Prochlorococcus and Synechococcus have Evolved Different Adaptive Mechanisms to Cope with Light and UV Stress.

International audienceProchlorococcus and Synechococcus, which numerically dominate vast oceanic areas, are the two most abundant oxygenic phototrophs on Earth. Although they require solar energy for photosynthesis, excess light and associated high UV radiations can induce high levels of oxidative stress that may have deleterious effects on their growth and productivity. Here, we compared the photophysiologies of the model strains Prochlorococcus marinus PCC 9511 and Synechococcus sp. WH7803 grown under a bell-shaped light/dark cycle of high visible light supplemented or not with UV. Prochlorococcus exhibited a higher sensitivity to photoinactivation than Synechococcus under both conditions, as shown by a larger drop of photosystem II (PSII) quantum yield at noon and different diel patterns of the D1 protein pool. In the presence of UV, the PSII repair rate was significantly depressed at noon in Prochlorococcus compared to Synechococcus. Additionally, Prochlorococcus was more sensitive than Synechococcus to oxidative stress, as shown by the different degrees of PSII photoinactivation after addition of hydrogen peroxide. A transcriptional analysis also revealed dramatic discrepancies between the two organisms in the diel expression patterns of several genes involved notably in the biosynthesis and/or repair of photosystems, light-harvesting complexes, CO(2) fixation as well as protection mechanisms against light, UV, and oxidative stress, which likely translate profound differences in their light-controlled regulation. Altogether our results suggest that while Synechococcus has developed efficient ways to cope with light and UV stress, Prochlorococcus cells seemingly survive stressful hours of the day by launching a minimal set of protection mechanisms and by temporarily bringing down several key metabolic processes. This study provides unprecedented insights into understanding the distinct depth distributions and dynamics of these two picocyanobacteria in the field

Light history influences the response of the marine cyanobacterium Synechococcus sp. WH7803 to oxidative stress.

International audienceMarine Synechococcus undergo a wide range of environmental stressors, especially high and variable irradiance, which may induce oxidative stress through the generation of reactive oxygen species (ROS). While light and ROS could act synergistically on the impairment of photosynthesis, inducing photodamage and inhibiting photosystem II repair, acclimation to high irradiance is also thought to confer resistance to other stressors. To identify the respective roles of light and ROS in the photoinhibition process and detect a possible light-driven tolerance to oxidative stress, we compared the photophysiological and transcriptomic responses of Synechococcus sp. WH7803 acclimated to low light (LL) or high light (HL) to oxidative stress, induced by hydrogen peroxide (H₂O₂) or methylviologen. While photosynthetic activity was much more affected in HL than in LL cells, only HL cells were able to recover growth and photosynthesis after the addition of 25 μM H₂O₂. Depending upon light conditions and H₂O₂ concentration, the latter oxidizing agent induced photosystem II inactivation through both direct damage to the reaction centers and inhibition of its repair cycle. Although the global transcriptome response appeared similar in LL and HL cells, some processes were specifically induced in HL cells that seemingly helped them withstand oxidative stress, including enhancement of photoprotection and ROS detoxification, repair of ROS-driven damage, and regulation of redox state. Detection of putative LexA binding sites allowed the identification of the putative LexA regulon, which was down-regulated in HL compared with LL cells but up-regulated by oxidative stress under both growth irradiances

Ultraviolet stress delays chromosome replication in light/dark synchronized cells of the marine cyanobacterium Prochlorococcus marinus PCC9511.

International audienceBACKGROUND: The marine cyanobacterium Prochlorococcus is very abundant in warm, nutrient-poor oceanic areas. The upper mixed layer of oceans is populated by high light-adapted Prochlorococcus ecotypes, which despite their tiny genome (approximately 1.7 Mb) seem to have developed efficient strategies to cope with stressful levels of photosynthetically active and ultraviolet (UV) radiation. At a molecular level, little is known yet about how such minimalist microorganisms manage to sustain high growth rates and avoid potentially detrimental, UV-induced mutations to their DNA. To address this question, we studied the cell cycle dynamics of P. marinus PCC9511 cells grown under high fluxes of visible light in the presence or absence of UV radiation. Near natural light-dark cycles of both light sources were obtained using a custom-designed illumination system (cyclostat). Expression patterns of key DNA synthesis and repair, cell division, and clock genes were analyzed in order to decipher molecular mechanisms of adaptation to UV radiation. RESULTS: The cell cycle of P. marinus PCC9511 was strongly synchronized by the day-night cycle. The most conspicuous response of cells to UV radiation was a delay in chromosome replication, with a peak of DNA synthesis shifted about 2 h into the dark period. This delay was seemingly linked to a strong downregulation of genes governing DNA replication (dnaA) and cell division (ftsZ, sepF), whereas most genes involved in DNA repair (such as recA, phrA, uvrA, ruvC, umuC) were already activated under high visible light and their expression levels were only slightly affected by additional UV exposure. CONCLUSIONS: Prochlorococcus cells modified the timing of the S phase in response to UV exposure, therefore reducing the risk that mutations would occur during this particularly sensitive stage of the cell cycle. We identified several possible explanations for the observed timeshift. Among these, the sharp decrease in transcript levels of the dnaA gene, encoding the DNA replication initiator protein, is sufficient by itself to explain this response, since DNA synthesis starts only when the cellular concentration of DnaA reaches a critical threshold. However, the observed response likely results from a more complex combination of UV-altered biological processes

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote Small Sub-Unit rRNA sequences with curated taxonomy

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR2, http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientist

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote Small Sub-Unit rRNA sequences with curated taxonomy

International audienceThe interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR2, http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing

International audienceAlthough protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date

A new approach to analyze genotypes of colony-forming cyanobacteria from environmental samples.

Several studies have shown the efficiency of sequences as rRNA-ITS, cpcBA, rbcLX and other housekeeping genes to study taxonomy [1, 2, 3], population, community structure of cyanobacteria, or for Multi Locus Sequence Analysis [4]. Recently, the genotypic analysis of single colonies and single filaments directly isolated from the environment has been carried out by other authors. It appears that different genotypes of Microcystis are present in one population in one lake. Besides, succession of toxic and non-toxic genotypes may have a critical influence on toxin concentrations during the blooms [5]. Genotypic analysis of colony-forming cyanobacteria requires enough DNA. So far, the genotypes of environmental single colonies of Microcystis were characterized on the basis of one or two PCR [6]. As the DNA content of one single colony only allows for a few PCR reactions, we have developed a new approach using Whole Genome Amplification with Phi29 polymerase to allow for the Multi Locus Sequences Typing analysis of a single colony or filament. For the first time, we were able to amplify and sequence more than one locus of the genome of a single colony of Microcystis. In addition, we have obtained the first sequences of rpoC1, rbcLX and rRNA-ITS from a single colony of the genus Woronichinia ( identified by microscopy). This approach allows to work with a small amount of DNA, and represents a concrete answer to the lack of data on non-cultivable cyanobacteria. This research is supported by the Belgian Science policy under the science for a sustainable development (SSD) and Fonds de la Recherche Scientifique-FNRS with a FRIA fellowship. References: [1] Otsuka S, et al (1999) FEMS Microbiology Letters 172 15-21 [2] Gugger M, et al (2002) Int J Syst Evol Microbiol 52 1867-1880 [3] Haverkamp T, et al (2008) Environmental Microbiology 10(1) 174-188 [4] Lodders N, et al (2005) Environmental Microbiology 7 (3) 434-442 [5] Kardinaal E, and Visser P (2005) In Harmfuf cyanobacteria, Springer Dordrecht pp 41-64 [6] Janse I, et al (2004) Appl Environm Microbiol 70 (7) 3979–398

Molecular diversity of planktonic cyanobacteria in belgian fresh waterbodies

Les développements massifs de cyanobactéries ou ‘blooms’ sont devenus un phénomène récurrent et de plus en plus important dans les eaux douces du monde entier durant la dernière décennie. Ces efflorescences présentent des risques potentiels majeurs pour la santé humaine et animale et interfèrent négativement avec l'utilisation des eaux de surface par exemple, pour le captage d'eau potable, les loisirs nautiques, l'irrigation, les exploitations piscicoles. Entre 25 et 70% des blooms sont toxiques. Comme beaucoup de pays la Belgique n'a pas échappé au problème des efflorescences de cyanobactéries toxiques, mais il y a encore relativement peu de données. Durant la dernière décennie, trois projets européens et nationaux (MIDICHIP 1999-2003, B-BLOOMS 2003-2005, B-BLOOMS 2 2007-2011) se sont intéressés à la diversité des cyanobactéries dans les eaux de surfaces belges. Nous présentons ici un arbre phylogénétique élaboré à partir d’ un pool de 249 séquences partielles du gène codant pour l’ARNr 16S obtenu à partir de 31 échantillons d’eaux belges issus de ces projets. Cet arbre représente la mise à jour d’une base de données qui constitue l’inventaire des cyanobactéries d’eaux douces belges. Cette base de données permet le suivi de l’évolution de la diversité observable de ces organismes en Belgique et la surveillance de l’apparition d’espèces tropicales comme conséquence aux changements climatiques globaux

Dynamique cyanobactérienne saisonnière dans un réservoir mésoeutrophe: comptages microscopiques et DGGE (Denaturating Gradient Gel Electrophoresis)

The seasonal planktic cyanobacterial dynamics was assessed during the year 2000 by microscopic and DGGE techniques, on the basis of 22 samples collected from the Haute-Sûre reservoir (Grand-Duchy of Luxembourg). Microscopic investigations were carried out according to the standard Utermöhl procedure while 16S rRNA gene fragments obtained by semi-nested PCR were subsequently separated by DGGE. Sequencing of selected excised bands was performed to genotypically define the cyanobacterial assemblages. The dynamics of cyanobacterial communities obtained by both approaches were compared. Several discordances were pointed out. The counting procedure failed to detect cyanobacteria with small dimensions or in very low abundances, whereas DGGE had a lower detection limit when cyanobacteria were scarce (e.g. in spring) and performed better for the study of picosized forms. Generally, only the dominant cyanobacteria were revealed by these two methods. Actually, both techniques appeared to be complementary rather than equivalent. This study underlines the necessity to use multidisciplinary approaches to obtain a more complete view of the microbial diversity and of the community structureMIDI-CHI