published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

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The regulatory gene for pyrimidine biosynthesis has been identified and designated pyrR. The pyrR gene product was purified to homogeneity and found to have a monomeric molecular mass of 19 kDa. The pyrR gene is located directly upstream of the pyrBC' genes in the pyrRBC' operon. Insertional mutagenesis of pyrR led to a 50- 70% decrease in the expression of pyrBC', pyrD, pyrE and pyrF while pyrC was unchanged. This suggests that PyrR is a positive activator. The upstream regions of the pyrD, pyrE and pyrF genes contain a common conserved 9 bp sequence to which the purified PyrR protein is proposed to bind. This consensus sequence is absent in pyrC but is present, as an imperfect inverted repeat separated by 11 bp, within the promoter region of pyrR. Gel retardation assays using upstream DNA fragments proved PyrR binds to the DNA of pyrD, pyrE, pyrF as well as pyrR. This suggests that expression of pyrR is autoregulated; moreover, a stable stem-loop structure was determined in the pyrR promoter region such that the SD sequence and the translation start codon for pyrR is sequestered. β-galactosidase activity from transcriptional pyrR::lacZ fusion assays, showed a two-fold in increase when expressed in a pyrR- strain compared to the isogenic pyrR+ strain. Thus, pyrR is negatively regulated while the other pyr genes (except pyrC) are positively activated by PyrR. That no regulation was seen for pyrC is in keeping with the recent discovery of a second functional pyrC that is not regulated in P. aeruginosa. Gel filtration chromatography shows the PyrR protein exists in a dynamic equilibrium, and it is proposed that PyrR functions as a monomer in activating pyrD, pyrE and pyrF and as a dimeric repressor for pyrR by binding to the inverted repeat. A related study discovered that the catabolite repression control (Crc) protein was indirectly involved in pyr gene regulation, and shown to negatively regulate expression of PyrR at the posttranscriptional level.

Methanococcus jannaschii is a thermophilic methane producing archaebacterium. In this organism genes encoding the aspartate transcarbamoylase (ATCase) catalytic (PyrB) and regulatory (PyrI) polypeptides were found. Unlike Escherichia coli where the above genes are expressed from a biscistronic operon the two genes in M. jannaschii are separated by 200-kb stretch of genome. Previous researchers have not been able to show regulation of the M. jannaschii enzyme by the nucleotide effectors ATP, CTP and UTP. In this research project we have genetically manipulated the M. jannaschii pyrI gene and have been able to assemble a 310 kDa E. coli like enzyme. By using the second methionine in the sequence we have shown that the enzyme from this organism can assemble into a 310 kDa enzyme and that this enzyme is activated by ATP, CTP and inhibited by UTP. Thus strongly suggesting that the second methionine is the real start of the gene. The regulation of the biosynthetic pathway in Pseudomoans aeruginosa has previously been impossible to study due to the lack of CTP synthase (pyrG) mutants. By incorporating a functional uridine (cytidine) kinase gene from E. coli it has been possible to isolate a pyrG mutant. In this novel mutant we have been able to independently manipulate the nucleotide pools and study its effects on the enzymes in the biosynthetic pathway. The enzyme asapartate transcarbamoylase was repressed 5-fold when exogenous uridine was high and cytidine was low. The enzyme dihydroorotate was repressed 9-fold when uridine was high. These results suggest that a uridine compound may be the primary repressing metabolite for the enzymes encoded by pyrB and pyrC. This is the first study to be done with the proper necessary mutants in the biosynthetic pathway of P aeruginosa. In the past it has been impossible to vary the internal UTP and CTP pools in this organism.

El presente trabajo investigó la adición de un inóculo de Pseudomonas aeruginosa en ciertos efluentes grasos recolectados de locales de expendio de comida rápida. Se consideró evaluar el comportamiento del inóculo en un medio apropiado que estimule o facilite la biodegradación de lípidos como parte de un futuro proceso de tratamiento de desechos. Para esto se utilizó el método de titulación con NaOH 0.05 N mediante el cual se cuantificó la cantidad de ácidos grasos liberados debido a la actividad de las lipasas de Pseudomonas. Los parámetros a evaluar fueron: temperatura, tiempo de incubación, pH inicial, concentración de sales y cantidad de inóculo (% V/V). Se comprobó que los mejores resultados (0,823, 0,747 y 0,781 U) se dieron con una temperatura de 37 ºC, un tiempo de 24 h y un pH inicial de 7. Los resultados mostraron que con tiempos mayores a 24 h (48 y 72 h) la actividad de enzima decrece y esto puede ser debido a la acción de otras enzimas por medio de los cuales Pseudomonas consume también los ácidos grasos liberados y por consiguiente se reduce la cantidad de NaOH usada para el punto final. Palabras clave: Efluentes grasos, Tratamiento de desechos grasos, Pseudomonas aeruginosa, lipasas.

Les bactéries à Gram négatif sont caractérisées par une organisation complexe de leur enveloppe,impliquant une membrane interne (ou cytoplasmique), un espace périplasmique et une membrane externe. Si le transport de petits composés chimiques se fait facilement, la sécrétion des protéines et des toxines nécessite par contre l’utilisation de machineries spécialisées : les systèmes de sécrétion.Chez Pseudomonas aeriginosa, une bactérie pathogène opportuniste, le système de sécrétion de Type II, ou sécrétion Xcp, constitue l’une des voies principales de la sécrétion. Ce sécréton Xcp est un complexe macromoléculaire de 12 protéines, nommées XcpAO et XcpPC-ZM, organisé en trois sous-complexes : une plateforme d’assemblage ancrée dans la membrane interne (XcpPC-SF etXcpYL-ZM), un pore localisé dans la membrane externe et formé par multimérisation de la sécrétine XcpQD, et un pseudopilus périplasmique impliquant les pseudopilines XcpTG-XK. Au travers de son introduction bibliographique, ce manuscrit présente les différents constituants de cette machinerie et leur implication dans la sécrétion. Un grand nombre de copies du constituant majoritaire de ce système, XcpTG, s’assemble sous forme d’un pseudopilus dont les cycles d’assemblage –désassemblage, semblables aux mouvements d’un piston, pourraient permettre la sécrétion des substrats à travers la membrane externe. Le travail effectué au cours de cette thèse a pour but d’approfondir la compréhension des conditions d’assemblage du pseudopilus, qui s’avère être une étape cruciale dans la sécrétion. Les résultats obtenus s’articulent autour de la détermination par RMN hétéronucléaire de la structure de XcpTG, le composant majoritaire du pseudopilus etre présente le premier constituant de la machinerie de type II de P. aeruginosa à voir sa structure résolue par RMN. / Gram negative bacteria are characterized by a complex organisation of their cell envelope, with aninner membrane (or cytoplasmic membrane), a periplasmic space and an outer membrane. Incontrast to the transport of chemical compounds, which is preformed usually by porins localized inthe impermeable cell envelop, secretion of proteins and toxins requires specialized machineries: thesecretion systems. In Pseudomonas aeruginosa, an opportunistic pathogen, among the wide rangeof section systems, the Type II secretion system, called Xcp secreton, is a major pathway for therelease of virulence factors. This Xcp secreton is a macromolecular complex involving 12 proteinscalled XcpA0 and XcpPC to XcpZM. This machinery is organized in 3 complexes, the assemblyplatform anchored in the inner membrane (implicating XcpPC,RE,SF,YL and ZM), the pore localizedin the outer membrane and formed by multimerization of the secretin XcpQD, and the periplasmicpseudopilus involving XcpTG-XK matured by the prepilin peptidase XcpAO. The introduction of thismanuscript presents all the components of the type II secretion system and their principal functionin the secretion process. XcpTG, the major components of this system, seems to polymerize to allowtransfer of secretion products across the outer membrane by a piston-like process. The workpresented in this manuscript is underlined by the idea of improving the understanding of themecanism of the type II secretion. The results articulate around the heteronuclear NMR solutionstructure determination of the XcpTG, which represents the first structure obtained for a componentof the type II secretion system of P. aeruginosa.

Biofilms are communities of microorganisms associated with surfaces encased in a protective extracellular matrix. These communities often pose clinical and industrial challenges due to their ability to tolerate biocidal treatments and removal strategies. Understanding the ecological interactions that take place during biofilm establishment is a key element for designing future treatment strategies. In this work, I utilized unique methods for studying factors contributing to cooperative antibiotic detoxification in a polymicrobial biofilm model. Subsequently, I tested a novel compound mixture that exhibited promising antibiofilm properties. Escapin is an L-amino acid oxidase that acts on lysine to produce hydrogen peroxide (H2O2), ammonia, and equilibrium mixtures of several organic acids collectively called Escapin intermediate products (EIP). Previous work showed that the combination of synthetic EIP and H2O2 functions synergistically as an antimicrobial toward diverse planktonic bacteria. To test the combination of EIP and H2O2 on bacterial biofilms, Pseudomonas aeruginosa was selected as a model, due to its role as an important opportunistic pathogen. Specifically, I examined concentrations of EIP and H2O2 that inhibited biofilm formation or fostered disruption of established biofilms. High-throughput assays of biofilm formation using microtiter plates and crystal violet staining showed a significant effect from pairing EIP and H2O2, resulting in inhibition of biofilm formation relative to untreated controls or to EIP or H2O2 alone. Similarly, flow cell analysis and confocal laser scanning microscopy revealed that the EIP and H2O2 combination reduced the biomass of established biofilms relative to controls. Area layer analysis of biofilms post-treatment indicated that disruption of biomass occurs down to the substratum. Only nanomolar to micromolar concentrations of EIP and H2O2 were required to impact biofilm formation or disruption, which are significantly lower concentrations than those causing bactericidal effects on planktonic bacteria. Micromolar concentrations of EIP and H2O2 combined enhanced P. aeruginosa swimming motility compared to either EIP or H2O2 alone. Collectively, these results suggest that the combination of EIP and H2O2 may affect biofilms by interfering with bacterial attachment and destabilizing the biofilm matrix.

Pseudomonas aeruginosa is found in numerous environments and is an opportunistic pathogen affecting those who are immunocompromised. Its large genome encodes tremendous metabolic and regulatory diversity that enables P. aeruginosa to adapt to various environments. We are interested in how P. aeruginosa senses and responds to the host-derived compounds, carnitine and acylcarnitines. Acylcarnitines can be hydrolyzed to carnitine, where the liberated carnitine and its catabolic product glycine betaine can be used as osmoprotectants, for induction of the virulence factor phospholipase C, and as sole carbon, nitrogen, and energy sources. P. aeruginosa is incapable of de novo synthesis of carnitine and acylcarnitines and therefore imports these compounds from exogenous source. Short-chain acylcarnitines are imported by the ABC transporter CaiX-CbcWV. Medium- and long-chain acylcarnitines are hydrolyzed extracytoplasmically and the liberated carnitine is transported through CaiX-CbcWV. Once in the cytoplasm, short-chain acylcarnitines are hydrolyzed by the L-enantiomer specific hydrolase, HocS. The transcriptional regulator CdhR is divergently transcribed from the carnitine catabolism operon and we have identified the upstream activating region, the binding site sequence, and essential residues required for CdhR binding and induction of the carnitine operon. Carnitine catabolism is repressed by glucose and glycine betaine at the transcriptional level. Furthermore, using two different cdhR translational fusions we show that CdhR enhances its own expression and that GbdR, a related transcription factor, contributes to cdhR expression by enhancing the level of basal expression. These studies are the first to determine the mechanism of O-acylcarnitine transport, metabolism, and the regulation of these processes, which contribute to utilization of these compounds for P. aeruginosa survival in diverse environments.

Somete a la cepa ambiental Pseudomonas aeruginosa 6K-11 a mutagénesis aleatoria mediante radiación ultravioleta (254 nm), consiguiéndose una producción de ramnolípidos de 32.3 g/l y la alteración en abundancia de las especies químicas producidas. Así mismo, se registra una reducción de 6 horas del tiempo de máxima producción en un bioproceso por lotes sumergido en un medio mineral suplementado con aceite de maíz como fuente de carbono. La cepa mutante muestra una menor producción de monorramnolípidos de cadenas hidrofóbicas de 10 y de 12 átomos de carbono (Rha-C10-C12, Rha-C12-C10) y un incremento sustancial de los siete dirramnolípidos estudiados y de los monorramnolípidos de hidroxialcanoatos de 8 y 10 carbonos (Rha-C8-C10, Rha-C10-C8, Rha-C10-C10, Rha-C10-C12:1). De forma paralela, la cepa Pseudomonas aeruginosa 6K-11 es curada de bacteriófagos temperados aplicando un protocolo que conjuga un inductor lítico de naturaleza química, ciprofloxacino, y otro físico, como es la radiación ultravioleta. / Tesis

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