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Artigos de revistas sobre o assunto "Production de mutants de trypanosomes"
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Villafraz, Oriana, Marc Biran, Erika Pineda, Nicolas Plazolles, Edern Cahoreau, Rodolpho Ornitz Oliveira Souza, Magali Thonnus et al. "Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline". PLOS Pathogens 17, n.º 3 (1 de março de 2021): e1009204. http://dx.doi.org/10.1371/journal.ppat.1009204.
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Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1–2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.
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Olayinka-Adefemi, Folayemi, Chukwunonso Onyilagha, Nipun Jayachandran, Sen Hou, Ping Jia, Jude E. Uzonna e Aaron J. Marshall. "The Function of Phosphatidylinositol 3-Kinase delta (PI3Kδ) Enzyme in Protective Immunity to Trypanosoma congolense Infection in Mice: The Role of Regulatory B cells". Journal of Immunology 204, n.º 1_Supplement (1 de maio de 2020): 82.39. http://dx.doi.org/10.4049/jimmunol.204.supp.82.39.
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Abstract The delta isoform of PI 3-kinase (PI3Kδ) has important functions in B cell activation. Trypanosoma parasites utilize several mechanisms to exploit and evade host B cell and antibody responses critical for immunity. We sought to determine the impact of PI3Kδ in immunity to Trypanosoma congolense infection. We found that PI3KδD910A mutant mice show a surprisingly enhanced control of parasitemia in early infection (7–9 days) when compared to wild-type (WT) C57BL/6 mice. Drug treatment with Idelalisib to acutely inhibit PI3Kδ during infection in WT mice led to improved early control of parasitemia, consistent with results from genetically deficient mice. Both mutant mice and Idelalisib-treated mice showed a delay in polyclonal B cell activation and CD80/86 expression. Analysis of cytokine levels in the blood and peritoneal cavity showed higher IFNg and lower IL-10 levels in the drug-treated group at early time points, indicating a more pro-inflammatory environment. B1 cells were identified as the major IL-10 producing cells in the peritoneal cavity in early infection, and B1 cell production of IL-10 was significantly impaired by PI3Kδ-inhibitor treatment. We further observed increased Nitric oxide production in drug-treated mice, which correlated with increased parasite killing in early infection. Despite the improved early parasite control, there was a 100% mortality in PI3KδD910A mutants and 25% mortality in Idelalisib treated mice, presumably due to compromised generation of parasite-specific antibodies required to clear the first wave of blood infection. Our findings suggest that PI3Kδ inhibition impacts both regulatory B cell functions, affecting the early innate response, and generation of critical protective antibodies.
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Beneke, Tom, Ross Madden, Laura Makin, Jessica Valli, Jack Sunter e Eva Gluenz. "A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids". Royal Society Open Science 4, n.º 5 (maio de 2017): 170095. http://dx.doi.org/10.1098/rsos.170095.
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Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major , Leishmania mexicana and bloodstream form Trypanosoma brucei ; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens.
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Sass, Gabriele, Laura C. Miller Conrad, Terrence-Thang H. Nguyen e David A. Stevens. "The Pseudomonas aeruginosa product pyochelin interferes with Trypanosoma cruzi infection and multiplication in vitro". Transactions of The Royal Society of Tropical Medicine and Hygiene 114, n.º 7 (20 de março de 2020): 492–98. http://dx.doi.org/10.1093/trstmh/trz136.
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Abstract Background Bacteria are sources of numerous molecules used in treatment of infectious diseases. We investigated effects of molecules produced by 26 Pseudomonas aeruginosa strains against infection of mammalian cell cultures with Trypanosoma cruzi, the aetiological agent of Chagas disease. Methods Vero cells were infected with T. cruzi in the presence of wild-type P. aeruginosa supernatants or supernatants of mutants with defects in the production of various virulence, quorum sensing and iron acquisition factors. Quantification of T. cruzi infection (percentage of infected cells) and multiplication (number of amastigotes per infected cell) was performed and cell viability was determined. Results Wild-type P. aeruginosa products negatively affected T. cruzi infection and multiplication in a dose-dependent manner, without evident toxicity for mammalian cells. PvdD/pchE mutation (loss of the P. aeruginosa siderophores pyoverdine and pyochelin) had the greatest impact on anti–T. cruzi activity. Negative effects on T. cruzi infection by pure pyochelin, but not pyoverdine, or other P. aeruginosa exoproducts studied, were quantitatively similar to the effects of benznidazole, the current standard therapy against T. cruzi. Conclusions The P. aeruginosa product pyochelin showed promising activity against T. cruzi and might become a new lead molecule for therapy development.
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Vassella, Erik, Peter Bütikofer, Markus Engstler, Jennifer Jelk e Isabel Roditi. "Procyclin Null Mutants ofTrypanosoma bruceiExpress Free Glycosylphosphatidylinositols on Their Surface". Molecular Biology of the Cell 14, n.º 4 (abril de 2003): 1308–18. http://dx.doi.org/10.1091/mbc.e02-10-0694.
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Procyclins are abundant, glycosylphosphatidylinositol (GPI)-anchored proteins on the surface of procyclic (insect) form trypanosomes. To investigate whether trypanosomes are able to survive without a procyclin coat, all four procyclin genes were deleted sequentially. Bloodstream forms of the null mutant exhibited no detectable phenotype and were able to differentiate to procyclic forms. Initially, differentiated null mutant cells were barely able to grow, but after an adaptation period of 2 mo in culture they proliferated at the same rate as wild-type trypanosomes. Analysis of these culture-adapted null mutants revealed that they were covered by free GPIs. These were closely related to the mature procyclin anchor in structure and were expressed on the surface in numbers comparable with that of procyclin in wild-type cells. However, free GPIs were smaller than the procyclin anchor, indicative of a lower number of poly-N-acetyllactosamine repeats, and a proportion contained diacylphosphatidic acid. Free GPIs are also expressed by wild-type cells, although to a lesser extent. These have been overlooked in the past because they partition in a solvent fraction (chloroform/water/methanol) that is normally discarded when GPI-anchored proteins are purified.
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Lüscher, Alexandra, Pinar Önal, Anne-Marie Schweingruber e Pascal Mäser. "Adenosine Kinase of Trypanosoma brucei and Its Role in Susceptibility to Adenosine Antimetabolites". Antimicrobial Agents and Chemotherapy 51, n.º 11 (13 de agosto de 2007): 3895–901. http://dx.doi.org/10.1128/aac.00458-07.
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ABSTRACT Trypanosoma brucei cannot synthesize purines de novo and relies on purine salvage from its hosts to build nucleic acids. With adenosine being a preferred purine source of bloodstream-form trypanosomes, adenosine kinase (AK; EC 2.7.1.20) is likely to be a key player in purine salvage. Adenosine kinase is also of high pharmacological interest, since for many adenosine antimetabolites, phosphorylation is a prerequisite for activity. Here, we cloned and functionally characterized adenosine kinase from T. brucei (TbAK). TbAK is a tandem gene, expressed in both procyclic- and bloodstream-form trypanosomes, whose product localized to the cytosol of the parasites. The RNA interference-mediated silencing of TbAK suggested that the gene is nonessential under standard growth conditions. Inhibition or downregulation of TbAK rendered the trypanosomes resistant to cordycepin (3′-deoxyadenosine), demonstrating a role for TbAK in the activation of adenosine antimetabolites. The expression of TbAK in Saccharomyces cerevisiae complemented a null mutation in the adenosine kinase gene ado1. The concomitant expression of TbAK with the T. brucei adenosine transporter gene TbAT1 allowed S. cerevisiae ado1 ade2 double mutants to grow on adenosine as the sole purine source and, at the same time, sensitized them to adenosine antimetabolites. The coexpression of TbAK and TbAT1 in S. cerevisiae ado1 ade2 double mutants proved to be a convenient tool for testing nucleoside analogues for uptake and activation by T. brucei adenosine salvage enzymes.
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Mensa-Wilmot, K., JH LeBowitz, KP Chang, A. al-Qahtani, BS McGwire, S. Tucker e JC Morris. "A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways". Journal of Cell Biology 124, n.º 6 (15 de março de 1994): 935–47. http://dx.doi.org/10.1083/jcb.124.6.935.
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The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI-PLC, cell-associated gp63 could not be detected in immunoblots. Pulse-chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.
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Guerra-Giraldez, Cristina, Luis Quijada e Christine E. Clayton. "Compartmentation of enzymes in a microbody, the glycosome, is essential in Trypanosoma brucei". Journal of Cell Science 115, n.º 13 (1 de julho de 2002): 2651–58. http://dx.doi.org/10.1242/jcs.115.13.2651.
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All kinetoplastids contain membrane-bound microbodies known as glycosomes,in which several metabolic pathways including part of glycolysis are compartmentalized. Peroxin 2 is essential for the import of many proteins into the microbodies of yeasts and mammals. The PEX2 gene of Trypanosoma brucei was identified and its expression was silenced by means of tetracycline-inducible RNA interference. Bloodstream-form trypanosomes, which rely exclusively on glycolysis for ATP generation, died rapidly upon PEX2 depletion. Insect-form (procyclic) trypanosomes do not rely solely on glycolysis for ATP synthesis. PEX2 depletion in procyclic forms resulted in relocation of most tested matrix proteins to the cytosol, and these mutants also died. Compartmentation of microbody enzymes is therefore essential for survival of bloodstream and procyclic T. brucei. In contrast, yeasts and cultured mammalian cells grow normally in the absence of peroxisomal membranes unless placed on selective media.
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Lillico, Simon, Mark C. Field, Pat Blundell, Graham H. Coombs e Jeremy C. Mottram. "Essential Roles for GPI-anchored Proteins in African Trypanosomes Revealed Using Mutants Deficient in GPI8". Molecular Biology of the Cell 14, n.º 3 (março de 2003): 1182–94. http://dx.doi.org/10.1091/mbc.e02-03-0167.
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The survival of Trypanosoma brucei, the causative agent of Sleeping Sickness and Nagana, is facilitated by the expression of a dense surface coat of glycosylphosphatidylinositol (GPI)-anchored proteins in both its mammalian and tsetse fly hosts. We have characterized T. brucei GPI8, the gene encoding the catalytic subunit of the GPI:protein transamidase complex that adds preformed GPI anchors onto nascent polypeptides. Deletion ofGPI8 (to give Δgpi8) resulted in the absence of GPI-anchored proteins from the cell surface of procyclic form trypanosomes and accumulation of a pool of non–protein-linked GPI molecules, some of which are surface located. Procyclic Δgpi8, while viable in culture, were unable to establish infections in the tsetse midgut, confirming that GPI-anchored proteins are essential for insect-parasite interactions. Applying specific inducible GPI8 RNAi with bloodstream form parasites resulted in accumulation of unanchored variant surface glycoprotein and cell death with a defined multinuclear, multikinetoplast, and multiflagellar phenotype indicative of a block in cytokinesis. These data show that GPI-anchored proteins are essential for the viability of bloodstream form trypanosomes even in the absence of immune challenge and imply that GPI8 is important for proper cell cycle progression.
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Ralston, Katherine S., Neville K. Kisalu e Kent L. Hill. "Structure-Function Analysis of Dynein Light Chain 1 Identifies Viable Motility Mutants in Bloodstream-Form Trypanosoma brucei". Eukaryotic Cell 10, n.º 7 (4 de março de 2011): 884–94. http://dx.doi.org/10.1128/ec.00298-10.
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ABSTRACT The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that is receiving increasing attention as a potential drug target and as a system for studying flagellum biology. RNA interference (RNAi) knockdown is widely used to test the requirement for a protein in flagellar motility and has suggested that normal flagellar motility is essential for viability in bloodstream-form trypanosomes. However, RNAi knockdown alone provides limited functional information because the consequence is often loss of a multiprotein complex. We therefore developed an inducible system that allows functional analysis of point mutations in flagellar proteins in T. brucei . Using this system, we identified point mutations in the outer dynein light chain 1 (LC1) that allow stable assembly of outer dynein motors but do not support propulsive motility. In procyclic-form trypanosomes, the phenotype of LC1 mutants with point mutations differs from the motility and structural defects of LC1 knockdowns, which lack the outer-arm dynein motor. Thus, our results distinguish LC1-specific functions from broader functions of outer-arm dynein. In bloodstream-form trypanosomes, LC1 knockdown blocks cell division and is lethal. In contrast, LC1 point mutations cause severe motility defects without affecting viability, indicating that the lethal phenotype of LC1 RNAi knockdown is not due to defective motility. Our results demonstrate for the first time that normal motility is not essential in bloodstream-form T. brucei and that the presumed connection between motility and viability is more complex than might be interpreted from knockdown studies alone. These findings open new avenues for dissecting mechanisms of flagellar protein function and provide an important step in efforts to exploit the potential of the flagellum as a therapeutic target in African sleeping sickness.
Teses / dissertações sobre o assunto "Production de mutants de trypanosomes"
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El, Kadri Mohammad. "Role(s) of glycerol metabolism in the biology of African trypanosomes". Electronic Thesis or Diss., Bordeaux, 2024. http://www.theses.fr/2024BORD0456.
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Trypanosoma brucei, un parasite extracellulaire responsable de la trypanosomiase africaine, doit s’adapter à différents environnements dans ses hôtes mammifères et l’insecte vecteur (la mouche tsétsé). Dans le sang des mammifères, le glucose est la principale source de carbone soutenant une croissance rapide du parasite, en alimentant son métabolisme et la production d'ATP. Lorsque les formes sanguines prolifératives « slender » atteignent des densités cellulaires élevées, un quorum sensing induit leur différenciation en formes non réplicatives « stumpy » (stumpy-QS). Ces formes stumpy-QS protègent l’hôte d’une parasitémie létale et sont également compétentes pour la transmission à la mouche tsétsé. Cependant, le glucose peut être remplacé par le glycérol pour alimenter le métabolisme central du parasite, suggérant un rôle significatif in vivo. Cela s'aligne avec la démonstration récente que les trypanosomes résident principalement dans les espaces extravasculaires de la peau et du tissu adipeux, où le glycérol interstitiel est 5 à 20 fois plus concentré que dans le plasma. Le glycérol est produit par les adipocytes via la glycolyse et la lipolyse, cette dernière induite par les trypanosomes, pourrait protéger l'hôte contre l'infection. Ces données suggèrent que les interactions entre les adipocytes et les trypanosomes, potentiellement médiées par le glycérol, jouent un rôle dans le cycle de vie du parasite.Cette thèse explore l’impact du glycérol sur les formes sanguines slender de Trypanosoma brucei. Nos résultats ont démontré que le glycérol induit la différenciation des formes prolifératives en formes non réplicatives (stumpy-Glyc) ressemblant aux formes stumpy-QS, mais avec une survie allongée. Des conditions similaires à celles des tissus (4 mM glucose, 0,2-0,5 mM glycérol) induisent la production de formes intermédiaires prolifératives (intermédiaire-Glyc) capables de se différencier in vitro en formes procycliques (parasites de l’intestin de l'insecte vecteur) et d’infecter les mouches tsétsé. De plus, le glycérol prolonge considérablement la durée de vie des formes stumpy-QS induites par le quorum sensing. Ces données nous ont conduit à proposer un modèle révisé de la transmission du parasite de l’hôte mammifère à l’insecte vecteur, où les formes stumpy-QS protègent l'hôte contre une parasitémie élevée, tandis que les formes intermédiaires-Glyc prolifératives et les formes stumpy à longue durée de vie, induites par le glycérol des adipocytes, assurent la transmission à l’insecte vecteur.Un autre aspect de ma thèse concerne la dissection de la voie de signalisation impliquée dans la différenciation induite par le glycérol. En exploitant la durée de vie prolongée des cellules stumpy-Glyc en culture, nous avons sélectionné des mutants résistants à la différenciation induite par le glycérol. L'analyse génomique comparative entre ces mutants a permis d'identifier des mutations candidates associées au phénotype de résistance. Une mutation a notamment été trouvée dans le gène de la sous-unité régulatrice de la protéine kinase A (PKAR), dont le rôle dans la voie de signalisation a été ensuite validé.Enfin, nous avons exploré la capacité de T. brucei à métaboliser le glycérol sécrété par les adipocytes, même en présence d'un excès de glucose. Pour ce faire, nous avons utilisé un système de co-culture in vitro permettant d'analyser les interactions entre les trypanosomes parentaux ou mutants et les adipocytes. Le profilage métabolique par résonance magnétique nucléaire (RMN) couplé à des approches de marquage au 13C a permis de tracer les métabolites produits par les adipocytes et les trypanosomes. Nos données ont montré que T. brucei utilise efficacement le glycérol sécrété par les adipocytes pour alimenter son métabolisme central, même en présence de grandes quantités de glucose.En conclusion, ces données ont démontré que le glycérol est un acteur clé dans la biologie de Trypanosoma bruceiTrypanosoma brucei, an extracellular parasite responsible for African trypanosomiasis, must adapt to distinct environments in its mammalian hosts and the tsetse fly vector. In the mammalian bloodstream, glucose serves as the primary carbon source, fueling the parasite's central carbon metabolism and ATP production, which supports its rapid growth. Once the parasites reach high cell densities, a quorum-sensing mechanism induces a transition from proliferative slender forms to growth-arrested stumpy forms (stumpy-QS). These stumpy forms help prevent host mortality by limiting parasitaemia and are primed for transmission to the tsetse fly. However, it has been demonstrated that glycerol can effectively replace glucose in feeding the parasite’s central carbon metabolism, suggesting a significant role in vivo. This aligns with findings that trypanosomes predominantly reside in the extravascular spaces of tissues such as the skin and adipose tissue, where interstitial glycerol concentrations are 5 to 20 times higher than in plasma. Glycerol is released from adipocytes through both lipolysis and lipolysis-independent processes such as glycolysis, and it has been suggested that trypanosome-induced adipocyte lipolysis may even protect the host against trypanosome infection. Together, these data suggest that interactions between adipocytes and trypanosomes, potentially mediated by glycerol, play a critical role in the parasite’s life cycle.This thesis explores the impact of glycerol on bloodstream form (BSF) Trypanosoma brucei. Our findings demonstrated that glycerol induces the differentiation of slender BSF into growth-arrested forms that resemble stumpy-QS, but with enhanced survival. Furthermore, under tissue-like conditions, characterized by glycerol levels between 0.2-0.5 mM and glucose at 4 mM, proliferative intermediate forms were generated, which were capable of differentiating into the insect vector stage (procyclics) and sustaining infections in tsetse flies. Additionally, glycerol extended the lifespan of quorum-sensing-induced stumpy forms, which normally have a limited lifespan of a few days. All these data led us to propose a revised model for transmission, in which quorum sensing-induced stumpy-QS forms protect the host from high parasitaemia, while glycerol from adipocytes induces intermediate-Glyc or long-lived stumpy forms that facilitate transmission to the fly.Another key aspect of my thesis concerns the dissection of the signalling pathway involved in glycerol-induced differentiation. By exploiting the extended lifespan of stumpy-Glyc cells in culture, we selected mutants resistant to glycerol-induced differentiation through extended in vitro culturing in a glycerol-containing medium. Comparative genomic analyses between these mutants and cells grown in glucose, which are sensitive to glycerol-induced differentiation, identified candidate mutations associated with the resistance phenotype. Notably, these mutations were found to affect the protein kinase A regulatory subunit (PKAR), whose role in the signalling pathway was validated.Finally, we explored whether T. brucei can metabolize glycerol secreted by adipocytes even in the presence of excess glucose. To investigate this, we used an in vitro co-culture system using a transwell assay, which allowed us to analyse the interactions between parental and mutant trypanosomes and adipocytes. We examined growth and exometabolome profiles using nuclear magnetic resonance (NMR)-based metabolite profiling, coupled with 13C-labeling to trace specific metabolites. Our data showed that T. brucei efficiently utilized glycerol secreted by adipocytes to support its central carbon metabolism, even when glucose was abundant.Together, these data demonstrated that glycerol is a key player in the biology of Trypanosoma brucei
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Fitzek, Elisabeth. "Production and characterization of mutants of UDP-glucose pyrophosphorylase". Thesis, Umeå University, Plant Physiology, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-26081.
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UDP-glucose pyrophosphorylase (UGPase) is a key component of carbohydrate production in plants, especially with respect to sucrose synthesis/ metabolism, by producing UDP-glucose, a key precursor to sucrose and to many polysaccharides in cell walls. UDP-glucose is also utilized in the synthesis of carbohydrate moiety of glycolipids, glycoproteins and a variety of secondary metabolites, among other functions. The UGPase enzyme may have a rate-limiting function in sugar biosynthesis, and its activity is now known to increase upon variety of abiotic stresses, with possible effects on an overall carbohydrate budget in stressed plants. The enzyme has been proposed to be regulated by (de)oligomerization and it has been estabished that only monomeric form of the enzyme is active. Based on mutant studies, the deoligomerization step (formation of monomers) was found as rate-limiting. A structural model of barley UGPase was recently suggested, based on homology to a human Antigen-X (AGX) protein that has a 40% protein sequence similarity to eukaryotic UGPase. The 3D model shows a bowl-shaped protein with three different domains: (a) N-terminal, (b) central part which includes the nucleotide binding loop (NB-loop) at the active centre and (c) C-terminal which includes an insertion loop (I-loop) that is possibly involved in dimer formation and stabilization. In this study, the model was used as a testable blueprint to verify details of the barley enzyme catalysis and substrate binding, as well as oligomerization process. In order to test the model, site-directed mutagenesis approaches and heterologous (E. coli) expression system were used to produce several UGPase mutants: Del-NB, lacking 4 amino acids (aa) at the NB region; Del-I-4 and Del-I-8, lacking respectively 4 and 8 aa of the I-loop; and Y192A, by replacing an active-site tyrosine into alanine. The Y192A mutant had about half the apparent activity of the wild-type (wt), whereas Del-I-8 and Del-I-4 had only 0.5 and 0.2 % activity, respectively, of the wt, and Del-NB showed no activity at all. Based on native-PAGE, both Y192A and Del-NB mutants had similar oligomerization status as the wt, i.e. existing as monomer only or a mixture of monomer, dimer and higher order oligomers, depending on incubation conditions. Both Del-I-8 and Del-I-4 were present in all conditions as higher order oligomers. Whereas Y192A mutant had similar Kms with both substrates as the wt protein, significant difference between the Del-I-4 and Del-I-8 mutants and wt could be detected. Both mutants had approximately 16-fold higher Kms for UDP-glucose, and the Kms with PPi were 735- and 1500-fold higher for Del-I-4 and Del-I-8, respectively, when compared to wt.The conclusion of those results: (A) Tyr-192 is not essential for activity and is not involved in substrate binding and/ or oligomerization of the enzyme. (B) The NB-loop is essential for catalysis, as evidenced by a complete lack of activity of the Del-NB mutant, and is not involved in oligomerization. On the other hand, (C) the region corresponding to central part of I-loop is located in the model far from active center, but deletion in this region does affect very strongly both catalysis and substrate binding parameters. This can be explained by the involvement of I-loop in formation of dimers (inactive) from monomers (active), as earlier proposed. Apparently, the Del-I-4 and Del-I-8 mutations lead to an enzyme form with a very high oligomerization ability. This affects both Kms and Vmaxs of the Del-I mutants. Taken together the results verify the essentiality of NB-loop for catalysis support the involvement of I-loop region in oligomerization and, overall, the importance of oligomerization status for enzymatic performance of UGPase.
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Niazy, Abdurahman. "Virulence Factor Production in PyrE Mutants of Pseudomonas Aeruginosa". Thesis, University of North Texas, 2010. https://digital.library.unt.edu/ark:/67531/metadc28458/.
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It has been shown previously in our lab that mutations in the pyrimidine pathway reduced the ability of Pseudomonas aeruginosa to produce virulence factors. Knockout mutations in pyrB, pyrC and pyrD genes of the pyrimidine pathway showed that virulence factor production was decreased. Pyoverdin, pyocyanin, hemolysin, iron chelation, motility, and adherence are all considered virulence factors. Here I further investigate the effects of mutations in the pyrimidine pathway by studying a pyrE mutant. I studied the effect of the pyrE mutation on the production of the above virulence factors. Just like the effect of pyrB, pyrC and pyrD mutations,the pyrE mutation also showed that the bacteria were deficient in producing virulence factors when compared to the wild type. The broader impact of this research would be the possibility of finding drugs that could treat patients infected with P. aeruginosa and possibly extend the lives of chronically infected patients with cystic fibrosis.
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Kuriakose, Shiby. "Molecular regulation of Trypanosoma congolense-induced proinflammatory cytokine production in macrophages and its modulation by diminazene aceturate (Berenil)". PLOS, Frontiers in Immunology, Elsevier, Sage, 2016. http://hdl.handle.net/1993/31677.
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African trypanosomiasis remains a major health problem to both humans and animals due to lack of effective treatment or vaccine to control the disease. Animal trypanosomiasis is considered one of the most important diseases affecting livestock production and agricultural development in sub-Saharan Africa. Although the use of trypanocides remain the most important method for controlling the disease in animals, the mechanisms of action of these compounds are not completely known. The overall aim of this thesis is to decipher the molecular mechanisms involved in Trypanosoma congolense-induced cytokine production and how this is modulated by the trypanocide, Diminazene aceturate (Berenil).
First, I investigated the molecular and biochemical mechanisms of action of Berenil to determine whether in addition to trypanolytic effect, it exerts a modulatory effect on the host immune system. Although it is known that T. congolense infection in mice is associated with increased production of pro-inflammatory cytokines by macrophages, the intracellular signaling pathways leading to the production of these cytokines remain unknown. Therefore, I investigated the innate receptors and intracellular signaling pathways that are involved in T. congolense-induced pro-inflammatory cytokine production in macrophages. Next I further determined whether the inhibitory effect of Berenil on proinflammatory cytokine production in macrophages is specific to T. congolense.
I found that Berenil treatment significantly reduced the immune activation and proinflammatory cytokine production in infected mice suggesting that in addition to its direct trypanolytic effect, Berenil also modulates the host immune response to the parasite. Next, I show that T. congolense induced pro-inflammatory cytokine production in macrophages is dependent on phosphorylation of mitogen-activated protein kinase (MAPK) and signal transducer and activation of transcription (STAT) proteins in a TLR2-dependent manner. I further show that Berenil treatment downregulates T. congolense as well as LPS induced cytokine production by affecting the phosphorylation of MAPK and STAT proteins.
Collectively, the results from this thesis provide novel insights into T. congolense-induced activation of the innate immune system and modulation of host immune response by Berenil. These findings are significant and could help in developing newer and better therapeutic strategies against the disease, in particular, and inflammatory responses in general.October 2016
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Zouheiry, Hassane. "La pénicilline amidase d'Escherichia coli : recherche des meilleurs conditions de production par bactéries recombinées, mise au point de production et de caractérisation de mutants, étude de ces mutants". Nancy 1, 1991. http://www.theses.fr/1991NAN10021.
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Ce travail, réalisé en collaboration avec Rhône-Poulenc santé, a consisté d'une part, à étudier la régulation de l'expression du gène cloné de la pénicilline amidase (PA) dans une souche mutante, d'autre part, à développer une technique de mutagenèse chimique adaptée à l'étude d'enzyme, à l'appliquer au gène PA et à caractériser les mutants obtenus. La souche transformée présente les caractéristiques suivantes par rapport à la souche non transformée: une exigence plus importante en source de carbone, un effet non pas négatif mais positif du glucose sur la production de PA, une expression en partie constitutive, une stimulation par l'acide phenylacetique n'est observée qu'en présence de glucose. La majorité des mutants PA obtenus est affectée dans le processus de maturation protéolytique interne. Un grand nombre des mutations responsables de ce phénotype est localise dans la région c-terminale de la grande sous-unité qui doit donc jouer un rôle important dans ce processus
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Zeyniyev, Araz. "Cephamycin C Production By Streptomyces Clavuligerus Mutants Impaired In Regulation Of Aspartokinase". Master's thesis, METU, 2006. http://etd.lib.metu.edu.tr/upload/12607641/index.pdf.
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Aspartokinase is the first enzyme of the aspartate family amino acids biosynthetic pathway. Cephamycin C is a β
-lactam antibiotic produced as a secondary metabolite via the enzymatic reactions in the lysine branch of this pathway in Streptomyces clavuligerus. The aspartokinase activity of S. clavuligerus is under concerted feedback inhibition by two of the end product amino acids, lysine plus threonine. It is also known that carbon flow through the lysine branch of the aspartate pathway is rate limiting step in the formation of cephamycin C. Therefore, genetic alterations in the regulatory regions of the aspartokinase enzyme are expected to lead to an increased cephamycin C production. The aim of this study was to obtain S. clavuligerus mutants that possess aspartokinase enzyme insensitive to feedback inhibition by lysine and threonine, identification of the mutation(s) accounting for the resistance being the ultimate goal. For this aim, chemical mutagenesis was employed to increase random mutation rate and a population of lysine anti-metabolite resistant S. clavuligerus mutants that can grow in the presence of S-(2-aminoethyl)-L-cysteine was obtained. The mutants were screened for their aspartokinase insensitivity via enzyme assays and one mutant exhibiting the highest level of deregulation was assessed for its cephamycin C production. The results revealed a 2-fold increase in specific production of the antibiotic. As a member of &
#946
-lactam class antibiotics, cephamycin C has an importance in medicine. Therefore, the mutant strain obtained might be a candidate for industrial production of the compound.
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Liu, Xiaoguang. "Production of butyric acid and hydrogen by metabolically engineered mutants of Clostridium tyrobutyricum". Connect to resource, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1124114266.
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Thesis (Ph. D.)--Ohio State University, 2005.Title from first page of PDF file. Document formatted into pages; contains xv, 220 p.; also includes graphics (some col.). Includes bibliographical references (p. 189-201). Available online via OhioLINK's ETD Center
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Wahab, Adbul. "Regulation of antibiotic production in Streptomyces coelicolor : phenotypic and transcriptomic analysis of AbsA mutants". Thesis, University of Surrey, 2007. http://epubs.surrey.ac.uk/843422/.
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The AbsA two-component system is a negative regulator of antibiotic biosynthesis in Streptomyces coelicolor. To gain further insight into the role of the AbsA system different in-frame deletion mutations were made in the absA1A2 operon in S. coelicolor MT1110, MT1110DeltacdaR and S. lividans 1326. Phenotypic analysis of DeltaabsA1A2 and DeltaabsA2 in the MT1110 derivatives showed that CDA was produced early on Oxoid nutrient agar relative to the parent strain. The absA mutants in S. lividans 1326 (both DeltaabsA1 and DeltaabsA1A2) did not show early CDA production. On solid R2YE both MT1110DeltaabsA1A2 and MT1110DeltaabsA2 produced undecylprodigiosin and actinorhodin earlier. In liquid R2YE medium MT1110DeltaabsA1A2 produced undecylprodigiosin and actinorhodin earlier compared to MT1110 wild-type. RNA was extracted from surface-grown cultures of MT1110 and its DeltaabsA1A2 derivative on R2YE agar at different stages of growth. Cy3-labelled cDNA and Cy5-labelled genomic DNA (gDNA) were hybridised on in-house printed 50-mer oligo arrays representing all S. coelicolor open reading frames. Differentially expressed genes were identified by the 'Rank Product' method. Transcriptomic analysis revealed that many genes encoding enzymes of central metabolism were up-regulated in MT1110DeltaabsA1A2 compared to MT1110 wild-type. Three genes, SCO4089, SCO3829 and SCO1270 encoding valine dehydrogenase, dihydrolipoamide acyltransferase component E2 and the alpha-subunit of pyruvate dehydrogenase complex respectively, were found to be more highly expressed in MT1110DeltaabsA1A2. These genes encode enzymes whose reactions produce possibly precursors for CDA, Act and Red biosynthesis. The gene encoding pyruvate kinase 2 (SCO5423) showed increased expression in the mutant at and after 24 h of growth. Genes encoding subunits of ATP synthases showed increased expression in the mutant that indicates that an increase oxidative metabolism is occurring in MT1110DeltaabsA1A2. A gene, SCO6195, encoding a stationary phase-active enzyme, acyl-coenzyme A synthetase was induced in MT1110DeltaabsA1A2 at 23 and 24 h of cultivation. Two genes in the cda cluster were found to be repressed in mutant: SCO3224 and SCO3229, encoding an ABC transporter ATP-binding protein and 4-hydroxymandelate synthase, respectively. The gene encoding AfsS (SCO4425) was found to be up-regulated in the DeltaabsA1A2 mutant at the late phase of the growth. Four genes encoding enzymes putatively involved in the pentose phosphate pathway were found to be up-regulated in MT1110DeltaabsA1A2 at certain time points. Genes encoding ribosomal protein showed a more relaxed growth phase dependent pattern of expression in the mutant while one r- protein encoding gene, rpsK (SCO4728), showed higher expression in the mutant except at stationary phase. It is deduced from the global gene expression analysis that the possible role of the AbsA system in S. coelicolor is to regulate precursor supply for antibiotic production rather than to directly regulate the genes of the respective antibiotic biosynthetic clusters.
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Xue, Liu. "HIGH-ACTIVITY MUTANTS OF HUMAN BUTYRYLCHOLINESTERASE FOR COCAINE ABUSE TREATMENT". UKnowledge, 2013. http://uknowledge.uky.edu/pharmacy_etds/40.
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Cocaine is a widely abused drug without an FDA-approved medication. It has been recognized as an ideal anti-cocaine medication to accelerate cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. butyrylcholinesterase (BChE)-catalyzed hydrolysis. However, the native BChE has a low catalytic activity against cocaine. We recently designed and discovered a set of BChE mutants with a high catalytic activity specifically for cocaine. An ideal, therapeutically valuable mutant of human BChE should have not only a significantly improved catalytic activity against cocaine, but also certain selectivity for cocaine over neurotransmitter acetylcholine (ACh) such that one would not expect systemic administration of the BChE mutant to interrupt cholinergic transmission. Through integrated computational-experimental studies, several BChE mutants were identified to have not only a considerably improved catalytic efficiency against cocaine, but also the desirable selectivity for cocaine over ACh. Representative BChE mutants have been confirmed to be potent in actual protection of mice from acute toxicity (convulsion and lethality) of a lethal dose of cocaine (180 mg/kg, LD100). Pretreatment with the BChE mutant (i.e. 1 min prior to cocaine administration) dose-dependently protected mice against cocaine-induced convulsions and lethality. The in vivo data reveal the primary factor, i.e. the relative catalytic efficiency, determining the efficacy in practical protection of mice from the acute cocaine toxicity and future direction for further improving the efficacy of the enzyme in the cocaine overdose treatment.
For further characterization in animal models, we successfully developed high-efficiency stable cell lines efficiently expressing the BChE mutants by using a lentivirus-based repeated-transduction method. The large-scale protein production enabled us to further characterize the in vivo profiles of the BChE mutant concerning the biological half-life and potency in accelerating cocaine clearance. In particular, it has been demonstrated that the BChE mutant can rapidly metabolize cocaine and completely eliminate cocaine-induced hyperactivity in rodents, implying that the BChE mutant may be developed as a promising therapeutic agent for cocaine abuse treatment.
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Merah, Mostefa. "Obtention et étude de mutants de Myxococcus xanthus affectés dans la production d'activités enzymatiques exocellulaires". Rouen, 1992. http://www.theses.fr/1992ROUES017.
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Myxococcus xanthus est une bactérie à gram-négatif qui sécrète de nombreuses activités enzymatiques durant la phase exponentielle de croissance. Nous avons tenté dans une première étape de cloner un gène de structure de l'une des trois activités sécrétées par cette bactérie: protéase, endoprotéase acide et glucanase en utilisant le promoteur mobile Ptac permettant de créer des phénotypes conditionnels inductibles par l'IPTG. Les résultats que nous avons obtenus ont montré que le clonage d'un fragment d'ADN de M xanthus situé en aval du Ptac dans E. Coli est faisable. Mais l'obtention conditionnelle par cette méthode n'a pas été possible faute de méthode de criblage suffisamment fiable. L'analyse des mutants d'insertion obtenus montre que la majorité sont affectés dans la production de certaines des activités seulement et sont vraisemblablement des mutants de régulation. Une du mutant paraît toucher le mécanisme de sécrétion et le cycle de développement multicellulaire et pourrait faire partie d'une famille comprenant au moins 6 gènes suggérant une relation entre mécanisme de sécrétion et le développement
Livros sobre o assunto "Production de mutants de trypanosomes"
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Simpson, Duncan Robert. Characterisation of morphological mutants arising during production of Quorn myco-protein. Manchester: University of Manchester, 1996.
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Stephens, Sarah Katharine. The production and characterisation of extracellular enzyme mutants in Coprinus bilanatus. Birmingham: University of Birmingham, 1988.
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Ikushujō, Hōshasen, ed. Production of mutants in tree crops: Report of symposium held on July 20-21, 1989. Ohmiya-machi, Naka-gun, Ibaraki-ken, Japan: Institute of Radiation Breeding, NIAR MAFF, 1991.
Encontre o texto completo da fonteCapítulos de livros sobre o assunto "Production de mutants de trypanosomes"
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Moguilevsky, N., F. Varsalona, J. P. Guillaume, C. Roobol e A. Bollen. "Recombinant Human Proapolipoprotein A-I: Experimental Strategies for the Production of an Authentic Molecule". In Human Apolipoprotein Mutants III, 145–63. Berlin, Heidelberg: Springer Berlin Heidelberg, 1993. http://dx.doi.org/10.1007/978-3-642-84634-2_14.
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Sarsu, Fatma, Suprasanna Penna e Ganesh C. Nikalje. "Strategies for Screening Induced Mutants for Stress Tolerance". In Mutation Breeding for Sustainable Food Production and Climate Resilience, 151–76. Singapore: Springer Nature Singapore, 2023. http://dx.doi.org/10.1007/978-981-16-9720-3_6.
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Awazuhara, Motoko, Akio Furuhashi, Toru Fujiwara e Mitsuo Chino. "Screening of Arabidopsis thaliana mutants sensitive to sulfur deficiency". In Plant Nutrition for Sustainable Food Production and Environment, 213–14. Dordrecht: Springer Netherlands, 1997. http://dx.doi.org/10.1007/978-94-009-0047-9_54.
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Park, Gyungsoon, Hildur V. Colot, Patrick D. Collopy, Svetlana Krystofova, Christopher Crew, Carol Ringelberg, Liubov Litvinkova et al. "High-Throughput Production of Gene Replacement Mutants in Neurospora crassa". In Methods in Molecular Biology, 179–89. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-040-9_13.
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Nishiwaki, Toshikazu, Takashi Sato, Hiroyuki Yashima, Taro Ikarashi, Takuji Ohyama, James E. Harper, Shoichiro Akao e Hiroshi Kouchi. "Changes in concentration of leghemoglobin components in hypernodulation mutants of soybean". In Plant Nutrition for Sustainable Food Production and Environment, 693–98. Dordrecht: Springer Netherlands, 1997. http://dx.doi.org/10.1007/978-94-009-0047-9_222.
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Pühler, A., M. F. Hynes, D. Kapp, P. Müller e K. Niehaus. "Infection Mutants of Rhizobium Meliloti are Altered in Acidic Exopolysaccharide Production". In Recognition in Microbe-Plant Symbiotic and Pathogenic Interactions, 29–37. Berlin, Heidelberg: Springer Berlin Heidelberg, 1986. http://dx.doi.org/10.1007/978-3-642-71652-2_3.
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Ruengjitchatchawalya, M., S. Chamutpong, M. Ponglikitmongkol, R. Chaiklahan e M. Tanticharoen. "Spirulina Platensis Mutants Defective in γ-Linolenic Acid Production: Molecular characterization". In Advanced Research on Plant Lipids, 105–8. Dordrecht: Springer Netherlands, 2003. http://dx.doi.org/10.1007/978-94-017-0159-4_23.
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Faull, J. L., e K. Graeme-Cook. "Characterization of Mutants of Trichoderma Harzianum with Altered Antibiotic Production Characteristics". In Biological Control of Plant Diseases, 345–51. Boston, MA: Springer US, 1992. http://dx.doi.org/10.1007/978-1-4757-9468-7_48.
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Bakshi, Suman, Johar Singh e Sanjay J. Jambhulkar. "Isolation and characterization of yellow rust resistant mutants in wheat." In Mutation breeding, genetic diversity and crop adaptation to climate change, 103–10. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781789249095.0010.
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Abstract Stripe rust, also known as yellow rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a major threat to wheat production leading to yield losses up to 84%. Due to climate change, new races of the yellow rust pathogen are appearing for which no durable source of resistance has been observed in the present high-yielding varieties. A mutation breeding programme was initiated in two popular varieties, namely PBW343 and HD2967, using gamma-ray and electron beam irradiation. Gamma-ray doses of 250, 300 and 350 Gy and electron beam doses of 150, 200 and 250 Gy were used for seed irradiation. The M2 population was screened in the field from seedling to adult plant stage by spraying a mixture of urediniospores of Pst pathotypes. Disease severity was recorded as the percentage of leaf area covered by the rust pathogen following a modified Cobb's scale. A total of 52 putative yellow rust resistant mutants in HD2967 and 63 in PBW343 were isolated. The number of mutants was higher in the electron beam irradiated population compared with gamma-rays. The absence of sporulation and spore production of the rust pathogen on the mutants indicated resistance. Mutant plants showing seedling resistance also showed resistance at adult plant stage. Seed yield and its contributing characters were better in the mutants compared with the parents. These rust resistant mutants could be novel sources of stripe rust or yellow rust resistance. The plant-to-row progenies of these mutants were confirmed and characterized in the M3 generation.
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Alvarez, W. Navarro. "Production of salt tolerant rice mutants using gamma rays and anther culture". In Mutations, In Vitro and Molecular Techniques for Environmentally Sustainable Crop Improvement, 93–96. Dordrecht: Springer Netherlands, 2002. http://dx.doi.org/10.1007/978-94-015-9996-2_9.
Texto completo da fonteTrabalhos de conferências sobre o assunto "Production de mutants de trypanosomes"
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Solarte, Ana Lucía, Rafael Jesús Astorga, Fabiana de Aguiar, Alfonso Maldonado, Ángela Galán-Relaño e Belén Huerta. "Induction of resistant mutants of Salmonella typhimurium under enrofloxacin and natural alternatives for control in pigs". In Safe Pork 2015: Epidemiology and control of hazards in pork production chain. Iowa State University, Digital Press, 2017. http://dx.doi.org/10.31274/safepork-180809-380.
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Gammanpila, H. W., e K. R. Manjula. "Exploring the Influence of Intermittent Heat Exposure on Spontaneous Mutations in Drosophila melanogaster: Assessing the Role of Vitamin C in Mitigating Heat Stress and Examining Inheritance Patterns of Induced Mutations". In SLIIT International Conference on Advancements in Sciences and Humanities 2023. Faculty of Humanities and Sciences, SLIIT, 2023. http://dx.doi.org/10.54389/thuh5711.
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Climate change poses a significant threat to the well-being of organisms. It has a detrimental impact on the survival of smaller organisms in response to climatic shifts, posing a substantial danger to biodiversity, which is already under stress due to habitat loss, emerging invasive species, and diseases. This study aimed to assess the influence of fluctuating temperatures on the physiology and behavior of Drosophila melanogaster, as well as to investigate whether such temperature fluctuations have any effect on phenotypic expression through potential spontaneous mutations. Genotypic changes were examined by observing cytological alterations in the salivary gland chromosomes. Drosophila melanogaster were exposed to intermittent heat conditions for a period of two weeks. The experimental setup was divided into four groups: a control group maintained at room temperature (25±2°C), a group at room temperature supplemented with vitamin C, a group exposed to heat at 38±2°C, and a group exposed to 38±2°C with vitamin C supplementation. Revival of the flies was noticeably better in the vitamin C supplemented group. These flies exhibited a higher revival rate even after exposure to the heat stress. Salivary gland chromosome analysis provided intriguing insights. More balbiani rings were observed, indicating elevated mRNA production during the heat exposure. Furthermore, an increase in the number of puffs in polytene chromosomes was noted, suggesting an overall increase in mRNA production in the heat-exposed flies. Additionally, the evaluation of wing mutants yielded important findings. It became evident that these mutations were not related to vestigial or curly wing traits. Instead, they indicated that heat exposure was damaging wing formation, resulting in abnormal wing patterns. These results suggest a substantial impact of temperature fluctuations on insect behavior, which can even lead to the induction of mutations. Generational studies further indicate that these mutations can be inherited.
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Alkotami, Linah, Brice Jarvis, Chaofu Lu, Doug Allen, Jianhui Zhang, John Sedbrook, Kathleen Schuler, Somnath Koley e Timothy Durrett. "Targeted genome editing of industrial oilseed crops to enhance synthesis of functional lipids". In 2022 AOCS Annual Meeting & Expo. American Oil Chemists' Society (AOCS), 2022. http://dx.doi.org/10.21748/orfd6797.
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Acetyl-triacylglycerols (acetyl-TAG) produced in the seeds of different Euonymus species are triacylglycerols (TAG) that possess an sn-3 acetate group instead of a long chain fatty acid. This unusual structure confers useful properties to acetyl-TAG, including reduced kinematic viscosity and improved cold temperature performance. Acetyl-TAG are synthesized by unique diacylglycerol acetyltransferases (DAcTs), expressed in the endosperm of Euonymus seeds, that use acetyl-CoA to acetylate the sn-3 position of diacylglycerol (DAG) molecules. Isolation and expression of DAcT enzymes from different Euonymus species has resulted in the successful accumulation of high levels of acetyl-TAG in different oil seed crops. For example, expression of EfDAcT isolated from E. fortunei caused acetyl-TAG levels of 81 mol% in camelina seeds and 51 mol% in pennycress. To increase acetyl-CoA supply for EfDAcT, CRISPR-based genome editing was used to generate mutations in FATTY ACID ELONGASE1 (FAE1) genes in both camelina and pennycress. FAE1 is a component of the fatty acid elongase complex that uses acetyl-CoA to produce the very long-chain fatty acids found in the seed oil of Brassicaceae species. Consistent with the hypothesis that eliminating FAE1 function results in more acetyl-CoA availability for acetyl-TAG synthesis, expression of EfDAcT in fae1 mutants resulted in very high levels of acetyl-TAG accumulation. This was particularly evident in pennycress fae1 lines where acetyl-TAG levels exceeded 95 mol% in the best transgenic lines. These results demonstrate the usefulness of genome editing to modify the genetic background of oilseed crops to enhance production of a targeted product.
Relatórios de organizações sobre o assunto "Production de mutants de trypanosomes"
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Ori, Naomi, e Jason W. Reed. Engineering parthenocarpic fruit production in tomato. Israel: United States-Israel Binational Agricultural Research and Development Fund, 2021. http://dx.doi.org/10.32747/2021.8134175.bard.
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Normally, fruits are formed only following fertilization. In tomato, fertilization is sensitive to extreme temperatures, resulting in reduced yield. Yield stability would therefore benefit from tomato varieties with parthenocarpic fruits, which develop independently of fertilization. The objective of the research was to generate parthenocarpic tomato lines by mutating PRC2 components, to investigate how PRC2 and auxin signaling regulate fruit initiation and growth, and to generate parthenocarpic lines for breeding. We reasoned that heterozygous prc2 mutations would generate parthenocarpic fruits with minimal vegetative effects, as they act in the female gametophyte. The specific objectives were : To generate (1) tomato PRC2 mutants and characterize them developmentally (2) and molecularly (3), and to test their performance in the field (4). Aim 1 proved challenging, and was achieved only during the third year. Therefore the research was extended for an additional 8 months, during which goals 2 and 4 were achieved. The research yielded mutations in 4 different PRC2 components, two of which were loss-of-function mutations that produced parthenocarpic fruits, Slfie and Slmsi1 mutants. Characterization of heterozygote Slfie mutants showed that they produce fruits independently of fertilization across a range of growth conditions. No homozygote Slfie mutants were obtained, likely due to failure of the mutant allele to transfer via the female gametopyte. Slfie/+ fruits were of good quality in contrast to most previously described parthenocarpic fruits. Initial characterization under heat stress showed a dramatic increase in yield under extreme heat, therefore providing yield stability. In addition, we characterized single and double mutants in tomato SlARF8a and SlARF8b, and found that these also gave plants with parthenocarpic fruit growth and increased yield stability. The research yielded genetic material that can be used in breeding programs to increase yield stability under unstable climate
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Horwitz, Benjamin A., e Barbara Gillian Turgeon. Fungal Iron Acquisition, Oxidative Stress and Virulence in the Cochliobolus-maize Interaction. United States Department of Agriculture, março de 2012. http://dx.doi.org/10.32747/2012.7709885.bard.
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Our project focused on genes for high affinity iron acquisition in Cochliobolus heterostrophus, a necrotrophic pathogen of maize, and their intertwined relationship to oxidative stress status and virulence of the fungus on the host. An intriguing question was why mutants lacking the nonribosomal peptide synthetase (NRPS) gene (NPS6) responsible for synthesis of the extracellular siderophore, coprogen, are sensitive to oxidative stress. Our overall objective was to understand the mechanistic connection between iron stress and oxidative stress as related to virulence of a plant pathogen to its host. The first objective was to examine the interface where small molecule peptide and reactive oxygen species (ROS) mechanisms overlap. The second objective was to determine if the molecular explanation for common function is common signal transduction pathways. These pathways, built around sensor kinases, response regulators, and transcription factors may link sequestering of iron, production of antioxidants, resistance to oxidative stress, and virulence. We tested these hypotheses by genetic manipulation of the pathogen, virulence assays on the host plant, and by following the expression of key fungal genes. An addition to the original program, made in the first year, was to develop, for fungi, a genetically encoded indicator of redox state based on the commercially available Gfp-based probe pHyper, designed for animal cell biology. We implemented several tools including a genetically encoded indicator of redox state, a procedure to grow iron-depleted plants, and constructed a number of new mutants in regulatory genes. Lack of the major Fe acquisition pathways results in an almost completely avirulent phenotype, showing how critical Fe acquisition is for the pathogen to cause disease. Mutants in conserved signaling pathways have normal ability to regulate NPS6 in response to Fe levels, as do mutants in Lae1 and Vel1, two master regulators of gene expression. Vel1 mutants are sensitive to oxidative stress, and the reason may be underexpression of a catalase gene. In nps6 mutants, CAT3 is also underexpressed, perhaps explaining the sensitivity to oxidative stress. We constructed a deletion mutant for the Fe sensor-regulator SreA and found that it is required for down regulation of NPS6 under Fe-replete conditions. Lack of SreA, though, did not make the fungus over-sensitive to ROS, though the mutant had a slow growth rate. This suggests that overproduction of siderophore under Fe-replete conditions is not very damaging. On the other hand, increasing Fe levels protected nps6 mutants from inhibition by ROS, implying that Fe-catalyzed Fenton reactions are not the main factor in its sensitivity to ROS. We have made some progress in understanding why siderophore mutants are sensitive to oxidative stress, and in doing so, defined some novel regulatory relationships. Catalase genes, which are not directly related to siderophore biosynthesis, are underexpressed in nps6 mutants, suggesting that the siderophore product (with or without bound Fe) may act as a signal. Siderophores, therefore, could be a target for intervention in the field, either by supplying an incorrect signal or blocking a signal normally provided during infection. We already know that nps6 mutants cause smaller lesions and have difficulty establishing invasive growth in the host. Lae1 and Vel1 are the first factors shown to regulate both super virulence conferred by T-toxin, and basic pathogenicity, due to unknown factors. The mutants are also altered in oxidative stress responses, key to success in the infection court, asexual and sexual development, essential for fungal dissemination in the field, aerial hyphal growth, and pigment biosynthesis, essential for survival in the field. Mutants in genes encoding NADPH oxidase (Nox) are compromised in development and virulence. Indeed the triple mutant, which should lack all Nox activity, was nearly avirulent. Again, gene expression experiments provided us with initial evidence that superoxide produced by the fungus may be most important as a signal. Blocking oxidant production by the pathogen may be a way to protect the plant host, in interactions with necrotrophs such as C. heterostrophus which seem to thrive in an oxidant environment.
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Gantt, Elisabeth, Avigad Vonshak, Sammy Boussiba, Zvi Cohen e Amos Richmond. Carotenoid-Rich Algal Biomass for Aquaculture: Astaxanthin Production by Haematococcus Pluvialis. United States Department of Agriculture, agosto de 1996. http://dx.doi.org/10.32747/1996.7613036.bard.
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The synthesis of carotenoids has been studied toward enhancing the production of ketocarotenoids, since fish and crustaceans raised by aquaculture require astaxanthin and other ketocaroteinoids in their feed for desirable pigmentation. Notable progress has been made in attaining the goals of determining improved conditions for ketocarotenoid production in Haematococcus pluvialis and in elucidating the carotenoid biosynthetic pathway. For production of astaxanthin a number of strains of the green alga Haematococcus were evaluated, a strain CCAG was found to be optimal for photoautotrophic growth. Of four mutants, selected for enhanced carotenoid production, two hold considerable promise because caroteinoid accumulation occurs without encystment. The biosynthetic pathway of carotenoids was elucidated in photosynthetic organisms by characterizing novel genes encoding carotenoid enzymes and by examining the function of these enzymes in a bacterial complementation system. Two cyclases (b- and e-) were cloned that are at a critical branch point in the pathway. One branch leads to the formation of b-carotene and zeaxanthin and astaxanthin, and the other to the production of a-carotene and lutein. Cyclization of both endgroups of lycopene to yield b-carotene was shown to be catalyzed by a single gene product, b-lycopene cyclase in cyanobacteria and plants. The formation of a-carotene was found to require the e-cyclase gene product in addition to the b-cyclase. By cloning a b-hydroxylase gene we showed that a single gene product forms zeaxanthin by hydroxylatin of both b-carotene rings. It is expected that a second hydroxylase is required in the synthesis of astaxanthin, since canthaxanthin rather than zeaxanthin is the precursor. Evidence, from inhibitor studies, suggests that astaxanthin is formed from canthaxanthin and that b-carotene is a major precursor. Feasibility studies with the photobioreactors have shown that a two-stage system is the most practical, where Haematococcus cultures are first grown to high cell density and are then switched to high light for maximal astaxanthin production. The basic knowledge and molecular tools generated from this study will significantly enhance Haematococcus as a viable model for enhanced astaxanthin production.
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Eshel, Amram, Jonathan P. Lynch e Kathleen M. Brown. Physiological Regulation of Root System Architecture: The Role of Ethylene and Phosphorus. United States Department of Agriculture, dezembro de 2001. http://dx.doi.org/10.32747/2001.7585195.bard.
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Specific Objectives and Related Results: 1) Determine the effect of phosphorus availability on ethylene production by roots. Test the hypothesis that phosphorus availability regulates ethylene production Clear differences were found between the two plants that were studied. In beans ethylene production is affected by P nutrition, tissue type, and stage of development. There are genotypic differences in the rate of ethylene production by various root types and in the differential in ethylene production when P treatments are compared. The acceleration in ethylene production with P deficiency increases with time. These findings support the hypothesis that ethylene production may be enhanced by phosphorus deficiency, and that the degree of enhancement varies with genotype. In tomatoes the low-P level did not enhance significantly ethylene production by the roots. Wildtype cultivars and ethylene insensitive mutants behaved similarly in that respect. 2) Characterize the effects of phosphorus availability and ethylene on the architecture of whole root systems. Test the hypothesis that both ethylene and low phosphorus availability modify root architecture. In common bean, the basal roots give rise to a major fraction of the whole root system. Unlike other laterals these roots respond to gravitropic stimulation. Their growth angle determines the proportion of the root length in the shallow layers of the soil. A correlation between ethylene production and basal root angle was found in shallow rooted but not deep-rooted genotypes, indicating that acceleration of ethylene synthesis may account for the change in basal root angle in genotypes demonstrating a plastic response to P availability. Short-time gravitropic response of the tap roots of young bean seedlings was not affected by P level in the nutrient solution. Low phosphorus specifically increases root hair length and root hair density in Arabidopsis. We tested 7 different mutants in ethylene perception and response and in each case, the response to low P was lower than that of the wild-type. The extent of reduction in P response varied among the mutants, but every mutant retained some responsiveness to changes in P concentration. The increase in root hair density was due to the increase in the number of trichoblast cell files under low P and was not mediated by ethylene. Low P did not increase the number of root hairs forming from atrichoblasts. This is in contrast to ethylene treatment, which increased the number of root hairs partly by causing root hairs to form on atrichoblasts. 3) Assess the adaptive value of root architectural plasticity in response to phosphorus availability. A simulation study indicated that genetic variation for root architecture in common bean may be related to adaptation to diverse competitive environments. The fractal dimension of tomato root system was directly correlated with P level.
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Antignus, Yehezkiel, Ernest Hiebert, Shlomo Cohen e Susan Webb. Approaches for Studying the Interaction of Geminiviruses with Their Whitefly Vector Bemisia tabaci. United States Department of Agriculture, julho de 1995. http://dx.doi.org/10.32747/1995.7604928.bard.
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The DNA of tomato yellow leaf curl virus (TYLCB) was detected in its whitefly vector, Bemisia tabaci, by dot spot hybridization as early as 1 h after acquisition access. The retention of the virus nucleic acid in the vector was at least 23 days after a 48 h acquisition access. However, the retention of TYLCV coat protein did not exceed 10 days. No replicative forms of TYLCV could be detected in B. tabaci, indicating a non-propagative relationship with the vector. Whiteflies were not able to accumulate naked virion ssDNA, virus cloned dsDNA, or virions with impaired coat protein. Deletion, frameshift, and single amino acid mutations were inserted into open reading frames (ORFs) V1 and V2 (Coat protein) of TYLCV. The ability of these mutants to replicate, to spread and to induce symptoms was tested both in leaf disks and in intact plants. No replication was found in tissues that were infected with a deletion mutant that lacked the carboxy half of the coat protein gene. Residual amounts of ssDNA and dsDNA were detected i tissues infected with a frameshift mutant in which an early termination at the extreme part of the protein. Two other mutants in which a single amino acid was changed in the overlapping part of V1 and V2 were able to spread systemically but infections remained symptomless and the production of ssDNA and dsDNA were significantly lower. These mutants were acquired and transmitted by Bemisia tabaci. Procedures for the the dissection, fixation and embedding of whiteflies were developed. The anatomy and ultrastructure of the salivary gland and the midgut of Bemisia tabaci and Trialeurodes vaporariorum (a vector and non-vector of geminiviruses respectively) was studied and described. Monoclonal antibodies against bean golden mosaic virus (BGMV) with narrow and broad spectrum were prepared. Transmission studies of tomato mottle geminivirus (TMoV) by B. tabaci were carried out. These studies were essential for a further work aimed to understand the interaction of geminiviruses with the insect and their localization in its tissues. To enable the production of transgenic plants procedures were developed for tomato transformation with both Agrobacterium and microparticle bombardment.
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Prusky, Dov, Noel T. Keen e Stanley Freeman. Elicitation of Preformed Antifungal Compounds by Non-Pathogenic Fungus Mutants and their Use for the Prevention of Postharvest Decay in Avocado Fruits. United States Department of Agriculture, janeiro de 1996. http://dx.doi.org/10.32747/1996.7570573.bard.
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C. gloeosporioides attacks unripe avocado fruits in the orchard. Germinated spores produce appressoria that germinate and breach the cuticle, but the resultant subcuticular hyphae become quiescent and do not develop further until fruit is harvested and ripens. Resistance of unripe avocado to attach by C. gloeosporioides is correlated with the presence of fungitoxic concentrations of the preformed antifungal compound, 1-acetoxy-2-hydroxy-4-oxoheneicosa-12, 15 diene in the pericarp of unripe fruits. The objective of this proposal was to study the signal transduction process by which elicitors induce resistance in avocado. It was found that abiotic elicitors, infection of avocado fruit with C. gloeosporioides or treatment of avocado cell suspension with cell-wall elicitor induced a significant production of reactive oxygen species (ROS). Ripe and unripe fruit tissue differ with regard to the ROS production. The unripe, resistant fruit are physiologically able to react and to produce high levels of ROS and increased activity of H+ATPase that can enhance the phenylpropanoid pathway ad regulate the levels of the antifungal compound-diene, inhibit fungal development, resulting in its quiescence. Interestingly, it was also found that growth regulators like cytokinin could do activation of the mechanism of resistance. Postharvest treatments of cytokinins strongly activated the phenylpropanoid pathway and induce resistance. We have developed non-pathogenic strains of C. gloeosporioides by Random Enzyme Mediated Integration and selected a hygromycin resistance, non-pathogenic strain Cg-142 out of 3500 transformants. This non-pathogenic isolate activates H+ATPase and induces resistance against Colletotrichum attack. As a basis for studying the importance of PL in pathogenicity, we have carried out heterologous expression of pel from C. gloeosporioides in the non-pathogenic C. magna and determine the significant increase in pathogenicity of the non-pathogenic strain. Based on these results we can state that pectate lyase is an important pathogenicity factor of C. gloeosporioides and found that fungal pathogenicity is affected not by pel but by PL secretion. Our results suggest that PH regulates the secretion of pectate lyase, and support its importance as a pathogenicity factor during the attack of avocado fruit by C. gloeosporioides . This implicates that if these findings are of universal importance in fungi, control of disease development could be done by regulation of secretion of pathogenicity factors.
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Alfano, James, Isaac Barash, Thomas Clemente, Paul E. Staswick, Guido Sessa e Shulamit Manulis. Elucidating the Functions of Type III Effectors from Necrogenic and Tumorigenic Bacterial Pathogens. United States Department of Agriculture, janeiro de 2010. http://dx.doi.org/10.32747/2010.7592638.bard.
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Many phytopathogenic bacteria use a type III protein secretion system (T3SS) to inject type III effectors into plant cells. In the experiments supported by this one-year feasibility study we investigated type III effector function in plants by using two contrasting bacterial pathogens: Pseudomonas syringae pv. tomato, a necrotrophic pathogen and Pantoea agglomerans, a tumorigenic pathogen. The objectives are listed below along with our major conclusions, achievements, and implications for science and agriculture. Objective 1: Compare Pseudomonas syringae and Pantoea agglomerans type III effectors in established assays to test the extent that they can suppress innate immunity and incite tumorigenesis. We tested P. agglomerans type III effectors in several innate immunity suppression assays and in several instances these effectors were capable of suppressing plant immunity, outputs that are suppressed by P. syringae effectors. Interestingly, several P. syringae effectors were able to complement gall production to a P. agglomerans pthGmutant. These results suggest that even though the disease symptoms of these pathogens are dramatically different, their type III effectors may function similarly. Objective 2: Construct P. syringae mutants in different combinations of type III-related DNA clusters to reduce type III effector redundancy. To determine their involvement in pathogenicity we constructed mutants that lack individual and multiple type III-related DNA clusters using a Flprecombinase-mediated mutagenesis strategy. The majority of single effector mutants in DC3000 have weak pathogenicity phenotypes most likely due to functional redundancy of effectors. Supporting this idea, Poly-DNAcluster deletion mutants were more significantly reduced in their ability to cause disease. Because these mutants have less functional redundancy of type III effectors, they should help identify P. syringae and P. agglomerans effectors that contribute more significantly to virulence. Objective 3: Determine the extent that P. syringae and P. agglomerans type III effectors alter hormone levels in plants. Inhibition of auxin polar transport by 2,3,5-triiodobenzoic acid (TIBA) completely prevented gall formation by P. agglomerans pv. gypsophilae in gypsophila cuttings. This result supported the hypothesis that auxin and presumably cytokinins of plant origin, rather than the IAA and cytokinins secreted by the pathogen, are mandatory for gall formation. Transgenic tobacco with pthGshowed various phenotypic traits that suggest manipulation of auxin metabolism. Moreover, the auxin levels in pthGtransgenic tobacco lines was 2-4 times higher than the control plants. External addition of auxin or cytokinins could modify the gall size in gypsophila cuttings inoculated with pthGmutant (PagMx27), but not with other type III effectors. We are currently determining hormone levels in transgenic plants expressing different type III effectors. Objective 4: Determine whether the P. agglomerans effectors HsvG/B act as transcriptional activators in plants. The P. agglomerans type III effectors HsvG and HsvB localize to the nucleus of host and nonhost plants and act as transcription activators in yeast. Three sites of adjacent arginine and lysine in HsvG and HsvB were suspected to act as Nuclear localization signals (NLS) domains. A nuclear import assay indicated two of the three putative NLS domains were functional NLSs in yeast. These were shown to be active in plants by fusing HsvG and HsvB to YFP. localization to the nucleus was dependent on these NLS domains. These achievements indicate that our research plan is feasible and suggest that type III effectors suppress innate immunity and modulate plant hormones. This information has the potential to be exploited to improve disease resistance in agricultural crops.
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Lindow, Steven, Yedidya Gafni, Shulamit Manulis e Isaac Barash. Role and In situ Regulation of Growth Regulators Produced in Plant-Microbe Interactions by Erwinia herbicola. United States Department of Agriculture, agosto de 1992. http://dx.doi.org/10.32747/1992.7561059.bard.
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The main objective of this work was to gain a better understanding of how some strains of Erwinia herbicola have evolved into serious plant pathogens while also commonly existing as epiphytes on the surface of healthy plants. The focus of our studies was to determine the nature of, and regulation, of virulence factors, including the phytohormones IAA and cytokinins, which are encoded on a large plasmid (pPATH) found in gall-forming strains of this species. In addition, the in situ regulation and contribution to epiphytic fitness of a second, chromosomal, IAA biosynthetic locus (ipdC) was determined to ascertain the relative contribution of the two redundant IAA-biosynthetic pathways to the biology of E. herbicola. Genes (pre-etz and etz) conferring production of cytokinins were clustered immediately 3' of the iaaM and iaaH genes conferring IAA boisynthesis on pPATH. A new insertion-like element, IS1327, was also found immediately 3' of etz on pPATH, suggesting that these virulence factors were all introduced onto pPATH from another pathogenic bacterium. Mutants of E. herbicola in which etz, iaaH, and iaaM, but not ipdC, were disrupted caused smaller galls to form on gypsophila plants. In contrast, ipdC but not iaaH or iaaM mutants of E. herbicola exhibited reduced ability to grow and survive on plant surfaces. Transcription of ipdC was induced when cells were on plants compared to in culture, suggesting that idpC may play a selective role in fitness on leaves.
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Splitter, Gary A., Menachem Banai e Jerome S. Harms. Brucella second messenger coordinates stages of infection. United States Department of Agriculture, janeiro de 2011. http://dx.doi.org/10.32747/2011.7699864.bard.
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Aim 1: To determine levels of this second messenger in: a) B. melitensiscyclic-dimericguanosinemonophosphate-regulating mutants (BMEI1448, BMEI1453, and BMEI1520), and b) B. melitensis16M (wild type) and mutant infections of macrophages and immune competent mice. (US lab primary) Aim 2: To determine proteomic differences between Brucelladeletion mutants BMEI1453 (high cyclic-dimericguanosinemonophosphate, chronic persistent state) and BMEI1520 (low cyclicdimericguanosinemonophosphate, acute virulent state) compared to wild type B. melitensisto identify the role of this second messenger in establishing the two polar states of brucellosis. (US lab primary with synergistic assistance from the Israel lab Aim 3: Determine the level of Brucellacyclic-dimericguanosinemonophosphate and transcriptional expression from naturally infected placenta. (Israel lab primary with synergistic assistance from the US lab). B. Background Brucellaspecies are Gram-negative, facultative intracellular bacterial pathogens that cause brucellosis, the most prevalent zoonosis worldwide. Brucellosis is characterized by increased abortion, weak offspring, and decreased milk production in animals. Humans are infected with Brucellaby consuming contaminated milk products or via inhalation of aerosolized bacteria from occupational hazards. Chronic human infections can result in complications such as liver damage, orchitis, endocarditis, and arthritis. Brucellaspp. have the ability to infect both professional and non-professional phagocytes. Because of this, Brucellaencounter varied environments both throughout the body and within a cell and must adapt accordingly. To date, few virulence factors have been identified in B. melitensisand even less is known about how these virulence factors are regulated. Subsequently, little is known about how Brucellaadapt to its rapidly changing environments, and how it alternates between acute and chronic virulence. Our studies suggest that decreased concentrations of cyclic dimericguanosinemonophosphate (c-di-GMP) lead to an acute virulent state and increased concentrations of c-di-GMP lead to persistent, chronic state of B. melitensisin a mouse model of infection. We hypothesize that B. melitensisuses c-di-GMP to transition from the chronic state of an infected host to the acute, virulent stage of infection in the placenta where the bacteria prepare to infect a new host. Studies on environmental pathogens such as Vibrio choleraeand Pseudomonas aeruginosasupport a mechanism where changes in c-di-GMP levels cause the bacterium to alternate between virulent and chronic states. Little work exists on understanding the role of c-di-GMP in dangerous intracellular pathogens, like Brucellathat is a frequent pathogen in Israeli domestic animals and U.S. elk and bison. Brucellamust carefully regulate virulence factors during infection of a host to ensure proper expression at appropriate times in response to host cues. Recently, the novel secondary signaling molecule c-di-GMP has been identified as a major component of bacterial regulation and we have identified c-di-GMP as an important signaling factor in B. melitensishost adaptation. C. Major conclusions, solutions, achievements 1. The B. melitensis1453 deletion mutant has increased c-di-GMP, while the 1520 deletion mutant has decreased c-di-GMP. 2. Both mutants grow similarly in in vitro cultures; however, the 1453 mutant has a microcolony phenotype both in vitro and in vivo 3. The 1453 mutant has increased crystal violet staining suggesting biofilm formation. 4. Scanning electron microscopy revealed an abnormal coccus appearance with in increased cell area. 5. Proteomic analysis revealed the 1453 mutant possessed increased production of proteins involved in cell wall processes, cell division, and the Type IV secretion system, and a decrease in proteins involved in amino acid transport/metabolism, carbohydrate metabolism, fatty acid production, and iron acquisition suggesting less preparedness for intracellular survival. 6. RNAseq analysis of bone marrow derived macrophages infected with the mutants revealed the host immune response is greatly reduced with the 1453 mutant infection. These findings support that microlocalization of proteins involved in c-di-GMP homeostasis serve a second messenger to B. melitensisregulating functions of the bacteria during infection of the host.
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Fromm, Hillel, Paul Michael Hasegawa e Aaron Fait. Calcium-regulated Transcription Factors Mediating Carbon Metabolism in Response to Drought. United States Department of Agriculture, junho de 2013. http://dx.doi.org/10.32747/2013.7699847.bard.
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Original objectives: The long-term goal of the proposed research is to elucidate the transcription factors, genes and metabolic networks involved in carbon metabolism and partitioning in response to water deficit. The proposed research focuses on the GTLcalcium/calmodulinbindingTFs and the gene and metabolic networks modulated by these TFs in Arabidopsis thaliana. The specific objectives are as follows. Objective-1 (USA): Physiological analyses of GTL1 loss- and gain-of-function plants under water sufficient and drought stress conditions Objective 2 (USA / Israel-TAU): Characterizion of GTL target genes and bioinformatic analysis of data to eulcidate gene-network topology. Objective-3 (Israel-TAU): Regulation of GTLmediated transcription by Ca²⁺/calmodulin: mechanism and biological significance. Objective-4 (Israel-BGU): Metabolic networks and carbon partitioning in response to drought. Additional direction: In the course of the project we added another direction, which was reported in the 2nd annual report, to elucidate genes controlling drought avoidance. The TAU team has isolated a few unhydrotropic (hyd) mutants and are in the process of mapping these mutations (of hyd13 and hyd15; see last year's report for a description of these mutants under salt stress) in the Arabidopsis genome by map-based cloning and deep sequencing. For this purpose, each hyd mutant was crossed with a wild type plant of the Landsberg ecotype, and at the F2 stage, 500-700 seedlings showing the unhydrotropic phenotype were collected separately and pooled DNA samples were subkected to the Illumina deep sequencing technology. Bioinformatics were used to identify the exact genomic positions of the mutations (based on a comparison of the genomic sequences of the two Arabidopsis thaliana ecotypes (Columbia and Landsberg). Background: To feed the 9 billion people or more, expected to live on Earth by the mid 21st century, the production of high-quality food must increase substantially. Based on a 2009 Declaration of the World Summit on Food Security, a target of 70% more global food production by the year 2050 was marked, an unprecedented food-production growth rate. Importantly, due to the larger areas of low-yielding land globally, low-yielding environments offer the greatest opportunity for substantial increases in global food production. Nowadays, 70% of the global available water is used by agriculture, and 40% of the world food is produced from irrigated soils. Therefore, much needs to be done towards improving the efficiency of water use by plants, accompanied by increased crop yield production under water-limiting conditions. Major conclusions, solutions and achievements: We established that AtGTL1 (Arabidopsis thaliana GT-2 LIKE1) is a focal determinant in water deficit (drought) signaling and tolerance, and water use efficiency (WUE). The GTL1 transcription factor is an upstream regulator of stomatal development as a transrepressor of AtSDD1, which encodes a subtilisin protease that activates a MAP kinase pathway that negatively regulates stomatal lineage and density. GTL1 binds to the core GT3 cis-element in the SDD1 promoter and transrepresses its expression under water-sufficient conditions. GTL1 loss-of-function mutants have reduced stomatal number and transpiration, and enhanced drought tolerance and WUE. In this case, higher WUE under water sufficient conditions occurs without reduction in absolute biomass accumulation or carbon assimilation, indicating that gtl1-mediated effects on stomatal conductance and transpiration do not substantially affect CO₂ uptake. These results are proof-of-concept that fine-tuned regulation of stomatal density can result in drought tolerance and higher WUE with maintenance of yield stability. Implications: Accomplishments during the IS-4243-09R project provide unique tools for continued discovery research to enhance plant drought tolerance and WUE.