Der Mensch besitzt ~25 funktionelle Bitterrezeptoren (TAS2R), die für die Wahrnehmung potenziell toxischer Substanzen in der Nahrung verantwortlich sind. Aufgrund der großen genetischen Variabilität der TAS2R-Gene könnte es eine Vielzahl funktionell unterschiedlicher TAS2R-Haplotypen geben, die zu Unterschieden der Bitterwahrnehmung führen. Dies konnte bereits in funktionellen Analysen und sensorischen Studien für einzelne Bitterrezeptoren gezeigt werden. In dieser Arbeit wurden die häufigsten Haplotypen aller 25 Bitterrezeptoren verschiedener Ethnien funktionell charakterisiert. Das Ziel war eine umfassende Aussage über die funktionelle Diversität der TAS2Rs, die die molekulare Grundlage für individuelle Bitterwahrnehmung bildet, treffen zu können. Fehlende Varianten wurden aus genomischer DNA kloniert oder durch gezielte Mutagenese bereits vorhandener TAS2R-Konstrukte generiert. Die funktionelle Analyse erfolgte mittels Expression der TAS2R-Haplotypen in HEK293TG16gust44 Zellen und anschließenden Calcium-Imaging-Experimenten mit zwei bekannten Agonisten. Die Haplotypen der fünf orphanen TAS2Rs wurden mit über hundert Bitterstoffen stimuliert. Durch die gelungene Deorphanisierung des TAS2R41 in dieser Arbeit, wurden für die 21 aktivierbaren TAS2Rs 36 funktionell-unterschiedliche Haplotypen identifiziert. Die tatsächliche funktionelle Vielfalt blieb jedoch deutlich hinter der genetischen Variabilität der TAS2Rs zurück. Neun Bitterrezeptoren wiesen funktionell homogene Haplotypen auf oder besaßen nur eine weltweit vorherrschende Variante. Funktionell heterogene Haplotypen wurden für zwölf TAS2Rs identifiziert. Inaktive Varianten der Rezeptoren TAS2R9, TAS2R38 und TAS2R46 sollten die Wahrnehmung von Bitterstoffen wie Ofloxacin, Cnicin, Hydrocortison, Limonin, Parthenolid oder Strychnin beeinflussen. Unterschiedlich sensitive Varianten, besonders der Rezeptoren TAS2R47 und TAS2R49, sollten für Agonisten wie Absinthin, Amarogentin oder Cromolyn ebenfalls zu phänotypischen Unterschieden führen. Wie für den TAS2R16 bereits gezeigt, traten Haplotypen des funktionell heterogenen TAS2R7 und TAS2R41 ethnien-spezifisch auf, was auf lokale Anpassung und verschiedene Phänotypen hinweisen könnte. Weiterführend muss nun eine Analyse der funktionell-variablen TAS2Rs in sensorischen Tests erfolgen, um ihre phänotypische Relevanz zu prüfen. Die Analyse der funktionsmodulierenden Aminosäurepositionen, z.Bsp. des TAS2R44, TAS2R47 oder TAS2R49, könnte weiterführend zum besseren Verständnis der Rezeptor-Ligand- und Rezeptor-G-Protein-Interaktion beitragen. / Bitter taste perception varies markedly from person to person, due to a high number of polymorphisms present in the 25 known functional bitter receptors (TAS2Rs). These polymorphisms lead to a number of haplotypes for each receptor, which are common in different populations, but vary in frequency. The individual combination of receptor variants seems to determine the person’s sensitivity of bitter perception, as could already be shown for single TAS2Rs. Bitter is an aversive taste quality, indicating the ingestion of harmful substances. Different sensitivity could have an impact on food choice. In order to characterize functional consequences of the genetic diversity, we performed calcium imaging experiments with all main haplotypes for the 25 bitter receptors. The obtained information about receptor properties enables us on the one hand to analyze structure-function relationships and on the other hand gives us the functional diverse candidates to focus on in psychophysical studies. The overall aim is to show genotype-phenotype correlation for bitter taste perception and their impact on food choice and therefore diet and health. Our first aim was to identify agonists for the 5 receptors, which could not be deorphaned in previous screens. We challenged all main haplotypes of these TAS2Rs with 106 bitter compounds and could identify the antibiotic chloramphenicol as agonist for bitter receptor TAS2R41. In total we identified 36 functionally different receptor variants of the 21 deorphaned TAS2Rs. Main haplotypes of nine TAS2Rs were functionally homogeneous while twelve TAS2Rs possessed between two and three functionally heterogeneous receptor variants. In summary the observed functional diversity is not as big as expected. Based on our in vitro findings the shown functional diversity of these twelve bitter receptors might be the molecular basis for individual differences in bitter taste perception and will be further analyzed in psychophysical studies.

Im Rahmen der vorliegenden Dissertation wurden die cDNAs der Hauptmyelinproteine IP2 und 36K aus dem ZNS der Regenbogenforelle isoliert, vervollständigt, molekularbiologisch charakterisiert und in verschiedenen Systemen heterolog exprimiert. Für das 36K Protein konnte eine Methode zur Proten-Aufreinigung etabliert werden. Für das IP-Protein konnte ein mimetisches Zellystem aufgebaut werden, welches erste experimentelle Hinweise auf eine Funktion als Adhäsionsmolekül der Myelinscheide ermöglichte. Die vorliegende Arbeit liefert den Grundstein für weiterführende Arbeiten auf dem Gebiet der Myelinproteine.

S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.

S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.

Die lösliche, NAD+-reduzierende Hydrogenase (SH) aus Ralstonia eutropha H16 ist eine Pyridinnukleotid-abhängige Hydrogenase. Das heißt, der Umsatz von H2 im Hydrogenasemodul des Enzyms ist an die Reduktion von NAD(P)+ im NAD(P)H:Akzeptor-Oxidoreduktasemodul gekoppelt. Die SH ist Vertreter des Subtyps, der auch in Gegenwart von O2 katalytisch aktiv ist. Dies wird ermöglicht durch eine reduktive Entfernung von O2, die nach dem aktuellen Modell abhängig ist vom rückläufigen e--Transport vom NADH:Akzeptor-Oxidoreduktasemodul zum aktiven [NiFe]-Zentrum in der großen Hydrogenaseuntereinheit. Der Einfluss des FeS-Clusters in der kleinen Hydrogenaseuntereinheit HoxY auf diesen Prozess wurde hier untersucht. Dabei konnte gezeigt werden, dass die vier hochkonservierten Cysteine C41, C44, C113 und C179 in HoxY an der Koordination des FeS-Zentrums beteiligt sind. Außerdem wurde das nahegelegene Cystein C39 als relevant für die Sauerstofftoleranz identifiziert. Weiterhin wurde gezeigt, dass das Tryptophan W42 aus HoxY essentiell für die Hydrogenaseaktivität der SH ist. Damit bestätigt sich, dass die Kinetik des rückläufigen e--Transports durch die Aminosäureumgebung des FeS-Clusters in HoxY beeinflusst ist. Ferner wurden in dieser Arbeit Ansätze zum Einsatz der SH aus R. eutropha in einer H2-produzierenden cyanobakteriellen Designzelle weiterverfolgt. Dazu wurden Hybridsysteme aus SH und cyanobakteriellem Photosystem I in vitro hinsichtlich ihrer Fähigkeit zur lichtgetriebenen H2-Produktion untersucht. Außerdem wurde an einem heterologen Expressionssystem der SH für Cyanobakterien gearbeitet. Weiterhin wurde die SH aus Rhodococcus opacus MR11 als komplementäres Modellsystem für O2-tolerante Pyridinnukleotid-abhängige Hydrogenasen etabliert. Dieser zur SH aus R. eutropha homologe Komplex hatte in früheren Arbeiten Vorteile für spektroskopische Studien offenbart und wurde hier erstmals im direkten Vergleich zur SH aus R. eutropha biochemisch und spektroskopisch charakterisiert. / The soluble, NAD+-reducing hydrogenase (SH) from Ralstonia eutropha H16 is a pyridine nucleotide-dependent hydrogenase. In these types of enzymes the conversion of H2 in the hydrogenase module of the complex is coupled to the reduction of NAD(P)+ in the NAD(P)H:acceptor oxidoreductase module. The SH belongs to a subtype that is catalytically active also in the presence of O2. This O2 tolerance is enabled by a reductive removal of O2, which according to the current model depends on a reverse e- flow from the NADH:acceptor oxidoreductase module to the active [NiFe] site in the large hydrogenase subunit. The impact of the FeS cluster in the small hydrogenase subunit HoxY on this process was analyzed in this study. Thereby it was shown that the four highly conserved cysteines C41, C44, C113 and C179 in HoxY are involved in the coordination of the FeS center. Further the nearby cysteine C39 was identified to be relevant for the O2 tolerance of the SH. Additionally we found the tryptophan W42 to be essential for the hydrogenase activity of the SH. Thus it was confirmed that the kinetic of the reverse e- transport is affected by the amino acid environment of the FeS cluster in HoxY. In addition, approaches for using the SH from R. eutropha in H2 producing cyanobacterial design cells were pursued. On one hand hybrid systems consisting of the SH and cyanobacterial photosystem I were analyzed in vitro for their capacity to produce H2 in a light dependent manner. On the other hand work on a heterologous expression system of the SH for Cyanobacteria was continued. Furthermore the SH from Rhodococcus opacus MR11 was established as complementary model system for O2-tolerant pyridine nucleotide-dependent hydrogenases. This complex, which is homologous to the SH from R. eutropha, has revealed advantages for spectroscopic analysis in earlier studies. Here it was characterized biochemically and spectroscopically for the first time in direct comparison with the SH from R. eutropha.

Die lichtaktivierte Kinase Phototropin aus Chlamydomonas reinhardtii, die photoaktivierte Adenylatcyclase (PAC) aus Euglena gracilis und das BLUF-Protein Slr1694 aus Synechocystis sp. PCC 6803 wurden in Hinblick auf die molekularen Details der primären photochemischen Prozesse sowie der Signalweiterleitung untersucht. Phototropin wurde mit Hilfe von Arginin aus Escherichia coli in Milligramm Mengen isoliert. Ohne Arginin wurde E. coli cAMP Rezeptorprotein assoziiert aufgefunden, welches eine hohe Homologie zu einer cAMP aktivierten Kinase aus C. reinhardtii besitzt. Volllängen Phototropin bildet wie einzelne LOV-Domänenkonstrukte ohne Kinasedomäne den Flavin-Triplettzustand und das kovalente Cysteinyl-Addukt. Der Zerfall des Signalzustandes ist in Anwesenheit von ATP beschleunigt und deutet auf Photorezeptor-Kinase Interaktion hin. Strukturelle Änderungen in der Kinasedomäne wurden durch FTIR-Differenzspektroskopie gezeigt. Über ELDOR-Spektroskopie wurde der Abstand der Photorezeptordomänen auf etwa 25 Angstrom bestimmt. Mutationen in Slr1694 an S28, N31 und W91 zeigten keine konservierten Einfluss auf die Dynamik des Signalzustands. Die Entfernung der Seitenkette von S28 führte zu einer 15 nm Rotverschiebung des Absorptionsspektrums aufgrund veränderter Wasserstoffbrückenkoordination des Kofaktors. Die Einführung von positiv geladenen Seitenketten an Stelle von N31 erhöhte die Kofaktorbindung von phosphorylierten Flavinen. Künstliche Kofaktoren wie Roseoflavin konnten in Slr1694 durch Koexpression eines prokaryotischen Flavintransporters erreicht werden. Die Rolle von M152 in PAC für die Signalweiterleitung wurde anhand der lichtaktivierten cAMP Synthese-Aktivität gezeigt. Durch ultraschnelle IR-Spektroskopie wurde die Beteiligung der Seitenketten von Y8 sowie Q50 bestätigt und eine genauere Beschreibung der Wasserstoffbrücken im langlebigen Signalzustand ermöglicht. / The light activated kinase Phototropin from Chlamydomonas reinhardtii, the photoactivated adenylylcyclase (PAC) from Euglena gracilis and the BLUF protein Slr1694 from Synechocystis sp. PCC 6803 were investigated concerning the molecular details of the primary photochemistry as well as signal transduction. Phototropin was isolated from Escherichia coli in mg amounts after solubilization with arginine. Without arginine E. coli cAMP receptor protein, which shows high homology to a cAMP activated kinase from C. reinhardtii, was copurified. Full length Phototropin shows similar photochemistry to LOV-domain containing proteins without the kinase including triplet and covalent cysteinyl adduct formation. Signaling state decay is accelerated in the presence of ATP and suggests photoreceptor-kinase interaction. FTIR spectroscopy showed light induced structural changes in the kinase domain. The distance of the photoreceptor domains of 25 Angstrom was determined by ELDOR spectroscopy. Mutation of the side chains of S28, N31 and W91 in Slr1694 showed no conserved influence on the dynamic of the signaling state. Removal of the hydroxyl group of S28 lead to a 15 nm red shift of the absorption spectrum as a result of altered hydrogen bond coordination of the cofactor. Introduction of positively charged side chains at the position of N31 strengthened the binding of phosphorylated flavins. An artificial flavin like roseoflavin was introduced in Slr1694 by coexpression of a bacterial flavin transporter. The essential role of M152 in PAC for signal transduction was shown by determination of light activated cAMP synthesis activity. Ultrafast IR spectroscopy confirmed the contribution of Y8 and Q50 in the photocycle and gave a more detailed description of the hydrogen bonding situation in the signaling state.

Background: Subtilin is a peptide antibiotic (lantibiotic) natively produced by Bacillus subtilis ATCC6633. It is encoded in a gene cluster spaBTCSIFEGRK (spa-locus) consisting of four transcriptional units: spaS (subtilin pre-peptide), spaBTC (modification and export), spaIFEG (immunity) and spaRK (regulation). Despite the pioneer understanding on subtilin biosynthesis, a robust platform to facilitate subtilin research and improve subtilin production is still a poorly explored spot. Results: In this work, the intact spa-locus was successfully integrated into the chromosome of Bacillus subtilis W168, which is the by far best-characterized Gram-positive model organism with powerful genetics and many advantages in industrial use. Through systematic analysis of spa-promoter activities in B. subtilis W168 wild type and mutant strains, our work demonstrates that subtilin is basally expressed in B. subtilis W168, and the transition state regulator AbrB strongly represses subtilin biosynthesis in a growth phase-dependent manner. The deletion of AbrB remarkably enhanced subtilin gene expression, resulting in comparable yield of bioactive subtilin production as for B. subtilis ATCC6633. However, while in B. subtilis ATCC6633 AbrB regulates subtilin gene expression via SigH, which in turn activates spaRK, AbrB of B. subtilis W168 controls subtilin gene expression in SigH-independent manner, except for the regulation of spaBTC. Furthermore, the work shows that subtilin biosynthesis in B. subtilis W168 is regulated by the two-component regulatory system SpaRK and strictly relies on subtilin itself as inducer to fulfill the autoregulatory circuit. In addition, by incorporating the subtilin-producing system (spa-locus) and subtilin-reporting system (PpsdA-lux) together, we developed “online” reporter strains to efficiently monitor the dynamics of subtilin biosynthesis. Conclusions: Within this study, the model organism B. subtilis W168 was successfully established as a novel platform for subtilin biosynthesis and the underlying regulatory mechanism was comprehensively characterized. This work will not only facilitate genetic (engineering) studies on subtilin, but also pave the way for its industrial production. More broadly, this work will shed new light on the heterologous production of other lantibiotics.