Bioactive compounds from cyanobacteria
Whole genome analyses
Genome driven discovery of new bioactive peptides
Non-ribosomal peptides
Ribosomal peptides
Detection methods for toxic cyanobacteria
Odorous metabolites
Akinete differentiation
Proteomics and transcriptomics
Eco – evolutionary dynamics in cyanobacterial populations
Cyanobacterial symbioses
Sediment research

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 couple of years we have found and described (1) the new potent protease inhibitors, spumigins, from Nodularia (Fewer et al. 2009), (2) an antitoxic cyclic heptapeptide for hepatotoxic microcystins (Jokela et al. 2010, Herfindal et al. 2011), (3) Novel lytic cyclic lipotetrapeptide from benthic Anabaena (Jokela et al. 2012), (4) new rare microcystin variants from lichen symbiotic Nostoc and Phormidium from Brazil (Shishido et al. unpub.), (5) novel antifungal cyclic glycolipononapeptide hassallidins from Anabaena (Vestola et al. unpub) and (6) new aeruginosin protease inhibitors from Nodularia and Nostoc (Fewer et al. unpub). Most recently we have found a new kind of antifungal compound from Nostoc and an antileukemic compound from Anabaena.

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 cyanobacterial culture collection is the basic resource used for screening new cyanobacterial compounds with interesting bioactivities. However, we are interested in finding new relevant targets for the 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 analyses

We have sequenced the complete genome of microcystin producing Anabaena sp. 90 in collaboration with the Beijing Genomics Institute, China. 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 XSP 2A and the kawaguchipeptin producing Microcystis aeruginosa NIES-88 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 are 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. 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.

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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. This diversity rivals that of antibiotic producing bacteria. 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. However, the end-products of the vast majority of these pathways are currently unknown.
We carry out genome driven discovery of new bioactive peptides from cyanobacteria. Through the use of genome sequences we discovered new families of protease inhibitors and antifungal peptides, new enzymatic machinery for making cyclic peptides. 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. We are developing methods 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 biosynthethic 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.

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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.

Peptide synthetase genes are common in cyanobacteria. 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 and anabaenopeptin synthetase gene cluster has been characterized from Anabaena sp. 90 in our group. Peptide synthetase gene clusters from Nostoc sp. 152 and Nodularia spumigena CCY9414 have also been characterized.

Non-ribosomal peptides with cytotoxic and antimicrobial effects from other cyanobacteria are also studied. 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. 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. 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, were recently found to be common in various Anabaena strains in our culture collection (Leikoski et al. 2010). More recently we have described unique geranylated cyanobactins, piricyclamides, from bloom forming cyanobacteria Microcystis. (Leikoski et al. unpub.). Our future research will focus on biosynthesis and detection of new cyanobactins in cyanobacteria through genome mining.

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Detection methods for toxic 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. Microcystinsare mainly produced by Anabaena, Microcystis, and Planktothrix in fresh waters, while nodularins are produced by Nodularia spumigena in brackish waters. 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 (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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Odorous metabolites

Geosmin and 2-methylisoborneol (2-MIB) are odorous metabolites produced by some cyanobacteria. These secondary metabolites cause earthy and/or musty odour and taste for example in freshwater, drinking water or fish. Humans have a very low odor threshold for these metabolites. The biosynthetic genes of geosmin and 2-MIB have been recently discovered from cyanobacteria. Research on odorous metabolites from cyanobacteria in our culture collection is underway.

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Akinete differentiation

The vegetative cells of certain filamentous cyanobacteria are capable of differentiating into resting cells called akinetes. Akinetes are large, thick-walled cells enabling organism to survive stress conditions. At the return of the more favourable conditions akinetes can germinate to produce new vegetative growth. Thus akinete formation and germination might play an important role in the life cycle of cyanobacterial strains, their survival and ability to form mass occurrances in the natural environments. Yet, little is known about these processes in Anabaena strains, which are typical to the Finnish fresh waters. In our research group we aim 1) to discover new genes and gene products, which at the cell level regulate akinete formation and germination. 2) to detect the changes in gene expression, transcription and translation, at the different stages of akinete differentiation.3) to study the environmental factors controlling akinete formation, survival and germination.

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Proteomics and transcriptomics

While genome is a relatively invariable reserve of potential functions, the proteome, i.e. the set of proteins expressed under defined physiological condition in an organism, reveals the active functions in the organism. Therefore, we want to apply proteomics approach to reveal which proteins are present at different stages of cyanobacterial life cycle. New proteins that appear during the transformation periods will be isolated and their structure revealed. The gene(s) responsible for the protein production and its function will be deduced from the available whole-genome sequences. The temporal changes in expression of the genes will be studied by using transcriptomics techniques, which allow quantification of RNA molecules produced at varying conditions.
We work especially on Finnish Anabaena strains to understand the regulation of bioactive compound and toxin production as well as akinete production in them. 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; its lack restricts their growth and microcystin production. Restricting the phosphorus emissions to water systems the toxic cyanobacteria blooms could be diminished.

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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. The methodological approach in the project resolving the “Metabolic adaptation of cyanobacteria in endosymbiosis.
In collaboration with prof. J. Rikkinen and M.Sc. 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 cyanobacteria. Findings demonstrate that lichens may pose a health threat to lichen-consuming herbivores and humans (Kaasalainen et al. 2012).

Photo I. Oksanen

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Sediment research

Sediment samples were collected from the Baltic Sea during Aranda r/v cruise  

Excessive phosphorus (P) and nitrogen loads increased anoxia of the bottom area of the Baltic Sea. Anoxia increases the P flux from the sediment, which boosts the growth of primary producers such as cyanobacteria. Cyanobacterial mass occurrences can be harmful to humans and animals, and lead to intoxication, as well as hinder recreational use of water and fishery. In addition biodegradation of sedimented mass occurrences of cyanobacteria increase further anoxy and nutrient flux from bottoms. Therefore also species biodiversity in the Baltic Sea decreases.
The sediment group aims to investigate by molecular biological (T-RFLP, cloning etc.), microbiological (strain isolation, microscopy etc.) and chemical tools (chemical extractions, Finnish Marine Institute) the microbial and chemical processes of phosphorus in the Baltic Sea sediment. The main goal is to find the key bacteria participating in P processes and determine the effect of the bacteria on these processes. We are studying especially sulphate, manganese and iron reducing bacteria putatively mobilising P from the Baltic Sea sediment. The microbiological and molecular biological data are combined with chemical and physical data and analysed by mathematical as well as statistical methods.

Culture collection

Cyanobacteria culture collection is part of the microbial HAMBI collection of the Microbiology Division (http://www.helsinki.fi/hambi/) and contains over cyanobacterial 1000 strains, one fifth of which are axenic. Majority of the strains originate from the Finnish lakes and from the Baltic Sea.

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