In numerous Gram-negative and Gram-positive bacteria as well as in Archaea SL proteins form the outermost layer of the cell envelope. SL (glyco)monomers self-assemble with oblique (p2), tetragonal (p4), or hexagonal (p3, p6) symmetries [12]. SL subunits interact with each other and with the underlying cell surface by relatively weak non-covalent forces such as hydrogen-bonds, ionic bonds, salt-bridges or hydrophobic interactions. This makes them easy to isolate by applying chaotropic agents like urea and guanidine hydrochloride (GuHCl), chelating chemicals, or by changing the pH of the environment [10]. Upon dialysis in an ambient buffer monomers recrystallize into regular arrays that possess the forms of flat sheets, open ended cylinders, or spheres on solid substrates, at air-water intefaces and on lipid films, making them appealing for nanobiotechnological applications [3, 18]. The aim of this study was to investigate the structure, thermal stability, in vivo self-assembly process, recrystallization and metallization of three different recombinant SL proteins (SslA-eGFP, mSbsC-eGFP and S13240-eGFP) expressed in yeast S. cerevisiae BY4741 which could be further used in nanobiotechnological applications. In order to fulfill this aim, I investigated the in vivo expression of SL proteins (SslA, SbsC, S13240) tagged with eGFP (SL-eGFP) in the yeast S. cerevisiae BY4141. First, I characterized the heterologous expression of SL fusion constructs with growth and fluorescence measurements combined with Western blot analyses. Fluorescence microscopy investigations of overnight grown cultures showed that SslA-eGFP fusion protein was expressed as fluorescent patches, mSbsC-eGFP as tubular networks, and S13240-eGFP as hollow-like fibrillar network structures, while eGFP did not show any distinct structure Thermal stability of in vivo expressed SL-eGFP fusion proteins were investigated by fluorescence microscopy and immunodetection. In vivo self-assembly kinetics during mitosis and meiosis was the second main issue. In parallel, association of in vivo mSbsC-eGFP structures with the cellular components was of interest. A network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton was observed. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis surface layer structures were formed de novo in the buds. Surface layer assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point. In order to get these in vivo SL assemblies stably outside the cells (in situ), cell distruption experiments were conducted. The tubular structures formed by the protein in vivo were retained upon bursting the cells by osmotic shock; however their average length was decreased. During dialysis, monomers obtained by treatment with chaotropic agents recrystallized again to form tube-like structures. This process was strictly dependent on calcium ions, with an optimal concentration of 10 mM. Further increase of the Ca2+ concentration resulted in multiple non-productive nucleation points. It was further shown that the lengths of the S-layer assemblies increased with time and could be controlled by pH. After 48 hours the average length at pH 9.0 was 4.13 µm compared to 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications. For example, such metalized protein nanotubes could be used in conductive nanocircuit technologies as nanowires.

No description available.

No description available.

Formation of segments during vertebrate embryogenesis is regulated by a biological clock. Models and experimental data indicate that the core of this clock consists of a cell- autonomous single cell oscillator. This oscillator likely involves a genetic feedback loop of transcriptional repressors belonging to the hairy gene family. In zebrafish, three her genes, her1, hes6 and her7, have been identified as core oscillator components. The main purpose of this project was to study the molecular mechanism of the hairy gene negative feedback oscillator in single cells. To determine whether a single cell oscillator is part of the zebrafish segmentation clock, a cell dissociation protocol was established to track the expression of Her1 ex vivo. Upon dissociation, Her1 expression continued to oscillate for up to three cycles. The period of oscillations was significantly slower than that of the segmentation clock, but appears to speed up in the presence of serum. To test whether the hairy gene interactions are sufficient to generate oscillations in single cells, a protocol was established that uses synthetic biology principles to design, construct and characterize hairy gene networks in yeast. First a library of network parts, containing hairy genes, promoters and Her binding sites was generated and subsequently assembled into simple devices to test their functionality in yeast. The three core oscillator components, Her1, Hes6 and Her7, were characterized and optimized for expression in yeast. In the SWITCH-OFF assay, the Her1 protein, modified with a MigED yeast repressor domain, was found to function as a transcriptional repressor in yeast, while Hes6 with the same modification can not. The dissociation of segmentation clock cells provides the first direct evidence that single cell oscillators exist in zebrafish. In this system, oscillator dynamics can be studied without the interactions of higher level clock components. In parallel, establishing a yeast chassis for hairy gene networks provides a novel technique to directly test predicted oscillator mechanisms by constructing them ’bottom up’.

The commonly used drug diclofenac is an important environmental anthropogenic pollutant. Currently, detection of diclofenac is mainly based on chemical and physical methods. Here we describe a yeast biosensor that drives the diclofenac-dependent expression of a recombinant fluorescent protein from the authentic promoter of the PDR5 gene. This key component of the pleiotropic drug response encodes a multidrug transporter that is involved in cellular detoxification. We analyse the effects on diclofenac sensitivity of artificial PDR5 promoter derivatives in wild-type and various yeast mutant strains. This approach enabled us to generate sensor strains with elevated drug sensitivity.

The commonly used drug diclofenac is an important environmental anthropogenic pollutant. Currently, detection of diclofenac is mainly based on chemical and physical methods. Here we describe a yeast biosensor that drives the diclofenac-dependent expression of a recombinant fluorescent protein from the authentic promoter of the PDR5 gene. This key component of the pleiotropic drug response encodes a multidrug transporter that is involved in cellular detoxification. We analyse the effects on diclofenac sensitivity of artificial PDR5 promoter derivatives in wild-type and various yeast mutant strains. This approach enabled us to generate sensor strains with elevated drug sensitivity.

The studies presented here were focused on the understanding of the principles for protein sorting from the Golgi to the cell surface. As a marker protein we used Fus1p, a type I plasma membrane protein that is O-glycosylated on the extracellular domain and plays a role in cell fusion during yeast mating. Additionally, we analyzed mechanisms responsible for asymmetric distribution of Fus1p in mating cells. We demonstrated that the glycans attached to the protein act as a sorting determinant for protein transport to the cell surface. In cells lacking PMT4, encoding a mannosyltransferase involved in the initial step of O-glycosylation, Fus1p was not glycosylated and accumulated in late Golgi structures. A similar defect in exocytosis was observed when a Fus1p mutant lacking the O-glycosylated domain was expressed in wild-type cells, however, the cell surface delivery could be rescued if the 33 amino acid portion of the Fus1p ectodomain, containing 15 potentially glycosylated sites was added to the protein. It was previously well documented in epithelial cells that different types of protein glycosylation and association with lipid rafts play a role of determinants for protein delivery to the apical plasma membrane. However, otherwise the machinery responsible for cargo sorting to the apical membrane is poorly understood. Our finding that also in yeast, protein glycosylation can function as a sorting determinant provides a new possibility to investigate underlying mechanisms...

Die Chorismatmutase (CM, EC 5.4.99.5), kodiert durch ARO7, katalysiert die Claisen-Umlagerung von Chorismat zu Prephenat in der Biosynthese von Tyrosin und Phenylalanin. Das relativ kleine, dimere Enzym der Hefe Saccharomyces cerevisiae wird allosterisch durch Tryptophan aktiviert und allosterisch durch Tyrosin inhibiert. In der vorliegenden Arbeit wurde die Theorie widerlegt, dass die Chorismatemutase an der Osmoregulation und Vakuolenentstehung beteiligt ist. Die Analyse einiger Stämme mit punktmutiertem oder deletiertem ARO7-Gen zeigte ausschließlich eine Funktion in der Aminosäure-Biosynthese. Die Fusion an das grün-fluoreszierende Protein ermöglichte die Lokalisierung der CM in Cytoplasma und Kern der Hefezelle.Auf Proteinebene wurde der intramolekulare Signalübertragungsweg von den allosterischen zu den aktiven Zentren näher untersucht. Es wurden Chimären-Enzyme hergestellt, in denen das molekulare Scharnier L220s zwischen der katalytischen und allosterischen Domäne ausgetauscht wurde gegen den entsprechenden Bestandteil homologer Pilzenzyme. Die kinetische Analyse zeigte, dass dieser Proteinteil essentiell ist für die Unterscheidung zwischen dem Signal Aktivierung bzw. Inhibierung. Diese Region ist auch für die Dimerisierung der CM von Bedeutung. Durch Austausch hydrophober Aminosäuren gegen geladene Reste in und in der Nähe dieses Scharniers wurde eine stabile, monomere Enzymvariante hergestellt. Diese CM zeigte reduzierte Aktivität und keine Regulation, aber das kodierende Gen komplementierte die Tyrosin- und Phenylalanin-Auxotrophie der Zellen. Diese Ergebnisse unterstützen die Theorie, dass das Hefeenzym durch gleichzeitige Evolution von Regulations- und Stabilisierungsmechanismen aus einem monomeren, unregulierten Vorläuferprotein entstanden ist, welches dem der Escherichia coli CM ähnlich war. Um weitere Erkenntnisse über die Prinzipien der Proteinstabilisierung zu erhalten, wurde auch die Chorismatmutase on Thermus thermophilus charakterisiert, nachdem das kodierende Gen kloniert war. Dieses Enzym ist ähnlich zu der strukturell einzigartigen Chorismatmutae aus Bacillus subtilis, wird aber, im Gegensatz zu letzterem durch Tyrosin in seiner Aktivität gehemmt. Modellierungsstudien zeigten, dass wie auch bei anderen Proteinen verstärkte Hydrophilität von Oberflächen, erhöhte Hydrophobizität innerhalb der Struktur wie auch die Versteifung von Loops in der Nähe des aktiven Zentrums zur Stabilisierung dieser Proteinfaltung beitragen.

Formation of segments during vertebrate embryogenesis is regulated by a biological clock. Models and experimental data indicate that the core of this clock consists of a cell- autonomous single cell oscillator. This oscillator likely involves a genetic feedback loop of transcriptional repressors belonging to the hairy gene family. In zebrafish, three her genes, her1, hes6 and her7, have been identified as core oscillator components. The main purpose of this project was to study the molecular mechanism of the hairy gene negative feedback oscillator in single cells. To determine whether a single cell oscillator is part of the zebrafish segmentation clock, a cell dissociation protocol was established to track the expression of Her1 ex vivo. Upon dissociation, Her1 expression continued to oscillate for up to three cycles. The period of oscillations was significantly slower than that of the segmentation clock, but appears to speed up in the presence of serum. To test whether the hairy gene interactions are sufficient to generate oscillations in single cells, a protocol was established that uses synthetic biology principles to design, construct and characterize hairy gene networks in yeast. First a library of network parts, containing hairy genes, promoters and Her binding sites was generated and subsequently assembled into simple devices to test their functionality in yeast. The three core oscillator components, Her1, Hes6 and Her7, were characterized and optimized for expression in yeast. In the SWITCH-OFF assay, the Her1 protein, modified with a MigED yeast repressor domain, was found to function as a transcriptional repressor in yeast, while Hes6 with the same modification can not. The dissociation of segmentation clock cells provides the first direct evidence that single cell oscillators exist in zebrafish. In this system, oscillator dynamics can be studied without the interactions of higher level clock components. In parallel, establishing a yeast chassis for hairy gene networks provides a novel technique to directly test predicted oscillator mechanisms by constructing them ’bottom up’.

In numerous Gram-negative and Gram-positive bacteria as well as in Archaea SL proteins form the outermost layer of the cell envelope. SL (glyco)monomers self-assemble with oblique (p2), tetragonal (p4), or hexagonal (p3, p6) symmetries [12]. SL subunits interact with each other and with the underlying cell surface by relatively weak non-covalent forces such as hydrogen-bonds, ionic bonds, salt-bridges or hydrophobic interactions. This makes them easy to isolate by applying chaotropic agents like urea and guanidine hydrochloride (GuHCl), chelating chemicals, or by changing the pH of the environment [10]. Upon dialysis in an ambient buffer monomers recrystallize into regular arrays that possess the forms of flat sheets, open ended cylinders, or spheres on solid substrates, at air-water intefaces and on lipid films, making them appealing for nanobiotechnological applications [3, 18]. The aim of this study was to investigate the structure, thermal stability, in vivo self-assembly process, recrystallization and metallization of three different recombinant SL proteins (SslA-eGFP, mSbsC-eGFP and S13240-eGFP) expressed in yeast S. cerevisiae BY4741 which could be further used in nanobiotechnological applications. In order to fulfill this aim, I investigated the in vivo expression of SL proteins (SslA, SbsC, S13240) tagged with eGFP (SL-eGFP) in the yeast S. cerevisiae BY4141. First, I characterized the heterologous expression of SL fusion constructs with growth and fluorescence measurements combined with Western blot analyses. Fluorescence microscopy investigations of overnight grown cultures showed that SslA-eGFP fusion protein was expressed as fluorescent patches, mSbsC-eGFP as tubular networks, and S13240-eGFP as hollow-like fibrillar network structures, while eGFP did not show any distinct structure Thermal stability of in vivo expressed SL-eGFP fusion proteins were investigated by fluorescence microscopy and immunodetection. In vivo self-assembly kinetics during mitosis and meiosis was the second main issue. In parallel, association of in vivo mSbsC-eGFP structures with the cellular components was of interest. A network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton was observed. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis surface layer structures were formed de novo in the buds. Surface layer assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point. In order to get these in vivo SL assemblies stably outside the cells (in situ), cell distruption experiments were conducted. The tubular structures formed by the protein in vivo were retained upon bursting the cells by osmotic shock; however their average length was decreased. During dialysis, monomers obtained by treatment with chaotropic agents recrystallized again to form tube-like structures. This process was strictly dependent on calcium ions, with an optimal concentration of 10 mM. Further increase of the Ca2+ concentration resulted in multiple non-productive nucleation points. It was further shown that the lengths of the S-layer assemblies increased with time and could be controlled by pH. After 48 hours the average length at pH 9.0 was 4.13 µm compared to 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications. For example, such metalized protein nanotubes could be used in conductive nanocircuit technologies as nanowires.