Flow cytometry (FCM) is a well established technique used for enumeration and characterization of marine biological particles, which fulfills the scientific demands of rapid cell counting automation. FCM allows the discrimination of pico- and nanoplankton populations regarding its abundance, cell size, and pigment content both by natural or induced fluorescence. The cyanobacterium Prochlorococcus is widespread in the euphotic zone of the tropical and subtropical oceans, and is considered the smallest and most abundant photosynthetic organism in the planet. Synechococcus, other important cyanobacterium genus present in the picoplankton, is highly diverse and is widely distributed in marine ecosystems from cold and mesotrophic, to warm, open ocean oligotrophic waters. Photosynthetic pico- and nanoeukaryotes display a range of physiologies and life strategies. Although its abundance is generally lower, the larger cell size leads to a significant contribution to the epipelagic community biomass. In this thesis I aimed to investigate the abundance and carbon biomass distribution of heterotrophic bacteria, Prochlorococcus, Synechococcus, autotrophic pico- and nanoeukaryotes in the Southwest Atlantic Ocean off Brazil, their relation to the different water masses and the influence of hydrodynamic (South Atlantic Central Water intrusion) and biological processes (Trichodesmium spp. and Mesodinium rubrum blooms) on such distributions. / A citometria de fluxo (FCM, sigla em inglês) é uma técnica bem estabelecida, usada para enumeração e caracterização de partículas biológicas marinhas, a qual supre a demanda científica por automação rápida de contagem de células. A FCM permite a discriminação de populações pico- e nanoplanctônicas no que concerne à sua abundância, tamanho de célula e pigmentação, tanto por fluorescência natural ou induzida. A cianobactéria Prochlorococcus é amplamente disseminada pela zona eufótica dos oceanos tropicais e subtropicais, sendo considerada o menor e mais abundante organismo fotossintético do planeta. Synechococcus, outro importante gênero de cianobactérias presente no picoplâncton, é diverso e amplamente distribuído em ecossistemas marinhos, de águas frias e mesotróficas à regiões oceânicas quentes e de águas oligotróficas. Pico- e nanoeucariotos fotossintéticos apresentam uma grande variedade de fisiologias e estratégias de vida. Embora a abundância destes grupos seja geralmente menor, o maior tamanho de suas células resulta numa contribuição significante dos mesmos para a biomassa da comunidade epipelágica. Na presente tese eu objetivei investigar a abundância e a distribuição da biomassa de carbono de bactérias heterotróficas, Prochlorococcus, Synechococcus, pico- e nanoeucariotos autotróficos no sudoeste do Oceano Atlântico ao largo do Brasil, sua relação com as diferentes massas d\'água e a influência de processos hidrodinâmicos (como a intrusão da Água Central do Atlântico Sul) e biológicos (florações de Trichodesmium spp. e Mesodinium rubrum) em tais distribuições.

Cyanobacteria are a model organism for photosynthesis and the Calvin cycle, and a promising chassis for 4th generation biofuel production. There are many ongoing efforts to improve the performance of cyanobacteria, in terms of CO2 fixation and production rate of biofuels. One straightforward way to improve CO2 fixation could be to overexpress the genes of limiting enzymes. In this project, we used a high-throughput method to test the overexpression of thousands of genes in cyanobacteria and measure the effect on growth rate. We created barcoded overexpression libraries, consisting of gene fragments from different cyanobacteria strains and transformed them into a model cyanobacterium, Synechocystis PCC 6803. We then cultivated the transformed cyanobacteria libraries and screened for effects of increased gene copy number on both maximum growth rate and robustness of growth under stress conditions. The cell populations were cultivated in a turbidostat, resulting in competitive growth between transformants. The relative abundance of each mutant was estimated using deep sequencing. Fitness scores, for each gene show how expression of that gene affects growth rate. This method, competitive growth and tracking of mutant populations with deep sequencing, is a high throughput method for screening a large proportion of genes from several organism at once, allowing the identification of trans-species effects as well as the effect of single genes on the metabolism of the host cell. / Cyanobakterier är en modellorganism för fotosyntes och Calvin-cykeln och ett lovande chassi för fjärde generationens biobränsleproduktion. Det finns många pågående ansträngningar för att förbättra cyanobakteriens prestanda med avseende på CO2-fixering och produktionshastighet för biobränslen. Ett enkelt sätt att förbättra CO2-fixering kan vara att överuttrycka generna för begränsande enzymer. I detta projekt använde vi en metod med hög kapacitet för att testa överuttryck av tusentals gener i cyanobakterier och mäta effekten på tillväxthastigheten. Vi skapade streckkodade överuttrycksbibliotek, bestående av genfragment från olika arter av cyanobakterier och transformerade in dem i en modellorganism för cyanobakterium, Synechocystis PCC 6803. Vi odlade sedan de transformerade biblioteken och screenade efter effekten av ökade antal genkopior på både maximal tillväxthastighet och robusthet hos tillväxt under stressförhållanden. Cellpopulationerna odlades i en Turbidostat, vilket resulterade i konkurrerande tillväxt mellan transformanter. Den relativa mängden av varje mutant uppskattades med användning av djup sekvensering. "Fitnesspoäng" för varje gen visar hur uttrycket av den genen påverkar tillväxthastigheten. Denna metod, konkurrerande tillväxt och spårning av mutantpopulationer med djup sekvensering, är en metod med hög genomströmning för att screena en stor andel gener från flera organismer samtidigt, vilket möjliggör identifiering av trans-art effekter såväl som effekten av enstaka gener på värdcellens metabolism.

No description available.

The Calvin-Benson-Bassham (CBB) pathway is the primary pathway for the entry of inorganic carbon in the biosphere. Autotrophic organisms use this cycle to ultimately convert CO2 into carbohydrates using a key enzyme known as ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). The gene that encodes for the large subunit of RubisCO is rbcL and detection of its expression can be used to determine the autotrophic organisms present in the environment. Recently, microarrays have been used to study functional gene expression from environmental samples such as those obtained from sediments and soil. The purpose of this thesis is to combine microarray technology and rbcL expression analysis to investigate phytoplankton populations in the Mississippi River Plume (MRP). Initially, a macroarray was constructed to determine its capabilities of quantifying gene expression in MRP. PCR amplicons were spotted onto a nylon membrane and labeled transcript RNA was hybridized to each array. Due to the large amount of cross hybridization that was observed, a microarray was used. Microarray analysis revealed large amounts of Synechococcus, pelagophyte and prymnesiophyte expression in the surface waters. Furthermore, there was no chlorophte or Prochlorococcus expression observed in the surface waters. Subsurface microarray data showed high levels of pelagophytes and other Form ID organisms. A significant chlorophyte signal was also observed in the subsurface. This study provides a third level of specificity at which phylogenetic diversity has been sampled in the MRP. Although a limited number of samples were analyzed by microarrays, this technology shows promise and this study was viewed as a pilot for their application. The rbcL probes designed were based upon published sequences from 2003 and we now have a much greater understanding of the diversity of rbcL-containing phytoplanktonic phylotypes. Future studies should employ this knowledge for judicious probe selection.

Part 1: Prophage induction has been demonstrated to be a sensitive indicator for a wide variety of toxic and mutagenic compounds and, as a consequence, has been utilized for biologically based carcinogen screenings. Fourteen marine bacterial isolates were screened for development into the Marine Prophage Induction Assay (MPIA), for marine samples. The selected isolate (P99-4S3) was identified by 16S rDNA sequencing as Pseudomonas aeruginosa. This isolate demonstrated a log-linear response to increasing dose of mutagens, and sensitivity to known environmental contaminants. Field-testing of the assay over two years demonstrated the MPIA would be a useful screening tool for environmental contamination. Part 2: The observed resistance of natural populations of Synechococcus to viral infection may be due to lysogeny with associated homoimmunity. A thirteen-month study of lysogeny in natural populations of Synechococcus demonstrated that lysogeny does occur and exhibits a seasonal pattern. Experiments were performed along a transect of the Mississippi River plume, which provided a variety of ambient nutrient regimes for comparison of lysogeny in Synechococcus. Nutrient amendments did not enable induction and often led to a decrease in viral production. Lysogeny in Synechococcus was primarily correlated with ambient host and cyanophage abundance. Cross-infectivity studies demonstrated cyanophage isolates possess variable virulence. The 35 isolates were examined by transmission electron microscopy (TEM), with 33 identified as myoviruses and two as podoviruses. This dominance of myovirus lytic cyanophage is consistent with prior observations. Twenty-five Synechococcus isolates were screened for prophage induction utilizing the inducing agent Mitomycin C. Eleven isolates demonstrated a statistically significant increase in virus-like particles (VLP’s) in treatment samples. No correlation was observed between their resistance to lytic viral infection and prophage induction. Isolate P99-14, with consistently high levels of prophage induction, was investigated further. In contrast to lytic cyanophage, the induced cyanophage is non-tailed. Differential staining and nuclease digestion experiments indicate that the induced particle contains single-stranded DNA. Environmental conditions potentially leading to prophage induction were investigated with Synechococcus cultures and natural populations. The isolate P99-14 demonstrated that high, continuous light caused prophage induction. Natural populations determined that shifts in salinity, temperature and phosphate are not triggers of prophage induction.

Within the content of this thesis, it was aimed to understand the changes in the biomass and abundance of heterotrophic bacteria and marine cyanobacteria Synechococcus in time with respect to multitude of ambient physical, chemical and biological factors. For this, monthly samples from discrete depths in the offshore (0-20-40-60-80-100-125-150-175-200 m) and in the near shore (surface and 10 m) stations over a period of one year in the Cilician Basin (eastern Mediterranean) were collected via rosette sampler. Epifluorescent microscope and the image analysis system were used to estimate abundance and biomass of both groups. Coastal station was more abundant and had much higher bacterial (heterotrophic bacteria) and cyanobacterial (Synechococcus) biomass than the offshore station as it receives substantial amount of freshwater from the nearby Lamas River throughout the year. The surface annual averages for bacterial and cyanobacterial abundance and biomass were 9600000 cells/ml - 56.5 microgram C/l and 400000 cells/ml - 24.1 microgram C/l, respectively, at the coastal station. The surface annual averages for bacterial and cyanobacterial abundance and biomass were 8100000 cells/ml &ndash / 49.1 microgram C/l and 210000 cells/ml &ndash / 10.6 microgram C/l, respectively, at the offshore station. Bacterial population always found to exceed Synechococcus abundance within the water column. In general, bacterial and cyanobacterial abundance and biomass tend to decrease with depth. On a seasonal basis, bacterial population was found excessively dominant at the surface or near-surface waters during the second half of the year. Synechococcus were also found more abundant during late summer and autumn. Temperature and nitrate concentration seemed to affect efficiently the abundance of both populations in the area. Based on Spearman Rank Correlation analysis, highly significant correlations between bacterial abundance as well as biomass and ambient temperature were observed at both stations. However, a significant correlation was found between Synechococcus and temperature only at the offshore station. Significant negative correlations are found between nitrate and bacterial abundance and biomass at both stations and between Synechococcus abundance and biomass only at the offshore station. At the offshore station, salinity was also found to be positively correlated with the bacterial and cyanobacterial abundance and biomass.

The eastern Mediterranean has been known as the most oligotrophic water body among the worlds oceans and as a result of limited nutrient inputs from terrestrial sources primary productivity and plankton succession are restricted by lack of nutrients especially by phosphorus and nitrogen. Within this domain, Mersin bay forms a so called hot spot (highly sensitive) area where a sharp contrast exists between the coastal area supplied by land-based nutrient sources and the nutrient limited open sea. Excess nutrient enrichment lead for eutrophication in the inner Mersin bay while altering the quality and quantity of flora from shore to offshore. Microorganisms are highly sensitive and profoundly affected by environmental disturbances and are widely used to assess the impact of environmental changes on ecosystem functioning. With this study, it is aimed to investigate responses of the smaller fractions of phytoplankton composed of heterotrophic bacteria, Synechococcus and flagellates to rapidly changing ambient biological, chemical and physical properties of shelf waters over an extended period between 2008 and 2010. Epiflourescence microscopy and image analysis setup were used to enumerate and measure size of cells for biomass estimates.

Chez la majorité des organismes vivants, depuis certaines bactéries, jusqu'aux mammifères, de nombreux processus biologiques sont rythmés, comme par exemple l'alternance veille sommeil, ou encore le cycle d'hibernation. Le contrôle de ces rythmes biologiques est effectué grâce à des horloges biologiques, qui sont dans certains cas des oscillateurs auto-entretenus programmés génétiquement. Les oscillateurs biologiques dont la période est de proche de 24h sont dits circadiens. La cyanobactérie Synechoccocus elongatus sp. PCC7942 est l'un des plus simples organismes possédant cette horloge circadienne. Malgré la division cellulaire (jusqu'à 3 divisions par cycle), l'oscillation circadienne persiste au sein d'une population. De plus, des mesures faites sur cellules individuelles montrent la persistance de l'oscillateur circadien avec un temps de corrélation de plusieurs mois. Nous nous intéressons ainsi à l'origine de la robustesse de l'oscillateur : comment conserver une oscillation aussi stable malgré les sources de bruits (fluctuations internes et environnementales) auxquelles est soumis le réseau génétique ? Une telle stabilité pourrait être intrinsèque à l'oscillateur, ou alors être due à une interaction entre oscillateurs. Notre étude s'intéresse à la 2ème hypothèse ; ainsi, nous avons cherché l'existence potentielle d'un couplage entre 2 populations d'oscillateurs circadiens. Pour accéder à l'oscillation de l'horloge circadienne, nous avons utilisé une souche sauvage dans laquelle a été introduit le gène rapporteur de la luciférase. La souche mutée ainsi obtenue va présenter une luminescence oscillante, traduisant l'expression de la luciférase sous le contrôle du promoteur correspondant. Les souches mutées et non mutées ont une activité génétique régulée par l'horloge circadienne. L'interaction est étudiée en réalisant des mélanges de deux populations -souche mutante +souche sauvage- de cyanobactéries ayant initialement des phases d'oscillation différentes, avec un rapport de concentration 1:20. L'évolution temporelle de la bioluminescence donne ainsi accès à l'oscillation circadienne de la population minoritaire. Les éventuels effets du couplage sont analysés en étudiant la phase d'oscillation de la population minoritaire. Notre étude a montré qu'après une quarantaine de jours d'interaction potentielle, la phase d'oscillation des minoritaires n'est pas affectée de façon significative par la présence d'oscillateurs ayant une autre phase initiale. Au vu de ces résultats, nous avons établi un modèle théorique de couplage afin de majorer l'éventuel couplage entre oscillateurs cyanobactériens. Notre modèle est principalement décrit par le modèle de Kuramoto, qui donne une description pertinente de nombreux oscillateurs biologiques. Des simulations numériques ont montré qu'il était possible de faire des hypothèses simplificatrices concernant la forme des distributions de phase au sein de chacune des populations en interaction. Ainsi, nous avons établi une relation simple entre le changement de phase, et la constante de couplage. En tenant compte des incertitudes sur la phase calculée expérimentalement, nous estimons une borne supérieure de la constante de couplage entre oscillateurs cyanobactériens.

Les picocyanobactéries marines des genres Prochlorococcus et Synechococcus sont les organismes photosynthétiques les plus abondants de la planète et ils contribuent de façon substantielle à la production primaire mondiale. Alors que le genre Prochlorococcus se caractérise par sa forte abondance dans les régions oligotrophes et son génome réduit, le genre Synechococcus se distingue par sa grande diversité génétique et pigmentaire ainsi qu'une aire de distribution plus étendue.Le principal objectif de ce travail a été de mettre en relation la diversité génétique de ces organismes avec leur niche écologique par des approches de génomique comparative et de métagénomique. Tout d'abord, le développement d'une méthode de scaffolding (WiseScaffolder) a permis de clore 32 nouveaux génomes de Synechococcus, lesquels ont été intégrés au système d'information Cyanorak, dédié à l'annotation de gènes orthologues. Ces nouveaux génomes, complétant les 65 génomes disponibles pour ces deux genres, ont fait l'objet d'analyses comparatives afin de mieux comprendre la diversité et l'évolution de ce phylum et de définir les gènes spécifiques de différents groupes phylogénétiques, potentiellement liés à leur adaptation à des niches écologiques distinctes.Ces génomes ont ensuite été utilisés comme référence, en conjonction avec le gène marqueur petB, pour analyser les données de métagénomique issues de l'expédition TARA-Océans. Ces analyses ont notamment mis en lumière la diversité génétique, la distribution et l'importance écologique de certains clades phylogénétiques. Ce travail soulève de nouvelles hypothèses quant au rôle des picocyanobactéries dans le fonctionnement global des océans. / Marine picocyanobacteria Prochlorococcus and Synechococcus genera are the most abundant photosynthetic organisms and contribute substantially to global primary production. While the genus Prochlorococcus is characterized by its high abundance in oligotrophic regions and its reduced genome, Synechococcus is characterized by a larger genetic and pigment diversity and a wider area of distribution.The main objective of this PhD thesis was to link the genetic diversity of these organisms to the environmental conditions of their ecological niche by comparative genomics and metagenomics approaches. Firstly, the development of a scaffolding method (WiseScaffolder) has allowed us to close 32 new genomes of Synechococcus which were integrated into the Cyanorak information system dedicated to the annotation of orthologous genes. These new genomes, supplementing the 65 genomes previously available for these two genera, allowed us to perform comparative analyses which led to a better understanding of the diversity and evolution of this phylum and to the definition of genes sets specific to different phylogenetic groups and thus potentially related to their adaptation to different ecological niches.These genomes were then used as reference, in conjunction with the marker gene petB gene, to analyze metagenomic data produced in the frame of the Tara-Oceans Expedition. In particular, these analyzes highlighted the genetic diversity, distribution and ecological importance of some phylogenetic clades. This work raises new hypotheses about the role of picocyanobacteria in the overall functioning of the oceans.

No description available.