Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.

Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.

Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.

Alginate biosynthesis is not only a major contributor to pathogenicity of P. aeruginosa but also an important factor in colonization of adverse environmental habitats by biofilm formation. The requirement of proteins Alg8 and Alg44, encoded by their respective genes in the alginate biosynthesis gene cluster, for alginate biosynthesis of P. aeruginosa was demonstrated, since deletion mutants were unable to produce or polymerise alginate. AlgX deletion mutants failed to produce the alginate characteristic mucoid phenotype, but showed low concentrations of uronic acid monomers in the culture supernatants. Complementation experiments using PCR based approaches were used to determine the complementing ORF and all deletion mutants could be complemented to at least wildtype levels by introducing a plasmid harbouring the respective gene. Increased copy numbers of Alg44 did not impact on the amount of alginate produced, whereas increased copy numbers of the alg8 gene led to an at least 10 fold stronger alginate production impacting on biofilm structure and stability. Topological analysis using reporter protein fusions and subsequent subcellular fractionation experiments revealed that Alg8 is located in the cytoplasmic membrane and contains at least 4 transmembrane helices, 3 of them at its C terminus. Its large cytosolic loop showed similarities to inverting glycosyltransferases and the similarities were used to generate a threading model using SpsA, a glycosyltransferase involved in spore coat formation of B. subtilis, as a template. Site-directed mutagenesis confirmed the importance of identified motifs commonly detected in glycosyltransferases. Inactivation of the DXD motif, which has been shown to be involved in nucleotide sugar binding, led to loss-offunction mutants of Alg8 and further replacements revealed putative candidates for the catalytic residue(s). Contradicting the commonly reported prediction of being a transmembrane protein, Alg44 was shown to be a periplasmic protein. The highest specific alkaline phosphatase activity of its fusion protein could be detected in the periplasmic fraction and not in the insoluble membrane fraction. Bioinformatical analysis of Alg44 revealed structural similarities of its N terminus to PilZ domains, shown to bind cyclic-di-GMP, and of its C terminus to MexA, a membrane fusion protein involved in multi-drug efflux systems. Thus, it was suggested that Alg44 has a regulatory role for alginate biosynthesis in bridging the periplasm and connecting outer and cytoplasmic membrane components. AlgX was shown to interact with MucD, a periplasmic serine protease or chaperone homologue, and is suggested to exert its impact on alginate production via MucD interaction. In vitro alginate polymerisation assays revealed that alginate production requires protein components of the outer and cytoplasmic membrane as well as the periplasm, and these data were used to construct a model describing a multi-enzyme, membrane and periplasm spanning complex for alginate polymerisation, modification and export.

Les répétitions en tandem sont constituées de successions de motifs d'ADN. Ces structures présentes dans tous les organismes, procaryotes comme eucaryotes, ont des applications dans de nombreux domaines. Depuis quelques années seulement, les répétitions en tandem sont étudiées chez les bactéries. Le polymorphisme associé à ces séquences peut être utilisé pour le génotypage de bactéries pathogènes, permettant une identification précise au niveau de la souche. Le polymorphisme des séquences répétées est de deux types : polymorphisme de longueur et mutations internes aux motifs. Les génomes des deux bactéries pathogènes responsables d'infections nosocomiales, Staphylococcus aureus et Pseudomonas aeruginosa, ont été étudiés dans le but d'identifier des séquences répétées polymorphes. Un ensemble de marqueurs polymorphes a été validé expérimentalement pour ces deux espèces permettant un typage dit MLVA (pour « Multiple Locus VNTR Analysis »). Le travail plus classique de typage par la taille de la répétition a été complété par un travail de séquençage de certains allèles. Les résultats obtenus montrent comment le typage « MLVA » complété si nécessaire par le séquençage d'allèles, pourraient constituer de nouvelles méthodes peu coûteuses participant au contrôle des infections bactériennes.

Pseudomonas aeruginosa est un bacille opportuniste responsable d'infections graves. Sa pathogénicité repose sur de nombreux facteurs de virulence dont le système de sécrétion de type III (SSTT). Ce système est activé par le contact de la bactérie avec une cellule ou une déplétion calcique et permet l'injection de toxines directement dans le cytosol de la cellule. Différents phénotypes sont observés lors d'une infection pulmonaire dans le cas de la mucoviscidose : un phénotype inductible par le contact cellulaire ou la déplétion calcique et un autre non inductible.<br />En l'absence de mutations, cette dualité de phénotype peut être envisagée sous un aspect épigénétique.<br />A l'aide d'un outil informatique, nous avons déterminé les dynamiques possibles d'un modèle du SSTT supportant l'hypothèse de bistabilité et mis en évidence l'existence possible d'épigénèse. Grâce à cette méthode nous avons également définit les expériences permettant de tester cette hypothèse. Nous avons démontré qu'une modification épigénétique pouvait être à l'origine d'une acquisition stable de l'inductibilité du SSTT in vitro. Ce changement héréditaire de phénotype a été confirmé, in vivo, à l'aide d'un modèle d'infection pulmonaire aiguë.<br />Dans un second temps, nous avons mis en évidence une répression du SSTT à densité cellulaire élevée. Celle-ci est induite par un signal produit et sécrété par la bactérie. L'utilisation de mutants a permis de montrer que les signaux connus du quorum sensing ne sont pas impliqués dans cette répression. Ainsi, l'expression du SSTT dépend de la densité bactérienne et la répression à densité cellulaire élevée est induite par un mécanisme de type quorum sensing non connu.

Pseudomonas aeruginosa is an opportunistic human pathogen that infects immunocompromised individuals such as those suffering from burns or the genetic disorder cystic fibrosis. This organism utilizes a cell-cell communication mechanism known as quorum sensing (QS) to coordinate virulence gene expression and biofilm formation. It has three interconnected QS systems, namely las, rhl and pqs. Each system is comprised of autoinducer synthesis genes, lasI, rhlI, and pqsABCDH, and the cognate regulatory genes, lasR, rhlR, and pqsR, respectively. Here, we primarily focused on understanding the regulatory mechanisms of QS, which we investigated at two levels. First, we sought to identify additional activators that regulate QS at the level of the las and rhl systems, and second, we investigated the regulation of downstream genes, particularly biofilm exopolysaccharide genes, by QS. For the first approach, we employed a mutagenesis screen to identify global QS activators. We screened a non-redundant transposon library for mutants deficient in QS-dependent phenotypes. We identified a novel regulator, GidA, a glucose-inhibited cell division protein, that selectively controls QS gene expression posttranscriptionally via RhlR-dependent and –independent pathways. For the second part, we established a regulatory link between QS and Pel exopolysaccharide. We showed that the las system represses Pel and modulates colony biofilm structure through the pqs pathway. LasR mediated colony rugosity via 4-hydroxy-2-alkylquinolines in a PqsR-independent manner, ascribing a novel function to this class of signaling molecules in P. aeruginosa. Taken together, our study highlights the complexity of QS, which involves integration of various regulatory pathways to control downstream processes in response to different environmental conditions. / Graduation date: 2012

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 150-153).

Pseudomonas aeruginosa is an opportunistic pathogen that is a serious problem for immuno-compromised patients. Toxins such as exoenzyme (Exo) S, ExoT, ExoY and ExoU are secreted and translocated from the bacteria into the eukaryotic cell via the bacterial encoded type III secretion system. Our research focuses on ExoS, a bifunctional toxin comprising a Rho-GTPase-activating protein domain (RhoGAP) and a 14-3-3 dependent ADP-ribosyltransferase domain. In addition, ExoS contains a membrane localization domain termed MLD. In this study, cell lines expressing activated forms of various components of the Ras signaling pathway have been used to understand the functional and mechanical activation of ExoS-ADP-ribosyltransferase activity and to reveal its cellular targets in the cell. Our observations suggested that Ras GTPase is the dominant target by which ExoS mediates cell death and activated Ras is able to protect cells against cell death, regardless of whether it has been ADP-ribosylated by ExoS. It has been reported that the 14-3-3 cofactor protein is required for ADP-ribosyltransferase activity of ExoS and a phosphorylation-independent interaction occurs between 14-3-3 and the C-terminal part of ExoS. We have undertaken a deeper analysis including structural and biological investigation of this interaction. Our results suggested that leucine-428 of ExoS is the most critical residue for ExoS enzymatic activity. Structural analysis showed that ExoS binds to 14-3-3 in a novel binding mode mostly relying on hydrophobic contacts. Our structure was supported by biochemical and cytotoxicity analyses, which revealed that the substitution of important residues of ExoS significantly weakens the ability of ExoS to modify endogenous targets such as RAS/RAP1 and to induce cell death. Further, mutation of key residues within the ExoS binding site for 14-3-3 impairs virulence in a mouse pneumonia model. Leucine residues-422, 423, 426, and 428 of ExoS are important for the interaction with the ″roof″ of the amphiphatic groove of 14-3-3. In conclusion, we show the mechanism of cell signal transduction pathways affected upon ExoS infection and also demonstrate that the hydrophobic residues of ExoS in 14-3-3 interaction motif have a significant role for ExoS enzymatic activity.

Arginine utilization in Pseudomonas aeruginosa with multiple catabolic pathways represents one of the best examples of metabolic versatility of this organism. To identify genes of this complex arginine network, we employed DNA microarray to analyze the transcriptional profiles of this organism in response to L-arginine. While most genes in arginine uptake, regulation and metabolism have been identified as members of the ArgR regulon in our previous study, eighteen putative transcriptional units of 38 genes including the two known genes of the arginine dehydrogenase (ADH) pathway, kauB and gbuA, were found inducible by exogenous L-arginine but independent of ArgR. The potential physiological functions of those candidate genes in L-arginine utilization were studied by growth phenotype analysis in knockout mutants. The insertion mutation of aruH encoding an L-arginine:pyruvate transaminase abolished the capability to grow on L-arginine of an aruF mutant devoid of a functional arginine succinyltransferase (AST) pathway, the major route of arginine utilization. The aruH gene was cloned and over-expressed in E. coli. Taking L-arginine and pyruvate as the substrates, the reaction products of recombinant enzyme were identified by MS and HPLC as 2-ketoarginine and L-alanine. Lineweaver-Burk plots of the data revealed a series of parallel lines characteristic of ping-pong kinetics mechanism, and the apparent Km and catalytic efficiency (Kcat/Km) were 1.6 ± 0.1 mM and 24.1 mM-1 s-1 for pyruvate and 13.9 ± 0.8 mM and 2.8 mM-1 s-1 for L-arginine. Recombinant AruH showed an optimal pH at 9.0 and substrate specificity with an order of preference being Arg > Lys > Met > Leu > Orn > Gln. These data led us to propose the arginine transaminase (ATA) pathway that removes the α-amino group of L-arginine via transamination instead of oxidative deamination by dehydrogenase or oxidase as originally proposed. In the same genetic locus, we also identified a two-component system, AruRS, for the regulation of arginine-responsive induction of the ATA pathway. Our latest DNA microarray experiments under D-arginine conditions also revealed PA3863 as the candidate gene encoding D-arginine dehydrogenase which might lead to the recognition of a wider network of arginine metabolism than we previously recognized.