L’agent pathogène Agrobacterium tumefaciens induit, chez les plantes, le développement de tumeurs dans lesquelles il prolifère, en intégrant un fragment de son plasmide Ti de virulence dans le génome de son hôte. Les tissus transformés synthétisent des composés originaux, appelés opines, qui sont utilisés comme nutriments spécifiques par la bactérie. Une vingtaine d’opines sont connues à ce jour, et chacune d’elle peut être métabolisée par des souches d’Agrobacterium tumefaciens possédant les gènes de transport et de catabolisme qui lui sont associés, ce qui apparait comme un avantage compétitif dans la colonisation de la tumeur. La présence de ces gènes dépend du type de plasmide Ti que la souche pathogène possède.Agrobacterium tumefaciens B6 possède un pTi de type octopine, qui porte les gènes de métabolisme des mannityl-opines, qui sont la mannopine, l’acide mannopinique, l’agropine et l’acide agropinique. La mannopine et l’acide mannopinique sont synthétisés par la même enzyme, et ont pour précurseurs respectivement la désoxy-fructosyl-glutamine (DFG) et le désoxy-fructosyl-glutamate (DFGA), tous deux opines de la famille de la chrysopine. La DFG est aussi un composé d’Amadori répandu et assimilable par de nombreux organismes. La mannopine sert de précurseur pour la synthèse de l’agropine. Enfin, la mannopine, l’acide mannopinique et l’agropine peuvent toutes trois se lactamiser spontanément en acide agropinique.Malgré la similarité chimique de ces quatre opines, chacune est transportée par une protéine périplasmique de fixation (PBP) associée à un transporteur ATP-binding Cassette (ABC) différent. La PBP sélectionne et fixe une opine pour l’apporter au transporteur ABC, qui permet le passage de l’opine dans le cytoplasme grâce à l’hydrolyse de deux molécules d’ATP. La spécificité du transporteur entier est déterminée par la PBP.Des études génétiques chez des souches possédant un pTi de type octopine ont montré que le système PBP-transporteur ABC AgaABCD est spécifique de l’acide agropinique, AgtABCD spécifique de l’agropine, MoaABCD spécifique de l’acide mannopinique et que MotABCD transporte la mannopine et également l’acide mannopinique. Chez la souche C58, qui ne possède pas un pTi de type octopine, le système de transport SocAB, codé par des gènes situés sur le plasmide cryptique At, transporte la DFG comme nutriment, et semble aussi capable d’importer la mannopine.Mon travail de thèse a permis, dans un premier temps, de caractériser les fortes affinités et la spécificité des PBP AgaA et AgtB pour l’acide agropinique, de la PBP MoaA pour l’acide mannopinique et de la PBP SocA pour la DFG, mais aussi la non spécificité de MotA pour la mannopine, l’acide mannopinique et la DFG, ce qui remet en question les affinités précédemment décrites pour AgtB et SocA. Dans un deuxième temps, ce travail a apporté les bases moléculaires et structurales des complexes PBP-mannityl-opines, complexes jamais caractérisés auparavant. Enfin, dans un troisième temps, la structure de la PBP AttC chez la souche C58, annotée comme mannopine-like, a été déterminée, et les expériences d’interaction ont montré qu’elle n’interagit avec aucune mannityl-opine, ce qui conduit à une révision de son annotation.Mes travaux apportent un éclairage nouveau sur l’import des mannityl-opines chez Agrobacterium tumefaciens. Le fait qu’aucun des transporteurs étudié ne permette l’import de l’agropine laisse penser qu’il existe une autre PBP ou un autre système de transport encore inconnu assurant cette fonction, ouvrant la voie vers de nouvelles études sur les pTi de type octopine et agropine. / Agrobacterium tumefaciens pathogenic agent confers the development of tumors in plants, in which it proliferates, integrating a fragment of its virulence Ti plasmid into its host genome. Transformed tissues synthesize original compounds, called opines, used as specific nutrients by the bacterium. More than twenty opines are known so far, and each one of them can be metabolized by A. tumefaciens strains possessing its associated transport and catabolism genes, which appears as a competitive advantage in the tumor colonization. The presence of these genes relies on the Ti plasmid type a pathogenic strain possesses. A. tumefaciens B6 possesses an octopine-type pTi, which harbors the metabolism genes of the mannityl-opines, which are mannopine, mannopinic acid, agropine and agropinic acid. Mannopine and mannopinic acid are synthesized by the same enzyme, and their precursors are deoxy-fructosyl-glutamine (DFG) and deoxy-fructosyl-glutamate (DFGA) respectively, both opines of the chrysopine family. DFG is also a wide-spread Amadori compound which can be uptaken by numerous organisms. Mannopine is a precursor for agropine synthesis. Finally, mannopine, mannopinic acid and agropine can spontaneously lactamize into agropinic acid.Despite the chemical similarity of these four opines, each one is transported by a different periplasmic binding protein (PBP) associated with an ATP-binding cassette (ABC) transporter. The PBP selects and binds one opine to bring it to the ABC transporter, which allows the passage of the opine to the cytoplasm due to two ATP molecules hydrolysis. The whole transporter specificity is determined by the PBP.Genetic studies in strains possessing an octopine-type pTi showed that AgaABCD PBP-ABC transporter system is specific to agropinic acid, AgtABCD to agropine, MoaABCD to mannopinic acid and that MotABCD transports mannopine and also mannopinic acid. In C58 strain, which do not possess an octopine-type pTi, SocAB transport system, coded by genes located on the cryptic pAt plasmid, allows the transport of DFG as a nutrient, and seems able to import mannopine too.My thesis work allowed, first, to characterize the strong affinities and the specificity of PBPs AgaA and AgtB to agropinic acid, PBP MoaA to mannopinic acid and PBP SocA to DFG, and also MotA unspecificity toward mannopine, mannopinique acid and DFG, which leads to a revision of the previously described affinities of AgtB and SocA. Secondly, this work brought molecular and structural basis of PBP-mannityl-opine complexes, never described before. Finally, the structure of PBP AttC, annotated as a mannopine binding-like protein in C58, was determined, and interactions experiments showed that it binds no mannityl-opines, leading to a revision of its annotation.My work sheds light on the mannityl-opines importation in Agrobacterium tumefaciens. The fact that none of the studied transport system allows agropine import lets think that there is another unknown PBP or another unknown whole transport system assuming this role, opening new ways to new studies about octopine- and agropine-type pTis.

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Symbiotic nitrogen fixation is the greatest potential input of nitrogen into terrestrial ecosystems. As a result, nitrogen fixation is critical to the functioning of the land carbon sink and its capacity to offset anthropogenic CO2 emissions and climate change. However, our understanding of the controls over nitrogen fixation rates and nitrogen fixing tree abundance is limited, resulting in paradoxes such as the relative absence of nitrogen fixing trees at high latitudes (where nitrogen is most limiting and it seems that nitrogen fixation should be most beneficial) and tropical forest nitrogen saturation, a mechanistically poor representation of nitrogen fixation in terrestrial biosphere models, and incomplete theory for variation in the successional trajectories of nitrogen fixing trees. This dissertation consists of four chapters that examine the drivers of symbiotic nitrogen fixation rates and the abundance of nitrogen fixing trees as they pertain to latitude, climate, and nitrogen fixation strategies. In chapter 1, I develop a method to measure coupled nitrogen fixation and plant carbon exchange in real-time, non-destructively, continuously, and at the whole plant scale. This permits a study of the controls of nitrogen fixation rates over timescales that range from seconds to months. In chapter 2 and 3, I apply the method developed in chapter 1 to determine the temperature response of nitrogen fixation rates and the timescales over which nitrogen fixation is regulated. For chapter 2 and 3, I grew nitrogen fixing tree species of tropical and temperate origin and representing the two types of nitrogen fixing symbioses (rhizobial and actinorhizal) across a 10 °C gradient of growing temperatures. In chapter 2, I show that nitrogen fixation depends on growing temperature and geographic origin and peaks at 30-38 °C, which is 5-13 °C higher than previous estimates based on other nitrogen fixing symbioses and 3-7 °C higher than net photosynthesis. These findings have direct implications for how nitrogen fixation is represented in terrestrial biosphere models and are in direct contrast to terrestrial biosphere model predictions of a decline in tropical nitrogen fixation with warming associated with climate change. In chapter 3, I show that nitrogen fixation takes 1-3 weeks to be down-regulated by 50% following an alleviation of nitrogen limitation, 1-5 weeks to be up-regulated by 50% following the initiation of nitrogen fixation when nitrogen becomes limiting, and up to 4 months for nitrogen fixation to start following a drastic reduction in soil nitrogen supply. Theory says that time-lags in regulating nitrogen fixation start becoming important for plant competition and losses of available nitrogen from ecosystems if they are between 1 day and 1 week. Thus, time-lags on the order of multiple weeks are a significant cost of a facultative nitrogen fixation strategy and resolve the tropical nitrogen forest nitrogen paradox characterized by high losses of available nitrogen at the ecosystem scale in spite of down-regulation of nitrogen fixation at the individual scale. In chapter 4, I show that nitrogen fixing tree abundance is bimodal in all regions of the contiguous United States except the Northeast and that founder effects can explain this pattern and the persistence of nitrogen fixing trees in old forests. Using theory, I show that founder effects are most probable at intermediate soil nitrogen supply, when nitrogen fixers have a high relative capacity to uptake available nitrogen, and when nitrogen fixing trees are facultative in their nitrogen fixation strategy. These chapters provide a new tool for studying nitrogen fixation, critical data for improving terrestrial biosphere models and our understanding of how nitrogen fixation and nitrogen cycling varies across latitude and how it will change with climate change, and new theory for the successional trajectories of nitrogen fixers.

This thesis describes the use of antibodies and immunofluorescence for subcellular localization of proteins. The key objective is the creation of an open-source atlas with information on the subcellular location of every human protein. Knowledge of the spatial distribution and the precise location of a protein within a cell is important for its functional characterization, and describing the human proteome in terms of compartment proteomes is important to decipher cellular organization and function.   Immunofluorescence and confocal microscopy of cultured cells were used for high-resolution detection of proteins on a high-throughput scale. Critical to immunofluorescence results are sample preparation and specific antibodies. Antibody staining of cells requires fixation and permeabilization, both of which can result in loss or redistribution of proteins and masking of epitopes. A high-throughput approach demands a standardized protocol suitable for the majority of proteins across cellular compartments. Paper I presents an evaluation of sample preparation techniques from which such a single fixation and permeabilization protocol was optimized. Paper II describes the results from applying this protocol to 4000 human proteins in three cell lines of different origin.   Paper III presents a strategy for application-specific antibody validation. Antibodies are the key reagents in immunofluorescence, but all antibodies have potential for off-target binding and should be validated thoroughly. Antibody performance varies across sample types and applications due to the competition present and the effect of the sample preparation on antigen accessibility. In this paper application-specific validation for immunofluorescence was conducted using colocalization with fluorescently tagged protein in transgenic cell lines. / <p>QC 20160509</p>

Mälaren är Sveriges tredje största sjö och även den dricksvattentäkt som försörjer flest människor i Sverige med vatten. Ett hot mot Mälarens vattenkvalité är algblomningar då vissa cyanobakterier producerar giftiga cyanotoxiner. Under 60-talet ledde övergödningen i Mälaren till kraftiga algblomningar och för att kunna råda bot på problemet infördes ett övervakningsprogram som nu har följt Mälarens utveckling i snart 60 år. I denna studie undersöktes med hjälp av detta övervakningsprogram vid vilka förhållanden cyanobakterier, framförallt de kvävefixerande Nostocales, frodas i Mälaren. Därefter önskades slutsatser kunna dras om vilka åtgärder som kan vara lämpliga för hålla nere populationen och därmed inte utgöra en risk för dricksvattenkvalitén.  Först genomfördes en långtidsstudie för att analysera vilka parametrar som historiskt har haft den tydligaste kopplingen till biovolymen växtplankton, cyanobakterier och Nostocales med hjälp av en multivariat regressionsmodell (PLS). Från detta framgick att de undersökta parametrarna kunde förklara 29 % av variationen i biovolymen för växtplankton, 41 % för cyanobakterier och 45 % för Nostocales. För växtplankton var pH och siktdjup de parametrar som kunde förklara störst del av variationen i biovolym och för cyanobakterier och Nostocales var istället vattentemperaturen, oorganiskt kväve/totalfosfor samt halten oorganiskt kväve de parametrar som hade tydligast koppling till biovolymen.  Därefter genomfördes en säsongsstudie för att kunna förklara nutida halter och säsongsvariationer för fem av Mälarens mätstationer (Ekoln, Galten, Granfjärden, Görväln och Södra Björkfjärden). Variationen av halten cyanobakterier kopplades närmast till temperaturen eftersom cyanobakterierna till stor del var begränsade till sommar och sensommar. Övriga växtplankton kunde däremot förekomma i höga halter även under våren. Att Ekoln hade betydligt högre halter oorganiskt kväve än Galten skulle kunna förklara skillnaden i andelen Nostocales mellan de två mätstationerna, där Galten hade en stor andel Nostocales och Ekoln en låg andel. Dock kunde kvävehalten inte förklara skillnader i fördelning mellan kvävefixerande och icke kvävefixerande cyanobakterier för de andra mätstationerna. Galtens stora tillrinning, grunda förhållanden och artrikedom skulle även kunna förklara bassängens höga halter av växtplankton under hela året.  Då temperaturen visat sig vara den parameter med tydligast koppling till cyanobakteriernas tillväxt är att begränsa den globala uppvärmningen den viktigaste åtgärden för att förhindra en ökad algblommning i framtiden. Kring resterande parametrar var slutsatser svåra att dra och i och med osäkerheten i frågan är en fortsatt noggrann miljöövervakning och handlingsplan när problemen väl uppstår av stor vikt. / Lake Mälaren is the third largest lake in Sweden and the source of drinking water for two million people. A threat to the water quality of Lake Mälaren is algal blooms since some cyanobacteria produce toxic cyanotoxins. In the 1960s, eutrophication in Lake Mälaren led to heavy algal blooms and in order to handle the problem, a monitoring program was introduced that has now followed Lake Mälaren's development for almost 60 years. In this study, the conditions under which cyanobacteria, especially the nitrogen-fixing Nostocales, thrive in Lake Mälaren was analysed using data from the monitoring program. Thereafter the goal was to draw conclusions regarding which measures may be needed to keep the population down so as not to pose a risk to drinking water quality. To begin with, a long-term study was conducted to analyse which parameters have historically had the clearest connection to the biovolume of phytoplankton, cyanobacteria and Nostocales using a multivariate regression model (PLS). This showed that the examined parameters could explain 29 % of the variation in phytoplankton biovolume, 41 % of the variation in the cyanobacteria biovolume and 45 % of the variation in Nostocales biovolume. For phytoplankton, pH and water transparency were the parameters that could explain most of the variation in biovolume, and for cyanobacteria and Nostocales, water temperature, the ratio between inorganic nitrogen and total phosphorus and the content of inorganic nitrogen were the parameters most clearly linked to the biovolume. Thereafter, a seasonal study was conducted to help explain current levels and seasonal variations for five of Lake Mälaren's measuring stations (Ekoln, Galten, Granfjärden, Görväln and Södra Björkfjärden). The variation of the cyanobacteria level was most closely linked to the temperature, as cyanobacteria was mostly limited to the summer. The ratio between inorganic nitrogen and total phosphorus and the content of inorganic nitrogen seemed to be an explanation for the difference between the high share of Nostocales in Galten where nitrogen was scarce, and the low proportions in Ekoln where nitrogen was abundant, but did not explain the distribution between nitrogen-fixing and non nitrogen-fixing cyanobacteria in the other basins. Galten's large inflow, shallow conditions and species richness could also explain the basin's high levels of phytoplankton throughout the year. With temperature as the parameter most clearly linked to the content of cyanobacteria, limiting global warming is the most important of measures to prevent increased algal blooms in the future. With regard to the remaining parameters, conclusions were difficult to draw and due to the uncertainty in the matter, continued close environmental monitoring and an action plan once the problems arise are of great importance.

The partnerships, or symbioses, between diatom hosts and cyanobacteria are widespread in the tropical and subtropical oceans. Many populate low nutrient waters where the heterocystous cyanobacteria fix N2 and provide reduced nitrogen (N) to the host. These types of symbioses are believed to be important in the global ocean biogeochemical cycle of N and carbon (C). The cyanobacteria morphology, cellular locations differ as well as genome size and content. The genome size and content are related to the cellular location: internal symbionts have smaller more eroded genomes, while external symbionts have larger genomes more similar to free-living cyanobacteria. Based on previously identified characteristics the aim of this report is to classify taxonomically, unidentified environmental metagenomic assembled genomes (MAGs) to the heterocystous symbionts of diatoms: Richelia and Calothrix. A select number of targeted gene sequences will be compared. MAGs and four draft genomes of Richelia and Calothrix were collected from public repositories (GTDB, NCBI and Tara project) and an initial comparison of GC-content and genome size was made. Based on this comparison, seven of the MAGs were determined as similar as the draft genomes of the known symbionts. After, a heatmap was created based on 27 targeted genes, some of which are highly conserved, to further characterize the phylogeny of the MAGs (Appendix 2). Results from the GC-content and genome size graph and the heatmap indicated similar trends which could connect one of the MAGs being most similar to the RintRC01 draft genome whereas the other five MAGs resembled the RintHH01 draft genome. Based on these results, conclusions can be drawn that the unknown MAGs are likely derived from symbionts of diatoms and could also possess similar characteristics, such as their cellular location, function and role in the N and C cycles.

Philosophiae Doctor - PhD / The tripartite symbiosis between Phaseolus vulgaris, arbuscular-mycorrhiza and the nodule bacteria, Rhizobia have been the focus of many studies ranging over a number of decades, however these studies have failed to answer certain questions relating the role of the symbionts in regard to host nutrition and the subsequent influence of these symbionts on the host C- economy. There is little doubt over the synergistic benefits involved in the dual inoculation of bean plants, as well as the resultant C-costs of maintaining the 2 symbionts, yet the specific contribution of the individual symbionts to the hosts overall nutrient and C-economy remain to be clarified. Thus the aim of this thesis is to help clarify these points by determining the symbiont induced photosynthetic, respiratory and nutritional changes taking place in the host. This was achieved by a series of experiments in which nodulated bean plants were split into two categories-those with and without AM colonized roots. These plants were then exposed to a range of growing conditions, including hi and low P, and a series of N treatments, ranging from zero N through to 3 mM NH/. Under these differing nutrient conditions growth, photosynthetic, respiratory, nutrient and amino acid responses were monitored, thus allowing for the determination of the symbionts influence on the host and the hosts reliance on the respective symbionts. Host reliance was noted most strongly under nutrient limiting conditions. Under low P treatment AM was the dominant symbiont as far as host C was concerned, allowing for the early establishment of the AM, thus ensuring the uptake of P for both host and nodule development. High P affected AM colonization to a greater extent than it did nodule dry weight and conversely the addition of N~ + led to a greater decrease in nodule dry weight than it did AM colonization. In spite of this decline, AM benefited the host by improving host N nutrition and relieving N-feedback inhibition of the export amino acid asparagine on BNF. These AM induced benefits did come at a cost to the host though, the dual inoculated plants had higher below ground respiratory costs and subsequently higher photosynthetic rates to compensate for the increased demand for C. The higher photosynthetic rates associated with dual inoculation were as a result of symbiont induced sink stimulation and not due to the improved nutrition of the host, as shown by the photosynthetic and nutrient response ratios. However, the respiratory costs associated with the uptake of soil nutrients were lower in AM colonized roots, thus showing an increased efficiency in nutrient gain by AM colonized roots. This improvement in host N nutrition as a result of AM colonization, coupled with the lower respiratory costs of AM nutrition led to the conclusion that under certain growing conditions nodules can become redundant and possibly parasitic.

In the Himmerfjärden Bay a large excess of nitrogen over phosphorus in the discharge from a large sewage treatment plant (STP) has suppressed growth of diazotrophic cyanobacteria in its inner parts. Implementation of nitrogen removal in the STP in 1997 drastically reduced nitrogen load and triggered growth of diazotrophs, mainly Aphanizomenon sp. This study is part of a long-term series of experiments with the overall aim to test how algal biomass and production in a receiving area can be reduced, without stimulating nitrogen fixation and biomass growth by diazotrophic cyanobacteria. Nitrogen removal was discontinued in the STP during two years (2007-8) and resumed in 2009, and the discharge shifted from 25 to 10 m depth, above the seasonal pycnocline. Cellular 15N showed that N2 was the most important N source for diazotrophic cyanobacteria, and that uptake of combined nitrogen was insignificant. As biomass was declining and at the end of the productive season, we could detect a small, but significant, increase in cellular δ15N at the inner bay stations (H3 and H4). However, this coincided with an increased proportion of Anabaena spp. of the total diazotrophic biomass. This may indicate that Anabaena spp. has a higher uptake of combined nitrogen compared with Aphanizomenon sp. or that declining populations of Aphanizomenon sp. take up combined nitrogen. We also found no evidence of uptake of combined nitrogen during the winter months when nitrogen supply is ample and Aphanizomenon sp. is devoid of heterocysts. During the first half of summer (week 21-27) heterocyst frequencies were higher at the outer stations B1 and H2, compared to the inner bay stations (H4 and H5). The lower frequencies at the inner bay stations are likely due to the reduced growth rate suffered by the Aphanizomenon sp. due to stronger competition for phosphorus by non-diazotrophs at these stations and hence lower need for heterocysts. Towards the end of summer conditions even out along the bay, as the surplus phosphorus from the spring bloom is used up at the outer stations and no heterocyst gradient is present. Heterocyst frequency varied significantly over the summer, with minimum values in the beginning of July, coinciding with the highest water temperatures, and higher frequencies in early and late summer. We suggest this is primarily due to a more efficiently functioning nitrogenase enzyme at high temperatures with a reduced need for “expensive” heterocysts. Spring heterocyst differentiation occurred around 4-6 weeks after depletion of dissolved inorganic nitrogen (DIN) and only when water temperature was 5-9 oC and a pycnocline established. It seems that temperature and light in concert will initiate growth, leading to an internal nitrogen deficiency which starts heterocyst differentiation. / Himmerfjärden eutrophication study

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