No description available.

The ecology, species distribution, and population structure of Armillaria was investigated in the forests of Massachusetts. From 64 plots at 16 sites, 640 isolates of Armillaria were collected from six forest types (northern hardwoods, mixed oak, pitch pine, white pine, white pine/mixed oak, and eastern hemlock). Armillaria gallica proved to be the most abundant species, making up 316/640 (52%) of all isolations. This was followed by A. solidipes (219/640; 34%), A. mellea (46/640; 7%), A. calvescens (36/640; 6%), A. gemina (16/640; 3%), and A. sinapina (7/640; 1%). Armillaria gallica was routinely encountered causing significant decay of the lower bole on living hardwood hosts, especially oaks. The population structure of 153 isolates of A. gallica collected from mixed oak forests was investigated using amplified fragment length polymorphisms (AFLPs). From a total sampling area of 4.51 ha, 38 AFLP genotypes were discovered, yielding a figure of eight genets per hectare with the average A. gallica genet occupying 0.13 ha. When the effects of hydrolyzable tannins on in vitro growth were compared between A. calvescens and A. gallica, it was A. gallica that appeared better at oxidizing and metabolizing commercial tannins (tannic acid and gallic acid) along with black oak root bark extracts. This was determined through measurements of colony area and dry biomass, and suggests that A. gallica may be a better adapted pathogen of oak. In order to more accurately distinguish between isolates of A. calvescens and A. gallica, a three-gene phylogeny was reconstructed, using partial sequences of the elongation factor 1-alpha (tef1), RNA polymerase II (rpb2) and nuclear large subunit (nLSU) genes. After comparing 12 isolates each of A. calvescens and A. gallica that originated from across northeastern North America, only the tef1 gene could accurately distinguish these two species. Five single nucleotide polymorphisms were present between the two species and maximum likelihood and maximum parsimony methods grouped A. calvescens and A. gallica into monophyletic clades.

No description available.

Abnormal expression of the eukaryotic translation elongation factor 1A (eEF1A) has been implicated in disease states such as motor neuron degeneration and cancer. Two variants of eEF1A are found in mammals, named eEF1A1 and eEF1A2. These two variants are encoded by different genes, produce proteins which are 92% identical but have very different patterns of expression. eEF1A1 is almost ubiquitously expressed while eEF1A2 is expressed only in specialised cell types such as motor neurons and muscle. A spontaneous mutation in eEF1A2 results in the wasted mouse phenotype which shows similar characteristics in the mouse to those seen in human motor neuron degeneration. This mutation has been shown to be a 15.8kb deletion resulting in the complete loss of the promoter region and first non coding exon of eEF1A2 which completely abolishes protein expression. The main aim of this project was to further investigate the role of eEF1A2 in motor neuron degeneration. Firstly, although the wasted phenotype is considered to be caused by a recessive mutation, I established a cohort of aged heterozygote mice to evaluate whether any changes are seen later in life that might model late onset motor neuron degeneration. A combination of behavioural tests and pathology was used to compare wild type and heterozygous mice up to 21 months of age. Whilst results indicate that there is no significant difference between ageing heterozygotes and wildtype controls, there is an indication that female heterozygote mice perform slightly worse that wildtype controls on the rotarod (a behavioural test for motor function). Secondly, I aimed to investigate the primary cause of the wasted pathology by generating transgenic wasted mice expressing neuronal eEF1A2 only. This would complement previous experiments in the lab which studied transgenic wasted mice expressing eEF1A2 in muscle only. Unfortunately the expression of eEF1A2 in the transgenic animals was not neuronal specific. However a transgenic line with expression of eEF1A2 in neurons and skeletal muscle but not cardiac muscle has been generated which clearly warrants further investigation. Thirdly, I wished to assess whether eEF1A2 has any role in human motor neuron degeneration. To achieve this, eEF1A2 expression was investigated in spinal cords from human motor neuron disease (MND) patients. Preliminary data suggests that motor neurons from some MND patients express significantly less eEF1A2 than motor neurons of control samples. Further work is required to confirm these findings. Finally, I investigated the individual roles of eEF1A1 and eEF1A2 in the heat shock response. I used RNAi to ablate each variant separately in cells and subsequently measured the ability of each variant individually to mount a heat shock response. Results indicate a clear role for eEF1A1 but not eEF1A2 in the induction of heat shock. This may explain in part why motor neurons exhibit a poor heat shock response as they express eEF1A2 and not eEF1A1. These experiments shed light on our understanding of the role of eEF1A2 in motor neuron degeneration and uncover many new avenues of future investigation.

Protein synthesis, translation, performed by the ribosome, is a fundamental process of life and one of the main targets of antibacterial drugs. This thesis provides structural and biochemical understanding of three aspects of bacterial translation. Elongation factor G (EF-G) is the target for the antibiotic fusidic acid (FA). FA binds to EF-G only on the ribosome after GTP hydrolysis and prevents EF-G dissociation from the ribosome. Point mutations in EF-G can lead to FA resistance but are often accompanied by a fitness cost in terms of slower growth of the bacteria. Secondary mutations can compensate for this fitness cost while resistance is maintained. Here we present the crystal structure of the clinical FA drug target, Staphylococcus aureus EF-G, together with the mapping and analysis of all known FA-resistance mutations in EF-G. We also present crystal structures of the FA-resistant mutant F88L, the FA-hypersensitive mutant M16I and the FA-resistant but fitness-compensated double mutant F88L/M16I. Analysis of mutant structures together with biochemical data allowed us to propose that fitness loss and compensation are caused by effects on the conformational dynamics of EF-G on the ribosome. Aminoglycosides are another group of antibiotics that target the decoding region of the 30S ribosomal subunit. Resistance to aminoglycosides can be acquired by inactivation of the drugs via enzymatic modification. Here, we present the first crystal structure an aminoglycoside 3’’ adenyltransferase, AadA from Salmonella enterica. AadA displays two domains and unlike related structures most likely functions as a monomer. Frameshifts are deviations the standard three-base reading frame of translation. -1 frameshifting can be caused by normal tRNASer3 at GCA alanine codons and tRNAThr3 at CCA/CCG proline codons. This process has been proposed to involve doublet decoding using non-standard codon-anticodon interactions. In our study, we showed by equilibrium binding that these tRNAs bind with low micromolar Kd to the frameshift codons. Our results support the doublet-decoding model and show that non-standard anticodon loop structures need to be adopted for the frameshifts to happen. These findings provide new insights in antibiotic resistance and reading-frame maintenance and will contribute to a better understanding of the translation elongation process.

Protein synthesis is a highly complex process executed by the ribosome in coordination with mRNA, tRNAs and translational protein factors. Several antibiotics are known to inhibit bacterial protein synthesis by either targeting the ribosome or the proteins factors involved in translation. Fusidic acid (FA) is a bacteriostatic antibiotic that blocks polypeptide chain elongation by locking elongation factor-G (EF-G) on the ribosome. Mutations in fusA, the gene encoding bacterial EF-G, confer high-level of resistance towards FA.  Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by acquiring secondary mutations. In order to understand the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that, the causes for fitness loss in the FA-resistant mutant F88L are resulting from significantly slower tRNA translocation and ribosome recycling. Analysis of the crystal structures, together with the results from our biochemical studies enabled us to propose that FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome. EF-G is a G-protein belonging to the GTPase super-family. In all the translational GTPases, a conserved histidine (H92 in E. coli EF-G) residue, located at the apex of switch II in the G-domain is believed to play a crucial role in ribosome-stimulated GTP hydrolysis and inorganic phosphate (Pi) release. Mutagenesis of H92 to alanine (A) and glutamic acid (E) showed different degree of defect in different steps of translation. Compared to wild type (WT) EF-G, mutant H92A showed a 10 fold defect in ribosome mediated GTP hydrolysis whereas the other mutant H92E showed a 100 fold defect. However, both the mutants are equally defective in single round Pi release (100 times slower than WT). When checked for their activity in mRNA translocation, H92A and H92E were 10 times and 100 times slower than WT respectively. Results from our tripeptide formation experiments revealed a 1000 fold defect for both mutants. Altogether, our results indicate that GTP hydrolysis occurs before tRNA translocation, whereas Pi release occurs probably after or independent of the translocation step. Further, our results confirm that, His92 has a vital role residue in ribosome-stimulated GTP hydrolysis and Pi release.

A selenocisteína (Sec, U) é o aminoácido que representa a principal forma biológica do elemento selênio e sua incorporação é um processo co-traducional em selenoproteínas como resposta ao códon UGA em fase e requer uma complexa maquinaria molecular. O repertório completo de genes envolvidos nessa via de síntese em procariotos é conhecido, porém algumas das interações moleculares ainda não foram totalmente esclarecidas. Este projeto visa à caracterização molecular nas interações entre o Fator de Elongação específico para incorporação de Sec (SelB) e Seril-tRNA sintetase (SerRS) com distintas construções do tRNASec de Escherichia coli afim de compreender a sua especificidade, seletividade e ordem de eventos. Para isso, medidas de Espectroscopia de Anisotropia de Fluorescência (FAS), Ultracentrifugação Analítica (AUC) e Calorimetria de Varredura Diferencial (DSC) foram utilizadas para determinação das constantes de interação desses complexos proteína-tRNA. Além disto, experimentos de Espalhamento de Raios-X a baixo ângulo (SAXS) e Microscopia eletrônica de transmissão por contraste negativo (NS-EM) foram realizados para elucidação estrutural destes complexos. Os estudos propostos irão auxiliar no entendimento do mecanismo de incorporação e de especificidade do tRNA para este aminoácido em bactérias bem como nos demais domínios da vida além de possibilitar um aumento na compreensão de complexos do tipo proteína-tRNA bem como salientar a importância dos elementos estruturais do tRNA para sua especificidade no processo de síntese de novas proteínas. / Selenocysteine (Sec, U) is an amino acid that represents the main biological form of the selenium element and its incorporation is a co-translational process in selenoproteins in response to the in-phase UGA codon and requires complex molecular machinery. The complete repertoire of genes involved in this pathway of synthesis in prokaryotes is known, although some of the molecular interactions have not yet been fully elucidated. This project aims at the molecular characterization in the interactions between the specific elongation factor for the incorporation of Sec (SelB) and Seril-tRNA synthase (SerRS) with different constructions of tRNASec from Escherichia coli in order to their specificity, selectivity and order of events. For this, measurements using Fluorescence Anisotropy Spectroscopy (FAS), Analytical Ultracentrifugation (AUC) and Differential Scanning Calorimetry (DSC) were employed to determine the interaction constants of the protein-tRNA complexes. In addition, Small Angle X-Ray Scattering (SAXS) experiments and negative stain transmission electron microscopy (NS-EM) were performed for structural elucidation of these complexes. The proposed studies will help to understand the mechanism of tRNA incorporation and specificity for this amino acid in bacteria as well as other domains of life. In addition, it allows an increase in the understanding of protein-tRNA-like complexes as well as emphasizing the importance of structural elements of tRNA for its specificity in the process of synthesis of new proteins.

A selenocisteína (Sec, U) é o aminoácido que representa a principal forma biológica do elemento selênio e sua incorporação é um processo co-traducional em selenoproteínas como resposta ao códon UGA em fase e requer uma complexa maquinaria molecular. O repertório completo de genes envolvidos nessa via de síntese em procariotos é conhecido, porém algumas das interações moleculares ainda não foram totalmente esclarecidas. Este projeto visa à caracterização molecular nas interações entre o Fator de Elongação específico para incorporação de Sec (SelB) e Seril-tRNA sintetase (SerRS) com distintas construções do tRNASec de Escherichia coli afim de compreender a sua especificidade, seletividade e ordem de eventos. Para isso, medidas de Espectroscopia de Anisotropia de Fluorescência (FAS), Ultracentrifugação Analítica (AUC) e Calorimetria de Varredura Diferencial (DSC) foram utilizadas para determinação das constantes de interação desses complexos proteína-tRNA. Além disto, experimentos de Espalhamento de Raios-X a baixo ângulo (SAXS) e Microscopia eletrônica de transmissão por contraste negativo (NS-EM) foram realizados para elucidação estrutural destes complexos. Os estudos propostos irão auxiliar no entendimento do mecanismo de incorporação e de especificidade do tRNA para este aminoácido em bactérias bem como nos demais domínios da vida além de possibilitar um aumento na compreensão de complexos do tipo proteína-tRNA bem como salientar a importância dos elementos estruturais do tRNA para sua especificidade no processo de síntese de novas proteínas. / Selenocysteine (Sec, U) is an amino acid that represents the main biological form of the selenium element and its incorporation is a co-translational process in selenoproteins in response to the in-phase UGA codon and requires complex molecular machinery. The complete repertoire of genes involved in this pathway of synthesis in prokaryotes is known, although some of the molecular interactions have not yet been fully elucidated. This project aims at the molecular characterization in the interactions between the specific elongation factor for the incorporation of Sec (SelB) and Seril-tRNA synthase (SerRS) with different constructions of tRNASec from Escherichia coli in order to their specificity, selectivity and order of events. For this, measurements using Fluorescence Anisotropy Spectroscopy (FAS), Analytical Ultracentrifugation (AUC) and Differential Scanning Calorimetry (DSC) were employed to determine the interaction constants of the protein-tRNA complexes. In addition, Small Angle X-Ray Scattering (SAXS) experiments and negative stain transmission electron microscopy (NS-EM) were performed for structural elucidation of these complexes. The proposed studies will help to understand the mechanism of tRNA incorporation and specificity for this amino acid in bacteria as well as other domains of life. In addition, it allows an increase in the understanding of protein-tRNA-like complexes as well as emphasizing the importance of structural elements of tRNA for its specificity in the process of synthesis of new proteins.

A leptospirose é uma zoonose causada por bactérias patogênicas do gênero Leptospira. A doença representa um grave problema de saúde pública nos países tropicais subdesenvolvidos. Mais de 500.000 casos graves de leptospirose são notificados a cada ano e a taxa de mortalidade excede 10% (World Health Organization, 1999). Os roedores são o principal reservatório urbano da doença, e eliminam leptospiras viáveis no meio ambiente ao longo de toda a vida. As bactérias entram no hospedeiro por abrasões na pele ou por membranas mucosas e rapidamente se espalham pelo organismo atingindo vários órgãos. A identificação de mecanismos de invasão e de evasão imune apresentados por leptospiras patogênicas é extremamente relevante e tem sido alvo de pesquisas recentes desenvolvidas por vários grupos. Nesse contexto, a caracterização funcional de proteínas de membrana externa de Leptospira, principais alvos de interação com moléculas do hospedeiro, é de grande importância. O Fator de Elongação Tu (EF-Tu), uma proteína bacteriana abundante envolvida na síntese protéica, pertence à categoria das proteínas conhecidas como \"moonlighting\". Tais moléculas possuem a capacidade de exercer mais de uma função e, normalmente, localizam-se em diferentes compartimentos da célula. Há relatos de que EF-Tu de agentes patogênicos possa atuar como um fator de virulência. No presente trabalho, demonstrou-se que EF-Tu de Leptospira está localizado na superfície da bactéria e possui funções adicionais, sendo receptor para moléculas presentes no plasma do hospedeiro. Tal proteína interage com vários componentes da matriz extracellular e também com plasminogênio, de maneira dosedependente. Resíduos de lisina são importantes para essa interação. Plasminogênio ligado a EF-Tu é convertido em sua forma ativa, plasmina, que, por sua vez, é capaz de clivar os substratos naturais C3b e fibrinogênio. EF-Tu de Leptospira também se liga a Fator H, principal regulador da via alternativa do sistema complemento, e este mantém sua atividade funcional ao agir como co-fator de Fator I na clivagem de C3b. O potencial imunoprotetor de EF-Tu em modelo animal foi avaliado, tendo em vista o alto grau de conservação da proteína em diferentes espécies de Leptospira. EF-Tu não conferiu proteção significativa e, portanto, não deve ser considerado como um candidato vacinal contra a leptospirose. Em suma, EF-Tu de Leptospira deve contribuir para o processo de invasão e evasão ao sistema imune inato do hospedeiro, inativando o sistema complemento. Tanto quanto é do nosso conhecimento, essa é a primeira descrição de uma proteína \"moonlighting\" em Leptospira. / Leptospirosis is a zoonosis caused by pathogenic bacteria from the genus Leptospira. The disease represents a serious public health problem in underdeveloped tropical countries. More than 500,000 cases of severe leptospirosis are reported each year, with mortality rates exceeding 10% (World Health Organization, 1999). Rodents are the main urban reservoir of the disease, shedding viable leptospires throughout their lives in the environment. Leptospires infect hosts through small abrasions in the skin or mucous membranes and they rapidly disseminate to target organs. The identification of invasion mechanisms and immune evasion strategies employed by pathogenic leptospires is of great relevance. In this context, functional characterization of leptospiral outer membrane proteins, which represent the main targets for interaction with host molecules, is extremely important. The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. In this work we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It interacts with several extracellular matrix components and also binds plasminogen in a dose-dependent manner. Lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires Factor H (FH), the main soluble regulator of the alternative pathway of the complement system. FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). Given the wide distribution of EF-Tu among Leptospira species, its immunoprotective potential was evaluated in an animal model. EF-Tu was not able to afford significant immunoprotection, and might not be considered a vaccine candidate against leptospirosis. In conclusion, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities.

Pseudomonas aeruginosa is an opportunistic human pathogen that is now the leading cause of morbidity and mortality in immunocompromised individuals. Those suffering with the genetic disease cystic fibrosis (CF) commonly encounter P. aeruginosa infections. P. aeruginosa infection can present itself as an acute infection, which is characterised by highly virulent, "free-swimming" bacteria, or as a chronic infection associated with the formation of surface-adhered bacterial communities known as biofilms. The labyrinth of interconnecting signalling networks has meant that the regulatory mechanisms behind biofilm formation and virulence are largely undefined. In this dissertation, a single nucleotide polymorphism was identified within the gene, fusA1, encoding elongation factor G (EF-G). The mutation introduced minor structural changes to the protein which were likely to have functional repercussions in its involvement in protein synthesis. Phenotypic analysis revealed that the mutation conferred changes in both resistance and sensitivity to various antibiotics, as well as changes in motility, exoenzyme production, quorum sensing, metabolism, synthesis of biofilm-associated proteins and exopolysaccharide production. Most notably was the up-regulation of a major virulence determinant, the type three secretion system, typically characteristic of cells comprising an acute infection. Proteomic and transcriptomic profiling of the mutant strain provided an insight into the genetic basis behind these phenotypes, identifying the up-regulation of multidrug efflux systems and modulations to the chemotactic systems. This study also found links between several biological processes that were modulated in the mutant strain, such as crosstalk between sulfur metabolism, iron uptake and the oxidative stress response. In summary, the work presented in this dissertation highlights the susceptibility of fusA1 to spontaneous mutation and identifies a novel role for EF-G in bacterial virulence and antibiotic sensitivity, both of which have worrying implications for infection within the CF lung.