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- Caatinga (1)
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- Pseudomonas aeruginosa (1)
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- Fachbereich Biologie (10) (remove)
Biological soil crusts (biocrusts) have been recognized as key ecological players in arid and semiarid regions at both local and global scales. They are important biodiversity components, provide critical ecosystem services, and strongly influence soil-plant relationships, and successional trajectories via facilitative, competitive, and edaphic engineering effects. Despite these important ecological roles, very little is known about biocrusts in seasonally dry tropical forests. Here we present a first baseline study on biocrust cover and ecosystem service provision in a human-modified landscape of the Brazilian Caatinga, South America's largest tropical dry forest. More specifically, we explored (1) across a network of 34 0.1 ha permanent plots the impact of disturbance, soil, precipitation, and vegetation-related parameters on biocrust cover in different stages of forest regeneration, and (2) the effect of disturbance on species composition, growth and soil organic carbon sequestration comparing early and late successional communities in two case study sites at opposite ends of the disturbance gradient. Our findings revealed that biocrusts are a conspicuous component of the Caatinga ecosystem with at least 50 different taxa of cyanobacteria, algae, lichens and bryophytes (cyanobacteria and bryophytes dominating) covering nearly 10% of the total land surface and doubling soil organic carbon content relative to bare topsoil. High litter cover, high disturbance by goats, and low soil compaction were the leading drivers for reduced biocrust cover, while precipitation was not associated Second-growth forests supported anequally spaced biocrust cover, while in old-growth-forests biocrust cover was patchy. Disturbance reduced biocrust growth by two thirds and carbon sequestration by half. In synthesis, biocrusts increase soil organic carbon (SOC) in dry forests and as they double the SOC content in disturbed areas, may be capable of counterbalancing disturbance-induced soil degradation in this ecosystem. As they fix and fertilize depauperated soils, they may play a substantial role in vegetation regeneration in the human-modified Caatinga, and may have an extended ecological role due to the ever-increasing human encroachment on natural landscapes. Even though biocrusts benefit from human presence in dry forests, high levels of anthropogenic disturbance could threaten biocrust-provided ecosystem services, and call for further, in-depth studies to elucidate the underlying mechanisms.
Micronuclei-based model system reveals functional consequences of chromothripsis in human cells
(2019)
Cancer cells often harbor chromosomes in abnormal numbers and with aberrant structure. The consequences of these chromosomal aberrations are difficult to study in cancer, and therefore several model systems have been developed in recent years. We show that human cells with extra chromosome engineered via microcell-mediated chromosome transfer often gain massive chromosomal rearrangements. The rearrangements arose by chromosome shattering and rejoining as well as by replication-dependent mechanisms. We show that the isolated micronuclei lack functional lamin B1 and become prone to envelope rupture, which leads to DNA damage and aberrant replication. The presence of functional lamin B1 partly correlates with micronuclei size, suggesting that the proper assembly of nuclear envelope might be sensitive to membrane curvature. The chromosomal rearrangements in trisomic cells provide growth advantage compared to cells without rearrangements. Our model system enables to study mechanisms of massive chromosomal rearrangements of any chromosome and their consequences in human cells.
The Atacama Desert is one of the driest and probably oldest deserts on Earth where only a few extremophile organisms are able to survive. This study investigated two terricolous and two epiphytic lichens from the fog oasis “Las Lomitas” within the National Park Pan de Azúcar which represents a refugium for a few vascular desert plants and many lichens that can thrive on fog and dew alone. Ecophysiological measurements and climate records were combined with molecular data of the mycobiont, their green algal photobionts and lichenicolous fungi to gain information about the ecology of lichens within the fog oasis. Phylogenetic and morphological investigations led to the identification and description of the new lichen species Acarospora conafii sp. nov. as well as the lichenicolous fungi that accompanied them and revealed the trebouxioid character of all lichen photobionts. Their photosynthetic responses were compared during natural scenarios such as reactivation by high air humidity and in situ fog events to elucidate the activation strategies of this lichen community. Epiphytic lichens showed photosynthetic activity that was rapidly induced by fog and high relative air humidity whereas terricolous lichens were only activated by fog.
Regulation of metabolism is complex and involves enzymes and membrane transporters, which form networks to support energy dynamics. Lactate, as a metabolic intermediate from glucose or glycogen breakdown, appears to play a major role as additional energetic substrate, which is shuttled between glycolytic and oxidative cells, both under hypoxic and normoxic conditions. Transport of lactate across the cell membrane is mediated by monocarboxylate transporters (MCTs) in cotransport with H+, which is a substrate, a signal and a modulator of metabolic processes. MCTs form a “transport metabolon” with carbonic anhydrases (CAs), which not only provide a rapid equilibrium between CO2, HCO3– and H+, but, in addition, enhances lactate transport, as found in Xenopus oocytes, employed as heterologous expression system, as well as in astrocytes and cancer cells. Functional interactions between different CA isoforms and MCTs have been found to be isoform-specific, independent of the enzyme’s catalytic activity, and they require physical interaction between the proteins. CAs mediate between different states of metabolic acidosis, induced by glycolysis and oxidative phosphorylation, and play a relay function in coupling pH regulation and metabolism. In the brain, metabolic processes in astrocytes appear to be linked to bicarbonate transport and to neuronal activity. Here, we focus on physiological processes of energy dynamics in astrocytes as well as on the transfer of energetic substrates to neurons.
In cyanobacteria and plants, VIPP1 plays crucial roles in the biogenesis and repair of thylakoid membrane protein complexes and in coping with chloroplast membrane stress. In chloroplasts, VIPP1 localizes in distinct patterns at or close to envelope and thylakoid membranes. In vitro, VIPP1 forms higher-order oligomers of >1 MDa that organize into rings and rods. However, it remains unknown how VIPP1 oligomerization is related to function. Using time-resolved fluorescence anisotropy and sucrose density gradient centrifugation, we show here that Chlamydomonas reinhardtii VIPP1 binds strongly to liposomal membranes containing phosphatidylinositol-4-phosphate (PI4P). Cryo-electron tomography reveals that VIPP1 oligomerizes into rods that can engulf liposomal membranes containing PI4P. These findings place VIPP1 into a group of membrane-shaping proteins including epsin and BAR domain proteins. Moreover, they point to a potential role of phosphatidylinositols in directing the shaping of chloroplast membranes.
Anisotropy of tracer-coupled networks is a hallmark in many brain regions. In the past, the topography of these networks was analyzed using various approaches, which focused on different aspects, e.g., position, tracer signal, or direction of coupled cells. Here, we developed a vector-based method to analyze the extent and preferential direction of tracer spreading. As a model region, we chose the lateral superior olive—a nucleus that exhibits specialized network topography. In acute slices, sulforhodamine 101-positive astrocytes were patch-clamped and dialyzed with the GJ-permeable tracer neurobiotin, which was subsequently labeled with avidin alexa fluor 488. A predetermined threshold was used to differentiate between tracer-coupled and tracer-uncoupled cells. Tracer extent was calculated from the vector means of tracer-coupled cells in four 90° sectors. We then computed the preferential direction using a rotating coordinate system and post hoc fitting of these results with a sinusoidal function. The new method allows for an objective analysis of tracer spreading that provides information about shape and orientation of GJ networks. We expect this approach to become a vital tool for the analysis of coupling anisotropy in many brain regions
The structural integrity of synaptic connections critically depends on the interaction between synaptic cell adhesion molecules (CAMs) and the underlying actin and microtubule cytoskeleton. This interaction is mediated by giant Ankyrins, that act as specialized adaptors to establish and maintain axonal and synaptic compartments. In Drosophila, two giant isoforms of Ankyrin2 (Ank2) control synapse stability and organization at the larval neuromuscular junction (NMJ). Both Ank2-L and Ank2-XL are highly abundant in motoneuron axons and within the presynaptic terminal, where they control synaptic CAMs distribution and organization of microtubules. Here, we address the role of the conserved N-terminal ankyrin repeat domain (ARD) for subcellular localization and function of these giant Ankyrins in vivo. We used a P[acman] based rescue approach to generate deletions of ARD subdomains, that contain putative binding sites of interacting transmembrane proteins. We show that specific subdomains control synaptic but not axonal localization of Ank2-L. These domains contain binding sites to L1-family member CAMs, and we demonstrate that these regions are necessary for the organization of synaptic CAMs and for the control of synaptic stability. In contrast, presynaptic Ank2-XL localization only partially depends on the ARD but strictly requires the presynaptic presence of Ank2-L demonstrating a critical co-dependence of the two isoforms at the NMJ. Ank2-XL dependent control of microtubule organization correlates with presynaptic abundance of the protein and is thus only partially affected by ARD deletions. Together, our data provides novel insights into the synaptic targeting of giant Ankyrins with relevance for the control of synaptic plasticity and maintenance.
Die Funktion der c-di-GMP modulierenden Membranproteine NbdA und MucR in Pseudomonas aeruginosa
(2019)
NbdA und MucR sind Multi-Domänenproteine aus Pseudomonas aeruginosa. Beide Proteine besitzen eine ähnliche Domänenorganisation mit einer N-terminalen, membranständigen
MHYT-Domäne sowie einer GGDEF- und einer EAL-Domäne im Cytoplasma. Die
cytosolischen Domänen von MucR sind beide aktiv, während NbdA neben der intakten
EAL-Domäne eine degenerierte GGDEF-Domäne mit dem Motiv AGDEF aufweist. Bioinformatischen
Vorhersagen zufolge soll die MHYT-Domäne eine sensorische Funktion für diatomische Gase wie Stickstoffmonoxid oder Sauerstoff vermitteln. Die phänotypische Charakterisierung der markerlosen PAO1-Deletionsmutanten \(Delta\)nbdA, \(Delta\)mucR und \(Delta\)nbdA \(Delta\)mucR zeigte, dass NbdA und MucR nicht in die NO-induzierte
Dispersion involviert sind. Ebenso konnte in einem neu etablierten heterologen in-vivo-System in E. coli keine NO-sensorische Funktion der Proteine detektiert werden. Im Weiteren wurde festgestellt, dass die MHYT-Domäne keinen ersichtlichen Einfluss auf die
Enzymaktivität von NbdA und MucR unter aeroben Bedingungen hat. Demzufolge fungiert
die Membrandomäne vermutlich weder als Sensor für Sauerstoff, noch für NO. Anhand heterologer Komplementationstests konnte eine PDE-Aktivität des NbdA-Volllängenproteins
nachgewiesen werden. Zudem wurde gezeigt, dass die degenerierte AGDEF-Domäne einen regulatorischen Effekt auf die EAL-Domäne hat, der essentiell für die in-
vivo-Aktivität von NbdA ist. In-vivo-Untersuchungen bestätigten die postulierte DGC-Aktivität von MucR. Weiterhin konnte belegt werden, dass MucR ein bifunktionelles Enzym
ist. Entgegen den Erwartungen scheint es jedoch im Planktonischen als DGC und im
Biofilm als PDE zu fungieren.
Ein weiterer Aspekt dieser Arbeit war die Charakterisierung der homologen Überexpression
von nbdA in P. aeruginosa, welche teilweise unerwartete Phänotypen ergab. Anhand
der homologen Überproduktion einer inaktiven NbdA-Variante stellte sich heraus, dass die
Hemmung der Motilität unabhängig von der Aktivität von NbdA auftritt. Massenspektrometrische
Analysen deuteten daraufhin, dass NbdA lokal c-di-GMP hydrolysiert. Diese
Ergebnisse implizieren, dass NbdA eine Trigger-PDE ist, deren primäre Funktion die Regulation
anderer makromolekularer Zielmoleküle ist. In Pseudomonas fluorescens Pf0-1 ist bekannt, dass das NbdA-Homolog Pfl01_1252 mit den Homologen von MucR (Pfl01_2525) und SadC (Pfl01_4451) interagiert. Ergebnisse einer früheren Arbeit lassen eine Interaktion
von NbdA und SadC ebenso in P. aeruginosa vermuten. Daher ist denkbar, dass sich
NbdA im gleichen Netzwerk wie MucR und SadC befindet und deren Aktivität reguliert.
Linking protistan community shifts along salinity gradients with cellular haloadaptation strategies
(2019)
Salinity is one of the most structuring environmental factors for microeukaryotic communities. Using eDNA barcoding, I detected significant shifts in microeukaryotic community compositions occurring at distinct salinities between brackish and marine conditions in the Baltic Sea. I, furthermore, conducted a metadata analysis including my and other marine and hypersaline community sequence data to confirm the existence of salinity-related transition boundaries and significant changes in alpha diversity patterns along a brackish to hypersaline gradient. One hypothesis for the formation of salinity-dependent transition boundaries between brackish to hypersaline conditions is the use of different cellular haloadaptation strategies. To test this hypothesis, I conducted metatranscriptome analyses of microeukaryotic communities along a pronounced salinity gradient (40 – 380 ‰). Clustering of functional transcripts revealed differences in metabolic properties and metabolic capacities between microeukaryotic communities at specific salinities, corresponding to the transition boundaries already observed in the taxonomic eDNA barcoding approach. In specific, microeukaryotic communities thriving at mid-hypersaline conditions (≤ 150 ‰) seem to predominantly apply the ‘low-salt – organic-solutes-in’ strategy by accumulating compatible solutes to counteract osmotic stress. Indications were found for both the intracellular synthesis of compatible solutes as well as for cellular transport systems. In contrast, communities of extreme-hypersaline habitats (≥ 200 ‰) may preferentially use the ‘high-salt-in’ strategy, i. e. the intracellular accumulation of inorganic ions in high concentrations, which is implied by the increased expression of Mg2+, K+, Cl- transporters and channels.
In order to characterize the ‘low-salt – organic-solutes-in’ strategy applied by protists in more detail, I conducted a time-resolved transcriptome analysis of the heterotrophic ciliate Schmidingerothrix salinarum serving as model organism. S. salinarum was thus subjected to a salt-up shock to investigate the intracellular response to osmotic stress by shifts of gene expression. After increasing the external salinity, an increased expression of two-component signal transduction systems and MAPK cascades was observed. In an early reaction, the expression of transport mechanisms for K+, Cl- and Ca2+ increased, which may enhance the capacity of K+, Cl- and Ca2+ in the cytoplasm to compensate possibly harmful Na+ influx. Expression of enzymes for the synthesis of possible compatible solutes, starting with glycine betaine, followed by ectoine and later proline, could imply that the inorganic ions K+, Cl- and Ca2+ are gradually replaced by the synthesized compatible solutes. Additionally, expressed transporters for choline (precursor of glycine betaine) and proline could indicate an intracellular accumulation of compatible solutes to balance the external salinity. During this accumulation, the up-regulated ion export mechanisms may increase the capacity for Na+ expulsion from the cytoplasm and ion compartmentalization between cell organelles seem to happen.
The results of my PhD project revealed first evidence at molecular level for the salinity-dependent use of different haloadaptation strategies in microeukaryotes and significantly extend existing knowledge about haloadaptation processes in ciliates. The results provide ground for future research, such as (comparative) transcriptome analysis of ciliates thriving in extreme-hypersaline habitats or experiments like qRT-PCR to validate transcriptome results.
Function of two redox sensing kinases from the methanogenic archaeon Methanosarcina acetivorans
(2019)
MsmS is a heme-based redox sensor kinase in Methanosarcina acetivorans consisting of alternating PAS and GAF domains connected to a C-terminal kinase domain. In addition to MsmS, M. acetivorans possesses a second kinase, MA0863 with high sequence similarity. Interestingly, MA0863 possesses an amber codon in its second GAF domain, encoding for the amino acid pyrrolysine. Thus far, no function of this residue has been resolved. In order to examine the heme iron coordination in both proteins, an improved method for the production of heme proteins was established using the Escherichia coli strain Nissle 1917. This method enables the complete reconstitution of a recombinant hemoprotein during protein production, thereby resulting in a native heme coordination. Analysis of the full-length MsmS and MA0863 confirmed a covalently bound heme cofactor, which is connected to one conserved cysteine residue in each protein. In order to identify the coordinating amino acid residues of the heme iron, UV/vis spectra of different variants were measured. These studies revealed His702 in MsmS and the corresponding His666 in MA0863 as the proximal heme ligands. MsmS has previously been described as a heme-based redox sensor. In order to examine whether the same is true for MA0863, redox dependent kinase assays were performed. MA0863 indeed displays redox dependent autophosphorylation activity, which is independent of heme ligands and only observed under oxidizing conditions. Interestingly, autophosphorylation was shown to be independent of the heme cofactor but rather relies on thiol oxidation. Therefore, MA0863 was renamed in RdmS (redox dependent methyltransferase-associated sensor). In order to identify the phosphorylation site of RdmS, thin layer chromatography was performed identifying a tyrosine as the putative phosphorylation site. This observation is in agreement with the lack of a so-called H-box in typical histidine kinases. Due to their genomic localization, MsmS and RdmS were postulated to form two-component systems (TCS) with vicinal encoded regulator proteins MsrG and MsrF. Therefore, protein-protein interaction studies using the bacterial adenylate two hybrid system were performed suggesting an interaction of RdmS and MsmS with the three regulators MsrG/F/C. Due to these multiple interactions these signal transduction pathways should rather be considered multicomponent system instead of two component systems.