Annual report of Unité des Cyanobactéries for year 2000

Director : TANDEAU de MARSAC Nicole (ntmarsac@pasteur.fr)

The cyanobacteria share with plants the capacity to perform oxygenic photosynthesis by using light and water for the reduction of CO₂. Endowed with capacities 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. Research work in the Unit is focussed on the development and valorisation of the collection of cyanobacteria (PCC), in particular strains potentially toxic, and on the study of the molecular mechanisms controlling the acclimation of these micro-organisms to changes in environmental parameters.

1. The PCC "Pasteur "Pasteur Culture Collection of Cyanobacteria", centre of research and documentation on cyanobacteria (R. Rippka and M. Herdman)

Our Unit possesses the largest collection of pure cyanobacterial cultures in the world.
The PCC has several missons:

preservation of biological resources (more than 720 pure strains);

consulting and sale of strains to research laboratories and industries all over the world; the number of strain requests has doubled in 2000 when compared with previous years;

isolation, purification and identification of strains by using a polyphasic approach; in 2000, emphasis has been put on potentially toxic planctonic strains;
search for and characterisation of bio-active molecules of pharmaceutical interests, including toxins; a transverse research programme (PTR), entitled "Neurotoxins and related compounds", cooordinated by M. Herdman, is ongoing (collaborations with H-O Nghiem from the Molecular Neurobiology unit, B. Baron from the Eukaryotic Gene Expression Unit and O. Barzu from the Laboratory of Structural Chemistry of Macromolecules).

Development of molecular tools for the detection, identification and survey of cyanobacteria in laboratory cultures or in the environment; new tools have ben designed for in situ hybridization and genotyping of potentially toxic strains.

In collaboration with J.M. Delattre (Department of Waters and Environment, IP-Lille), expert tests have been set up for the identification and the determination of hepatotoxins in samples from surface waters. Several databases are in constant evolution:

a database of the properties of cyanobacteria is available on the Web, via the server of the Institut Pasteur (http://www.pasteur.fr/recherche/banques/PCC/);
a database of 16S rRNA sequences, representative of the biodiversity of cyanobacteria, consists of more than 500 aligned sequences;
a database of ITS consists of 30 aligned sequences and 300 entries of their number and size; the development of this database that will permit a rapid identification of the strains will be a very valuable tool for quality control tests.

A second edition of the Bergey's Manual of Systematic Bacteriology has been published in which the descriptions of the cyanobacterial strains are largely based on studies performed on strains from the PCC.

2. Molecular mechanisms of the acclimation of cyanobacteria to the environment (N. Tandeau de Marsac)

Light and nutrients act in a number of circuits regulating cyanobacterial metabolism, and may, in some strains belonging to the genus Calothrix, control the differentiation of cells specialized for nitrogen fixation (heterocysts), and that of motile minifilaments (hormogonia), and/or regulate changes in pigment composition (complementary chromatic adaptation).

Cyanobacterial phytochromes, a family of photoreceptors ancestors of the plant phytochromes

Two cyanobacterial phytochromes, CphA and CphB, have been genetically characterized in Calothrix PCC 7601. These light-regulated histidine kinases act at an early step in the transmission of the red/far red light signal by a mechanism of phosphorylation/dephosphorylation of the response regulators RcpA and RcpB. In contrast to CphA that carries a covalently bound chromophore, CphB carries a non-covalently bound one. No cross-talk has been detected between CphA-RcpA and CphB-RcpB, indicating that these couples are specific. Based on studies using a bacterial two-hybrid system, the RcpA protein forms homodimers. The 3D-structure of RcpB has been established, that of RcpA is under way. The biochemical and spectral properties of different mutants are being studied to elucidate the structure-function relationships of these four proteins (collaborations with W. Gärtner, T.Hübschmann and T. Börner, Germany, and with D. Ladant and G. Karimova, IP).

P_II, a signal transducer that coordinates carbon and nitrogen metabolism

The primary structure of the P_II (GlnB) protein is very conserved among procaryotes and plants, its function, however, differs depending on organisms. In order to get deeper insights into the role of this protein in cell metabolism, different cyanobacteria are being studied. In the strains Synechococcus PCC7942, obligate photoautotroph, and Synechocystis PCC 6803, facultative heterotroph, the phosphorylated form of P_II inhibits the uptake of nitrate/nitrite ions. This inhibition is relieved when P_II is liganded to 2-oxoglutarate and phosphorylated, i.e. in the presence of high carbon and low nitrogen concentrations. In Synechocystis, this modified form of P_II inhibits the high affinity transport system for bicarbonate ions. Finally, the phosphorylation level of P_II in both Synechococcus and Synechocystis depends on the intracellular redox potential and, consequently, the photosynthetic activity of the cell (collaboration with S. Bédu and R. Jeanjean, LCB, Marseille). This regulatory process allows cells to maintain a proper N/C balance in response to changes in light and nutrients (nitrogen and carbon) in the environment.
In the frame of an European Programme (PROMOLEC "Prochlorococcus molecular ecology", programme MAAST III, 1998-2001), some characteristics of Prochlorococcus marinus, a cyanobacterium extremely abundant in oligotrophic tropical and subtropical oceans, have been studied. This strain has the smallest cell size (diameter 0.5-0.6mm), the smallest genome (1,67 Mb), the lowest percentage G+C (31 %) among cyanobacteria, and possesses atypical light harvesting antennae that consist of protein complexes containing divinyl-chlorophylls a and b but no phycobiliproteins.
P. marinus has a cyanobacterial-type P_II that remains unphosphorylated, although its primary and quaternary structures are well conserved (collaborations with S. Loiseaux-De Goër, CNRS, Roscoff, and A. Blondel, IP). The presence of a high affinity transport system for bicarbonate ions that is active even in the presence of a high CO₂ concentration and the lack of an active transport system for nitrate/nitrite coïncide with the presence of a P_II protein which remains dephosphorylated under these conditions (collaboration with S. Bédu, CNRS, Marseille).

3. Cyanobacterial genomes (N. Tandeau de Marsac)1000 short tagged sequences (STS) of the genome of the hepatotoxic strain Microcystis PCC 7806 have been sequenced in the Laboratory of F. Kunst and P. Glaser (IP) and analyzed in collaboration with our unit. The genome of this cyanobacterium displays a low level of reiteration and noticingly differs from that of the non-toxic strain Synechocystis PCC 6803. In silico analyses of the sequence of the latter genome revealed the presence of repeated motives whose function(s) remain(s) to be determined (collaboration with M. F. Sagot, IP).

Legend of the photograph:
In situ hybridations (FISH) of hepatotoxic cyanobacteria belonging to the genus Microcystis (green) and of eukaryotic organisms (amoebae; red) from a water sample. Detection with two different fluorochromes coupled to tyramide and observation under confocal laser scanning microscopy (W. Shönhuber and M. Herdman).

More informations on our web site

Publications of the unit on Pasteur's references database

	Office staff	Researchers	Scientific trainees	Other personnel
	LEFEBVRE Jacqueline, IP	HERDMAN Michael, CNRS RIPPKA-HERDMAN Rosmarie, IP	ITEMAN Isabelle, Post-doc LALOUI Wassila, Post-doc MARCEL Anne, Stagiaire DESS SCHÖNHUBER Wilhelm, Post-doc WU Tianfu, Post-doc	CASTETS Anne-Marie, CNRS COURSIN Thérèse, IP LAURENT Thierry, IP

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