<p> Malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate with the simultaneous reduction of a nicotinamide cofactor. It was previously reported that the nitrogen-fixing bacterium, Sinorhizobium meliloti, has two malic enzymes, a diphosphopyridine-dependent malic enzyme (DME) and a triphosphopyridine-dependent malic enzyme (TME). The dme gene is essential for symbiotic nitrogen-fixation in alfalfa root nodules and this symbiotic requirement cannot be met through increased expression of tme. In order to determine if a metabolic difference exists between the dme and tme mutants which might explain the symbiotic phenotypes, we conducted an analysis of intracellular and extracellular polar metabolomes. Differences noted between the intracellular profiles of the dme and tme mutant strains hinted at osmotic stress or a disturbance in central carbon metabolism. Extracellular studies indicated that dme mutant cells excreted at least 10-fold greater concentrations of both malate and fumarate. When considered together, the metabolic data implies that the DME enzyme is primarily responsible for the conversion of malate to pyruvate to generate acetyl-CoA whereas the TME enzyme must serve a secondary function within the cell.</p> <p> While the C-terminal 320 amino acid regions from both DME and TME are similar in sequence to phosphotransacetylase enzymes, enzyme assays with DME and TME proteins have failed to detect PTA activity. Here we report that the chimeric malic enzyme structure is conserved among various gram negative bacteria including Agrobacterium tumefaciens, Escherichia coli, Bradyrhizobium japonicum and Porphyromonas gingivalis. Moreover these chimeric proteins are also present in the archaebacteria. Halobacterium salinarum and Haloarcula marismortui. To further our understanding of the functions of DME and TME in S. meliloti, we have fused protein domains from DME to an affinity tag consisting of strepII and a calmodulin binding peptide. To identify proteins interacting with this fusion, we expressed these protein fusion constructs in S. meliloti, prepared extracts containing the soluble proteins and passed these through tandem affinity chromatography columns. All proteins that coeluted with the fusion proteins appeared to be interacting with antibodies specific for the DME protein and so may have been aggregates or break-down products of DME.</p> / Thesis / Master of Science (MSc)

Xylella fastidiosa é uma bactéria limitada ao xilema de plantas, sendo transmitida principalmente por cigarrinhas da subfamília Cicadellinae (Hemiptera: Cicadellidae). No Brasil, é o agente causal da Clorose variegada dos citros (CVC), doença que afeta laranja-doce [Citrus sinensis (L.) Osbeck]. O objetivo deste trabalho foi caracterizar o comportamento alimentar da cicadelíneo vetor Bucephalogonia xanthophis (Berg) em citros e correlacionar suas atividades estiletares com a transmissão de X. fastidiosa. Inicialmente, testes de escolha e análises de excreção de honeydew foram realizadas para determinar os locais e períodos preferidos para alimentação em mudas de citros. B. xanthophis preferiu a haste dos ramos novos, na parte superior da muda. Esta cigarrinha ingeriu seiva do xilema e apresentou maior volume médio de excreção e maior percentual de indivíduos que excretaram durante a fotofase. Assim, estudou-se a penetração estiletar do vetor na haste de brotações cítricas, durante a fotofase, pela técnica de \"Electrical Penetration Graph\" (EPG, sistema DC). Os principais padrões de EPG foram correlacionados com análises histológicas e de \"honeydew\" para determinação da posição exata dos estiletes no tecido vegetal e atividades envolvidas. Seis padrões foram descritos: (S) secreção de bainha salivar e caminhamento dos estiletes através de células da epiderme ou parênquima; (R) estiletes inseridos na planta, porém sem nenhuma atividade aparente; (X) contato dos estiletes com os vasos do xilema; (Xi) ingestão ativa no xilema; (I) breve interrupção durante X ou Xi; (W) retirada dos estiletes da planta. Durante uma prova, a seqüência de eventos com maior probabilidade de ocorrência foi penetração estiletar através da epiderme e parênquima (S) (100% dos insetos), seguida de contato com o xilema (X) (67,6%). Entre os indivíduos que exibem o padrão X, 88,3% passam para Xi. Os vasos do xilema foram localizados pelo inseto após uma média de 2,2 provas. O tempo médio para atingir o xilema (X) e iniciar ingestão (Xi) após o início da primeira prova foi 27.8 min e 34,2 min, respectivamente. Entretanto, verificou-se ingestão prolongada no xilema (Xi > 5 min) somente após 39,8 min, em média. Em um outro estudo, investigou-se a relação dos padrões S, X e Xi com os processos de aquisição e inoculação de X. fastidiosa em citros. B. xanthophis adquiriu X. fastidiosa somente no padrão Xi. Após 1 h de ingestão no xilema, esta cigarrinha transmitiu a bactéria para plantas-teste com uma eficiência de 7,7%. A inoculação ocorreu durante os padrões S, S+X e S+X+Xi, com eficiência de 3,5; 7,1 e 7,4%, respectivamente. Um fato intrigante foi a ocorrência de inoculação de X. fastidiosa pelo inseto antes de atingir os vasos do xilema, durante o padrão S. Contudo, as maiores taxas de transmissão ocorreram após o contato com o xilema (S+X ou S+X+Xi). Por fim, estudou-se o efeito da infecção sintomática e assintomática por X. fastidiosa em plantas cítricas sobre a penetração estiletar de B. xanthophis. O comportamento alimentar foi semelhante em plantas sadias e infectadas sem sintomas. A infecção sintomática não afetou a capacidade de B. xanthophis localizar os vasos do xilema, mas reduziu o tempo gasto por este inseto ingerindo seiva dos mesmos. Estes resultados sugerem que a aquisição de X. fastidiosa pode ser mais eficiente em plantas infectadas assintomáticas do que em plantas com sintomas severos de CVC. As informações sobre penetração estiletar do vetor B. xanthophis em citros são importantes para estudos mais avançados de mecanismos de transmissão de X. fastidiosa, bem como para estabelecer estratégias que visem interferir neste processo. / Xylella fastidiosa is a xylem-limited bacterium transmitted mainly by leafhoppers (Hemiptera: Cicadellidae) in the subfamily Cicadellinae. In Brazil, it is the causal agent of Citrus variegated chlorosis, a disease that affects sweet orange [Citrus sinensis (L.) Osbeck]. The goal of this research was to characterize the feeding behavior of the cicadeline vector Bucephalogonia xanthophis (Berg) in citrus and correlate its feeding activities with transmission of X. fastidiosa. Initially, choice tests and honeydew excretion analyses were carried out to determine preferred feeding sites and periods on citrus nursery trees. B. xanthophis preferred the stems of young shoots, in the upper part of the plant. This species ingested sap from the xylem vessels and showed larger excretion volume and higher proportion of excreting individuals during the day. Thus, vector stylet penetration was studied on the stem of citrus shoots in the photophase, by using the Electrical Penetration Graph (EPG, DC system) technique. The main EPG waveforms were correlated with histological and honeydew excretion analyses to determine the precise stylet position in the plant tissue and feeding activities. Six waveforms and proposed activities are described: (S) secretion of salivary sheath and intracellular pathway; (R) stylets inserted into the plant, without any apparent activity; (X) contact of stylets with xylem vessels; (Xi) active xylem ingestion; (I) interruption between X and Xi; and (W) stylet withdrawal from the plant. During a probe, the most likely sequence of events is stylet pathway (S) through epidermal and parenchymal cells (all individuals), followed by contact with xylem (X) (67.6% of all individuals) and then active ingestion (Xi) (88.3% of those that exhibit waveform X). The xylem was reached after an average of 2.2 probes. The mean time to contact the xylem (X) and initiate ingestion (Xi) after onset of the first probe was 27.8 and 34.2 min, respectively. However, sustained xylem ingestion (Xi > 5 min) was established only after 39.8 min, on average. In a second study, the waveforms S, X and Xi were correlated with X. fastidiosa acquisition and inoculation in citrus. Acquisition of X. fastidiosa from infected plants occurred only after onset of the pathogen to 7.7% of test plants. In healthy plants, inoculation took place during waveform Xi (xylem ingestion); but with just 1 h in Xi, B. xanthophis subsequently transmitted waveforms S (salivary sheath formation and stylet pathway), S+X (X= first xylem contact by stylets) and S+X+X1, with efficiencies of 3.5, 7.1 and 7.4%, respectively. Although higher transmission rates were recorded after the first contact with xylem (S+X and S+X+Xi), it is intriguing the fact that inoculation of this xylem-limited bacterium also occurred before that (during S). Finally, the effect of X. fastidiosa infection on the feeding behavior of B. xanthophis was studied by comparing stylet penetration on: a) healthy citrus; b) symptomless infected citrus; c) infected citrus with CVC symptoms. Based on the analysis of 26 EPG parameters, no significant differences were found in stylet penetration on healthy versus asymptomatic infected citrus. Symptomatic infection did not affect the ability of B. xanthophis to locate xylem vessels, but reduced the time spend by this vector ingesting xylem sap. These results suggest that X. fastidiosa acquisition may be more efficient on symptomless infected plants than on citrus with severe CVC symptoms. Information on vector feeding behavior is basic for future studies on transmission mechanisms of X. fastidiosa and to establish control strategies aimed to interfere with this process.

A colonização da mucosa respiratória é a primeira etapa na patogênese de Streptococcus pneumoniae, bactéria causadora de pneumonia, meningite, e otite média, responsável por mais de um milhão de mortes por ano no mundo. As proteínas de superfície PsaA e PspA têm sido investigadas como candidatos vacinais para estas doenças. CTB é a porção não tóxica da toxina colérica (CT), responsável pela ligação da toxina ao receptor celular GM1 e descrita como adjuvante de mucosas. Neste trabalho, estes genes de S. pneumoniae foram clonados em pAE, um vetor de expressão de E. coli, que utiliza o promotor T7, ou a 3\' de ctxB no plasmídeo pAE-ctxB. As proteínas recombinantes CTB, PsaA, CTB-PsaA, PspA1, CTB-PspA1, PspA3 e CTB-PspA3 foram expressas em E. coli BL21 (SI) e purificadas através de coluna carregada com níquel. Ensaios de ligação ao receptor GM1 mostraram que CTB e a porção CTB das proteínas de fusão foram obtidas na forma funcional. Imunização por via intranasal com CTB-PsaA e, por via intranasal e intradérmica com CTB-PspA1 e CTB-PspA3 induziu a produção de IgG no soro. Em compensação, somente a imunização com a fusão CTB-PsaA induziu produção de IgA nas secreções de mucosa. Ensaios de colonização da nasofaringe de camundongos BALB/C e C57BL/6 mostraram que a imunização intranasal com CTB-PsaA resulta em diminuição de colonização por S. pneumoniae. Desafios letais com linhagem virulenta de S. pneumoniae mostraram que a imunização intradérmica com CTB-PspA3, ao contrário da imunização intranasal, é capaz de proteger os animais. Uma vez que estas proteínas de fusão induziram resposta imune protetora, estas deverão ser investigadas como componentes de uma nova vacina para infecções causadas por S. pneumoniae. / The colonization of the respiratory mucosa is the first step in the pathogenesis of Streptococcus pneumoniae, bacterium that causes pneumonia, meningitis and otitis media. It is responsible for more than one million deaths per year worldwide. The surface proteins PsaA and PspA have been investigated as vaccine candidates against these diseases. CTB is the non-toxic portion of cholera toxin (CT), responsible for the toxin binding to the cellular receptor GM1 and described as mucosal adjuvant. In this study, these genes from S. pneumoniae were cloned in pAE, an E. coli expression vector that uses T7 promoter, or downstream to ctxB gene in the pAE-ctxB plasmid. The recombinant proteins CTB, PsaA, CTB-PsaA, PspA1, CTB-PspA1, PspA3 and CTB-PspA3 were expressed in E. coli BL21 (SI) and purified through a chelating resin charged with nickel. GM1 binding assays showed that CTB and CTB portion of the fusion proteins were functional. Intranasal immunization with CTB-PsaA and, intranasal and intradermal administration of CTB-PspA1 and CTB-PspA3 induced IgG production in the serum. On the other hand, only CTB-PsaA fusion protein induced IgA in the mucosal secretions. Nasopharyngeal colonization assays in BALB/C and C57BL/6 mice showed that intranasal immunization with CTB-PsaA results in a decrease of colonization by S. pneumoniae. Lethal challenges with S. pneumoniae virulent strains indicated that intradermal immunization with CTB-PspA3, in contrast to the intranasal immunization, is able to protect mice. Since the fusion proteins induced a specific immune response, they should be further investigated as components of a new vaccine against infections caused by S. pneumoniae.

A partir de 24 amostras de solos foram isoladas 461 colônias bacterianas, sendo que destas 190 foram identificadas como Bacillus thuringiensis (Bt). A relação entre as características químicas das amostras de solos e a presença do patógeno pode ser expressa por iBt = -0,4 + 0,6Ca + 0,07Cu + 0,009Fe - 0,53Mg -0,12Mn + 1,26Zn. Dentre os 83 isolados de Bt testados para o controle de Spodoptera frugiperda, o ESALQ 3.7 mostrou-se mais promissor para o controle desta praga, causando 86,6% de mortalidade em lagartas provenientes da população de São Paulo, com uma CL50 estimada de 0,749 x 108 esporos/mL. Esse isolado possui as toxinas Cry1Ac, Cry1C e Cry1E que justificam sua eficiência para S. frugiperda. Toxinas da classe Cry1 foram encontradas em 72% dos isolados testados contra este inseto. Foram observadas diferenças na suscetibilidade entre as três populações (São Paulo, Minas Gerais e Rio Grande do Sul) da lagarta-do-cartucho do milho para 49,4% dos isolados testados e na população de São Paulo, alguns isolados afetaram os parâmetros biológicos das lagartas sobreviventes aos tratamentos. A persistência do Dipel foi superior às outras formulações de Bt, até 27 horas depois da aplicação dos tratamentos (65.772 esporos/mL), porém sua meia vida (p = 17,54 horas) foi inferior ao Ecotech e Bac-Control. O estudo da persistência do isolado ESALQ 3.7 foi prejudicado por fatores bióticos e abióticos tanto em campo como em casa-de-vegetação, que comprometeram a validação dos resultados. Não foi verificada diferença estatística significativa nas notas da escala de dano causado pela lagarta-do-cartucho na testemunha e o tratamento com o isolado ESALQ 3.7. Nos estudos de interação com outros entomopatógenos observou-se que entre Bt e Heterorhabditis sp. ocorreu interação positiva, variando de efeito aditivo a sinergismo subaditivo, de acordo com a concentração do nematóide. Entre Bt e os fungos entomopatogênicos Beauveria bassiana e Nomuraea rileyi foi verificada interação negativa (antagonismo) e entre um vírus de poliedrose nuclear e Bt a interação foi negativa e positiva (efeito aditivo), dependendo da concentração do vírus. / From 24 soil samples about 461 bacterial colonies have been isolated, and 190 were identified as Bacillus thuringiensis (Bt). The relation between soil chemical characteristics and the presence of this pathogen may be expressed by the equation iBt = -0,4 + 0,6Ca + 0,07Cu + 0,009Fe - 0,53Mg -0,12Mn + 1,26Zn. Among 83 Bt isolates assayed against Spodoptera frugiperda, the ESALQ 3.7 was the most effective to control this pest, causing 86.6% of mortality to São Paulo larvae, with an estimated LC 50 of 0.749 x 108 spores/mL. This isolate has the toxins Cry1Ac, Cry1C e Cry1E that justify its efficiency against S. frugiperda. The Cry1 toxins were found in 72% of isolates assayed for this pest. There have been observed differences in the susceptibility among three fall armyworm populations (São Paulo, Minas Gerais e Rio Grande do Sul) to 49.4% of the assayed isolates, and some of them affected the biological parameters of the São Paulo larvae. The Dipel persistence was higher than the others Bt based products, until 27 hours after application (65.772 spores/mL), but its half life (p = 17.54 hours) was lower than Ecothech and Bac-Control. The study of ESALQ 3.7 persistence was prejudiced by biotic and abiotic factors in field condition and at the greenhouse conditions, that consequently constrained the data validation. About the isolate ESALQ 3.7 efficiency in the field, there was no statistical differences in the damage caused by fall armyworm in the control and treatment. In the interaction studies with other entomopathogens, it was observed a positive interaction between Bt and Heterorhabditis sp. This interaction varied from na additive effect to a subadditive sinergism, according to nematode concentration. Between Bt and entomopathogenic fungi Beauveria bassiana e Nomuraea rileyi it was observed a negative interaction (antagonism). Also, the interaction between a poliedrosis nuclear vírus and Bt varied according to the virus concentration.

O diagnóstico rápido e eficiente da mastite subclínica é importante para reduzir a persistência da doença e os prejuízos decorrentes. O objetivo do estudo foi avaliar a técnica de espectrometria de massas (MS) por ionização e dessorção a laser assistida por matriz - tempo-de-vôo (MALDI-TOF) para identificação de bactérias causadoras de mastite subclínica bovina por dois métodos: 1) a partir de bactérias isoladas por cultivo icrobiológico; 2) a partir da recuperação de bactérias diretamente do leite, visando eliminar completamente a necessidade de cultivo microbiológico para identificação dos patógenos. O estudo foi composto por dois experimentos (1 e 2), no experimento 1 foram utilizadas 33 amostras de leite provenientes de animais das raças Gir e Holandesa de quatro fazendas leiteiras para a identificação icrobiológica e MALDI-TOF MS. As amostras com resultados conflitantes foram confirmadas por sequenciamento do gene 16S rRNA. Os resultados de cultura microbiológica foram Staphylococcus aureus (n = 13), Streptococcus agalactiae (n = 10) e Estafilococos coagulase negativo (ECN) (n = 10). Para todas as amostras de Streptococcus agalactiae, resultados similares foram observados para a identificação microbiológica e por MALDI-TOF MS. De 13 isolados de Staphylococcus aureus, 11 foram igualmente identificados por MALDITOF, 1 isolado foi identificado como Staphylococcus haemolyticus por sequenciamento do gene 16S rRNA, e o outro isolado isolado com resultado conflitante foi caracterizado como cultura mista de Staphylococcus aureus e Enterococcus faecalis. Em relação às amostras de ECN, todas as amostras do grupo foram identificadas por MALDI-TOF em nível de gênero (S. simulans, S. epidermidis, S. Haemolyticus, S. Chromogens e S. aureus coagulase negativa). No experimento 2 foi avaliado o método de recuperação de bactérias presentes em leite e sua identificação por MALDI-TOF MS utilizando o método de contaminação experimental de leite com Escherichia coli, Enterococcus faecalis e Staphylococcus aureus. A identificação de patógenos recuperados diretamente do leite foi possível quando a concentração de E. faecalis e S. aureus foi de 106 ufc/mL, e de 107 ufc/mL para E.coli. Concluímos que o uso de MALDI-TOF MS pode acelerar a identificação de patógenos causadores de mastite subclínica bovina podendo contribuir para a adoção de medidas de controle e tratamento mais adequado. / Rapid and efficient diagnosis of subclinical mastitis is important to reduce the persistence of the disease and its losses. This study aimed to evaluate the technique of mass spectrometry (MS) and desorption ionization by matrix assisted laser time-offlight (MALDI-TOF) for identification of bacteria causing bovine subclinical mastitis by two methods: 1) from bacteria isolated by microbiological culture, 2) from bacteria recovered directly from milk, to eliminate completely the need for microbiological culture for identification of pathogens. The study consisted of two experiments (1 and 2).In experiment 1, 33 milk samples from Gir and Holstein animals collected in four dairy farms were used for microbiological identification and MALDI-TOF MS. The samples with different results were confirmed by sequencing the 16S rRNA. These samples were identified by microbiological culture as Staphylococcus aureus (n = 13), Streptococcus agalactiae (n = 10) and Staphylococcus coagulase negative (SCN) (n = 10). For all strains of Streptococcus agalactiae, similar results were observed for microbiological identification and MALDI-TOF MS. From 13 isolates of Staphylococcus aureus, 11 were also identified by MALDI-TOF, one isolate was identified as Staphylococcus haemolyticus by 16S rRNA sequencing , and the other discrepant sample was charactezided a mixed culture of Staphylococcus aureus and Enterococcus faecalis. Regarding the SCN samples, all samples of this group were identified by MALDI-TOF at gender level (S. simulans, S. epidermidis, S. haemolyticus, S. Chromogens and S. aureus coagulase negative). In experiment 2, we evaluated the method of recovery of bacteria present in milk and their identification by MALDI-TOF MS using experimental method of contamination of milk with Escherichia coli, Enterococcus faecalis and Staphylococcus aureus. The identification of pathogens recovered directly from milk was possible when the concentration of E. faecalis and S. aureus was 106 ufc/mL and 107 ufc/mL for E.coli. We conclude that the use of MALDI-TOF MS can accelerate the identification of pathogens causing bovine subclinical mastitis may contribute to the adoption of control measures and appropriate treatment.

Xylella fastidiosa é uma bactéria fitopatogênica, responsável por doenças em diversas plantas de importância econômica. Diversas cepas têm sido estudadas, porém, pouco se sabe a respeito da resposta a estresses ambientais em X. fastidiosa. Utilizando a tecnologia de microarranjos de DNA, verificou-se a resposta global aos estresses térmico, salino e osmótico em nível de transcrição. Os experimentos foram realizados em séries temporais, os perfis de expressão gênica dos genes diferencialmente expressos foram agrupados e validados por RT-PCR quantitativo. No choque térmico, 261 genes foram induzidos (9,7%) e 222 genes foram reprimidos (8,3%). Dentre os genes altamente induzidos, destacam-se os que codificam proteínas de choque térmico (Hsps), que previnem a desnaturação e a formação de agregados protéicos ou degradam polipeptídeos irreversivelmente desnaturados. A partir da determinação do início de transcrição de seis genes altamente induzidos no choque térmico, propôs-se um consenso para promotores dependentes do fator sigma alternativo que controla a resposta ao choque térmico, sigma32. Observou-se também a indução de genes relacionados ao estresse extracitoplasmático, que são regulados pelo fator sigma alternativo sigmaE. No choque osmótico e salino, os genes codificando a maioria das Hsps foram reprimidos na exposição prolongada a esses estresses, indicando que a resposta não é mediada por sigma32 ou sigmaE. Dos 142 genes induzidos tanto no estresse salino como osmótico, 57% codificam proteínas hipotéticas ou hipotéticas conservadas, indicando uma possível função na resposta a estes estresses. Observou-se a repressão de genes relacionados à síntese protéica e ao metabolismo intermediário nos três estresses analisados, além da indução de genes relacionados à virulência como toxinas e adesinas, revelando a complexa rede de genes envolvida na resposta a estresses ambientais. Para auxiliar a análise de dados de microarranjos de DNA, foram desenvolvidas três ferramentas de bioinformática: HTself, utilizada na determinação de genes diferencialmente expressos; BayGO, utilizada na análise categorias funcionais altamente representadas dentre os genes de interesse e SpotWhatR, uma plataforma que integra programas utilizados nas diversas etapas da análise e pré-processamento de dados de microarranjos, com uma interface de fácil utilização. Estas ferramentas foram utilizadas com sucesso e estão disponíveis livremente para outros pesquisadores. / Xylella fastidiosa is a phytopathogenic bacterium responsible for diseases in many economically important crops. Although different strains have been studied, little is known about X. fastidiosa stress responses. To investigate X. fastidiosa genes involved in heat, salt and osmotic shock responses, we performed a whole genome microarray analysis in time-course experiments. The expression profiles of the differentially expressed genes were grouped and their expression patterns were validated by quantitative RT-PCR experiments. During heat shock, 261 genes were induced (9.7%) and 222 genes were repressed (8.3%). Among the differentially expressed genes, the ones presenting the highest induction ratios encode heat shock proteins (Hsps), which prevents protein misfolding and aggregation or promote the degradation of the irreversibly denatured polypeptides. We determined the transcription start sites of six heat shock inducible genes and analyzed their promoter regions, which allowed us to propose a putative consensus for sigma32 promoters in X. fastidiosa. We also observed the induction of genes related to the extracytoplasmic stress response, that are regulated by the alternative sigma factor sigmaE. During prolongued exposure to salt and osmotic stress, genes encoding most of the Hsps were repressed, indicating that the response is not mediated by sigma32 or sigmaE. Among the 142 genes induced by both salt and osmotic stress, 57% encode hypothetical or conserved hypothetical proteins, indicating a possible role of these genes in the stress response. In addition, we observed the repression of genes related to protein biosynthesis and intermediary metabolism during the three stresses tested, besides the induction of genes related to virulence such as toxins and adhesins, revealing the complex network of genes that work together in response to environmental stresses. To facilitate the microarray data analysis process, we developed three bioinformatics tools: HTself, which is used to determine the differentially expressed genes; BayGO, which aims at finding over-represented gene categories and SpotWhatR, a system that integrates programs used in different steps of microarray data analysis in a user-friendly interface. These tools were successfully used and are freely available to the research community.

This thesis was performed within the research project “TrophinOak”, which addresses the impact of multitrophic interactions on the pedunculate oak (Quercus robur) clone DF159. In this frame, the present work focuses on the genetic and physiological mechanisms ruling the interaction of the mycorrhiza helper bacterium (MHB) Streptomyces sp. AcH 505 with microcuttings of DF159 either alone or in presence of the ectomycorrhizal fungus Piloderma croceum or the fungal leaf pathogen oak powdery mildew Microsphaera alphitoides. The work consists of 3 chapters. Chapter 1 characterises the growth of AcH 505 and P. croceum in a soil-based culture system used within the TrophinOak project. Besides the establishment and evaluation of quantification methods of these microorganisms by quantitative real-time PCR, the impact of the soil microbial community and the oak on the bacterium-fungus interaction was investigated, and AcH 505 and P. croceum were visualized by scanning electron microscopy. It was observed that the presence of the soil microorganisms and the oak both affect the bacterium-fungus interaction, and that P. croceum enhances the growth of AcH 505. Chapter 2 presents a study with the oak, AcH 505 and the EM fungus P. croceum, enabling to disentangle the direct effect of the MHB on the oak from the indirect one via the EM symbiosis. The used approach was transcriptomic based on RNA sequencing. It was shown that i) differential gene expression occurred between root and the distant leaf tissues (local vs. systemic effects), different developmental stages and treatments, suggesting that oak specifically coordinates its gene expression patterns, and ii) that genes related to plant growth, defence and DNA modification were dominant among the differential expressed genes, suggesting that these processes play essential roles in both symbiotic interactions investigated. Chapter 3 represents a second transcriptome study, addressing how AcH 505 suppresses powdery mildew infection in oak by analysing RNA Sequencing data from singly- and coinoculated oaks. This study combined the systemic impact of the root associated bacterium with local effects of the leaf pathogen, thereby linking belowground and aboveground interactions. Systemic defence response is induced by the bacterium and further enhanced upon pathogen challenge, suggesting that on the leaf level, some bacterial effectors are recognized as harmful for the plant.

The cytosolic protein degradation pathway, performed by ATP-dependent proteases and ATP-independent peptidases, plays important roles in several cellular activities, e.g. cell division, cell cycle progression, intracellular signaling, MHC class I antigen presentation, host-pathogen interactions, etc. The roles of ATP-dependent proteases during stress and infection have been studied in great detail but the functional roles of ATP-independent peptidases are not clearly understood. In this study, the functional roles of E. coli or S. typhimurium encoded Peptidase N (PepN), an ATP-independent enzyme belonging to theM1 family of metallopeptidases, were investigated. The thesis will address four different aspects. (i) In the first part, the utility of using E coli ∆pepN to identify and characterize novel peptidases will be shown. It is known that deletion of pepN leads to inability to cleave the majority of in vitro peptidase substrates in E. coli and S. typhimurium. To study the differences between two closely related paralogs of the M17 family, E. coli encoded pepA and pepB were cloned in pBAD24 vector and introduced in E. coli ∆pepN. Peptidase A (PepA) and Peptidase B (PepB) expression increases the cleavage of several aminopeptidase substrates and partially rescues growth of ∆pepN during nutritional downshift and high temperature stress (NDHT), a dual stress involving growth in minimal media at 42°C. Purified PepA and PepB enzymes display broad substrate specificity; however, distinct differences are observed between these two paralogs: PepA is more stable at high temperature whereas PepB displays broader substrate specificity as it cleaves Asp and Insulin B chain peptide. The strategy utilized in this study, i.e. overexpression of peptidases in ∆pepN followed by screening for substrate specificities in total cell extracts, may be used to rapidly identify the substrate preferences of novel peptidases encoded in genomes of different organisms. (ii) The second aspect investigates the functional roles of PepN during stress and infection in S. typhimurium. PepN has two conserved signature motifs of the M1 family, GAMEN and HEXXH, which play roles in substrate recognition and catalysis. To address the roles of catalytic activity of PepN, the residue E-298, which is present in the HEXXH motif and acts as a general base during catalysis, was mutated to A-298 by site-specific mutagenesis and introduced into ∆pepN (pBR322/pepNE298A). Biochemical and biophysical analysis of purified PepN (WT and E298A) revealed loss of catalytic activity of E298A but no major structural changes were observed in comparison to the WT protein. The functional roles of this mutation using ∆pepN expressing pBR322/pepN or pBR322/pepNE298A were investigated using two conditions: (i) Nutritional downshift high temperature (NDHT)stress and (ii) systemic infection in mice. Monitoring growth profiles of different strains demonstrated the requirement of the enzymatic activity of PepN for adaptation and growth to NDHT stress. Earlier studies have shown that S. typhimurium ∆pepN hyper proliferates in peripheral organs during systemic infection in mice. However, expression of wild type (WT)or E298A PepN led to lower colony forming units (CFU), demonstrating that the decrease in CFU is independent of catalytic activity. These observations are consistent with lower serum amounts of inflammatory cytokines, lower tissue damage and increase in survival of mice infected with S. typhimurium expressing WT or E298A PepN. (iii) Although pathogen encoded peptidases are known to be important during infection, their roles in modulating host responses in immunocompromised individuals are not well studied. In the third part of this thesis, the roles of S. typhimurium encoded PepN were studied in mice lacking Interferon-γ (Ifnγ), a cytokine important for immunity. S. typhimurium lacking pepN displays enhanced CFU compared to WT in peripheral organs during systemic infection in C57BL/6 mice. However, Ifnγ-/-mice show higher CFU compared to C57BL/6 mice, resulting in lower fold differences between WT and ∆pepN. Concomitantly, reintroduction of pepN in ∆pepN reduces CFU, demonstrating pepN dependence. In addition, three distinct differences were observed between infection ofC57BL/6 and Ifnγ-/-mice upon infection with different S. typhimurium strains: (i) cytokine profiles, (ii) histological analysis and (iii) mice survival. Overall, the roles of the host encoded Ifnγ during infection with S. typhimurium strains with varying degrees of virulence will be highlighted. (iv) The final aspect of this study reveals differences in gene expression between S. typhimurium grown in rich medium (Luria-Bertani) versus NDHT stress. This adaptation affects several pathways and the gene expression of secretory proteins that are important for virulence in S. typhimurium are greatly reduced during NDHT stress. Also, analysis of secretory protein amounts in different media conditions shows reduction during growth in minimal media plus high temperature stress. The functional consequences of this reduction in secretory protein amounts lead to lower bacterial replication after infection of RAW cells or mice infected via the oral route. In addition, the differences in gene expression between WT and ∆pepN during these conditions were studied. Interestingly, there is reduction in expression of flagellar genes whereas the genes involved in nitrogen metabolism are upregulated in ∆pepN upon exposure to NDHT stress. Further studies were performed by quantifying the motility of different S. typhimurium strains grown in a variety of culture conditions. Overall, this part of the study attempts to compare and contrast the possible adaptive responses of WT and ∆pepN to NDHT stress. Together, this thesis addresses multiple aspects of the biochemistry and roles of the enigmatic PepN during stress and infection.

This project was aimed to use a purple non-sulphur bacterium, Rhodopseudomonas palustris, as a biocatalyst for hydrogen production, from the waste of biodiesel manufacturing, crude glycerol. The goal of this project was to understand the fundamentals relevant to scaling up the process and developing an off the shelf product. The first objective was to determine the ability of R. palustris to generate hydrogen by non-growing cells in comparison to that by growing cells. Similar average hydrogen production rates and energy conversion were found for both processes but a significant difference in the hydrogen yield was observed. Hydrogen production reached ~ 80 % of the theoretical maximum hydrogen yield by non-growing R. palustris, about eight-fold of that reached by growing R. palustris. The high yield suggested that it is economically appealing to use non-growing R. palustris as the biocatalyst for continuous hydrogen production. To accomplish the proposed scale-up systems, understanding its product formation kinetics is the key. It was found that the hydrogen production rate is not growth-associated and depends solely on the dry cell mass with a non-growth associated coefficient of 2.52 (Leudeking–Piret model dP/dt=2.52 X). Light is vital for hydrogen production by non-growing R. palustris, in terms of light intensity and wavelength range. It was found that excessive or insufficient light intensity may constrain the performance. Only photons of light with appropriate wavelengths can excite cytochrome bacteriochlorophyll complexes II in R. palustris to generate hydrogen. Among white LEDs, infrared LEDs, and incandescent light bulbs, at the same light intensity, infrared LEDs gave the best results in the H2 production rate and energy conversion by non-growing cells, 22.0 % ± 1.5 % higher than that with white LEDs and around 25-30 times of that by incandescent light bulbs. It was found that non-growing R. palustris can be immobilised in alginate beads to give similar H2 production rates as that by cells suspended in media. This preliminary result pointed the direction of developing an off the shelf product of immobilised non-growing R. palustris as a biocatalyst for continuous hydrogen production.

Made available in DSpace on 2016-12-08T16:24:22Z (GMT). No. of bitstreams: 1 PGCA14MA179.pdf: 979505 bytes, checksum: 7ba008b4597749c8a754e3db15d5dc33 (MD5) Previous issue date: 2014-12-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to determine differences in the expression of virulence genes (invA) and antibiotic resistance (blaCTXM-2) from S. Heidelberg isolates resistant to antimicrobials and disinfectants. To do this, first isolates of S. Heidelberg (54), S. Enteritidis (54) and S. Typhimurium (54) were assessed by the disk diffusion test using ten antibiotics routinely applied in veterinary and human medicine. S. Heidelberg (17), S. Enteritidis (2) and S. Typhimurium (1) isolates resistant to cephalosporins (ceftiofur and ceftriaxone) were also evaluated by the technique of Minimum Inhibitory Concentration (MIC). Moreover, isolates of S. Heidelberg (17) resistant to ceftiofur by disk diffusion and MIC were subjected to dilution test tubes to determine the efficacy of disinfectants quaternary ammonium base, sodium hypochlorite and glutaraldehyde. Screening efflux was performed using ethidium bromide. The technique of antibiotic susceptibility showed 18, 16,9 and 9,6% of resistance for S. Heidelberg, S. Typhimurium and S. Enteriditis, respectively. High levels of resistance were observed for nalidixic acid in S. Heidelberg (67%), S. Typhimurium (65%), and S. Enteritidis (68.5%), as well as for tetracycline in S. Heidelberg (69%) and S. Typhimuirum (63%). The gentamicin resistance was also high for S. Typhimurium (31.5%) followed by S. Heidelberg (7%). Of great importance to the poultry industry, our results show significant resistance for ceftiofur among S. Heidelberg isolates (32%), differently from that observed for S. Enteritidis (3,7%), and S. Typhimuirum (1,9%). Only isolates of S. Heidelberg (9%) and S. Enteritidis (3,7%) showed resistance against ceftriaxone. By grouping the antibiotics used, it is observed that those belonging to the quinolone, penicillin and cephalosporin classes showed higher levels of resistance 68, 47 and 16%, respectively. The resistance pattern most commonly found was C on 10% of the isolates. It was also observed that 22,8% of the isolates were multidrug-resistant or resistant to more than three drugs. In relation to disinfectants, only one isolate of S. Heidelberg was resistant to the association of ammonium quaternary and glutaraldehyde. The isolates subjected to screening efflux system did not show the presence of this mechanism. All isolates of S. Heidelberg (17) expressed the invA gene with quantitative differences and the samples from the field showed higher expression (2.53) when compared to samples from the slaughterhouse (0.56). On the other hand, for the blaCTX-M2 gene, related to antibiotic resistance, the expression was 4.94 times greater in poultry meat samples. The expression of the gene invA allows us to conclude that S. Heidelberg isolates have pathogenic potential. But the pattern of resistance to ceftiofur and ceftriaxone associated with multidrug resistance and the positive results of gene expression, demonstrate the presence of ESBL producing bacteria, which could difficult the treatment of salmonellosis, as there is the potential for direct transmission of Salmonella from meat products to humans. . New studies in the future will allow in vivo evaluation to acess pathogenicity / O objetivo deste trabalho foi determinar as diferenças na expressão dos genes de virulência (invA) e de resistência aos antibióticos (blaCTXM-2) de isolados de S. Heidelberg resistentes aos antimicrobianos e desinfetantes. Para isso, primeiramente os isolados de S. Heidelberg (54), S. Enteritidis (54) e S. Typhimurium (54) foram submetidos ao teste do disco difusão frente a dez antibióticos utilizados rotineiramente na medicina veterinária e humana com intuito de avaliar o padrão fenotípico de resistência e multirresistência antimicrobiana. Os isolados de S. Heidelberg (17), S. Enteritidis (2) e S. Typhimurium (1) resistentes as cefalosporinas (ceftiofur e ceftriaxone) foram também avaliados pela técnica de Concentração Inibitória Mínima (MIC). Além disso, os isolados de S. Heidelberg (17) resistentes ao ceftiofur pelo disco-difusão e MIC foram submetidos ao teste de diluição em tubos para averiguar a eficiência dos desinfetantes a base de amônia quaternária, glutaraldeído e hipoclorito de sódio. Ainda foi feita a triagem do sistema de efluxo, utilizando o brometo de etídio. Pela técnica do antibiograma, os isolados de S. Heidelberg, S. Typhimurium e S. Enteriditis apresentaram 18, 16,9 e 9,6% de resistência, respectivamente. Os maiores níveis de resistência foram observados para o ácido nalidíxico nos isolados de S. Heidelberg (67%), S. Typhimurium (65%), e S. Enteritidis (68,5%), assim como para a tetraciclina nos isolados de S. Heidelberg (69%) e S. Typhimuirum (63%). A resistência à gentamicina também foi elevada para os isolados S. Typhimurium (31,5%), seguido da S. Heidelberg (7%). De grande importância para a indústria avícola, nossos resultados mostram significativa resistência para o ceftiofur entre os isolados S. Heidelberg (32%) diferente do observado para isolados de S. Enteritidis (3,7%) e S. Typhimurium (1,9%). Apenas os isolados de S. Heidelberg (9%) e S. Enteritidis (3,7%) apresentaram resistência frente ao ceftriaxone. Ao agrupar os antibióticos utilizados, observou-se que os pertencentes às classes quinolona, penicilina e cefalosporinas apresentaram os maiores níveis de resistência com 68, 47 e 16%, respectivamente. O padrão de resistência mais encontrado foi C em 10% dos isolados. Observou-se ainda que 22,8% dos isolados são multirresistentes, ou seja, resistentes a mais do que três drogas. Em relação aos desinfetantes, apenas um isolado de S. Heidelberg (ID 81) mostrou-se resistente a associação glutaraldeído-amônia quaternária. Os isolados submetidos a triagem do sistema de efluxo não mostraram a presença deste mecanismo. Todos os isolados de S. Heidelberg (17) expressaram o gene invA com diferenças quantitativas, sendo que as amostras provenientes do campo mostraram maior expressão (2,53) quando comparadas as de frigorífico (1,00). Por outro lado, para o gene blaCTX-M2, relacionado a resistência antimicrobiana, a expressão foi 4.94 vezes maior nas amostras advindas do frigorífico. A expressão do gene invA permitem concluir que as S. Heidelberg apresentam potencial patogênico. Já o padrão de resistência observado ao ceftiofur e ceftriaxone associado à multirresistência e aos resultados positivos da expressão gênica comprovam a existência de bactérias produtoras de ESBL, o que dificultaria o tratamento das salmoneloses, já que há o potencial de transmissão direta da Salmonella advinda dos produtos cárneos para cadeia alimentar humana, gerando danos à saúde pública. Novos estudos a serem conduzidos no futuro permitirão a melhor avaliação in vivo do grau de patogenicidade ao testar os isolados resistentes