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

Als &amp;quot;surface-layer&amp;quot; (S-Layer, SL) bezeichnet man die regelmäßig strukturierten Hüllproteinlagen auf der Oberfläche von etwa 80 % aller bisher bekannten Bakterienspezies. Sie entstehen durch Selbstassemblierung von identischen Proteinuntereinheiten, die wiederum zumeist durch nichtkovalente Wechselwirkungen mit der darunterliegenden Zellwandkomponente verknüpft sind. Trotz ihrer Diversität auf der Ebene der Primärstruktur weisen S-Layer verschiedener Bakterienarten einheitliche physikochemische Merkmale auf. Dazu zählt u.a. die Wiedereinnahme einer hochgradig strukturierten, porösen Proteinschicht nach reversibler Denaturierung. Infolge der Reassemblierung entstehen sowohl in Lösung als auch an Phasengrenzen Proteinassemblate, deren Porenanordnungen die gleiche regelmäßige Symmetrie aufweisen, wie die nativen Hüllproteine auf der Bakterienzelle. Das in seiner Domänenstruktur aufgeklärte Hüllprotein SbsC des mesophilen Bakterienstammes Geobacillus (G.) stearothermophilus ATCC 12980 zeichnet sich durch eine ausgezeichnete Synthetisierbarkeit in E. coli aus. C-terminale Fusionen, die im Falle des verstärkt grün fluoreszierenden Proteins (EGFP) bis zu 240 Aminosäuren umfassen, führten nicht zu einem Verlust der Selbstassemblierung. Darüber hinaus zeigen in vitro gebildete SbsC-Assemblate eine außergewöhnliche Stabilität gegenüber hohen Ethanolkonzentrationen. Die durch gerichtete Mutagenese erzeugten SbsC-Fusionsproteine SbsC(aa 31-1099)-HspA und SbsC(aa 31-1099)-12His besitzen in assemblierter Form im Vergleich mit dem unmodifizierten Protein eine bis zu zweimal höhere Bindungsaffinität gegenüber Platinionen. In denaturierter Form waren beide Fusionsproteine in der Lage, Nickelionen zu komplexieren. In der vorliegenden Arbeit wurde erstmals ein SL-Protein in einem eukaryontischen Mikroorganismus produziert. Das in der Hefe S. cerevisiae gebildete Fusionsprotein SbsC(aa 31-1099)-EGFP assembliert dabei im Cytosol der Wirtszellen zu röhrenförmigen Assemblaten mit regelmäßiger Symmetrie. Das bisher unbekannte SL-Protein des Stammes G. stearothermophilus DSM 13240 wurde erfolgreich heterolog in E. coli exprimiert. Die Vorläuferform besitzt im Vergleich zum maturen Protein ein 31 aa umfassendes Sekretionssignal am extremen N-Terminus. Sowohl das authentische Protein als auch das heterolog in E. coli exprimierte Vorläuferprotein zeigen eine dem SbsC-Protein vergleichbare Reassemblierungscharakteristik. Im Gegensatz dazu führte die Verkürzung der N-terminalen 30 Aminosäuren des als S13240 bezeichneten Hüllproteins im heterologen System zu einem irreversiblen Verlust der Fähigkeit zur Selbstassemblierung.

<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>

Indiana University-Purdue University Indianapolis (IUPUI) / The discovery and isolation of naturally occurring fluorescent proteins, FPs, have provided much needed tools for molecular and cellular level studies. Specifically the cloning of green fluorescent protein, GFP, revolutionized the field of biotechnology and biochemical research. Recently, a red fluorescent protein, DsRed, isolated from the Discosoma coral has further expanded the pallet of available fluorescent tools. DsRed shares only 23 % amino acid sequence homology with GFP, however the X-ray crystal structures of the two proteins are nearly identical. DsRed has been subjected to a number of mutagenesis studies, which have been found to offer improved physical and spectral characteristics. One such mutant, DsRed-Monomer, with a total of 45 amino acid substitutions in native DsRed, has shown improved fluorescence characteristics without the toxic oligomerization seen for the native protein. In our laboratory, we have demonstrated that DsRed proteins have a unique and selective copper-binding affinity, which results in fluorescence quenching. This copper-binding property was utilized in the purification of DsRed proteins using copper-bound affinity columns. The work presented here has explored the mechanism of copper-binding by DsRed-Monomer using binding studies, molecular biology, and other biochemical techniques. Another focus of this thesis work was to demonstrate the applications of DsRed-Monomer in biochemical studies based on the copper-binding affinity and fluorescence properties of the protein. To achieve this, we have focused on genetic fusions of DsRed-Monomer with peptides and proteins. The work with these fusions have demonstrated the feasibility of using DsRed-Monomer as a dual functional tag, as both an affinity tag and as a label in the development of a fluorescence assay to detect a ligand of interest. Further, a complex between DsRed-Monomer-bait peptide/protein fusion and an interacting protein has been isolated taking advantage of the copper-binding affinity of DsRed-Monomer. We have also demonstrated the use of non-natural amino acid analogues, incorporated into the fluorophore of DsRed-Monomer, as a tool for varying the spectral properties of the protein. These mutations demonstrated not only shifted fluorescence emission compared to the native protein, but also improved extinction coefficients and quantum yields.

Als &amp;quot;surface-layer&amp;quot; (S-Layer, SL) bezeichnet man die regelmäßig strukturierten Hüllproteinlagen auf der Oberfläche von etwa 80 % aller bisher bekannten Bakterienspezies. Sie entstehen durch Selbstassemblierung von identischen Proteinuntereinheiten, die wiederum zumeist durch nichtkovalente Wechselwirkungen mit der darunterliegenden Zellwandkomponente verknüpft sind. Trotz ihrer Diversität auf der Ebene der Primärstruktur weisen S-Layer verschiedener Bakterienarten einheitliche physikochemische Merkmale auf. Dazu zählt u.a. die Wiedereinnahme einer hochgradig strukturierten, porösen Proteinschicht nach reversibler Denaturierung. Infolge der Reassemblierung entstehen sowohl in Lösung als auch an Phasengrenzen Proteinassemblate, deren Porenanordnungen die gleiche regelmäßige Symmetrie aufweisen, wie die nativen Hüllproteine auf der Bakterienzelle. Das in seiner Domänenstruktur aufgeklärte Hüllprotein SbsC des mesophilen Bakterienstammes Geobacillus (G.) stearothermophilus ATCC 12980 zeichnet sich durch eine ausgezeichnete Synthetisierbarkeit in E. coli aus. C-terminale Fusionen, die im Falle des verstärkt grün fluoreszierenden Proteins (EGFP) bis zu 240 Aminosäuren umfassen, führten nicht zu einem Verlust der Selbstassemblierung. Darüber hinaus zeigen in vitro gebildete SbsC-Assemblate eine außergewöhnliche Stabilität gegenüber hohen Ethanolkonzentrationen. Die durch gerichtete Mutagenese erzeugten SbsC-Fusionsproteine SbsC(aa 31-1099)-HspA und SbsC(aa 31-1099)-12His besitzen in assemblierter Form im Vergleich mit dem unmodifizierten Protein eine bis zu zweimal höhere Bindungsaffinität gegenüber Platinionen. In denaturierter Form waren beide Fusionsproteine in der Lage, Nickelionen zu komplexieren. In der vorliegenden Arbeit wurde erstmals ein SL-Protein in einem eukaryontischen Mikroorganismus produziert. Das in der Hefe S. cerevisiae gebildete Fusionsprotein SbsC(aa 31-1099)-EGFP assembliert dabei im Cytosol der Wirtszellen zu röhrenförmigen Assemblaten mit regelmäßiger Symmetrie. Das bisher unbekannte SL-Protein des Stammes G. stearothermophilus DSM 13240 wurde erfolgreich heterolog in E. coli exprimiert. Die Vorläuferform besitzt im Vergleich zum maturen Protein ein 31 aa umfassendes Sekretionssignal am extremen N-Terminus. Sowohl das authentische Protein als auch das heterolog in E. coli exprimierte Vorläuferprotein zeigen eine dem SbsC-Protein vergleichbare Reassemblierungscharakteristik. Im Gegensatz dazu führte die Verkürzung der N-terminalen 30 Aminosäuren des als S13240 bezeichneten Hüllproteins im heterologen System zu einem irreversiblen Verlust der Fähigkeit zur Selbstassemblierung.

Les systèmes d’efflux tripartite de type Resistance, Nodulation and cell-Division (RND) sont essentiels dans le maintien de phénotypes de résistance multidrogues et contre les métaux lourds dans nombreuses bactéries Gram-négatives. Le transport de ces composés toxiques hors de la cellule est permis par l’assemblage d’une protéine de type antiporteur cation/proton (unité RND) insérée dans la membrane interne, connectée à une protéine insérée dans la membrane externe, pour former un canal de sorti qui traverse l’entièreté de l’enveloppe cellulaire. Le troisième composant du système, la protéine de type membrane fusion protein (MFP) qui est aussi appelée periplasmic adaptor protein (PAP), est requis pour permettre l’assemblage de tout ce complexe à trois composants. Cependant, les MFPs sont supposées jouer un rôle important et actif dans le mécanisme d’efflux du substrat. Pour mieux comprendre le rôle des MFPs au sein des systèmes d’efflux de type RND, nous avons étudié les protéines ZneB (précédemment appelée HmxB) et SilB, les composants périplasmiques des systèmes ZneCBA et SilABC responsables de la résistance aux métaux lourds chez Cupriavidus metallidurans CH34. Nous avons identifié la spécificité de liaison au substrat de ces protéines, montrant leur capacité à fixer le zinc (ZneB), ou le cuivre et l’argent (SilB). De plus, nous avons résolu la structure cristalline de ZneB à une résolution de 2.8 Å dans la forme apo- et avec un ion zinc fixé. La structure de ZneB possède une architecture générale composée de quatre domaines caractéristiques des MFPs, et la présence du site de coordination au zinc dans une région très flexible à l’interface des domaines β-barrel et membrane proximal. Les modifications structurales que la protéine subit lors de la fixation du zinc on été observée dans le cristal mais aussi en solution, ce qui suggère un rôle actif des MFPs dans le mécanisme d’efflux des métaux, vraisemblablement via la fixation et le relargage de l’ion à l’antiporteur. Les études de sélectivité de transport des antiporteurs ZneA et SilA montre que ces dernières et leurs protéines périplasmiques respectives ont des affinités similaires pour les métaux lourds. De plus, les études de transport ont apportés des arguments en faveur de l’hypothèse de capture cytoplasmique du substrat par l’antiporteur, tandis que la capacité des protéines périplasmiques à fixer les métaux lourds a apporté des arguments en faveur de l’hypothèse de capture périplasmique du substrat par l’antiporteur. Les deux modes de capture pourraient en réalité coexister ;cependant, le débat autour du compartiment cellulaire de capture du substrat par l’antiporteur est complexe et requiert de plus amples efforts afin d’être cerné. / Tripartite resistance nodulation cell division (RND)-based efflux complexes are paramount for multidrug and heavy metal resistance in numerous Gram-negative bacteria. The transport of these toxic compounds out of the cell is driven by the inner membrane proton/substrate antiporter (RND protein) connected to an outer membrane protein to form an exit duct that spans the entire cell envelope. The third component, a membrane fusion protein (MFP) also called periplasmic adaptor protein, is required for the assembly of this complex. However, MFPs are also proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we studied ZneB (formerly HmxB) and SilB, the MFP components of the ZneCAB and SilABC heavy metal RND-driven efflux complexes from Cupriavidus metallidurans CH34. We have identified the substrate binding specificity of the proteins, showing their ability to selectively bind zinc (ZneB), or copper and silver cations (SilB). Moreover, we have solved the crystal structure of the apo- and the metal-bound forms of ZneB to 2.8 Å resolution. The structure of ZneB displays a general architecture composed of four domains characteristic of MFPs, and it reveals the metal coordination site at the very flexible interface between the β-barrel and the membrane proximal domains. Structural modifications of the protein upon zinc binding were observed in both the crystal structure and in solution, suggesting an active role of MFPs in substrate efflux possibly through binding and release. The selectivity assays of the antiporter proteins ZneA and SilA demonstrated similar specificities in relation to their cognate MFPs toward heavy metal cations. Moreover, antiporter transport assays provide evidence for cytoplasmic substrate capture by this protein, whereas MFP substrate binding provides evidence for periplasmic substrate capture. Therefore, both modes of capture might co-exist; nevertheless, the substrate capture issue is a complex topic still needing consequent efforts to understand it. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

This report summarizes the work on the cloning, expression, and purification of PerR, a metal sensing regulator from Saccharopolyspora erythraea and the subsequent characterization using small angle X-ray scattering and other biochemical methods. The report aims to provide an insight into prokaryotic metal homeostasis, provide a better understanding of how PerR works and provide valuable information for the continued work on the crystallization of PerR.