Pseudomonas aeruginosa est une bactérie pathogène opportuniste qui peut développer deux modes d’infection. Les infections aigües sont associées à la production et à la sécrétion de toxines qui peuvent avoir un effet cytotoxique, alors que dans le contexte d’une infection chronique la bactérie a tendance à s’établir sous la forme d’un biofilm. Un biofilm est une population de microorganismes organisée en une communauté et attachée sur une surface. Cette surface peut être biotique ou abiotique. Les biofilms bactériens ont des caractéristiques intrinsèques qui les rendent plus résistants à des conditions environnementales difficiles (pH, oxygène, UV, flux, etc…). Dans le cadre d’une infection l’établissement du biofilm résulte aussi en une population bactérienne difficile à éliminer par le système immunitaire mais également résistante aux antibiotiques. Des déterminants moléculaires majeurs qui interviennent dans la formation du biofilm sont le flagelle, les pili de type IV et les fimbriae Cup pour l’attachement, ou bien encore les exopolysaccharides qui sont avec l’ADN des composants essentiels de la matrice extracellulaire du biofilm qui englobe la population bactérienne. Chez P. aeuginosa, si l’alginate est le polysaccharide majeur, il a été montré que des souches non-mucoides forment également des biofilms et que dans ce contexte la synthèse et la sécrétion du polysaccharide majeur de la matrice sont dépendantes des gènes pel. Mon travail a consisté dans l’étude de l’organisation structurale du système Pel et le contrôle de son activité. / Pseudomonas aeruginosa is a human opportunistic pathogen, and in the course of an infection can develop two lifestyles. One of them is involved in secretion of toxins and results in acute infection, and the other one causes chronic infections and is characterized by formation of a biofilm. A biofilm is a highly organized bacterial population, organized as a complex community that is attached to a surface. Bacterial biofilm shows higher resistance to different enviromental factors including physical factors, antimicrobials or host immune response. Major components taking part in biofilm formation are flagella, type IV pili, cup fimbriae as well as exopolysaccharides. The latter provides a protective matrix for the biofilm, together with proteins and DNA. In non-mucoid strains the major exopolysaccharide of biofilm matrix is synthetised and secreted by pel gene cluster. My work concentrates on the structural organisation of Pel polysaccharide secretion machinery, regulation of its activity as well as detailed characterisation of the Pel polysaccharide itself.

It was demonstrated that induction of a major outer protein, HI, was associated with increased resistance to chelators of divalent cations such as EDTA and to the cationic antibiotics polymyxins and aminoglycosides. Outer membrane protein HI was the major cellular protein in cells grown in Mg²⁺-deficient medium (0.02 mM Mg²⁺) and in mutants selected for resistance to polymyxin. Increase in protein HI was associated with decrease in cell envelope Mg²⁺. Induction of protein HI was prevented by supplementation of Mg²⁺-deficient medium with 0.5 mM Mg²⁺, Ca²⁺, Mn²⁺ or Sr²⁺, but not by Zn²⁺, Ba²⁺, or Sn²⁺. Cells grown in Ca²⁺, Mn²⁺ or Zn²⁺ showed enhanced levels of these cations as main major cell envelope associated cation. Only cells grown in the presence of those cations which failed to prevent HI induction were resistant to chelators, polymyxin B and gentamicin. Protein HI overproducing cells also demonstrated altered streptomycin uptake. It was further demonstrated that aminoglycosides could interact with the outer membrane so as to make it more permeable to other substances. Mg²⁺ inhibited aminoglycoside-mediated permeabilization. Both aminoglycosides and polymyxin B could be shown to displace a small amount of Mg²⁺ from the cell envelope. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Bactérias aclimatadas a cobre foram isoladas a partir de amostras de solo e água coletadas na região da Mina de Sossego (Vale, Carajás, PA). Pseudomonas putida sp. foi escolhida, pois, apresentou a maior capacidade sortiva de Cu2+, Q = 40 mg/g. O cultivo em regime de batelada, meio mineral, com glicerol como fonte de carbono, resultou fator de conversão de glicerol a células, YX/S, de 0,49 g/g e velocidade específica máxima de crescimento, mmax, de 0,11 h-1. Alta concentração celular, 90 g/L, foi alcançada em cultivos em regime de batelada alimentada. Promoveu-se sorção de cobre pelas células, por meio de adição contínua ou em pulsos, de solução de CuSO4. A maior sorção específica de cobre, Q, de 30 (mg de Cu2+/g de células), foi verificada na adição por pulsos. Fotos de MET da bactéria na ausência e presença de Cu2+ mostram acúmulo de cobre na membrana e internamente, caracterizando biossorção e bioacumulação. / Bacteria acclimated to copper were isolated from soil and water samples collected in Mina de Sossego (Vale, Carajás, PA). Pseudomonas putida sp. was chosen as it had the highest sorptive capacity for Cu2+, Q = 40 mg/g. The fed-batch culture in mineral medium with glycerol as the carbon source resulted in a glycerol-to-cell conversion factor, YX/S of 0.49 g/g and maximum specific growth rate, mmax of 0.11 h-1. High cell concentration, 90 g/L, was achieved in cultures in fed-batch regimen. Cooper sorption by cells was promoted, by continuous or pulse addition of CuSO4 solution. The highest specific copper sorption, Q, 30 (mg Cu+2/g of cells) was seen with the addition by pulses. TEM photos of the bacteria in the absence and presence of Cu+2 show copper accumulation in the membrane and internally, featuring biosorption and bioaccumulation.

Utilization of cyanide as the sole nitrogen source by Pseudomonas fluorescens NCIMB 11764 (Pf11764) occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated oxygenolytically by an enzyme referred to as cyanide oxygenase (CNO), which exhibits properties of a pterin-dependent hydroxylase. The pterin requirement for Pf11764 CNO was satisfied by supplying either the fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds at catalytic concentrations (0.5 µM). These compounds included, for example, biopterin, monapterin and neopterin, all of which were also identified in cell extracts. A related CNO-mediated mechanism of cyanide utilization was identified in cyanide-degrading P. putida BCN3. This conclusion was based on (i) the recovery of CO2 and NH3 as enzymatic reaction products, (ii) the dependency of substrate conversion on both O2 and NADH, and (iiii) utilization of cyanide, O2 and NADH in a 1:1:1 reaction stoichiometry. In contrast to findings reported for Pf11764, it was not possible to demonstrate a need for exogenously added pterin as a cofactor for the PpBCN3 enzyme system. However, results which showed that cells of PpBCN3 contained approximately seven times the amount of pterin as Pf11764 (of which a significant portion was protein-bound) were interpreted as indicating that sufficient bound CNO-cofactor exists, thus eliminating any need for a supplemental source.

A pyrA mutation in Pseudomonas putida was isolated using transposon mutagenesis for the first time. Transposon Tn5 was used to inactivate the pyrA gene for carbamoylphosphate synthetase in these mutants. Accordingly, these mutants were defective in pyrimidine and arginine biosynthesis. The suicide vector, pM075, from Pseudomonas aeruginosa, was used to introduce the transposon into the cells. Tn5 was subsequently used to supply homology so that the plasmid pM075 could be introduced in its entirety into the Pseudomonas putida chromosome at the locus of the Tn5 insertion in the pyrA gene. Consequently, these strains exhibited high frequency of recombination and were capable of chromosome mobilization.

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Pseudomonas aeruginosa est l’agent pathogène principal causant des infections pulmonaires chroniques chez les patients atteints de fibrose kystique (FK). Le mutant STM_modA dérivé de la souche P. aeruginosa PAO1 a été obtenu par mutagénèse par étiquette-signature et est donc incapable de persister dans le poumon d’un modèle de rat. Le mutant STM_modA présente une mutation par insertion interrompant le cadre de lecture du gène modA, dont le produit fait partie du transporteur ModABC qui permet l’internalisation du molybdate depuis l’espace périplasmique. Le molybdate est la forme environnementale et assimilable du molybdène, essentiel pour l’activité des molybdoenzymes. Il a été démontré par les présents travaux que l’activité du transporteur ModABC est essentielle pour l’infection pulmonaire chronique chez le rat, pour la formation du biofilm, pour la résistance à la prédation par l’amibe Dictyostelium discoideum, pour la dénitrification et la croissance anaérobie subséquente, ainsi que pour l’expression du transcriptome en conditions anaérobies. / Pseudomonas aeruginosa is the main pathogen causing chronic lung infections in cystic fibrosis patients. The genome of the laboratory reference strain PAO1 was sequenced revealing its highly complex virulence regulatory network and a large part of genes of unknown function. A PCR-based signature tagged mutagenesis (STM) allowed the identification of 148 genes essential for chronic lung infection in a rat model. The PAO1-derivated strain STM_modA was obtained using this technique and is thus unable to persist in the rat lug in competition with a pool of strains. This strain carries an insertional mutation interrupting the open reading frame of the modA gene. This gene codes with the co-transcribed modB and modC genes for an ATP-binding cassette transporter (ModABC) responsible for the internalization of molybdate from the periplasmic space. Molybdate is the environmental molybdenum-containing ion, which is essential for the activity of the molybdoenzymes, a group of enzymes involved in a wide range of metabolic functions. The present work demonstrates that the ModABC transporter activity is essential for chronic lung infection in a rat model, for biofilm formation, for resistance to predation by the amoeba Dictyostelium discoideum as well as for denitrification and subsequent anaerobic growth. Whole transcriptome shotgun sequencing demonstrated major changes in the gene transcription levels of STM_modA in comparison with wild-type PAO1 in anaerobic conditions. This work highlights the ModABC transporter as a potential target for the inhibitors development, a new strategy for antimicrobial research.

Pseudomonas aeruginosa souche LESB58 est une bactérie pathogène connue pour sa versatilité, son antibiorésistance et son caractère opportuniste émergent qui le rend responsable d'infections nosocomiales importantes en milieu hospitalier. Dans le cadre de ce travail, nous posons l’hypothèse qu’il est possible d’étudier le génome de la bactérie pathogène P. aeruginosa souche LESB58 par une approche de modélisation métabolique et analyse in silico à partir des annotations de son génome séquencé. Nous utilisons une approche basée sur les contraintes nommée « Fc (FBA) » pour analyser le modèle in silico. Comme le génome de P. aeruginosa n’est pas très caractérisé et qu’il contient un nombre considérable de gènes sans annotation fonctionnelle, nous n’avons pas terminé la reconstruction à l’échelle génomique. Comme un nombre important de relations « génotype-phénotype » n’ont pu être établies, nous avons débuté une analyse de génomique comparative de quatre souches séquencées de P. aeruginosa, soit les souches LESB58, PAO1, PA7 et PA14, afin de cibler les gènes impliqués dans les phénotypes connus de P. aeruginosa. Le but de ce travail a été de s’initier aux méthodes d’analyses métaboliques in silico et de recherches de relations « génotype-phénotype » pouvant reconstruire un modèle in silico.