Pseudomonas fluorescens does not appear to regulate the enzymes of de novo pyrimidine biosynthesis at the level of gene expression. Little or no apparent repression of pyr gene expression is observed upon addition of exogenous pyrimidines to the growth medium. The Escherichia coli pyrBI genes for aspartate transcarbamoylase (ATCase) were sized down and cloned into the broad host range plasmid, pKT230. Upon introduction into a P.fluorescenspyrB mutant strain, ATCase showed repression in response to exogenously fed pyrimidine compounds. Thus, it was possible to bring about changes in pyrimidine nucleotide pool levels and in transcriptional regulation of gene expression at the same time.

The applicability of an isotopically labelled assay system to determine the lipase production in Pseudomonas aeruginosa was evaluated. Supernatant from cultures of Pseudomonas aeruginosa grown in a medium containing olive oil was incubated with a substrate containing labelled trioleate. Fatty acids were isolated by means of a liquid-liquid partition system. Enzyme activity was determined by measuring the amounts of free fatty acid by liquid scintillation counting. Findings indicate that the isotopicallylabelled, liquid-liquid partitioning assay is reliable, sensitive and adaptable to rapid assay conditions. It was also determined that different strains of Pseudomonas aeruginosa produce varying amounts of lipase. Partial purification of supernatant by gel filtration produced two protein peaks showing enzymatic activity.

Évolution expérimentale aux limites de l'extinction : Le cas de Pseudomonas fluorescens soumise au stress. Les stress environnementaux : ces perturbations d'origine biotiques ou abiotiques des conditions de vie qui ont un impact sur les populations par l'altération des capacités de reproduction et/ou de survie des organismes, peuvent parfois mener une espèce à l'extinction si celle-ci n'est pas en mesure de s'adapter suffisamment rapidement. Avec l'augmentation de l'impact des activités humaines, le taux d'extinction des espèces est aujourd'hui estimé être de plusieurs ordres de grandeur au dessus du taux d'extinction basal. Il est donc important de comprendre les facteurs écologiques et évolutifs, à l'échelle de l'espèce comme de la communauté, qui déterminent la survie ou l'extinction d'une espèce soumise à un stress sévère.Au cours de cette thèse, nous utiliserons les outils de l'évolution expérimentale avec la bactérie Pseudomonas fluorescens soumise à un stress antibiotique, ainsi que des outils théoriques, pour étudier les facteurs déterminants les conditions d'un sauvetage évolutif : événement d'évolution rapide d'une population en situation d'effondrement démographique sous l'effet d'un stress létal. Nous étudierons aussi le rôle d'un stress plus modérés : une carence en ressource, sur deux aspects de la structure des communautés liés à la stabilité de ces dernières face aux perturbations environnementales : La topologie d'un réseau trophique bactérie-phage, ainsi que la distribution des tailles de populations, décrite par la loi de Taylor, pour des populations bactériennes en mono-culture ou en compétition sur un gradient de ressource. Nos travaux ont permis de mieux comprendre le sauvetage évolutif, en montrant notamment le rôle déterminant de la taille des populations et de leur diversité génétique initiale dans la capacité d'adaptation à un stress létal. Mais aussi en proposant une meilleure description des dynamiques de populations typiques de cette situation. Nous avons aussi montré la capacité d'une limitation des ressources à altérer la structure des réseaux trophiques et ainsi leur stabilité face aux perturbations. / Experimental Evolution at the Edge of Extinction, The Case of Pseudomonas fluorescens in Stressful Environments.Environmental stresses : the biotic or abiotic perturbations of life conditions which have an impact on populations through impaired organism's ability to survive and/or reproduce, can sometimes result in species extinction if rapid evolutionary adaptation doesn't occur. With the increasing impact of human activities, the rate of species extinction is now estimated to be several orders of magnitudes above the background rate. It is therefore crucial to understand the ecological and evolutionary factors, at species and community levels, which determine the survival or extinction of species exposed to severe stresses. All along this thesis, we will use experimental evolution tools with the bacterium Pseudomonas fluorescens exposed to antibiotic stress, as well as theoretical tools, to study the factors driving evolutionary rescue : the rapid evolutionary adaptation occurring in populations experiencing demographic collapse under a lethal stress. Then we will study how a moderate stress (low resource availability) drives different aspects of communities structure related to their stability against environmental perturbations : The topology of a bacteria-bacteriophage trophic network, and the distribution of population sizes along a resource gradient, described by Taylor's law, for bacterial populations either in mono-culture or in competition. Our results led to a better understanding of evolutionary rescues. In particular, we show the crucial role of initial population size and genetic diversity in the ability to evolve adaptation to an initially lethal stress. And we refined the description of populations dynamics in such cases. We show also how resource limitation can disturb trophic network structure, resulting in a lower stability against environmental perturbations.

La recherche de nouvelles molécules pour combattre Pseudomonas.aeruginosa est d'une grande importance. L'utilisation des antibiotiques a spectre large a grandement accru la résistance de P.aeruginosa aux antibiotiques. Malgré cette situation, aucune nouvelle drogue active sur P.aeruginosa n'a été introduite en pratique clinique durant les 2 dernières décennies. Le monoxyde de carbone (CO) pourrait agir comme un inhibiteur efficace de la chaîne respiratoire de P.aeruginosa mais l'utilisation pratique de ce gaz comme molécule antibactérienne est gênée par sa toxicité et les difficultés de manipulation. Une avancée fondamentale récente dans le domaine de la recherche sur le CO a été la découverte des « carbon monoxide releasing molecules » (CO-RMs), qui servent de transporteur et délivre des quantités contrôlées de CO aux systèmes biologiques.Nous montrons ici que les CO-RMs possèdent des propriétés antibactériennes contre P.aeruginosa. Cet effet antibactérien des CO-RMs à lieu à des concentrations non toxiques pour les cellules eucaryotes et passe par une interaction du CO libérer par le transporteur avec la chaîne respiratoire bactérienne. Nous présentons des résultats in vivo montrant que les CO-RMs diminuent l'inoculum bactérien et augmentent la survie des souris après une bactériémie à P.aeruginosa. La comparaison de 4 CO-RMs ayant différente structures chimiques suggère que la précence d'un métal de transition joue un rôle important dans l'activité antibactérienne des CO-RMs. Une autre découverte importante présentée dans ce travail est l'inhibition de l'activité antibactérienne de certain CO-RMs par les molécules contenant des résidus thiols. Cette découverte limite la possibilité d'utiliser les CO-RMs concernés comme des agents anti-infectieux.En considérant les résultats présentés dans ce travail, l'inhibition de la chaîne respiratoire pourrait être considérée comme un nouveau mécanisme prometteur pour la recherche de nouveaux agents pharmaceutique pour combattre les infections à P.aeruginosa. / The search of new molecules to fight Pseudomonas.aeruginosa is of paramount importance. The use of broad spectrum antibiotics has greatly increased the antibiotic resistance of P.aeruginosa. In spite of this situation, no new drug against P.aeruginosa has been successfully introduced into the clinic in the past 2 decades. Carbon monoxide (CO) could act as an effective inhibitor of the respiratory chain in P. aeruginosa but the practical use of this gas as an antibacterial molecule is hampered by its toxicity and difficulty to manipulate. A recent fundamental development in the field of CO research has been the discovery of carbon monoxide-releasing molecules (CO-RMs), which serve as carriers for the delivery of controlled amounts of CO in biological systems.Here, we show that CO-RMs possesse bactericidal properties against P.aeruginosa. This antimicrobial effect of CO-RMs occurs at non toxic concentrations for eukaryotic cells and is mediated by an interaction of CO liberated by the carrier with bacterial respiratory chain. We present in vivo results showing that CO-RMs decrease bacterial inoculum and increase survival in mice following P.aeruginosa bacteraemia. A comparison of 4 CO-RMs with different chemical structures suggests that the presence of a transition metal center plays an important role in the antibacterial activity of CO-RMs. Another important finding presented in this work is the inhibition of the antibacterial activity of some CO-RMs by thiol containing molecules. This finding could deserve the possibility to use concerning CO-RMs as anti-infective agent.Considering results presented in this work, inhibition of respiratory chain could be considered as a promising new mechanism for the research in new pharmaceutical agent to fight P.aeruginosa infections.

Les composés polyaromatiques polysoufrés représentent une classe de molécules peu étudiées, malgré leurs propriétés intéressantes. La présence du soufre influence les propriétés redox, photophysiques et de complexation. Nous avons préparé une série de ces composés par des réactions de substitution nucléophile aromatique. Ceux-ci peuvent être fonctionnalisés par des unités polyaromatiques, tels que l’anthracène ou le pyrène, ou par des glycosides. Leurs propriétés de luminescence ont été étudiées en particulier sous l’angle de l’émission induite par l’agrégation, où les composés deviennent émissifs lorsqu’ils sont immobilisés en phase solide ou fortement refroidis. Ils permettent également de stabiliser des nanoparticules de fer/platine, où une forte densité d’atomes de soufre divalent joue un rôle important en nanosciences. Les dérivés glycosylés ont été testés chez différentes lectines comme ConA, PAIL, PAIIL et Bc2lA, les trois dernières étant impliquées dans les infections bactériennes chez les patients atteints de mucoviscidose. Les études comprennent les propriétés photophysiques, de réticulation et des mesures d’affinité par des méthodes biophysiques (SPR, ITC et HIA). Ceci a ammené à la production de nouveaux biocapteurs ou sondes luminescentes basés sur des interactions sélectives lectine-sucre dans l’éventualité future de dispositifs de diagnostics et de détection à partir d’expression de lectines (ex. cancer, infections bactériennes, etc.).Une autre classe de composés polyaromatiques a été étudiée, les hélicènes. Ces molécules hélicoïdales chirales sont retrouvées sous deux formes énantiomériques, selon le pas d’hélice. Nous présentons ici une nouvelle méthode de synthèse des hélicènes par une voie organométallique de réactions d’annulation et d’insertions C-H. Une étude poussée des conditions réactionnelles a été réalisée. Nous avons préparé des hélicènes fonctionnalisés. Les dérivés bromés et cyanés du [5]-hélicène ont été déposés sur la surface de Suzuki, afin d’étudier par nc-AFM les propriétés de structuration de ces produits sur une surface isolante. Le [5]-hélicène a aussi été fonctionnalisé par des unités mannosylés, dans les mêmes perspectives que pour les astérisques soufrés glycosylés. Ces hélicènes glycosylés sont les premiers de la sorte décrit dans la littérature. Ils ont présenté des propriétés convenables vers des sondes luminescentes chirales et d’autres biocapteurs, qui sont basés sur les interactions lectine-sucre. / Polysulfurated polyaromatic compounds represent a class of molecules that are not extensively studied, despite their interesting properties. The presence of sulfur affects their redox, photophysical and complexation properties. We prepared a series of these compounds by using some aromatic nucleophilic substitutions. They can be further functionalized by some polyaromatic units, such as anthracene or pyrene, or by some glycosides.Their luminescence properties have been especially studied for their agregation induced emission properties, where the emission of the compounds is turned on when they are immobilized in solid phase or strongly cooled. These compounds can also be used to stabilize iron/platinum nanoparticles, where a high density of divalent sulfur atoms play an important role in nanosciences. Glycosylated derivatives have been tested with different lectins like ConA, PAIL, PAIIL and Bc2lA, the last three being involved in bacterial infections found in patient suffering from cystic fibrosis. Studies include photophysical and reticulation properties, and also affinitiy assays by using biophysical methods (SPR, ITC, and HIA). It provided some novel biosensors or luminescent sensors based on some selective lectin-carbohydrate interactions, in the event of future diagnostic devices and biological detection originating from lectins expressions (ex. cancer, bacterial infection, etc.).Another class of polyaromatic compounds has been studied, the helicenes,. Those chiral helical molecules can be found under two enantiomeric forms, according to the sense of the helical pitch.We herein present a new synthetic method of helicenes based on an organometallic route for some annulation reactions and C-H insertions. An exhaustive study of reaction conditions has been performed. We have prepared some functionnalized helicenes. Brominated and cyanated [5]-helicene derivatives have been deposited on a Suzuki surface, in order to study their structuration properties by nc-AFM on a non conductive surface. [5]-helicene was further functionnalized with some mannosylated units, with the same perspectives as for the glycosylated sulfurated asterisks. Those glycosylated helicenes are the first of their kind described in the literature. They provided some adequate properties toward new chiral and luminescent sensors, as well as other biosensors, which are based on lectin-carbohydrate interactions.

Pseudomonas aeruginosa (PA) représente un véritable problème de santé publique étant l'une des principales causes d'infections nosocomiales et de mortalité chez les patients atteints de fibrose cystique. Cette bactérie provoque des pathologies respiratoires chroniques qui persistent malgré une thérapie antibiotique agressive à cause de l'émergence de souches résistantes et de la formation du biofilm. Une stratégie prometteuse consiste à inhiber les facteurs de virulence de PA tels que PA-IL qui est une lectine soluble impliquée dans la reconnaissance des résidus galactose et qui semble jouer un rôle dans l'adhésion de la bactérie au glycocalyx autour de la cellule hôte ainsi que dans le développement du biofilm. Alors que les interactions lectine-carbohydrate sont caractérisées par une spécificité élevée, l'affinité entre les lectines et les saccharides simples est faible et une présentation multivalente des unités saccharidiques est généralement requise pour atteindre une interaction significative d'un point de vue physiologique. Ce manuscrit décrit la synthèse de glycooligonucléotides dont l'affinité envers PA-IL a été étudiées par DNA Direct Immobilisation microarray. Les blocs de construction saccharidiques ont été assemblés sur des échafaudages phosphorylés en utilisant une combinaison de synthèse en phase solide d'ADN et « click chemistry » (cycloaddition 1,3-dipolaire azide/alcyne). Grâce à la technologie glycoarray, les glycomimétiques ont été analysés à une échelle nanomolaire. Les résultats expérimentaux ont permis d'établir des relations structure-activités précises. En outre, des études des docking ont confirmé les résultats expérimentaux. La synthèse des candidats les plus affins envers PA-IL a été conduite sans l'étiquette d'ADN et à plus grande échelle pour vérifier par des analyses biologiques leurs propriétés anti-adhésives ou inhibitrices du biofilm. / Pseudomonas aeruginosa (PA) is a major public health issue due to its impact on nosocomial infections as well as its impact on cystic fibrosis patient mortality. It often leads to chronic respiratory infection despite aggressive antibiotic therapy due to the emergence of resistant strains and to the formation of biofilm. A promising approach is to inhibit the virulence factors of PA such as PA-IL which is a soluble lectin implicated in the recognition of galactose residues that seems to be involved in the adhesion of the bacterium to the glycocalyx surrounding host's cells as well as in the biofilm development.If carbohydrate-lectin interactions proceed with high specificity, the affinity between lectins and simple saccharides is low and a multivalent display of saccharidic units is generally required to attain physiologically significant association. This manuscript reports the synthesis of high affinity glycooligonucleotides toward PA-IL for its inhibition and their bindings properties were studied on a DNA direct immobilisation microarray. Glycoside building blocks were assembled on phosphorylated scaffolds using a combination of DNA solid phase synthesis and microwave assisted « click chemistry » (copper (I) catalyzed 1,3-dipolar cycloaddition). Thanks to glycoarray technology the glycomimetics were studied at the nanomole scale. The experimental results have been used to assess structure binding relationships. In addition, docking studies have confirmed ours experimental results. The synthesis of the best hits as anti-biofilm or anti-adhesive molecules have been synthesized at upper scale without their DNA tag for biological studies as anti-adhesive compounds and biofilm inhibitors of PA.

La lysine décarboxylase (LDC) appartient à une famille d'enzymes décamériques dépendantes du cofacteur PLP qui sont connus pour catalyser la réaction transformant la L-Lysine en cadavérine tout en consommant un proton. Dans les entérobactéries comme Escherichia coli, nous trouvons deux paralogues, LdcI et LdcC. LdcI permet de faire face à la bactérie au conditions hostile de pH acide lors du passage à travers du tract gastro-intestinale. LdcC est produite pendant la phase stationnaire et aussi quand les bactéries font face aux traitements antibiotiques. La cadavérine produite par les LDCs est connue pour protéger les bactéries du stress oxydant. Cela s’explique par le fait que la cadavérine bloque les porines de la membrane externe, réduisant ainsi la perméabilité des molécules responsables du stress acides et oxydant. L'activité des LDCs chez E. coli est coordonnée avec la réponse stringente qui est mise en place lorsque les microorganismes sont dans des conditions pauvres en nutriments, afin d’éviter l’épuisement intracellulaire de la L-Lysine nécessaire pour la synthèse des protéines. Cependant, cette inhibition peut être levée par la formation d'un complexe en forme de cage avec son partenaire RavA, permettant ainsi aux bactéries de faire face aux stress multiples. Etant donné que la réponse au stress est importante pour que les bactéries puissent exhiber leur pathogénicité, nous nous sommes demandés si la bactérie opportuniste Pseudomonas aeruginosa pourrait employer LdcA pour contrer des conditions de stress qui ont déjà été décrites pour LdcI chez les entérobactéries. Au cours de ma thèse, nous avons abordé cette question en utilisant différentes approches complémentaires. Tout d'abord, nous avons utilisé des fusions promoteur-gène et de l'analyse par Western-blot pour déterminer les conditions dans lesquelles le gène ldcA a été exprimé et sa protéine synthétisée. Nous avons pu observer que ldcA est exprimé sur la phase stationnaire de croissance dans des conditions aérobies en milieux riches et également pendant des conditions anaérobies de respiration avec nitrate. Nous avons également confirmé que l'expression de ldcA est régulée par ArgR et elle est induite complètement lorsque l’acide aminé L-arginine est présente dans le milieu de croissance. Même si nous avons trouvé que les conditions de stress n'induisent pas l'expression de ldcA, nous avons obtenu de nouvelles données suggérant que d'autres mécanismes de régulation tels que le système de quorum sensing dépendant des quinolones (PQS) pourraient être impliqués dans l'expression de ldcA. En utilisant des souches mutantes de ldcA et son complémentée, nous avons évalué si LdcA était impliqué dans la réponse au stress acide et oxydatif. Bien que les données obtenues à l'aide des expériences dans notre laboratoire et des technologies à haut débit (Biolog) aient révélé que LdcA ne présente pas les mêmes fonctions que LdcI, nous avons découvert que la cadavérine produite par LdcA est nécessaire pour la croissance en milieu minimal avec L- Glutamate comme source de carbone. Nous avons également examiné si la présence de LdcA modifie la résistance aux antibiotiques et nous montré que les rends moins persistants face aux carbenicillines. Enfin, en combinant l'analyse phylogénétique et structurelle, nous avons découvert que LdcA appartient à un sous-groupe différent de LDCs bactériennes. Les alignements de séquences montrent que les résidus clés nécessaires pour lier le ppGpp ne sont pas présents dans le site de liaison prédit ce qui a été confirmer par l'analyse biochimique. Notre travail montre que, malgré le fait que LdcA catalyse la même réaction enzymatique et partage les mêmes caractéristiques structurelles que LdcI et LdcC, elle ne joue pas le même rôle que ses homologues. Son rôle est lié aux effets physiologiques de la cadavérine et à la relation entre la L-lysine et le catabolisme de la L-arginine. / The lysine decarboxylase (LDC) belongs to a family of decameric PLP-dependent enzymes that catalyse the reaction transforming L-Lysine into cadaverine while consuming a proton. They are known to be involved in polyamine metabolism and during acid and oxidative stress responses.In enterobacteria like Escherichia coli, two paralogs are present, LdcI and LdcC. LdcI takes part in acid stress response by buffering bacterial cytoplasm. LdcC is produced during stationary phase and also when bacteria face fluoroquinolone treatment. The cadaverine produced by LDCs is known to scavenge reactive oxygen species (ROS) and is capable of blocking outer membrane proteins, thus reducing the permeability of molecules responsible for acid and oxidative stresses. The activity of the LDCs from E. coli is coordinated with the stringent response (nutrient starvation) in order to prevent intracellular L-Lysine depletion. The stringent response signal molecule ppGpp is able to bind directly to LDCs and inhibit their enzymatic activity. However, the inhibition of the LdcI can be prevented by the formation of a cage-like complex with its partner RavA allowing bacteria to face the challenge of both acid and nutrient stresses.Since mechanisms allowing bacteria to counter stress challenges are important for displaying full virulence, we wondered if the opportunistic bacterium Pseudomonas aeruginosa could be using LdcA to counter stress conditions that have already been described for LdcI in enterobacteria. During my PhD, we addressed this question by using different but complementary approaches.First of all, we used promoter-gene fusions and western-blot analysis to determine the conditions in which ldcA was expressed and its product synthesized. We could observe that ldcA is expressed on stationary phase under aerobic conditions in rich media and also during nitrate-respiring anaerobic conditions. As previously described in literature, we also confirmed that ldcA expression is regulated by ArgR and fully induced when L-Arginine is present in the growth medium. Even though we found out that acid and oxidative stress conditions do not induce the expression of ldcA, we obtained new data suggesting that other regulation mechanisms such as the quinolone signal system (PQS) could be involved in ldcA expression.In paralell, we constructed an ldcA mutant and its complemented strain to understand whether LdcA was involved in acid and oxidative stress response. Although the data obtained by using manual screenings and high-throughput technologies (Biolog) revealed that LdcA is not displaying the same functions as LdcI, we discovered that the cadaverine produced by LdcA is needed for full growth fitness when growing in minimal medium using L-glutamate as carbon source. Since slow growing phenotypes are linked to heightened bacterial persistence and because cadaverine has been shown to reduce the persisters population, we also examined if the presence of LdcA is modifying the amount of persisters during carbenicillin treatment. Our data has confirmed that this is indeed the case.Finally, by combining phylogenetic and structural analysis, we discovered that LdcA belongs to a different subgroup of bacterial LDCs. Sequence alignments show that key residues needed for binding ppGpp are not present in the predicted binding site which also suggests that the enzymatic activity is not inhibited by this molecule. And biochemical analysis has confirmed that this is indeed the case as it is the case for Arginine decarboxylases.Our work shows that, in spite of the fact that LdcA catalyses the same enzymatic reaction and shares the same structural fold than LdcI and LdcC, it is not implicated in acid stress or oxidative stress responses. Its role is linked to physiological effects of cadaverine and to the relationship between L-lysine and L-Arginine catabolism.

Os ramnolipídeos (RLs) são biossurfactantes glicolipídeos que podem ser produzidos por diferentes espécies bacterianas. P. aeruginosa produz RLs ricos em 3-hidroxidecanoato, já o RL produzido por B. thailandensis apresenta elevada proporção de 3-hidroxitetradecanoato. A enzima RhlA sintetiza o 3-(3-hidroxialcanoiloxi)alcanoato (HAA), porção lipídica de RLs, e apresenta diferenças estruturais nas espécies de P. aeruginosa e B. thailandensis. Esse trabalho concentrou-se na clonagem e expressão heteróloga dos genes rhlA da linhagem P. aeruginosa LFM634 e Burkholderia thailandensis E264. Os HAAs e RLs produzidos pelas linhagens recombinantes foram caracterizados. Além disso, genes quiméricos foram sintetizados com a finalidade de modificar a especificidade das enzimas RhlAs. Os resultados obtidos neste trabalho demonstraram que a expressão de genes rhlA de diferentes espécies modificou a composição dos HAAs e/ou RLs produzidos em P. aeruginosa, B. thailandensis e E. coli. Os dados indicam que a enzima RhlA desempenha um papel importante na composição dos HAAs produzidos, porém o metabolismo bacteriano também é responsável pela composição desses tensoativos. Além disso, os resultados sugerem que a enzima RhlB, responsável pela ligação de ramnose ao HAA, também apresenta diferenças de especificidade. Quando as enzimas quiméricas foram avaliadas, detectou-se um comportamento semelhante à RhlA de B. thailandensis indicando que a estrutura de RhlA de P. aeruginosa é muito específica para funcionar adequadamente junto com a enzima RhlB. Por fim, as propriedades tensoativas demonstraram diferenças quando há modificações na composição dos 3-hidroxiácidos incorporados aos RLs. / Rhamnolipids (RLs) are glycolipid biosurfactants that can be produced by different bacterial species. P. aeruginosa produces RLs rich in 3-hydroxydecanoate, whereas the RL produced by B. thailandensis presents a high proportion of 3-hydroxytetradecanoate. RhlA enzyme synthesizes 3-(3-hydroxyalkanoyloxy)alkanoate (HAA), lipid portion of RLs, and presents structural differences in P. aeruginosa and B. thailandensis. HAAs and RLs produced by the recombinant strains were characterized. In addition, chimeric genes were synthesized for the purpose of modifying the specificity of the RhlAs enzymes. The results obtained in this study demonstrated that the expression of rhlA genes from different species modified the composition of HAAs and/or RLs produced by P. aeruginosa, B. thailandensis and Escherichia coli. The data also strongly suggest that the composition of the RLs produced depends on the 3-hidroxiacids supplies by the cellular metabolism. Futhermore, the results suggest that the RhlB enzyme that binds to the molecule of HAA a d-TDP-L-rhamnose, also exhibits differences in specificity. When the chimeric enzymes were evaluated, a RhlA-like behavior of B. thailandensis was detected indicating that the RhlA structure of P. aeruginosa is very specific to function properly together with the RhlB enzyme. Finally, the results also indicate differences in the tensoactive properties of the RLs with different compositions.

Pseudomonas aeruginosa é uma gamaproteobactéria com capacidade de colonizar diversos tipos de ambiente e infectar hospedeiros filogeneticamente distintos. Em humanos, comporta-se como um patógeno oportunista,estando frequentemente relacionada à infecções em indivíduos imunocomprometidos e indivíduos portadores de fibrose cística. Um mecanismo importante para a versatilidade de P. aeruginosa é o sistema de percepção de quórum (QS), onde a bactéria pode vincular expressão gênica à densidade populacional e às características do ambiente. Atualmente, sabe-se que muitos outros reguladores estão interligados com QS, entre eles, a proteína reguladora RsmA e os pequenos RNAs RsmZ e RsmY. Além disso, diversos fatores importantes para a patogenicidade da bactéria são reguladas por QS. Em P. aeruginosa PA14, um fator importante para a patogenicidade em diversos hospedeiros é a proteína KerV, cujo envolvimento com QS foi descrito pela primeira vez neste trabalho. A linhagem D12, que possui uma deleção no gene kerV, mostrou alterações em fenótipos regulados por QS, como a maior produção de piocianina, composto que contribui para virulência e persistência das infecções causada por P. aeruginosa. Por ser facilmente detectável e pela regulação de sua síntese não ter sido completamente explorada em PA14, a expressão dos genes responsáveis pela produção de piocianina é um interessante repórter na investigação do possível envolvimento de KerV com QS. Além de piocianina, D12 apresenta níveis reduzidos de ramnolipídeos. Esses fenótipos somados se assemelham aos fenótipos da mutação de rsmA, sugerindo o envolvimento de KerV com os sistemas QS e Gac-Rsm direta ou indiretamente. Neste trabalho, mostramos que KerV exerce um efeito negativo na regulação dos operons phz1 e phz2, responsáveis pela síntese de piocianina, alterando a expressão desses genes. KerV exerce também um efeito positivo na expressão da proteína RsmA, responsável pela repressão de diversos genes alvos, onde RsmA se liga ao sítio de ligação ao ribossomo no mRNA, impedindo a tradução. Ensaios de gel shift mostraram que a ligação direta de RsmA na sequência líder de phzA1 e phzA2 ocorre, elucidando a maneira pela qual KerV está envolvido na regulação da expressão dos operons phz em P. aeruginosa PA14. Mostramos também que phz2 é ativo e contribui para a síntese de piocianina, pois na ausência de phz1, os níveis do pigmento são maiores do que aqueles detectados em PA14. Isso sugere uma maior expressão de phz2 e uma regulação diferencial dos operons de acordo com as condições ambientais como possível estratégia para manter os níveis desse composto. Uma evidência dessa regulação diferencial é vista no mutante lasR. Na fase inicial de crescimento, esse mutante não produz piocianina, porém quando exposto a tempos mais longos de cultivo, a produção de piocianina é maior quando comparada a PA14. Isso é reflexo da ativação da expressão de phz1 no mutante lasR em fase estacionária tardia, enquanto phz2 permanece não expresso. Isso indica que phz2 é dependente de LasR, ainda que indiretamente. Já phz1, embora tenha sua expressão influenciada por LasR no estágio inicial de crescimento, na fase estacionária é regulado por outros fatores independentes de las. / Pseudomonas aeruginosa is a gammaproteobacterium that colonizes several environments and infects phylogenetically distinct hosts. It behaves as an opportunistic pathogen in humans, often related to infection in immunocompromised individuals and cystic fibrosis patients. An important mechanism for P. aeruginosa versatility is the quorum sensing (QS) network, that allows bacteria to link gene expression to population density and environmental traits. Several additional regulators are interconnected with QS, as the regulatory mRNA binding protein RsmA and the non-coding small RNAs RsmZ and RsmY. Futhermore, key factors for pathogenicity are QS-regulated. In P. aeruginosa PA14, an important pathogenicity-related factor is the KerV protein, described for the first time here as involved in QS. D12 strain, that harbor a deletion in the kerV gene, shows alterations in QS-regulated phenotypes, such as high production of pyocyanin, a compound that contributes to virulence and persistence of P. aeruginosa infections. As the production of pyocyanin is easily detected and all mechanisms involved in its synthesis regulation are not fully described, the expression of genes responsible for production of this pigment is a good reporter to investigate KerV involvement in the QS network. Additionally, D12 also shows lower levels of rhamnolipids, another QS-regulated trait. Taken together, these phenotypes resemble the effects of a rsmA mutation, suggesting KerV involvement with QS and Gac-Rsm systems. In this work, we propose that KerV exerts a negative effect in the regulation of phz1 and phz2 operons, responsible for pyocyanin synthesis, by alterating the expression of these genes. KerV also has a positive effect on rsmA expression, responsible for the repression of several genes by blocking the ribosome binding site preventing the translation. Gel shift assays showed that RsmA binds directly in the leader sequence of phzA1 and phzA2, elucidating the manner in which KerV is involved in the regulation of phz operons expression in P. aeruginosa PA14. We also demonstrate that phz2 is actively expressed and contributes to pyocyanin production in PA14, since in the phz1 mutant the levels of pyocyanin are even higher than in the wild type strain. This suggests a phz2 higher expression and a differential regulation of phz operons according to environmental changes as a mechanism to maintain the levels of pyocyanin synthesis. An evidence for this regulation is the synthesis of pyocyanin by the lasR mutant, which does not make pyocyanin at early growth stages. However, at late stationary phase, pyocyanin production is even higher than in the wild-type strain, reflecting the LasR-independent regulation of phz1 expression, while phz2 operon remains silent.

Surfactantes de origem química e biológica são substâncias anfipáticas que se localizam em interfaces e diminuem a tensão intererficial. Este trabalho objetivou isolar microrganismos do gênero Pseudomonas, a partir de amostras ambientais; selecionar, dentre os isolados, aqueles que são capazes de produzir biossurfactante e caracterizar os biossurfactantes encontrados de acordo com suas propriedades. Foram isoladas 1713 linhagens, destas, 944 foram capazes de crescer em meio PIA. A análise do sobrenadante para presença de ácidos graxos normalmente encontrados em ramnolipídios demonstrou que em 31 linhagens, das 95 que apresentaram atividades tensoativas, foi detectada a presença de 3-hidroxidecanoato (3HD) e outras 56 não apresentaram picos característicos de ácidos graxos característicos de ramnolipídios. Foram selecionadas 10 linhagens para caracterização dos biossurfactantes produzidos por elas. Foi confirmada a produção de ramnolipídios por 3 linhagens e foi possível identificar um novo composto com tensoatividade, o 2-acetilfurano. / Biological and chemical surfactants are anphipatic compounds whitch reduce interfaces tension. The present work aimed to isolate microorganisms of samples from diverse environments, chosing among the isolated ones, those capaple of producing biosurfactants and characterizing it according to some of their properties. From 1713 isolated strains, 944 were capable of growing in PIA medium. The supernatant analysis for the presence of fatty acids usually found in rhamnolipds showed that 31 of the 87 strains that presented tensoactive activities, had 3-hydroxyalkanoates (3HD) detected in their supernatant. Other 56 strains did not present characteristic rhamnolipid fatty acids peaks. Based in this resoults 10 isolates were selected for a better characterization of their biosurfactants. The producton os RHLs were confirmed for 3 diferent strains. It was possible to identify a new compound called 2-acethylfuran that was not mentioned by other authors as a possible biosurfactant or as produced by Pseudomonas.