Dissertations / Theses on the topic 'Rhizobium leguminosarum'
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Finnie, Christine. "Protein secretion by Rhizobium leguminosarum." Thesis, University of East Anglia, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361420.
Full textThorne, Stephen Howard. "Stationary phase survival of Rhizobium leguminosarum." Thesis, Imperial College London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265401.
Full textSeaman, Jonathan. "Signature-tagged mutagenesis in Rhizobium leguminosarum." Thesis, University of Reading, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.499374.
Full textHeinrich, Keith. "Ecological and molecular studies on rhizobial rhizopines." Title page, contents and summary only, 1999. http://web4.library.adelaide.edu.au/theses/09PH/09phh469.pdf.
Full textSimpkins, Sean A. "The DnaK molecular chaperone of Rhizobium leguminosarum." Thesis, University of East Anglia, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302035.
Full textLusby, John. "Hemin Utilization in Rhizobium leguminosarum ATCC 14479." Digital Commons @ East Tennessee State University, 2021. https://dc.etsu.edu/etd/3897.
Full textMcKay, Ian. "Carbon metabolism in Rhizobium leguminosarum MNF 3841." Thesis, McKay, Ian (1988) Carbon metabolism in Rhizobium leguminosarum MNF 3841. PhD thesis, Murdoch University, 1988. https://researchrepository.murdoch.edu.au/id/eprint/51790/.
Full textDelgutte, Dominique. "Introduction dans un sol agricole d'une souche de Rhizobium leguminosarum biovar viciae marquée génétiquement : étude de sa survie, de sa multiplication, de sa dissémination et du transfert de gènes à d'autres bactéries." Dijon, 1991. http://www.theses.fr/1991DIJOS027.
Full textMiller, Lance Delano. "Characterization of the Chemotaxis System of the Endosymbiotic Bacterium Rhizobium leguminosarum." Diss., Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/19707.
Full textGray, Kathryn Margaret. "Regulation of oxidative stress responses of rhizobium leguminosarum." Thesis, Imperial College London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408404.
Full textLuca, Nicola de. "The regulation of iron acquisition in Rhizobium leguminosarum." Thesis, University of East Anglia, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267474.
Full textKrehenbrink, Martin. "Protein secretion in Rhizobium leguminosarum biovar viciae 3841." Thesis, University of East Anglia, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432434.
Full textHawkins, Fiona K. L. "Studies on the nifA gene of Rhizobium leguminosarum." Thesis, University of East Anglia, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.329054.
Full textWilkinson, Adam. "N-acyl-homoserine lactone signalling in Rhizobium leguminosarum." Thesis, University of East Anglia, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267721.
Full textWhite, James. "Amino acid transport and metalobism by Rhizobium leguminosarum." Thesis, University of Reading, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.440075.
Full textFry, Judith. "myo-Inositol utilisation by Rhizobium leguminosarum biovar viciae." Thesis, University of Reading, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326187.
Full textHood, Graham. "Physiological response of Rhizobium leguminosarum during bacteroid development." Thesis, University of East Anglia, 2013. https://ueaeprints.uea.ac.uk/48693/.
Full textMazurier, Sylvie. "Diversité de populations naturelles nodulantes de Rhizobium leguminosarum." Lyon 1, 1989. http://www.theses.fr/1989LYO10067.
Full textHill, Brian D. "Characterization of TonB in Rhizobium leguminosarum ATCC 14479." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etd/2379.
Full textCarson, Kerry C. A. "Iron nutrition of Rhizobium leguminosarum biovar viciae MNF710." Thesis, Carson, Kerry C.A. (1993) Iron nutrition of Rhizobium leguminosarum biovar viciae MNF710. PhD thesis, Murdoch University, 1993. https://researchrepository.murdoch.edu.au/id/eprint/51953/.
Full textWong, Cheryl. "Genetic and physiological studies on the entry of aromatic substrates into cells of Rhizobium leguminosarum biovar viciae and Rhizobium leguminosarum biovar trifolii." Thesis, Wong, Cheryl (1993) Genetic and physiological studies on the entry of aromatic substrates into cells of Rhizobium leguminosarum biovar viciae and Rhizobium leguminosarum biovar trifolii. PhD thesis, Murdoch University, 1993. https://researchrepository.murdoch.edu.au/id/eprint/51971/.
Full textYost, Christopher K. "Characterization of Rhizobium leguminosarum genes homologous to chemotaxis chemoreceptors." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ31082.pdf.
Full textVenter, Alexandra Patricia. "Bacteriocin production in Rhizobium leguminosarum bv. viciae strain 306." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0016/MQ55252.pdf.
Full textMulley, Geraldine. "Nitrogen regulation of ABC transport systems in Rhizobium leguminosarum." Thesis, University of Reading, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.529974.
Full textMavridou, Annoula. "Genetic loci of Rhizobium leguminosarum affecting nod gene expression." Thesis, University of East Anglia, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316102.
Full textMarie, Corinne. "Roles of two Rhizobium leguminosarum glucosamine synthases in symbiosis." Thesis, University of East Anglia, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334333.
Full textStevens, James B. "The molecular genetics of iron uptake in rhizobium leguminosarum." Thesis, University of East Anglia, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323075.
Full textRossen, L. "Molecular analysis of the nodulation genes of Rhizobium leguminosarum." Thesis, University of East Anglia, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.370396.
Full textLodwig, Emma Mary. "Regulation of carbon and nitrogen metabolism in Rhizobium leguminosarum." Thesis, University of Reading, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368874.
Full textBahar, Masoud. "Molecular biology of rhizopine genes in Rhizobium leguminosarum br. viciae /." Title page, table of contents and summary only, 1997. http://web4.library.adelaide.edu.au/theses/09PH/09phb1508.pdf.
Full textLethbridge, Benjamin James. "The structure of trifolitoxin : A bacteriocin from Rhizobium leguminosarum biovar trifolii strain T24 /." Title page, table of contents and summary only, 1989. http://web4.library.adelaide.edu.au/theses/09PH/09phl647.pdf.
Full textBurn, Joanne Elizabeth. "Analysis of the regulatory nodulation gene nodD of rhizobium leguminosarum." Thesis, University of East Anglia, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.329095.
Full textMudd, E. A. "Transcription and translation from a symbiotic plasmid of Rhizobium leguminosarum." Thesis, University of East Anglia, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.355533.
Full textShearman, C. A. "Structure, function and regulation of nodulation genes of Rhizobium leguminosarum." Thesis, University of East Anglia, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376087.
Full textDiÌaz-Mireles, Edith. "Mur, a manganese-responsive, Fur-like regulator in Rhizobium leguminosarum." Thesis, University of East Anglia, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.423802.
Full textBarr, Michelle. "Development of an in vivo selection system for Rhizobium leguminosarum." Thesis, University of Reading, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424255.
Full textWalshaw, David L. "The general amino acid permease of Rhizobium leguminosarum biovar viciae." Thesis, University of Reading, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283765.
Full textPalmer, Katharine M. "Rhizobium leguminosarum biovar viciae populations from arable and grass lands." Thesis, University of York, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323685.
Full textMiller, Deborah Ann. "The characterisation of bacteriocin production and resistance in Rhizobium leguminosarum." Thesis, University of Bristol, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393082.
Full textPereira, Sofia Isabel Almeida. "Estudo da tolerância de Rhizobium leguminosarum bv. viciae ao cádmio." Master's thesis, Universidade de Aveiro, 2004. http://hdl.handle.net/10773/16726.
Full textA contaminação do meio ambiente por metais pesados é muito comum, e pode resultar de uma variedade de fontes antropogénicas, entre as quais indústrias metalúrgicas, descargas de efluentes, fundições, minas e aplicação pesticidas e fertilizantes contaminados. A quantidade de metais acumulada no solo, resultante da poluição ambiental, depende da escala de emissão, do transporte do metal a partir da fonte e também da retenção do metal no solo. Os microrganismos do solo são sensíveis a exposições prolongadas a metais pesados. Assim, neste trabalho pretendeu-se isolar Rhizobium de diferentes locais que sofreram contaminação por metais pesados durante longos anos, por forma a determinar a sua tolerância ao cádmio em meio artificial suplementado com diferentes concentrações de CdCl2. Além disso, determinaram-se alguns parâmetros físico-químicos que influenciam a biodisponibilidade dos metais e as concentrações destes elementos nos solos. A variabilidade de tolerâncias verificada entre os isolados de Rhizobium sugere que por detrás da maquinaria metabólica básica, existem outros mecanismos que permitem os isolados mostrarem diferentes respostas ao stresse provocado pelo cádmio. Este facto levou-nos a aprofundar algumas características macromoleculares dos isolados com o objectivo de identificar mecanismos de tolerância e/ou características intrínsecas dos próprios isolados que lhes possam conferir essa tolerância ao metal. De acordo com os resultados obtidos podemos dizer que VE e AS foram os locais que apresentaram concentrações mais elevadas de metais, sendo por isso os mais contaminados. A concentração máxima permitida por lei para o Zn foi largamente ultrapassada em VE, e em AS a concentração verificada esteve muito próxima do limite máximo permitido. Os resultados obtidos evidenciaram que a tolerância dos isolados de Rhizobium foi diferente entre os isolados estudados. De acordo com a CMT os isolados podem ser divididos em 4 categorias: os sensíveis (0-125 μmol.l-1 CdCl2), os medianamente tolerantes (125 e 210 μmol.l-1 CdCl2), os tolerantes (250 e 500 μmol.l-1 CdCl2) e os extremamente tolerantes que toleraram concentrações ≥ 750 μmol.l-1 de cádmio. Os isolados de BTA foram os mais tolerantes uma vez que 46,7% destes toleraram concentrações entre 250 e 500 μmol.l-1 de cádmio, seguindo-se os isolados de AS e SB. No entanto, para concentrações ≥ 750 μmol.l-1, esta tendência apenas foi verificada para BTA. Os isolados de BTB são na sua maioria medianamente tolerantes (70%), enquanto que os isolados de MA são medianamente tolerantes e tolerantes, ao contrário dos isolados de MB que são todos sensíveis. Os perfis polipeptídicos dos isolados de Rhizobium foram bastante diferentes entre si, evidenciando uma grande variabilidade entre os isolados estudados. A presença de cádmio induziu o aumento/diminuição de alguns polipeptídeos, sugerindo uma adaptação das células às condições de stresse. Na sua maioria, as alterações provocadas pelo cádmio corresponderam a diminuições da expressão proteica, sugerindo que o metal afecta o metabolismo básico das células, inibindo passos metabólicos importantes para a sobrevivência e crescimento do rizóbio. vi Os isolados de Rhizobium estudados também apresentaram diferentes perfis plasmídicos. A maioria dos isolados tolerantes apresentaram 2 plasmídeos de pesos moleculares iguais a 485 e 415 kb, sugerindo que a tolerância destes isolados está relacionada com a presença de plasmídeos. Todos os isolados estudados apresentaram concentrações de cádmio intracelular significativamente inferiores ao cádmio adsorvido à parede. Nos isolados sensíveis, medianamente tolerantes e tolerantes a quantidade de cádmio adsorvida foi em muitos casos 100 vezes superior à verificada no interior da célula. Estes resultados sugerem a existência de mecanismos de exclusão ou sequestração extracelular do metal nos isolados medianamente tolerantes e tolerantes. Nos isolados extremamente tolerantes, a quantidade de cádmio adsorvida à parede foi em média 15 vezes superior à concentração de cádmio intracelular, isto significa que muito provavelmente existem mecanismos de sequestração intracelular. Em suma, podemos concluir que a resposta destas bactérias ao stresse provocado pelo cádmio é um fenómeno complexo, estando a tolerância de Rhizobium dependente de vários mecanismos que se desencadeiam paralelamente. Com efeito, parece evidente a existência de um mecanismo de tolerância comum a todos os isolados, que se caracteriza pela presença e/ou indução do aumento de LPS que contribuem para a sequestração extracelular dos metais. Contudo, apesar dos LPS conferirem um certo grau de tolerância, estes não são suficientes para suportar níveis mais elevados de stresse. Assim, a presença de outros mecanismos é essencial. De facto, nos isolados tolerantes a existência de 2 plasmídeos (485 e 415 kb) indica-nos um possível envolvimento destes na tolerância ao cádmio. Por outro lado, nos isolados extremamente tolerantes, podemos inferir que a tolerância se deve à presença de agentes intracelulares que impedem o metal de interferir com os processos metabólicos mais importantes. Os isolados tiveram diferentes origens e estiveram sujeitos a diferentes tipos de contaminação, no entanto estes factores não influenciaram os mecanismos de tolerância ao cádmio.
Environment heavy metals contamination is now widespread. Soils may become contaminated from a variety of anthropogenic sources such as smelters, mining, industry and application of metal-containing pesticides and fertilizers. The amounts of cadmium accumulated in soils from environmental pollution will depend on the scale of emission, the transport of the metal from the source to the site and the retention of the metal once it has reached the soil. The soil microorganisms are very sensitive to long exposures to moderate heavy metal concentrations. Therefore, the present work aimed to isolate Rhizobium from different locations, which have suffered heavy metal contamination since years ago, in order to determine cadmium tolerance in artificial media supplemented with different concentrations of CdCl2. Furthermore, we have determined some physic-chemical parameters that influence the availability of metals, and also their concentrations in soil. The heavy metal tolerance variability among Rhizobium isolates suggests that beyond basic metabolic machinery there also exist variations that allow isolates to display distinct tolerance responses to cadmium stress. This fact, leads us to investigate some macromolecular characteristics from isolates in order to identify tolerance mechanisms and/or intrinsically characteristics that permit isolates to enhance tolerance. In agreement with results we can say that VE and AS presented the highest concentrations of metals, and are therefore the most polluted soils. The maximum permissible concentration to zinc was largely overcome in VE, and in AS the registered concentration was near the limit. Our results showed different tolerances among Rhizobium isolates, according CMT, the isolates can be divided in 4 groups: sensitive (0-125 μmol.l-1 CdCl2); moderately tolerant (125 –210 μmol.l-1 CdCl2); tolerant (250-500 μmol.l-1 CdCl2); and extremely tolerant (≥ 750 μmol.l-1 CdCl2). BTA isolates were the most tolerant ones since 46,7 % of them tolerated concentrations between 250 e 500 μmol.l-1 of CdCl2, followed by AS and SB isolates. However, in concentrations up to 750 μmol.l-1 this tendency was only checked to BTA. The isolates from BTB are mainly moderately tolerant (70%), while MA isolates are moderately tolerant and tolerant, unlike MB isolates that are all sensitive. The electrophoretic patterns of whole-cell soluble proteins of Rhizobium isolates were very distinct, showing high variability between them. Cadmium induced increases/decreases of some polypeptides, which indicates an attempt of cells to adjust to the new adverse conditions. Most of the alterations corresponded to decreases in protein expression, suggesting a deleterious effect on basic cell metabolism, which imposed an inhibition of important metabolic pathways hence inhibiting survival and development of Rhizobium. The studied Rhizobium isolates also presented different plasmid profiles. Most tolerant isolates presented 2 plasmids with molecular weights of 485 and 415 kb, suggesting the possible involvement of these plasmids in cadmium tolerance. All isolates presented smaller quantities of cadmium inside the cell when compared with concentrations adsorbed to cell wall. viii In sensitive, moderately and tolerant isolates the quantity of metal adsorbed was, in many cases 100 times higher than inside cell. These results suggest the existence of exclusion mechanisms or extracelular sequestration of metal in moderately tolerant and tolerant isolates. In extremely tolerant isolates, the amount of cadmium adsorbed to cell wall was, on average, 15 times higher than cadmium absorbed, which probably means that exists intracellular sequestration mechanisms. In summary, we can conclude that the response of these bacteria to cadmium stress is a complex phenomenon, and the tolerance of Rhizobium is dependent of a variety of mechanisms, which unchained parallel. It seems that there is a common tolerance mechanism to all isolates, which is characterized by the presence and /or induction of LPS. These molecules contribute to extracelular sequestration of metals. However, even though LPS conferred a degree of tolerance, they are not sufficient to support higher stress. Thus, the presence of others mechanisms are essential. In fact, in tolerant isolates, smaller quantity of cadmium intracellular and the existence of 2 plasmids (485 and 415 kb), indicated the possible involvement of these plasmids in cadmium tolerance. On the other hand, in extremely tolerant isolates, we can assume tolerance is related with the presence of intracellular agents that prevent metal to interfere with the most metabolic processes. Even though isolates were originated from different locations and had been exposed to different kinds of heavy metal contamination the tolerance mechanisms were not influenced.
Hui, Kailin. "Metabolism and host specificity in the Rhizobium leguminosarum species complex." Thesis, University of York, 2014. http://etheses.whiterose.ac.uk/7017/.
Full textWorsley, Penelope S. "Sidero[p]hore synthesis by Rhizobium leguminosarum biovar viciae WSM710." Thesis, Worsley, Penelope S. (2000) Sidero[p]hore synthesis by Rhizobium leguminosarum biovar viciae WSM710. PhD thesis, Murdoch University, 2000. https://researchrepository.murdoch.edu.au/id/eprint/52061/.
Full textAusmees, Nora. "Structure and function of cellulose fibers and lectins in Rhizobium leguminosarum and regulation of their synthesis /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 2001. http://epsilon.slu.se/avh/2001/91-576-5811-0.pdf.
Full textAndam, Cheryl Marie Palacay. "Role of lateral gene transfer in the evolution of legume nodule symbionts." Diss., Online access via UMI:, 2007.
Find full textGould, Phillip Spencer. "Regulation and role of the three chaperonin operons of Rhizobium leguminosarum." Thesis, University of Birmingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273921.
Full textSindhu, Satyavir Singh. "Molecular analysis of lipopolysaccharide and membrane associated proteins in Rhizobium leguminosarum." Thesis, University of East Anglia, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.256988.
Full textLithgow, James Kennett. "Quorum-sensing in Rhizobium leguminosarum : the role of the cinRI locus." Thesis, University of East Anglia, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302200.
Full textReid, Colm J. "The regulation of the DCT system in Rhizobium leguminosarum biovar viciae." Thesis, University of Reading, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.284031.
Full textCorticeiro, Sofia Caçoilo. "O papel da glutationa na tolerância ao cádmio em Rhizobium leguminosarum." Master's thesis, Universidade de Aveiro, 2006. http://hdl.handle.net/10773/8957.
Full textO crescente aumento de metais pesados no solo, devido a poluiçâo ambiental, afecta directamente a comunidade microbiana bem como as relações simbióticas que alguns destes microrganismos estabelecem com leguminosas. Uma dos metais pesados mais prejudiciais e tóxicos é o cádmio. Devido à sua elevada toxicidade, este metal constitui uma grave ameaça, não só para as comunidades bacterianas presentes nos solos, mas também para os outros organismos, uma vez que a transferência de iões metálicos ao longo da cadeia alimentar e uma realidade e um problema sério de saúde pública. Sendo a glutationa um importante agente antioxidante e quelante de metais, este trabalho foi delineado de modo a perceber qual o papel deste tripéptido na tolerância ao cádmio em Rhizobium leguminosarum. Inicialmente alguns isolados de R. leguminosarum foram expostos a diferentes concentrações de cádmio e foi testada a influência da glutationa no seu crescimento. Dos isolados que apresentaram um aumento significativo de tolerância na presença desse tio1 (4 em 5), foram seleccionados dois com graus de tolerância distintos, E20-8 (tolerante) e NI-2 (sensível), para a realizaçâo do restante trabalho experimental. Posteriormente foi avaliado o nivel de stresse oxidativo dos dois isolados, através da determinação dos níveis de GSH e de GSSG intracelulares e das actividades de algumas das principais enzimas envolvidas no stresse oxidativo (SOD, CAT, GR e GPX). Os resultados demonstraram que a exposição ao cádmio induziu stresse oxidativo nas células, o que foi comprovado pelo aumento na concentração de GSSG e pela indução de actividade das enzimas estudadas. A adição de glutationa ao meio de crescimento teve um efeito protector, principalmente no isolado sensível. Os dados obtidos sugerem que o aumento de tolerância ao cádmio em R. leguminosarum não está relacionado com uma maior eficiência do mecanismo antioxidante per si, mas com níveis mais elevados de glutationa intracelular. Mostrou-se ainda que o fornecimento de glutationa ao isolado sensível aumentou a sua tolerância. Tendo já sido demonstrado que a capacidade de aumentar a síntese de glutationa estava relacionada com uma maior tolerância ao cádmio. Assim é plausível concluir que a glutationa desempenhe um papel fundamental na tolerância ao cádmio nesta espécie e que esse papel não se limite á sua actividade como antioxidante. Assim, foi estudado o papel da glutationa como agente quelante nesta espécie e, através da adaptação de um protocolo inicialmente desenvolvido para complexos de fitoquelatinas. As diferentes fracções de cádmio extraídas foram separadas: o cádmio fracamente ligado a parede foi removido por extracção aquosa em banho de ultrasons. O cádmio intracelular foi obtido por extracções sequenciais com tampão HEPES, de modo a manter a estabilidade de possíveis complexos. O cádmio ligado á membrana e a parede celular foi extraído com ácido. O cádmio intracelular foi separado por cromatografia de exclusão molecular e as fracções correspondentes aos picos de cadmio foram colhidas e analisadas por HPLC. Verificou-se que a percentagem de cadmio retido na parede celular era semelhante nos dois isolados, demonstrando a importância deste mecanismo, mas confirmando contudo que esta não pode justificar a diferença de tolerância entre os isolados. O isolado tolerante apresentou uma maior concentração de cadmio intracelular. A análise por HPLC revelou que a glutationa era o principal agente quelante de cádmio em Rhizobium, sendo responsável pela complexação de 75% do cádmio intracelular no isolado tolerante e por 28% no isolado sensivel. A adição de glutationa ao meio de crescimento aumentou a eficácia da complexação de cádmio, passando para 90% no isolado tolerante e para 53% no sensível. Os dados apresentados nesta tese contribuem certamente para a compreensão da tolerância aos metais pesados em bactérias. No entanto, os resultados deste trabalho não se cifram só em respostas, mas também em questões. A razão pela qual a adição de glutationa diminui o cádmio intracelular na estirpe tolerante e qual o destino dos complexos nas células são alguns dos pontos que terão de ser esclarecidos em trabalhos futuros.
The increase in the anthropogenic activities has introduced new types of stresses to soil bacteria communities as well as to symbiotic associations between microbes and legumes. Heavy metal contamination, particularly by cadmium, constitutes a problem. Because of its high toxicity, even at low concentrations, cadmium imposes a serious threat to organisms and food-chain transfer of metal ions has become a major public concern. Since glutathione is an important antioxidant and rnetal chelator. this study was undertaken to determine the role of this tripeptide in cadmium tolerance of Rhizobiurn legurninosarurn. Two strains expressing different degrees of tolerance to cadmium stress were used and the influente of the addition of extracelular GSH to the growth media was determined. Oxidative stress and ROS scavenging enzymes were evaluated, as well as changes in GSH and GSSG levels. Results confirmed that cadmium imposes oxidative stress in Rhizobiurn legurninosarurn, which was characterized by an increase in GSSG formation and an induction of SOD, GPX. GR and CAT activities. Addition of reduced glutathione to the growth media had a protective effect, particularly in the sensitive strain. These findings show that increased tolerance in Rhizobiurn legurninosarum is not related to a higher efficiency of the oxygen scavengers per se, but with higher levels of intracellular glutathione. Furthermore, we demonstrated that glutathione suply to the sensitive strain enhanced tolerance, concluding that glutathione plays a crucial role in cadmium tolerance in this species. In a previous report, it was demonstrated that glutathione played an important role in cadmium detoxification in Rhizobiurn cells. but the molecular role of this tripeptide remained to be elucidated. In this work, we report an efficient extraction of GSH-Cd complexes in two R. legurninosarum strains with different levels of tolerance to cadmium. through a modified protocol, originally developed for metal-phytochelatins complexes. The different sub-cellular Cadmium fractions were extracted: loosely bound cadmium was extracted with water, in an ultrasonic bath; intracellular cadmium was sequentially extracted with HEPES buffer; and wall-bound cadmium was extracted with acid. Intracellular cadmium was separated through size exclusion chromatography, and the amount of cadmium in collected fractions was analysed. Peptide peaks containing the higher cadmium concentrations were analysed by HPLC. In both strains, the same percentage of cadmium was retained in cell walls, hence demonstrating an effective avoidance mechanism, but that was not responsible for the obse~edto lerance differences, displayed by the two strains. Intracellular cadmium accumulation was higher in the tolerant strain and rnetal ions were mainly chelated by small-weight peptides. HPLC analysis revealed that glutathione was the main cadmium chelator in Rhizobirim. being responsible for sequestering 75% of intracellular cadmium in the tolerant strain. The sensitive strain presented a less effective cadmium complexation; only 28% of intracellular cadmium was sequestered by glutathione The presence of glutathione increased the efficiency of this mechanism: the tolerant strain was able to sequester 90% of the intracellular cadmium and the sensitive strain complexed 53%. Our findings add a novel and important aspect to the proposed role of glutathione in heavy metal coping for bacteria. These results can be useful in developing biotechnological strategies for cadmium bioremediation procedures and open novel perspectives for the irnprovement of metal tolerance in soil bacteria. However these findings have raised severa1 questions. The reason why glutathione supply decreases the intracellular cadmium in the tolerant strain and the fate of the complexes within the cell are some of the issues that remain to be elucidated.
Corticeiro, Sofia Caçoilo. "Study of the Cd complexation mechanism by glutathione in Rhizobium leguminosarum." Doctoral thesis, Universidade de Aveiro, 2012. http://hdl.handle.net/10773/10078.
Full textA associação simbiótica de plantas leguminosas com bactérias do género Rhizobium é o maior e mais eficiente contribuinte de azoto fixado biologicamente (Somasegaran e Hoben, 1994; Zahran, 1999). No entanto, o constante aumento da poluição em solos agrícolas, nomeadamente a contaminação por metais devido à aplicação de fertilizantes e de lamas, está a tornar-se um problema ambiental cada vez mais preocupante (Alloway, 1995a; Giller et al., 1998; Permina et al., 2006; Thorsen et al., 2009; Wani et al., 2008), influenciando de forma negativa a persistência destas bactérias nos solos agrícolas, assim como a sua eficácia de nodulação (Broos et al., 2005; Wani et al., 2008;. Zhengwei et al., 2005). Desta forma, o estudo dos mecanismos de tolerância de Rhizobium a metais tornou-se uma área de investigação de elevada importância. Com o trabalho apresentado nesta tese pretendeu-se perceber melhor a tolerância Rhizobium leguminosarum ao cádmio (Cd), dando particular atenção a um mecanismo de tolerância previamente descrito em R. leguminosarum (Lima et al., 2006): a complexação intracelular de Cd pelo tripéptido glutationa (GSH). Assim, o principal objectivo deste trabalho foi perceber melhor qual a importância deste mecanismo nos níveis de tolerância de rizóbio ao Cd. Como já tinha sido descrito em trabalhos anteriores (Figueira et al., 2005; Lima et al., 2006), foi possível verificar que a estirpe mais tolerante ao metal apresenta níveis mais elevados de Cd e GSH intracelulares. Demonstrou-se ainda que a tolerância ao Cd está dependente da maior eficiência no mecanismo de complexação observada na estirpe tolerante, logo durante as primeiras 12 h de crescimento. Gomes et al. (2002) verificou que a acumulação de complexos GSH-Cd no citoplasma inibe a entrada de metal na célula. Como neste trabalho se observou um aumento nos níveis de Cd intracelular na estirpe tolerante ao longo do tempo, surgiu a hipótese dos complexos serem excretados para o espaço periplasmático. Os elevados níveis de GSH e de Cd determinados no espaço periplasmático corroboraram esta hipótese. Neste trabalho demonstrou-se ainda que a eficácia do mecanismo de complexação, depende da actividade enzimática de uma isoforma específica de GST, que apresentou um elevado acréscimo de actividade na presença do metal. Desta forma, os resultados desta tese indicam que, a maior tolerância de R. leguminosarum ao Cd, depende da capacidade das estirpes para induzir a síntese de GSH na presença de Cd e, simultaneamente aumentar a actividade enzimática da GST específica, optimizando assim o mecanismo de complexação de Cd intracelular.
The symbiotic association of leguminous plants and rhizobia also has a crucial impact on the nitrogen cycle: estimates are that rhizobial symbioses, with a number greater than 100 important agronomical legumes, contribute nearly half to the annual quantity of nitrogen fixed biologically entering soil ecosystems (Somasegaran and Hoben, 1994; Zahran, 1999). Nevertheless, the permanent increased of metal pollution in agriculture soils, due to the current use of fertilizers and industrial and domestic sludge, is becoming one of the most troublesome environmental problems (Alloway, 1995a; Giller et al., 1998; Permina et al., 2006; Thorsen et al., 2009; Wani et al., 2008), has a negative impact in rhizobia persistence in agricultural soils and its ability to form nitrogen-fixing nodules (Broos et al., 2005; Wani et al., 2008; Zhengwei et al., 2005). So, the study of the tolerance mechanisms of soil bacteria to metals arises as a research area with great importance. In this thesis it was intended to draw attention to the tolerance to Cd of Rhizobium leguminosarum, a specific soil bacteria that establish symbiotic associations with legumes. The present study focused on a particular strategy that Rhizobium leguminosarum possess to tolerate Cd: the intracellular chelation of Cd by the tripetide GSH. Thus the elucidation of the role of GSH as a metal chelating agent in bacteria tolerance to Cd was considered to be the main aim of this work. Higher intracellular Cd and GSH levels were observed in the tolerant strain as it had already been demonstrated by others authors (Figueira et al., 2005; Lima et al., 2006). It was also demonstrated that Rhizobium leguminosarum tolerance to Cd was dependent on the higher efficiency of the chelation mechanism exhibited by the tolerant strain. This mechanism was activated at the beginning of the lag phase (12 h of growth). As the accumulation of GSH-Cd complexes may inhibit intracellular Cd uptake (Gomes et al., 2002), which was not observed in the tolerant strain, the GSHCd complexes were suggested to be transported into the periplasmic space. The results point out this hypothesis as the most viable as high levels of GSH and Cd were found in the periplasmic space but only a small percentage of GSH-Cd complexes were quantified. It was also established that the chelation mechanism occurred in the cytoplasm, and its efficiency appeared to be dependent on the enzymatic activity of a specific GST isoform. So, the ability of the tolerant strain to induce GSH synthesis under Cd exposure and, simultaneously, to increment the activity of a specific GST was point out as the main reasons behind the differences the tolerance to Cd observed between the two strains.