Unit: Cyanobacteria - CNRS URA 2172
Director: TANDEAU de MARSAC Nicole
Cyanobacteria, oxygenic photosynthetic procaryotes, largely contribute 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. For the last decades, the occurrence of waterblooms of planktonic cyanobacteria greatly increased in continental aquatic ecosystems as a consequence of pollution generated by humans. These proliferations disrupt ecosystem equilibrium and may be harmful to animals and Man due to the large number of secondary metabolites (hepato- and/or neurotoxins) some cyanobacteria may produce.
Our research programme aims at increasing our knowledge of the biodiversity and physiology of cyanobacteria, in particular toxic strains. These studies from gene to ecosystem apply a multidisciplinary approach that relies on the analysis of environmental samples and on The PCC, a collection of axenic strains that our laboratory has developed over the years.
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, M. Herdman, T. Coursin and T. Laurent)
The PCC houses 750 axenic strains representing about sixty morphotypes isolated from very diverse ecosystems; 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. A large number of cyanobacteria are cryosensitive even in the presence of a cryoprotectant such as dimethylsulfoximide. We have shown that addition of desferrioxamine, an iron chelator that indirectly acts as an antioxydant, improves the survival of two strains of the genus Arthrospira ("Spirulina"). This chelator is presently being tested on other strains in order to generalize its use if possible.
Consultation and sale of strains (R. Rippka)
In 2005, about 300 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.
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 PCC appeared in 2005.
Research and characterisation of toxic and bioactive secondary metabolites
Neurotoxic alkaloids (A. Méjean, C. Peyraud-Thomas and R. Rippka). Cyanobacteria synthesize different types of neurotoxic alkaloids, harmful to animals and humans. In order to quantify anatoxin-a in neurotoxic cyanobacterial strains and in environmental samples, we have synthesized in vivo anatoxin-a labelled with 13C and 15N isotopes using the strain Oscillatoria PCC 6506. Collection of environmental samples was conducted in summer in the South of France where mortality of dogs has been reported each year since 2002. The presence of anatoxin-a has been detected by gas chromatography coupled to mass spectrometry in most of the samples. Several filamentous nonheterocystous cyanobacteria have been isolated from these samples, including two strains belonging to the genera Geitlerinema and Oscillatoria. These strains have been axenized in order to determine their toxicity.
Toxic or bioactive oligopeptides (S. Cadel, M. Welker, V. Gaget, I. Iteman, A.M. Castets et N. Tandeau de Marsac). Cyanobacteria of the genus Microcystis synthesize hepatotoxic oligopeptides (microcystins) and inhibitors of serine proteases (cyanopeptolins and aeruginosins) by nonribosomal pathways. Cyanopeptolins also display cytotoxic activity and promote cell differentiation. In the context of the European project PEPCY "Toxic and bioactive peptides in cyanobacteria" (http://www.pepcy.de/) and in collaboration with the team of E. Dittmann (Humboldt Universität, Berlin, Germany), we have characterized peptide synthetases and polyketide synthases involved in the synthesis of these oligopeptides and constructed mutants in order to elucidate their role. Depending on strains, cyanopeptolins and aeruginosins are chlorinated or non-chlorinated and, in a given strain, chlorination of one of these two types of oligopeptides is exclusive. Accordingly, the genes encoding halogenases are present or absent in the genome. When present, these genes are flanked by direct and inverted repeated sequences characteristic of mobile elements, while in their absence, a genomic print of these elements remain. Cyanobacterial halogenases form a monophyletic group among bacteria suggesting their unique and ancient acquisition during evolution (Collaboration with C. Dauga, Plate-form 4 - Integration et Genome analysis). Moreover, the further division in two sub-groups indicates the high specificity of these enzymes for their respective substrate: 4-hydroxyphenyl lactate for the halogenase of aeruginosins and tyrosine for that of cyanopeptolins. These enzymes are FADH2-dependent and belong to the halogenase family that chlorinates phenols and pyrrol derivatives. The biochemical characterization of these halogenases and the determination of their function in Microcystis are underway. Finally, in the context of a Research and Development Project (Tripartite contract Veolia-Eau/Genesystems/Institut Pasteur), we are developing a system for the rapid and reliable detection and identification of three genera of potentially hepatotoxic cyanobacteria (Microcystis, Anabaena and Planktothrix). This device will be used to monitor health risk in drinking-water reservoirs.
2. Biodiversity and ecology of aquatic microbial communities (J.F. Humbert)
Monitoring of the Planktothrix rubescens populations in lake of Bourget: by a comparative approach, we have shown that red-pigmented Planktothrix strains that colonize lake of Bourget are particularly well adapted to their ecological niche. Indeed, this ecotype displays high competitiveness at low temperature and under low light enriched in green wavelengths. Moreover, since zooplankton do not graze on these cyanoabacteria, predation does not seem to directly control the dynamics of these populations (Collaboration with Pr. B. Pinel-Alloul, Université du Québec, Montréal, Canada).
Composition and structure of bacterial communities in continental aquatic ecosystems: we have determined the relative influence of the trophic control and the volumetric characteristics of water reservoirs, as well as other parameters such as the climate, on the composition and structure of bacterial communities in continental aquatic ecosystems. Several libraries of clones have been constructed using amplicons of 16S rRNA sequences obtained by PCR amplification of total DNA extracted from water samples collected from Alpine lakes and African reservoirs. Results indicate that all these communities are dominated by actinobacteria. It also appears that the bacterial composition in small-size West-African ecosystems may deeply vary (drift effect?). In contrast, in large Alpine lakes, despite contrasted trophic status, no significant difference has been observed in the structure of these communities (Collaboration with P. Cecchi and M. Bouvy, IRD, France).
3. Genome structure and acclimation in cyanobacteria (P. Quillardet, A.M. Castets, D. Mourier des Gayets et N. Tandeau de Marsac)
In the environment, planktonic cyanobacteria of the genus Microcystis form waterblooms potentially harmful to animals and Man. In order to elucidate the developmental cycle of these microorganisms and their acclimation capabilities to variations in environmental parameters, we have chosen the hepatotoxic strain Microcystis aeruginosa PCC 7806 as a model system. The finishing of its genome sequence is underway in collaboration with the teams of C. Bouchier (Plate-form 1 - Génomics), and L. Frangeul and S. Bun (Plate-form 4 - Integration et Genome Analysis). Mining of the genome sequence has already revealed genes encoding proteins of particular interest: a lectin (microvirin) and a RuBisCO-like protein (RbcLIV). Lectins are targets for intercellular signalling in bacteria that form biofilms. Similarly, the interactions between microcystins, lectins, and lipopolysaccharides may be involved in the formation and/or the maintenance of colonies, structures that largely contribute to the high competitiveness of these cyanobacteria in their environment (Collaboration with the team of E. Dittmann, Humboldt Universität, Berlin, Germany). RbcLIV is a form IV member of the superfamily of ribulose-1,5-diphosphate carboxylases/oxygenases (RuBisCOs). It shares similarities with the large subunit of the RuBisCO of form I that fixes carbon dioxide in photosynthetic organisms. In Microcystis cells, the expression of rbcLIV increases under sulphur limitation suggesting a role of this protein in sulphur metabolism. Moreover, this enzyme has no carboxylase but an enolase activity. Therefore it perfoms only the first of the three reactions catalysed by the RubisCO of form I. Finally, complementation of a MtnW mutant in B. subtilis indicated that it might be involved in the recycling of methionine (Collaboration with A. Yokota, Nara Institute of Science and Technology, Japan). This metabolic pathway so far unknown in cyanobacteria may benefit to Microcystis cells exposed to high light intensity and to elevated oxygen concentration at the surface of dense waterblooms. Cyanobacteria are presently the only microorganisms in which both a photosynthetic RubisCO and a RuBisCO-like protein of form IV are demonstrated to coexist.
Cyanobacteria belonging to the genera Prochlorococcus and Synechococcus are ubiquitous and dominant in the marine phytoplankton. They highly contribute to the photosynthetic primary production on our planet. In central regions of members of Prochlorococcus are more abundant than those of Synechococcus. Conversely, the latter are preponderant in nutrient-rich and coastal areas. In 2005, the sequence of the genome of Synechococcus RCC307 (http://www.cns.fr/externe/Francais/Projets/Projet_HP/organisme_HP.html), a strain isolated from an oligotrophic area, 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). The genome sequence of the coastal strain Synechococcus WH7803 determined last year has been annotated. Comparative analysis of ten genomes of marine Synechococcus strains (from upwelling zones and central or coastal areas) and three Prochlorococcus members revealed the structure diversity of their genome (different degrees of compactness or plasticity), as well as specific traits of their evolution depending on the ecological niches they colonize.
Legend of the pictures : Anabaena sp. (left), Microcystis sp. (centre), Planktothrix sp. (right).