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.

Pseudomonas fluorescens NCIMB 11764 (Pf11764) is uniquely able to grow on the poison cyanide as its sole nitrogen source. It does so by converting cyanide oxidatively to carbon dioxide and ammonia, the latter being assimilated into cellular molecules. This requires a complex enzymatic machinery that includes nitrilase and oxygenase enzymes the nature of which are not well understood. In the course of a proteomics analysis aimed at achieving a better understanding of the proteins that may be required for cyanide degradation by Pf11764, an unknown protein of 17.8 kDa was detected in cells exposed to cyanide. Analysis of this protein by ESI-coupled mass spectrometry and bioinformatics searches gave evidence of strong homology with a protein (Hyp1) of unknown function (hypothetical) present in the bacterium Photorhabdus luminescens subsp. laumondii TTO1 (locus plu_1232). A search of available microbial genomes revealed a number of Hyp1 orthologs the genes of which are found in a conserved gene cluster known as Nit1C. Independent studies revealed that in addition to Hyp1, Pf11764 possesses a gene (nit) specifying a nitrilase enzyme whose closest homologue is a nitrilase found in Nit1C gene clusters (77% amino acid identity). DNA sequence analysis has further revealed that indeed, hyp1Pf11764 and nitPf11764 are contained in a cluster that includes also a gene specifying an oxygenase. Given the possible connection of Nit1C-endoded nitrilase and oxygenase enzymes to enzymatic cyanide degradation, there is strong reason for thinking that the genes specifying these enzymes contribute to bacterial growth on cyanide in those bacteria containing the Nit1C cluster. Because the biological function of the Hyp1 protein is currently unknown, it was cloned and the protein expressed in E. coli so that its properties could further be explored. Unfortunately, the expression of the protein in an insoluble form complicated these analyses. However, at least two lines of evidence suggest a possible role as a regulator of gene expression. First, over-expression of the protein was accompanied by the parallel elevation of the putative vector-encoded b-lactamase, implying that Hyp1Pf11764 can affect the expression of other genes. Second, a comparison of the amino acid sequence of select peptide fragments of Hyp1Pf11764, by conducting searches for homology with proteins in the existing nonredundant protein database, consistently revealed motifs in common with those present in bacterial response regulators that are part of two-component signal transduction systems widely distributed in bacteria.

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.

The aim of this thesis was to gain an understanding of the molecular and ecological basis for the biological control of Pythium by fluorescent pseudomonads. A fluorescent pseudomonad biocontrol agent (BCA), Pseudomonas fluorescens 54/96, identified as a potential candidate for commercial development, was analysed together with transposon induced mutants in a variety of assays for anti-fungal activity (Chapter 2). It was revealed that 54/96 had a fungistatic effect generated by a number of different mechanisms, which included nutrient competition and antibiosis. The synecology of this organism with Pythium was then compared to a similar organism (P. fluorescens SBW25) demonstrating a similar degree of anti-fungal activity (Chapter 3). The similarity of the population dynamics of these two strains prompted an examination of the genetic basis for the anti-fungal activity of the second strain, with the intention of comparing with 54/96 (Chapter 4). Again this revealed a multifactorial mode of action of SBW25 against Pythium. Whilst some mutants with reduced anti-fungal activity were deficient in growth on seed exudate others were unaffected, but the mechanisms appeared to be different to those utilized by 54/96. The comparison of strains was expanded to a larger collection of pseudomonad BCAs which were contrasted by a number of phenotypic and genotypic methods (Chapter 5). Various markers were identified which showed commonality within the different classes of BCA, the most useful of which was cyclopropanated fatty acids. These may prove to be a useful marker when screening for new pseudomonad BCAs. It was concluded that a greater understanding of the molecular, physiological and ecological basis of anti-fungal activity of bacterial will lead to the development of biocontrol strategies with improved efficacy.

The success of biocontrol bacteria is often restrained due to their low persistence in the rhizosphere and fluctuations in expression of antagonistic compounds. In the first part of this thesis the ability of the biocontrol agents (BCAs) Pseudomonas brassicacearum DF41 and Pseudomonas chlororaphis PA23 to resist grazing by the bacterivorous nematode Caenorhabditis elegans was investigated. We found that both BCAs are capable of killing the nematodes through exposure to toxic metabolites. We discovered that in addition to HCN, pyrrolnitrin (PRN) is a potent nematicide produced by PA23. Unique for a pseudomonad, DF41 was also found to kill the nematodes by forming biofilms on the nematode anterior, causing starvation. Biofilm formation was dependent upon the Gac two-component system and NaCl concentration of the media. Co-culturing these BCAs in the presence of nematodes increased expression of a number of genes associated with biocontrol. We observed elevated exoproduct formation, consistent with our gene expression analysis. The nematicidal activity exhibited by DF41 and PA23 towards C. elegans bodes well for their persistence in the environment. In the second part of this thesis the role of hydrogen cyanide (HCN) and the anaerobic regulator ANR in PA23 biocontrol was explored. An hcn mutant was created and in vitro antifungal (AF) assays revealed the involvement of HCN in Sclerotinia sclerotiorum suppression. Addition of glycine promoted both AF activity and HCN production. In addition, HCN was found to be positively regulated by quorum sensing (QS). Besides a phz box, an anr box was identified in the hcnA promoter, suggesting a role for ANR in regulating hcnA. An anr mutant was generated and phenotypic characterization revealed that ANR is a key regulator governing PA23 secondary metabolite production. Through gene expression analysis, ANR was shown to positively regulate phzI/phzR and PhzR negatively regulate anr. Furthermore, expressing anr in trans partially complemented the QS-deficient phenotype with respect to several biocontrol genes and exoproducts. Overall, the global regulator ANR is vital for PA23-mediated biocontrol and a significant overlap exists between the QS and ANR regulons. / October 2016

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.

The nucleotide sequence for the pDKl TOL plasmid region encoding toluate-1,2-dioxygenase (Xy1XYZ, TO) was determined. TO is the first enzyme in the meta-cleavage operon, responsible for the conversion of toluates and benzoates to their carboxy-substituted diols. DNA sequence analysis revealed the presence of three open reading frames (ORF). The three ORFs correspond to xylX (1353 bp), xylY (486 bp) and xylZ (1008 bp), encoding predicted protein products of 51370 Da, 19368 Da and 36256 Da, respectively.

Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in pyrimidine biosynthesis. Bacterial ATCases are divided into three classes, A, B and C. Class A ATCases are largest at 450-500, are. dodecamers and represented by Pseudomonas ATCase. The overlapping pyrBC' genes encode the Pseudomonases ATCase, which is active only as a 480 kDa dodecamer and requires an inactive pyrC'-encoded DHOase for ATCase activity. ATCase has been studied in two non-pathogenic members of Mycobacterium, M. smegmatis and M. phlei. Their ATCases are dodecamers of molecular weight 480 kDa, composed of six PyrB and six PyrC polypeptides. Unlike the Pseudomonas ATCase, the PyrC polypeptide in these mycobacteria encodes an active DHOase. Moreover, the ATCase: DHOase complex in M. smegmatis is active both as the native 480 kDa and as a 390 kDa complex. The latter lacks two PyrC polypeptides yet retains ATCase activity. The ATCase from M. phlei is similar, except that it is active as the native 480 kDa form but also as 450,410 and 380 kDa forms. These complexes lack one, two, and three PyrC polypeptides, respectively. By contrast,.ATCases from pathogenic mycobacteria are active only at 480 kDa. Mycobacterial ATCases contain active DHOases and accordingly. are placed in class A1 . The class A1 ATCases contain active DHOases while class A2 ATCases contain inactive DHOases. ATCase has also been purified from Burkholderia cepacia and from an E. coli strain in which the cloned pyrB of B. cepacia was expressed. The B. cepacia ATCase has a molecular mass of 550 kDa, with two different polypeptides, PyrB (52 kDa) and PyrC of (39 kDa). The enzyme is active both as the native enzyme at 550 kDa and as smaller molecular forms including 240 kDa and 165 kDa. The ATCase synthesized by the cloned pyrB gene has a molecular weight of 165 kDa composed of three identical PyrB and no PyrC polypeptides. Nucleotide effectors ATP, CTP, and UTP inhibited all forms of enzymes. Because of its size and its activity as a trimer and smaller than native forms, the B. cepacia enzyme is placed in a new class.

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