Department of Food and Environment Sciences
Division of Microbiology
Viikki Biocenter, (Viikinkaari 9)
FIN-00014 Helsinki University
Fax: +358-9-191 59322
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.
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.
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.
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.
Anabaena strain 90
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).
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.
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.
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.
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.
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.
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.
Photo I. Oksanen
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.
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.