High–resolution multidimensional proton-detected NMR was used to study the solvent-exposed regions of a seven-helical integral membrane proton pump proteorhodopsin (PR). Fully deuterated PR samples with protons reintroduced to solvent-accessible sites through back exchange were prepared and found to produce NMR spectra with acceptable proton resolution (~0.2 ppm). Novel three-dimensional proton-detected chemical shift correlation spectroscopy was used for the identification and resonance assignment of the solvent–exposed regions of PR. Though most of the observed residues were located at the membrane interface there were notable exceptions, particularly in helix G. This helix contains the Schiff base-forming Lys231 and many conserved polar residues in the extracellular half. Solvent accessibility of helix G supports the hypothesis that high mobility of the F-G loop could transiently expose a hydrophilic cavity in the extracellular half of PR, and implies that such a cavity may be part of the protein’s proton-conduction pathway. / Natural Sciences and Engineering Research Council, Ontario Ministry of Training, Colleges, and Universities, Canadian Foundation for Innovation, Ontario Ministry of Research and Innovation, University of Guelph

Marine bacteria play an important role in the marine nutrition cycles. About half of all sea-living bacteria can use light as an energy source, in addition to organic carbon compounds, which is important for survival as the seas becomes acidic and low in nutrition due to changing climate. The Flavobacteriaceae is a bacterial family that plays an important role in the degradation of complex organic compounds in natural environments. Dokdonia donghaensis MED134 belongs to the Flavobacteriaceae family and use both chemotrophy and phototrophy as a source of energy. For its phototrophic ability, the bacteria use proteorhodopsin, which is a membrane-bound proton pump. This allows the bacteria to survive and grow in nutrient-poor environments. The purpose of the study was to investigate with qPCR if there was a difference in gene expression for proteorhodopsin and isocitrate dehydrogenase in Dokdonia donghaensis MED134 depending on whether the bacteria had grown in constant light or darkness. Analysis with qPCR showed a significantly greater gene expression for proteorhodopsin in the bacteria grown in constant light for seven days, compared to those grown in constant light for three and four days and those grown in constant darkness. No significant difference could be demonstrated in gene expression for isocitrate dehydrogenase. This indicates that Dokdonia donghaensis MED134 in nutritional deficiency can use light as a source of energy for survival and further growth. By simulating climate change an adaptability could be seen through increased gene expression. Through this, further understanding of the role of bacteria in the marine ecosystem can be obtained and further research can be conducted as the marine climate issue becomes more relevant.

Oceans are powered by active, metabolically diverse microorganisms, which are important in regulating biogeochemical cycles on Earth. Most of the ocean surface is often limited by nutrients, influencing bacterial growth and activities. Bacterial adaptation to fluctuating environmental conditions involves extensive reprogramming, and redirection of bacterial metabolism and physiology. In this thesis, I investigated the molecular mechanisms of bacterial adaptation strategies to sustain their growth and survival, focusing on the regulation of gene and protein expression in heterotrophic marine bacteria. Comparative proteomics analyses of the growth and non-growth conditions, uncovered central adaptations that marine bacteria employ to allow them to change their metabolism to support exponential growth in response to nutrients and to readjust to stationary phase under nutrient limitation. Our results highlight that during nutrient rich conditions three distinct bacteria lineages have great similarities in their proteome. On the other hand, we observed pronounced differences in behavior between taxa during stationary phase. Analyses of the proteorhodopsin containing bacterium Vibrio sp. AND4 during starvation showed that significantly improved survival in the light compared to darkness. Notably, proteins involved in promoting cell vitality and survival had higher relative abundance under light. In contrast, cells in the dark need to degrade their endogenous resources to support their basic cellular demands under starvation. Thus, light strongly influences how PR-containing bacteria organize their molecular composition in response to starvation. Study of alternative energy generation metabolisms in the Alphaproteobacteria Phaeobacter sp. MED193 showed that the addition of thiosulfate enhanced the bacterial growth yields. Concomitantly, inorganic sulfur oxidation gene expression increased with thiosulfate compared to controls. Moreover, thiosulfate stimulated protein synthesis and anaplerotic CO2 fixation. These findings imply that this bacterium could use their lithotrophic potential to gain additional energy from sulfur oxidation for both improving their growth and survival. This thesis concludes that analyses in model organisms under defined growth conditions gives invaluable knowledge about the regulatory networks and physiological strategies that ensure the growth and survival of heterotrophic bacteria. This is critically important for interpreting bacterial responses to dynamic environmental changes. Moreover, these analyses are crucial for understanding genetic and proteomic responses in microbial communities or uncultivated organisms in terms of defining ecological niches of planktonic bacteria

O aproveitamento da energia luminosa por bactérias que produzem hidrogenases poderia aumentar a eficiência do processo de produção de biohidrogênio. Neste trabalho, foi realizada a clonagem do gene que codifica a proteorrodopsina (PR) do isolado metagenômico SAR86 num plasmídeo de expressão para bactérias Gram-negativas. PR é uma proteína ligada ao cromóforo retinal, que, sob iluminação, promove o efluxo de prótons através da membrana celular. O excesso de prótons na face externa da membrana pode servir como substrato para a hidrogenase, resultando em maior eficiência na produção de hidrogênio (2H+ + 2e→ H2). O plasmídeo contendo o gene da PR foi utilizado na transformação genética das bactérias Cupriavidus necator e Escherichia coli, que produzem diversas hidrogenases. Enquanto a PR não se mostrou funcional em C. necator, na linhagem recombinante de E. coli, cultivada em presença de luz e retinal, foi obtido um aumento de até 2,17 vezes na produção de H2 em relação ao cultivo no escuro, desde que a linhagem estivesse produzindo a hidrogenase endógena HYD-4. / The utilization of light energy by hydrogenase producing bacteria could increase the efficiency of the biohydrogen production process. In the present work, the gene coding for proteorhodopsin (PR) of the SAR86 metagenomic lineage was cloned in an expression plasmid for Gram-negative bacteria. PR is an apoprotein linked to the chromophore retinal, which, upon illumination, promotes proton efflux across the cell membrane. The excess of protons on the plasma membrane surface may serve as a substrate for hydrogenases, resulting in a higher efficiency of hydrogen production (2H+ + 2e→ H2). The plasmid containing the PR gene was used to transform the Gram-negative bacteria Cupriavidus necator and Escherichia coli which produce several hydrogenases. Whereas PR did not display functionality in C. necator, in the recombinant E. coli cells, grown under illumination in the presence of retinal, an enhancement up to 2.17 fold in H2 production was found, relative to cells grown under darkness, provided that the cells were expressing the endogenous HYD-4 hydrogenase.

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.

O aproveitamento da energia luminosa por bactérias que produzem hidrogenases poderia aumentar a eficiência do processo de produção de biohidrogênio. Neste trabalho, foi realizada a clonagem do gene que codifica a proteorrodopsina (PR) do isolado metagenômico SAR86 num plasmídeo de expressão para bactérias Gram-negativas. PR é uma proteína ligada ao cromóforo retinal, que, sob iluminação, promove o efluxo de prótons através da membrana celular. O excesso de prótons na face externa da membrana pode servir como substrato para a hidrogenase, resultando em maior eficiência na produção de hidrogênio (2H+ + 2e→ H2). O plasmídeo contendo o gene da PR foi utilizado na transformação genética das bactérias Cupriavidus necator e Escherichia coli, que produzem diversas hidrogenases. Enquanto a PR não se mostrou funcional em C. necator, na linhagem recombinante de E. coli, cultivada em presença de luz e retinal, foi obtido um aumento de até 2,17 vezes na produção de H2 em relação ao cultivo no escuro, desde que a linhagem estivesse produzindo a hidrogenase endógena HYD-4. / The utilization of light energy by hydrogenase producing bacteria could increase the efficiency of the biohydrogen production process. In the present work, the gene coding for proteorhodopsin (PR) of the SAR86 metagenomic lineage was cloned in an expression plasmid for Gram-negative bacteria. PR is an apoprotein linked to the chromophore retinal, which, upon illumination, promotes proton efflux across the cell membrane. The excess of protons on the plasma membrane surface may serve as a substrate for hydrogenases, resulting in a higher efficiency of hydrogen production (2H+ + 2e→ H2). The plasmid containing the PR gene was used to transform the Gram-negative bacteria Cupriavidus necator and Escherichia coli which produce several hydrogenases. Whereas PR did not display functionality in C. necator, in the recombinant E. coli cells, grown under illumination in the presence of retinal, an enhancement up to 2.17 fold in H2 production was found, relative to cells grown under darkness, provided that the cells were expressing the endogenous HYD-4 hydrogenase.

Les réchauffements climatiques associés aux activités anthropiques ont soumis les écosystèmes arctiques à des changements rapides qui menacent leur stabilité à court terme. La diminution dramatique de la banquise arctique est une des conséquences les plus concrètes de ce réchauffement. Dans ce contexte, comprendre et prédire comment les systèmes arctiques évolueront est crucial, surtout en considérant comment les flux de carbone (C) de ces écosystèmes - soit des puits nets, soit des sources nettes de CO2 pour l'atmosphère - pourraient avoir des répercussions importantes sur le climat. Le but de cette thèse est de dresser un portrait saisonnier de l’activité bactérienne afin de déterminer l’importance de sa contribution aux flux de carbone en Arctique. Plus spécifiquement, nous caractérisons pour la première fois la respiration et le recours à la photohétérotrophie chez les microorganismes du golfe d’Amundsen. Ces deux composantes du cycle du carbone demeurent peu décrites et souvent omises des modèles actuels, malgré leur rôle déterminant dans les flux de C non seulement de l’Arctique, mais des milieux marins en général. Dans un premier temps, nous caractérisons la respiration des communautés microbiennes (RC) des glaces de mer. La connaissance des taux de respiration est essentielle à l’estimation des flux de C, mais encore limitée pour les milieux polaires. En effet, les études précédentes dans le golfe d’Amundsen n’ont pas mesuré la RC. Par la mesure de la respiration dans les glaces, nos résultats montrent des taux élevés de respiration dans la glace, de 2 à 3 fois supérieurs à la colonne d'eau, et une production bactérienne jusqu’à 25 fois plus importante. Ces résultats démontrent que la respiration microbienne peut consommer une proportion significative de la production primaire (PP) des glaces et pourrait jouer un rôle important dans les flux biogéniques de CO2 entre les glaces de mer et l’atmosphère (Nguyen et Maranger, 2011). Dans un second temps, nous mesurons la respiration des communautés microbiennes pélagiques du golfe d’Amundsen pendant une période de 8 mois consécutif, incluant le couvert de glace hivernal. En mesurant directement la consommation d'O2, nous montrons une RC importante, mesurable tout au long de l’année et dépassant largement les apports en C de la production primaire. Globalement, la forte consommation de C par les communautés microbiennes suggère une forte dépendance sur recyclage interne de la PP locale. Ces observations ont des conséquences importantes sur notre compréhension du potentiel de séquestration de CO2 par les eaux de l’Océan Arctique (Nguyen et al. 2012). Dans un dernier temps, nous déterminons la dynamique saisonnière de présence (ADN) et d’expression (ARN) du gène de la protéorhodopsine (PR), impliqué dans la photohétérotrophie chez les communautés bactérienne. Le gène de la PR, en conjonction avec le chromophore rétinal, permet à certaines bactéries de capturer l’énergie lumineuse à des fins énergétiques ou sensorielles. Cet apport supplémentaire d’énergie pourrait contribuer à la survie et prolifération des communautés qui possèdent la protéorhodopsine. Bien que détectée dans plusieurs océans, notre étude est une des rares à dresser un portrait saisonnier de la distribution et de l’expression du gène en milieu marin. Nous montrons que le gène de la PR est présent toute l’année et distribué dans des communautés diversifiées. Étonnamment, l’expression du gène se poursuit en hiver, en absence de lumière, suggérant soit qu’elle ne dépend pas de la lumière, ou que des sources de photons très localisées justifie l’expression du gène à des fins sensorielles et de détection (Nguyen et al., soumis au journal ISME). Cette thèse contribue à la compréhension du cycle du C en Arctique et innove par la caractérisation de la respiration et de l’efficacité de croissance des communautés microbiennes pélagiques et des glaces de mer. De plus, nous montrons pour la première fois une expression soutenue de la protéorhodopsine en Arctique, qui pourrait moduler la consommation de C par la respiration et justifier son inclusion éventuelle dans les modélisations du cycle du C. Dans le contexte des changements climatiques, il est clair que l'importance de l’activité bactérienne a été sous-estimée et aura un impact important dans le bilan de C de l'Arctique. / Arctic ecosystems are undergoing rapid changes, primarily due to unprecedented climatic warming as a function of anthropogenic activities, which threaten their short-term stability. One of the most dramatic impacts has been the loss and change in annual sea ice. Understanding and predicting how these systems will evolve is crucial, especially if considering how carbon (C) fluxes from these ecosystems – either net sinks or net CO2 sources for the atmosphere – could have important repercussions on global climate. The objective of this thesis is to establish a seasonal portrait of bacterial activity to characterize its contribution to Arctic carbon fluxes. Specifically, we quantify for the first time microbial respiration in sea-ice and the water column and explore the use of photoheterotrophy by microorganism over an annual cycle in the Amundsen Gulf of the Arctic Ocean. These components of carbon cycling remain poorly understood and infrequently directly measured. As a consequence they are either extrapolated or omitted from models, despite their significant role in C dynamics not only in the Arctic, but also in marine systems in general. First, we characterise respiration in sea-ice microbial communities (CR). An understanding of respiration rates is essential for accurate estimation of C fluxes, but the role of respiration in sea ice is poorly understood. This work represents the first comprehensive evaluation of respiration in polar sea ice to date. Using novel O2 consumption measurements in sea-ice, we found high respiration rates in sea-ice, 2 to 3 times higher than in the water column and bacterial production rates up to 25 times higher. These results show that microbial respiration can consume a significant portion of sea ice primary production (PP) and play a key role in biogenic CO2 fluxes between sea-ice and the atmosphere (Nguyen and Maranger, 2011). Second, we measure respiration of pelagic microbial communities of Amundsen Gulf over an eight-month period, including under the winter ice-cover. By measuring directly O2 consumption, we show high CR, measurable over the whole year and greatly surpassing C inputs from PP. Globally, high C consumption by microbial communities supports a high reliance on internal recycling of local PP. These observations have important consequences on our understanding of the CO2 sequestering potential of the Arctic Ocean (Nguyen et al., 2012) Finally, we describe the seasonal patterns in presence (DNA) and expression (RNA) of the proteorhodospin (PR) gene, involved in bacterial photoheterotrophy. The PR gene, combined with the retinal chromophore, allows bacteria to capture energy from light towards energetic or sensory purposes. This additional energy source could contribute to the survival and proliferation of bacterial communities expressing the gene in the highly variable polar environment. Although PR has been found in many oceans, this study represents a unique time-series that follows the seasonal distribution and expression of the gene in a natural marine system. We show that the PR gene was present over the whole study period and widely distributed in diverse bacterial communities. Surprisingly, we observed continued PR expression over winter, in the absence of sunlight. This suggests either that the PR’s expression does not depend on light or, that other very localized photon sources could justify PR expression for detection and sensory functions (Nguyen et al., submitted to the ISME journal). This thesis contributes to the understanding of Arctic carbon cycling and includes several novel elements such as the characterization of respiration and bacteria growth efficiency in both pelagic and sea-ice habitats. The use of an alternative C pathway by bacteria in the Polar ocean was also explored for the first-time in a time-series. The observed sustained expression of the PR gene in the Arctic could modulate C consumption by respiration and justify its inclusion in future models of C cycling. In a context of climate change, it is clear that bacterial activity has been underestimated and how this will change in a warmer Arctic will have a significant impact in the ecosystem’s overall C budget.