Düsseldorf, Univ., Diss., 2004. / Computerdatei im Fernzugriff.

Düsseldorf, Universiẗat, Diss., 2004.

Staphylococcus xylosus et Staphylococcus carnosus sont utilisés comme ferments de salaisons. S. xylosus est fréquemment isolé dans l'environnement des ateliers de transformation alimentaire alors que S. carnosus l'est rarement. Ainsi, nous avons étudié la capacité de ces deux espèces à former des biofilms sur divers supports abiotiques. Les souches de S. carnosus sont hydrophiles, adhèrent à des supports hydrophiles mais ne forment pas de biofilms. Certaines souches de S. xylosus sont hydrophiles, d'autres moyennement hydrophobes et elles forment des biofilms quelque soit le support. S. xylosus C2a, choisie comme souche d'étude, forme des biofilms denses avec des agrégats intercelluaires séparés par des canaux et englués dans une matrice constituée de polysaccharides dont la synthèse ne semble pas liée au gène icaA. Nous avons mis en evidence les gènes atl et bap codant des protèines de surface impliquées dans la formation de biofilm chez S. aureus. Pour étudier l'ensemble des protéines de surface impliquées, nous avons développé une méthode d'analyse des protéines pariétales et membranaires. Un total de 101 protéines a été identifié dont 51 sont prédites comme protéines de surface et seulement 9 sont cmmunes aux deux fractions. La comparaison des profils protéiques des fractions pariétales, membranaires et intracellulaires a révélé une expression différentielle de 115 protéines dont 74 sont surexprimées en mode sessile et 41 en planctonique. Cette étude a révélé la modification de nombreuses voies métabolliques. Leur analyse permettra de mieux appréhender les mécanismes mis en jeu par S. xylosus lors de sa croissance en biofilm

The family 1 cellulose-binding modules (CBM1) form a groupof small, stable carbohydrate-binding proteins. These modulesare essential for fungal cellulosedegradation. This thesisdescribes both functional studies of the CBM1s as well asprotein engineering of the modules for several objectives. The characteristics and specificity of CBM1s from theTrichoderma reeseiCel7A and Cel6A, along with severalother wild type and mutated CBMs, were studied using bindingexperiments and transmission electron microscopy (TEM). Datafrom the binding studies confirmed that the presence of onetryptophan residue on the CBM1 binding face enhances itsbinding to crystalline cellulose. The twoT. reeseiCBM1s as well as the CBM3 from theClostridium thermocellumCipA were investigated by TEMexperiments. All three CBMs were found to bind in lineararrangements along the sides of the fibrils. Further analysesof the bound CBMs indicated that the CBMs bind to the exposedhydrophobic surfaces, the so called (200) crystalline face ofValoniacellulose crystals. The function and specificity of CBM1s as a part of an intactenzyme were studied by replacing the CBM from the exo-actingCel7A by the CBM1 from the endoglucanase Cel7B. Apart fromslightly improved affinity of the hybrid enzyme, the moduleexchange did not significantly influence the function of theCel7A. This indicates that the two CBM1s are analogous in theirbinding properties and function during cellulosehydrolysis. The CBM1 was also used for immobilization studies. Toimprove heterologous expression of a CBM1-lipase fusionprotein, a linker stability study was carried out inPichia pastoris. A proline/threonine rich linker peptidewas found to be stable for protein production in this host. Forwhole bacterial cell immobilization, theT. reeseiCel6A CBM1 was expressed on the surface of thegram-positive bacteria,Staphylococcus carnosus. The engineeredS. carnosuscells were shown to bind cellulosefibers. To exploit the stable CBM1 fold as a starting point forgenerating novel binders, a phage display library wasconstructed. Binding proteins against an amylase as well asagainst a metal ion were selected from the library. Theamylase-binding proteins were found to bind and inhibit thetarget enzyme. The metal binding proteins selected from thelibrary were cloned on the surface of theS. carnosusand clearly enhanced the metal bindingability of the engineered bacteria. <b>Keywords</b>: cellulose-binding, family 1carbohydrate-binding module, phage display, bacterial surfacedisplay, combinatorial protein library, metal binding, proteinengineering,Trichoderma reesei, Staphyloccus carnosus.

<p>The family 1 cellulose-binding modules (CBM1) form a groupof small, stable carbohydrate-binding proteins. These modulesare essential for fungal cellulosedegradation. This thesisdescribes both functional studies of the CBM1s as well asprotein engineering of the modules for several objectives.</p><p>The characteristics and specificity of CBM1s from the<i>Trichoderma reesei</i>Cel7A and Cel6A, along with severalother wild type and mutated CBMs, were studied using bindingexperiments and transmission electron microscopy (TEM). Datafrom the binding studies confirmed that the presence of onetryptophan residue on the CBM1 binding face enhances itsbinding to crystalline cellulose. The two<i>T. reesei</i>CBM1s as well as the CBM3 from the<i>Clostridium thermocellum</i>CipA were investigated by TEMexperiments. All three CBMs were found to bind in lineararrangements along the sides of the fibrils. Further analysesof the bound CBMs indicated that the CBMs bind to the exposedhydrophobic surfaces, the so called (200) crystalline face of<i>Valonia</i>cellulose crystals.</p><p>The function and specificity of CBM1s as a part of an intactenzyme were studied by replacing the CBM from the exo-actingCel7A by the CBM1 from the endoglucanase Cel7B. Apart fromslightly improved affinity of the hybrid enzyme, the moduleexchange did not significantly influence the function of theCel7A. This indicates that the two CBM1s are analogous in theirbinding properties and function during cellulosehydrolysis.</p><p>The CBM1 was also used for immobilization studies. Toimprove heterologous expression of a CBM1-lipase fusionprotein, a linker stability study was carried out in<i>Pichia pastoris</i>. A proline/threonine rich linker peptidewas found to be stable for protein production in this host. Forwhole bacterial cell immobilization, the<i>T. reesei</i>Cel6A CBM1 was expressed on the surface of thegram-positive bacteria,<i>Staphylococcus carnosus</i>. The engineered<i>S. carnosus</i>cells were shown to bind cellulosefibers.</p><p>To exploit the stable CBM1 fold as a starting point forgenerating novel binders, a phage display library wasconstructed. Binding proteins against an amylase as well asagainst a metal ion were selected from the library. Theamylase-binding proteins were found to bind and inhibit thetarget enzyme. The metal binding proteins selected from thelibrary were cloned on the surface of the<i>S. carnosus</i>and clearly enhanced the metal bindingability of the engineered bacteria.</p><p><b>Keywords</b>: cellulose-binding, family 1carbohydrate-binding module, phage display, bacterial surfacedisplay, combinatorial protein library, metal binding, proteinengineering,<i>Trichoderma reesei, Staphyloccus carnosus</i>.</p>

Certaines espèces de Staphylocoques à Coagulase Négative (SCN) sont naturellement présentes ou employées comme ferments de salaison. La diversité de cette flore au niveau des ateliers de salaisons reste mal connue en raison notamment de la difficulté à identifier les espèces de SCN. Nous avons développé une PCR spécifique pour identifier Staphylococcus xylosus, principale espèce présente dans les produits carnés fermentés. Une PCR multiplex ciblant S. xylosus et trois espèces de staphylocoques pathogènes opportunistes a été développée permettant l'identification de ces espèces et du genre Staphylococcus. Ces méthodes et des méthodes complémentaires basées sur la caractérisation des souches par analyse en PFGE après restriction de l'ADN, par séquençage du gène sodA et par hybridation avec des sondes spécifiques ont permis l'étude de la diversité des SCN d'ateliers de salaisons traditionnelles, artisanales ou industrielles. Nous avons pu montrer que, dans un atelier traditionnel n'utilisant aucun ferment, S. equorum et S. succinus ont colonisé majoritairement les produits et l'environnement alors que les espèces S. xylosus et S. carnosus habituellement utilisées comme ferments sont minoritaires et n'ont pas été retrouvées au niveau des produits finis. Le suivi de S. carnosus et S. xylosus, utilisés comme ferments dans un atelier artisanal et un industriel, a révélé qu'ils ne colonisent pas l'environnement et que S. xylosus devient dominant dans les produits. Il pourrait être intéressant de développer de nouveaux ferments appartenant aux espèces S. equorum ou S. succinus qui semblent adaptées à l'environnement carné.

The engineering of bacterial surfaces has in recent yearsattracted a lot of attention with applications in manydifferent areas of bioscience. Here we describe the use of twodifferent surface display systems for the gram-positivebacteria Staphylococcus carnosus and Staphylococcus xylosus invarious biotechnological applications. Environmental microbiology currently attracts a lot ofattention since genetically engineered plants and bacteriamight be used as bioadsorbents for sequestration of toxicmetals. Bacterial surface display of metal-binding peptidesmight enable recycling of the biomass by desorption ofaccumulated heavymetals. In an attempt to recruitstaphylococcal display systems for bioremediation purposes,polyhistidyl peptides were successfullly displayed on thesurface of recombinant S. carnosus and S. xylosus cells.Whole-cell Ni2+-binding assays demonstrated that therecombinant cells had gained metal-binding capacity compared towild-type cells. Tailor-made, metal-binding staphylococci was created using apreviously constructed phage-display combinatorial proteinlibrary based on a fungal cellulose-binding domain (CBD)derived from the cellobiohydrolase Cel7A of Trichoderma reseii.Novel metal-binding CBDs were generated through a phagemediated selection procedure. Selected CBD variants, now devoidof cellulose binding, were randomly selected and sequenceanalysis of selected variants revealed a marked preference forhistidine residues at the randomized positions. Surface displayof these novel CBD variants resulted in recombinantstaphylococci with increased metal-binding capacity compared tocontrol strains, indicating that this could become a generalstrategy to engineer bacteria for improved binding to specificmetal ions. Directed immobilization of cells with surface displayedheterologous proteins have widespread use in modernbiotechnology. Among other things they could provide aconvenient way of generating biofilters, biocatalysts orwhole-cell diagnostic devices. It was therefore investigatedwhether directed immobilization of recombinant staphylococci oncotton fibers could be achieved by functional display of afungal cellulose-binding domain (CBD). Recombinant S. carnosuscells with surface anchored CBDs from Trichoderma reseii Cel6Awere found to efficiently bind to cotton fibers creating almosta monolayer on the fibrous support. The co-expression of thisCBD together with previously described metal-binding proteinson the surface of our staphylococci would create means fordeveloping effective bioadsorbents for remediationpurposes. The original plasmid vector, designed for heterologoussurface display on recombinant S. carnosus cells has exhibitedproblems related to structural instability, possibly due to thepresence of a phage f1 origin of replication in the vectorsequence. This would be a problem if using the vector systemfor library display applications. Therefore, novel surfacedisplay vectors, lacking the phage ori were constructed andevaluated by enzymatic and flow cytometric whole-cell assays.One such novel vector, pSCXm, exhibited dramatically increasedplasmid stability with the retained high surface density ofexpressed heterologous proteins characteristic for the originalS. carnosus display vector, thus making it potentially moresuitable for library display applications. The successful engineering of our staphylococcal displaysystem encouraged us to further evaluate the potential to usethe staphylococcal system for display of combinatorial proteinlibraries and subsequent affinity based selections using flowcytometric cell sorting. A model system of recombinant S.carnosus cells with surface displayed engineered protein Adomains was constructed. It was demonstrated that target cellscould be sorted essentially quantitatively from a moderateexcess of background cells in a single sorting-step.Furthermore, the possibility of using staphylococcal surfacedisplay and flow cytometric cell sorting also for specificenrichment of very rare target cells by multiple rounds ofcell-sorting and in between amplification was demonstrated. <b>Key words:</b>affibody, albumin binding protein, bacterialsurface display, cell immobilization, bioremediation,combinatorial protein engineering, flow cytometry,Gram-positive, metal binding, staphylococcal protein A,Staphylococcus carnosus, Staphylococcus xylosus, whole-celldevices

<p>The engineering of bacterial surfaces has in recent yearsattracted a lot of attention with applications in manydifferent areas of bioscience. Here we describe the use of twodifferent surface display systems for the gram-positivebacteria Staphylococcus carnosus and Staphylococcus xylosus invarious biotechnological applications.</p><p>Environmental microbiology currently attracts a lot ofattention since genetically engineered plants and bacteriamight be used as bioadsorbents for sequestration of toxicmetals. Bacterial surface display of metal-binding peptidesmight enable recycling of the biomass by desorption ofaccumulated heavymetals. In an attempt to recruitstaphylococcal display systems for bioremediation purposes,polyhistidyl peptides were successfullly displayed on thesurface of recombinant S. carnosus and S. xylosus cells.Whole-cell Ni2+-binding assays demonstrated that therecombinant cells had gained metal-binding capacity compared towild-type cells.</p><p>Tailor-made, metal-binding staphylococci was created using apreviously constructed phage-display combinatorial proteinlibrary based on a fungal cellulose-binding domain (CBD)derived from the cellobiohydrolase Cel7A of Trichoderma reseii.Novel metal-binding CBDs were generated through a phagemediated selection procedure. Selected CBD variants, now devoidof cellulose binding, were randomly selected and sequenceanalysis of selected variants revealed a marked preference forhistidine residues at the randomized positions. Surface displayof these novel CBD variants resulted in recombinantstaphylococci with increased metal-binding capacity compared tocontrol strains, indicating that this could become a generalstrategy to engineer bacteria for improved binding to specificmetal ions.</p><p>Directed immobilization of cells with surface displayedheterologous proteins have widespread use in modernbiotechnology. Among other things they could provide aconvenient way of generating biofilters, biocatalysts orwhole-cell diagnostic devices. It was therefore investigatedwhether directed immobilization of recombinant staphylococci oncotton fibers could be achieved by functional display of afungal cellulose-binding domain (CBD). Recombinant S. carnosuscells with surface anchored CBDs from Trichoderma reseii Cel6Awere found to efficiently bind to cotton fibers creating almosta monolayer on the fibrous support. The co-expression of thisCBD together with previously described metal-binding proteinson the surface of our staphylococci would create means fordeveloping effective bioadsorbents for remediationpurposes.</p><p>The original plasmid vector, designed for heterologoussurface display on recombinant S. carnosus cells has exhibitedproblems related to structural instability, possibly due to thepresence of a phage f1 origin of replication in the vectorsequence. This would be a problem if using the vector systemfor library display applications. Therefore, novel surfacedisplay vectors, lacking the phage ori were constructed andevaluated by enzymatic and flow cytometric whole-cell assays.One such novel vector, pSCXm, exhibited dramatically increasedplasmid stability with the retained high surface density ofexpressed heterologous proteins characteristic for the originalS. carnosus display vector, thus making it potentially moresuitable for library display applications.</p><p>The successful engineering of our staphylococcal displaysystem encouraged us to further evaluate the potential to usethe staphylococcal system for display of combinatorial proteinlibraries and subsequent affinity based selections using flowcytometric cell sorting. A model system of recombinant S.carnosus cells with surface displayed engineered protein Adomains was constructed. It was demonstrated that target cellscould be sorted essentially quantitatively from a moderateexcess of background cells in a single sorting-step.Furthermore, the possibility of using staphylococcal surfacedisplay and flow cytometric cell sorting also for specificenrichment of very rare target cells by multiple rounds ofcell-sorting and in between amplification was demonstrated.</p><p><b>Key words:</b>affibody, albumin binding protein, bacterialsurface display, cell immobilization, bioremediation,combinatorial protein engineering, flow cytometry,Gram-positive, metal binding, staphylococcal protein A,Staphylococcus carnosus, Staphylococcus xylosus, whole-celldevices</p>