Plants produce a large number of specialized or secondary compounds that aid in their reproduction and protection against biotic and abiotic stress. In this work I investigated the metabolism and function of terpenes, the largest class of specialized metabolites, in switchgrass and carrot. Switchgrass (Panicum virgatum L.), a perennial C4 grass of the Tallgrass Prairie, represents an important species in natural and anthropogenic grasslands of North America. Its natural resilience to abiotic and biotic stress has made switchgrass a preferred bioenergy crop. I have investigated the metabolism of terpenes in switchgrass leaves and roots in response to herbivory or defense hormone treatments and the application of drought. With a focus on volatile terpene metabolites, I functionally characterized over thirty genes (terpene synthases, TPSs), of which one third could be correlated with the production and release of volatile monoterpenes and sesquiterpenes that likely function in direct chemical defense or in the attraction of insect predators or parasitoids. Drought stress application caused switchgrass roots to accumulate a larger amount of oxygenated terpenes and presumably non-volatile terpenes, the function of which in direct or indirect drought stress protection requires further investigation. I also examined the metabolic dynamics and role of the monoterpene borneol, which accumulates at high concentrations in the roots of switchgrass and to a lower extent in the roots of the close relative Setaria viridis, in root microbe interactions. Although we demonstrated a successful RNAi based knock down of the borneol terpene synthase TPS04, we found no immediate evidence that borneol significantly modifies bacterial communities in the root. Further studies on Setaria and equivalent RNAi lines in switchgrass will provide more detailed and needed insight to decipher the role of monoterpene accumulation in grasses interactions with mutualists, pathogens, and pests. In an applied project, I investigated terpene specialized metabolism in carrot (Daucus carota L.) to identify genetic determinants of carrot aroma and flavor. To determine central enzymes which contribute to the terpene component of carrot volatile blends, we first analyzed tissue specific expression patterns of carrot terpene synthase genes (TPS) in the genomic model carrot (cv. DH1) and in roots of four aromatically unique colored carrot genotypes (orange-4943B, red-R6637, yellow-Y9244A and purple-P7262). We selected nineteen key biosynthetic enzymes involved in terpene formation and compared in vitro products from recombinant proteins with native volatile profiles obtained from DH1 and colored carrot genotypes. We biochemically characterized several highly expressed TPSs with direct correlations to major compounds of carrot flavor and aroma including germacrene-D (DcTPS11), (DcTPS30) and -terpinolene (DcTPS03). Random forest analysis of colored carrot volatiles revealed that nine terpene compounds are sufficient for distinguishing the flavor and aroma of raw colored carrots. Interestingly, accumulation of specific terpene compounds rather than chemical diversity is responsible for differences in sensory quality traits in colored genotypes. As accumulations of specific terpene compounds can contribute to the undesired flavor in carrot, our report provides a detailed roadmap for future breeding efforts to enhance carrot flavor and aroma. / Doctor of Philosophy / Plants produce a large number of chemicals that are important for growth, defense, flavor, and aroma. While chemical production has been studied in some major food crops (corn, tomato, rice), knowledge of the formation and function of chemicals in switchgrass and carrot is still limited. Switchgrass (Panicum virgatum L.), a grass of the Tallgrass Prairie, represents an important species grasslands of North America. Its natural resilience to stress has made switchgrass a preferred bioenergy crop. I found that switchgrass produces many compounds in the chemical class of terpenoids in roots and leaves that likely serve as a defense against damage from pests. In addition, I found that drought stress leads to the production of terpenoid compounds that may have roles in protection when water is limited. My research also demonstrates that roots of switchgrass and the related grass Setaria maintain substantial levels of the essential oil compound borneol. This terpenoid compound can act as a nutrient source for specific bacteria and/or an antimicrobial agent. Therefore, I proposed that switchgrass and Setaria roots produce borneol to establish a distinct root microbiome by recruitment of beneficial bacteria and deterrence of harmful microorganisms. To test this hypothesis, we genetically engineered plants to reduce borneol formation and accumulation in roots. Using these plants, we evaluated changes in the root microbiome in response to altered borneol levels. We found that interfering with borneol production in Setaria roots has limited influence on the microbiome inside roots. Although a similar approach was used for switchgrass, we were unable to significantly reduce borneol V formation in roots. Results from this study provide a better understanding of belowground plant-microbe interactions, and potential for enhancing resistance traits into other crop species. I also investigated the flavor and aroma compounds produced in carrots, which are considered a key supplemental vegetable due to high nutritional value and pleasant taste. Surprisingly, little has been known about the genetic factors that control flavor and aroma traits in colored carrot varieties. Therefore, I performed a robust characterization of the biosynthesis of terpenoids, which are the predominant aroma and flavor compounds in carrot. I identified several enzymes in carrot that can produce a diverse blend of terpenoids which are associated with sweet, spicy and bitter tastes. In addition, I discovered that carrot stems and leaves also maintain a rich chemistry of terpenoids similar to that in roots. Results from this work provide a baseline for engineering enhanced flavor in carrot and provide a deeper insight into essential oil formation in root crops.

Bacteria of the Neisseria are predominately commensal, though N. gonorrhoeae and N. meningitidis are capable of causing disease. Both of these species often asymptomatically colonize humans, a trait reminiscent of their commensal cousins. The factors that shift the balance between asymptomatic carriage and disease are unknown. Pathogenic Neisseria use retractile surface structures called Type IV pili to coordinate community behavior and to initiate and sustain infection. Previously, the contributions of pilus retraction have been studied by deleting the pilus retraction motor, PilT. Recent findings suggest the speed and force exerted by pilus retraction is responsive to environmental cues. By examining several PilT mutants that maintain the ability to retract pili, I show retraction, per se, is not required for N. gonorrhoeae social interactions with bacteria or with human cells. Furthermore, Type IV pilus retraction by the commensal N. elongata affects the host cell differently than retraction by N. gonorrhoeae. These observations collectively suggest pilus retraction properties shape the host cell response to Neisseria colonization and could tip the balance of asymptomatic colonization to symptomatic disease.

Adapting the tools of optical microscopy to the large-scale dynamic systems encountered in the development of multicellular organisms provides a path toward understanding the physical processes necessary for complex life to form and function. Obtaining quantitatively meaningful results from such systems has been challenging due to difficulty spanning the spatial and temporal scales representative of the whole, while also observing the many individual members from which complex and collective behavior emerges. A three-dimensional imaging technique known as light sheet fluorescence microscopy provides a number of significant benefits for surmounting these challenges and studying developmental systems. A thin plane of fluorescence excitation light is produced such that it coincides with the focal plane of an imaging system, providing rapid acquisition of optically sectioned images that can be used to construct a three-dimensional rendition of a sample. I discuss the implementation of this technique for use in larva of the model vertebrate Danio rerio (zebrafish). The nature of light sheet imaging makes it especially well suited to the study of large systems while maintaining good spatial resolution and minimizing damage to the specimen from excessive exposure to excitation light. I show the results from a comparative study that demonstrates the ability to image certain developmental processes non-destructively, while in contrast confocal microscopy results in abnormal growth due to phototoxicity. I develop the application of light sheet microscopy to the study of a previously inaccessible system: the bacterial colonization of a host organism. Using the technique, we are able to obtain a survey of the intestinal tract of a larval zebrafish and observe the location of microbes as they grow and establish a stable population in an initially germ free fish. Finally, I describe a new technique to measure the fluid viscosity of this intestinal environment in vivo using magnetically driven particles. By imaging such particles as they are oscillated in a frequency chirped field, it is possible to calculate properties such as the viscosity of the material in which they are embedded. Here I provide the first known measurement of intestinal mucus rheology in vivo. This dissertation includes previously published co-authored material.

O comércio mundial de enzimas industriais estava estimado em 2.3 bilhões de dólares entre detergentes (U$ 789 milhões), aplicações alimentícias (U$ 634 milhões), agricultura (U$ 237 milhões), entre outros. Neste contexto, as enzimas lipolíticas estão atraindo enorme atenção devido ao seu potencial biotecnológico, visto que estas podem catalisar múltiplas reações (hidrólise, acidólise, interesterificação e glicerólise). Enzimas lipolíticas de origem microbiana são economicamente atrativas por serem biodegradáveis, atuarem normalmente em condições brandas, e serem quimio-seletivas propiciando à indústria farmacêutica a obtenção de drogas com efeito colateral reduzido. Neste projeto, quatro genes potenciais codificadores de esterases/lipases, advindos de uma biblioteca metagenômica de um consórcio microbiano degradador de óleo diesel, foram clonados em vetores de expressão e expressos em Escherichia coli BL21 (DE3), e as proteínas correspondentes foram submetidas a ensaios funcionais e estruturais. / The global trade of industrial enzymes is estimated at 2.3 billion U.S. dollars, divided mainly between detergents (US$ 789 million), food applications (US$ 634 million), and agriculture (US$ 237 million). Within this trade, lipolytic enzymes have attracted enormous attention because of their biotechnological potential as catalysts of multiple reaction types (including hydrolysis, acidolysis, interesterification and glycerolysis). Lipolytic enzymes of microbial origin are economically attractive because they are easily biodegradable, usually act in mild conditions, and are chemo-selective, providing the pharmaceutical industry a method for obtaining drugs with reduced side effects. In this project, four individual genes encoding putative esterases/lipases identified in a metagenomic library obtained from a microbe consortium isolated from diesel oil-contaminated soil were cloned into expression vectors and expressed in Escherichia coli BL21 (DE3), and their corresponding recombinant proteins were used for functional and structural studies.

All animals harbor microorganisms that interact with each other and with their hosts. These microorganisms play important roles in health, disease, and defense against pathogens. The microbial communities in the intestine are particularly important in preventing colonization by pathogens; however, this defense mechanism and the means by which pathogens overcome it remain largely unknown. Moreover, while the composition of animal-associated microbial communities has been studied in great depth, the spatial and temporal dynamics of these communities has only recently begun to be explored. Here, we use a transparent model organism, larval zebrafish, to study how a human pathogen, Vibrio cholerae, invades intestinal communities. We pay particular attention to a bacterial competition mechanism, the type VI secrection system (T6SS), in this process. In vivo 3D fluorescence imaging and differential contrast imaging of transparent host tissue allow us to establish that V. cholerae can use the T6SS to modulate the intestinal mechanics of its host to displace established bacterial communities, and we demonstrate that one part of the T6SS apparatus, the actin crosslinking domain, is responsible for this function. Next, we develop an automated high-throughput light sheet fluorescence microscope to allow rapid imaging of bacterial communities and host cells in live larval zebrafish. Light sheet fluorescence microscopy (LSFM) has been limited in the past by low throughput and tedious sample preparation, and our new microscope features an integrated fluidic circuit and automated positioning and imaging to address these issues and allow faster collection of larger datasets, which will considerably expand the use of LSFM in the life sciences. This microscope could also be used for future experiments related to bacterial communities and the immune system. The overarching theme of the work in this dissertation is the use and development of advanced imaging techniques to make new biological discoveries, and the conclusions of this work point the way toward understanding pathogenic invasion, maximizing the use of LSFM in the life sciences, and gaining a better grasp of host-associated bacterial community dynamics. This dissertation includes previously published and unpublished co-authored material.

Les rhamnolipides produits par Pseudomonas aeruginosa sont des glycolipides ayant des propriétés élicitrices des réactions de défense de la vigne et d'Arabidopsis thaliana dont le mode de perception est inconnu. En tant que composés amphiphiles, uneinteraction avec la membrane plasmique a été proposée. Au niveau fondamental, il est démontré ici par une approche de biophysique utilisant la RMN du solide et des simulations de dynamique moléculaire in silico, que le caractère amphiphile des RLs favorise leur insertion au sein de modèles lipidiques de membranes de plantes, qui ne sont pas déstabilisés par leur présence. De plus il a été observé, par la réalisation de puces à ADN sur A. thaliana, que la perception de ces composés induisait une reprogrammation transcriptionnelle précoce de grande ampleur. En vue de démontrer l'intérêt agronomique des RLs pour la protection d'une plante de grande culture, leur activité sur le colza (Brassica napus) a également été étudiée. Des marqueurs caractéristiques de la mise en place des réponses de défense ont été observés, ainsi qu'un effet de protection detissus foliaires contre le pathogène B. cinerea, ce qui renforce le potentiel des RLs comme agents de biocontrôle pour la protection des Brassicacées / Pseudomonas aeruginosa rhamnolipids are glycolipids known to trigger defense responses in grapevine and Arabidopsis thaliana but their mode of perception by plants is still unknown. As amphiphilic compounds, rhamnolipids have been proposed to interactdirectly with plasma membrane lipids. Here we show, by a biophysical approach involving solid state NMR and in silico molecular dynamic simulations, that the insertion of rhamnolipids does not disturb the dynamic of plant plasma membrane models. Inorder to characterize early gene expression modifications triggered by rhamnolipids a micro-array study on A. thaliana was realized, revealing a large transcriptional change. The potential of rhamnolipids to protect the agronomical plant Brassica napus was also investigated. Rhamnolipid triggering of chemical and physical defenses associated with efficient protection against the opportunistic pathogenic fungus Botrytis cinerea, used as a model, was shown. Those results highlight a real potential of RLs as biocontrol agents for Brassicaceae protection

Microbial communities influence the kinetics and pathways of reactions involved in the dissolution of a number of minerals (Ehrlich 1996). On a smaller scale these interactions can affect substrate permeability, porosity, and create highly localized biogeochemical conditions. However, a mechanistic understanding of the consequences of microbial surface colonization on calcite dissolution rate has yet to be achieved. More specifically, little is known about the impact of sulfur-oxidizing bacteria activity on the rate of carbonate mineral dissolution, or the nature of the microbe-limestone attachment and interaction. Through a series of laboratory and field experiments the effect of mineral surface colonization by microbial communities, obtained from an active sulfuric acid cave (Lower Kane Cave (LKC), Big Horn Basin, WY), on the dissolution rate of Madison Limestone was quantified. Results from laboratory experiments showed that a microbial biofilm, composed primarily of Epsilonproteobacteria and Gammaproteobacteria growing on a limestone surface oxidized thiosulfate and increased carbonate dissolution rates up to 3.3 times faster than abiotic rates. When all thiosulfate substrate was withheld the community oxidized stored intracellular sulfur, continuing to accelerate limestone dissolution and decreasing pH. This process is sensitive to O2 limitations. Characterization of this aggressive sub-biofilm corrosion was more closely examined by SEM imaging. By comparing mineral surface morphology of colonized chips to non-colonized chips of various carbonate substrates, it was shown that even under conditions near equilibrium with calcite, aggressive dissolution of carbonate substratum occurs exclusively beneath the biofilm. These findings support the hypothesis that (1) sulfur-oxidizing microbial communities aggressively dissolve carbonates in order to buffer the production of excess acidity by neutrophilic communities and (2) biofilm presence affects carbonate mineral dissolution by physically separating a bulk stream water from the sub-biomat environment. Furthermore, it was found that mineralogy affects the degree of establishment of microbial communities in this environment. Results from a series of four laboratory and one in situ reactor experiment showed that limestone and dolostone substratum consistently had higher biomass accumulations than silicate minerals or pure Iceland spar calcite in the same reactor. These results provide evidence to support the hypothesis that mineralogy influences microbial accumulation in sulfuric-acid karst systems. Particularly, neutrophilic sulfur-oxidizing communities accumulate in greater quantities on solid substrates that buffer metabolically-generated acidity. These results also demonstrated the dependence of microorganisms on colonization of a particular mineral surface, possibly in order to gain access to micronutrients bound within solid substrates when exposed to nutrient-limited conditions. / text

Thèse (Ph. D.)--Université Laval, 2007. / Bibliogr.: f. 120-138. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Thesis (Ph. D.) -- Lincoln University, 2009. / Also available via the World Wide Web.

Thesis (Ph. D.)--Johns Hopkins University. / Includes bibliographical references.