1,721,186 research outputs found
Novel shades of inositol pyrophosphates – in vitro and in cellulo evaluation of inositol pyrophosphate probes
No full textInositol pyrophosphates are an important class of second messengers that recently became an important point of cell signaling due to their presence and abundance in eukaryotic cells. They have been implicated in the regulation of diverse cellular activities ranging from phosphate homeostasis, DNA repair, telomere length, ribosome synthesis and apoptosis to chemotaxis, vesicle trafficking and exocytosis. However, the entire extent of their metabolic relevance and biological significance has yet to be described.In the presented work, a method for the delivery of inositol pyrophosphates into mammalian cells using a prometabolite approach following metal dioxide extraction for subsequent recovery and analysis by polyacrylamide gel electrophoresis or capillary electrophoresis was established. With this method in hand, we identified the presence of several inositol polyphosphates in peripheral blood mononuclear cells and CD8+ T cells, respectively. Besides, the presumed role of 1-PP-InsP5 and 1,5-(PP)2-InsP4 in innate immune responses by phosphorylating the interferon regulatory factor 3 could be refuted. However, the major part of this thesis was concentrated on the role of inositol pyrophosphates on β-cell physiology. With the discovery of 1,5-(PP)2-InsP4 regulating Ca2+ oscillations in pancreatic β-cells, we suggested an impact of 1,5-(PP)2-InsP4 on insulin-containing vesicle exocytosis. This was furthermore confirmed by the finding that intracellular release of 1,5-(PP)2-InsP4 induces the translocation of the C2AB domain of granuphilin, which is known to be involved in SNARE-mediated exocytosis. Isothermal titration calorimetry analyses proved an actual binding of 1,5-(PP)2-InsP4 as well as other inositol polyphosphates to both C2 domains, leading to the design of a corresponding C2AB-based FRET (FÖRSTER Resonance Energy Transfer) sensor to develop an in vivo method for the detection of inositol pyrophosphates
Studies on the biosynthesis of urdamycin A and generation of novel natural compounds by tools of molecular biology
No full textUrdamycin A, das Hauptprodukt von Streptomyces fradiae Tü2717, gehört zur Gruppe der Angucyclin-Antibiotika und zeigt antitumorale sowie antibakterielle Aktivitäten. Es enthält eine C-glycosidisch gebundene D-Olivose und drei weitere O-glycosidisch gebundene Desoxyzucker: zwei L-Rhodinosen und eine weitere D-Olivose.
Um Gene identifizieren zu können, die an der Biosynthese der Desoxyzucker sowie an Tailoring Reaktionen beteiligt sind, wurde ein 8kb großes Fragment aus dem Biosynthesegencluster von Urdamycin A kloniert und sequenziert. Dabei konnten 7 neue Offene Leserahmen (urdL, urdM, urdJ2, urdZ1, urdGT2, urdG und urdH) detektiert werden. Deren abgeleitete Genprodukte zeigten Homologien zu bekannten Cyclasen (UrdL), Oxygenasen (UrdM), Transportern (UrdJ2), Desoxyzucker-Biosynthesegenen (UrdZ1, UrdG, UrdH) und Glycosyltransferasen (UrdGT2).
Um Aufschluß über die mögliche Funktion der Gene urdM, urdGT2 und urdZ1 zu erhalten, wurden Geninaktivierungsexperimente durchgeführt. Die Inaktivierung von urdZ1 über frame-shift Mutation führte zu einer Mutante, die Urdamycinon B produziert. Dies läßt vermuten, daß urdZ1 für eine dNDP-Hexose-3,5-Epimerase codiert, die an der Biosynthese des Desoxyzuckers L-Rhodinose beteiligt ist. Die Inaktivierung von urdM über in-frame Deletion resultierte in einer Mutante, die überwiegend Rabelomycin akkumuliert. Daraus kann gefolgert werden, daß UrdM die Oxygenierung an Position 12b des Urdamycin A katalysiert. Die Mutante, die durch Inaktiavierung von urdGT2 über in-frame Deletion erhalten wurde, produzierte die Urdamycine I, J und K anstelle von Urdamycin A. Diese neuen Urdamycine weisen an Position C-9 keine C-C-verknüpfte D-Olivose auf. Es kann deshalb angenommen werden, daß UrdGT2 den C-Glycosyltransfer einer NDP-D-Olivose katalysiert.
Die heterologe Expression ganzer Gencluster oder einzelner Gene in verschiedenen Wirtsstämmen, bzw. die Expression verschiedener Glycosyltransferasegene in der urdGT2-Mutante führte zur Produktion nUrdamycin A, the major product of Streptomyces fradiae Tü2717, belongs to the group of angucycline antibiotics and exhibits antitumoral and antibacterial activity. It contains a C-glycosidically linked D-olivose and three additional O-glycosidically linked desoxysugars: two L-rhodinoses and another D-olivose.
To characterize genes beeing involved in the biosynthesis of desoxysugars and tailoring reactions, a 8 kb fragment of the urdamycin A biosynthetic gene cluster has been cloned and sequenced. 7 new ORFs (urdL, urdM, urdJ2, urdZ1, urdGT2, urdG and urdH) have been detected, their deduced products showing similarities to known cyclases (UrdL), oxygenases (UrdM), transporters (UrdJ2), desoxysugar biosynthetic genes (UrdZ1, UrdG, UrdH) and glycosyltransferases (UrdGT2).
To determine the function of urdM, urdGT2 and urdZ1, targeted gene inactivation experiments were performed. Inactivation of urdZ1 by frame-shift mutation led to a mutant producing urdamycinone B. This might indicate that urdZ1 encodes a dNDP-hexose-3,5 epimerase which is involved in the biosynthesis of the desoxysugar L-rhodinose. The in-frame deletion of urdM generated a mutant strain accumulating predominantly rabelomycin. UrdM might be involved in oxygenation at position 12b of urdamycin A. The mutant, in which urdGT2 had been deleted by in-frame deletion produced urdamycin I, urdamycin J and urdamycin K instead of urdamycin A. All these new derivatives are missing the C-C connected D-olivose at position C-9, indicating that UrdGT2 catalyses the C-glycosyltransfer of one NDP-D-olivose.
Heterologous expression of entire gene clusters or single genes in different host strains or expression of different glycosyltransferase genes in the urdGT2-mutant, respectively, resulted in the production of novel natural compounds. However, due to the very low production level, the structure of these compounds could not be determined
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Diversität und Selektivität pflanzlicher Carboligasen am Beispiel der Pyruvatdecarboxylasen und der Phenolkupplung
No full textIn dieser Arbeit wurde eine Enzymkaskade etabliert, die die Synthese des Arzneistoffs (1R,2R)-Pseudoephedrin ausgehend von Benzaldehyd und Pyruvat ermöglicht; zudem wurde Ephedrin in Spuren gebildet. In dieser Kaskade sind eine Pyruvatdecarboxylase (PDC), die das Hydroxyketon (R)-Phenylacetylcarbinol ((R)-PAC) bildet, und eine Iminreduktase (IR21) aus Paenibacillus ehimensis, die das in situ gebildete PAC-Imin (2-(Methylimino)-1-phenylpropan-1-ol) reduziert, beteiligt. PDCs katalysieren bevorzugt die Decarboxylierung von Pyruvat, die Carboligation ist eine Nebenreaktion. In dieser Arbeit wurde gezeigt, dass heterolog produzierte pflanzliche PDCs in der Lage sind, die Carboligation von Benzaldehyd und Pyruvat zu (R)-PAC zu katalysieren. PDCs aus Pflanzen weisen untereinander hohe Sequenzähnlichkeiten auf. Daher wurde aus Arabidopsis thaliana, deren Genom sequenziert ist, die Gene, die für die Enzyme At-PDC1 und At-PDC4 codieren, amplifiziert. Beide Enzyme wurden heterolog in E. coli produziert und katalysierten die Bildung von (R)-PAC (ee >97%). Um die Selektivität der PDCs aus Ephedra zu untersuchen, wurde eine PDC aus E. viridis, eine Pflanze die vermutlich keine Amphetaminanaloga bildet, und eine PDC aus E. sinica (Es-PDC) heterolog in E. coli produziert. Beide Enzyme katalysierten die Bildung von (R)-PAC, lediglich der Enantiomerenüberschuss war mit Es-PDC geringer im Vergleich zu den anderen drei PDCs aus Pflanzen (ee >86%). Es wurde mit allen pflanzlichen PDCs die selektive Bildung des Carboligaseproduktes (R)-PAC gezeigt. Für den zweiten Schritt der Pseudoephedrinsynthese wurden Iminreduktasen aus unterschiedlichen Organismen auf ihre Fähigkeit getestet, das PAC-Imin zu reduzieren. Es wurde gezeigt, dass IR21 aus P. ehimensis das in situ produzierte Imin zu (1R,2R)-Pseudoephedrin reduziert (de >93%). Letztlich wurden beide Enzymsysteme in einem Reaktionsgefäß vereint und es wurden bis zu 19mol% Pseudoephedrin gebildet.Im zweiten Teil beschäftigte sich diese Arbeit mit der Phenolkupplung in Pflanzen. Während auf nicht-enzymatischen Wegen die Dimerisierung von Anthrachinonen unselektiv verläuft, werden in Pflanzen bestimmte Regioisomere stereoselektiv produziert. Wie Pflanzen die Reaktion katalysieren und die Stereoselektivität kontrollieren, sollte untersucht werden. Hierzu wurde das Transkriptom von Cassia angustifolia, ein Produzent der Sennoside, analysiert. Die Transkriptom¬daten wurden mit bioinformatischen Methoden untersucht und lieferte 10 mögliche CYP-Enzyme, von denen drei ausgewählt und in E. coli Zellen produziert wurden. Die Enzyme wurden experimentell auf ihre Fähigkeit die Dimerisierung zweier monomerer Verbindungen S2 und Q6 zu katalysieren, untersucht. Es wurde keine Dimerisierung beobachtet. Zudem wurde eine putative NADPH-abhängige CYP Reduktase (CPR) aus C. angustifolia identifiziert, die die Aktivität der CYP-Enzyme beeinflussen könnte. Sechs Laccasen wurden identifiziert, die die Phenolkupplung ebenfalls katalysieren könnten.In this work the enzymatic-synthesis of (1R,2R)-pseudoephedrine and of ephedrine are described. At first (R)-phenylacetylcarbinol ((R)-PAC) was produced with pyruvate-decarboxylase (PDC), benzaldehyde and pyruvate. Secondly, imine reductase (IR21) from Paenibacillus ehimensis reduced the in situ produced PAC-Imin (2-(Methylimino)-1-phenylpropan-1-ol) to ephedrine and pseudoephedrine. PDCs catalyze the decarboxylation of pyruvate, whereas the carboligation is only a side reaction. In this work it is shown that heterologously produced PDCs from plants can also catalyze the reaction from benzaldehyde and pyruvate to form (R)-PAC. Due to the high sequence homology of plant PDCs two PDCs from Arabidiopsis thaliana (At-PDC1 and 4) were chosen for production in E. coli. Both At-PDCs showed the formation of enantiopure (R)-PAC (ee >97%). Two further PDCs from genus Ephedra were chosen for heterologous production in E. coli, to test for their selectivity. PDCs from E. viridis, a presumably amphetamine non-producing plant, and from E. sinica produced both (R)-PAC, although Es-PDC showed a lower enantiomeric excess (ee >86%). Hence, the selective synthesis of (R)-PAC with all four plant PDCs was shown, regardless of the original plant species. For the second step imine reductases from different organisms were tested to reduce the PAC-imine. Imin reductase 21 (IR21) from P. ehimensis catalyzed the formation of (1R,2R)-pseudoephedrine (de >93%). The combination of these two steps in a one-pot-reaction was shown also. It was possible to produce up to 19mol% of (1R,2R)-pseudoephedrine from benzaldehyde and pyruvate. The second part of this work deals with investigations of the intermolecular oxidative phenol-coupling in the biosynthesis of secondary metabolites from plants. Non-enzymatic reactions of anthraquinones are unselective. Plants, however, produce dimeric anthraquinones in a regio- and stereoselective manner. The transcriptome of Cassia angustifolia, a sennoside producing plant, was analyzed. The data were analyzed with bioinformatics tools. Ten potential CYP enzymes were identified and three of them were chosen for further studies. They were produced in E. coli and tested for the dimerization of the two dimeric compounds S2 and Q6. However, no dimerization was observed. Moreover, one putative NADPH-dependent CYP reductase (CPR) from C. angustifolia was identified. This enzyme might influence the activity of the CYP enzymes. Furthermore, six potential laccases were identified, which could also be potential phenol-coupling enzymes
Biocatalytic phenol coupling of [gamma]-naphthopyrones and biosynthesis of ibotenic acid
No full textThe present work deals with the identification and investigation of enzymes involved in the biosynthesis of fungal natural products. It is composed of two parts. Part I covers the biosynthesis of dimeric γ-naphthopyrone metabolites in ascomycetes. Part II covers the biosynthesis of ibotenic acid in the mushroom Amanita muscaria. I. Dimeric γ-naphthopyrones are produced by many strains of filamentous fungi. Their chemical diversity emerges from differing regiochemistries of the biaryl bond. Moreover, the hindered rotation around this bond gives rise to atropisomers. Typically, one fungal strain produces dimeric γ-naphthopyrones with one defined regio- and stereochemistry. Therefore, a highly selective enzymatic system responsible for the oxidative coupling of the monomeric γ-naphthopyrones was expected.To identify the enzymes responsible for the biosynthesis of the γ-naphthopyrones, genomics, transcriptomics, and retrobiosynthesis were combined. Despite the high structural similarity of the various γ-naphthopyrone dimers, two unrelated groups of enzymes were shown to catalyze the oxidative phenol coupling reaction. Ustilaginoidin A is biosynthesized by laccase enzymes in fungi such as Ustilaginoidea virens. Bifonsecin-type products such as nigerone and bifonsecin B are formed by the action of an entirely new group of CYP enzymes in Aspergillus species. Representative enzymes of both groups were heterologously produced and experimentally investigated.The ustilaginoidin laccases UstL, CheL, and MytL were shown to regio- and atropselectively catalyze the coupling of the achiral monomeric γ-naphthopyrone, nor-rubrofusarin. CheL and MytL were highly selective for production of (P)-ustilaginoidin A, with enantiomeric excesses of above 95%. UstL could even be tuned to either M- or P-atropselectivity by the reaction conditions. Overall, this is the first report of stereoselectivity in laccases.Likewise, the bifonsecin CYP enzymes AunB and BfoB were shown to catalyze selective phenol coupling reactions. Their native substrates fonsecin B and rubrofusarin B were each homodimerized regioselectively; AunB yielded the 7,10′-dimers, whereas BfoB yielded the 10,10′-dimers. However, the degree of atropselectivity was dependent on the monomeric substrate. Both enzymes coupled rubrofusarin B with moderate atropselectivity, while fonsecin B was converted to nearly atropisomerically pure dimers.The unexpected identification of two separate enzymatic systems for the dimerization of γ-naphthopyrones represents a notable case of convergent evolution, and highlights the importance of phenol coupling for the generation of chemical diversity in nature.II. Ibotenic acid is the primary toxic metabolite of the mushroom Amanita muscaria, or fly agaric. Despite its interesting pharmacology and long history, the biosynthetic origin of ibotenic acid was hitherto uninvestigated. Using retrobiosynthesis and transcriptomics, a candidate biosynthetic gene cluster for the formation of ibotenic acid was identified in A. muscaria. Deducing from the genes present in the gene cluster, probable biosynthetic pathways were inferred. The putative first biosynthetic step is the introduction of a hydroxyl group at position 3 of glutamic acid.Accordingly, the enzyme proposed to catalyze this reaction, IboH, was heterologously produced and experimentally investigated. It was shown that it is a 2 oxoglutarate-dependent enzyme which catalyzes stereoselective hydroxylation of L glutamic acid to threo-3 hydroxyglutamic acid. This suggested that the ibotenic acid biosynthetic gene cluster was correctly identified. The results will enable the complete reconstruction of the biosynthetic pathway, eventually facilitating the exploitation of the involved enzymes for various possible applications, such as biotechnological production of ibotenic acid and its analogs
Biochemische Untersuchungen zu NirN und NirF aus Pseudomonas aeruginosa
No full textPseudomonas aeruginosa ist ein ubiquitär verbreitetes Bakterium und kommt besonders häufig in Böden vor. Als Denitrifizierer ist P. aeruginosa Teil des Stickstoffkreislaufs, der alle Lebewesen mit essenziellen Stickstoffverbindungen versorgt. Während der Denitrifikation kann P. aeruginosa unter anaeroben Bedingungen Stickoxide als terminale Elektronenakzeptoren verwenden. Für diese Aufgabe besitzt P. aeruginosa u. a. die Cytochrom cd1 Nitritreduktase NirS, die einen speziellen und einzigartigen Cofaktor besitzt: Häm d1. Häm d1 ist das katalytische Zentrum von NirS, an dem Nitrit zu Stickstoffmonoxid reduziert wird. Der Schwerpunkt dieser Arbeit liegt auf den Proteinen NirN und NirF, die beide Teil der Biosynthese von Häm d1 sind. Der erste Abschnitt der Arbeit befasst sich mit NirN. NirN katalysiert den letzten Biosyntheseschritt von Dihydrohäm d1 zu Häm d1, bei dem eine Acrylatseitenkette entsteht (Adamczack et al. 2014). NirN wurde in dieser Arbeit rekombinant in Escherichia coli hergestellt und mehreren analytischen Verfahren unterzogen. Ein Teil der Untersuchungen bestand aus der Erzeugung und Untersuchung von NirN Varianten, Cyclovoltammetrie sowie ESR-Messungen. Dadurch konnten wichtige Erkenntnisse bezüglich der Katalyse, der Thermodynamik und des Reaktionsmechanismus von NirN erlangt werden.Im letzten Teil der Arbeit wurde die Funktion von NirF untersucht. NirF ist ein periplasmatisches Protein, dessen Aufgabe in der Biosynthese von Häm d1 nicht geklärt ist. Durch die Untersuchung des Cofaktorgehalts einer P. aeruginosa RM301 ∆nirF-Mutante und eines isolierten Cofaktors aus NirF einer P. aeruginosa RM361 ∆nirN-Mutante konnten mehrere Tetrapyrrole gefunden werden. Die einzelne Identifizierung steht noch aus und könnte dazu beitragen, mehr zur Rolle von NirF zu erfahren
Functional Characterization of the Two Gene Operons msmeg_1709-1712 and msmeg_0243/0244/0246 in Mycobacterium smegmatis mc2155
No full textATP-binding cassette (ABC) transporters have been described as one of the essential transport systems used by Mycobacterium tuberculosis (M. tuberculosis), the major causative agent of tuberculosis, for the uptake of a variety of necesseray nutrients (lipids, ions, amino acids, iron, carbohydrates, etc). Therefore, inhibitory studies targeting these transporters might represent a general strategy for killing mycobacteria under limited nutrient conditions. In this thesis, a simple and rapid lipoproteomics protocol was constructed to identify ABC transporters of carbohydrates in M. smegmatis mc2155. The constructed protocol is based on three conditions: (1) the identified transport systems have a high-affinity periplasmic substrate binding protein (SBP); (2) SBPs could be significantly induced by a respective carbohydrate; (3) SBPs belong to the class of lipoproteins, which can be extracted via using the detergent Triton X-114. The constructed workflow is time-saving and easier to handle. With the help of the constructed protocol, the ABC transporter of L-arabinose from M. smegmatis mc2155 could be identified, which was verified by biochemical and microbiological experiments. Theoretically, the constructed lipoproteomics protocol is also applicable to other carbohydrates or nutrients.In order to survive and adapt under a variety of environmental stimuli and changes, M. tuberculosis requires two-component regulatory systems which have been identified as a potential target for new drug discovery based on their vital functions in mycobacterial viability, virulence, and their lack in human. In this work, the potential three-component system containing a heme binding protein MSMEG_0243 and one PrrA/PrrB two-component regulatory system (MSMEG_0244/0246) was studied in M. smegmatis mc2155. Lipid profiles revealed the accumulations of diacyl phosphatidylinositol dimannoside (Ac2PIM2) and diacyl phosphatidylinositol hexamannoside (Ac2PIM6) in the absence of msmeg_0243-PrrA/PrrB resulting in changes of cell morphology, biofilm formation, and sliding motility. The results indicated that a heme binding protein MSMEG_0243 (a potential PrrA/PrrB accessory protein) and a PrrA/PrrB two-component regulatory system (MSMEG_0244/0246) are potentially involved in the regulation of biosynthesis of phosphatidylinositol mannosides (PIMs), especially in the regulation of acylation of PIMs. Further investigation could explore the interaction between MSMEG_0243 and PrrA/PrrB and the regulated mechanism in PIMs biosynthesis, especially on the level of acylation
The regulation of septins in ciliogenesis and septin functions beyond the diffusion barrier
No full textPrimary cilia are fundamentally important organelles involved in organ development, signaling pathways and cancer. The septin family of small GTP-binding proteins have been linked to cilia and ciliopathies but their function in ciliogenesis remains elusive. A detailed understanding of the process of cilia formation is critical for therapeutic approaches in the future.This thesis demonstrated that cilia formation depends on the presence and dynamics of septins. Knockdown of septins and the inhibition of septin dynamics via forchlorfenuron (FCF) resulted in significantly decreased ciliogenesis. In addition, septins were visualized at the base of primary cilia or along the full length of the axoneme. In the first part, it was shown that septins are regulated by the Rho GTPases Cdc42 and TC10 via their downstream effectors of the Borg family. Here it was shown that septin filaments associate with Borg proteins in the cytoskeleton as well as within cilia. Dominant active and negative Cdc42 inhibit cilia formation indicating that the free cycling of Cdc42 between its active and inactive state is essential for Borg and septin regulation during ciliogenesis. Even though Cdc42 and TC10 are close homologues, their functions during ciliogenesis are not redundant. The expression of dominant active TC10 inhibited cilia formation, whereas dominant negative TC10 had no influence on ciliogenesis. Knockdown experiments demonstrated that Cdc42 is essential for ciliogenesis but TC10 is not. Also the double knockdown of Cdc42 and TC10 showed no additive effect on ciliogenesis. However, an influence of TC10 on ciliary length was detected. Moreover, two distinct cellular distributions at cilia were observed: active Cdc42 was predominantly located as a spot at the base whereas a TC10 accumulation surrounded the base of cilia, suggesting different functions during ciliogenesis.The second part demonstrated that septins are involved in exocytotic processes during ciliogenesis. Septins colocalize with the exocyst complex at the base of cilia. Rab10 vesicles were shown to accumulate at the base of cilia and to colocalize with septins. GST pull-down experiments revealed that septins interact with Rab10, independent of the nucleotide-binding state. Knockdown experiments revealed that Rab10 is important for cilia formation. Moreover, the inhibition of septin dynamics via FCF hindered the traffic of a cilia-targeted protein to the cilium. This thesis investigated how septins are regulated via Borg proteins and their upstream Rho GTPases. Moreover, it presents a novel prospective for the role of septins in the process of ciliogenesis, away from the propagated diffusion barrier theory
Effects of c-di-GMP on the biosynthesis of natural products in streptomyces
No full textMultiresistente Bakterien sind ein globales Gesundheitsproblem, da die Entwicklung neuer Antibiotika mit der Zunahme resistenter Stämme nicht Schritt hält. Eine vielversprechende Lösung für dieses Problem liegt in den von Bakterien produzierten Naturstoffen, mit antimikrobieller Aktivität, die in Zukunft als Arzneimittel eingesetzt werden könnten. Diese Arbeit untersucht Streptomyceten als Quelle bioaktiver Naturstoffe. Ihr Genom enthält dutzende kryptische biosynthetische Gencluster (BGCs), die neue antimikrobielle Verbindungen synthetisieren könnten. In der vorliegenden Arbeit wurden die Signalprozesse des NukleotidSecond-Messengers Bis-(3',5')-cyclisches dimeres Guanosinmonophosphat (c-di-GMP) untersucht, um weitere kryptische BGCs aktivieren. Frühere Studien haben gezeigt, dass Veränderungen des c-di-GMPSpiegels tatsächlich kryptische BGCs aktivieren können; wobei die zugrunde liegenden Mechanismen weiter aufgeklärt werden müssen. Die c-di-GMP-Signalübertragung ist ein komplexes Netzwerk. Die erste Ebene der c-di-GMPSignalübertragung ist die Adaption der Streptomyceten an Umweltsignale. Diese Arbeit untersucht c-di-GMPmetabolisierende Enzyme, die c-di-GMP synthetisieren oder abbauen, und dessen sensorischen Domänen, die auf Umweltsignale reagieren. Streptomyceten sind Umweltbedingungen wie Temperaturschwankungen und osmotischen Veränderungen durch Regen und Trockenheit ausgesetzt. Gegenstand der vorliegenden Studie ist die Analyse des c-di-GMP-metabolisierenden Enzyms CdgA aus Streptomyces ghanaensis. Dieses Enzym enthält Domänen sowohl für die Synthese als auch für den Abbau von c-di-GMP sowie für eine häm-bindende PAS-Domäne als sensorische Domäne. Analysen mittels ultraviolet/visible (UV/Vis)-Spektroskopie und Enzym-tests zeigen, dass die Diguanylat-Cyclase (DGC)-Domäne von CdgA in reduzierter, häm-gebundener Form aktiv ist; in oxidierter oder häm-freier Form jedoch inaktiv bleibt. Im Gegensatz dazu ist die Phosphodiesterase (PDE)-Domäne in allen Zuständen aktiv. CdgA reguliert den c-di-GMP-Spiegel in Abhängigkeit von Redox-Signalen (Sauerstoffmangel oder osmotische Veränderungen) und Häm-Spiegeln (Eisenverfügbarkeit). Die zweite Regulierungsebene umfasst c-di-GMP-Effektorproteine, deren Aktivität durch c-di-GMP-Bindung moduliert wird. In der vorliegenden Arbeit wird die Identifizierung neuer Effektorproteine angestrebt, wobei ein c-di-GMP-Capture Compound als Identifikationsmethode eingesetzt wird. Zu diesem Zweck wurden zwei Verbindungen synthetisiert und in Streptomyces sp. Tu6071 eingesetzt. Die Methode führte zur Identifizierung zahlreicher potenzieller c-di-GMP-bindender Proteine, von denen zwei durch Microscale Thermosphoresis (MST) als c-di-GMP-bindende Proteine bestätigt wurden: Der Transkriptionsregulator GlnR und eine putative Oxidoreduktase (STTU_0845). GlnR ist am Stickstoffmetabolismus beteiligt und wurde bereits mit der Regulierung von BGCs in Verbindung gebracht. Die Funktion der putativen Oxidoreduktase bedarf weiterer Klärung, jedoch weisen andere c-di-GMP-bindende Oxidoreduktasen darauf hin, dass dieses Protein eine regulatorische Funktion als enzymatische Aktivität aufweist. Weitere Analysen, einschließlich Massenphotometrie und Proteinkristallographie, lieferten zusätzlich Einblicke in die Funktion dieser Proteine. Der dritte Abschnitt der Untersuchung widmet sich der Analyse der Auswirkungen von Änderungen des c-diGMP-Spiegels. Zu diesem Zweck wurden drei Gendeletionsstämme von S. sp. Tu6071 mit Deletionen in c-diGMP-metabolisierenden Enzymen erzeugt. Ziel war es, die Auswirkungen einer Veränderung des c-di-GMPSpiegels auf die Biosynthese von Naturstoffen zu untersuchen. Die Gendeletionen umfassten die beiden Gene rmdA und cdgA, die für bifunktionale Enzyme codieren, sowie das Gen rmdB das für eine PDE codiert. Die Veränderung des c-di-GMP-Spiegels führte zu einem Anstieg der Produktion von Phenalinolacton A in MSMedium und zahlreicher neuer Verbindungen in SG+ und YMPG-Medium. Zur umfassenden Analyse dieser Veränderungen wurde ein molecular-Network-Ansatz verwendet, mit dem die Veränderungen in den Biosyntheseprofilen der Naturstoffe untersucht wurden. Zusammenfassend betonen die Ergebnisse dieser Studie die Relevanz der c-di-GMP-Signalübertragung in den Signalwegen der Naturstoffproduktion in Streptomyceten. Es konnte nachgewiesen werden, dass c-di-GMPmetabolisierende Enzyme befähigt sind, Umweltsignale wahrzunehmen und infolgedessen ihre Aktivität als Anpassungsmechanismus zu modulieren. Die Identifizierung neuartiger c-di-GMP-Effektorproteine hat zu neuen Erkenntnissen bezüglich der Verbindung zwischen c-di-GMP und der Regulierung von BGCs geführt. Dies unterstreicht das Potenzial von c-di-GMP bei der Identifizierung neuer, von Streptomyceten produzierten Naturstoffen mit antimikrobieller Wirkung.Multi-drug-resistant bacteria are an emerging global health problem. In the last decades, the development of novel antibiotics lagged behind the growing resistance problem, causing millions of deaths per year today and even more in the future. A potential solution to this problem lies in the natural products produced by bacteria, which may contain antimicrobial compounds that could be used as drugs in the future. This work investigates the potential of Streptomyces as a source of bioactive natural products. These bacteria harbor dozens of inactive, so-called cryptic, biosynthetic gene clusters (BGCs) in their genome that have the potential to synthesize new antimicrobial natural products upon activation. This work investigates the signaling processes of the nucleotide second messenger bis-(3’,5’)-cyclic dimeric guanosine monophosphate (c-di-GMP), activating further cryptic BGCs. Prior studies have demonstrated that disruptions in the intracellular c-di-GMP level can indeed activate cryptic BGCs; however, the underlying mechanisms require further elucidation. C-di-GMP signaling is a complex network involving multiple layers of regulation. Nucleotide second messengers have been shown to integrate incoming signals, leading to specific outcomes that are necessary for cells to adapt to their environment. The first layer of the c-di-GMP signaling encompasses the sensing of incoming signals. This work investigates the activity of c-di-GMP metabolizing enzymes, the enzymes that make and break c-di-GMP. Many of these enzymes possess sensory domains to be able to adapt the activity to environmental signals. Streptomyces live in diverse environments and experience rapid temperature fluctuations over the course of a year, as well as osmotic changes through rain and drought. This study focuses on investigations of the c-di-GMP-metabolizing enzyme CdgA from Streptomyces ghanaensis. This enzyme harbors domains for both the synthesis and degradation of c-di-GMP as well as a heme-binding PAS domain as a sensory domain. The analysis of CdgA was conducted with ultraviolet/visible (UV/Vis) spectroscopy, and in vitro enzyme activity assays were performed. The results demonstrated that CdgA can activate its c-di-GMP synthesizing diguanylate cyclase (DGC) domain in the reduced and heme-bound form. Conversely, in its oxidized or heme-free form, the DGC domain is inactive, while the phosphodiesterase (PDE) remains active in all states. The regulation of CdgA activity enables the modulation of the c-di-GMP level in response to two distinct signals: The integration of redox signals is necessary for sensing oxidative stress that results from lack of oxygen or osmotic changes, while the integration of heme levels is essential for sensing iron availability. The second layer of regulation is characterized by the c-di-GMP effector proteins. C-di-GMP has been demonstrated to bind to proteins, thereby altering their activity. Besides the known c-di-GMP effector proteins, this work aimed to identify novel effector proteins using a c-di-GMP capture compound. These compounds were chemically synthesized and then utilized in Streptomyces sp. Tu6071. The method yielded numerous potential c-di-GMP-binding proteins, two of which were confirmed as c-di-GMP-binding proteins using microscale thermophoresis (MST): The transcriptional regulator GlnR and a putative oxidoreductase STTU_0845. GlnR is involved in nitrogen metabolism and was also previously linked to regulating the biosynthesis of natural products. The role of the putative oxidoreductase remains to be elucidated, but based on other c-di-GMP-binding oxidoreductases, this protein rather has a regulatory function than enzymatic activity. Further investigations involved mass photometry to determine changes in the oligomerization state through c-di-GMP binding and protein crystallography. The third part of the investigation entailed the analysis of the effects that changing c-di-GMP levels have. Three gene deletion strains of S. sp. Tu6071 with a deletion in c-di-GMP-metabolizing enzymes were created to investigate the effects of changing c-di-GMP levels on the biosynthesis of natural products. These gene deletions included the two bifunctional genes rmdA and cdgA as well as the PDE rmdB. The alteration of c-diGMP levels led to an increase in phenalinolactone A production in MS media and numerous novel compounds in SG+ and YMPG media. To further investigate these alterations, a molecular networking approach was employed to comprehensively analyze the changes in the natural product biosynthesis profiles. In summary, the results of this study underscore the significance of c-di-GMP signaling in the signaling pathways of natural product biosynthesis in Streptomyces. The results demonstrate that c-di-GMP metabolizing enzymes are capable of sensing environmental signals, thereby modulating their activity as a mechanism of adaptation. The identification of novel c-di-GMP-effector proteins leads to the establishment of novel connections between c-di-GMP and the regulation of BGCs. This emphasizes the potential of c-di-GMP in the identification of novel natural products produced by Streptomyces with antimicrobial activity
- …
