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Thermoanaerobacter kivui is an acetogenic model organism that reduces CO2 with electrons derived from H2 or CO, or from organic substrates in the Wood–Ljugdahl pathway (WLP). For the calculation of ATP yields, it is necessary to know the electron carriers involved in coupling of the oxidative and reductive parts of metabolism. Analyses of key catabolic oxidoreductases in cell-free extract (CFE) or with purified enzymes revealed the physiological electron carriers involved. The glyceraldehyde-3-phosphate dehydrogenase (GA3P-DH) assayed in CFE was NAD+-specific, NADP+ was used with less than 4% and ferredoxin (Fd) was not used. The methylene-THF dehydrogenase was NADP+-specific, NAD+ or Fd were not used. A Nfn-type transhydrogenase that catalyzes reduced Fd-dependent reduction of NADP+ with NADH as electron donor was also identified in CFE. The electron carriers used by the potential electron-bifurcating hydrogenase (HydABC) could not be unambiguously determined in CFE for technical reasons. Therefore, the enzyme was produced homologously in T. kivui and purified by affinity chromatography. HydABC contained 33.9 ± 4.5 mol Fe/mol of protein and FMN; it reduced NADP+ but not NAD+. The methylene-THF reductase (MetFV) was also produced homologously in T. kivui and purified by affinity chromatography. MetFV contained 7.2 ± 0.4 mol Fe/mol of protein and FMN; the complex did neither use NADPH nor NADH as reductant but only reduced Fd. In sum, these analysis allowed us to propose a scheme for entire electron flow and bioenergetics in T. kivui.
Currently one of the biggest challenges for society is to combat global warming. A solution to this global threat is the implementation of a CO2-based bioeconomy and a H2-based bioenergy economy. Anaerobic lithotrophic bacteria such as the acetogenic bacteria are key players in the global carbon and H2 cycle and thus prime candidates as driving forces in a H2- and CO2-bioeconomy. Naturally, they convert two molecules of CO2 via the Wood-Ljungdahl pathway (WLP) to one molecule of acetyl-CoA which can be converted to different C2-products (acetate or ethanol) or elongated to C4 (butyrate) or C5-products (caproate). Since there is no net ATP generation from acetate formation, an electron-transport phosphorylation (ETP) module is hooked up to the WLP. ETP provides the cell with additional ATP, but the ATP gain is very low, only a fraction of an ATP per mol of acetate. Since acetogens live at the thermodynamic edge of life, metabolic engineering to obtain high-value products is currently limited by the low energy status of the cells that allows for the production of only a few compounds with rather low specificity. To set the stage for acetogens as production platforms for a wide range of bioproducts from CO2, the energetic barriers have to be overcome. This review summarizes the pathway, the energetics of the pathway and describes ways to overcome energetic barriers in acetogenic C1 conversion.
Energy-converting hydrogenases (Ech) are ancient, membrane-bound enzymes that use reduced ferredoxin (Fd) as an electron donor to reduce protons to molecular H2. Experiments with whole cells, membranes and vesicle-fractions suggest that proton reduction is coupled to proton translocation across the cytoplasmatic membrane, but this has never been demonstrated with a purified enzyme. To this end, we produced a His-tagged Ech complex in the thermophilic and anaerobic bacterium Thermoanaerobacter kivui. The enzyme could be purified by affinity chromatography from solubilized membranes with full retention of its eight subunits, as well as full retention of physiological activities, i.e., H2-dependent Fd reduction and Fd2--dependent H2 production. We found the purified enzyme contained 34.2 ± 12.2 mol of iron/mol of protein, in accordance with seven predicted [4Fe-4S]-clusters and one [Ni-Fe]-center. The pH and temperature optima were at 7 to 8 and 66 °C, respectively. Notably, we found that the enzymatic activity was inhibited by N,N′-dicyclohexylcarbodiimide, an agent known to bind ion-translocating glutamates or aspartates buried in the cytoplasmic membrane and thereby inhibiting ion transport. To demonstrate the function of the Ech complex in ion transport, we further established a procedure to incorporate the enzyme complex into liposomes in an active state. We show the enzyme did not require Na+ for activity and did not translocate 22Na+ into the proteoliposomal lumen. In contrast, Ech activity led to the generation of a pH gradient and membrane potential across the proteoliposomal membrane, demonstrating that the Ech complex of T. kivui is a H+-translocating, H+-reducing enzyme.
The pyruvate:ferredoxin oxidoreductase of the thermophilic acetogen, Thermoanaerobacter kivui
(2021)
Pyruvate:ferredoxin oxidoreductase (PFOR) is a key enzyme in bacterial anaerobic metabolism. Since a low-potential ferredoxin (Fd2−) is used as electron carrier, PFOR allows for hydrogen evolution during heterotrophic growth as well as pyruvate synthesis during lithoautotrophic growth. The thermophilic acetogenic model bacterium Thermoanaerobacter kivui can use both modes of lifestyle, but the nature of the PFOR in this organism was previously unestablished. Here, we have isolated PFOR to apparent homogeneity from cells grown on glucose. Peptide mass fingerprinting revealed that it is encoded by pfor1. PFOR uses pyruvate as an electron donor and methylene blue (1.8 U·mg−1) and ferredoxin (Fd; 27.2 U·mg−1) as electron acceptors, and the reaction is dependent on thiamine pyrophosphate, pyruvate, coenzyme A, and Fd. The pH and temperature optima were 7.5 and 66 °C, respectively. We detected 13.6 mol of iron·mol of protein−1, consistent with the presence of three predicted [4Fe–4S] clusters. The ability to provide reduced Fd makes PFOR an interesting auxiliary enzyme for enzyme assays. To simplify and speed up the purification procedure, we established a protocol for homologous protein production in T. kivui. Therefore, pfor1 was cloned and expressed in T. kivui and the encoded protein containing a genetically engineered His-tag was purified in only two steps to apparent homogeneity. The homologously produced PFOR1 had the same properties as the enzyme from T. kivui. The enzyme can be used as auxiliary enzyme in enzymatic assays that require reduced Fd as electron donor, such as electron-bifurcating enzymes, to keep a constant level of reduced Fd.
For palaeotropical regions, only a few anecdotal reports had been published on the existence of 'ant-gardens' before this study started. As opposed to this, 'ant-house epiphytes' (i.e. domatiabearing epiphytes) were reported to be highly abundant in Southeast Asia and were presumed to be a second type of ant-epiphyte interaction. In the much better studied neotropical regions the situation seemed to be the reverse: Many reports on AGs in contrast to very few reports on anthouse epiphytes. In this study, I have presented extensive data which may help towards a better understanding of the 'Southeast Asian part' of this 'ant-epiphyte puzzle'. In Peninsular Malaysia, Borneo, Java, and Southern Thailand, a great variety of formerly unknown AG systems were discovered. 18 ant species (from 5 genera, 4 subfamilies) were identified as true AG ants, i.e. these ants actively retrieved seeds of certain epiphyte species into their carton nests. Another 49 ant species inhabited AGs as secondary, opportunistic settlers. On the epiphyte side, 84 plant species were found growing on AGs, 51 (19 genera, 12 families) of which were probably true AG epiphytes, i.e. ants retrieved the seeds to their arboreal carton nests, on which the epiphytes were then cultivated. Most of the epiphyte flora of lowland forests in Peninsular Malaysia (except for ferns, orchids and facultative epiphytes) seemed to be totally dependent on ants for their establishment in the canopy. Together with the high number of opportunistic AG inhabitants (ants, epiphytes, and many arthropod guests), these facts suggest that AGs function as pioneers in the canopy of Southeast Asian rain forests. Moreover, AG-associations might even have accounted for the unusual species richness in the epiphyte genera Dischidia, Hoya (Asclepiadaceae), Myrmecodia, and Hydnophytum (Rubiaceae). The definition of the term ant-garden only describes the basic interactions. In the ant-garden associations investigated in this study, interactions going beyond these basic ones varied depending on ant and epiphyte species. Ant-gardens initiated by Diacamma spKfmA111 were regarded as the 'most primitive' type, because this ponerine was totally dependent on preformed cavities for nest establishment, did not tend any trophobionts, and was the least selective in its seed-retrieving behavior. On the other end of the scale, Crematogaster spKfmA18 and Camponotus spKfmA9 were rated as 'most advanced' because both lived in free (i.e. cavityindependent) AGs, tended trophobionts underneath their nests, were associated with a couple of other organisms, and were highly selective in their seed-retrieving behavior. Moreover, Camponotus spKfmA9 occurred preferentially with one single epiphyte species, Hoya elliptica (Asclepiadaceae), and Crematogaster spKfmA18 was specialized on some species of giant bamboo as phorophyte. Philidris spKfmA160, which occupied a medium position in relation to the other AGs was particularly interesting for several reasons. This ant species was mainly associated with ant- house epiphytes and occurred in the heath forests of Borneo. However, the major part of the colonies, including the queen, was located underneath carton structures near the surface of the host tree and not inside the domatia of the associated plants. Moreover, very young Philidris spKfmA160 colonies had only small seedlings growing on their carton nests. The ant workers actively retrieved the seeds of their epiphyte partners into the nests. These results indicate that associations with ant-house epiphytes must be regarded as a special case of ant-gardens. I therefore suggest using the term 'ant-house' only to describe the epiphytes, but not to describe the association, and to include this type of association in the group of AGs. Strict species-specificity never occurred, but some epiphytes showed great preference for growing on the nests of certain ant species, while others occurred over a wider range. Vice versa, most ant species had several epiphytes growing on their nests, while others were mostly found with one or very few epiphyte species. These patterns were shown to be the effect of different factors, including common microclimatic preferences of ants and epiphytes, interspecific competition of epiphytes, and selective seed retrieval of AG ants. The main behavioral trait responsible for the establishment of AGs was the selectivity shown by the ants in the epiphyte seeds they carried. However, details of the mechanisms, i.e. what characteristics of the seeds are important and what motivates the ants to retrieve them, varied widely. In many cases, seed compounds located on the surface triggered carrying behavior. Detailed experimental investigations combined with literature data from the two other known 'myrmecochory systems', terricolous myrmecochores and neotropical AGs, suggested that myrmecochory is frequently triggered by a two-stage system. One relatively unspecific compound (or a combination of such compounds) constitutes the basic attractiveness for a number of ant species. Other seed characteristics (elaiosomes, mechanical properties, other surface-compounds) modulate this basic signal, accounting for species-specific preferences of ants towards certain plant species. A comparison of AGs in Southeast Asia and the neotropics shows that the numbers of AG ant and epiphyte species in each case are almost equal. Southeast Asian AG epiphytes might even turn out to outnumber the neotropical ones. Thus, not only was it possible to break down the distinction between ant-house and AG associations, but also to show that AGs in Southeast Asia are present in such high diversity and abundance as to diminish the apparent contrast between the two biogeographical regions yet further. These data help to solve at least the Southeast Asian part of the 'ant-epiphyte puzzle'.
This work addresses the investigation of the biosynthesis mechanisms of type II polyketide synthase (PKS) and fatty acid synthase (FAS) derived specialized metabolites (SMs) from Photorhabdus laumondii.
The elucidation of the biosynthetic pathway of the bacterial 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) was one of the major topics of this thesis. IPS exhibits several bioactive characteristics as it inhibits the phenoloxidase of insects, acts antibacterial, but also influences the soluble epoxide hydrolase which is involved in inflammatory reactions. It was recently approved as a treatment against psoriasis by the FDA and is the first Photorhabdus derived drug.
The stilbene generation in Photorhabdus requires the formation of the two acyl-carrier-protein (ACP) bound 5-phenyl-2,4-pentadienoyl- and isovaleryl-β-ketoacyl-moieties. The ketosynthase (KS)/cyclase StlD catalyzes a ring formation via a Michael-addition between the two intermediates which is then further processed by an aromatase. The formation of 5-phenyl-2,4-pentadienoyl-ACP was shown via in vitro assays with purified proteins by proving the influence of the KS FabH, ketoreductase FabG and dehydratase FabA or FabZ of the fatty acid metabolism. While E. coli was able to complement most of these enzymes in attempts to produce IPS in the heterologous host, the Photorhabdus derived FabH was not replaceable despite 73 % sequence identity with the E. coli based isoenzyme, acting as a gatekeeper enzyme for cinnamic acid (CA) moieties. Furthermore, the ability to incorporate meta-substituted halogenated CA-derivatives was shown in order to produce 3-chloro- and 3-bromo-IPS. While studying the stilbene biosynthesis, the ability of Photorhabdus and Xenorhabdus to produce hydrazines was also discovered.
The second investigated biosynthesis was the formation of benzylideneacetone (BZA). BZA is produced by Photorhabdus and Xenorhabdus strains acting as a suppressor for the immune cascade of insects and has also antibiotic activities towards Gram-negative bacteria. Due to its structural similarity towards CA and the intermediates during the stilbene formation, a shared mechanism for Photorhabdus and Xenorhabdus budapestensis was proposed due to their ability to produce CA. The production of BZA was also dependent on the stilbene related CoA-ligase, the ACP and FabH. It was verified in vitro and in vivo in E. coli yielding a 150-fold increase of the BZA production compared to the Photorhabdus and Xenorhabdus wildtype (WT) strains.
The second part of this work deals with the optimization of P. laumondii strains regarding the production titers of IPS. Therefore, several deletions of other SM related genes as well as promoter exchanges in front of stilbene related genes were carried out. These approaches were combined with the upregulation of the phenylalanine by heterologous plasmid expression, since it is the precursor of CA. Another approach applied in parallel was the optimization of the cultivation conditions with different media and supplementation with XAD-resins. It was proved that media rich on fatty acids or peptides led to higher optical densities of the cultures and thus to higher titers of stilbenes. Since IPS is inhibiting the phenoloxidase, an enzyme important for the insect immunity, it was hypothesized that cultivation in media containing insects might enhance the output of this SM. Starting from 23 mg/l of IPS in the P. laumondii WT strain, it was possible to increase the production levels to more than 860 mg/l by utilizing the mentioned approaches.
The last topic of this thesis focuses on the production of epoxidated IPS (EPS) and its derivatives. Under laboratory conditions, only a low titer of EPS was observed for the wildtype strain. However, the optimized IPS strains and IPS-production conditions could also be applied for EPS which led to higher productions and also to the detection of many new derivatives. Most of the EPS derivatives were amino acid or peptide derived acting as nucleophiles to open the epoxide ring and yielding β-amino-alcohols. However, purification and chemical synthesis attempts to obtain EPS failed due to its poor stability. Epoxides were utilized in in vitro assays with amino acids, peptides and proteins to get insights whether epoxidations might act as posttranslational modification in Photorhabdus. The reactions were performed with styrene oxide and stilbene oxide replacing EPS based on their structural similarity. The modifications were executed successfully although proteomics approaches with in vivo data are required to confirm these findings. During the purification attempts of EPS, further derivatives were detected. The structures of dimerized stilbenes, a cis-isomer of IPS and another derivative that might incorporate an amino-group in the resveratrol ring were proposed on the basis of the HPLC-MS data.
The main aim of this thesis work was to elucidate the catalytic mechanism of several enzyme complexes on the basis of their three-dimensional structure. All investigated enzyme complexes occur in the anaerobic energy metabolism and have an essential function by the challenging degradation of aromatic compounds and the flavin-based electron bifurcation (FBEB)/confurcation, an energy-coupling mechanism. More specifically, I studied the phthaloyl-CoA decarboxylase of Thauera chlorobenzoica (Pcd) involved in phthalate ester decomposition, the FBEB protein complexes lactate dehydrogenase/electron-transfer flavoprotein (Ldh/EtfAB) of Acetobacterium woodii, the heterodisulfide-related subunit HdrA of the sulfur- oxidizing bacteria Hyphomicrobium denitrificans (sHdrA). In addition, I contributed to the structure determination of the caffeyl-CoA reductase- EtfAB complex of A. woodii and the naphthoyl-CoA reductase of the sulfate-respiring enrichment culture N47 (mentioned in the Appendix E and F).
Oscillating magnetic field disrupts magnetic orientation in Zebra finches, Taeniopygia guttata
(2009)
Background Zebra finches can be trained to use the geomagnetic field as a directional cue for short distance orientation. The physical mechanisms underlying the primary processes of magnetoreception are, however, largely unknown. Two hypotheses of how birds perceive magnetic information are mainly discussed, one dealing with modulation of radical pair processes in retinal structures, the other assuming that iron deposits in the upper beak of the birds are involved. Oscillating magnetic fields in the MHz range disturb radical pair mechanisms but do not affect magnetic particles. Thus, application of such oscillating fields in behavioral experiments can be used as a diagnostic tool to decide between the two alternatives. Methods In a setup that eliminates all directional cues except the geomagnetic field zebra finches were trained to search for food in the magnetic north/south axis. The birds were then tested for orientation performance in two magnetic conditions. In condition 1 the horizontal component of the geomagnetic field was shifted by 90 degrees using a helmholtz coil. In condition 2 a high frequently oscillating field (1.156 MHz) was applied in addition to the shifted field. Another group of birds was trained to solve the orientation task, but with visual landmarks as directional cue. The birds were then tested for their orientation performance in the same magnetic conditions as applied for the first experiment. Results The zebra finches could be trained successfully to orient in the geomagnetic field for food search in the north/south axis. They were also well oriented in test condition 1, with the magnetic field shifted horizontally by 90 degrees. In contrast, when the oscillating field was added the directional choices during food search were randomly distributed. Birds that were trained to visually guided orientation showed no difference of orientation performance in the two magnetic conditions.
An die Signalübertragung im ZNS werden in bestimmten Entwicklungsstadien sehr unterschiedliche Anforderungen gestellt. Im adulten Gehirn dient sie nicht nur der Nachrichtenübermittlung, sondern auch der aktivitätsbasierten Umgestaltung neuronaler Verbindungen bei Lernprozessen und der regenerativen Umgestaltung nach Verletzungen. Im sich entwickelnden Gehirn werden schon frühzeitig Nervenzellen elektrisch erregbar und spontan aktiv. Diese elektrische Aktivität und die dadurch verursachte Transmitterausschüttung spielen bei der selektiven Stabilisierung von Synapsen und damit bei der Ausgestaltung des neuronalen Netzwerks eine große Rolle. Im ZNS von Vertebraten beruht die Wirkung des Neurotransmitters und Neuromodulators Acetylcholin auf den nikotinischen und muskarinischen Acetylcholinrezeptoren. Muskarinische Acetylcholinrezeptoren (mAChRen) koppeln, abhängig von den fünf identifizierten Rezeptorsubtypen (M1- M5), an unterschiedliche intrazelluläre Signalketten an. Dabei interagieren für gewöhnlich die M1, M3 und M5 Rezeptoren mit einem G-Protein des Typs Gq/11, während M2 und M4 ein G-Protein des Typs Gi/o aktivieren. Der Colliculus inferior (IC) ist eine wichtige Verschaltungsstation im auditorischen Mittelhirn von Säugetieren. Inhibitorische und exzitatorische Eingänge werden dort während der Entwicklung mit großer Präzision angelegt und konvergieren auf einzelne ICNeurone. Die physiologische Bedeutung von mAChRen im IC ist weitgehend unerforscht und die Subtypen die im juvenilen IC eine Rolle spielen wurden noch nicht charakterisiert. Es war das Ziel der vorliegenden Arbeit mittels elektrophysiologischer Untersuchungen im IC der juvenilen Ratte (P5-P12) folgende Fragen zu klären: i) Gibt es im IC der jungen Ratte eine Modulation der GABAergen Transmission durch muskarinische Acetylcholinrezeptoren? ii) Welcher muskarinische Rezeptorsubtyp spielt dabei eine Rolle? iii) Welcher intrazelluläre Signalmechanismus ist der Aktivierung des muskarinischen Acetylcholinrezeptors nachgeschaltet? Unter Wirkung von Muskarin kam es bei 41,2% der untersuchten Neurone des Colliculus inferior zu einer Erhöhung der Frequenz der spontanen IPSCs. Die sIPSCs wurden durch Bicucullin blockiert, somit handelt es sich um GABAerge IPSCs. Die Wirkung von Muskarin nahm zu, wenn die Tiere älter als 9 Tage waren. Es wurde gezeigt, dass nAChRen keine Rolle spielen bei der Erhöhung der sIPSC-Frequenz, während eine selektive Blockade der M3-(M5-) mAChRen durch 4-DAMP die Muskarinwirkung blockierte. In der Regel sind die M3-Rezeptoren über folgende Signalkaskade aktiv: Phospholipase C, Kalzium-Calmodulin, NO-Synthase, Guanylatzyklase, die NO-Konzentration und cGMP. Alle Glieder dieser Kaskade wurden untersucht, sie hatten aber keinen Einfluss auf die Muskarinwirkung. Im Gegensatz dazu führte die Erhöhung des intrazellulären cAMP-Spiegels zu einer vergleichbaren Frequenzsteigerung der sIPSCs wie sie unter der Wirkung von Muskarin gemessen wurde. Weiterhin ließ sich die Erhöhung der sIPSC-Frequenz durch Muskarin vollständig aufheben, wenn die intrazelluläre Adenylatzyklase blockiert wurde. Es ist bekannt, dass der M3-(M5-) mAChR über verschiedenste Signalwege den intrazellulären cAMP-Spiegel verringern oder erhöhen kann und dass die Spezifizierung des Rezeptors vom Grad der Rezeptorexpression, dem Zelltyp und der Kombination der Effektormoleküle abhängig ist. Die Abweichung der juvenilen Kaskade vom im erwachsenen Tier üblichen Signalweg hat dabei den Vorteil, dass eine Aufgabentrennung zwischen Netzwerkbildung und Synapsenstabilisierung einerseits und Signalweiterleitung andererseits erfolgt. Beim juvenilen Tier steht die Netzwerkbildung im Vordergrund, beim erwachsenen Tier wird die Signalweiterleitung moduliert. Der sekundäre Botenstoff NO, der weit diffundieren kann, spielt beim juvenilen Signalweg keine Rolle und dies ermöglicht eine klare Trennung der aktivierten von den umgebenden Synapsen. Dadurch wird eine starke Selektion innerhalb der vorhandenen Synapsen ermöglicht und eine aktivitätsbasierte Netzwerkbildung vereinfacht. Die Etablierung funktioneller GABAerger Synapsen ist ausschlaggebend für die Entwicklung des neuronalen Netzwerks. Innerhalb der ersten postnatalen Wochen führt die Aktivierung von GABAA-Rezeptoren zur Depolarisierung der IC-Neurone und zu einer Erhöhung der intrazellulären Ca2+-Konzentration. Acetylcholin potenziert also letztlich den GABA-aktivierten Einstrom von Ca2+ und führt damit zur Erhöhung der intrazellulären Ca2+ Konzentration im postsynaptischen Neuron des IC. Diese modulatorische Wirkung von Acetylcholin könnten damit eine wichtige Rolle bei der Entwicklung und Stabilisierung von inhibitorischen Synapsen im sich entwickelnden IC der Ratte spielen.
BMPs control postnatal dendrite growth and complexity in sympathetic neurons / von Afsaneh Majdazari
(2012)
The vertebrate nervous system is a complex network of billions of neurons connected by dendrites and axons, integrated to functional circuits and areas/organs in the central and peripheral nervous system. The cells of the nervous system origin from common progenitors, which take on different cell fates based on intrinsic and extrinsic factors. These factors determine general neuronal traits, but also the morphology and the type of connections made to other cells. Mechanisms underlying axonal and dendritic growth are well described in contrast to the initiation of neurite growth, which remains to be fully elucidated, especially concerning dendrite formation. Recently BMPs have been identified as candidate dendrite inducing factors in sympathetic, cortical and hippocampal neurons. Here we focus on the in vivo role of BMPs on dendrite growth in sympathetic neurons as their development and differentiation processes have been analyzed in detail.