An increasing appreciation for the role of small RNA regulators prompted us to investigate the scope of RNA regulation in the bacterium, Streptomyces coelicolor. Our search revealed an antisense RNA that corresponds to the upstream region of four genes encoding cell wall cleavage enzymes (cell wall hydrolases), and a previously uncharacterized population of transfer RNA (tRNA) cleavage products. Further characterization of the 'tRNAs led to the discovery that S. coelicolor tRNAs are cleaved into 'tRNA halves' in a developmentally regulated fashion. All tRNAs seem to be susceptible to tRNA cleavage, although a bias was detected for tRNAs specifying highly used codons. To date, our work is the sole description of 'tRNA half production in a bacterium, and recent studies suggest that it is a widespread phenomenon among eukaryotic organisms. In a separate line of investigation, we noticed that a previous study had predicted that the genes associated with the antisense RNA are under the control of a riboswitch- a regulatory RNA element that directly controls gene expression in response to specific conditions. Our multifaceted characterization of this system began with the construction and phenotypic analyses of deletion mutant strains for several of the cell wall hydrolase-encoding genes. We demonstrate that S. coelicolor cell wall hydrolases are involved in germination, vegetative growth, and sporulation. Finally, we studied the potential for riboswitch regulation of one of the cell wall hydrolase-encoding genes, rpfA. RpfA is a resuscitation: Qromoting factor protein that is important for the revival of dormant bacteria, including the human pathogen and S. coelicolor relative - Mycobacterium tuberculosis. Our investigation uncovered evidence suggesting that the riboswitch region is involved in the regulation of rpfA, and we identified specific conditions under which it is repressed. This work represents a novel paradigm in the regulation of cell wall hydrolase expression. / Thesis / Doctor of Philosophy (PhD)

Mycobacteria are atypical bacteria possessing unusual cell envelopes comprised of an outer membrane, covalently linked to an arabinogalacatan-peptidoglycan structure via waxy mycolic acids, in addition to the conventional inner membrane. This thick and highly impermeable cell envelope is a major deterrent to antibiotic treatment of clinically relevant mycobacterial pathogens, including Mycobacterium tuberculosis (Mtb), which infects a third of the world’s population and kills millions each year. Thus, the regulation of mycobacterial cell envelope biosynthesis is of great interest for the development of more effective therapeutics for treating Mtb infections. Using the model organism Mycobacterium smegmatis (M. smegmatis), we identified a novel protein, Spe2, with an unknown role in the biosynthesis of the cell envelope glycolipids lipomannan (LM) and lipoarabinomannan (LAM). Based on the observation that Δspe2 mutants produce truncated LM/LAM, I speculated Spe2 might enhance the elongation of these products. Here, I use biochemical assays to show Spe2 is localized to the periplasm where it can directly interact with the LM/LAM biosynthetic pathway. I further utilize a genetic approach to demonstrate that Spe2 acts at the stage in which the mannosyltransferase MptA mediates periplasmic LM elongation. Moreover, native polyacrylamide gel electrophoresis (PAGE) and co-immunoprecipitation techniques failed to reveal Spe2 protein binding partners. Together, these data suggest Spe2 is a periplasmic protein involved in regulating LM/LAM biosynthesis, perhaps through direct interactions with LM intermediates.

No description available.

Le potentiel de valorisation, aussi bien pour l'alimentation animale que pour la production bioénergétique, du maïs est limité par la lignification et la réticulation des parois. L'amélioration de la dégradabilité enzymatique des lignocelluloses est un objectif important. Nous nous sommes attachés dans ce travail à l'étude approfondie de la mise en place de la lignification dans la paroi chez le maïs ainsi qu'aux facteurs biochimiques et anatomiques pariétaux qui peuvent avoir un impact sur la dégradabilité des parois. Dans la première partie de ma thèse, 8 lignées recombinantes de maïs ont été sélectionnées sur la base d'une teneur en lignine comparable pour évaluer l'impact des facteurs biochimiques sur la dégradabilité des parois et sur les performances agronomiques des maïs. Ces lignées recombinantes et leurs parents ont été analysés biochimiquement de façon approfondie. Le rendement β-O-4 au sein des lignines et la teneur en acide p-coumarique estérifié sont les deux facteurs les plus corrélés négativement et significativement à la dégradabilité des parois. Une analyse par régression multiple a montré que plus de 80% des variations de la dégradabilité de paroi, dans ces dix lignées, sont expliqués par un modèle retenant comme meilleurs régresseurs la teneur en lignines combinée au pourcentage d'unité S acylées par l'acide p-coumarique. L'étude morphologique que nous avons réalisée nous a montré que les facteurs biochimiques limitant la dégradabilité de paroi peuvent par contre s'avérer favorables pour assurer la performance agronomique de la plante via des mécanismes de plasticité pariétale. Cependant et de façon très encourageante, nous avons aussi montré qu'il était tout à fait possible de retenir du matériel amélioré en termes de qualité de paroi et qui conservait de bonnes performances agronomiques. Dans la deuxième partie de ma thèse, nous avons étudié le schéma de mise en place des parois au cours du développement des plantes pour des lignées présentant des teneurs en lignine comparables au stade ensilage mais des dégradabilités de parois contrastées. Nous avons caractérisé biochimiquement les entrenoeuds au cours du développement. En parallèle, nous avons développé une méthode de quantification de la variation de distribution spatiale des lignines au long de la coupe afin de compléter notre étude au cours du développement par une caractérisation histologique détaillée. D'un point de vue temporel, les différentes composantes pariétales sont incorporées graduellement, en fonction du stade de développement. Trois étapes majeures de développement ont été identifiées. D'un point de vue spatial, au sein de l'entrenoeud, le développement des parois est différent selon les régions. Une importante variabilité génétique a été observée pour le développement sur ces deux axes et ces variations sont associées à la variation de la dégradabilité de paroi. L'ensemble des résultats de ce travail est discuté d'une part en termes de contribution à la compréhension de la lignification des parois ainsi qu'à l'impact des différents facteurs biochimiques et histologiques sur la dégradabilité et d'autre part en termes d'application afin de proposer des solutions pour améliorer de la qualité des parois. / Maize potential value, as well as animal feed or as bioenergetics resources, is limited by the lignification of the cell wall and by the cross-linking between cell wall components. Improvement of cell wall enzymatic degradability is an important goal. In the present work we focused on the study of cell wall lignification including its establishment and the biochemical or anatomical cell wall related factors in order to investigate the impact of all these characteristics on cell wall degradability. In the first part of my work, 8 maize recombinant inbred lines were selected on the basis of their comparable lignin content and their contrasted cell wall degradability in order to assess the impact of biochemical traits on both the cell wall degradability and the plant agronomic performances independently of the main “lignin content” factor. These recombinant inbred lines and their parents were analyzed for esterified and etherified p-hydroxycinnamic acid content, for lignin content, composition and structure and for in vitro cell wall degradability. Lignin structure and esterified p-coumaric acid content were highly and significantly correlated with in vitro cell wall degradability. A multiple regression analysis showed that more than 80 % of cell wall degradability variations were explained by a regression model including two main explanatory factors: the lignin content and the estimated proportion of S lignin units esterified by p-coumaric acid. The morphological study showed that the biochemical factors limiting cell wall degradability were on the contrary favorable for ensuring agronomic performances. In the second part of my work, we studied the cell wall developmental pattern during maize internode growth. We compared the developmental pattern of 3 maize lines which presented quite similar lignin content but very different cell wall degradability at silage stage. We first characterized biochemically internodes sampled throughout plant development. Subsequently, we developed a method to quantify the variation of lignin spatial distribution within the whole stem section. At last, we completed our study of cell wall establishment by an anatomical characterization. From a temporal point of view, the different wall components were incorporated gradually during the development and three major steps were identified. From a spatial point of view, in the internode, the development of cell wall is different according to the considered region. Clearly genetic variations were observed for developmental pattern in both the two axes and were found to be associated with the variation of cell wall degradability. The results of this work were discussed firstly in terms of contribution to the understanding of cell wall lignification and of relationships between biochemical and anatomical factors with cell wall degradability variations and secondly in terms of application to propose solutions to improve cell wall quality.

In the yeast Saccharomyces cerevisiae, stress on the cell wall is caused by various external influences (e.g. exposure to chemicals, oxidative stress, osmotic changes, pH changes or heat shock) which trigger the cell wall integrity signalling pathway (CWI). The aim of my work was to investigate the effect of the CWI pathway on yeast colony morphogenesis. Using strains with deletions in genes of the CWI pathway derived from two parental strains BR-F-Flo11p-GFP and PORT, I have found that differences in genetic background influences the process and activation of this pathway. Among the strains derived from BR-F-Flo11p-GFP, only the strain with the deletion of MID2 affects the appearance of colonies. MID2 encodes a cell-surface sensor of CWI pathway. In all deletion strains derived from PORT, the disruption of the CWI pathway causes a slower development of colonies growing on glycerol medium supplemented with 0,05 mM selenate inducing fluffy colony morphology. The largest effect has deletion of gene MTL1 which also encodes a cell-surface sensor with homology to Mid2. I have confirmed that strains with deletions in genes of CWI pathway have altered sensitivity to inhibitors disrupting cell wall integrity (Calcofluor white, Congo red, zymolyase). By means of zymolyase assay, I have confirmed the...

Evidence suggests that root cell walls are a target of low pH stress. Severe low pH stress causes cell death in the root tip. The walls of these cells are highly dynamic. Our hypothesis is that in these cells low pH causes stress in the cell wall due to excessive loosening. Thus, a certain level of turgor pressure should be required to cause cell death. Here, we aimed to investigate the role of the cell wall in low pH stress leading to cell death. We looked for the possible involvement of players such as class III peroxidases and ethylene signaling, which could promote changes in the cell wall and cause differential sensitivity to low pH. Arabidopsis thaliana and mutants in the genetic background of Col-0 were grown in a medium containing agar (0.8%) and half the concentration of Hoagland\'s nutrient medium. Five-day-old seedlings were exposed to low pH in a solution composed of 0.5 mM CaCl2 and 0.6 mM Homopipes buffer. Treatment of roots at pH 4.6 caused death of cells in the transition zone (TZ) and meristematic zone (MZ). However, cell death was negligible when plants were treated at pH 4.6 in an hyperosmotic solution (Ψs = -0.37 MPa), thereby decreasing cell wall tension. Also, an hypoosmotic treatment (HO) caused cell death at pH 5.8 in TZ. Cell death was accelerated when HO was performed in a low pH solution. The mutant of a cell wall integrity sensor protein, wak-1, displayed reduced cell death when exposed to low pH. Also, cell death seems to occur through a programmed cell death mechanism. Thus, low-pH induced cell death appears to be triggered by perception of cell wall stress. We examined published data to search for class III peroxidases possibly involved in cell death due to low pH. The gene for AtPrx62 is induced 8.37-fold in low pH exposed roots. The atprx62 KO mutant was less sensitive to low pH than Col-0 roots. The mRNA of AtPRX62 accumulated in the same zone that cell death occurred due to low pH. This strongly suggests that AtPRX62 is positive regulator of low-pH induced cell death. Also, ethylene pretreatment induced subsequent tolerance of roots to low pH and this was dependent of its receptor ETR1. Together we show that a cell wall stress caused by low pH causes cell death. This death was in part due AtPRX62 activity and was also suppressed by ethylene. / Evidencias recentes sugerem que a parede celular é um alvo direto do estresse por baixo pH em raízes. Estresse severo por baixo pH rapidamente causa a morte de células do ápice radicular, onde a parede é altamente dinâmica. Nossa hipótese é de que nessas células, o baixo pH cause mudanças na parede celular, como afrouxamento excessivo. Assim, a pressão de turgor sobre a parede deve ser necessária para causar danos que levam à morte das células. Neste trabalho, nós investigamos o papel da parede celular no estresse por baixo pH e na consequente morte de células radiculares. Além disso, tambem foi investigado o papel de peroxidases de classe III e sinalização por etileno, que promovem mudanças na parede celular as quais podem gerar sensibilidade diferenciada a baixo pH. Plântulas de Arabidopsis thaliana e mutantes no background de Col-0 foram crescidas em meio contendo ágar (0.8%) e metade da concentração dos nutirentes do meio de Hoagland. Plântulas com 5 dias de idade foram expostas a baixo pH em uma solução composta por 0.5 mM de CaCl2 e 0.6 mM de tampão Homopipes. O tratamento de raízes a pH 4.6 causou morte em células da zona de transição (TZ) e zona meristemática (MZ). Entretanto, a morte celular foi negligível quando as plantas foram tratadas a pH 4.6 simultaneamente com a diminuição da tensão na parede celular, através de solução com potencial de - 0.37 MPa. Além disso, um choque repentino na pressão de tugor por intermédio de tratamento hiposmótico (HO) causou morte celular a pH 5.8 na TZ. A morte celular foi acelerada quando HO foi realizado em uma solução a baixo pH. A morte celular foi reduzida no mutante wak-1 exposto a baixo pH. WAK-1 é um receptor de parede que atua no sistema de monitoramento de integridade da parede celular. A morte das células provavelmente ocorreu por meio de morte celular programada. Juntos, esses dados trazem evidências que a parede celular é crucial para percepção do estresse causado por baixo pH e essa percepção possivelmente está envolvida em repostas que causam a morte celular. Nós examinamos dados publicados procurando por peroxidases classe III possivelmente envolvidas com a morte celular devido baixo pH. O gene codante para AtPRX62 foi induzido 8.37 vezes em raízes expostas a baixo pH. O mutante KO atprx62 foi menos sensível a baixo pH que raízes de Col-0. O mRNA de AtPRX62 acumulou-se na mesma zona de morte celular devido baixo pH em raízes de Col-0. Isso sugere que a atividade de AtPRX62 está relacionada com a morte celular devido baixo pH. Além disso, o pré-tratamento com etileno induziu tolerância de raízes à exposição subsequente a baixo pH. Esta indução foi dependente de sinalização via ETR1. No conjunto, nós mostramos que um estresse causado na parede celular pelo baixo pH causa a morte celular. Essa morte é em parte devido a atividade de AtPRX62 mas pode ser aliviada por etileno.

No description available.

Made available in DSpace on 2015-03-26T13:51:58Z (GMT). No. of bitstreams: 1 texto completo.pdf: 3002168 bytes, checksum: 936461b9e52c3ed25ff8924c9843452e (MD5) Previous issue date: 2013-10-29 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Basidiospores of the ectomycorrhizal fungus P. microcarpus are characterized by impermeable, hydrophobic cell walls. These features are possibly related to the low germination percentages of these propagules and make it difficult the isolation of monokaryons and the use of these spores as inoculants. Sodium hypochlorite can be used as a permeabilizing agent of the cell wall of fungal spores. The aim of this study was to evaluate the effects of permeabilization treatments with commercial bleach on the cell wall ultrastructure and hydrophobicity, and on the viability and germination of P. microcarpus basidiospores. For this, fungal basidiospores, from fruiting bodies associated with Eucalyptus spp., were collected and permeabilized using different concentrations of bleach and times of exposure. After permeabilization, the basidiospores were analyzed by scanning and transmission electron microscopy. Surface hydrophobicity, viability, and germination of these propagules were also analyzed. The percentage of permeabilized basidiospores of P. microcarpus was proportional to the increases in bleach concentration and the exposure time. Basidiospores from different fruiting bodies differed significantly in their susceptibility to the permeabilization treatments with bleach and in the decrease of cell surface hydrophobicity after permeabilization. Changes in the ultrastructure of permeabilized basidiospores were observed at bleach concentrations of 15 and 50 %, with an exposure time of 40 s. For one basidiocarp, after permeabilization with bleach at 5 % for 40 s, 80% of the permeabilized basidiospores were viable. The plating of basidiospores permeabilized with 10% bleach for 40 s resulted in the production of colonies of P. microcarpus. The colonies appeared after 15 days of incubation of the permeabilized basidiospores in the presence of the host plant, E. citriodora. The germination percentage obtained, 0,001 %, was similar to those reported for non-permeabilized basidiospores. This work is the first report on the ultrastructure of the cell wall of basidiospores of P. microcarpus and contributes to the understanding of the recalcitrance of these propagules to germination. / Basidiósporos do fungo ectomicorrízico P. microcarpus apresentam parede celular impermeável e hidrofóbica. Essas características estão possivelmente relacionadas às baixas porcentagens de germinação desses propágulos, dificultando a obtenção de monocários e a utilização desses esporos em inoculantes. O hipoclorito de sódio pode ser usado como agente permeabilizador da parede celular de esporos fúngicos. O objetivo deste estudo foi avaliar os efeitos do tratamento de permeabilização com água sanitária sobre a ultraestrutura e hidrofobicidade da parede celular, a viabilidade e a capacidade de germinação de basidiósporos de P. microcarpus. Para isso, basidiósporos fúngicos, oriundos de corpos de frutificações associados a plantas de Eucalyptus spp., foram coletados e permeabilizados utilizando-se diferentes concentrações de água sanitária e tempos de exposição ao composto. Após a permeabilização, os basidiósporos foram analisados por microscopia eletrônica de varredura e de transmissão. A hidrofobicidade superficial, a viabilidade e a germinação desses propágulos também foram analisadas. A porcentagem de basidiósporos de P. microcarpus permeabilizados foi proporcional ao aumento na concentração de água sanitária e ao tempo de exposição. Basidiósporos oriundos de diferentes basidiocarpos diferiram de forma significativa na susceptibilidade ao tratamento de permeabilização com água sanitária e na redução da hidrofobicidade da superfície celular após esse tratamento. Alterações da ultraestrutura dos basidiósporos permeabilizados foram observadas nas concentrações de 15 e 50 % de água sanitária pelo tempo de exposição de 40 s. Para um dos basidiocarpos avaliados e após a permeabilização com água sanitária a 5 % por 40 s, 80 % dos esporos permeabilizados encontravam-se viáveis. O inóculo dos basidiósporos permeabilizados com água sanitária a 10 % por 40 s resultou na produção de algumas colônias de P. microcarpus. As colônias apareceram após 15 dias de incubação dos basidiósporos permeabilizados na presença da planta hospedeira, Eucalyptus citriodora. A porcentagem de germinação obtida, 0,001%, foi semelhante àquelas relatadas na literatura para basidiósporos não-permeabilizados. Este trabalho é o primeiro relato sobre a ultraestrutura da parede celular dos basidiósporos de P. microcarpus e contribui para a compreensão da recalcitrância desses propágulos à germinação.

Self-assembling protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and many additional processes, which makes them appealing targets for therapeutics and biotechnological applications, including live vaccines, liposome drug delivery and biosensors. Unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of specialized S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Characterization of this interaction in the Gram-positive model organism Paenibacillus alvei CCM 2051T reveals that, remarkably, binding-site switches can occur between two distinct SLH-domain SCWP receptor-site grooves in the S-layer protein SpaA, possibly as part of a mechanism to alleviate strain in the S-layer. To date, however, analysis of this novel mechanism has been limited to the terminal SCWP monosaccharide and the internal SCWP repeat disaccharide ligand analogues, leaving open the role of subsequent SCWP sugar residues in binding, as well as whether the two receptor sites are also suited to accommodate longer SCWP ligands that better approximate the biological target at the surface of P. alvei. To address this, the objective of this work aims to uncover and characterize the details of the SpaA SLH-domain (SpaASLH¬) SCWP-interaction by determining the co-crystal structures of SpaASLH¬, and single (SpaASLH/G109A) and the corresponding double (SpaASLH/G46A/G109A) mutants in complex with synthetic terminal disaccharide and trisaccharide analogues of the P. alvei CCM 2051T SCWP target. These structural characterizations have been supplemented with disaccharide and trisaccharide binding data, which was obtained through thermodynamic ITC analyses carried out by collaborators. The co-crystal structures of P. alvei SpaASLH with synthetic, terminal SCWP disaccharide and trisaccharide analogues, together with previously published monosaccharide-bound SpaASLH structures, reveal that while the SLH trimer accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly-exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations for longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division. In addition, to further clarify the steps involved in SCWP biosynthesis, we present a crystal structure of the unliganded UDP-GlcNAc 2-epimerase enzyme MnaA, which catalyzes the interconversion of UDP-GlcNAc into UDP-ManNAc—an essential building block of the P. alvei SCWP target. The P. alvei MnaA epimerase adopts a GT-B fold that is consistent with the architecture of previously published structures of other bacterial non-hydrolyzing UDP-GlcNAc 2-epimerase enzymes for which substrate binding is observed in the cleft located between the two domains. Characterization of this structure, coupled with an analysis of the sequence of the MnaA protein, reveals the presence of conserved residues that define the catalytic and allosteric sites in homologous enzymes from different organisms. These residues are positioned to accommodate substrate within the MnaA binding cleft in much the same manner as the published enzyme homologues, suggesting that allosteric regulation as a mechanism for enzyme regulation is conserved in P. alvei MnaA. These investigations are part of a greater effort toward understanding SLH domain-mediated SCWP-interactions in Gram-positive organisms, and provide insight into the structure and putative function of this SCWP biosynthetic enzyme. By understanding these processes, this knowledge may contribute to providing a platform for the rational design of Gram-positive inhibitors. Such inhibitors could selectively target, for example, the bacterial S-layer SCWP-binding interaction, or perhaps the essential biosynthetic enzymes involved in producing the exclusive targets that these S-layer proteins recognize and bind, and would thus represent a new class of antimicrobial therapeutics. / Graduate

<p>Phospho-MurNAc-pentapeptide translocase (MraY) is a conserved membrane-spanning enzyme involved in the biosynthesis of bacterial cell walls. MraY generates lipid I by transferring the phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl-phosphate. MraY is a primary target for antibiotic development because it is essential in peptidoglycan synthesis and targeted by 5 classes of natural product antibiotics. The structure of this enzyme will provide insight into the catalytic mechanism and a platform for future antibiotic development. MraY genes from 19 bacteria were cloned, expressed, purified and assayed for biochemical stability. After initial crystallization screening, I found that MraY from Aquifex aeolicus (MraYAA) produced diffracting crystals. Recombinant MraYAA is functional and shows inhibition by the natural inhibitor capuramycin. After extensive optimization of crystallization conditions, we extended the resolution limit of the crystal to 3.3 Å. The crystal structure, the first structure of the polyprenyl-phosphate N-acetyl hexosamine 1-phosphate transferase (PNPT) superfamily, reveals the architecture of MraYAA and together with functional studies, allow us to identify the location of Mg2+ at the active site and the putative binding sites of both substrates. My crystallographic studies provide insights into the mechanism of how MraY attaches a building block of peptidoglycan to the carrier lipid.</p><p>Voltage-gated Na+ (NaV) channels initiate action potentials in neurons and cardiac myocytes. NaV channels are composed of a transmembrane domain responsible for voltage-dependent Na+ conduction and a cytosolic C-terminal domain (CTD) that regulates channel function through interactions with many auxiliary proteins including members of the fibroblast growth factor homologous factor (FHF) family and calmodulin (CaM). Through the collaboration between our lab and Geoffrey Pitt's lab, we report the first crystal structure of the ternary complex of the human NaV1.5 CTD, FGF13, and Ca2+-free CaM at 2.2 Å. Combined with functional experiments based on structural insights, we present a platform to understand roles of these auxiliary proteins in NaV channel regulation and the molecular basis of mutations that lead to neuronal and cardiac diseases. Furthermore, we identify a critical interaction that contributes to the specificity between individual NaV CTD isoforms and distinctive FHFs.</p> / Dissertation