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Caulobacter Crescentus --- Periplasm

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Although Escherichia coli is probably one off the best characterized organisms, very little is known about the mechanisms governing the biogenesis of its outer membrane and preserving its integrity during the bacterial cell cycle. For example, we have only a very limited knowledge of the way in which the various components of this membrane, namely phospholipids, lipopolysaccharides and β barrel proteins, are carried across the periplasm and then inserted in the membrane after synthesis in the cytoplasm or in the inner membrane.
My host team focuses on coming to a better understanding of the mechanisms permitting the folding and insertion of β barrel proteins in the outer membrane. In addition to the fundamental interest of studying these mechanisms, this research should lead to the development of new antibiotics that are effective against Gram negative bacteria. Indeed, the physicochemical properties of the outer membrane make these bacteria very resistant to many bacterial agents.
The β barrel proteins of the E. coli cell envelope are synthesized in the cytoplasm, and then carried through the inner membrane as unfolded polypeptides. They must then cross the periplasm before reaching the outer membrane as unfolded polypeptides. They must then cross the periplasm before reaching the outer membrane where they are assembled. According to the current model, β barrel proteins are escorted in the periplasm by chaperones that are present in this compartment. The precise role of these chaperones has however not been characterized.
FkpA is one of the four periplasmic chaperones identified so far. Its structure and in vitro chaperone activity have been well studied. However, we know nothing about FkpA’s in vivo role and about the nature of its substrates. The purpose of my work was therefore to characterize the function of this chaperone in the periplasm and to identify its physiological substrates.
To do this, I used a multidisciplinary approach. First, I carried out microbiological studies to characterize the phenotype of different bacterial strains lacking FkpA. The results of these studies have shown that strains lacking both DegP, the primary periplasmic protease, and FkpA were particularly sensitive to elevated temperatures. I’ve also shown that FkpA interacts with several β barrel proteins when purified from strains lacking one of the chaperones involved in the folding of these proteins. Finally, the in vitro tests that I carried out suggest that FkpA is capable of protecting these β barrel proteins from aggregation, without however participating actively in their folding. Put together, my results suggest that FkpA plays a role in the quality control of secreted proteins. Bien qu’Escherichia coli soit probablement l’un des organismes les mieux caractérisés, les mécanismes permettant la biogenèse de sa membrane externe et le maintien de l’intégrité de celle-ci durant le cycle cellulaire bactérien restent très peu connus. Par exemple, nous n’avons qu’une connaissance très partielle de la façon avec laquelle les différents constituants de cette membrane, à savoir les phospholipides, les lipopolysaccharides et les protéines en tonneau β, sont transportés au travers du périplasme puis insérés au sein de celle-ci après avoir été synthétisés dans le cytoplasme ou au niveau de la membrane interne.
Mon laboratoire d’accueil s’intéresse particulièrement aux mécanismes qui permettent le repliement et l’insertion des protéines en tonneau β dans la membrane externe. Outre l’intérêt fondamental que présente l’étude de ces mécanismes, ces recherches devraient ouvrir la voie au développement de nouveaux antibiotiques efficaces contre les bactéries à Gram négatif. En effet, les propriétés physicochimiques de leur membrane externe les rendent peu perméable à de nombreux bactéricides.
Les protéines en tonneau β de l’enveloppe d’E. coli sont synthétisées dans le cytoplasme, puis transportées sous forme non repliée au travers de la membrane interne. Elles doivent ensuite traverser le périplasme avant de gagner la membrane externe où elles sont assemblées et acquièrent leur conformation native. Selon le modèle actuel, les protéines en tonneau β seraient escortées au travers du périplasme par des chaperonnes présentes dans ce compartiment. Le rôle précis de ces dernières n’a cependant pas été caractérisé.
FkpA est lune des quatre chaperonnes périplasmiques identifiées jusqu’à présent. Sa structure et son activité chaperonne in vitro ont bien été étudiées. Cependant, on ne connaît rien du rôle que FkpA joue in vitro et de la nature de ses substrats. L’objectif de mon mémoire était donc de caractériser la fonction exercée par cette chaperonne au sein du périplasme et d’en identifier les substrats. L’objectif de mon mémoire était donc de caractériser la fonction exercée par cette chaperonne au sein du périplasme et d’en identifier les substrats physiologiques.
Pour ce faire, j’ai utilisé une approche multidisciplinaire. Tout d’abord, j’ai réalisé des études microbiologiques afin de caractériser le phénotype de différentes souches bactériennes dépourvues de FkpA. Les résultats de ces études ont fait apparaître que les souches dépourvues de FkpA et de la principale protéase périplasmique, DegP, étaient particulièrement sensibles à un stress thermique. J’ai aussi montré que FkpA interagit avec plusieurs protéines en tonneau β en purifiant la protéine. Pour finir, les tests in vitro réalisés suggèrent que FkpA est capable de protéger ces protéines en tonneau β de l’agrégation, sans toutefois participer activement à leur repliement. Mis ensemble, mes résultats suggèrent que FkpA joue un rôle dans le « contrôle qualité » des protéines sécrétées

Gram-Negative Bacterial Infections --- Cell Membrane --- Biogenesis --- FkpA protein, E coli --- Escherichia coli Proteins --- Periplasm

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Biotransformation has accompanied mankind since the Neolithic community, when people settled down and began to engage in agriculture. Modern biocatalysis started in the mid-1850s with the pioneer works of Pasteur. Today, biotransformations have become an indispensable part of our lives, similar to other hi-tech products. Now, in 2019, biocatalysis “received” the Nobel Prize in Chemistry due to prof. Frances H. Arnold’s achievements in the area of the directed evolution of enzymes. This book deals with some major topics of biotransformation, such as the application of enzymatic methods in glycobiology, including the synthesis of hyaluronan, complex glycoconjugates of N-acetylmuramic acid, and the enzymatic deglycosylation of rutin. Enzymatic redox reactions were exemplified by the enzymatic synthesis of indigo from indole, oxidations of β-ketoesters and the engineering of a horse radish peroxidase. The enzymatic reactions were elegantly employed in biosensors, such as glucose oxidase, in the case of electrochemical glucose sensors. Nitrilases are important enzymes for nitrile metabolism in plants and microorganisms have already found broad application in industry—here, these enzymes were for the first time described in Basidiomyceta. This book nicely describes molecular biocatalysis as a pluripotent methodology—“A jack of all trades...”—which strongly contributes to the high quality and sustainability of our daily lives.

Technology: general issues --- E. coli --- recombinant horseradish peroxidase --- site-directed mutagenesis --- periplasm --- glycosylation sites --- Aspergillus niger --- quercetin --- rutin --- rutinose --- rutinosidase --- “solid-state biocatalysis” --- hyaluronic acid --- in vitro synthesis --- one-pot multi-enzyme --- optimization --- enzyme cascade --- Basidiomycota --- Agaricomycotina --- nitrilase --- cyanide hydratase --- nitrile --- substrate specificity --- overproduction --- homology modeling --- substrate docking --- phylogenetic distribution --- indigo --- MISO library --- flavin --- monooxygenase --- FMO --- β-N-acetylhexosaminidases --- transglycosylation --- Glide docking --- Talaromyces flavus --- muramic acid --- non-reducing carbohydrate --- glucose oxidase --- direct electron transfer --- amine-reactive phenazine ethosulfate --- glucose sensor --- glycemic level monitoring --- Pseudomonas putida MnB1 --- biogenic manganese oxides --- abiotic manganese oxides --- α-Hydroxy-β-keto esters --- whole-cell biocatalysis --- surface display --- cell wall anchor --- Lactobacillus plantarum --- whole-cell biocatalyst --- n/a --- Fe(II)/2-ketoglutarate-dependent dioxygenase --- 2-ketoglutarate generation --- regio- and stereo-selective synthesis --- hydroxy amino acids --- sequential cascade reaction --- "solid-state biocatalysis"

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Biotransformation has accompanied mankind since the Neolithic community, when people settled down and began to engage in agriculture. Modern biocatalysis started in the mid-1850s with the pioneer works of Pasteur. Today, biotransformations have become an indispensable part of our lives, similar to other hi-tech products. Now, in 2019, biocatalysis “received” the Nobel Prize in Chemistry due to prof. Frances H. Arnold’s achievements in the area of the directed evolution of enzymes. This book deals with some major topics of biotransformation, such as the application of enzymatic methods in glycobiology, including the synthesis of hyaluronan, complex glycoconjugates of N-acetylmuramic acid, and the enzymatic deglycosylation of rutin. Enzymatic redox reactions were exemplified by the enzymatic synthesis of indigo from indole, oxidations of β-ketoesters and the engineering of a horse radish peroxidase. The enzymatic reactions were elegantly employed in biosensors, such as glucose oxidase, in the case of electrochemical glucose sensors. Nitrilases are important enzymes for nitrile metabolism in plants and microorganisms have already found broad application in industry—here, these enzymes were for the first time described in Basidiomyceta. This book nicely describes molecular biocatalysis as a pluripotent methodology—“A jack of all trades...”—which strongly contributes to the high quality and sustainability of our daily lives.

Technology: general issues --- E. coli --- recombinant horseradish peroxidase --- site-directed mutagenesis --- periplasm --- glycosylation sites --- Aspergillus niger --- quercetin --- rutin --- rutinose --- rutinosidase --- "solid-state biocatalysis" --- hyaluronic acid --- in vitro synthesis --- one-pot multi-enzyme --- optimization --- enzyme cascade --- Basidiomycota --- Agaricomycotina --- nitrilase --- cyanide hydratase --- nitrile --- substrate specificity --- overproduction --- homology modeling --- substrate docking --- phylogenetic distribution --- indigo --- MISO library --- flavin --- monooxygenase --- FMO --- β-N-acetylhexosaminidases --- transglycosylation --- Glide docking --- Talaromyces flavus --- muramic acid --- non-reducing carbohydrate --- glucose oxidase --- direct electron transfer --- amine-reactive phenazine ethosulfate --- glucose sensor --- glycemic level monitoring --- Pseudomonas putida MnB1 --- biogenic manganese oxides --- abiotic manganese oxides --- α-Hydroxy-β-keto esters --- whole-cell biocatalysis --- surface display --- cell wall anchor --- Lactobacillus plantarum --- whole-cell biocatalyst --- Fe(II)/2-ketoglutarate-dependent dioxygenase --- 2-ketoglutarate generation --- regio- and stereo-selective synthesis --- hydroxy amino acids --- sequential cascade reaction