water in the pond, 2009 IV CENA 272 Brazil

Gloeotrichia sp. CENA 285 Piauí soil, Brazil 2009 IV Gloeotrichia sp. CENA 288 Ceará soil, Brazil 2009 IV

Hapalosiphon Hapalosiphon BZ-3-1 Soil or freshwater mud sample, unknown EU151900 II III hibernicus Oahu, Hawaii

Leptolyngbya Leptolyngbya sp. Açude São Mateus/CE water used 2009 IV CENA 229 by population (lake), Brazil Leptolyngbya sp. Sodrelândia/BA water in the pond, 2009 IV CENA 254 Brazil Leptolyngbya sp. Sodrelândia/BA water in the pond, 2009 IV CENA 256 Brazil Leptolyngbya sp. CENA 282 Piauí soil, Brazil 2009 IV Leptolyngbya sp. CENA 292 Ceará soil, Brazil 2009 IV Leptolyngbya sp. CENA 299 Rio Grande do Norte soil, Brazil 2009 IV Hiidenvesi, Kirkkojärvi sediment, IV Leptolyngbya sp. HIID A16A 1999 Finland Hiidenvesi, Kirkkojärvi sediment, IV Leptolyngbya sp. HIID A19B 1999 Finland Hiidenvesi, Kirkkojärvi sediment, II IV Leptolyngbya sp. HIID A25A 1999 Finland Hiidenvesi, Mustionselkä IV Leptolyngbya sp. HIID B15A 1999 sediment, Finland Hiidenvesi, Kiihkelyksenselkä IV Leptolyngbya sp. HIID D2A 1999 sediment

Limnothrix Limnothrix sp. Morro Branco/CE water 2009 IV CENA 217 accumulated, Brazil

Merismopedia Merismopedia sp. CENA 264 Icó/CE water accumulated, Brazil 2009 IV

Microchaete Microchaete sp. Rio Camarão/CE river water, 2009 IV CENA 251 Brazil

Microcystis Microcystis NPLJ-4 Brazil unknown JQ771624 I aeruginosa Microcystis sp. RST 9501 Brazil 1995 JQ771625 I

Myxosarcina Myxosarcina sp. Cisterna/Canindé/CE water in the 2009 IV CENA 240 cistern, Brazil Myxosarcina sp. Cisterna/Canindé/CE water in the 2009 IV CENA 241 cistern, Brazil Myxosarcina sp. Cisterna/Canindé/CE water in the 2009 IV CENA 242 cistern, Brazil Myxosarcina sp. Cisterna/Canindé/CE water in the 2009 IV CENA 244 cistern, Brazil

Nodularia Nodularia BECID 27 The Baltic Sea, epiphytic 2001 II III harveyana Nodularia BECID 29 The Baltic Sea, epilithic 2001 II III harveyana Nodularia sp. BECID 36 The Baltic Sea, epilithic 2002 II III

Nodularia Bo53 The Baltic Sea Shallow coastal 1992 II III harveyana water Nodularia BY1 The Baltic Sea planktonic 1986 AF268004 II III spumigena Brackish water, Baltic Sea AF268005 III Nodularia sp. HEM planktonic 1986 Nodularia Hübel 1983/300 The Baltic Sea 1983 II harveyana Nodularia PCC 7804 France benthic 1966 II III sphaerocarpa Nodularia PCC 73104/1 Canada soil 1972 II sphaerocarpa

Nostoc Nostoc sp. 1tu14s8 Lake Tuusulanjärvi, Finland 2001 II Nostoc fc. calcicola VI Field, Dobre' Pole, Czech Republic 1998 II III field Nostoc V Field, Nezamyslice, Czech 1990 II III ellipsosporum Republic field Nostoc calcicola 6 sf Calc Dobre Pole 1998 + IV Nostoc sp. 102I IV Nostoc sp. N107.3 Lichen associated + IV Nostoc sp. 113.5 Lichen associated unknown KF631395 II Nostoc sp. 116.6.23 IV Nostoc sp. 116.7.5 IV Nostoc sp. 152 Lake Sääksjärvi, Finland AJ133161 IV Nostoc sp. 159 Lake Haukkajärvi, Finland 1986 KF631398 II IV Nostoc sp. 342/7 Kemiö, Pederså III Nostoc sp. ATCC 53789 Arron Island, Scotland; lichen- unknown II III associsted Nostoc sp. BECID 2 The Baltic Sea, Gulf of Finland 2001 II III Nostoc sp. BECID 32 The Baltic Sea, Gulf of Finland, 2001 II (JL28102001/5) Pikku-Kallahti, Finland, epilithic Nostoc sp. BIR L1S7 The Gulf of Finland IV Morro Branco/CE water + IV Nostoc sp. CENA 219 accumulated 2009 Morro Branco/CE water 2009 IV Nostoc sp. CENA 221 accumulated, Brazil Açude São Mateus/CE water used 2009 IV Nostoc sp. CENA 227 by population (lake), Brazil Açude São Mateus/CE water used 2009 IV Nostoc sp. CENA 234 by population (lake), Brazil Lagoa Povoado Nova Aurora/CE 2009 IV Nostoc sp. CENA 237 water pond used by cattle, Brazil Lagoa Povoado Nova Aurora/CE 2009 IV Nostoc sp. CENA 238 water pond used by cattle, Brazil Lagoa Povoado Nova Aurora/CE 2009 IV Nostoc sp. CENA 239 water pond used by cattle, Brazil Sodrelândia/BA water in the pond, 2009 IV Nostoc sp. CENA 257 Brazil Sodrelândia/BA water in the pond, 2009 IV Nostoc sp. CENA 258 Brazil Sodrelândia/BA water in the pond, 2009 IV Nostoc sp. CENA 259 Brazil Sodrelândia/BA water in the pond, 2009 IV Nostoc sp. CENA 261 Brazil Paulista/PA water in the pond, 2009 IV Nostoc sp. CENA 269 Brazil Paulista/PA water in the pond, 2009 IV Nostoc sp. CENA 271 Brazil Nostoc sp. CENA 274 Bahia soil, Brazil 2009 IV Nostoc sp. CENA 275 Bahia soil, Brazil 2009 IV

Nostoc sp. CENA 277 Bahia soil, Brazil 2009 IV Nostoc sp. CENA 278 Bahia soil, Brazil 2009 IV Nostoc sp. CENA 293 Ceará soil, Brazil 2009 IV Nostoc sp. CENA 296 Ceará soil, Brazil 2009 IV Nostoc sp. CENA 298 Paraíba soil, Brazil 2009 + IV Kobben, Hanko, Finland small 2012 + IV Nostoc sp. HAN 10/1 pond on a rock Kobben, Hanko, Finland small 2012 + IV Nostoc sp. HAN 11/1 pond on a rock Kobben, Hanko, Finland biofilm 2012 + IV Nostoc sp. HAN 22 growth on rock waterline Nostoc sp. HAN 24/1 Kobben, Hanko, Finland epilithic 2012 + IV Hanko Casino sea shore, Hanko, 2012 + IV Nostoc sp. HAN 27 Finland small rock pond Hiidenvesi, Nummelanselkä IV Nostoc sp. HIID C18A 1999 sediment Nostoc sp. IO-102-I Rock in Sysmä, Finland; lichen- 2000 AY566855 II III associated Nostoc sp. N115.3.2 Lichen, unknown IV Nostoc sp. N122.4.B Lichen, unknown IV Nostoc sp. N123.4 Lichen, unknown IV Nostoc sp. N124.3.B Lichen, unknown IV Nostoc sp. N135.9.1 Lichen, unknown IV Nostoc sp. N990622Ш Lichen, unknown IV Nostoc sp. N990653.17 Lichen, unknown IV Nostoc sp. N138 Lichen, unknown IV Nostoc sp. PCC 7120 Unknown unknown NR_074310 IV Australia root section, Macrozamia 1973 AF027655 IV Nostoc punctiforme PCC 73102 sp. Oulunkylä, Helsinki, Finland IV

Nostoc sp. UK1 lichen Peltigera sp. Oulunkylä, Helsinki, Finland + IV Nostoc sp. UK2aImI Lichen Peltigera praetextata IV Nostoc sp. UK 3 Itä-Pakila, Helsinki, Finland lichen 2006 II III Itä-Pakila, Helsinki, Finland lichen IV

Nostoc sp. UK4 Peltigera spuria Itä-Pakila, Helsinki, Finland IV Nostoc sp. UK7BIIm Lichen Peltigera canina? Autti, Finland Lichen Peltigera 2006 + IV Nostoc sp. UK18AI leucophlebia Autti, Finland lichen Peltigera IV

Nostoc sp. UK18AШ leucophlebia Autti, Finland Lichen Peltigera IV Nostoc sp. UK18AIV leucophlebia Autti, Finland Lichen Peltigera IV Nostoc sp. UK18BI leucophlebia Autti, Finland Lichen Peltigera IV Nostoc sp. UK18BП leucophlebia Autti, Finland Lichen Peltigera IV Nostoc sp. UK18BШ leucophlebia Autti, Finland Lichen Peltigera IV Nostoc sp. UK18BIV leucophlebia Nostoc sp. UK18bV Autti; lichen-associated, unknown II III Nostoc sp. UK35 lichen-associated, Finland IV Nostoc sp. UK89IIa Finland unknown JQ771626 I II Hiidenvesi, Kirkkojärvi sediment, IV Nostoc sp. XHIID A1 1999 Finland Hiidenvesi, Mustionselkä III IV Nostoc sp. XHIID A6 1999 sediment, Finland Hiidenvesi, Nummelanselkä IV Nostoc sp. XHIID C1 1999 sediment, Finland

Hiidenvesi, Nummelanselkä IV Nostoc sp. XHIID C2 1999 sediment, Finland Hiidenvesi, Nummelanselkä IV Nostoc sp. XHIID C3 1999 sediment, Finland Hiidenvesi, Nummelanselkä IV Nostoc sp. XHIID C4 1999 sediment, Finland Hiidenvesi, Nummelanselkä IV Nostoc sp. XHIID C5A 1999 sediment, Finland Hiidenvesi, Nummelanselkä IV Nostoc sp. XHIID C5B 1999 sediment, Finland Nostoc sp. XHIID C12 Hiidenvesi, Nummelanselkä, 1999 II III Finland Hiidenvesi, Kiihkelyksenselkä IV Nostoc sp. XHIID D7 1999 sediment, Finland Hiidenvesi, Kiihkelyksenselkä IV Nostoc sp. XHIID D8 1999 sediment, Finland Hiidenvesi, Kiihkelyksenselkä IV Nostoc sp. XHIID D12 1999 sediment, Finland Hiidenvesi, Kiihkelyksenselkä IV Nostoc sp. XHIID D13 1999 sediment, Finland Hiidenvesi, Kiihkelyksenselkä IV Nostoc sp. XHIID D14 1999 sediment, Finland Nostoc sp. XPORK 4A The Baltic Sea coast, Porkkala 1999 II III cape, Finland benthic Nostoc sp. XPORK 5A The Baltic Sea coast, Porkkala 1999 + II III IV cape, Finland epithytic Nostoc sp. XPORK 14A The Baltic Sea coast, Porkkala 1999 II III cape, Finland benthic Nostoc sp. XPORK 15C The Baltic Sea coast, Porkkala 1999 II III cape, Finland Nostoc sp. XPORK 24A The Baltic Sea coast, Porkkala 1999 II III cape, Finland Nostoc sp. XPORK 24B The Baltic Sea coast, Porkkala 1999 II III cape, Finland

Phormidium Phormidium sp. CENA 270 Brazil 2009 JQ771627 I IV Phormidium sp. DVL1003c USA unknown JQ771628 I Phormidium sp. HIID B22B Hiidenvesi, Mustionselkä sediment 1999 IV

Planktothrix Planktothrix sp. 126/8 Lake Vesijärvi, Finland 1984 AJ133166 III Povoado Novo Aurora/CE water in 2009 IV Planktothrix sp. CENA 236 the pond, Brazil Lake Gjersjøen, Norway III CYA 18 Planktothrix sp. planktonic 1971

Pseudanabaena Povoado Novo Aurora/CE water in 2009 IV Pseudanabaena sp. CENA 235 the pond, Brazil Paulista/PA water in the pond, 2009 IV Pseudanabaena sp. CENA 266 Brazil Paulista/PA water in the pond, 2009 IV Pseudanabaena sp. CENA 267 Brazil Pseudanabaena sp. CENA 287 Piauí soil, Brazil 2009 IV Hiidenvesi, Kirkkojärvi sediment, IV Pseudanabaena sp. HIID A13A 1999 Finland Hiidenvesi, Mustionselkä IV Pseudanabaena sp. HIID B19 1999 sediment, Finland Hiidenvesi, Nummelanselkä IV Pseudanabaena sp. HIID C15A 1999 sediment, Finland

Rivularia Rivularia sp. BECID 10 Baltic Sea, Kirkkonummi, Finland; 1999 II III sediment benthic, epilithic Helsinki Hettoniemi, Gulf of III Rivularia sp. BECID 14 Finland, epilithic 2001 Rivularia sp. PCC 7116 Sea water, Baja California, USA 1968 II Rivularia sp. XPORK 3A The Baltic Sea coast, Porkkala 1999 II III cape, Finland periphytic, benthic Rivularia sp. XPORK 16B The Batic Sea coast, Porkkala 1999 II III cape, Finland, epilithic

Synechococcus Synechococcus sp. Morro Branco/CE water 2009 IV CENA 222 accumulated, Brazil Synechococcus sp. Açude São Mateus/CE water used 2009 IV CENA 232 by population (lake), Brazil Synechococcus sp. Açude São Mateus/CE water used 2009 IV CENA 233 by population (lake), Brazil Synechococcus sp. Rio Camarão/CE river water, 2009 IV CENA 249 Brazil Synechococcus sp. Sodrelândia/BA water in the pond, 2009 IV CENA 253 Brazil

Synechocystis Hiidenvesi, Mustionselkä IV Synechocystis sp. HIID B11B 1999 sediment, Finland

Scytonema Paulista/PA water in the pond, 2009 IV Scytonema sp. CENA 268 Brazil Kobben, Hanko, Finland green 2012 IV Scytonema sp. HAN 3/2 biofilm in the pond Scytonema sp. PCC 7110 Limestone cave, Crystal Cave, 1971 II III IV Bermuda limestone

Snowella Snowella sp. 0tU37S4 Lake Tuusulanjärvi, Finland 2000 III

Stigonema Stigonema sp. CENA 291 Ceará soil, Brazil 2009 IV Stigonema sp. CENA 295 Ceará soil, Brazil 2009 IV

Tolypothrix Tolypothrix sp. BIR MGR4 The Gulf of Finland IV Tolypothrix sp. HAN 25/1 Kobben, Hanko, Finland epilithic 2012 + IV Hanko Casino sea shore, Hanko, 2012 + IV Finland Black biofilm, Hanko Tolypothrix sp. HAN 26/2 Casino sea shore, Hanko, Finland Tolypothrix sp. PCC 7101 Soil, Borneo 1950 II IV unknown Tolypothrix sp. PCC 7415 Greenhouse soil, Stockholm, 1972 II IV Sweden Tolypothrix sp. PCC 7504 Aquarium, Stockholm, Sweden 1972 II IV Tolypothrix sp. PCC 9009 Watkins Glen state Park, New unknown PRJNA6342 II York, USA 5 Tolypothrix sp. TOL328 University of Kuopio, Green 1999 II III IV house, Finland soil Tolypothrix sp. XPORK 34B Porkkala cape IV

Trichormus Trichormus 1 Unknown unknown II III doliolum Trichormus azollae BAI/1983 Unknown 1983 II III Trichormus GREIFSWALD Unknown 1992 II III variabilis Trichormus sp. HIID B6.A Freshwater, Hiidenvesi, 1999 II III IV Mustionselkä, Finland

Hiidenvesi, Kiihkelyksenselkä IV Trichormus sp. HIID D3 1999 sediment, Finland

Non classified cyanobacteria Baixão dos Bois/BA water 2009 IV Chroococcales CENA 263 accumulated, Brazil Chroococcales CENA 276 Bahia soil, Brazil 2009 IV CENA 281 Piauí soil, Brazil 2009 IV Nostocales CENA 294 Ceará soil, Brazil 2009 IV Kobben, Hanko, Finland green 2012 IV HAN 3/1 biofilm in the pond Kobben, Hanko, Finland Growth 2012 IV HAN 6/1 on rock waterline Kobben, Hanko, Finland 2012 IV HAN 15/3 Gastropod from waterline Kobben, Hanko, Finland 2012 IV HAN 19/3 black/green biofilm from waterline Kobben, Hanko, Finland sediments 2012 IV HAN 20/1 10 cm under water Kobben, Hanko, Finland 2012 IV HAN 21/2 Gastropod 10 cm under water Kobben, Hanko, Finland Black 2012 IV HAN22/2 biofilm Hanko Casino sea shore, Hanko, 2012 IV HAN 29/3 Finland green biofilm on the rock HAN 31/a Hanko, Finland biofilm 2012 IV Hanko Casino sea shore, Hanko, 2012 IV HAN 32/2 Finland epilithic Hanko Casino sea shore, Hanko, 2012 IV HAN 40/1 Finland epilithic HIID A5.A Hiidenvesi, Kirkkojärvi, Finland 1999 II HIID A7 Hiidenvesi, Kirkkojärvi, Finland 1999 II HIID A18.A Hiidenvesi, Kirkkojärvi, Finland 1999 II HIID B4.B Hiidenvesi, Mustionselkä, Finland 1999 II HIID B11.B Hiidenvesi, Mustionselkä, Finland 1999 II HIID B16.A Hiidenvesi, Mustionselkä, Finland 1999 II HIID B22.A Hiidenvesi, Mustionselkä, Finland 1999 II PORK 10 The Baltic Sea coast, Porkkala 1999 II cape, Finland UHCC 0006 Pernajanlahti, Finland 2009 II UHCC 0007 Pernajanlahti, Finland 2009 II UHCC 0020 Bonäsbåset, Finland 2009 II UHCC 0021 Lake Puujärvi, Finland 2009 II XHIID A6 Hiidenvesi, Kirkkojärvi, Finland 1999 II XHIID C1 Hiidenvesi, Nummelanselkä, 1999 II Finland XHIID D4 Hiidenvesi, Kiihtelyksenselkä, 1999 II Finland XHIID D8 Hiidenvesi, Kiihtelyksenselkä, 1999 II Finland XHIID D14 Hiidenvesi, Kiihtelyksenselkä, 1999 II

Finland XPORK 2C The Baltic Sea coast, Porkkala 1999 II cape, Finland XPORK 14F The Baltic Sea coast, Porkkala 1999 II cape, Finland XSPORK 15B The Baltic Sea coast, Porkkala 1999 II cape, Finland XSPORK 15C The Baltic Sea coast, Porkkala 1999 II cape, Finland XSPORK 20A The Baltic Sea coast, Porkkala 1999 II cape, Finland XSPORK 22A The Baltic Sea coast, Porkkala 1999 II cape, Finland XSPORK 24A The Baltic Sea coast, Porkkala 1999 II cape, Finland XSPORK 34A The Baltic Sea coast, Porkkala 1999 II cape, Finland + 16S rRNA sequences are not available in the NCBI.