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1
ADAMO, GIUSY MANUELA. "Evolution of copper tolerance in yeast cells." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2012. http://hdl.handle.net/10281/29458.
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For all living organisms, copper (Cu) is an essential micronutrient taking part, with its redox chemistry, to several metabolic and regulatory cellular events. However, the same redox properties that make Cu essential are responsible for its toxicity. Indeed, Cu participates in reactions that generate Reactive Oxygen Species (ROS). ROS target main cellular macromolecules (proteins, lipids, DNA and RNA), leading to cellular dysfunctions and in the extreme case, to cell death. All living organisms evolved molecular mechanisms for Cu homeostasis. Indeed, uptake, transport and detoxification systems that actively prevent both Cu deficiency and poisoning are well conserved along the phylogenetic tree. Among eukaryotes, these mechanisms have been mainly investigated in the yeast Saccharomyces cerevisiae used as a model organism.
Evolutionary engineering is a rational approach that uses the evolutionary principles to direct the selection of organisms with a desired set of phenotypes, allowing for the improvement of microbial properties. This approach can be exploited to obtain Cu-tolerant and Cu-accumulating yeast cells, with potential application in nutraceutics, as nutritional supplements, as well as in bioremediation, for the removal or recovery of metal ions. At the same time, evolutionary engineering is a valuable strategy to gain more insight into the molecular aspects of Cu tolerance in microbial cells.
In the present work is described an evolutionary engineering strategy to improve Cu tolerance of natural yeasts. Strains of Saccharomyces cerevisiae and of Candida humilis originally endowed with different sensitivity and tolerance toward Cu have been exposed to increasing concentrations of Cu during cell cultivation in liquid medium. This treatment stably improved Cu tolerance of all strains. One evolved strain for each yeast species was then chosen to analyze in detail the physiological response to Cu. Compared with the original Cu-sensitive strains the two evolved strains showed improved cell viability and attenuated production of ROS. A reshaping of the profile of antioxidant enzymes and Cu-binding proteins was observed in both strains as a specific response to copper.
Further investigations carried out on S. cerevisiae strains demonstrated a pivotal role of the CUP1 gene, encoding for a metallothionein. A 7-fold amplification of this gene was found associated with evolution of Cu tolerance.
Finally, Cu tolerance in C. humilis cells was studied by proteomic analyses. Changes were observed in the levels of several proteins involved in the oxidative stress response (such as glycolytic enzymes), heat shock proteins, proteins involved in protein synthesis and energy production, proteins with a role in phospholipids synthesis. Cu exposure resulted in differential protein expression, in both non-evolved and Cu evolved cells. In general, changes in protein levels detected in evolved cells were smaller. On this basis, it was hypothesized that in the evolved cells copper tolerance relies only partly on the molecular mechanisms associated with the oxidative stress response. This work shows once again that evolutionary engineering is a powerful strategy to drive the gain of stable phenotypic traits. The evolved strains might found direct application in several biotechnological fields, and provide a kind of “molecular platform” for the investigation on the mechanisms of stress tolerance. The availability of data about the S. cerevisiae genome allowed a focused investigation on the molecular actors involved in Cu tolerance. In the case of C. humilis, the use of a proteomic approach allowed to compensate for the poor information available on the determinants of Cu tolerance.
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Wadey, P. "The physiology of copper tolerance in Mimulus guttatus." Thesis, University of Liverpool, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.233878.
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Harper, Frances Anne. "The cost of copper tolerance in Mimulus guttatus." Thesis, University of Exeter, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307278.
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Strange, J. "The physiology of copper tolerance in Mimulus guttatus." Thesis, University of Exeter, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.383346.
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Lloyd, Deborah. "Reproductive isolation associated with the copper tolerance locus in Mimulus guttatus." Thesis, University of Exeter, 2010. http://hdl.handle.net/10036/3173.
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The evolution of reproductive isolating barriers that prevent gene flow between species is essential to the process of speciation. One such barrier is intrinsic postzygotic isolation, which proceeds as hybrid sterility or inviability, and is commonly attributed to Dobzhansky-Muller genic incompatibilities. Here, deleterious interlocus interactions occur between incompatible alleles of complementary genes when brought together in the genome of a hybrid. Although these hybrid incompatibilities are widespread, having been identified in mammals, fish, plants and fungi, still relatively little is known about the nature of the genes involved. In the model plant species Mimulus, a Dobzhansky-Muller incompatibility exists between two populations of the yellow monkey flower, Mimulus guttatus, in which the interaction between a single gene from a copper tolerant population, Copperopolis, and a small number of polymorphic genes from a second non-tolerant population, Cerig-y-drudion, results in hybrid necrosis in the F1. Hybrid necrosis, a form of hybrid inviability with phenotypic characteristics strongly similar to those of plants responding to pathogen attack, is a common barrier preventing hybridization in plants. As well as being of interest in terms of evolution, hybrid necrosis has practical implications in plant breeding as it prevents the combining of desirable traits from related species in commercial cultivars. In the cross between Copperopolis and Cerig-y-drudion, copper tolerance, conferred by a single major gene, and hybrid necrosis are tightly linked but the independent or synonymous nature of the gene(s) in the Copperopolis population that contribute to these two characteristics is unknown. A key aim of this thesis was to establish the nature of the single gene in Copperopolis that contributes to hybrid necrosis with regards to its linkage to copper tolerance. The gene for hybrid necrosis was found to be tightly linked to, but discrete from, the gene controlling copper tolerance. Three candidate genes for this hybrid necrosis locus were indentified: a Jumonji-domain containing protein with probable function as a methyltransferase, a glycosyltransferase and a possible phosphatase. Interestingly, the latter two have potential functional roles in the plant immune system. The second key aim of this thesis was to perform the first investigation into the small number of genes in the Cerig-y-drudion population that contribute to the crossing barrier. Two QTLs for hybrid necrosis were identified. One QTL on Chromosome 9 is responsible for around 20% of the hybrid necrosis whilst the second QTL on Chromosome 12 acts as an enhancer of the first QTL causing an additional 10% of necrosis. Interestingly both these QTLs contain R genes, further implicating the possible involvement of the plant immune system in this crossing barrier.
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Fuentealba, I. C. "Cellular mechanisms of toxicity and tolerance in the copper-loaded rat." Thesis, University of Liverpool, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.383477.
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Gonzalez-Chavez, Ma del Carmen. "Arbuscular mycorrhizal fungi from As/Cu polluted soils, contribution to plant tolerance and importance of the external mycelium." Thesis, University of Reading, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312514.
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Metaxas, Anna. "Effect of the interaction between two species of marine diatoms on their individual copper tolerance." Thesis, University of British Columbia, 1989. http://hdl.handle.net/2429/27597.
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The importance of species interactions in studies assessing the trace metal tolerance of individual species, has been largely overlooked. Most toxicity assessments involve single-species tests. Although relevant in some cases, this approach does not incorporate the biological enviroment of an organism as a factor that might influence its survival. On the other hand, this factor is incorporated in studies at the community level. However, the latter, not examining the mechanisms that drive the interactions in the community, do not allow for prediction of the outcome after the addition of a stress inducer, such as trace metals. The purpose of this study was to examine the effect of the interaction of two species of marine diatoms (Skeletonema costatum (Cleve) Greville and Nitzschia thermalis (Ehrenberg) Auerswald) on their individual copper tolerances.
The two species were grown in unialgal cultures in order to determine their copper tolerance. 5. costatum did not exhibit growth above 5 x 10⁻⁷ M (pCu = 8.46) and N. thermalis above 6 x 10⁻⁷M added total copper (pCu = 8.36). Skeletonema exhibited increased growth rate and lag phase with increasing copper concentration (and decreasing pCu). On the other hand, Nitzschia demonstrated decreased growth rate. No effect on lag phase was observed for this species.
No difference between the tolerances of two strains of S. costatum was observed. Both strains demonstrated unaffected growth at 1 x 10⁻⁷M (pCu = 9.16) and no growth at 1 x 10⁻⁵ M added total copper (pCu = 7.16).
Subsequently, Skeletonema costatum and Nitzschia thermalis were grown together at three copper concentrations (1 x 10⁻⁹ M, 4 x 10⁻⁷M and 5 x 10⁻⁷M added total copper). In the unialgal cultures that were used as controls, the two species grew as predicted from their tolerance tests. However, in the mixed cultures, Nitzschia was the only species that exhibited growth, regardless of the copper concentration in the medium. This inhibition of Skeletonema in the presence of the second diatom, was attributed to a toxic Nitzschia exudate. The effect of the exudate appeared to be temporary, as demonstrated by the extended lag phase and subsequent satisfactory exponential growth rate of Skeletonema. It is suggested that exponential growth rate was resumed because the exudate degrades within a period of five days (= lag phase).
It is shown that the interaction between the two diatom species is more important in determining the survival of S. costatwn than its individual copper tolerance. This is not the case for N. thermalis. Such interactions would be unaccounted for in single-species toxicity tests. On the other hand, if they are known, prediction of how a community that includes these two species would respond to copper additions becomes possible.Science, Faculty of
Earth, Ocean and Atmospheric Sciences, Department of
Graduate
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Pinto, Cecilia de Agrela. "The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus." Master's thesis, Faculdade de Ciências e Tecnologia, 2012. http://hdl.handle.net/10362/10062.
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Dissertation for the Master’s Degree in Structural and Functional BiochemistryThe majority of bacterial heavy metal resistance systems are regulated by twocomponent signal transduction systems. Stimuli from the environment interact with the histidine kinase, which in turn activates the response regulator by phosphorylation. The effector domain of the response regulator then binds to DNA, eliciting the specific response. Analysis of the Marinobacter hydrocarbonoclasticus genome revealed the presence of genes, copXAB, that code for proteins associated with copper response. The biochemical characterization of the two-component signal transduction system, copSR, is of interest due to the vital role it plays in the regulation of expression of the copXAB operon. The genes that encode for the CopR and CopS_C (cytosolic sensor domain of CopS) proteins were heterologously expressed in E.coli and expression was optimized for the production of soluble protein using LB medium. Due to solubility problems, the genes that code for these proteins were cloned as hexahistidine or glutathione S-transferase fusion proteins. CopR and its domains were optimally expressed at 16°C for 16 and 3 h after induction, respectively, whilst CopS_C was expressed at 37°C during 3 h after induction. Proteins were purified using different chromatographic strategies, most of them using affinity chromatography. The yields of pure protein per liter of growth culture obtained after complete purification from the soluble cellular extract were: 0.14 to 0.23 mg/L for CopR; 0.42 mg/L, CopR_NHis6; for the CopR_CHis6 it was 0.16 mg/L and 4.2 mg/L of CopS_C. The molecular mass of each protein was determined by gel filtration, 31 kDa for CopR, 17.5 kDa for CopR_NHis6, 15.1 kDa for CopR_CHis6 and 38.2 kDa for CopS_C. In the case of CopS_C there is the possibility that a dimer is formed, which should be evaluated. From the evaluation of disulfide bonds, using SDS PAGE and PAGE gels, all proteins or protein domains appeared to be monomers when in the presence of β-MEtOH. Circular dichroism evaluated the state of folding of the CopS_C and CopR proteins, which were shown to be folded in which the α-helix structures predominate. A model structure for CopR was also determined which agrees with this analysis. However, in the case of the CopR domains, the data obtained merely indicate folding, due to the low concentrations of the proteins. Phosphorylation and electrophoresis mobility shift assays of the CopR protein were, for the most part, inconclusive. However, in the absence of BSA, formation of the CopR:DNA complex in a gel filtration column is observed, though requires additional evaluation.
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Saint-Pierre, Édith. "Mise en évidence d'un mécanisme de tolérance chez scenedesmus quadricauda suite à une intoxication à long terme au cuivre /." Thèse, Chicoutimi : Université du Québec à Chicoutimi, 1992. http://theses.uqac.ca.
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Mémoire (M.Ress.Renouv.)-- Université du Québec à Chicoutimi, 1992.Résumé disponible sur Internet. CaQCU Bibliogr.: f. 69-73. Document électronique également accessible en format PDF. CaQCU
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Paraskevopoulou, Angeliki T. "An investigation into the copper and lead tolerance of populations of Buddleia davidii and Chamaenerion angustifolium." Thesis, Sheffield Hallam University, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367035.
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Hussain, Noor Hana. "Inducible tolerance and sensitivity to stress responses in 'Escherichia coli' with particular reference to copper and pH." Thesis, University College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244279.
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Watkins, A. J. D. "Within-mine differentiation in degree of copper and arsenic tolerance in Agrostis capillaris L. (Agrosis tenuis sibth)." Thesis, University of Exeter, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.353993.
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Gruhn, Christine Mae. "Effect of a heavy metal on ecto- and vesicular-arbuscular mycorrhizal fungi: the physiology, ultrastructure, and ecology of copper stress and tolerance." Diss., Virginia Polytechnic Institute and State University, 1989. http://hdl.handle.net/10919/54531.
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This work consists of an introduction, six chapters dealing with various aspects of the response of mycorrhizal fungi to copper, and a brief conclusion. The first chapter examines the enzyme tyrosinase in several ectomycorrhizal fungi and shows that its activity is altered in these fungi in response to copper. Polyamines are also examined in this chapter, and it is shown that their levels are altered in some ectomycorrhizal fungi due to copper stress but not in others. The second chapter uses transmission electron microscopy to demonstrate that copper is bound to the hyphae of ectomycorrhizal fungi grown on solid media, but the location of the binding varies between fungal species. In vitro copper tolerances of a number of ectomycorrhizal species are compared in this chapter and differences in tolerance are evident between species and between different isolates of the same species. In the third chapter, four ectomycorrhizal fungi and one nonmycorrhizal fungus are evaluated for their ability to improve the growth of Japanese Red Pine under conditions of copper stress. Improvement of pine seedling growth is not correlated with in vitro copper tolerance of the fungus, but is related to the degree of compatibility between host and fungus. Despite differences in in vitro tolerance between three isolates of the same species, there are no differences in the effect of the isolates on the tree host under conditions of copper stress. Ectomycorrhizal fungi were also inoculated in pairs on pine seedlings and the competitive abilities of the fungi are compared under stressed and nonstressed conditions. The fourth chapter discusses the results of inoculation of pine with a nonhost fungus which stimulates dichotomous branching of the root system. The compound responsible for the branching is demonstrated to be indole-3-acetic acid (IAA), a plant growth hormone. The final two chapters deal with endomycorrhizal fungi. In the first of the two, inoculation of onion with an endomycorrhizal fungus demonstrates that the fungus probably plays no direct role in the response of the plant to heavy metals, based on biomass production, nutrient uptake, and photosynthetic rate. The last chapter demonstrates that the vascular plants found on abandoned mines in Virginia and North Carolina are well colonized by endomycorrhizal fungi; thus, an absence of these fungi is not a reason for the limited natural recolonization of the mine spoils.Ph. D.
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Gonçalves, Sara Isabel Bastos. "Zinc and copper impacts on freshwater diatoms: physiological, biochemical and metabolomic response of Tabellaria flocculosa." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/18812.
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Mestrado em Biologia Molecular e CelularOs ecossistemas dulçaquícolas encontram-se sob a ameaça constante de pressões antropogénicas, nomeadamente contaminação por metais. As diatomáceas são utilizadas como indicadores de qualidade da água, contudo a influência de micronutrientes, como zinco (Zn) e cobre (Cu), e os seus possíveis impactes são pouco compreendidos. Os objetivos deste estudo passam por elucidar o nível de tolerância, os alvos e repostas celulares para contradizer a toxicidade dos metais Zn e Cu em diatomáceas de água doce, expondo Tabellaria flocculosa (TFLO), isolada de um local contaminado, a 30, 500 e 1000 μg Zn/L e 0,3, 6 e 10 μg Cu/L. Diferentes abordagens bioquímicas, fisiológicas e metabolómicas foram utilizadas. Concentrações de Zn e Cu que ocorrem em ambientes contaminados tem efeitos tóxicos nesta espécie. O Cu, este é tóxico para TFLO a concentrações comuns no ambiente que não são considerados contaminados (0.3 μg Cu/L) e a sua toxicidade aumenta com a concentração. TFLO mostrou ainda ter estratégias distintas para sobreviver à exposição a diferentes níveis de stress impostos por Zn e Cu. TFLO sobrevive a elevadas concentrações intracelulares de Zn e Cu pelo aumento das enzimas antioxidantes (SOD, CAT) e recorrendo a compostos antioxidantes de baixo peso molecular (GSH). Estes mecanismos são suportados pela elevada produção de energia (atividade ETS e ainda no caso do cobre, açucares e lípidos). Às concentrações de 1000 μg Zn/L e 6 e 10 μg Cu/L, todos estes processos metabólicos mostraram ser especialmente aumentados em acréscimo aos processos de imobilização extracelular. O aumento da imobilização extracelular (EPS e frustulinas) parece ser uma estratégia comum de combate à toxicidade do Cu. Desta forma as células procuram restringir e mitigar o stress oxidativo gerado pelo aumento das concentrações intracelulares de Zn e Cu. Contudo, estes mecanismos não foram suficientes para proteger as células de danos em membranas e proteínas, incluindo do aumento do numero de valvas com teratologias a elevadas concentrações de Zn (500 e 1000 μg Zn/L) e em todas as concentrações de Cu. Mais ainda, uma diminuição nos compostos como a sacarose e especialmente o lumicromo deveriam ser estudados futuramente como marcadores específicos da toxicidade do Zn. No caso do Cu, a diminuição do composto hidroxilamina e de ácidos gordos (FA) insaturados e o aumento dos FA saturados, 2-palmitoilglicerol, glicerol e compostos diterpenos assim como o conteúdo em clorofila c devem ser testados como marcadores específicos de exposição ao Cu. Esta informação pode suportar o melhor entendimento do modo de ação de Zn e Cu a predição da resposta da comunidade de diatomáceas de água doce em diferentes ambientes contaminados com Cu e Zn, incluindo ambientes altamente contaminados, como na exploração mineira, pode ainda ajudar no desenvolvimento de novos índices para contaminação por metais, tendo em conta a existência de espécies tolerantes e ajudando políticas de avaliação de risco ambiental.
Freshwater ecosystems are under threatening anthropogenic pressures worldwide, namely metals. Diatoms are used as water quality indicators, but the impacts of micronutrients such as zinc (Zn) and copper (Cu) on diatoms are poorly understood. Our study aimed to elucidate the tolerance level, the cellular targets and the responses to counteract Zn and Cu toxicity of freshwater diatoms by exposing Tabellaria flocculosa (TFLO), isolated from a contaminated stream, to 30, 500 and 1000 μg Zn/L and 0.3, 6 and 10 μg Cu/L. Biochemical, physiological and metabolomic approaches were used. It was demonstrated that Zn and Cu are toxic to TFLO at concentrations occurring in contaminated environments. Cu was already toxic to TFLO at concentrations common in non-contaminated environments (0.3 μg Cu/L), and toxicity increased with Cu concentration. Distinct strategies to cope with Zn and Cu were shown. TFLO cells cope with intracellular high Zn and Cu concentrations by increasing antioxidant enzymes (SOD, CAT) and using low molecular weight antioxidants (GSH). These mechanisms are fuelled by a high energy production (ETS activity, and in Cu exposure sugars and lipids oxidation). At the highest Zn concentration (1000 μg/L) and 6 and 10 μg Cu/L, these metabolic processes were specially enhanced in addition to extracellular immobilization (EPS, frustulins), in an attempt to restrain the oxidative stress generated by high intracellular Zn and Cu concentrations. However, these mechanisms were not able to fully protect cells and damage in membranes and proteins occurred, including the increase of teratological valves at high Zn concentrations (500 and 1000 μg Zn/L) and Cu exposure from low to high concentrations (0.3 to 10 μg Cu/L). Additionally, the response of certain compounds was especially promising as potential markers for metals stress. For example, the decrease in sucrose and especially lumichrome should be tested as new specific markers of Zn toxicity. Additionally, the decrease of hydroxylamine and unsaturated fatty acids (FA) and the increase of saturated FA, 2-palmitoylglycerol, glycerol and diterpenoid compounds and chlorophyll c should be tested as new specific markers of Cu toxicity in future studies. This information supports the better understanding of Zn and Cu mode of action and prediction of diatom response in different Zn or Cu contamination levels, including highly impacted environments, such as mining scenarios, and may help develop new indices, taking into account species’ tolerance strategies and assist in environmental risk assessment policies.
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Azzouzi, Asma. "L’homéostasie du cuivre chez la protéobactérie Rubrivivax gelatinosus." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA112078/document.
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L’adaptabilité des cellules aux changements environnementaux repose sur leur capacité à mettre en place des complexes enzymatiques qui régulent leurs différentes voies métaboliques. Ces complexes protéiques nécessitent des cofacteurs (hème et métaux) comme le cuivre. En effet, le cuivre est un oligoélément essentiel pour la survie des organismes vivants, il est notamment présent dans des complexes tels que la cytochrome c oxydase de la chaine respiratoire aérobie. Cependant, l’excès de Cu est aussi néfaste pour la cellule. Il génère du stress oxydatif responsable de divers dommages cellulaires. Il est donc nécessaire aux cellules de contrôler la prise en charge du cuivre afin d’assurer la régulation de sa concentration intracellulaire. Dans ce travail, nous avons étudié le système de tolérance au cuivre chez Rubrivivax gelatinosus. J’ai identifié plusieurs gènes impliqués dans ce système, un de ces gènes code pour un transporteur de cuivre CopA (homologue aux ATP7A/ATP7B de l’homme). Les autres gènes codent pour : un régulateur de ce système sensible aux variations du cuivre CopR et deux protéines solubles (CopI et CopJ) fortement induites dans des conditions d’excès de cuivre. J’ai également montré que malgré l’homologie de séquence entre les pompes à cuivre CopA et CtpA (identifiées auparavant au laboratoire) ; elles remplissent deux rôles physiologiques différents dans la cellule. CopA est vitale pour la tolérance au Cu alors que CtpA a un rôle dans l’insertion du Cu au sein des cuproprotéines. Par ailleurs, j’ai montré qu’en absence de CopA, la toxicité du cuivre est plus importante dans les conditions de croissance en microaérobie ou en photosynthèse anaérobe. En effet, l’excès du Cu affecte la voie de biosynthèse des porphyrines (hèmes et bactériochlorophylles) reflété par l’accumulation d’un intermédiaire de cette voie, la coproporphyrine III. Cette accumulation résulte de l’effet de l’excès du cuivre sur la coproporphyrine III oxydase (HemN) probablement en déstabilisant son cluster [4Fe-4S]. Ces résultats sont soutenus par le fait que l’effet toxique du cuivre sur les tétrapyrolles n’est pas observé à forte aération en raison de la substitution de HemN par une enzyme à centre bimétalliques HemF. Cette dernière tolère et utilise l’oxygène, notre étude suggère qu’elle tolère aussi le cuivre. Les résultats obtenus sur les cibles du cuivre en anaérobie au cours de cette étude ainsi que celle publiés par Macomber (Macomber and Imlay 2009), suggèrent que les métaux ont pu éventuellement jouer un rôle dans l’émergence des enzymes à clusters bimétalliques lors du passage à une atmosphère oxygénique permettant une adaptation à l’augmentation de la toxicité de certains métaux. L’ensemble des résultats obtenus à partir de l’analyse des différents mutants au cours de cette étude sur la tolérance au cuivre chez R. gelatinosus me permet de proposer un modèle d’efflux du cuivre différent de celui d’E. coli. Je propose d’attribuer un rôle crucial aux protéines solubles CopI et CopJ qui fixeraient probablement le cuivre grâce aux nombreux résidus histidines et méthionines. Leurs rôles seraient soit de séquestrer le cuivre au niveau du périplasme, soit de l’oxyder ou encore de l’excréter vers un système d’efflux de la membrane externe afin de le chasser de la celluleThe ability of Rubrivivax to adapt to its environment (aerobic versus anaerobic) relay on its ability to assemble different complexes involved respiration or photosynthesis pathways. These complexes require cofactors such as heme or chlorophylls, and metals such as magnesium, iron and copper. In particular, copper (Cu) is an essential trace element required for the assembly and the activity of the cytochrome c oxidase in the aerobic respiratory chain. Excess Cu however, is toxic and can originate in various cellular damages. In the absence of a tight control of copper entrance in the cells, bacteria have evolved different efflux systems to control copper concentration within the cytoplasm and the membrane. Very few data are available on the copper homeostasis systems in photosynthetic bacteria. We therefore studied the copper homeostasis system in Rubrivivax gelatinosus to understand how these microorganisms can deal with excess copper. In this work, I have identified several genes involved in copper tolerance. Central to this system, the P1B-type Cu⁺-ATPase CopA plays a major role in copper tolerance and translocates copper from the cytoplasm to the periplasm. The outlet of copper in the periplasm varies depending on the species. Cu can be sequestrated, oxidized or released outside the cells. Here I describe the identification CopI, a periplasmic protein present in many proteobacteria including Pseudomonas and Cupriavidus and show its requirement for copper tolerance in Rubrivivax under both aerobic and anaerobic conditions. Expression of both CopA and CopI is induced under excess copper and is regulated by CopR, a MerR regulator sensitive to changes in copper concentration. Rubrivivax genome encodes two P1B-type Cu⁺-ATPases, CopA and CtpA. My work confirmed that despite the sequence homology between these copper ATPases, they fulifill two different physiological roles in the cell. CopA is vital for tolerance to Cu while CtpA has a role in the insertion of Cu within cuproproteines. Furthermore, I showed that excess copper in the copA⁻ null mutant resulted in a substantial decrease of the cytochrome c oxidase and the photosystem under microaerobic and anaerobic conditions together with the extrusion of coproporphyrin III. Analyses of the mutant indicated that copper targeted the tetrapyrrole biosynthesis pathway at the level of the coproporphyrinogen III oxidase HemN and thereby affects the heme and chlorophyll containing complexes, the oxidase and the photosystem. These results, as well as published work by Macomber (Macomber and Imlay 2009) suggest that Cu target the 4Fe-4S clusters and that this metal may have played a role in the emergence of bimetallic enzymes to replace 4Fe-4S clusters during the appearance of oxygen in the atmosphere.Analyses of CopI expression and the copI⁻ null mutant, demonstrate that CopI is required for copper tolerance, and in the absence of an E. coli Cus-like copper efflux system in R. gelatinosus, my results strongly suggest that CopI is the major copper handling protein within the membrane. Altogether, my results allowed me to draw a comprehensive picture of the copper tolerance system within the purple photosynthetic bacterium Rubrivivax gelatinosus and probably other proteobacteria that possess a homologue of copI gene
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Lambert, Anne-Sophie. "Influence de la température sur la réponse de communautés microbiennes périphytiques à une exposition métallique : cas du cuivre." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10235/document.
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De nombreux cours d’eau sont exposés aux polluants métalliques. Dans ces milieux, les communautés microbiennes structurées sous forme de biofilms peuvent être affectées par ces polluants. Or, une des questions prégnantes en écotoxicologie concerne l’évaluation des impacts toxiques dans le cas de combinaisons multi-stress. Dans ce contexte, l’objectif principal de ce travail était d’évaluer l’influence de la température sur la réponse du biofilm à une exposition au cuivre (Cu). Cet objectif a été abordé en considérant l’influence de la température, d’une part, sur la sensibilité des communautés au Cu, et d’autre part, sur l’exposition des communautés de biofilm à ce métal. Ces travaux ont été réalisés en microcosmes de laboratoires et menés sur des biofilms prélevés à différentes saisons. Nous avons tout d’abord optimisé les protocoles utilisés pour mesurer i) les niveaux de tolérance des communautés au Cu et ii) la bioaccumulation du Cu dans les biofilms. Les résultats des expérimentations ont révélé que la température pouvait moduler l’impact structural et fonctionnel du Cu sur les biofilms, du fait notamment d’une diminution de la bioaccumulation aux plus fortes températures. En complément, les résultats ont mis en évidence l’influence d’une hausse de la température sur la sensibilité des communautés au Cu, qu’elles aient été préalablement exposées (PICT) ou non à ce métal. Cependant, nous avons observé que les effets de la température sur la réponse du biofilm étaient variables d’une étude à l’autre, suggérant une influence de nombreux paramètres, tels que la composition initiale de la communauté, les niveaux de stress appliqués (température et Cu), le compartiment microbien étudié (phototrophe ou hétérotrophe), ou les fonctions considéréesMany streams are impacted by metallic pollution. In such ecosystems, microbial communities, which grow preferentially as biofilms, can be affected by these pollutants. However, there is a need to better assess the impact of toxic substances under multi-stress interactions. In this context, the main aim of this work was to evaluate the effect of temperature on biofilm response to copper (Cu) exposure. Accordingly, we addressed the influence of temperature on microbial community sensitivity to Cu on the one hand, and its influence on biofilm exposure to this metal on the other hand. Microcosm experiments were performed under laboratory conditions using biofilms sampled in winter or summer. This work allowed us to set up new methodological developments, which were applied to improve the protocols used to measure (i) Cu tolerance levels of biofilm microbial communities and (ii) Cu bioaccumulation in biofilms. Results showed that temperature could modulate structural and functional effects of Cu on biofilms, notably due to a decrease in Cu bioaccumulation at higher temperatures. In addition, our findings indicate that temperature increase could influence the sensitivity of microbial communities to Cu, whether they have been previously chronically exposed (PICT) or not to this metal. Nonetheless, temperature effects on biofilms varied among experiments, revealing that the influence of temperature on the effects of Cu on microbial biofilms depends on many parameters, such as the initial composition of communities, the level of the applied stresses (temperature and Cu), the microbial compartment studied (i.e. phototrophic or heterotrophic), or the measured functional parameters
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Орлова, М. В., and M. V. Orlova. "Поиск и отбор генотипов растений рода Alissum на устойчивость к никелю, меди и цинку : магистерская диссертация." Master's thesis, б. и, 2020. http://hdl.handle.net/10995/94208.
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Цель работы – отобрать генотипы растений рода Alissum устойчивые к повышенным концентрациям ионов никеля, меди и цинка. Были выбраны 3 вида растений из рода Alissum sp. семейства Brassicaceae, а именно: A. litvinovii, A. tortuosum, A. оbovatum. В ходе эксперимента из семян были получены растения, которых подвергли микроклональному размножению. Растения культивированы на средах, содержащих Cu2+, Ni2+ или Zn2+ в концентрации 50 – 100 – 200 µМ/л. В течение 6 недель проводили морфологические измерения и описания. После эксперимента было определено содержание ионов Ni2+ в сырой биомассе с использованием реактива Чугаева. Отобраны генотипы растений, устойчивые к избытку тяжелых металлов в среде и способные к активному росту в условиях стресса, генотип A. tortuosum № 6 превысил контроль в 2,6 раза, достигнув размера 46,7 мм (200 µМ Zn2+). Растения показали аккумулирующие способности в отношении ионов никеля в условиях in vitro. Генотип № 5 A. оbovatum аккумулирует 1,59 мг/г Ni2+ сырой массы, что в 8 раз превысило контроль (200 µМ Ni2+). Данное исследование подтверждает перспективность использования растений из рода Alyssum для фиторемедиации. Выпускная квалификационная работа магистра – 61 стр., 21 рис., 5табл., 81 литературных источников, из которых русскоязычных 50, англоязычных 31.The purpose of the work is to select plant genotypes of the genus Alissum that are resistant to increased concentrations of nickel, copper and zinc ions. Three species of plants from the genus Alissum sp. Brassicaceae family, namely: A. litvinovii, A. tortuosum, A. obovatum. During the experiment, plants were obtained from seeds, which were subjected to microclonal propagation. Plants were cultivated on media containing Cu2+, Ni2+ or Zn2 + at a concentration of 50 – 100 – 200 µM/L. Morphological measurements and descriptions were performed over 6 weeks. After the experiment, the content of Ni2+ ions in the raw biomass was determined using the Chugaev reagent. Plant genotypes that are resistant to an excess of heavy metals in the medium and capable of active growth under stress were selected; A. tortuosum genotype № 6 exceeded the control by 2.6 times and reached a size of 46.7 mm (200 µM Zn2+). Plants showed an accumulating ability with respect to nickel ions in vitro. Genotype № 5 A. obovatum accumulates 1.59 mg/g Ni2+ wet weight, which is 8 times higher than the control (200 µM Ni2+). This study confirms the promise of using plants from the genus Alyssum for phytoremediation. The final qualifying work of the master is 61 pages, 21 figures, 5 tables, 81 literary sources, of which 50 are Russian-speaking and 31 are English-speaking.
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Torres, Urquidy Oscar Hernando. "Copper Resistant Bacteria Better Tolerate Commercially Available Antimicrobial Treatments Based in Silver and Silver-Copper Ions." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/202734.
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In the current study, the antibacterial efficacy of zeolites containing silver or copper ions or a combination of these metals was assessed against several diverse copper resistant (CuR) and copper sensitive (CuS) strains of clinically relevant bacterial species. CuR Pseudomonas putida was significantly reduced in comparison to the unamended zeolite control. Unexpectedly, a CuS P. putida strain with no reported metal resistance appeared to be more resistant to the zeolite containing either Ag or Ag/Cu than the CuR strain. Contrary to expectations, after three and six hours of exposure, the CuS Escherichia coli displayed significantly more resistance to the Ag/Cu and Cu treatments than the reportedly CuR E. coli. All three reportedly CuR strains of Salmonella enterica exhibited resistance to Cu and Ag, as well as to the combination of the two metals after three and six hours of exposure. The reductions observed after 24 hours for all three CuR strains with Cu alone were still statistically significant compared to that of the CuS S. enterica strain. In addition, two of the CuR strains were more resistant to silver after 24 hours of exposure, suggesting a shared resistance mechanism such a copper efflux pump that also removes silver ions from the cell. Both the CuR and CuS strains of E. faecium were highly resistant to all of the treatments. In general, after comparison of all the resistances with all the treatments, E. faecium was the most resistant species, P. putida was the least resistant species, and the Salmonella strains were more resistant than E. coli in most cases.
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Geiss, Loren V. "Freeze tolerance and cryoprotection of erythrocytes from Dryophytes chrysoscelis." University of Dayton / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=dayton1533074960877566.
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Tambosi, Reem. "Stress and toxicity of metal in photosynthetic bacteria : multi-scale study of the effects and the targets of metal ions and nanoparticles Silver and Copper Acute Effects on Membrane Proteins and Impact on Photosynthetic and Respiratory Complexes in Bacteria Silver Effect on Bacterial Cell Membrane Structure Investigated by Atomic Force and Scanning Electron Microscopes Cadmium and Copper Cross-tolerance. Cu+ alleviates Cd2+ toxicity, and both cations target heme and chlorophyll biosynthesis pathway in Rubrivivax gelatinosus Additive effects of metal excess and superoxide, a highly toxic mixture in bacteria." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL070.
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L’usage intensif des métaux et des ions métalliques dans l'industrie et l'agriculture représente une menace sérieuse pour l'environnement et pour tous les êtres vivants en raison de la toxicité aiguë de ces ions. Cependant, cela peut aussi être un outil prometteur. En effet, les ions comme les nanoparticules d'argent sont très utilisés dans diverses applications médicales, industrielles et sanitaires. L'effet antimicrobien de ces nanoparticules est en partie lié aux ions Ag⁺ libérés et à leur capacité à interagir avec les membranes bactériennes. L'objectif de ce projet est d'étudier l'interaction entre un objet biologique (les bactéries) et des objets physiques (métaux), pour comprendre l'impact des métaux sous différentes formes (ions, nanoparticules et nanostructures) sur les cellules bactériennes en utilisant différentes approches: de physiologie, biochimie, génétique et de biologie cellulaire. Nous avons utilisé comme modèles biologiques, principalement la bactérie photosynthétique pourpre Rubrivivax (R.) gelatinosus, mais aussi Escherichia coli; et pour les objets physiques, nous avons utilisé l'argent comme métal principal mais aussi d'autres métaux (cuivre, cadmium et nickel) à titre de comparaison. Les principaux objectifs de ce travail sont: 1- d'étudier l'impact et les mécanismes de toxicité de ces ions métalliques / NPs sur les métabolismes bactériens respiratoire et photosynthétique. 2- Identifier des gènes bactériens impliqués dans la réponse à un excès d'ions Ag⁺. 3- Etudier l'internalisation et l'interaction des ions métalliques et des NP au sein des membranes biologiques. Ainsi, nous avons pu identifier, à la fois in vitro et in vivo, des cibles spécifiques d'ions Ag⁺ et Cu²⁺ dans la membrane des bactéries. Cela inclut des complexes impliqués dans la photosynthèse, mais également des complexes de la chaine respiratoire. Il a été démontré que les ions Ag⁺ et Cu²⁺ ciblent spécifiquement une bactériochlorophylle exposée au solvant dans les antennes de collecte de lumière du photosystème de la bactérie. Ceci présente également, à notre connaissance, la première preuve directe de dommages induits par des ions Ag⁺ sur les protéines membranaires impliquées dans ces métabolismes. Par ailleurs, nous avons également réalisé une étude comparative par microscopie (AFM/ MEB) de l'effet de l'Ag⁺ en solution ou des Ag-NPs synthétisés dans notre laboratoire, sur la morphologie des cellules bactériennesThe extensive use of metal ions in industry and agriculture represents a serious threat to the environment and to all living being because of the acute toxicity of these ions. However, it can also be a promising tool, silver ions and nanoparticles are some of the most widely used metals in various industrial and health applications. The antimicrobial effect of these nanoparticles is in part related to the released Ag⁺ ions and their ability to interact with bacterial membranes. The goal of this project is to study the interaction between biological subject (the bacteria) and physical objects (metals), and more specifically to understand the impact of metals in different forms (ions, nanoparticles and nanostructures) on the growth of the bacterial cells using different approaches : physiology, biochemistry, genetics and cell biology. We used as biological models, principally the purple photosynthetic bacterium Rubrivivax (R.) gelatinosus, but also Escherichia coli; and for physical objects, we used silver as main metal but also other metals (copper, cadmium and nickel) for comparison. The main objectives are: 1- to study the impact and the mechanisms of toxicity of these metallic ions/NPs on the bacterial respiratory and photosynthesis metabolisms. 2- To identify the bacterial genes involved in response to excess silver. 3- To study the internalization and interaction of metals ions and NPs within biological membranes. The results showed that we were able to identify, both in vitro and in vivo, specific targets of Ag⁺ and Cu²⁺ ions within the membrane of bacteria. This include complexes involved in photosynthesis, but also complexes involved in respiration. Ag⁺ and Cu²⁺ were shown to specificaly target a solvent exposed bacteriochlorophyll in the light harvesting antennae of the photosystem. This also presents, in our knowledge, the first direct evidence of silver ions damages to membrane proteins involved in these metabolisms. We also carried out a microscopy (AFM/ SEM) comparative study of the effect of Ag⁺ ions or Ag-NPs synthesized in our laboratory, on the bacterial cell morphology
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Watt, Grace R. "Impact of Device Parametric Tolerances on Current Sharing Behavior of a SiC Half-Bridge Power Module." Thesis, Virginia Tech, 2020. http://hdl.handle.net/10919/96559.
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This paper describes the design, fabrication, and testing of a 1.2 kV, 6.5 mΩ, half-bridge, SiC MOSFET power module to evaluate the impact of parametric device tolerances on electrical and thermal performance. Paralleling power devices increases current handling capability for the same bus voltage. However, inherent parametric differences among dies leads to unbalanced current sharing causing overstress and overheating. In this design, a symmetrical DBC layout is utilized to balance parasitic inductances in the current pathways of paralleled dies to isolate the impact of parametric tolerances. In addition, the paper investigates the benefits of flexible PCB in place of wire bonds for the gate loop interconnection to reduce and minimize the gate loop inductance. The balanced modules have dies with similar threshold voltages while the unbalanced modules have dies with unbalanced threshold voltages to force unbalanced current sharing. The modules were placed into a clamped inductive DPT and a continuous, boost converter. Rogowski coils looped under the wire bonds of the bottom switch dies to observe current behavior. Four modules performed continuously for least 10 minutes at 200 V, 37.6 A input, at 30 kHz with 50% duty cycle. The modules could not perform for multiple minutes at 250 V with 47.7 A (23 A/die). The energy loss differential for a ~17% difference in threshold voltage ranged from 4.52% (~10 µJ) to -30.9% (~30 µJ). The energy loss differential for a ~0.5% difference in V_th ranged from -2.26% (~8 µJ) to 5.66% (~10 µJ). The loss differential was dependent on whether current unbalance due to on-state resistance compensated current unbalance due to threshold voltage. While device parametric tolerances are inherent, if the higher threshold voltage devices can be paired with devices that have higher on-state resistance, the overall loss differential may perform similarly to well-matched dies. Lastly, the most consistently performing unbalanced module with 17.7% difference in V_th had 119.9 µJ more energy loss and was 22.2°C hotter during continuous testing than the most consistently performing balanced module with 0.6% difference inV_th.Master of Science
This paper describes the design, construction, and testing of advanced power devices for use in electric vehicles. Power devices are necessary to supply electricity to different parts of the vehicle; for example, energy is stored in a battery as direct current (DC) power, but the motor requires alternating current (AC) power. Therefore, power electronics can alter the energy to be delivered as DC or AC. In order to carry more power, multiple devices can be used together just as 10 people can carry more weight than 1 person. However, because the devices are not perfect, there can be slight differences in the performance of one device to another. One device may have to carry more current than another device which could cause failure earlier than intended. In this research project, multiple power devices were placed into a package, or "module." In a control module, the devices were selected with similar properties to one another. In an experimental module, the devices were selected with properties very different from one another. It was determined that the when the devices were 17.7% difference, there was 119.9 µJ more energy loss and it was 22.2°C hotter than when the difference was only 0.6%. However, the severity of the difference was dependent on how multiple device characteristics interacted with one another. It may be possible to compensate some of the impact of device differences in one characteristic with opposing differences in another device characteristic.
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Privett, Sean. "Some physiochemical factors associated with filamentous freshwater algal growths in the Breede river, and the derivation of a rapid test for screening their copper tolerances." Thesis, University of Cape Town, 1994. http://hdl.handle.net/11427/25866.
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Aelst, Sébastien van. "Etude fonctionnelle des gènes plasmidiques de résistance au cuivre de Cupriavidus metallidurans: aspects physiologique, biochimique et écologique." Doctoral thesis, Universite Libre de Bruxelles, 2008. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210529.
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Cupriavidus metallidurans CH34 est la bactérie Gram négative considérée comme organisme-modèle pour l’étude de la résistance aux métaux lourds. Notre travail a porté sur sa résistance au cuivre, codée par les gènes cop du plasmide pMOL30. Ces gènes, responsables des différentes étapes de la résistance (compartimentation des systèmes d’efflux entre périplasme et cytoplasme, modification de valence, et d’autres fonctions totalement inconnues) ont suscité notre intérêt.On distingue dans l’îlot cop des gènes codant pour des fonctions de résistance proprement dite (essentiellement par détoxication active du cytoplasme et du périplasme). En effet, les mutants de copSRABCD, copF, et dans une moindre mesure copJ et copE deviennent sensibles. Les phénotypes des mutants divergent toutefois suivant que la mutation soit sur un cosmide qui ne porte que l’îlot (pMOL1024) ou dans son plasmide d’origine (pMOL30). Un second groupe de mutants (copVTMK, copG, copL, copQ) se distingue par un phénotype plus résistant ou identique à la souche parente, sauf autour de la CMI. Ces gènes interviendraient donc à la CMI pour assurer la résistance la plus élevée et le maintien d'un état viable latent.
La présence de l’îlot cop permet de contenir le taux d’oxygène radicalaire qui reste à un taux basal lorsque les cellules sont adaptées au cuivre environnent. Après un choc de Cu (ou stress aigu), l’îlot cop répond de façon « explosive » au stress, en consommant l’énergie du potentiel membranaire et en augmentant fortement l’activité de la chaîne respiratoire.
La résistance au cuivre est inductible, mais de façon différenciée pour la souche sauvage (CH34) et celle qui ne porte qu l’îlot cop (AE1744) :la CMI de CH34 triple après adaptation au cuivre, alors que celle d’AE1744 est inchangée. Après un choc de Cu, la résistance au cuivre est plus fortement induite pour AE1744 que pour CH34. Ces observations suggèrent que l’îlot cop ait été sélectionné pour sa capacité à répondre à un stress aigu puis intégré dans un ensemble de gènes plus vaste qui répond à des impératifs de stress chronique.
L’analyse biochimique de CopI, une petite protéine bleue à cuivre, montre qu’elle porte un site analogue à celui des oxydases multicuivre. Son rôle pourrait dès lors être celui d’une réductase multicuivre. La protéine CopK lie de façon très spécifique le Cu(I) et il semble que la liaison du cuivre modifie sa structure. L’analyse écologique a montré que des homologues de copK pourraient être présents dans l’ADN extrait de la terre de biotopes chargés en cuivre, et dans les souches cuprorésistantes qu’on y trouve.
La contribution majeure de cette thèse est de montrer que l’effet d’un stress métallique ne se résume pas à deux états physiologiques « mort ou vif ». Il y a lieu de considérer des états transitoires (choc de Cu, adaptation au métal, survie autour de la CMI, persistance) où interviennent des gènes spécifiques dans un ou plusieurs états donnés. Les résultats biochimiques et physiologiques ne nous éclairent pas encore assez sur les interconversions Cu(I)/Cu(II) ni sur les flux de cations notamment vers l'espace extracellulaire. Cette thèse ouvre des perspectives sur des mécanismes (protection à la CMI, phénotype persistant) assurant la survie des bactéries ou leur potentiel de recolonisation lors d'une diminution de la pression toxique :les gènes copT, copV, copK, copM, copB, copG, copL et copQ semblent impliqués dans ces fonctions.
Doctorat en Sciences agronomiques et ingénierie biologique
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Faucon, Michel-Pierre. "Ecologie et biologie de la conservation des métallophytes: le cas de Crepidorhopalon perenniset C. tenuis (Scrophulariaceae) des sols cupro-cobaltifères du Katanga." Doctoral thesis, Universite Libre de Bruxelles, 2009. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/210267.
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La province du Katanga est connue pour ses affleurements naturels de roches riches en cuivre et en cobalt. Plus d’une centaine de « collines de cuivre », isolées géographiquement et écologiquement possèdent une flore très originale qui comprend plus de 600 espèces dont 32 endémiques. Les métallophytes du Katanga sont des plantes fascinantes aux points de vue écologique, biogéographique, physiologique et évolutif. Elles restent très peu étudiées et leur conservation est très problématique compte tenu de l’extension des activités minières. L’objectif général de la thèse est de contribuer à une meilleure connaissance des métallophytes du Katanga, dans une perspective explicite de conservation de leur biodiversité.Dans ce contexte, la thèse représente une des toutes premières approches de la biologie et l’écologie des populations de deux métallophytes apparentées. L’objectif est de préciser la distribution et la niche des deux taxons, et la variation de leur capacité d’accumuler et de tolérer les métaux lourds. Les résultats sont utilisés pour discuter la valeur conservatoire des cuprophytes du Katanga, leur vulnérabilité et envisager des stratégies de conservation.
Nous avons montré que les sols des affleurements de roche cuprocobaltifère se distinguer édaphiquement d’autres types de sols métallifères dans le monde. La révision phytogéographique et taxonomique a permis de valider 32 taxons endémiques et de mettre en évidence 23 couples de cuprophytes étroitement apparentés. La proportion élevée de couples de cuprophytes apparentés nous laisse suggérer que des événements de spéciation auraient existés au sein même des habitats cuprifères. Un isolement écologique pourrait s’exercer au niveau des zones les plus riches en cuivre. Ces couples d’espèces cuprophytes constitueraient un outil de recherche idéal pour l’étude de la spéciation.
Les propriétés hyperaccumulatrices de certaines espèces ont été exagérées, sans doute par suite de problèmes méthodologiques. L’accumulation du Cu ne se produit que dans des conditions édaphiques particulières, très difficiles à obtenir en culture. Cependant, nous avons montré que C. tenuis et C. perennis étaient hautement tolérantes au cuivre. Chez ces deux espèces, le cuivre provoquait une augmentation des performances de certaines populations métallicoles en milieux stérile. Ces populations posséderaient des besoins élevés en cuivre. Dans ce cas, la cuprophilie apparente de l’espèce in natura pourrait être expliquée par un coût de la tolérance au cuivre. Cette cuprophilie semble être un phénomène rare chez les espèces tolérantes au cuivre et confère ainsi à C. perennis et C. tenuis une valeur biologique remarquable.
Actuellement, parmi les 32 taxons endémiques stricts des habitats cuprifères du Katanga, 6% sont éteints (EX), 73% sont en danger critique d’extinction (CR), 9% sont en danger d’extinction (EN) et 3% sont vulnérables (VU). De plus, 18 taxons présentant une affinité particulièrement élevée pour les sols cuprifères, sans y être totalement confinés (fréquence sur Cu > 75%) sont réellement menacés d’extinction (22 % EN et 78% VU). Néanmoins, 18 % de ces endémiques menacées sont capables de coloniser les habitats métallifères anthropogènes. Comme C. perennis, certaines endémiques se développaient uniquement sur des sols perturbés par l’activité minière. C. tenuis possède aussi son optimum écologique sur les sols anthropogènes cuprifères. Cela indique que certaines espèces de la flore du cuivre du Katanga considérées en danger critique d’extinction sont en fait moins menacées parce qu’elles sont en mesure de se développer sur les habitats anthropogènes métallifères. L’exploitation minière est toujours destructive mais les habitats anthropogènes récents semblent avoir une valeur de conservation pour certains métallophytes rares. Une partie des déchets de l’exploitation minière et les sols contaminés pourraient être gérés de manière à fournir des habitats artificiels pour la colonisation ou la conservation ex situ de ces espèces. Dans la discussion, nous examinons les stratégies de conservation de ces espèces.
Doctorat en Sciences
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Bell, Francesca Y. "Copper tolerance of Listeria monocytogenes strain DRDC8." Thesis, 2010. http://hdl.handle.net/2440/65482.
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Listeria monocytogenes is one of the most important food-borne pathogens due to the severity of the disease it can cause. While the virulence factors required for effective colonisation and infection of mammalian hosts have been well described, other genes may modulate disease persistence. For L. monocytogenes strain DRDC8, the ctpA gene encodes a copper transporting P-type ATPase that apparently maintains intra-cellular copper ion
homeostasis (Francis & Thomas, 1997a) and is also required for persistent infection of the liver and spleens of mice (Francis & Thomas, 1997b). However, the distribution of this gene is apparently limited to non-clinically derived environmental L. monocytogenes isolates (Bell, 2002). This may be explained by carriage of ctpA on plasmid DNA (Bell, 2002). Based on predictions of function and proximity to the ctpA gene (pCT0020), ORFs pCT0017, pCT0018, pCT0019 and ctpA were identified as a putative a cop-like operon involved in copper ion transport in L. monocytogenes (Bell, 2002). Southern hybridisation analysis was used to confirm that the ctpA gene is carried on plasmid pCT100 in strain DRDC8. In addition, evidence to suggest that ctpA was encoded by bacteriophage DNA was not obtained. Furthermore, sequence analysis of DNA flanking ctpA identified ORFs that encode polypeptide sequences similar to proteins involved in plasmid replication and other plasmid-associated functions. Mating experiments provided evidence to show that plasmid pCT100 is not conjugative. This suggested that lateral transfer of this plasmid between cohabitating organisms may be limited. Sequence analysis of a 37.279 kbp region of plasmid pCT100 from L. monocytogenes strain DRDC8 (GenBank Accession U15554) showed this plasmid had regions of gene content and organisation similar to that of other characterised Listeria plasmids, particularly plasmid pLI100 from L. innocua CLIP11262 and plasmid pLM80 from L. monocytogenes strain 4b H7858. Gene’s common to these plasmids included those implicated in plasmid DNA replication, DNA transposition/insertion and heavy metal (cadmium) transport. Sequence analysis of plasmid pCT100 also identified regions of DNA absent from other Listeria sequences. For example, a DNA region encoding a series of polypeptide sequences similar to chromosomally-encoded proteins involved in copper transport in other Gram-positive bacteria was identified. The ORFs encoded by this region (pCT0017, pCT0018, pCT0019 and pCT0020 (ctpA), pCT0023, pCT0024, pCT0025, pCT0026, pCT0027) represent a novel cluster of genes implicated in copper homeostasis/tolerance that had not been previously described for other Listeria spp. PCR analysis was used to show that carriage of this copper gene cluster may be restricted to only some Australian ctpA positive L. monocytogenes isolates, typically of dairy and poultry origin. In addition to these plasmid-encoded ORFs, PCR and sequence analysis identified a chromosomal ORF (cutR) also implicated in copper homeostasis/tolerance for strain DRDC8. cutR encodes a polypeptide similar to chromosomally-encoded coppertranslocating P-type ATPases from other Listeria species. The role of ORFs cutR, pCT0017, pCT0018 and pCT0019 in copper tolerance was assessed by comparison of the ability of wild type parent strain DRDC8 and variants containing independent mutations (pCT0017::erm, pCT0018::erm, pCT0019::erm or cutR::erm) to tolerate copper ion stress. The impact of loss of these genes (as a result of curing strain DRDC8 and cutR::erm derivatives of plasmid pCT100) on copper tolerance by DRDC8 was also examined. Minimal inhibitory concentration (MIC) and growth experiments showed that inactivation of cutR, pCT0018 or pCT0019, or removal of plasmid-encoded genes by curing DRDC8 of plasmid DNA, had a significant effect on copper tolerance. In addition, loss of plasmid DNA combined with disruption of cutR was shown to render cells completely incapable of growth in high levels of copper (14 mM CuSO4). This data indicated that pCT0018, pCT0019 and cutR are involved in copper tolerance of L. monocytogenes strain DRDC8. MIC experiments also provided evidence to show that ORFs cutR and pCT0018 may play an additional role in tolerance to cadmium. Interestingly, a L. monocytogenes mutant carrying an erm insertion within pCT0017 could not be constructed. However, evidence that showed that ORF pCT0017 encodes a CopY-like negative repressor protein directly implicated this ORF in copper tolerance. DNA gel shift experiments were used to show that pCT0017 protein binds to
two ‘cop box-like’ nucleotide sequences located upstream of the pCT0017 translation start site. Binding occurs in a copper-dependant manner that is consistent with published models of CopY-like protein function. Thus pCT0017 protein may regulate expression of ORFs pCT0017, pCT0018, pCT0019 and ctpA in a copper responsive manner. This is consistent with the view that these ORFs form a cop-like operon involved in copper homeostasis. In conclusion, L. monocytogenes strain DRDC8 displayed an exceptional tolerance to high concentrations of copper ions. The data obtained suggested that both chromosomal and plasmid-encoded genes are involved in copper homeostasis/tolerance of DRDC8. This particular strain may have acquired multiple genes involved in copper tolerance from a cohabitating Gram-positive bacterium in response to exposure to high levels of copper within the environment. Given that strain DRDC8 is an Australian dairy isolate, these genes may provide a selective advantage for survival of other L. monocytogenes strains in associated environments.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2010
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Louro, Filipe José Afonso Sabino de Sousa. "Heritability and inheritance of tolerance to copper in Daphnia magna." Master's thesis, 2013. http://hdl.handle.net/10316/24615.
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Dissertação de mestrado em Ecologia apresentada ao Departamento Ciências da Vida da Faculdade de Ciências e Tecnolofgia da Universidade de CoimbraGenetic erosion is a case of microevolution where genetic information is lost through the loss of genotypes. Although there are many factors that may contribute to the loss of genotypes, contaminants originating from human activities such as mining and agriculture are among the most concerning. Acid mine drainage (AMD) is a product of mining activity that is a very acidic effluent with high concentrations of various metals, contamination which may lead a population to genetic erosion. In Daphnia sp., tolerance to copper was found to be correlated with tolerance to AMD and can be used to study AMD driven genetic erosion. To better understand it two specific objectives were delineated with the present work, to calculate the heritability and inheritance of the tolerance to copper in Daphnia magna. Ephippia of D. magna were obtained from the following two different sources: (1) a commercial source producing ephippia from uni-clonal mass cultures and (2) a natural pond where ephippia were collected once in time. Heritability of the tolerance to copper was studied, based on time to death toxicity tests on over 200 clones from two different sources, where values of 71 and 82% were calculated for clones from source M and B, respectively. Relative spread was also calculated for each clone and compared with the tolerance of the respective clones, showing that more extreme clones (the most tolerant and the most sensitive) have smaller variabilities. To assess inheritance, sexual reproduction was attempted by stimulating ephippia production within a group of 24 selected clones, 12 clones from each source, being the six most sensitive and the six most tolerant to copper. However, iv ephippia production was not successfully achieved. Therefore it was not possible to study the inheritance of tolerance to copper in D. magna. Overall, the wide distribution of tolerances, the lower variability in extreme clones, and the high heritability show that tolerance to copper can easily be involved in microevolution episodes. Although not all objectives proposed were achieved, this work still represents new information and one significant contribution to the study of the microevolution of tolerance in Daphnia magna.
A erosão genética é um caso de microevolução em que a informação genética é perdida através da perda de genótipos. Apesar de haverem muitos fatores que podem contribuir para a perda de genótipos, contaminantes oriundos de atividades humanas como a indústria mineira e a agricultura estão entre os mais preocupantes. A drenagem ácida de minas (AMD) é um produto da atividade mineira que é um efluente acídico com altas concentrações de vários metais, contaminação esta que pode levar à erosão genética de populações. Em Daphnia sp., foi descoberto que a tolerância ao cobre está correlacionada com a tolerância à AMD e pode ser utilizada para estudar a erosão genética provocada pela AMD. Para melhor perceber dois objetivos específicos foram delineados no presente trabalho, calcular a heritabilidade e a herança da tolerância ao cobre em Daphnia magna. Ephippia de D. magna foram obtidos de duas fontes diferentes: (1) uma fonte comercial que produz ephippia apartir de culturas de massa uni-clonais e (2) um lago natural onde ephippia foram colhidas de uma única vez. A heritabilidade da tolerância ao cobre foi estudada, baseada em testes de toxicidade de tempo até à morte em mais de 200 clones de duas fontes diferentes, onde foram obtidos valores de 71 e 82% de heritabilidade para os clones oriundos da fonte M e B, respetivamente. O relative spread foi também calculado para cada clone e comparado com a tolerância do respetivo clone, mostrando que os clones mais extremos (os mais tolerantes e os mais sensíveis) têm variabilidades menores. vi Para avaliar a herança, a indução da reprodução sexuada foi tentada através da estimulação da produção de ephippia num grupo de 24 clones selecionados, 12 clones de cada fonte, sendo os 6 mais sensíveis e os 6 mais tolerantes ao cobre. No entanto, não tenha sido conseguida qualquer produção de ephippia. Assim, não foi possível estudar a herança da tolerância ao cobre em D. magna. Em suma, a larga distribuição de tolerâncias, as variabilidades baixas em clones extremos e a alta heritabilidade mostram que a tolerância ao cobre pode facilmente ser envolvida em episódios de microevolução. Apesar de em todos os objetivos propostos terem sido alcançados, este trabalho representa nova informação e uma contribuição significante para o estudo da microevolução da tolerância em Daphnia magna.
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Inglis, Colleen. "The effect of copper on kairomone-mediated responses by wild Daphnia pulicaria clones from along a copper gradient." Thesis, 2009. http://hdl.handle.net/1974/5372.
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Kairomones are infochemicals that benefit a heterospecific receiver. Chaoborus spp. release a kairomone from their gut when feeding on Daphnia spp. Daphnia respond to kairomone by shifting life history parameters or producing neonates with induced morphological defenses, increasing their chance of survival. When laboratory-cultured Daphnia are exposed to environmentally-relevant metal concentrations, a kairomone response is not induced, increasing predation vulnerability.
Currently Daphnia live in metal-contaminated lakes in Sudbury, ON. It is possible the extant population is tolerant of relatively high copper (Cu) concentrations and can still induce a kairomone response. In comparison, it is hypothesized clones found in lakes isolated from anthropogenic sources of metal-contamination would be less tolerant as they have not been exposed to high Cu concentrations.
The purpose of this study was to examine how multiple clones of D. pulicaria obtained from lakes along a Cu gradient respond to kairomone in the absence and presence of copper. Several different clones from Ontario lakes located in the Canadian Shield were exposed to environmentally-relevant Cu concentrations and Chaoborus kairomone. Neonates were collected and measured to assess predator-induced defenses.
Results indicate that kairomone-mediated responses and Cu-tolerance vary among D. pulicaria clones. Clones from the Sudbury area were able to induce a response to kairomone when exposed to Cu, indicating a Cu-tolerance. However, this was not true for all Sudbury clones. In contrast, most clones from clean lakes did not respond to kairomone when exposed to Cu; while some clones exhibited a Cu-tolerance. Clones that were not tolerant of Cu were affected at concentrations much lower than those predicted by the Biotic Ligand Model (BLM) that induce acute toxicity. Predictions generated by the BLM can be conservative making them useful when creating water quality criteria; however, my results indicate these predictions can also be under-protective. Chemosensory cues mediate vital life processes that are essential for survival. Populations may be devastated if metals interfere with chemosensory cues. Overall, the results of my study suggest that genetic variation is important for population establishment and maintenance, specifically when exposed to multiple stressors, and that directional selection may result in stress tolerance.Thesis (Master, Biology) -- Queen's University, 2009-12-23 09:50:46.152
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張嘉渝. "The effect of heat pretreatment on copper-tolerance of Chlamydomonas reinhardtii." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/84792089548384193460.
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Lee, Kuo-ting, and 李國婷. "The MAP Kinase Kinase MKK2 Pathway Affects Copper Tolerance in Arabidopsis." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/05461840430876509934.
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碩士國立成功大學
生命科學系碩博士班
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Eukaryotic mitogen-activated protein kinase (MAPK) signalling pathway acts downstream of receptors or sensors to transduce extracellular stimuli, including abiotic and biotic stresses, into adaptive, intracellular responses. A MAPK pathway minimally consists of a MAPKKK-MAPKK-MAPK module. The Arabidopsis thaliana MKK1 and MKK2 MAP kinase kinases have been implicated in biotic and abiotic stress responses, but the role of the MKK2 in metal signalling pathway is unknown. In this study, we evaluated the role of the MKK2 in regulating copper stress responses in Arabidopsis . Overexpressing of MKK2 enhanced Arabidopsis resistance to copper stress. When wild type, mkk2 and MKK2-EE4 seedlings were subjected to copper treatment for 7 days, the root elongation of MKK2-EE4 plants were significantly higher than the wild type and mkk2 mutant plants. Copper induced cell death was reduced and reactive oxygen species (ROS) production was accumulated in MKK2-EE4 plants compared with wild type and mkk2 mutant plants. In addition, 200 μM copper supply resulted in higher copper concentration in mkk2 mutant roots as compared to wild type, and the copper concentration in MKK2-EE4 roots was lower than the wild type plants. Taken together these results demonstrate that MKK2 signalling is involved in Arabidopsis copper tolerance and the uptake and storage of copper are also affected by this signalling pathway.
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Neves, Simão Pedro Branco da Silva. "Tolerance response of Solanum cheesmaniae and Solanum lycopersicum to copper: the role of the antioxidant system and copper transporters." Master's thesis, 2015. http://hdl.handle.net/1822/44420.
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Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em PlantasCopper (Cu) is an abundant heavy metal (HM) in the environment due to anthropogenic activities and natural sources. In excess, Cu easily become phytotoxic to most species, once this HM can interfere with essential nutrient uptake from rhizosphere. Therefore, Cu accumulation in plants is considered a threat to the environment and public health since it can easily enter food chains. Thus, the development of new strategies that ensure both crop yield and nutritional quality is of most importance. The present study aimed to compare the physiological and molecular responses of Solanum cheesmaniae and Solanum lycopersicum to Cu stress, in particular those related to antioxidant defense and to Cu transport and compartmentation. Some studies are already available regarding the responses of S. lycopersicum to Cu excess, so we wanted to address the hypothesis that S. cheesmaniae is more adapted to Cu stress than S. lycopersicum, since the former is more resistant to salt stress than the latter. Results showed that the germination rate of S. lycopersicum seeds were not affected by up to 2.5 mM Cu 7 days after treatment, while higher Cu concentrations negatively affected the growth of the seedlings and induced signs of necrosis and cell death. The application of 250 μM Cu negatively affected biometric parameters of both species cultivated in 0.25 x Hoagland solution for 28 days, but the biomass and length of both roots and shoots of S. lycopersicum plants were more affected by Cu stress. Cu treatment did not significantly alter chlorophylls and carotenoids levels in both species. Biomarkers of oxidative status (TBARS, H2O2 and O2.- levels) revealed a more pronounced imbalance in the redox homeostasis in shoots of S. lycopersicum plants treated with excess Cu than in S. cheesmaniae. Quantification of total phenols and flavonoids demonstrated an activation of the non-enzymatic antioxidant system in both species in response to excess Cu. Furthermore, the activity of key antioxidant enzymes clearly differed in both species in response to Cu, apart superoxide dismutase (SOD) whose activity increased in the roots of S. cheesmaniae and S. lycopersicum. Ascorbate peroxidase (APX) activity increased in S. cheesmaniae shoots and decreased in S. lycopersicum roots, while catalase (CAT) activity decreased in both species, except in S. cheesmaniae shoots. Cu quantification performed by flame-atomic absorption spectroscopy showed that both species preferentially accumulated Cu in the radicular system, though a great increase in the aerial parts of S. lycopersicum was also measured. The response to Cu excess of S. lycopersicum and S. cheesmaniae COPT4, COPT5 and COPT6, which are small integral membrane proteins that catalyze Cu uptake into cells, was also investigated by qPCR analysis. Results showed that SlCOPT5 expression increased by 30-fold in roots of S. lycopersicum cultivated with excess Cu, suggesting that it may have a pivotal role in Cu homeostasis in roots. Overall, results validated the hypothesis that S. cheesmaniae is more tolerant to excess Cu than S. lycopersicum and the new knowledge will facilitate future work on the development of breeding strategies.
O cobre (Cu) é um metal pesado (MP) abundante na natureza, estando os seus níveis a aumentar devido a atividades antropogénicas e a causas naturais. Em excesso, o Cu torna-se facilmente fitotóxico para a maior parte das espécies, uma vez que este MP é capaz de interferir com a assimilação e absorção de nutrientes essenciais a partir da rizosfera. Neste sentido, a acumulação de Cu pelas plantas é já considerada uma grande ameaça para o ambiente e saúde pública, dado que este elemento consegue entrar nas cadeias alimentares de uma forma relativamente rápida. Assim, torna-se crucial desenvolver novas estratégias que permitam garantir a produtividade e qualidade nutricional das culturas. O principal objetivo do presente trabalho consistiu no estudo comparativo de algumas respostas moleculares e fisiológicas de Solanum cheesmaniae e Solanum lycopersicum expostas a elevados níveis de Cu, em particular ao nível do sistema antioxidante e dos mecanismos de transporte e compartimentalização do Cu. Estudos anteriores evidenciaram algumas das respostas de S. lycopersicum ao excesso de Cu, pelo que no presente trabalho pretendemos validar a hipótese que S. cheesmaniae é uma espécie mais tolerante ao Cu que S. lycopersicum, uma vez que é mais resistente ao stress salino. A taxa de germinação de sementes de S. lycopersicum não foi afetada por concentrações crescentes de Cu até 2,5 mM avaliada após 7 dias de exposição, ainda que para as concentrações superiores o crescimento das plântulas tenha sido inibido e se tenham desenvolvido diversos sinais de necrose e morte celular. Os nossos resultados demonstraram que os parâmetros biométricos de ambas as espécies, irrigadas com uma solução nutritiva de 0.25 x Hoagland durante 28 dias, foram afetados pela aplicação de Cu a 250 μM, embora a produção de biomassa e o comprimento da raiz e da parte aérea de S. lycopersicum tenham sido claramente mais afetados. O tratamento com Cu não alterou de forma significativa o teor em clorofilas e carotenoides. Os marcadores de stress oxidativo (conteúdo de TBARS, H2O2 e O2.-) revelaram um maior desequilíbrio da homeostasia redox na parte aérea de plantas de S. lycopersicum tratadas com excesso de Cu, quando comparadas com S. cheesmaniae. A quantificação dos fenóis totais e dos flavonoides revelou a ativação da componente não enzimática do sistema antioxidante em ambas as espécies. Para além disso, a atividade das principais enzimas antioxidantes em reposta ao excesso de Cu diferiu bastante entre as duas espécies, com a exceção da superóxido dismutase (SOD), cuja atividade aumentou nas raízes de S. cheesmaniae e S. lycopersicum. A atividade da peroxidase do ascorbato (APX) aumentou na parte aérea de S. cheesmaniae e decresceu significativamente nas raízes de S. lycopersicum, enquanto a atividade da catalase (CAT) registou uma diminuição em ambas as espécies, à exceção da parte aérea de S. cheesmaniae. A quantificação do Cu nos tecidos vegetais, através espectroscopia de absorção atómica de chama, revelou que ambas as espécies acumularam mais Cu no sistema radicular, ainda que se tenha também verificado um aumento considerável na parte aérea de plantas de S. lycopersicum. Paralelamente foi avaliado por qPCR o envolvimento dos transportadores de Cu COPT4, COPT5 e COPT6 de S. lycopersicum e S. cheesmaniae em resposta ao excesso de Cu. Os resultados mostraram que a expressão relativa do gene SlCOPT5 aumentou cerca de 30 vezes em raízes de plantas de S. lycopersicum cultivadas na presença de excesso de Cu, sugerindo que este transportador tem um papel de destaque na homeostasia do Cu ao nível dos tecidos da raiz. No seu conjunto, os resultados permitiram validar a hipótese que S. cheesmaniae é uma espécie mais tolerante ao Cu do que S. lycopersicum e obter evidências experimentais importantes para o desenho de estratégias de melhoramento.
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Sousa, Filipa Rodrigues de. "Wrack as a strategy to increase Hordeum vulgare L. tolerance to copper contaminated soils." Master's thesis, 2020. https://hdl.handle.net/10216/132033.
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Sousa, Filipa Rodrigues de. "Wrack as a strategy to increase Hordeum vulgare L. tolerance to copper contaminated soils." Dissertação, 2020. https://hdl.handle.net/10216/132033.
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(9780491), Karina Griffin. "Copper tolerance in Pseudomonas syringae pv. tomato isolates from tomato crops in Eastern Australia." Thesis, 2019. https://figshare.com/articles/thesis/Copper_tolerance_in_Pseudomonas_syringae_pv_tomato_isolates_from_tomato_crops_in_Eastern_Australia/13409477.
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Pseudomonas syringae pv. tomato causing bacterial speck disease in tomatoes is a significant threat to commercial field tomato production in most growing regions of Australia and globally. Infection of crops with this pathogen can cause significant reductions in fruit quality and yields. There are limited pesticide control options available for bacterial diseases in tomato, with copper-based bactericides currently one of the few registered products globally. The state of Queensland (QLD) in Australia produces approximately 69% of Australia’s fresh market outdoor tomatoes estimated at a value of AUD$122 billion. P. syringae pv. tomato consistently threatens tomato production in QLD and other Eastern Australian states and many producers report copper products fail to adequately control disease progression. To date no studies have tested for copper tolerance in P. syringae pv. tomato in QLD Australia, despite reports of tolerance in many other countries. This study found that 100% of the P. syringae pv. tomato isolates collected were tolerant to copper and this tolerance was linked to the presence of cop genes in their genetic profiles. This is the first systematic study of copper tolerance prevalence in Eastern Australia, particularly QLD, and the first study analysing the genetic basis of copper tolerance in Australian P. syringae pv. tomato.
Published copper tolerance and copper efficacy studies on bacterial disease control report a wide range of response data, generated under varying field and laboratory conditions, making it difficult to draw strong conclusions from individual studies. Therefore, a systematic literature review was completed, investigating the prevalence of copper tolerance, the relative efficacy of copper for the control of disease and the identification of key emerging alternative products to copper for disease control. Results highlighted that copper tolerance is a global issue, which is affecting the usefulness of copper-based products for the control of bacterial diseases. A large range of alternative products for disease control were identified and the efficacy of eight key products were evaluated. However, there was a limited volume of published efficacy data available of for these alternative products, particularly for the control of disease caused by P. syringae pv. tomato.
The systematic literature review also identified inconsistencies with in vitro copper tolerance screening methodology for P. syringae pv. tomato in current literature, particularly in relation to the appropriate media to use, copper tolerance thresholds and inadequate reporting of media pH and/or pH adjustment steps. The effect of media and pH on copper tolerance results was therefore investigated, including the use of a pH buffering agent. Copper tolerance thresholds with different media were found to vary significantly and outside of a specific pH range, copper tolerance data was unreliable. A recommended methodology for copper tolerance screening was developed and published. This refined methodology was used to screen P. syringae pv. tomato isolates from a number of geographically distinct regions of QLD, New South Wales (NSW) and Victoria (VIC).
To date, no published studies are available on copper tolerance stability in P. syringae pv. tomato. Understanding the stability or biological fitness of copper tolerance in P. syringae pv. tomato can provide valuable insights into how copper-based disease control programs could be modified to mediate or even reduce the prevalence copper tolerance. Therefore, the stability of copper tolerance in study isolates was investigated through in vivo experiments. Findings suggested that copper tolerance may not be stable in all isolates when copper selection pressure is removed in vivo.
Despite a general consensus that plasmid cop genes are essential for copper tolerance in P. syringae pv. tomato, the gene and protein characterisation work undertaken to form these conclusions is solely based on isolates collected in America. Additionally, Australian P. syringae pv. tomato are yet to be genetically characterised. Polymerase Chain Reaction (PCR) assays and genomic analysis were used to explore the genetic basis of copper tolerance in Australian isolates, with a particular focus on the cop genes. Genetic analysis identified putative Cop-protein coding regions on a Cop operon and a CopA/B complex in Australian isolates. The analysis also suggested that the Cop operons may be located on either plasmid or chromosomal DNA, depending on the isolate studied. This study is the first detailed investigation of the genetic basis of tolerance in this species outside of America.
This study presents a range of novel findings which are of significance to both the scientific community and the agricultural industry. The presence of widespread copper tolerance has serious implications for commercial tomato producers. Bacterial disease management programs need to be revised to mediate resistance development and provide a more environmentally sustainable approach to crop production.
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Rousos, Penelope Ann. "The identification and physiological basis of tolerance of cabbage to copper toxicity in solution culture." 1986. http://catalog.hathitrust.org/api/volumes/oclc/14695929.html.
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Hii, Mei Mei. "Role of ORF pCT0018 for copper homeostasis in Listeria monocytogenes strain DRDC8." 2009. http://hdl.handle.net/2440/57906.
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Sequence analysis of part of a large plasmid carried by Australian environmental isolate of Listeria monocytogenes strain DRDC8 has lead to identification of an islet of genes that encode proteins similar to copper binding and transport genes found in other Gram positive bacteria. Comparative sequence analysis showed that there are at least four genes (pCT0017, pCT0018, pCT0019 and ctpA) on this islet predicted to be involved in copper homeostasis. One of these, ctpA, is predicted to encode a P-type ATPase with a function analogous to CopA, a copper transporting gene in Enterococcus hirae. ORF pCT0017 is likely to be a CopY-like regulatory protein which could control the expression of ctpA. ORF pCT0019 is predicted to be a Cu²⁺ binding protein. In addition, two genes located downstream of the ctpA are predicted to encode a two component regulatory system region. The predicted function of ORF pCT0018 is not clear. A related chromosomal gene (cutR) is predicted to also encode a copper transporting P-type ATPase. To investigate the role of the protein encoded by pCT0018, the growth behavior of L. monocytogenes strain DRDC8, other strains carrying mutations within pCT0018, pCT0019, cutR and ctpA, as well as strains cured of the large plasmid, were grown under conditions of copper stress and starvation. The growth data showed that with the exception of strain DRDC8 and other strains carrying ctpA, most were unable to grow at higher copper concentration (>15 mM CuSO₄) and suggested that the copper homeostasis genes located on the large plasmid are associated with tolerance to high levels of copper. Strain DSE955PL, which carries a cutR mutation and is cured of the large plasmid, was the most sensitive (<5 mM CuSO₄). This indicated that proteins encoded by plasmid genes work synergistically to confer tolerance to copper. Of most interest was the fact that a pCT0018 mutant was more sensitive (<15 mM CuSO₄) to high levels of copper than the wild type parent DRDC8 (<20 mM CuSO₄). This suggested that ORF pCT0018 was necessary for copper tolerance. To investigate the effects of insertion mutations in pCT0017, pCT0018 and ctpA on copper uptake and export, the levels of copper accumulated by these strains was assessed using atomic absorption spectroscopy. A significant difference in copper accumulation among the bacteria strains was observed when either LEB or BHI media were used to culture the bacteria. This data suggested that the growth medium chemicals influence the levels of copper accumulated by cells. However, the effect of these media on bacteria growth rates during copper stress was not significant. Atomic absorption analysis of intracellular copper accumulation suggested that DSE955PL and DSE955 (a chromosome mutant) were able to accumulate copper (80 - 110 mg.gˉ¹ dry weight of cells), whereas DRDC8 and strains carrying mutations in pCT0018, ctpA, and strains cured of the large plasmid, were less able to accumulate copper (30 - 70 mg.gˉ¹ dry weight of cells). This data suggested that cutR may encode a copper export system and that ctpA is involved in copper uptake. To investigate the gene expression profile for pCT0018 under elevated copper, reverse transcriptase PCR was used to detect transcripts encoding pCT0017, pCT0018, pCT0019 and pCT0020 from RNA extracted from L. monocytogenes strain DRDC8 following culture at elevated levels of copper. Although transcripts for each of the target genes were detected, transcription was not responsive to copper, nor was the pattern of transcription consistent with that expected for a single operon. To directly determine whether the protein encoded by the pCT0018 open reading frame was able to bind copper, this gene was cloned in pET15b in frame with an N-terminal Histag and expressed in E. coli. The expressed protein was purified with a Ni-NTA column and shown to contain copper. Attempts to directly show that protein pCT0018 could bind copper by Cu-IMAC were unable to unequivocally show that the protein was immobilized on the column. Purified protein was used to raise a polyclonal antiserum in rabbit and the antiserum was used for Western analysis to test expression of pCT0018 by wild type L. monocytogenes DRDC8 and specific gene mutants. Although the antiserum bound to purified protein, it was not possible to demonstrate binding to native pCT0018 in cell lysates prepared from L. monocytogenes DRDC8. SDS-PAGE of cytoplasmic and cell envelope proteins isolated from L. monocytogenes strains was used to identify proteins expressed in response to copper stress and starvation. No significant differences in protein profiles for cytoplasmic protein were observed. However, copper-immobilized metal affinity chromatography (Cu-IMAC) showed that expression of a number of copper binding proteins were differentially expressed by DRDC8 following growth in copper stress and starvation conditions. Three of these proteins were selected for amino sequence analysis by MALDI-TOFF MS. Two were confirmed to be L. monocytogenes non-heme iron-binding ferritin and a thiol peroxidase, both of which bind copper. The other protein was similar to an unknown protein from L. monocytogenes. Interestingly, no proteins directly implicated with the copper homeostasis islet were identified.http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374407
Thesis (M.Sc.) - University of Adelaide, School of Molecular and Biomedical Science, 2009
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BRESNEHAN, AMANDA. "FLEEING PREDATION: THE EFFECT OF COPPER EXPOSURE ON INDUCIBLE ANTIPREDATOR DEFENSES IN DAPHNIA PULICARIA CLONES FROM A HISTORICALLY METAL CONTAMINATED LAKE." Thesis, 2012. http://hdl.handle.net/1974/7045.
Full textAbstract:
Antipredator defenses are ubiquitous in aquatic ecosystems. In the widely studied Chaoborus-Daphnia predator-prey system, Daphnia elicit a variety of phenotypically plastic responses to Chaoborus including: morphological, life history, and behavioral responses. While these inducible defenses benefit the prey, metal contaminants have been shown to interfere with chemosensory functions, thereby inhibiting antipredator defenses and decreasing survivorship. However, in lakes with a history of metal contamination, such as Kelly Lake in Sudbury, Ontario, there is evidence to suggest that Daphnia may have adapted to high, ambient copper concentrations. Using seven distinct Daphnia clones that were hatched from resting eggs from Kelly Lake, we examined morphological and life history traits when clones were exposed to either a nominal concentration of copper, kairomone, or a combination of both. As expected, clones displayed a variety of inducible responses in both kairomone-control and kairomone-copper treatments, which was attributed to genetic variability. Expected trade-offs in life history traits were not always observed, suggesting that inducible traits may be coupled. Furthermore, in contradiction to life history theory, one clone exhibited both increased somatic growth and increased reproductive output, indicating that clones likely adopted adaptive strategies to stressors rather than elicitng trade-offs in traditional traits. Our results indicate that environmentally relevant copper concentrations do not inhibit the induction of antipredator defenses in Daphnia from Kelly Lake, and we conclude that Kelly Lake Daphnia have developed an adaptive tolerance to copper. Adaptation to copper contamination may have implications for resilience in natural Kelly Lake populations.Thesis (Master, Biology) -- Queen's University, 2012-04-03 19:33:59.137
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GIORNI, ELISABETTA. "Responses to environmental stresses in metallicolous and non metallicolous Silene paradoxa L. populations." Doctoral thesis, 2017. http://hdl.handle.net/2158/1080136.
Full textAbstract:
Due to the high heavy metal concentrations, metalliferous soils, both of
natural and anthropogenic origin, provide a very restrictive and selective
environment for plant life. Some plants, named “metallophytes”, have evolved
tolerance mechanisms that allow them to cope with toxic heavy metal
concentrations.
A good model for comparative studies of metal tolerance is the specie Silene
paradoxa L. (Caryophyllaceae), an excluder facultative metallophyte that
presents populations living in metalliferous and non-metalliferous soils.
This project aims to investigate responses to different environmental
stresses, both biotic and abiotic, in metallicolous and non-metallicolous
populations of this specie.
At the root level, we investigated how Cu affects the morphology and
composition of such organ in a non-metallicolous and a Cu-tolerant Silene
paradoxa population. We found out that in the Cu-tolerant population some of
the possible Cu exclusion strategies could be the mucilage and lignine production
and the reduction of subapical zone of the root. Passing to the shoot level, we
investigated the different effects of Cu excess on photosynthetic parameters in
the same two populations. The Cu-tolerant population showed a more efficient
photosynthetic activity in respect to the non-metallicolous population and a
different nature of photosynthetic limitations, being mostly stomatal, compared
to non-metallicolous mostly diffusional and biochemical limitations.
Metalliferous soils are characterized by low macronutrient concentrations
and availability, if compared to non-metalliferous ones, so that metallophytes
have to adapt even to nutrient scarcity. We compared the ability to use nutrients
in a non-metallicolous, a Cu-tolerant and a serpentine Ni-tolerant population
exposed to Cu and Ni excess. We found out that metallicolous populations have evolved mechanisms to adapt to Ca and Mg scarcity in a metal-dependent way,
optimizing nutrient utilization.
Responses to biotic stresses in excluder metallophytes are poorly studied.
These plants, unlike hyperaccumulators, cannot rely on the accumulation of the
metal to defend themselves from pathogens. Therefore, we quantified the
responses to pathogen attack in a metallicolous and Cu-tolerant Silene paradoxa
population exposed to Cu. As an elicitor of defense responses, we used a purified
fungal protein with PAMP activity, called cerato-platanin (CP). An overproduction
of phytoalexins was recorded for the Cu-tolerant population exposed
to Cu, suggesting that adaptation to metalliferous soils can affect plant response
to biotic stress. Remaining in the same outline and with the same experimental
setting, we further investigated whether the pathway leading to induction of
defense responses is dependent on ROS (reactive oxygen species) production or
not. Our results showed incompatibility between the ordinary ROS-mediated
response to fungal attack and the acquired mechanisms of preventing oxidative
stress in the Cu-tolerant population. Therefore, the same incompatibility of
hyperaccumulators in ROS-mediated biotic stress signals seemed to be exhibited
by this excluder metallophyte, but without the advantage of being able to rely on
the elemental defense for plant protection from natural enemies.
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Wright, Kevin Matthew. "Theoretical and Emperical Investigations into Adaptation." Diss., 2010. http://hdl.handle.net/10161/2475.
Full textAbstract:
The problem is two fold: how does natural selection operate on systems of interacting genes and how does natural selection operate in natural populations. To address the first problem, I have conducted a theoretical investigation into the evolution of control and the distribution of mutations in a simple system of interacting genes, a linear metabolic pathway. I found that control is distributed unevenly between enzymes, with upstream enzymes possessing the greatest control and accumulating the most beneficial mutations during adaptive evolution. To address the second problem, I investigated the evolution of copper tolerance in the common yellow monkeyflower, Mimulus guttatus. I genetically mapped a major locus controlling copper tolerance, Tol1. A Dobzhansky-Muller incompatibility was hypothesized to also be controlled by Tol1, however, we have demonstrated that it maps to another, tightly linked locus, Nec1. Finally, we investigated the parallel evolution of copper tolerance in multiple new discovered mine populations. We found that copper tolerance has evolved in parallel multiple times via at least two distinct physiological mechanisms. In four mine populations, there was a strong signal of selection at markers linked to Tol1, implying that copper tolerance has evolved via the same genetic mechanisms in these populations.
Dissertation
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Garton, Jeffrey Earl. "Evaluation of race and copper tolerant strains of Xanthomonas axonopodis pv. vesicatoria, causal agent of bacterial leaf spot of bell pepper in Georgia." 2009. http://purl.galileo.usg.edu/uga%5Fetd/garton%5Fjeffrey%5Fe%5F200912%5Fms.
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