University of Helsinki
Faculty of Agriculture and Forestry
 
The Cyanobacteria Group
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Contact information:

Address:
Department of Food and Environment Sciences
Division of Microbiology and Biotechnology
P.O.Box 56
Viikki Biocenter, (Viikinkaari 9)
FIN-00014 Helsinki University
Finland
Fax: +358 2 941 911

Projects

Bioactive compounds from cyanobacteria
Whole genome sequencing
Genome driven discovery of new bioactive peptides
Nonribosomal peptides
Ribosomal peptides
Detection methods for toxin and odorous metabolite producing cyanobacteria
Research on Baltic Sea and Lakes
Eco – evolutionary dynamics in cyanobacterial populations
Cyanobacterial symbioses
Culture collection

Bioactive compounds from cyanobacteria

Cyanobacteria are a rich source of natural products with interesting biological activities. Cyanobacteria strains from our culture collection are studied for the presence of toxins and other bioactive compounds. We determined the structure of over 20 novel microcystins variants in collaboration with Prof. M. Namikoshi (Tokyo Univ. of Fisheries, Japan), Prof. K. Rinehart (Univ. of Illinois, USA), Prof. W. Carmichael (Wright State Univ., USA) and solved the 3D-structure of nodularin with Dr. A. Annila & Prof. T. Drakenberg (VTT). More recently we characterized several cyclic and linear peptides from cyanobacteria in collaboration with Prof. K. Harada (Meijo Univ., Japan).

During the last years we have described (1) new potent serine protease inhibiting linear tetrapeptides, spumigins, aeruginosins and pseudoaeruginosins from Nodularia (Fewer et al. 2009, 2013, Liu et al. 2015), pseudospumigins from Brasilian Nostoc (Jokela et al. in preparation) and linear tripeptide nostosins from Nostoc (Liu et al. 2014). (2) An antitoxic cyclic heptapeptide nostocyclopeptide M1, cyclic decapeptide nostoweipeptin W1 and nonapeptidic nostopeptolides L1-4 for hepatotoxic microcystins (Jokela et al. 2010, Herfindal et al. 2011, Liu et al. 2015). (3) Novel lytic cyclic lipotetrapeptides anabaenolysins A and B from benthic Anabaena (Jokela et al. 2012). (4) Novel acetylated cyclodextrins from the benthic Anabaena which are synergistically antifungal with anabaenolysins (Shishido et al. 2015 ) (5) New rare microcystin variants from lichen symbiotic Nostoc and Phormidium from Brazil and USA (Shishido et al. 2013, 2015 in preparation). (6) Novel antifungal cyclic glycolipononapeptide hassallidins from Anabaena (Vestola et al. 2014). (6) A number of ribosomally produced cyanobactins which were structurally so novel that the definition of cyanobactins have to be revised (Leikoski et al. 2010, 2012, 2013). (7) Novel variants of antifungal compounds hassallidins and scytophycins from new cyanobacterial strains (Shishido et al. 2015).

LC-MS facility of the research center of excellence

Further screening (e. g. cell assays, enzymes tests) and characterization of novel bioactive compounds from our culture collection as well as new isolates of cyanobacteria are underway. Our recent studies indicate the frequent production of antifungal and antileukemia activities in cyanobacterial extracts which remain to be identified (Liu et al. 2014, Humisto et al. 2015, Shishido et al. 2015). Our cyanobacterial culture collection is an important basic resource used for screening new cyanobacterial compounds with interesting bioactivities. However, we are interested in finding new relevant clinical targets for the bioactive compound screenings and therefore we are happy to welcome new partners and collaborations to work with us. The discovery of new bioactive metabolites by using a combination of mass spectrometry and genome mining is getting a more and more important approach.

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Whole genome sequencing

We have sequenced a number of model toxin producing cyanobacteria from Finnish lakes and the Baltic Sea from our culture collection. We have sequenced the complete genome of microcystin producing Anabaena sp. 90 in collaboration with the Beijing Genomics Institute, China (Wang et al. 2012). This strain was isolated from Lake Vesijärvi and produces microcystins. The whole genome of anatoxin-a producing Anabaena strain 37 was initiated in collaboration with Institute of Biomedical Technologies, Italy. The akinete forming Anabaena strain ITU33S10, anabaenolysin producing Anabaena strain XSPORK 2A, XPORK13A andXPORK15F are currently being sequenced. These genome projects will provide further insights into the proliferation of cyanobacteria in Finnish lakes and the production of bioactive compounds by cyanobacteria. We were also involved in the genome project of Nodularia spumigena CCY9414 which was isolated from near Bornholm in the Baltic Sea and produces the hepatotoxin nodularin (Voss et al. 2013). The genome project was initiated by Lucas Stal at NIOO in Holland and sequenced at the Craig Venter Institute in the US as part of the Moore Foundations Marine Microbiology initiative. We have now obtained genome sequences for 60 further strains from benthic and planktonic environments of the Baltic Sea and Finnish lakes. Analysis of these genome sequences have provided new insights into the ecology of these organisms, how they are adapted to their environment and the types of bioactive compounds they produce (Wang et al. 2012, Voss et al. 2013, Leikoski et al. 2013, Calteau et al. 2014, Vestola et al. 2014, Shishido et al. 2015).

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Genome driven discovery of new bioactive peptides

Much of the natural product chemical diversity observed in nature is attributed to versatile non-ribosomal peptide synthase (NRPS) and polyketides synthetase (PKS) biosynthetic pathways. We have shown that cyanobacterial genomes encode a huge diversity of these biosynthetic pathways (Shih et al. 2013, Wang et al. 2011, 2012, 2014, Shih et al. 2013). Likewise, ribosomal gene clusters, recently shown to produce complex peptides through the post-translational modification of short precursor proteins, are very common in cyanobacterial genomes (Leikoski et al. 2010, 2013). However, the end-products of the vast majority of these pathways are currently unknown (Calteau et al. 2015, Dittmann et al. 2015). Our genome mining studies at the phylum level have demonstrated the unexpected widespread distribution and biological diversity of secondary metabolite biosynthetic gene clusters (Shih et al. 2013, Wang et al. 2014, Calteau et al. 2014).

We carry out genome driven discovery of new bioactive peptides from cyanobacteria (Wang et al. 2014). Through the use of genome sequences we discovered new families of protease inhibitors and antifungal peptides, new enzymatic machinery for making cyclic peptides (Vestola et al. 2015, Shishido et al. 2015). However, the lack of genetic system and slow growth times of cyanobacteria and most other bioactive compound producing organisms is a bottleneck in natural product discovery and slows the pace at which research can be carried out (Dittmann et al. 2015). We are developing methods to overcome these limitations and accelerate the discovery of new natural products from cyanobacteria. We are also interested in understanding how cyanobacteria make natural products and in understanding how the enzymatic machinery for producing peptides functions. Biochemical investigations shed light on how these enzymes work together to create cyclic peptides. We use phylogenetic analyses of the biosynthetic machinery to show how natural products evolve in nature and this ultimately may provide clues to how cyanobacteria can be rewired to make new natural products (Calteau et al. 2014, Dittmann et al. 2015).

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Nonribosomal peptides

Several cyclic, branched, or linear bioactive peptides of bacteria and lower eukaryotes are produced non-ribosomally by multidomain peptide synthetases, employing a thiotemplate mechanism. Different domains of peptide synthetases act as independent enzymes whose function is to join one amino acid to the growing polypeptide chain and make possible modifications. The specific order of the domains forms the protein template that defines the sequence of the incorporated amino acids. Our genome mining studies conducted at the phylum and domain level have demonstrated that peptide synthetase genes are common in cyanobacteria (Wang et al. 2014, Calteau et al. 2014, Dittmann et al. 2015). One hepatotoxic strain, Anabaena 90, isolated from a Finnish lake, Vesijärvi, was selected as a model strain to study the peptide synthetase system. Three classes of cyclic peptides have been isolated and characterized from Anabaena sp. 90: two types of heptapeptides, microcystins and anabaenopeptilides, and one type of hexapeptides, anabaenopeptins. The microcystin, anabaenopeptilide, anabaenopeptin, hassallidin and anabaenolysin synthetase gene clusters have been characterized from Anabaena sp. 90 or other Anabaena strains in our group (Rouhiainen et al. 2000, 2004, 2010, Vestola et al. 2014, Shishido et al. 2015). Peptide synthetase gene clusters from Nostoc sp. 152 (Fewer et al. 2011, 2013) and Nodularia spumigena CCY9414 have also been characterized (Fewer et al. 2009, 2013, Liu et al. 2015).

 

We have also studied non-ribosomal peptides with cytotoxic and antimicrobial effects from other cyanobacteria. Information about genes involved in the synthesis of these compounds, occurrence of these genes and production of these compounds are investigated. Through phylogenetic analysis it is possible to analyze the distribution of the genes among distant or close related cyanobacterial strains (Wang et al. 2014, Calteau et al. 2014). Eventually, bioassays trying to solve the ecological role or analysis of mechanism of action of these compounds will be performed.

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Ribosomal peptides

Anabaena strain 90


Cyanobactins are small cyclic peptides recently described from cyanobacteria (Sivonen et al. 2010). They are formed through the proteolytic cleavage and post-translational modification of short precursor proteins and exhibit anti-tumor, cytotoxic or multi-drug reversing activities. Novel cyanobactins, anacyclamides, piricyclamides and linear aeruginosamide and viridisamide were recently found to be common in various cyanobacteria (Leikoski et al. 2010, 2012, 2013). Our future research will focus on biosynthesis and detection of new cyanobactins in cyanobacteria through genome mining. Bacteriocin gene cluster were found to be common in cyanobacteria (Wang et al. 2011, Shih et al. 2013). Our work has shown that ribosomal peptide biosynthetic gene clusters are very common in cyanobacteria (Wang et al. 2011, Shih et al. 2013). However, the vast majority of these biosynthetic pathways have no known end product associated with them. Work is now underway to unravel the complex distribution of ribosomal peptides in cyanobacteria and develop novel methods to express silent gene clusters in heterologous hosts. This work has led to the discovery of novel peptide with unusual posttranslational modifications and antimicrobial bioactivities.

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Detection methods for toxin and odorous metabolite producing cyanobacteria

The most common toxins are the cyclic peptide hepatotoxins, microcystins and nodularins. Another prominent group of toxins are alkaloid neurotoxins, including e.g. anatoxin-a and homoanatoxin-a. Microcystins are mainly produced by Anabaena, Microcystis, and Planktothrix in freshwater, while nodularins are produced by Nodularia spumigena in brackish water. The main producers of anatoxin-a and homoanatoxin-a are Anabaena, Oscillatoria, and Aphanizomenon. Toxins pose a risk for water users, especially when cyanobacteria form dense mass occurrences (blooms) in water bodies. A bloom can comprise both toxic and nontoxic strains and include several potentially toxigenic genera. However, toxic strains look alike nontoxic strains, and cannot be recognized by conventional microscopy. This makes it difficult to assess the risk for the water users. The genes encoding the biosynthetic enzymes required for toxin production offer a practical way for differentiation and identification of potential toxin producers. We developed molecular detection methods for microcystin (mcy), nodularin (nda) and anatoxin-a (ana) synthetase genes to detect and identify hepatotoxin- and anatoxin-producing cyanobacteria in environmental samples. The methods include general and genus-specific PCR assays for the mcyE, ndaF, and anaC genes (Vaitomaa et al. 2003, Rantala et al. 2006, Koskenniemi et al. 2007, Rantala-Ylinen et al. 2011). The DNA-chip and PCR-RFLP assays were used for simultaneous identification of potential microcystin/nodularin (Rantala et al. 2008) and anatoxin-a producers (Rantala-Ylinen et al. 2011), respectively. Genus-specific qPCR assays were designed to explore the quantity of microcystin-producing Anabaena, Microcystis and Planktothrix (Vaitomaa et al. 2003, Sipari et al. 2010), nodularin-producing Nodularia (Koskenniemi et al. 2007), and anatoxin-a producing Anabaena and Oscillatoria (Suurnäkki et al. unpublished results) present in water samples. In addition, active transcription of the both Anabaena- and Microcystis-mcyE genes in Lake Tuusulanjärvi samples could be confirmed by the chip and qPCR assays (Sipari et al. 2010).

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Research on Baltic Sea and Lakes

Microbes play a key role in nutrient cycles in water reservoirs. Knowledge about changes in structure and function of bacterial communities under varying environmental conditions helps us to understand, which environmental factors can effect on the distribution of the bacterial species, and why certain biological processes may be enhanced in prevailing conditions. Our aim is to evaluate the environmental status on the Baltic Sea based on indicators reflecting the biodiversity and genetic functional profiles of microbes in sea water in our BONUS program project "BLUEPRINT – Biological lenses using gene prints". To achieve our objectives, laboratory and field studies with next-generation sequencing, bioinformatics and modeling are combined to get wide view of the environmental status of the Baltic Sea. Our main role in this project is to study harmful cyanobacteria in a purpose to find new marker genes for varying environmental conditions which can later be used in monitoring and modeling purposes. Based on previous studies and genomic information our major interests are to unravel the importance of different phosphorus sources and salinity gradient on the function and bloom formation of the toxic cyanobacterial genera from the Baltic Sea (Nodularia spumigena and Anabaena). The question will be addressed by analyzing transcriptomics and proteomics profiles together with chemical and biological rates.

Similarly we work especially on Finnish Anabaena strains originating from the lakes to understand the regulation of bioactive compound and toxin production as well as akinete production. We try to find out cellular processes that are affected by changes in environmental factors, e.g. nutrient concentrations, or gene mutations leading to non-functional bioactive compound machinery. Phosphorus is an essential nutrient for cyanobacteria and major nutrient to be studied since Anabaena cyanobacteria are nitrogen fixers. Our recent research showed that alkaline phosphatase is not the only marker relevant for studies on phosphorus status of Anabaena (Teikari et al. 2015).

Cyanobacteria and their phages: Cyanobacteria like most organisms, are strongly influenced by ecological interactions such as competition, predation and parasitism. It has been shown that parasitic viruses can reduce cyanobacterial biomass and that they generate a selective force for the host genotypes. This interaction can have also a wider impact on the nutrients in environment. Nitrogen is the limiting nutrient for phytoplanktonic and non-nitrogen fixing cyanobacterial growth in the Baltic Sea thus rapid input of nitrogen can be important by modifying the planktonic community structure. In our laboratory, we study the release of nitrogen by phage-induced cyanobacterial cell lysis and the diversification of the cyanobacterial host through an experimental approach.

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Eco – evolutionary dynamics in cyanobacterial populations

Rapid contemporary evolution of toxicity in cyanobacteria can affect multispecies ecological dynamics in aquatic food webs. In this project we will apply recent eco- evolutionary theory to the relatively well-studied field of harmful cyanobacteria, with a focus on the Baltic Sea, where the cyanobacteria have wide socioeconomic impacts.

Parasites and predators, besides being a major factor driving ecological dynamics of cyanobacterial populations, can also generate a huge selective force for the emergence of new genotypes. Research during recent decades has demonstrated that evolutionary change can happen rapidly and on the same time scale as ecological processes. Furthermore, as cyanobacteria often form blooms in resource-enriched conditions, there might be an interesting yet untested link between observed cyanobacterial dominance and resource dependent trade-offs in life-history characters. To understand the potentially complex drivers of cyanobacterial blooms and the evolution of toxicity, there is a need to integrate various community level processes with rapid evolutionary dynamics occurring in the communities.

Methods used in project include field and chemostat experiments, LC-MS toxin analyses and analyses of genotype frequencies with quantitative PCR. Empirical work is also bound to a strong theoretical component where we model community dynamics.

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Cyanobacterial symbioses

Cyanobacteria are known to enter symbiosis with eukaryotes ranging from fungal phyla and marine sponges to lower and higher plant divisions. There are no other microbes today that have the capacity to form symbiosis with such a wide range of eukaryotes. Neither the genetic background, nor changes in the proteomes are known for which could be the underpinning molecular mechanisms – from recognition, through infection to the intra-cellular adaptation – in the cyanobacterial symbioses.

In collaboration with prof. J. Rikkinen and Dr. U. Kaasalainen (Faculty of Biological and Environmental Sciences, University of Helsinki) we have found that microcystins and nodularin are produced by terrestrial cyanobacteria in lichens. We detected substantial amounts of these liver toxins from several lichens of the 800 lichen specimens collected all around the world. Three lichen specimens contained nodularin, previously known only from aquatic, bloom-forming, brackish water cyanobacteria. Findings demonstrate that lichens may pose a health threat to lichen-consuming herbivores and humans (Kaasalainen et al. 2012, 2013).

Toxin-producing cyanolichen. A, Peltigera leucophlebia in natural habitat (scale bar 1 cm). B, Cephalodia (fungal structures containing symbiotic cyanobacteria) on the upper surface of the lichen thallus (scale bar: 1 mm). The white spots on the lichen surface are pine pollen. C, Isolated microcystinproducing Nostoc sp. strain UK18 in pure culture (scale bar: 20 µm). (Kaasalainen et al. 2009)

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Culture collection

Our culture collection was founded in 1985 and now contains over 1000 strains. These strains are isolated mainly from benthic and planktonic environments of Finnish lakes and the Baltic Sea. One fifth of the strains are maintained in pure axenic cultures. The collection also contains cyanobacterial strains isolated from lichen symbiosis and from a variety of habitats in Brazil as part of an ongoing the Academy of Finland and Brazilian FAPESP funded collaboration with Prof. Marli de Fátima Fiore (Univ. of Sao Paolo). The culture collection has formed the basis for important publications advancing the knowledge of toxic cyanobacteria as well as the bioactive compounds they produce. The collection also forms the basis for experiments to understand the physiology, ecology and genomics of these important toxic cyanobacteria from Finnish lakes and the Baltic Sea. The collection has already lead to over 150 publications in international peer reviewed journals. The culture collection is now part of the microbial HAMBI collection of the Microbiology Division (http://www.helsinki.fi/hambi/).

Brazilonema from Brazil (two upper pictures, photo by Lyudmila Saari) and several Anabaena strains from our culture collection.

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