The last half-century has seen a dramatic and alarming rise in the incidence of autoimmune disease in industrialized nations too rapid to be accounted for by genetics alone. Among those, Inflammatory Bowel Disease (IBD) has risen from a western disease affecting industrialized populations to an emerging global threat affecting diverse populations around the world. IBD is a complex disease that combines genetic susceptibility and environmental exposure, but one aspect appears to be clear: the involvement of the gut microbiome. Current thought holds that IBD is an autoimmune attack on commensal microbiota, causing extensive collateral damage to the host intestinal tissues in the process. However, it has remained unclear in the field whether the changes observed in the IBD microbiome are causative in nature or whether the microbiome is responding to already-underway inflammatory processes within the host. This dissertation investigates one host factor in particular with regard to the microbiome and the development of inflammation: sodium-hydrogen exchange at the brush border, mediated by sodium hydrogen exchanger 3 (NHE3). NHE3 is inhibited during active IBD, but its loss in knockout animals is also enough to promote spontaneous colitis in a microbiome-dependent fashion. This dissertation investigates the specific contribution of the microbiome in NHE3 knockout animals to determine whether loss of NHE3 may be mediating the onset of colitis through pro-inflammatory changes in the microbiome. Our results suggest that the microbiome fostered in an NHE3-deficient environment may accelerate the onset and severity of experimental colitis, though likely in concert with additional host factors.

Duas enzimas do clone PL14.H10 de biblioteca metagenômica de consórcio microbiano degradador de óleo diesel, EST3, da família IV, e EST5, da família V das enzimas lipolíticas, foram caracterizadas. Os modelos estruturais mostraram cap-domínio e grande cavidade interna. A EST3 hidrolisou pNP-ésteres de até 12 carbonos, com Kcat/Km para pNP-acetato foi 1533,27 s-1.mM-1. Já a EST5 hidrolisou pNP-ésteres de 2 a 14 átomos de carbono, com maior atividade em pNP-valerato, e Kcat/Km de 1732,3 s-1.mM-1. Ambas apresentaram o fenômeno da ativação térmica. A atividade máxima da EST5 se deu a 45°C e em pH 7,5. A EST3 exibiu atividade máxima em pH 6,0, a 41°C. A atividade das enzimas aumentou na presença de quase todos os íons testados, e a EST5 quase não foi influenciada por detergentes. Foram relativamente estáveis em solventes orgânicos. Um dos cristais da proteína EST5 difratou a 2,311 Å e espera-se resolver a estrutura desta proteína. Os resultados deste trabalho revelaram interessantes características da EST3 e EST5 para possíveis aplicações biotecnológicas. / Two enzymes of PL14.H10 clone from a metagenomic fosmid library from a microbial consortium specialized for diesel oil degradation, EST3, from family IV, and EST5, from the V of lipolytic enzymes were characterized. Structural models showed cap-domain and large internal cavity. EST3 hydrolyzed pNP-esters of up to 12 carbons, with Kcat/Km in pNP-acetate of 1533.27 s-1.mM-1. EST5 hydrolyzed pNP-esters from 2 to 14 carbon atoms, with greater activity on pNP-valerate, and Kcat/Km of 1732.3 s-1.mM-1. Both showed the thermal activation phenomenon. EST5 activity was optimal at 45°C and pH 7.5. EST3 exhibited maximum activity at pH 6.0, 41°C. The esterases activity increased in the presence of almost all ions tested, and EST5 was not influenced by detergents. They were relatively stable in organic solvents. One of the crystals of EST5 protein diffracted at 2.311 Å and is expected to solve the structure of this protein. The results of this study revealed interesting features of the EST3 and EST5 proteins for possible biotechnological applications.

One of the major challenges for agricultural research in the 21st century is to increase crop productivity to meet the growing demand for food and feed. Biotic (e.g. plant pathogens) and abiotic stresses (e.g. soil salinity) have detrimental effects on agricultural productivity, with yield losses being as high as 60% for major crops such as barley, corn, potatoes, sorghum, soybean and wheat, especially in semi-arid regions such as Saudi Arabia. Plant growth promoting bacteria isolated from pioneer desert plants could serve as an eco-friendly, sustainable solution for improving plant growth, stress tolerance and health. In this dissertation, culture-independent amplicon sequencing of bacterial communities revealed how native desert plants influence their surrounding bacterial communities in a phylogeny-dependent manner. By culture-dependent isolation of the plant endosphere compartments and a number of bioassays, more than a hundred bacterial isolates with various biochemical properties, such as nutrient acquisition, hormone production and growth under stress conditions were obtained. From this collection, five phylogenetically diverse bacterial strains were able to promote the growth of the model plant Arabidopsis thaliana under salinity stress conditions in a common mechanism of inducing transcriptional changes of tissue-specific ion transporters and lowering Na+/K+ ratios in the shoots. By combining a number of in vitro bioassays, plant phenotyping and volatile-mediated inhibition assays with next-generation sequencing technology, gas chromatography–mass spectrometry and bioinformatics tools, a candidate strain was presented as a multi-stress tolerance promoting bacterium with potential use in agriculture. Since recent research showed the importance of microbial partners for enhancing the growth and health of plants, a review of the different factors influencing plant-associated microbial communities is presented and a framework for the successful application of microbial inoculants in agriculture is proposed. The presented work demonstrates a holistic approach for tackling agricultural challenges using microbial inoculants from desert plants by combining culturomics, phenomics, genomics and transcriptomics. Microbial inoculants are promising tools for studying abiotic stress tolerance mechanisms in plants, and they provide an eco-friendly solution for increasing crop yield in arid and semi-arid regions, especially in light of a dramatically growing human population and detrimental effects of global warming and climate change.

Under the extreme arid conditions of deserts, long periods of drought, nutrient-poor soils and high temperatures severely challenge the primary productivity of the ecosystem. Desert plants have evolved morphological and physiological adaptations against abiotic stresses. Along with these adaptation strategies they can recondition their surrounding soil, which will result in the enrichment of nutrients and moisture in the soil surrounding the plant. Although such self-fertilization may support the growth of other sympatric plant species under the plant, competitive exclusion mechanisms (i.e., allelopathy) reduce this possibility. Consequently, this will affect the diversity and functionality of the edaphic microbial communities. I hypothesize that desert xerophytes recondition the soils surrounding their body along with combining the ‘fertility’ and ‘allelopathy’ mechanisms to create a favorable new niche in desert ecosystem. I tested this hypothesis on the soil reconditioned by Welwitschia mirabilis growing in its native environment, the Namib Desert, Namibia. The collected soils were first used to confirm that Welwitschia manipulates the surrounding soil creating a ‘fertile’ but ‘exclusive’ soil area around the plant. Along with evaluating the effect of the reconditioned soil on the germination and plant development under normal irrigation and controlled drought condition, using barley as phytometer. The physio-chemical (i.e., WHC and WP) and microbial community analyses demonstrate that W. mirabilis reconditions the surrounding soil creating an environmental gradient around itself, in which the fertility is increased, through the accumulation and incorporation of shed reproductive parts of the plants (i.e., cones) in the surrounding soil, that will stimulate the plant growth under drought stress. Along with the fertilization effect, soil reconditioning also favor the antagonist effect (i.e., allelopathy) against plant competitors (e.g., new germinating seeds) to protect its ecological niche. Furthermore, the microorganisms and/or soluble/thermolabile molecules contribute to the allelopathic effect activated by the soil-reconditioning around W. mirabilis. The interactions among W. mirabilis, soil and microbes highlight an adaptive strategy that combines soil fertilization and allelopathy that I defined as “Alleolofertility” strategy. This allelofertility island surrounding the W. mirabilis may contributes to explain the evolutionary success of such a ‘living fossil’.

Climate change can alter ecosystem processes and organismal phenology through both long-term, gradual changes and alteration of disturbance regimes. Because microbes mediate decomposition, and therefore the initial stages of nutrient cycling, soil biogeochemical responses to climate change will be driven by microbial responses to changes in temperature, precipitation, and pulsed climatic events. Improving projections of soil ecological and biogeochemical responses to climate change effects therefore requires greater knowledge of microbial contributions to decomposition. This dissertation examines soil microbial and biogeochemical responses to the long-term and punctuated effects of climate change, as well as improvement to decomposition models following addition of microbial parameters. First, through a climate change mesocosm experiment on two soils, I determined that biogeochemical losses due to warming and snow reduction vary across soil types. Additionally, the length of time with soil microbial activity during plant dormancy increased under warming, and in some cases decreased following snow reduction. Asynchrony length was positively related to carbon and nitrogen loss. Next, I examined soil enzyme activity, carbon and nitrogen biodegradability, and fungal abundance in response to ice storms, an extreme event projected to occur more frequently under climate change in the northeastern United States. Enzyme activity response to ice storm treatments varied by both target nutrient and, for nitrogen, soil horizon. Soil horizons often experienced opposite response of enzyme activity to ice storm treatments, and increasing ice storm frequency also altered the direction of the microbial response. Mid-levels of ice storm treatment additionally increased fungal hyphal abundance. Finally, I added explicit microbial parameters to a global decomposition model that previously incorporated climate and litter quality. The best mass loss model simply added microbial flows between litter quality pools, and addition of a microbial biomass and products pool also improved model performance compared to the traditional implicit microbial model. Collectively, these results illustrate the importance of soil characteristics to the biogeochemical and microbial response to both gradual climate change effects and extreme events. Furthermore, they show that large-scale decomposition models can be improved by adding microbial parameters. This information is relevant to the effects of climate change and microbial activity on biogeochemical cycles.

<div>Host-pathogen interactions are the result of long term evolutionary processes due to the conflicting goals of the host and the infections pathogens in their quest for survival, creating an interplay of co-evolution as various adaptation are acquired by one and then in turned adapted to by the other. Selection of the host’s antimicrobial strategies and the resultant adaptations of infectious microorganisms leads to the development of complex and dynamic relationships ranging from symbiotic to commensal to pathogenic. In an effort to understand the selective process and identify unique mechanisms of antimicrobial defense, sera from 18 species (7 invertebrate, 11 vertebrate) were tested for antimicrobial potential against 20 Gram-negative and 11 Gram-positive bacteria. <i>Alligator mississippiensis</i> sera exhibited the strongest inhibitory potential. A transposon mutagenesis screen performed on the resistant bacterium <i>Serratia marcescens</i> identified several genes, including <i>glnL</i>, as necessary for defense. The <i>glnL</i> gene encodes for the sensory histidine kinase/phosphatase NtrB, controlling the expression of regulatory genes in response to nitrogen limitation. Attenuated growth of the Tn::<i>glnL</i> mutant in the presence of alligator serum and minimal media was rescued with nitrogen supplementation, suggesting the existence of a mechanism for nitrogen limitation as an antimicrobial strategy in alligator sera. Utilization of a <i>Drosophila melanogaster</i> oral model of infection showed that <i>glnL</i> is required for <i>S. marcescens</i> virulence, and nitrogen supplementation rescued the phenotype, as measured by fly mortality and bacterial cfu recovery. S. marcescens, an environmentally ubiquitous Gram-negative bacterium, is an opportunistic pathogen in several species, including alligators and Drosophila. Subsequent <i>in vitro</i> testing of the antimicrobial potential of invertebrate hemolymph utilizing the Tn::<i>glnL</i> mutant showed a nitrogen-dependent growth inhibition of species in the order Dipteria. Combined, these results support a model of evolutionary convergence of nitrogen limitation as an antimicrobial mechanism. This work not only identifies a novel antimicrobial strategy that could be used in the development of therapeutics, and a novel virulence factor in <i>S. marcescens</i>, but has broad mplications for bacterial management and can provide insight into the evolutionary history of host-pathogen interactions.</div>

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20438号 / 農博第2223号 / 新制||農||1049(附属図書館) / 学位論文||H29||N5059(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 矢﨑 一史, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Legumes are unique among plants for their ability to fix atmospheric nitrogen with the help of soil bacteria rhizobia. Medicago truncatula is used as a model legume to study different aspects of symbiotic nitrogen fixation. M. truncatula, in association with its symbiotic partner Sinorhizobium meliloti, fix atmospheric nitrogen into ammonia, which the plant uses for amino acid biosynthesis and the bacteria get reduced photosynthate in return. M. truncatula NPF1.7 previously called MtNIP/LATD is required for symbiotic nitrogen fixing root nodule development and for normal root architecture. Mutations in MtNPF1.7 have defects in these processes. MtNPF1.7 encodes a member of the NPF family of transporters. Experimental results showing that MtNPF1.7 functioning as a high-affinity nitrate transporter are its expression restoring chlorate susceptibility to the Arabidopsis chl1-5 mutant and high nitrate transport in Xenopus laevis oocyte system. However, the weakest Mtnip-3 mutant allele also displays high-affinity nitrate transport in X. laevis oocytes and chlorate susceptibility to the Atchl1-5 mutant, suggesting that MtNPF1.7 might have another biochemical function. Experimental evidence shows that MtNPF1.7 also functions in hormone signaling. Constitutive expression of MtNPF1.7 in several species including M. truncatula results in plants with a robust growth phenotype. Using a synthetic auxin reporter, the presence of higher auxin in both the Mtnip-1 mutant and in M. truncatula plants constitutively expressing MtNPF1.7 was observed. Previous experiments showed MtNPF1.7 expression is hormone regulated and the MtNPF1.7 promoter is active in root and nodule meristems and in the vasculature. Two potential binding sites for an auxin response factors (ARFs) were found in the MtNPF1.7 promoter. Chromatin immunoprecipitation-qRT-PCR confirmed MtARF1 binding these sites. Mutating the MtARF1 binding sites increases MtNPF1.7 expression, suggesting a mechanism for auxin repression of MtNPF1.7. Consistent with these results, constitutive expression of an ARF in wild-type plants partially phenocopies Mtnip-1 mutants’ phenotypes.

Hepatitis B virus (HBV) is a leading cause of liver disease. Its success as a human pathogen is related to the immense production of subviral envelope particles (SVPs) contributing to viral persistence by interfering with immune functions. To explore cellular pathways involved in SVP formation and egress, we investigated host–pathogen interactions. Yeast-based proteomics revealed Sec24A, a component of the coat protein complex II (COPII), as an interaction partner of the HBV envelope S domain. To understand how HBV co-opts COPII as a proviral machinery, we studied roles of key Sec proteins in HBV-expressing liver cells. Silencing of Sar1, Sec23, and Sec24, which promote COPII assembly concomitant with cargo loading, strongly diminished endoplasmic reticulum (ER) envelope export and SVP secretion. By analysing Sec paralog specificities, we unexpectedly found that the HBV envelope is a selective interaction partner of Sec24A and Sec23B whose functions could not be substituted by their related isoforms. In support, we found that HBV replication upregulated Sec24A and Sec23B transcription. Furthermore, HBV encountered the Sec24A/Sec23B complex via an interaction that involved the N-terminal half of Sec24A and a di-arginine motif of its S domain, mirroring a novel ER export code. Accordingly, an interference with the COPII/HBV cross-talk might display a tool to effectively inhibit SVP release.

No description available.