Cyanobacteria that appeared on our planet about 3 billion years ago contribute even today, by their ability to perform oxygenic photosynthesis, to the balance between CO2 and O2 in the atmosphere. Capable of acclimation 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 develop, by using 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 and M. Herdman)

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; in 2002, new strains assignable to the genera Gloeocapsa and Aphanizomenon (a saxitoxin producer) entered the PCC; 450 strains are presented in a catalogue ( http://www.pasteur.fr/recherche/banques/PCC/ ).

• Consultation and sale of strains to research laboratories and industries all over the world, including teaching institutions in France.

• Development of molecular tools for the detection, identification and survey of cyanobacteria in laboratory cultures or in the environment;

• Search for and characterisation of bio-active molecules of pharmaceutical interest (Collaboration with V. Bultel, Muséum National d'Histoire Naturelle, Paris); in the frame of a transverse research programme (PTR), entitled "Neurotoxins and related alkaloids" and coordinated by M. Herdman, development of a micro-detection method for neurotoxins; 10 % of the PCC strains tested were found to be neurotoxic.

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 that permit to choose oligonucleotide primers and probes; database (software GelCompare) of profiles generated by ITS-RFLP and amplification with HIP1 extended primers.

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 the frame of the European contract COBRA "The Conservation of a vital European scientific and biotechnological resource: microalgae and cyanobacteria" (Programme "Quality of Life"), different cryopreservation methods are presently tested, as well as a PCR method using the repetitive repeat sequences HIP1, with variable extensions, for amplification and control of strain identity.

2. Molecular mechanisms of the acclimation of cyanobacteria to the environment

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.

Cyanobacterial photoreceptors, ancestors of the plant phytochromes(Collaborations with W. Gärtner (Max Planck Institut, Mülheim, Germany), B. Quest (Max Planck Institut, Martinsried, Germany) and G. Karimova (Biochimie des Interactions Macromoléculaires, Institut Pasteur).

In Calothrix sp. PCC 7601, two systems of photoperception, the phytochrome-like CphA and CphB associated to the response regulators, RcpA and RcpB, respectively, display strong similarities to bacterial two-component regulatory systems. In vitro, a chromophore of the phycocyanobilin-type covalently binds to the residue Cys259 of CphA. In contrast, in CphB a chromophore of the biliverdin IXa -type binds not non-covalently to the Leu266 residue. The holoprotein CphB recovers spectral properties similar to the holoprotein CphA when Leu266 is replaced by a cysteinyl residue. CphA and CphB act at an early step of the transduction of the light signal by a mechanism of phosphorylation/dephosphorylation of RcpA and RcpB, respectively. The 3D structure of RcpA and RcpB is closely related to that of the response regulators of the CheY family, but they possess a novel dimerisation motif.

Carbon and nitrogen metabolism, floatation and toxicity(Collaboration with E. Dittmann, Humboldt University, Berlin, Germany)

Microcystis aeruginosa PCC 7806 is a representative of planktonic hepatotoxic cyanobacteria that synthesize gas vesicles (intracellular floatation devices) and under our latitude form blooms at the surface of water bodies during summer time. A research programme has been initiated to establish the relationships between the metabolism of carbon and nitrogen, the formation of gas vesicles and the production of hepatotoxins (microcystins). As in other unicellular cyanobacteria from freshwater previously studied in the Unit, a PII protein, whose phosphorylation state depends on the availability of carbon and nitrogen, has been evidenced in strain PCC 7806. This protein is a key element of a control system that coordinates the metabolism of nitrogen and carbon in response to variations of environmental parameters. This system also includes the global transcriptional regulator NtcA. A cluster of genes (gvpA1A2A3CNJKF/L), encoding components involved in gas vesicle synthesis, has been characterized. Some of these genes are organized in operons whose transcription is segmented. The promoter regions of some gvp genes and of gene modules involved in the non-ribosomal synthesis of microcystins contain a DNA binding site for the regulator NtcA, suggesting that their expression is controlled by nitrogen. The genes encoding the two sub-units of ribulose biphosphate carboxylase/oxygenase (Rubisco), a key enzyme of the Calvin cycle of CO2 fixation, have also been characterized. A new gene coding for a protein sharing similarity with Rubisco has been discovered. The structure and function of the corresponding gene product overexpressed in E. coli are under study.

Cyanobacterial genomes (N. Tandeau de Marsac and I. Iteman)

Sequencing of the genome of the hepatotoxic cyanobacterium Microcystis aeruginosa PCC 7806 is ongoing at the genopole from the Institut Pasteur (Collaborations with S. Cole and C. Bouchiez, Plate-forme Technique 1-Genomics, P. Glaser and F. Kunst, Laboratoire de Génomique des Micro-organismes Pathogènes).

A web server for the genomes of two cyanobacteria, Synechocystis PCC 6803 and Anabaena PCC 7120 has been established (http://genolist.pasteur.fr/CyanoList/). A multi-organism database, including bacterial and cyanobacterial genomes is in progress (Collaboration with I. Moszer, Plate-forme Technique 4-Annotation et Analyse de Génomes).

The annotation of the genome of the marine cyanobacterium Prochlorococcus SS120 is in progress (Collaboration with the Génoscope, Evry, France). This cyanobacterial species is very abundant at 200 m depth (low-light ecotype). The genome of SS120 can now be compared to those of other ecotypes of the genus Prochlorococcus (MED4 and MIT 9313) and to that of the marine Synechococcus WH8102 (sequences available at the DOE Joint Genome Institute, USA).

Photos : Toxic planktonic cyanobacteria: Oscillatoria PCC 6506, producer of neurotoxins (left) et Planktothrix PCC 10106, producer of hepatotoxins (right)

Keywords: Collection of cyanobacteria (PCC), Databases, Biodiversity, Toxins, Photoperception and signal transduction, Genome