As part of a large study aimed at searching for patterns of diversity in the genus Persephonella along the north to south geochemical gradient of the ELSC, ten novel strains of Alphaproteobacteria were isolated unexpectedly. Using defined media under microaerophilic conditions to enrich for Persephonella from chimney samples collected at the seven vent fields on the ELSC and the dilution to extinction by serial dilution method to purify cultures, a total of ten strains belonging to the Alphaproteobacteria were isolated. Two of these isolates, designate MN-5 and TC-2 were chosen for further characterization and are proposed as two new species of a novel genus to be namedThermopetrobacter. Both strains are aerobic, capable of chemoautotrophic growth on hydrogen and grow best at 55°C, pH 6 and 3.0% NaCl. Strain MN-5 is capable of heterotrophic growth on pyruvate and malate and TC-2 is only able to grow heterotrophically with pyruvate. The GC content of MN-5 is 69.1 and TC-2 is 67 mol%. GenBank BLAST results from the 16S rRNA gene reveal the most closely related sequence to MN-5 is 90% similar and the most closely related sequence to strain TC-2 is 89% similar. Sampling at a shallow marine vent on the coast of Vulcano Island, Italy in 2007 led to the isolation of a novel species of Hydrogenothermus, a genus within the Hydrogenothermaceae family. This isolate, designated NV1, represents the secondHydrogenothermusisolated from a shallow marine vent. NV1 cells are rod-shaped, approximately 1.5μm long and 0.7μm wide, motile by means of a polar flagellum and grow singularly or in short chains. Cells grow chemoautotrophically using hydrogen or thiosulfate as electron donors and oxygen as the sole electron acceptor. Growth was observed between 45 and 75°C with an optimum of 65°C (doubling time 140 min), pH 4.0-6.5 and requires NaCl (0.5-6.0% w/v). The G+C content of total DNA is 32 mol%.

Les océans couvrent plus de 70% de la surface de la Terre (planète bleue) et la productivité primaire nette (PPN) marine est équivalente à celle terrestre. Alors qu‟il ne représente que 1% de la biomasse totale d‟organismes photosynthétiques de la planète, le phytoplancton est responsable d‟environ 45% de la PPN globale. Le terme phytoplancton décrit un assemblage polyphylétique comprenant des eucaryotes et procaryotes photosynthétiques dérivant avec les courants. Dans les océans contemporains, les diatomées constituent un groupe d‟eucaryotes unicellulaires autotrophes très abondant, responsable de 40% de la PPN marine. Les diatomées appartiennent à la lignée des straménopiles qui sont issus d‟un évènement d‟endosymbiose entre une algue rouge et un hôte hétérotrophe. Elles sont classifiées en deux groupes majeurs : les centriques qui son apparues il y a environ 200 millions d‟années (Ma), et les pennées qui ont évolué il y a environ 90 Ma. Deux génomes de diatomées ont récemment été séquencés : celui de la diatomée centrique Thalassiosira pseudonana (32 Mb), et celui de la diatomée pennée Phaeodactylum tricornutum (27 Mb). Mon sujet de doctorat s‟est focalisé sur l‟étude de différents aspects de la régulation de l‟expression génique ainsi que sur la dynamique et l‟évolution de ces génomes. L‟expression des gènes est régulée à différents niveaux: trancriptionel, post-transcriptionel, et épigénétique. Dans le cadre de mon doctorat, une étude de la régulation transcriptionelle chez les diatomées a été effectuée et comprend l‟identification et l‟analyse in silico des facteurs de transcription (FT). Cela a permis par exemple d‟établir qu‟une classe spécifique de FT, les Heat Shock Factors, sont particulièrement abondants chez les diatomées par rapport aux autres eucaryotes. L‟analyse de la représentation des FT identifiés dans différentes librairies d‟EST élaborées à partir de cultures ayant subi divers stress a permis de détecter certaines spécificités d‟expression. L‟évolution des génomes eucaryotes est largement impactée par les effets directs et secondaires des éléments transposables (ET) qui sont des éléments génétiques mobiles se trouvant dans le génome de la plupart des organismes. Dans le but d‟étudier la dynamique des génomes de diatomées, la recherche de différents types d‟ET a permis d‟établir qu‟une certaine classe, les rétrotransposons de type Copia, est la plus abondante dans ces génomes et constitue un part significativement plus importante du génome de P. tricornutum (5,8%) par rapport à T. pseudonana (1%). D‟autre part, des analyses phylogénitiques ont montré que les rétrotransposons de type copia forment deux classes distinctes et éloignées de la lignée Copia. L‟analyse de leurs niveaux d‟expression a montré que la transcription de deux éléments s‟active en réponse à des stress spécifiques comme la limitation en nitrate dans le milieu de culture. Cette activation est accompagnée par un hypométhylation de l‟ADN et l‟analyse de profils d‟insertions chez différents écotypes de P. tricornutum ainsi que l‟étude d‟autres phénomènes suggèrent que les rétrotransposons de type Copia ont joué un rôle important dans l‟évolution des diatomées. Mon grand intérêt pour les ET m‟a ensuite amené à chercher à les caractériser dans d‟autres génomes récemment séquencés tels celui de l‟algue brune Ectocarpus siliculosus. La recherche in silico de différents gènes codant des protéines capables d‟introduire ou de stabiliser des états épigénétiques telle que la modification des histones et la méthylation de l‟ADN a montré leur présence chez P. tricornutum ainsi que leurs particularités. La présence de certaines modifications d‟histones spécifiques d‟une conformation compacte ou ouverte de la chromatine dans le proteome de P. tricornutum a été montrée. De plus, la mise au point de la technique d‟immunoprécipitation de la chromatine chez P. tricornutum a permis d‟établir que les nucléosomes enrobés d‟éléments transposables étaient marqués par des modifications spécifiques. D‟autres expériences ont permis d‟établir que l‟ADN de différents types d‟éléments transposables est marqué par la méthylation de cytosines chez P. tricornutum. Une expérience permettant l‟analyse du profil de méthylation à l‟échelle de génome en utilisant une puce à ADN a été lancée et permettra de découvrir si certains gènes portent aussi des traces de méthylation. Enfin, les ARN interférents constituent un troisième mode de régulation de l‟expression se situant à l‟interface de la régulation transcriptionelle, post-transcriptionelle et épigénétique. Les mécanismes d‟interférences chez les diatomées ont été étudié par la recherche in silico d‟enzymes clés impliquées dans ce processus ainsi qu‟en établissant expérimentalement un lien direct avec la méthylation de l‟ADN.

A poluição, alteração física do habitat e a introdução de espécies invasoras via água de lastro, representam os maiores impactos antropogênicos para os ambientes costeiros. Foram pesquisadas em amostras da região costeira de S. Paulo, regiões portuárias brasileiras e de tanques de lastro de navios, bactérias das espécies Vibrio cholerae (Vc), V. parahaemolyticus (Vp) e V. vulnificus (Vv) que são as que têm maior implicação na saúde pública. As amostras foram avaliadas levando-se em conta parâmetros físico-químicos e microbiológicos e suas relações com a presença de Vc, Vp e Vv. As relações clonais foram verificadas através das técnicas de ERIC, BOX e REP-PCR. Foram identificadas 90 cepas de Vp e 11 de Vc. Foram observadas correlações entre alguns parâmetros microbiológicos e a presença de vibrios. A análise clonal permitiu verificar a alta diversidade das cepas. Concluiu-se que Vc e Vp são autóctones do ambiente costeiro brasileiro e podem ser tornar reservatórios para determinados fatores associados à virulência, gerando cepas com potencial epidêmico. / Pollution, physical alteration of habitat and the introduction of alien species through ballast water constitute the biggest anthropogenic impacts on coastal environments. We examined samples taken from the coastal region of S. Paulo state, Brazilian ports and ship ballast tanks, for bacteria of the species Vibrio cholerae (Vc), V. parahaemolyticus (Vp) and V. vulnificus (Vv) which have the most significant implication for public health. The samples were evaluated for microbiological and physical-chemical parameters as well as the presence of Vc, Vp and Vv. Clonal relationships of bacterial isolates were determined through ERIC, BOX and REP-PCR. A total of 90 strains of Vp and 11 of Vc were identified. Correlations between some microbiological parameters and the presence of vibrios were observed. The clonal analysis revealed extensive strain diversity. We concluded that Vc and Vp are autochthonous bacteria of the Brazilian coastal environment that can become reservoirs for factors associated with virulence, and are capable of generating strains with epidemic potential.

A ressurgência da massa de Água Central do Altântico Sul (ACAS) é o principal processo oceanográfico de disponibilização de nutrientes na zona eufótica da plataforma continental sudeste do Brasil (PCSE). Isto gera um acréscimo na população de organismos autotróficos e consequente de heterotróficos que produzem e oxidam a matéria orgânica gerada. O que possibilita um aumento no fluxo de carbono e consequentemente a manutenção dos recursos pesqueiros na região durante o verão. A abundância de micro-organismos autotróficos e heterotróficos planctônicos e a relação destes com as massas de água da PCSE foram avaliadas no verão de 2013. Para o tal, os micro-organismos autotróficos, menores que 20 µm foram quantificados por citometria de fluxo. Foram realizadas análises de abundância do gene 16S de bactérias, archaeas e do clado Sar11 e a expressão dos genes rubisco e proteorodpsina por meio de PCR em tempo real. O valor médio obtido de micro-organismos autrotróficos, Prochlorococcus spp. e Synechococcus spp. foi de 15.151 e de 8.313 células.ml-1, respectivamente. E de bactérias heterotróficas de 7.4x105 células.ml-1. Em relação aos domínios, a distribuição de Bacteria ocorreu na superfície e de Archaea no fundo da região oceânica. Os resultados indicam que a distribuição dos micro-organismos ocorreu conforme as características físicas e a disponibilidade de nutrientes das massas de água. Bactérias heterotróficas foram abundantes em regiões costeiras provavelmente devido a maior disponibilidade de matéria orgânica, o que corrobora com o resultado encontrado de maior biomassa de carbono em região costeira. / The resurgence of South Atlantic Central Waters (ACAS) is the main oceanographic process that involves availability of nutrients in the photic zone of the southeast continental shelf in Brazil. This causes an increase in the population of autotrophs and heterotrophic organisms (that produce and oxidizes the organic matter). The carbon flux increase and consequently the maintenance of fish resource in the region during the 2013 summer. The abundance of autotrophic and heterotrophic planktonic microorganisms was evaluated and also was studied their relation with the masses of water of the continental shelf. Thus, autotrophic microorganisms, smaller than 20 microns were quantified by flow cytometry. Using real time PCR were analysed 16S abundance of bacteria, archaea and SAR11 clade and the expression of the rubisco and proteorhodopsin gene. Autotrophic microorganisms, Prochlorococcus spp. and Synechococcus spp. had a mean value of 15.151 and 8.313 cell.ml-1, respectively. Heterotrophic bacteria had a mean value of 7.4x105 cell.ml-1. In relation to the domains, the distribution of bacteria occurred in the surface and the Archaea in the deep of the ocean region. The results indicate that the distribution of micro-organisms occur as the physical characteristics and the availability of nutrients of water masses. Heterotrophic bacteria were abundant in coastal areas probably due to increased of organic matter, which corroborates the result of higher biomass carbon in coastal region.

Bacterioplankton are keystone organisms in marine ecosystems. They are important for element cycles, by transforming dissolved organic carbon and other nutrients. Bacterioplankton community composition and productivity rates change in surface waters over spatial and temporal scales. Yet, many underlying biological processes determining when, why and how bacterioplankton react to changes in environmental conditions are poorly understood. Here, I used experiments with model bacteria and natural assemblages as well as field studies to determine molecular, physiological and ecological responses allowing marine bacteria to adapt to their environment. Experiments with the flavobacterium Dokdonia sp. MED134 aimed to determine how the metabolism of bacteria is influenced by light and different organic matter. Under light exposure, Dokdonia sp. MED134 expressed proteorhodopsin and adjusted its metabolism to use resources more efficiently when growing with lower-quality organic matter. Similar expression patterns were found in oceanic datasets, implying a global importance of photoheterotrophic metabolisms for the ecology of bacterioplankton. Further, I investigated how the composition and physiology of bacterial assemblages are affected by elevated CO2 concentrations and inorganic nutrients. In a large-scale experiment, bacterioplankton could keep productivity and community structure unaltered by adapting the gene expression under CO2 stress. To maintain pH homeostasis, bacteria induced higher expression of genes related to respiration, membrane transport and light acquisition under low-nutrient conditions. Under high-nutrient conditions with phytoplankton blooms, such regulatory mechanisms were not necessary. These findings indicate that open ocean systems are more vulnerable to ocean acidification than coastal waters. Lastly, I used field studies to resolve how bacterioplankton is influenced by environmental changes, and how this leads to seasonal succession of marine bacteria. Using high frequency sampling over three years, we uncovered notable variability both between and within years in several biological features that rapidly changed over short time scales. These included potential phytoplankton-bacteria linkages, substrate uptake rates, and shifts in bacterial community structure. Thus, high resolution time series can provide important insights into the mechanisms controlling microbial communities. Overall, this thesis highlights the advantages of combining molecular and traditional oceanographic methodological approaches to study ecosystems at high resolution for improving our understanding of the physiology and ecology of microbial communities and, ultimately, how they influence biogeochemical processes.

Thesis (S.M.)--Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Dept. of Civil and Environmental Engineering; and the Woods Hole Oceanographic Institution), 2008. / Includes bibliographical references (leaves 52-56). / Functional screening of environmental genomic libraries permits the identification of clones expressing activities of interest without requiring prior knowledge of the genes responsible. In this study, protocols were optimized for the construction of mid-size DNA insert, inducible expression environmental genomic plasmid libraries for this purpose. A library with a mean insert size of 5.2 kilobases was constructed with environmental DNA isolated from surface ocean water collected at Hawaii Ocean Time-series station ALOHA in plasmid cloning vector pMCL200 under the inducible control of the PLAC promoter. To begin to evaluate the utility of such libraries for gene expression-based screens, this library was screened phenotypically for clones expressing genes that confer fluorescence or distinctive coloration on colonies of host Escherichia coli cells, and results were compared to those for a fosmid library constructed from the same marine microbial DNA sample. Ecologically relevant sequences were identified in both libraries, and each was observed to offer both advantages and disadvantages. Results of this study suggest that mid-size insert plasmid libraries under the control of inducible promoters can provide a useful and complementary approach for both functional screening and shotgun sequencing of environmental genomic libraries. / by Jennifer C. Braff. / S.M.

Continental shelves located along eastern boundary currents occupy relatively small volumes of the world’s oceans, yet are responsible for a large proportion of global primary production. The Oregon coast is among these ecosystems. Recent analyses of dissolved oxygen at shallow depths in the water column has suggested increasing episodes of hypoxia and anoxia, events that are detrimental to larger macro-faunal species. Microbial communities, however, are metabolically diverse, capable of utilizing alternative electron donors and acceptors, and can withstand transient periods of low dissolved oxygen. Understanding the phylogenetic and metabolic diversity of microorganisms in these environments is important for assessing the impact hypoxic events have on local and global biogeochemistry. Several molecular ecology tools were used to answer questions about the distribution patterns and activities of microorganisms residing along the coast of Oregon in this dissertation. Ribosomal rRNA fingerprinting and sequence analyses of samples collected during 2007-2008 suggested that bacterial community structure was not substantially influenced by changes in dissolved oxygen. However, substantial depth dependent changes were observed, with samples collected in the bottom boundary layer (BBL) displaying significant differences from those collected in the surface layer. Phylogenetic analyses of bacterial rRNA genes revealed novel phylotypes associated with this area of the water column, including groups with close evolutionary relationships to putative or characterized sulfur oxidizing bacteria (SOB). Analysis of metagenomes and metatranscriptomes collected during 2009 suggested increasing abundances of chemolithoautrophic organisms and their activities in the BBL. Thaumarchaea displayed significant depth dependent increases during the summer, and were detected at maximal frequencies during periods of hypoxia, suggesting that nitrification maybe influenced by local changes in dissolved oxygen. Metagenomic analysis of samples collected from 2010 revealed substantial variability in the metabolic potential of the microbial communities from different water masses. Samples collected during the spring, prior to upwelling clustered independently of those collected during the summer, during a period of upwelling, and did not display any clear stratification. Samples collected during the summer did cluster based on depth, consistent with previous observations, and increases in the relative abundances of chemolithotrophic gene suites were observed in the BBL during stratified conditions, suggesting that the metabolic potential for these processes is a repeatable feature along the Oregon coast. Overall, these observations suggest that depth impacts microbial community diversity, metabolic potential, and transcriptional activity in shallow areas of the Northeast Pacific Ocean. The increase in lithotrophic genes and transcripts in the BBL suggests that this microbial community includes many organisms that are able to use inorganic electron donors for respiration. We speculate that the dissolved organic material in the BBL is semi-labile and not available for immediate oxidation, favoring the growth for microorganisms that are able to use alternative electron donors. / Graduation date: 2012

Metagenomics has revealed that the marine microbial biosphere is immensely more diverse than originally considered, and is an almost untapped reservoir for the potential discovery of microbial natural products. Despite numerous advances in culturing, biosynthetic engineering and genomic-based screening efforts to uncover much of this diversity in relatively accessible environments, a high rediscovery rate has resulted in the investigation of unique, relatively unexplored ecosystems harboring phylogenetically diverse communities of marine organisms. The focus of this research was to establish a culture repository of microorganisms collected from the Red Sea and from deep-sea hydrothermal vents, and to assess their biosynthetic potential for the production of new chemical scaffolds. Cultivation of marine cyanobacteria from the Red Sea has led to the identification of five new cyclic depsipeptides, apratoxin H, grassypeptolides D and E, Ibu-epidemethoxylyngbyastin 3 and leptochelin, the latter possessing a unique chemical scaffold capable of binding metals. A collection of deep-sea hydrothermal vent sediment and microbial mat samples led to the isolation of 64 unique bacterial strains, with eight assigned as members of the order Actinomycetales. Importantly, these isolates, along with a collection of deep-vent invertebrates and microbes, have led to the development of methods for the collection, culturing and biological screening of organisms from this extreme environment for future natural products research. / Graduation date: 2013