Unit: Cyanobacteria - CNRS URA 2172

Director: TANDEAU de MARSAC Nicole

Cyanobacteria appeared on our planet about 3 billion years ago and contribute even today, by their ability to perform oxygenic photosynthesis, to the balance between CO2 and O2 in the atmosphere. Adapted to a wide range of environmental conditions, including extreme ones, they colonize most aquatic and terrestrial ecosystems. Some of them produce hepatotoxins or neurotoxins harmful to animals and Man. The main objective of our research programme is to increase, by applying a polyphasic approach, our knowledge of the biodiversity, the physiology and the genome of cyanobacteria, to elucidate their modes of acclimation to the environment, the bases of their toxicity, as well as to exploit their biotechnological potential.

1. The PCC or "Pasteur Culture Collection of Cyanobacteria" (R. Rippka, I. Iteman, M. Welker, M. Herdman, K. Comte, A. Trottet, S. Cadel, T. Coursin, T. Laurent)

The PCC is internationally recognized both for the quality of the strains (axenic cultures) and for the diversity of their morphological and physiological properties.

The PCC has several missions that include both service and research activities:

- Preservation and development of biological resources (750 strains representing 57 genera); strain identification by a polyphasic approach; 475 strains are presented in a catalogue (http://www.pasteur.fr/recherche/banques/PCC/); three strains of Nostoc ellipsosporum, potentially producing cyanovirin-N, an antiviral protein, and one strain of Calothrix were purified and included in the PCC. In the frame of the European project COBRA "The Conservation of a vital European scientific and biotechnological resource: microalgae and cyanobacteria" (http://www.cobra.ac.uk/introduction/html/about_COBRA.cfm), cryopreservation methods were tested. A PCR-method using primers based on the repetitive repeat sequence HIP1 was validated for rapid genotyping of strains from different culture collections.

- Consultation and sale of strains (about 200 in 2003) to research and industrial laboratories all over the world, including teaching institutions in France.

- Development of molecular tools for identification of cyanobacteria in culture and their detection and monitoring in the environment. A DNA/DNA hybridization technique in micro-titre plates, easier and faster to perform than the traditional method in liquid phase, has been optimized. Members of the genus Trichodesmium are marine, non-heterocystous cyanobacteria that as diazotrophs play an important role in the biogeochemical cycles of the South-east Pacific Ocean. The morphology of trichomes originating from three regions of New Caledonia (Nouméa, Bourail and Thio) has been analysed by light microscopy. This permitted to distinguish two species of Trihodesmium, T. erythraeum and T. thiebautii, as well as two species of the genus Katagnymene, K. spiralis and K. pelagica. Several tools of genotyping were applied to confirm the identification based on microscopic examination and to evaluate the genetic biodiversity of these marine cyanobacteria (Coll. LeBorgne, IRD, Nouméa, New-Caledonia).

- Search for and characterisation of bioactive molecules of pharmaceutical interest. In collaboration with V. Bultel, (Muséum National d'Histoire Naturelle, Paris), a cytotoxic molecule, the cylindrocyclophan, produced by strain PCC 7417 has been characterized. In the frame of the European project PEPCY " Toxic and Bioactive Peptides in Cyanobacteria "  (http://www.umweltbundesamt.de/pepcy/), two new clusters of genes encoding polyketide synthases in Microcystis aeruginosa PCC 7806 were identified. Analyses are in progress to correlate the presence of these genes with the corresponding products in other planktonic strains of the PCC.

Several databases in constant evolution are maintained in the Unit:

- "CYANOBANK" (Windows 98, Microsoft Access): properties of the strains in the PCC; "ITS size database": number and size of ITS amplicons (300 entries); "Photographic database" (610 entries); "Storage database": list of strains preserved in liquid nitrogen; the last three modules interact with Cyanobank.

- Databases independent of CYANOBANK: "ITS sequence database" with 296 aligned sequences; "Cyanobacterial 16S rRNA sequence database" (software ARB) with more than 900 aligned sequences; "Bacterial 16S rRNA sequence database" with more than 13000 sequences, permitting the design of oligonucleotide primers and probes; database (software GelCompare) of profiles generated by ITS-RFLP and amplification with HIP1 extended primers.

Anticipating the future integration of the PCC in the Centre for Biological Resources at the Pasteur Institute, steps towards quality control and transfer of the strain information from the PCC databases to the ARPAS software are in progress.

Expertises of cyanobacterial identification and determination of hepatotoxins in samples from surface waters are performed in collaboration with J.M. Delattre (Department of Waters and Environment, IP-Lille). In 2003, fifty-five expertises have been performed for DDASS in diverse regions of France.

2. Molecular mechanisms of the acclimation of cyanobacteria to the environment (N. Tandeau de Marsac, A. Mlouka, K. Comte, A.M. Castets)

Light and nutrients act in a number of regulatory circuits in cyanobacterial metabolism, and play a key role in the formation of water blooms in planktonic species.

PII, a key protein for the coordination of the carbon and nitrogen metabolism in cyanobacteria

PII (GlnB) is a signal transducer protein found in eubacteria and archaebacteria, as well as in plants and algae. The tridimensional structure of PII from Synechococcus PCC 7942 and Synechocystis PCC 6803 were determined by X-ray crystallography. Although the PII proteins from cyanobacteria and from E. coli react with different receptors, the core of the molecule is highly conserved. In contrast, the T- and C- loops differ in agreement with the hypothesis that these regions are important for specific receptor recognition. The residue Gln57, only present in cyanobacterial PII sequences, seems also to be involved in such recognition events.(Coll. S.G. Vasudevan, James Cook University and D.L. Ollis, Australian National University, Australia).

Twelve genes are required for gas vesicle formation in Microcystis aeruginosa

Cyanobacteria of the genus Microcystis are planktonic and most of them are hepatotoxic. Due to an abundant synthesis of intracellular buoyant structures, the gas vesicles ( GV), they form under our latitude very dense blooms at the surface of water bodies during summer times. For the first time, a cluster of 12 genes encoding components involved in gas vesicle synthesis of Microcystis has been characterized. Ten of these genes are organized in two operons whose transcription is segmented, and two genes are independently transcribed. Analyses of four GV- mutants revealed DNA rearrangements due to insertion elements of the IS1, IS4 and IS5 families. Three of them were located in gvpN, gvpV or gvpW, the fourth one led to the deletion of the gvpAI-AIII region. The gvpA gene encoding the major structural protein remains transcribed but the corresponding protein is not synthesized in the three mutants, GvpN-, GvpV- and GvpW-. gvpV and gvpW are two new genes involved in the synthesis of GV in bacteria and cyanobacteria.

An ancestral Rubisco in Microcystis aeruginosa

Like all photosynthetic organisms that fix atmospheric CO2, M. aeruginosa synthesizes a ribulose diphosphate carboxylase/oxygenase (Rubisco) of type I, whose structure is L8S8. The new gene identified in M. aeruginosa PCC 7806 encodes a Rubisco of type IV whose in vitro structure is a dimer. In spite of the variations in the primary sequences, the secondary and tertiary structures of the Rubisco of type IV are similar to the Rubisco of types I, II and III described so far. The Rubisco of type IV is widely distributed among Microcystis strains, but absent in the ten cyanobacterial genomes sequenced to date (Coll. E. Dittmann, Humboldt University, Berlin, Germany).

3. Biosynthesis and detection of neurotoxins (A. Méjean, A. Coquin)

Cyanobacteria synthesize different types of neurotoxins that are lethal for animals and dangerous for Man. Several structural homologs of anatoxin-a and homoanatoxin-a have been characterized, following analyses of cyanobacterial cell extracts. Anatoxin-a and homoanatoxin-a labelled with 13C were biosynthesized for use as standards in metabolic studies or for quantification of these neurotoxins in environmental samples. A study of the relationship between neurotoxicity and the presence of genes encoding polyketide synthases in PCC strains is in progress.

4. Cyanobacterial genomes (N. Tandeau de Marsac, P. Quillardet, A.M. Castets)

- Sequencing of the genome of the hepatotoxic cyanobacterium Microcystis aeruginosa PCC 7806 is pursued at the Génopole, Institute Pasteur (Coll. S. Cole and C. Bouchier, Plate-forme Technique 1-Genomics, P. Glaser and F. Kunst, Laboratoire de Génomique des Micro-organismes Pathogènes). A declaration of invention has been deposited in Germany, following the discovery of a gene encoding microvirin, an antiviral protein (Coll. E. Dittmann, Humboldt Universität, Germany).

- The sequence the genome of Prochlorococcus marinus SS120 is now available on the web sites: http://www.bart.sb-roscoff.fr/blast/blastSS120.html or http://www.kazusa.or.jp/cyano/ (Coll. F. Partensky, Station Biologique, Roscoff, and the Génoscope, Evry, France). This marine cyanobacterium, very abundant in tropical and subtropical oceans, is the smallest photosynthetic microorganism (0.6 μm). Compared to other cyanobacteria, the genome of SS120 (1.75 Mbp, about 1900 genes) is remarkable by its compactness and low number of genes involved in regulatory systems or the perception of environmental signals. This genome permits to identify the minimal set of genes required to perform photosynthesis.

Photos : Marine cyanobacteria of the genera Trichodesmium (left) and Prochlorococcus (right)

Keywords: Collection of cyanobacteria (PCC), Databases, Biodiversity, Toxins, Metabolism, Genome

Activity Reports 2003 - Institut Pasteur

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