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 colonise most aquatic and terrestrial ecosystems. Some of them form waterblooms, disrupting ecosystem equilibrium, and produce hepatotoxins or neurotoxins, secondary metabolites 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 genome and the physiology of cyanobacteria, to elucidate their modes of acclimation to the environment and the bases of their toxicity, as well as to exploit their biotechnological potential.

1. The PCC or "Pasteur Culture Collection of Cyanobacteria"

The PCC is internationally recognised 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 (R. Rippka, I. Iteman, K. Comte, M. Herdman, T. Coursin and T. Laurent)

The PCC houses 750 strains representing 57 genera; 475 strains are presented in a catalogue (http://www.pasteur.fr/recherche/banques/PCC/). Anticipating the future integration of the PCC in the Centre for Biological Resources at the Institut Pasteur (CRBIP), steps towards quality control are in progress. 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/) several methods of cryopreservation, including in the presence of antioxidants (α-tocopherol or desferrioxamine), confirmed the cryosensitivity of strains of the genus Arthrospira ("Spirulina"). Different methods of genotyping were applied to cyanobacteria of the PCC, in particular to strains of the genera Arthrospira, Spirulina, Planktothrix, Microcystis and Nostoc, in order to determine their internal genetic relationships, as well as to strains of Phormidium recently isolated from extreme ecosystems (Coll. J. Elster and M. Sabacka, University of South Bohemia, Trebon, Czech Republic). The phylogenetic clustering observed for isolates from both Arctic and Antarctic regions may result from fortuitous dispersal by Man or is due to bird migration between the poles.

Consultation and sale of strains (R. Rippka)

In 2004, 236 strains have been provided to research and industrial laboratories all over the world, including teaching institutions in France. Consultation is another important activity associated with the PCC.

Expert analyses (I. Iteman and T. Laurent)

This activity includes the identification of cyanobacteria and the determination of hepatotoxins in samples from surface waters (Coll. J.M. Delattre, Department of Waters and Environment, Institut Pasteur Lille). In 2004, about hundred analyses have been performed for the DDASS in diverse regions of France. Monitoring of waterbodies for the presence of potentially toxic cyanobacteria has increased significantly since the publication of a decree on the sanitary control of bathing waters.

Databases (I. Iteman, R. Rippka and M. Herdman)

"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. Other databases are independent of CYANOBANK: "ITS sequence database" with ~300 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; photographic database of environmental samples (~700 entries). A majority of the strain information from the PCC has been transferred from CYANOBANK to a new database shared by all the culture collections of the CRBIP and a new edition of the catalogue of the cyanobacterial strains will appear in 2005.

Research development and characterisation of toxic and bioactive secondary metabolites

- Neurotoxic alkaloids (A. Méjean, C. Monard and I. Iteman)

Cyanobacteria synthesise different types of neurotoxic alkaloids, harmful for animals and humans. As previously shown in the laboratory by R. Araoz and coll. (Microbiology, in press) three PCC strains of the genus Oscillatoria synthesise anatoxin-a and homoanatoxin-a. The presence of either of the toxins depends on growth conditions. Three genes encoding the ketoacylsynthase domain of polyketide synthases have been identified in the neurotoxic strain Oscillatoria PCC 6506. Analysis of the relationship between the production of neurotoxic molecules and the presence of theses genes in fourteen strains of the PCC demonstrated that one of the genes is a good candidate for use as a molecular marker for neurotoxicity. This marker should permit the early detection of neurotoxic cyanobacteria in environmental samples.

- Bioactive oligopeptides (M. Welker, S. Cadel, A.M. Castets and N. Tandeau de Marsac). In the frame of the European project PEPCY "Toxic and bioactive peptides in cyanobacteria"  (http://www.pepcy.de/), two new clusters of genes encoding polyketide synthases in Microcystis aeruginosa PCC 7806 were characterised. These genes are involved in the synthesis of two secondary metabolites: cyanopeptolin and aeruginosin. These two non-ribosomal peptides are inhibitors of serine proteases. In addition, cyanopeptolin is cytotoxic and promotes cell differentiation. These peptides have been detected by HPLC and mass spectrometry (MALDI-TOF) in more than half of the strains from the PCC. New peptides and new congeners of known peptides, including mono- and di-chlorinated ones, have been identified in these strains (Coll. with H. Van Döhren, Technische Universität, Berlin, Germany).

2. Acclimation to the environment of the hepatotoxic cyanobacterium Microcystis (P. Quillardet, A.M. Castets, D. Ciumac and N. Tandeau de Marsac)

Light and nutrients act in a number of regulatory circuits in cyanobacterial metabolism, and play a key role in the formation of water blooms and in the intracellular content of toxins in planktonic species of the genus Microcystis. To perform a global analysis of the acclimation of these organisms, we have chosen the hepatotoxic strain Microcystis aeruginosa PCC 7806. The finishing of its genome sequence is in progress at the Génopole of the Institut Pasteur (C. Bouchier and coll., Plate-form 1 - Genomics; L. Frangeul and coll., Plate-form 4 – Integration et Genome Analysis). At present, we have at our disposal a series of large contigs covering almost the entire genome. Macroarrays with a selection of genes of interest have been prepared for transcriptome analyses in function of fluctuating environmental parameters, such as light and nutrients. These analyses are in progress (Coll. E. Dittmann, Humboldt University, Berlin, Germany).

The role of the hepatotoxins synthesised by a number of Microcystis strains is unknown. Microcystins, however, might play a role in the regulation of processes important for cell life. We have indeed shown that the presence of these oligopeptides influences in particular the synthesis of gas vesicles (intracellular structures proving buoyancy) and of the RuBisCo (enzyme for CO2 fixation). The genome sequence permitted the identification by proteomics of other proteins that respond to microcystins. One of them is an ortholog of a plant protein; others are unique, or at least absent from the cyanobacterial genomes sequenced. A Microcystis-specific lectin has also been characterised. This lectin might play a role in cell-cell attachment and therefore in the formation of colonies, a feature often observed for cyanobacteria of this genus (Coll. with E. Dittmann, Y. Zilliges and J.C. Kehr, Humboldt Universität, Berlin, Germany).

3. Comparative genomics: marine cyanobacteria and nitrogen metabolism (N. Tandeau de Marsac)

Cyanobacteria of the genera Prochlorococcus and Synechococcus dominate in marine phytoplankton and make a major contribution to the global photosynthetic primary production. In central regions of the oligotrophic oceans, members of Prochlorococcus are more abundant than those of Synechococcus. Conversely, the latter are preponderant in nutrient-rich and coastal areas. In 2004, the sequence of the genome of the coastal strain Synechococcus WH7803 has been determined at the Génoscope (Evry, France), by an international consortium of five laboratories including ours (Coordination: F. Partensky, Station Biologique de Roscoff, France). Comparison of the five genomes of Prochlorococcus (MED4, SS120 et MIT9313) and Synechococcus (WH8102 and WH7803) shows that, except for a proteobacterial-like nitrite transporter present in MIT9313, the genes involved in nitrogen metabolism have not been acquired by horizontal gene transfer but originate from an ancestral cyanobacterium. During evolution, some of the non-essential genes were lost giving rise to genotypes specific of the different ecological niches typically inhabited by Prochlorococcus and Synechococcus populations (Coll. with JM García-Fernández, Universidad de Córdoba, Córdoba, Spain). These changes were accompanied by a simplification of the regulatory networks and a remarkable reduction of genome sizes.

Photos:

Photo 1: Axenic culture of Arthrospira sp. PCC 9450 (Bar marker: 20 μm).

Photo 2: Aphanizomenon sp., Microcystis sp., Limnothrix sp. and Planktothrix sp. in a sample collected from a freshwater lake in France (Bar marker: 20 μm).

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


Activity Reports 2004 - Institut Pasteur
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