Refine
Year of publication
Document Type
- Article (1154)
- Doctoral Thesis (836)
- Preprint (76)
- Book (59)
- Contribution to a Periodical (44)
- Conference Proceeding (10)
- Diploma Thesis (10)
- Review (8)
- diplomthesis (4)
- Report (3)
Has Fulltext
- yes (2205)
Is part of the Bibliography
- no (2205)
Keywords
- Podospora anserina (17)
- aging (17)
- mitochondria (12)
- autophagy (10)
- Archaea (9)
- Haloferax volcanii (9)
- Saccharomyces cerevisiae (9)
- Phylogeny (8)
- climate change (8)
- gene expression (8)
Institute
- Biowissenschaften (2205) (remove)
An exploration of the relationship between recruitment communication and foraging in stingless bees
(2021)
Social information is widely used in the animal kingdom and can be highly adaptive. In social insects, foragers can use social information to find food, avoid danger, or choose a new nest site. Copying others allows individuals to obtain information without having to sample the environment. When foragers communicate information they will often only advertise high-quality food sources, thereby filtering out less adaptive information. Stingless bees, a large pantropical group of highly eusocial bees, face intense inter- and intra-specific competition for limited resources, yet display disparate foraging strategies. Within the same environment there are species that communicate the location of food resources to nest-mates and species that do not. Our current understanding of why some species communicate foraging sites while others do not is limited. Studying freely foraging colonies of several co-existing stingless bee species in Brazil, we investigated if recruitment to specific food locations is linked to 1) the sugar content of forage, 2) the duration of foraging trips, and 3) the variation in activity of a colony from 1 day to another and the variation in activity in a species over a day. We found that, contrary to our expectations, species with recruitment communication did not return with higher quality forage than species that do not recruit nestmates. Furthermore, foragers from recruiting species did not have shorter foraging trip durations than those from weakly recruiting species. Given the intense inter- and intraspecific competition for resources in these environments, it may be that recruiting species favor food resources that can be monopolized by the colony rather than food sources that offer high-quality rewards.
Obligate endoparasitic oomycetes are known to ubiquitously occur in marine and freshwater diatoms, but their diversity is still largely unexplored. Many of these parasitoids are members of the early-diverging oomycete lineages (Miracula, Diatomophthora), others are within the Leptomitales of the Saprolegniomycetes (Ectrogella, Lagenisma) and some have been described in the Peronosporomycetes (Aphanomycopsis, Lagenidium). Even though some species have been recently described and two new genera were introduced (Miracula and Diatomophthora), the phylogeny and taxonomy of most of these organisms remain unresolved. This is contrasted by the high number of sequences from unclassified species, as recently revealed from environmental sequencing, suggesting the presence of several undiscovered species. In this study, a new species of Miracula is reported from a marine centric diatom (Minidiscus sp.) isolated from Skagaströnd harbor in Northwest Iceland. The morphology and life cycle traits of this novel oomycete parasite are described herein, and its taxonomic placement within the genus Miracula is confirmed by molecular phylogeny. As it cannot be assigned to any previously described species, it is introduced as Miracula islandica in this study. The genus Miracula thus contains three described holocarpic species (M. helgolandica, M. islandica, M. moenusica) to which likely additional species will need to be added in the future, considering the presence of several lineages known only from environmental sequencing that clustered within the Miracula clade.
The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.
Acinetobacter baumannii is an opportunistic pathogen, which has become a rising threat in healthcare facilities worldwide due to increasing antibiotic resistances and optimal adaptation to clinical environments and the human host. We reported in a former publication on the identification of three phopholipases of the phospholipase D (PLD) superfamily in A. baumannii ATCC 19606T acting in concerted manner as virulence factors in Galleria mellonella infection and lung epithelial cell invasion. This study focussed on the function of the three PLDs. A Δpld1-3 mutant was defect in biosynthesis of the phospholipids cardiolipin (CL) and monolysocardiolipin (MLCL), whereas the deletion of pld2 and pld3 abolished the production of MLCL. Complementation of the Δpld1-3 mutant with pld1 restored CL biosynthesis demonstrating that the PLD1 is implicated in CL biosynthesis. Complementation of the Δpld1-3 mutant with either pld2 or pld3 restored MLCL and CL production leading to the conclusion that PLD2 and PLD3 are implicated in CL and MLCL production. Mutant studies revealed that two catalytic motifs are essential for the PLD3-mediated biosynthesis of CL and MLCL. The Δpld1-3 mutant exhibited a decreased colistin and polymyxin B resistance indicating a role of CL in cationic antimicrobial peptides (CAMPs) resistance.
The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.
The extraordinary desiccation resistance of the opportunistic human pathogen Acinetobacter baumannii is a key to its survival and spread in medical care units. The accumulation of compatible solute such as glutamate, mannitol and trehalose contributes to the desiccation resistance. Here, we have used osmolarity as a tool to study the response of cells to low water activities and studied the role of a potential inorganic osmolyte, K+, in osmostress response. Growth of A. baumannii was K+-dependent and the K+-dependence increased with the osmolarity of the medium. After an osmotic upshock, cells accumulated K+ and K+ accumulation increased with the salinity of the medium. K+ uptake was reduced in the presence of glycine betaine. The intracellular pools of compatible solutes were dependent on the K+ concentration: mannitol and glutamate concentrations increased with increasing K+ concentrations whereas trehalose was highest at low K+. After osmotic upshock, cells first accumulated K+ followed by synthesis of glutamate; later, mannitol and trehalose synthesis started, accompanied with a decrease of intracellular K+ and glutamate. These experiments demonstrate K+ uptake as a first response to osmostress in A. baumannii and demonstrate a hierarchy in the time-dependent accumulation of K+ and different organic solutes.
Acinetobacter baumannii is outstanding for its ability to cope with low water activities and therefore its adaptation mechanism to osmotic stress. Here we report on the identification and characterization of five different secondary active compatible solute transporters, belonging to the betaine-choline-carnitine transporter (BCCT) family. Our studies revealed two choline-specific and three glycine betaine-specific BCCTs. Activity of the BCCTs was differentially dependent to the osmolality: one choline and one betaine transporter were osmostress-independent. Addition of choline to resting cells of Acinetobacter grown in the presence of the co-substrate choline or with phosphatidylcholine as sole carbon source led to ATP synthesis in the wild type but not in the BCCT quadruple mutant. This indicates that the BCCTs are essential to transport the energy substrate choline. The role of the different BCCTs in osmostress resistance and in metabolic adaptation of A. baumannii to the human host is discussed.
A1AO ATP synthases with a V-type c subunit have only been found in hyperthermophilic archaea which makes bioenergetic analyses impossible due to the instability of liposomes at high temperatures. A search for a potential archaeal A1AO ATP synthase with a V-type c subunit in a mesophilic organism revealed an A1AO ATP synthase cluster in the anaerobic, acetogenic bacterium Eubacterium limosum KIST612. The enzyme was purified to apparent homogeneity from cells grown on methanol to a specific activity of 1.2 U·mg−1 with a yield of 12%. The enzyme contained subunits A, B, C, D, E, F, H, a, and c. Subunit c is predicted to be a typical V-type c subunit with only one ion (Na+)-binding site. Indeed, ATP hydrolysis was strictly Na+-dependent. N,N′-dicyclohexylcarbodiimide (DCCD) inhibited ATP hydrolysis, but inhibition was relieved by addition of Na+. Na+ was shown directly to abolish binding of the fluorescence DCCD derivative, NCD-4, to subunit c, demonstrating a competition of Na+ and DCCD/NCD-4 for a common binding site. After incorporation of the A1AO ATP synthase into liposomes, ATP-dependent primary transport of 22Na+ as well as ΔµNa+-driven ATP synthesis could be demonstrated. The Na+ A1AO ATP synthase from E. limosum is the first ATP synthase with a V-type c subunit from a mesophilic organism. This will enable future bioenergetic analysis of these unique ATP synthases.
Understanding hominin expansions requires the comprehension of movement processes at different scales. In many models of hominin expansion these processes are viewed as being determined by large-scale effects, such as changes in climate and vegetation spanning continents and thousands or even millions of years. However, these large-scale patterns of expansions also need to be considered as possibly resulting from the accumulation of small-scale decisions of individual hominins. Moving on a continental scale may for instance involve crossing a water barrier. We present a generalized agent-based model for simulating the crossing of a water barrier where the agents represent the hominin individuals. The model can be configured to represent a variety of movement modes across water. Here, we compare four different behavioral scenarios in conjunction with a set of water barrier configurations, in which agents move in water by either paddling, drifting, swimming or rafting. We introduce the crossing-success-rate (CSR) to quantify the performance in water crossing. Our study suggests that more focus should be directed towards the exploration of behavioral models for hominins, as directionality may be a more powerful factor for crossing a barrier than environmental opportunities alone. A prerequisite for this is to perceive the opposite shore. Furthermore, to provide a comprehensive understanding of hominin expansions, the CSR allows for the integration of results obtained from small-scale simulations into large-scale models for hominin expansion.
Young trees of deciduous Quercus robur and Q. petraea and evergreen Q. ilex were grown together in a competition lysimeter experiment to assess i) – whether the observed growth differences between evergreen Q. ilex and the deciduous Q. robur and Q. petraea on sandy soil in the field and ii) – whether the different natural distribution of Q. robur and Q. petraea could be attributed to physiological differences between the species under experimental drought stress (DS). Half of the plants were subjected to long-term DS in two consecutive years and monitored for physiological and growth parameters. In the first year, water withholding for more than three months did not lead to significant drought stress, probably because of a sufficient residual water volume in the lysimeter for the relatively small plants. However, in the second year, 2018, which was warmer, the bigger plants now competed for the residual water and clear drought stress symptoms developed for more than two months in all trees in the DS lysimeter basin. Growth was only moderately (and mostly not significantly) affected by the DS in the second year, except for a smaller total leaf area in DS Q. ilex as compared to DS Q. robur and Q. petraea and smaller root collar diameter in DS Q. ilex compared to DS Q. robur. Under DS, the deciduous species revealed significant decreases in ΔVIP, indicating a negative effect on electron transport through PS I. Pn, PIabs and water relations parameters (ΨPD and LWC) all decreased to various extents under DS in all three species, leading to clear separation of the deciduous from the evergreen species by PCA. However, PCA did not separate the two deciduous species from each other. It is concluded that longer root growth in the two deciduous species as compared to Q. ilex ameliorates DS effects in Q. robur and Q. petraea and may be the key to understand the better performance of deciduous oaks on sandy soil in the field.
The Southern Ocean (SO) continental shelf and deep sea are environments characterised by different benthic communities. Their structure and composition are driven and shaped by different variables: whilst on the continental shelf physical environmental variables are the main drivers shaping faunal abundance, structure and composition, the deep-sea fauna is most problably driven by biological variables such as predation and competition. Among shelf and deep-sea benthic communities, peracarids (e.g. amphipods and isopods) are one of the most dominant groups, showing high levels of abundance and diversity in both environments. Knowledge on their assemblage structure and composition in the SO remains limited, as well as the knowledge of the environmental variables that influence them. Therefore, the aim of our study was to investigate peracarid assemblages from the SO continental shelf and deep sea and to assess the main drivers shaping their assemblage structure along a wide bathymetric gradient (from 160 m to about 6000 m depth) and at a large geographic scale. We analysed the spatial distribution of 183,606 peracarids sampled using an epibenthic sledge (EBS) during nine different expeditions in the SO, covering a latitudinal range of 77° to 41° South. Depth was identified as the main driver shaping peracarid abundance pattern, their assemblage structure from the continental shelf (<1499 m) was dissimilar to that from the deep sea (>1500 m). Also, depth was differently correlated with different peracarid orders: while isopod abundances increased with depth, amphipods and mysids were negatively correlated; no correlation was found with cumaceans and tanaidaceans. The dissimilar peracarid assemblage structure between the SO continental shelf and the SO deep sea can be due to the assumption that there are different driving forces shaping benthic assemblages from these two environments (physical variables on the continental shelf, biological interactions in the deep sea). As a result, we also suggest that environmental changes due to climate change (e.g. temperature, ice coverage, productivity) would have different consequences depending on the bathymetric range considered.
Chemosensory impairments have been established as a specific indicator of COVID-19. They affect most patients and may persist long past the resolution of respiratory symptoms, representing an unprecedented medical challenge. Since the SARS-CoV-2 pandemic started, we now know much more about smell, taste, and chemesthesis loss associated with COVID-19. However, the temporal dynamics and characteristics of recovery are still unknown. Here, capitalizing on data from the Global Consortium for Chemosensory Research (GCCR) crowdsourced survey, we assessed chemosensory abilities after the resolution of respiratory symptoms in participants diagnosed with COVID-19 during the first wave of the pandemic in Italy. This analysis led to the identification of two patterns of chemosensory recovery, partial and substantial, which were found to be associated with differential age, degrees of chemosensory loss, and regional patterns. Uncovering the self-reported phenomenology of recovery from smell, taste, and chemesthetic disorders is the first, yet essential step, to provide healthcare professionals with the tools to take purposeful and targeted action to address chemosensory disorders and their severe discomfort.
FAD synthase is the last enzyme in the pathway that converts riboflavin into FAD. In Saccharomyces cerevisiae, the gene encoding for FAD synthase is FAD1, from which a sole protein product (Fad1p) is expected to be generated. In this work, we showed that a natural Fad1p exists in yeast mitochondria and that, in its recombinant form, the protein is able, per se, to both enter mitochondria and to be destined to cytosol. Thus, we propose that FAD1 generates two echoforms—that is, two identical proteins addressed to different subcellular compartments. To shed light on the mechanism underlying the subcellular destination of Fad1p, the 3′ region of FAD1 mRNA was analyzed by 3′RACE experiments, which revealed the existence of (at least) two FAD1 transcripts with different 3′UTRs, the short one being 128 bp and the long one being 759 bp. Bioinformatic analysis on these 3′UTRs allowed us to predict the existence of a cis-acting mitochondrial localization motif, present in both the transcripts and, presumably, involved in protein targeting based on the 3′UTR context. Here, we propose that the long FAD1 transcript might be responsible for the generation of mitochondrial Fad1p echoform.
In recent years, several neuronal differentiation protocols were published that circumvent the requirement of embryoid body (EB) formation under serum-deprivation and simplified medium conditions. But a neuronal default model to establish an approach that works efficiently for all pluripotent cells and neuronal precursors is still lacking. Whether such a default neural mechanism exist and how this is implemented across a broad spectrum of cell source, is addressed in several studies and still controversially discussed. It was proposed that the default neuronal fate is initiated in the absence of extrinsic signals and is achieved by eliminating extracellular inhibitors of neuroectodermal fate and suppressing cell-cell signalling through limited cell density. Previous studies reported that ESC and ECC grown at low density and in absence of exogenous factors or feeder layers die within 24 h but acquire a neural identity as indicated by expression of the neural marker Nestin. Thus, this application is not suitable for generating neural cultures. Furthermore, it was reported that P19 cells survive and express neuroectodermal marker genes in serum-free DMEM/F12 medium containing transferrin, insulin, and selenite, although no neurites were identified.
Based on this background, in this study, a novel approach to induce neuronal differentiation in vitro was developed that implements a nutrient-poor environment, which, in contrast to previous studies, ensures the survival of neuronally differentiated cells over a long period of time and allows normal formation of neurites. Neither the formation of free-floating aggregates nor supplementation of growth factors or known inducers was required to establish a reliable neuronal differentiation protocol. A simple medium, consisting of DMEM/F12+N2 that was highly diluted in salt solution, was sufficient to drive a fast neuronal differentiation in monolayer cultures. Serum deprivation and strong dilution of DMEM/F12+N2 medium cause a nutrient-poor environment in which the influence of growth factors and inducers is minimized. This medium creates a metabolically defined environment that is presumably free of extrinsic signals that prevent the decision of neuronal fate. Analysis of the medium components discovered no actual inducer. Hence, it was suggested that the metabolic composition of the medium exclusively covers specific cell requirements of neurons, therefore ensures their survival, and drives the switch from pluripotent cells to neurons. The self-developed method was established by usage of the murine embryonal carcinoma cell line P19 and could be transferred to murine ESC. Consequently, the method could provide a feasible protocol for a generally valid neuronal default model.
The established protocol provides several advantages such as the possibility to generate stable pure neuronal cultures by a fast, simple, and highly reproducible one-step induction under defined medium conditions with a minimum of exogen effectors. The method is characterised by clear and steady medium conditions that makes the investigation of specific cell requirements during differentiation accessible. It is therefore expected to be a useful tool to investigate the molecular basis of neuronal differentiation as well as for high throughput screenings. The phenotype of mature postmitotic neurons was arising within one week and cultures were shown to stay stable at least for three weeks. The neuronal identity was confirmed by expression of neuronal markers through immunofluorescence staining and mass spectrometry analysis. Furthermore, increased levels of axon markers were detected in early neuronal differentiation and functionality of the synapses of the P19-derived neurons was ascertained by detection of calcium activity. Axonal laser ablation, immediately followed by fast regrowth of connections in the neuronal network, revealed a strong regeneration potential under the given conditions. Furthermore, the generated neurons showed a morphologically distinct phenotype and the formation of neural rosettes. Immunofluorescence staining demonstrated the generation of pure and homogeneous neuronal cultures, free of glial cells.
Retinoic acid (RA) plays an essential role in cell signalling during embryogenesis and efficiently induces neuronal differentiation in vitro in a concentration dependent manner. Neither retinol nor retinoic acid was included in any of the components of the self-prepared medium in this work. However, I observed, dependence on RARβ- and/or RARγ-regulated RA signalling in serum-free monolayer cultures. Nevertheless, neuronal differentiation in serum-free monolayer cultures was assumed to be RARα-independent because (i) RARα was slightly downregulated after neuronal induction, (ii) the truncated RARα of the RAC65 mutant had no effect on induction efficiency, and (iii) a pan-RAR inhibitor suppressed neuronal differentiation. In contrast to serum-free monolayer cultures, the truncated RARα prevented neuronal differentiation by application of the conventional protocol where cells are grown in free floating cell aggregates in serum-containing medium. Proteome analysis of P19 cells, treated by the self-developed differentiation protocol over five days showed increased levels of cellular RA binding proteins that mediate the cellular RA transport and are involved in canonical as well as non-canonical RA signalling.
...
Acetogenic bacteria are a polyphyletic group of organisms that fix carbon dioxide under anaerobic, non-phototrophic conditions by reduction of two mol of CO2 to acetyl-CoA via the Wood–Ljungdahl pathway. This pathway also allows for lithotrophic growth with H2 as electron donor and this pathway is considered to be one of the oldest, if not the oldest metabolic pathway on Earth for CO2 reduction, since it is coupled to the synthesis of ATP. How ATP is synthesized has been an enigma for decades, but in the last decade two ferredoxin-dependent respiratory chains were discovered. Those respiratory chains comprise of a cytochrome-free, ferredoxin-dependent respiratory enzyme complex, which is either the Rnf or Ech complex. However, it was discovered already 50 years ago that some acetogens contain cytochromes and quinones, but their role had only a shadowy existence. Here, we review the literature on the characterization of cytochromes and quinones in acetogens and present a hypothesis that they may function in electron transport chains in addition to Rnf and Ech.
Im Alter von 77 Jahren verstarb am 5.7.2014 der Mikrobiologe Prof. Friedrich Willi Pons. Nach einem Studium der Biologie und Chemie spezialisierte er sich auf Genetik in der Pionierzeit der Molekularen Biologie in einem sehr guten Umfeld mit den Kollegen B. Rajewsky, Th. Wieland, G. Pfleiderer, R. W. Kaplan, A. Kleinschmidt, H. Zahn. Seine Promotion zur Untersuchung der DNS einiger Serratia-Stämme und deren Phagen bei Prof. R. W. Kaplan fand 1965 sehr viel wissenschaftliche Beachtung.
[Nachruf] Abbas Gholami
(2013)
Dr. Abbas Gholami ist am 28. August 2013 verstorben. Geboren 1945 und aufgewachsen in Quchan, Persien, führte ihn seine Sehnsucht als 18-Jährigen nach Deutschland, nach Frankfurt. Hier nahm er das Chemiestudium an der Goethe-Universität auf und beendete es als Diplomchemiker. Eine Dissertation auf dem Alkaloidgebiet bei Prof. Teuber folgte und 1979 wurde er promoviert.
Zottelige Landschaftspfleger
(2013)
[Nachruf] Willy Hilgenberg
(2013)
Plastic products leach chemicals that induce in vitro toxicity under realistic use conditions
(2021)
Plastic products contain complex mixtures of extractable chemicals that can be toxic. However, humans and wildlife will only be exposed to plastic chemicals that are released under realistic conditions. Thus, we investigated the toxicological and chemical profiles leaching into water from 24 everyday plastic products covering eight polymer types. We performed migration experiments over 10 days at 40 °C and analyzed the migrates using four in vitro bioassays and nontarget high-resolution mass spectrometry (UPLC-QTOF-MSE). All migrates induced baseline toxicity, 22 an oxidative stress response, 13 antiandrogenicity, and one estrogenicity. Overall, between 17 and 8681 relevant chemical features were present in the migrates. In other words, between 1 and 88% of the plastic chemicals associated with one product were migrating. Further, we tentatively identified ∼8% of all detected features implying that most plastic chemicals remain unknown. While low-density polyethylene, polyvinyl chloride, and polyurethane induced most toxicological endpoints, a generalization for other materials is not possible. Our results demonstrate that plastic products readily leach many more chemicals than previously known, some of which are toxic in vitro. This highlights that humans are exposed to many more plastic chemicals than currently considered in public health science and policies.
1. Das Wachstum und die Fähigkeit zur Butyratproduktion von E. callanderi KIST612 wurde in geschlossenen Batch-Kulturen mit den Substraten Glukose, Methanol, Formiat, H2 + CO2 und CO untersucht. E. callanderi KIST612 zeigte sich nur bei Wachstum auf 20 mM Glukose oder 20 mM Methanol in der Lage, Butyrat in größeren Mengen (3,7 – 4,3 mM) zu produzieren. Das Hauptprodukt bei allen untersuchten Wachstumssubstraten war jedoch Acetat.
2. In bioinformatischen Analysen des Genoms von E. callanderi KIST612 konnte nur eine A1AO-ATP-Synthase gefunden werden, welche eine V-typ c-Untereinheit bestehend aus 4 TMH‘s mit nur einer Na+-Bindestelle aufweist. Diese konnte aus gewaschenen Membranen von E. callanderi durch Saccharose-Dichtegradientenzentrifugation, Anionenaustausch-Chromatographie (DEAE) sowie einer Größenausschluss-Chromatographie (Superose 6) bis zur apparenten Homogenität gereinigt werden. Nach Produktion einzelner Untereinheiten (A, B, C, D, E, F und H) in E. coli und Generierung von Antikörpern, konnten alle Untereinheiten (A, B, C, D, E, F, H, a sowie c) in der gereinigten Enzympräparation immunologisch oder mittels „Peptide-Mass-Fingerprinting“ nachgewiesen werden. Es konnte somit erstmals eine A1AO-ATP-Synthase aus einem mesophilen Organismus ohne Verlust von Untereinheiten gereinigt werden.
3. Der Gesamtkomplex wies unter nativen Bedingungen eine molekulare Masse von ca. 670 kDa auf. In elektronenmikroskopischen Aufnahmen zeigte sich anhand der hantelförmigen Strukturen, dass die A1AO-ATP-Synthase als intakter Gesamtkomplex gereinigt werden konnte.
4. Die gereinigte A1AO-ATP-Synthase wurde zunächst anhand ihrer ATP-Hydrolyse-Aktivität biochemisch charakterisiert. Die ATP-Hydrolyse-Aktivität hatte ein pH-Optimum von 7 – 7,5 und ein Temperaturoptimum bei 37 °C. Durch Messung der ATPase-Aktivität in Abhängigkeit von verschiedenen Mengen an Na+ konnte die vorhergesagte Na+-Abhängigkeit des Enzyms nachgewiesen werden. Zudem zeigten Hemmstoffexperimente mit DCCD, dass dieser Inhibitor mit Na+ um die gemeinsame Bindestelle in der c-Untereinheit konkurriert. Dies bestätigte nochmals, dass das Enzym funktionell gekoppelt gereinigt werden konnte.
5. Zur weiteren Untersuchung der Ionenspezifität wurde der an die ATP-Hydrolyse gekoppelte Ionentransport durch Rekonstitution des Enzyms in Liposomen und anschließender Messung des Na+- oder H+-Transports gemessen. In den Proteoliposomen konnte mit Hilfe von 22Na+ gezeigt werden, dass das Enzym Natriumionen translozieren kann. Während in Anwesenheit des Natriumionophors ETH 2120 kein 22Na+-Transport beobachtet werden konnte, führte die Anwesenheit des Protonophors TCS zu einer geringfügigen Stimulation der 22Na+-Translokation. Insgesamt konnte ein primärer Na+-Transport nachgewiesen werden, welcher von der A1AO-ATP-Synthase aus E. callanderi katalysiert wird.
6. Durch Rekonstitution der A1AO-ATP-Synthase aus E. callanderi in Liposomen konnte erstmals biochemisch nachgewiesen werden, dass ein solches Enzym trotz seiner V-Typ c-Untereinheit in der Lage ist, ATP zu synthetisieren. Durch die Zugabe von Ionophoren (ETH 2120 und TCS) konnte der elektrochemische Ionengradient aufgehoben werden, wodurch keine ATP-Synthese beobachtet werden konnte. Der erstmalige Nachweis der ATP-Synthese wurde bei einem ΔµNa+ von 270 mV erbracht.
7. Die ATP-Synthese zeigte sich ebenfalls abhängig von der Na+-Konzentration. Der KM-Wert lag bei 1,1 ± 0,4 mM und war vergleichbar mit dem für die ATP-Hydrolyse ermittelten Wert. Ebenso konnte für die ATP-Synthese-Richtung gezeigt werden, dass DCCD mit Na+ um die gemeinsame Bindestelle in der c-Untereinheit konkurriert.
8. Um den biochemischen Nachweis zu erbringen, dass die A1AO-ATP-Synthase auch unter physiologisch relevanten Potentialen zur ATP-Synthese befähigt ist, wurde der energetische Schwellenwert der ATP-Synthese bestimmt. Dieser betrug 87 mV als Triebkraft für ΔpNa, 94 mV als Triebkraft für Δψ und 90 mV als Triebkraft für ΔµNa+. Erstaunlicherweise konnte die ATP-Synthese der A1AO-ATP-Synthase aus E. callanderi KIST612 sowohl durch Δψ als auch ΔpNa angetrieben werden. Unterschiedliche Kombinationen von Δψ und ΔpNa führten zu dem gleichen energetischen Schwellenwert; Δψ und ΔpNa waren im Enzym aus E. callanderi KIST612 äquivalente Triebkräfte.
9. Der energetische Schwellenwert der A1AO-ATP-Synthase aus E. callanderi KIST612 wurde mit dem der F1FO-ATP-Synthasen aus A. woodii, E. coli und P. modestum verglichen. Dazu wurden die Enzyme im ATP-Synthase-defizienten E. coli-Stamm DK8 produziert und anschließend durch Ni2+-NTA-Affinitätschromatographie gereinigt. Nach Einbau der Enzyme in Liposomen waren alle Enzyme in der Lage, ATP als Reaktion auf ΔµNa+ (A. woodii und P. modestum) oder ΔµH+ (E. coli) zu synthetisieren. Im Vergleich zum Enzym aus E. callanderi zeigten sich zwei auffällige Unterschiede. Erstens war keine der F1FO-ATP-Synthasen in der Lage, ΔpNa/ΔpH als alleinige Triebkraft zu nutzen. Während die ATP-Synthese in den Enzymen aus E. coli und P. modestum nur durch ΔµH+ bzw. ΔµNa+ angetrieben werden konnte, konnte das Enzym aus A. woodii zusätzlich auch durch Δψ als einzige Triebkraft angetrieben werden.
...
Nature's non-material contributions to people are difficult to quantify and one aspect in particular, nature's contributions to communication (NCC), has so far been neglected. Recent advances in automated language processing tools enable us to quantify diversity patterns underlying the distribution of plant and animal taxon labels in creative literature, which we term BiL (biodiversity in literature). We assume BiL to provide a proxy for people's openness to nature's non-material contributions enhancing our understanding of NCC. We assembled a comprehensive list of 240,000 English biological taxon labels. We pre-processed and searched a subcorpus of digitised literature on Project Gutenberg for these labels. We quantified changes in biodiversity indices commonly used in ecological studies for 16,000 books, encompassing 4,000 authors, as proxies for BiL between 1705 and 1969. We observed hump-shape patterns for taxon label richness, abundance and Shannon diversity indicating a peak of BiL in the middle of the 19th century. This is also true for the ratio of biological to general lexical richness. The variation in label use between different sections within books, quantified as β-diversity, declined until the 1830s and recovered little, indicating a less specialised use of taxon labels over time. This pattern corroborates our hypothesis that before the onset of industrialisation BiL may have increased, reflecting several concomitant influences such as the general broadening of literary content, improved education and possibly an intensified awareness of the starting loss of biodiversity during the period of romanticism. Given that these positive trends continued and that we do not find support for alternative processes reducing BiL, such as language streamlining, we suggest that this pronounced trend reversal and subsequent decline of BiL over more than 100 years may be the consequence of humans’ increasing alienation from nature owing to major societal changes in the wake of industrialisation. We conclude that our computational approach of analysing literary communication using biodiversity indices has a high potential for understanding aspects of non-material contributions of biodiversity to people. Our approach can be applied to other corpora and would benefit from additional metadata on taxa, works and authors.
This work comprises the investigation of four different biosynthesis gene clusters from Xenorhabdus. Xenorhabdus is an entomopathogenic bacterium that lives in mutualistic symbiosis with its Steinernema nematode host and together they infect and kill insect larvae. Xenorhabdus is well known for the production of so-called specialised metabolites and many of these compounds are synthesised by non-ribosomal peptide synthetases (NRPSs) or NRPS-polyketide synthase (PKS)-hybrids. These enzymes are organised in a modular manner and produce structurally very diverse molecules, often with the help of modifying domains and tailoring enzymes. In general, the genes involved in the biosynthesis are organised in so-called biosynthetic gene clusters (BGCs) in the genome of the producing strain. Exchanging the native promoter with an inducible promoter, e.g. PBAD, allows the targeted activation of the BGC and in turn the analysis of the biosynthesis product via LC-MS analysis.
The first BGC investigated in this work is responsible for the biosynthesis of xenofuranones. Based on gene deletions, this work shows that the NRPS-like enzyme XfsA produces a carboxylated furanone intermediate which is subsequently decarboxylated by XfsB to yield xenofuranone B. The next step in xenofuranone biosynthesis is the O-methylation of xenofuranone B to yield xenofuranone A. A comparative proteomics approach allowed the identification of four methyltransferase candidates and subsequent gene deletions confirmed one of the candidates to be responsible for methylation of xenofuranone B. The proteome analysis was based on the comparison of X. szentirmaii WT and X. szentirmaii Δhfq because distinct levels of the methylated xenofuranone A were observed when the xfs BGC was activated in either WT or Δhfq strain. Hfq is a global transcriptional regulator whose deletion is associated with the down regulation of natural product biosynthesis in Xenorhabdus. The strong PBAD activation of the xfs BGC also allowed the detection of two novel xenofuranone derivatives which arise from incorporation of one 4-hydroxyphenylpyruvic acid as first or second building block, respectively.
PBAD based activation of the second BGC addressed in this work lead to the detection of a novel metabolite and compound purification allowed NMR-based structure elucidation. The molecule exhibits two pyrrolizidine moieties and was named pyrrolizwilline (pyrrolizidine + twin (German: “Zwilling”)). The BGC comprises seven genes and single gene deletions as well as heterologous expression in E. coli and NRPS engineering were conducted to investigate the biosynthesis. The first two genes xhpA and xhpB encode a bimodular NRPS and a monooxygenase which synthesise a pyrrolizixenamide-like structure, similar to PxaA and PxaB in pyrrolizixenamide biosynthesis. It is suggested that the acyl side chain incorporated by XhpA is removed by the α,β-hydrolase XhpG. The keto function is then reduced by two subsequent two electron reductions catalysed by XhpC and XhpD. One of these two reduced pyrrolizidine units most likely is extended with glyoxalate prior to non-enzymatic dimerisation with the second pyrrolizidine moiety. To finally yield pyrrolizwilline, L-valine is incorporated, probably by the free-standing condensation domain XhpF.
The third BGC investigated is responsible for the production of a tripeptide composed of β-D-homoserine, α-hydroxyglycine and L-valine and is referred to as glyoxpeptide. This work demonstrates that the previously observed glyoxpeptide derivative is derived from glycerol present in the culture medium. Furthermore, this work shows that the monooxygenase domain, which is found in an unusual position between motifs A8 and A9 within the adenylation domain, is responsible for the α-hydroxylation of glycine. It is suggested that the α-hydroxylation of glycine renders the tripeptide prone to hydrolysis via hemiacetal formation. Hence, the XgsC_MonoOx domain might be an interesting candidate for further NRPS engineering.
The fourth BGC addressed is responsible for the production of xildivalines and this work describes two additional derivatives which are detected only when the promoter is exchanged and activated in the X. hominickii WT strain but not in X. hominickii Δhfq. Deletion of the methyltransferase encoding gene xisE results in the production of non-methylated xildivalines. It remains to be determined when the N-methylation of L-valine takes place. It is discussed that the methyltransferase could act on the NRPS released product but also during the assembly. The peptide deformylase is not involved in the proposed biosynthesis as xildivaline production is detected in a ΔxisD strain. The PKS XisB features two adjacent, so-called tandem T domains. The inactivation of the first or the second T domain by point mutation causes decreased production titres of detected xildivalines in the respective mutant strain when compared to the wild type.
Natural products (NPs) from microorganisms have been important sources for discovering new therapeutic and chemical entities. While their corresponding biosynthetic gene clusters (BGCs) can be easily identified by gene-sequence-similarity-based bioinformatics strategies, the actual access to these NPs for structure elucidation and bioactivity testing remains difficult. Deletion of the gene encoding the RNA chaperone, Hfq, results in strains losing the production of most NPs. By exchanging the native promoter of a desired BGC against an inducible promoter in Δhfq mutants, almost exclusive production of the corresponding NP from the targeted BGC in Photorhabdus, Xenorhabdus and Pseudomonas was observed including the production of several new NPs derived from previously uncharacterized non-ribosomal peptide synthetases (NRPS). This easyPACId approach (easy Promoter Activated Compound Identification) facilitates NP identification due to low interference from other NPs. Moreover, it allows direct bioactivity testing of supernatants containing secreted NPs, without laborious purification.
Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure–function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.
Acetogenic bacteria are a group of strictly anaerobic bacteria that may have been first life forms on Earth since they employ an ancient pathway for CO2 fixation into acetyl-CoA that is coupled to the synthesis of ATP, the Wood–Ljungdahl pathway. Electrons for CO2 reduction are derived from oxidation of H2 or CO and thus, these bacteria can grow lithotrophically on gases present on early Earth. Among the organic molecules present on early Earth is acetaldehyde, a highly volatile C2 compound. Here, we demonstrate that the acetogenic model bacterium Acetobacterium woodii grows on acetaldehyde. Acetaldehyde is dismutated to ethanol and acetyl-CoA, most likely by the bifunctional alcohol dehydrogenase AdhE. Acetyl-CoA is converted to acetate by two subsequent enzymes, phosphotransacetylase and acetate kinase, accompanied by the synthesis of ATP by substrate-level phosphorylation. Apparently, growth on acetaldehyde does not employ the Wood–Ljungdahl pathway. Our finding opens the possibility of a simple and ancient metabolic pathway with only three enzymes that allows for biomass (acetyl-CoA) and ATP formation on early Earth.
Frontal areas of the mammalian cortex are thought to be important for cognitive control and complex behaviour. These areas have been studied mostly in humans, non-human primates and rodents. In this article, we present a quantitative characterization of response properties of a frontal auditory area responsive to sound in the brain of Carollia perspicillata, the frontal auditory field (FAF). Bats are highly vocal animals, and they constitute an important experimental model for studying the auditory system. We combined electrophysiology experiments and computational simulations to compare the response properties of auditory neurons found in the bat FAF and auditory cortex (AC) to simple sounds (pure tones). Anatomical studies have shown that the latter provides feedforward inputs to the former. Our results show that bat FAF neurons are responsive to sounds, and however, when compared to AC neurons, they presented sparser, less precise spiking and longer-lasting responses. Based on the results of an integrate-and-fire neuronal model, we suggest that slow, subthreshold, synaptic dynamics can account for the activity pattern of neurons in the FAF. These properties reflect the general function of the frontal cortex and likely result from its connections with multiple brain regions, including cortico-cortical projections from the AC to the FAF.
Clean water is fundamental to human health and ecosystem integrity. However, water quality deteriorates due to novel anthropogenic pollutants present at microgram per liter concentrations in urban water cycles (termed micropollutants). Wastewater treatment plants (WWTP) have been identified as major point sources for aquatic (micro-)pollutants. Chemical and ecotoxicological analyses have shown that conventional biological WWTPs do not fully remove micropollutants and associated toxicities, which is often because of mobile, polar and/or recalcitrant compounds and transformation products (TPs). To minimize possible environmental risks, advanced wastewater treatment (AWWT) technologies could be a promising mitigation measure. Multiple processes are therefore being developed and evaluated such as ozonation and ozonation followed by granulated activated carbon (GAC) or biological filtration. Assessing the performance of these combined AWWTs was the focus the TransRisk project. Within this project, this thesis accomplished four major goals.
Firstly, the preparation of (waste)water samples was optimised for in vitro bioassays. Acidification, filtration and solid phase extraction (SPE) were tested for their impact on environmentally relevant in vitro endocrine activities, mutagenicity, genotoxicity and cytotoxicity. Significantly different outcomes of these assays were detected comparing neutral and acidified samples. Sample filtration had a lesser impact, but in some cases retention of particle-bound compounds could have caused significant toxicity losses. Out of three SPE sorbents the Telos C18/ENV at sample pH 2.5 extracted highest toxicity, some undetected in aqueous samples. These results indicate that sample preparation needs to be optimised for specific sample matrices and bioassays to avoid false-positive or -negative detects in effect-based analyses.
Secondly, the above listed in vitro toxicities were monitored in a protected region for drinking water production in South-West Germany (2012-2015). Out of 30 sampling sites surface water and groundwater were the least polluted. Nonetheless, a few groundwater samples induced high anti-estrogenic activity that prompted further monitoring. The latter included a waterworks in which no toxicity was detected. Hospital wastewater also had elevated in vitro toxicities and hospitals are, thus, relevant intervention points for source control. The biological WWTPs were effective in removing most of the detected toxicity, and the selected bioassays proved to be pertinent tools for water quality assessment and prioritisation of pollution hotspots.
Thirdly, the in vivo bioassay ISO10872 based on Caenorhabditis elegans (C. elegans) was adapted for this thesis. Using this model, a median effect concentration (EC50) for reproductive toxicity of the polycyclic aromatic hydrocarbon β-naphthoflavone (β- NF) of 114 µg/L was computed which is slightly lower than reported in the scientific literature. β-NF induced cyp-35A3::GFP (a biomarker in transgenic animals) in a time and concentration dependent manner (≤ 21.3–24 fold above controls). β-NF spiked wastewater samples supported earlier hypotheses on particle-bound pollutants. Reproductive toxicity (96 h) and cyp-35A3 induction (24 h) of biologically treated and/or ozonated wastewater extracts and growth promoting effects of GAC/biologically filtered ozonated wastewater extracts were observed. This suggested the presence of residual bioactive/toxic chemicals not included in the targeted chemical analysis. It also highlighted the importance of integrating multiple (apical and molecular) endpoints in wastewater assessments.
Fourthly, five in vitro and the adapted C. elegans bioassay were integrated into a wastewater quality evaluation (developed within TransRisk). Out of the five AWWT options, ozonation (at 1 g O3,applied/g DOC, HRT ~ 18 min) combined with nonaerated GAC filtration was rated most effective for toxicity removal. All five AWWTs largely removed estrogenic and (anti-)androgenic activities, but not anti-estrogenic activity and mutagenicity, which even increased during ozonation. This has been observed in related studies and points towards toxic TPs. These results also emphasized the need for implementing an effective post-treatment for ozonation. The results from a parallel in vivo study with Lumbriculus variegatus and Potamopyrgus antipodarum conducted on site at the WWTP (using flow through systems) were in accordance with the C. elegans results. In this context, it is suggested to further implement C. elegans as sensitive, feasible and ecologically relevant model.
In conclusion, this thesis shows how optimised sample preparation, long-term (in vitro) environmental monitoring, sensitive and ecologically relevant (in vivo) bioassays as well as innovative evaluation concepts, are pivotal in improving the removal of micropollutants and their toxicities with AWWTs. Future research should further develop and evaluate measures at sewer systems, conventional biological, tertiary and other advanced treatment technologies, as well as sociopolitical strategies (e.g., source control or natural conservation) and restoration projects. The effect-based tools optimised in this thesis will support assessing their success.
Extremophilic prokaryotes live under harsh environmental conditions which require far-reaching cellular adaptations. The acquisition of novel genetic information via natural transformation plays an important role in bacterial adaptation. This mode of DNA transfer permits the transfer of genetic information between microorganisms of distant evolutionary lineages and even between members of different domains. This phenomenon, known as horizontal gene transfer (HGT), significantly contributes to genome plasticity over evolutionary history and is a driving force for the spread of fitness-enhancing functions including virulence genes and antibiotic resistances. In particular, HGT has played an important role for adaptation of bacteria to extreme environments. Here, we present a survey of the natural transformation systems in bacteria that live under extreme conditions: the thermophile Thermus thermophilus and two desiccation-resistant members of the genus Acinetobacter such as Acinetobacter baylyi and Acinetobacter baumannii. The latter is an opportunistic pathogen and has become a world-wide threat in health-care institutions. We highlight conserved and unique features of the DNA transporter in Thermus and Acinetobacter and present tentative models of both systems. The structure and function of both DNA transporter are described and the mechanism of DNA uptake is discussed.
he ubiquity of microplastics in aquatic ecosystems has raised concerns over their interaction with biota. However,microplastics research on freshwater species, especially mollusks, is still scarce. We, therefore, investigated the factorsaffecting microplastics ingestion in the freshwater musselDreissena polymorpha. Using polystyrene spheres (5, 10, 45,90μm), we determined the body burden of microplastics in the mussels in relation to 1) exposure and depuration time, 2)body size, 3) food abundance, and 4) microplastic concentrations.D. polymorpharapidly ingested microplastics and ex-creted most particles within 12 h. A few microplastics were retained for up to 1 wk. Smaller individuals had a higher relativebody burden of microplastics than larger individuals. The uptake of microplastics was concentration‐dependent, whereas anadditional food supply (algae) reduced it. We also compared the ingestion of microplastics byD. polymorphawith 2 otherfreshwater species (Anodonta anatina,Sinanodonta woodiana), highlighting that absolute and relative uptake depends onthe species and the size of the mussels. In addition, we determined toxicity of polystyrene fragments (≤63μm,6.4–100 000 p mL–1) and diatomite (natural particle, 100 000 p mL–1)inD. polymorphaafter 1, 3, 7, and 42 d of exposure,investigating clearance rate, energy reserves, and oxidative stress. Despite ingesting large quantities, exposure to poly-styrene fragments only affected the clearance rate ofD. polymorpha. Further, results of the microplastic and diatomiteexposure did not differ significantly. Therefore,D. polymorphais unaffected by or can compensate for polystyrene fragmenttoxicity even at concentrations above current environmental levels.Environ Toxicol Chem2021;40:2247–2260. © 2021 TheAuthors.Environmental Toxicology and Chemistrypublished by Wiley Periodicals LLC on behalf of SETAC.Keywords:Microplastics; Toxic effects; Mollusk toxicity
Aquatic ecosystems are globally contaminated with microplastics (MP). However, comparative data on MP levels in freshwater systems is still scarce. Therefore, the aim of this study is to quantify MP abundance in water and sediment of the German river Elbe using visual, spectroscopic (Fourier-transform infrared spectroscopy) and thermo analytical (pyrolysis gas chromatography mass spectrometry) methods. Samples from eleven German sites along the German part of the Elbe were collected, both in the water and sediment phase, in order to better understand MP sinks and transport mechanisms. MP concentrations differed between the water and sediment phase. Sediment concentrations (mean: 3,350,000 particles m−3, 125–5000 μm MP) were in average 600,000-fold higher than water concentrations (mean: 5.57 particles m−3, 150–5000 μm MP). The abundance varied between the sampling sites: In sediments, the abundance decreased in the course of the river while in water samples no such clear trend was observed. This may be explained by a barrage retaining sediments and limiting tidal influence in the upstream parts of the river. Particle shape differed site-specifically with one site having exceptionally high quantities of spheres, most probably due to industrial emissions of PS-DVB resin beads. Suspended MP consisted predominantly of polyethylene and polypropylene whereas sediments contained a higher diversity of polymer types. Determined MP concentrations correspond well to previous results from other European rivers. In a global context, MP levels in the Elbe relate to the lower (water) to middle section (sediment) of the global range of MP concentrations determined for rivers worldwide. This highlights that elevated MP levels are not only found in single countries or continents, but that MP pollution is an issue of global concern.
Zur Evolution der Hirnmorphologie und Anpassungen an Extremhabitate im Taxon Poecilia (Teleostei)
(2020)
Diese Dissertation befasst sich mit den Auswirkungen kontrastierender Umweltbedingungen auf die Gehirnmorphologie von neotropischen Fischen der Gattung Poecilia, welche unterschiedlichen abiotischen sowie biotischen Stressoren ausgesetzt sind. Da das Gehirn der Teleostei ein energetisch kostspieliges Organ und viel plastischer ist als z. B. bei Säugetieren, stellt sich die Frage, wie die Gehirnanatomie durch divergierende ökologische Faktoren in verschiedenen Umgebungen geformt wird, die ´extreme´, ´ressourcenbeschränkte und günstige´ Umgebungen repräsentieren. Zur Beantwortung dieser Frage wurden intraspezifische Studien an freilebenden und Laborindividuen von Poecilia-Arten durchgeführt, um die evolutionäre und ökologische Formgebung des Gehirns besser verstehen zu lernen. Im ersten Teil der Arbeit wurden Gehirnvolumina verglichen zwischen reproduktiv isolierten Populationen des neotropischen Fisches Poecilia mexicana (Ntotal = 95), die in Dunkelheit leben (Cueva Luna Azufre), in einem nahegelegenen Oberflächenhabitat (El Azufre), welcher giftigen Schwefelwasserstoff enthält und einer Kombination aus beiden Stressoren Dunkelheit und H2S (Cueva del Azufre). In einer zweiten Studie wurde auf anatomische („konvergente“) Veränderungen im Teleost-Gehirn entlang eines natürlichen Gradienten von Sulfidkonzentrationen getestet. Hierfür wurden Gehirne (Ntotal = 100) von P. mexicana verglichen, die in drei Flusssystemen im Süden Mexikos unabhängig voneinander eine erhöhte Toleranz gegenüber Schwefelwasserstoff (H2S) entwickelt haben. Dazu gehörten eine phylogenetisch alte H2S-adaptierte Form (P. sulphuraria) und zwei P. mexicana Formen, welche frühere Stufen der Anpassung an H2S darstellen. Zur Überprüfung des Einflusses anderer abiotischer und biotischer Faktoren auf die Morphologie der Gehirnregionen wurde eine weitere Studie durchgeführt. Hierbei wurden die phänotypischen Variationen der Gehirnregionen und der Körpermorphologie von Poecilia vivipara-Populationen (Ntotal = 211) aus Lagunen des Restinga de Jurubatiba Nationalpark untersucht, die sich in abiotischen Umgebungsbedingungen, insbesondere in Salzgehalt, Wassertransparenz, Phosphat und Nitrat sowie biotischen Faktoren wie Prädatorendichte unterschieden. Die erste Studie zeigte lebensraumabhängige Unterschiede bei freilebenden Fischen. Bei Fischen, die in Dunkelheit ohne H2S (LA) oder in Oberflächenhabitaten mit H2S lebten, wurden vergrößerte telenzephale Lappen, kleinere Augen und optische Tekta gefunden. Fische aus der sulfidischen Höhle (CA) zeigten zusätzlich vergrößerte Corpus cerebelli. Der Vergleich mit den Gehirnen von Labor aufgezogenen weiblichen Fischen (Ntotal = 25) zeigt eine allgemeine Verringerung der Gehirngröße sowie eine geringe Abweichung der Gehirngröße zwischen Labor aufgezogenen und freilebenden Fischen. Auch in der zweiten Studie zeigten alle in H2S-haltigen Lebensräumen lebenden Fische kleinere Augen, ein kleineres optisches Tektum und ein kleineres Gehirnvolumen, jedoch größere Corpus cerebelli und Hypothalamusvolumen als Fische aus nicht-sulfidischen Lebensräumen. Flusssystem-spezifische Effekte wurden für die telenzephalen Lappen, das gesamte Gehirn und die Augengröße festgestellt, da die Geschlechter je nach Quelle des Flusssystems unterschiedlich auf das Vorhandensein von H2S reagierten. Die dritte Studie zeigt auch, dass andere Umwelteinflüsse bemerkenswerte Verschiebungen im Gehirn und in den Gehirnregionen verursachen können. Fische, die im Süßwasser leben, zeigten eine verringerte Gesamthirngröße, telenzephale Lappen, Corpus cerebelli und Hypothalamusvolumen. Darüber hinaus zeigten Fische aus Salzwasserlagunen (hypersalin), ein verringertes Volumen des optischen Tektum, während telenzephale Lappen, Corpus cerebelli und Hypothalamusvolumen im Vergleich zu Süßwasserfischen vergrößert waren. Im Brackwasser lebende Fische wiesen im Vergleich zu Süß- und Salzwasserfischen die größten Gehirnregion-Volumen auf. Darüber hinaus zeigten die Ergebnisse über die Lagunen hinweg auch Unterschiede in der Morphologie der Kopf- und Augendurchmesser. Bei Augengröße, Kopfgröße, optischem Tektum Volumen, Hypothalamusvolumen und dem Gesamthirnvolumen wurde ein sexueller Dimorphismus beobachtet. Die dargestellten Ergebnisse verdeutlichen, dass die gefundenen Muster nahezu mit denen von H2S-Fischen identisch sind. Die ausgeprägten Unterschiede in den Hirnregionen zwischen freilebenden Fischen können als Teil der Mosaikentwicklung interpretiert werden. Die Ergebnisse der Laborpopulation zeigen jedoch eine hohe phänotypische Plastizität. Diese Studie unterstreicht damit die Bedeutung der Kombination der Untersuchung von freilebenden mit im Labor lebenden Individuen zur Beantwortung von Fragen der Gehirnentwicklung. Kleinere Augen und ein kleineres optisches Tektum, aber größere telenzephale Lappen wurden auch bei Fischen aus einem sulfidischen Oberflächenhabitat in der Nähe einer der Höhlen gefunden und sind den Ergebnissen zufolge das Resultat begrenzter Sehkraft in trüben sulfidischen Lebensräumen.
...
Ziel dieser Arbeit war es, einen genaueren Einblick in die Rolle von PaCLPXP für den Energiemetabolismus von P. anserina zu erhalten und mögliche Komponenten zu identifizieren, welche wichtig für die Langlebigkeit der PaClpP-Deletionsmutante sind. Folgende neue Erkenntnisse konnten hierbei gewonnen werden:
1. Die Substrat-Analyse durch eine Cycloheximid-Behandlung und anschließender Proteom-Analyse legte erfolgreich eine Reihe potentieller bisher nicht bekannter Substrate von PaCLPP offen. Interessanterweise waren unter den identifizierten Proteinen viele ribosomale Untereinheiten und Komponenten verschiedener Stoffwechselwege des Energiemetabolismus zu finden. Am auffälligsten unter diesen Substraten war die extreme Anreicherung eines Retikulon-ähnlichen Proteins, das einen neuen Aspekt der möglichen molekularbiologischen Rolle von PaCLPP in P. anserina andeutet.
2. Durch die Zugabe von Butyrat zum Medium, konnte erfolgreich die Autophagie sowohl im P. anserina Wildtyp als auch in der PaClpP-Deletionsmutante reduziert werden. Diese Verminderung der Autophagie sorgt bei ΔPaClpP für eine Verkürzung der Lebensspanne. Dieser Effekt ist spezifisch für die PaClpP-Deletionsmutante, während die Auswirkung von Butyrat auf den Wildtyp nur marginal ist. Dieses Ergebnis untermauert frühere Analysen dieser Deletionsmutante, welche besagen, dass die Langlebigkeit von ΔPaClpP Autophagie abhängig ist (Knuppertz und Osiewacz, 2017).
3. Die Metabolom-Analyse von ΔPaClpP im Vergleich zum Wildtyp zeigt, dass das Fehlen der PaCLPP zu Veränderungen in der Menge der Metaboliten der Glykolyse und des Citratzyklus kommt. Außerdem sind die Mengen der meisten Aminosäuren und der Nukleotide betroffen. Diese Analyse beweist, dass das Fehlen dieser mitochondrialen Protease weitreichende Folgen für die ganze Zelle hat. Durch die signifikante Verringerung von ATP und die Anreicherung von AMP in jungen ΔPaClpP-Stämmen und durch den Umstand der gesteigerten Autophagie in dieser Mutante, fiel das Augenmerk auf die AMPK. Dieses veränderte AMP/ATP-Verhältnis ist ein Indiz für eine gesteigerte AMPK-Aktivität und könnte auch den Umstand der gesteigerten Autophagie in ΔPaClpP erklären.
4. Das Gen codierend für die katalytische α-Untereinheit der AMPK (PaSnf1) konnte erfolgreich in P. anserina deletiert werden. Das Fehlen von PaSNF1 führt zu einer reduzierten Wuchsrate, eine beeinträchtige weibliche Fertilität und eine verzögerte Sporenreifung. Es konnte gezeigt werden, dass die Autophagie infolge einer PaSnf1-Deletion nicht gänzlich unterdrückt wird, PaSNF1 allerdings für die Stress-induzierte Autophagie notwendig ist. Überraschenderweise führt die Abwesenheit von PaSNF1 zu einer verlängerten Lebensspanne im Vergleich zum Wildtyp. Die meisten Effekte infolge einer PaSnf1-Deletion konnten durch die Einbringung eines FLAG::PaSNF1-Konstrukts komplementiert werden.
5. Eine gleichzeitige PaSnf1 und PaClpP-Deletion führt zu eine unerwarteten, extremen Lebenspannenverlängerung, die die Verlängerung der Lebensspanne bei der PaClpP-Deletionsmutante noch übertrifft. Interessanterweise geht dieser Phänotyp nicht mit einer erhöhten Autophagie einher. Des Weiteren konnte beobachtet werden, dass das Fehlen von PaSNF1 sowohl in ΔPaSnf1 als auch in ΔPaSnf1/ΔPaClpP zu einer veränderten Mitochondrien-Morphologie im Alter führt. Die Abwesenheit von PaSNF1 verursacht, dass die Stämme auch im Alter (20d) noch überwiegend filamentöse Mitochondrien aufweisen. Zudem zeigen die drei analysierten Deletionsstämme (ΔPaSnf1, ΔPaClpP und ΔPaSnf1/ΔPaClpP) massive Einschränkungen wenn sie auf die mitochondriale Funktion angewiesen sind.
6. Auffallend war, dass bei ΔPaSnf1, ΔPaClpP und bei ΔPaSnf1/ΔPaClpP die Stämme mit dem Paarungstyp „mat-“ langlebiger sind als die Stämme mit dem Paarungstyp „mat+“. Dieser Effekt ist bei der ΔPaSnf1/ΔPaClpP-Doppelmutante am stärksten ausgeprägt. Weitere Untersuchungen dazu ergaben, dass die Paarungstypen immer dann eine Rolle spielen, wenn die Stämme mitochondrialem Stress ausgesetzt, oder aber auf die mitochondriale Funktion angewiesen sind. Verantwortlich für diese Unterschiede sind zwei rmp1-Allele, die mit den unterschiedlichen Paarungstyp-Loci gekoppelt sind und mit dem jeweiligen Paarungstyp-Locus vererbt werden (rmp1-1 mit „mat-“; rmp1-2 mit „mat+“).
Extremophilic prokaryotes live under harsh environmental conditions which require far-reaching cellular adaptations. The acquisition of novel genetic information via natural transformation plays an important role in bacterial adaptation. This mode of DNA transfer permits the transfer of genetic information between microorganisms of distant evolutionary lineages and even between members of different domains. This phenomenon, known as horizontal gene transfer (HGT), significantly contributes to genome plasticity over evolutionary history and is a driving force for the spread of fitness-enhancing functions including virulence genes and antibiotic resistances. In particular, HGT has played an important role for adaptation of bacteria to extreme environments. Here, we present a survey of the natural transformation systems in bacteria that live under extreme conditions: the thermophile Thermus thermophilus and two desiccation-resistant members of the genus Acinetobacter such as Acinetobacter baylyi and Acinetobacter baumannii. The latter is an opportunistic pathogen and has become a world-wide threat in health-care institutions. We highlight conserved and unique features of the DNA transporter in Thermus and Acinetobacter and present tentative models of both systems. The structure and function of both DNA transporter are described and the mechanism of DNA uptake is discussed.
Seit den 1950er Jahren hat sich Plastik als unverzichtbare Ressource im menschlichen Alltag etabliert. Als negative Folge dieses Booms wird seit einigen Jahrzehnten jedoch eine zunehmende Belastung aquatischer Ökosysteme mit Plastikmüll bzw. dessen Degradationsprodukten, sogenanntes „Mikroplastik“ (MP, < 5 mm) bzw. „Nanoplastik“ (NP, < 1 µm), beobachtet. Ziel dieser Arbeit war die Untersuchung des aktuellen Vorkommens von MP in limnischen Gewässern, die Analyse der Interaktion zwischen MP und limnischen Wirbellosenarten und der daraus resultierenden Toxizität sowie eine erste Risikoabschätzung.
Das Vorkommen von Mikroplastik in limnischen Gewässern wurde exemplarisch anhand der Elbe als großes Fließgewässer in Deutschland untersucht. Durch die Auswertung von elf Probestellen entlang des Verlaufs der Mittel- und Unterelbe konnte gezeigt werden, dass die MP-Konzentrationen im Sediment (2,26x10^4 – 2,27x10^7 P m^-3) im Mittel fast 150.000-fach höher sind als in der Wasserphase (0,88–13,24 P m^-3). Sedimente sind somit eine Senke für MP. Die Zusammensetzung der Polymerarten sowie MP-Formen deuten zudem an, dass die Partikel sowohl aus diffusen wie auch aus Punktquellen (z.B. Industrieabwässer) stammen. Im globalen Vergleich können die MP-Konzentrationen in deutschen Fließgewässern z. Z. als durchschnittlich betrachtet werden. Allerdings muss insgesamt davon ausgegangen werden, dass die bisher bestimmten MP-Umweltkonzentrationen die realen Konzentrationen möglicherweise unterschätzen. So zeigte die Elbestudie, dass die Sedimentfeinfraktion < 100 µm einen bedeutenden Polymeranteil enthielt. Die meisten bisher durchgeführten Studien zur Bestimmung von MP-Partikeln in Flüssen haben Partikel < 100 µm jedoch nicht in ihrer Analyse berücksichtigt.
Die Interaktion von MP mit limnischer Biota wurde anhand der Artgruppen der Muscheln (Bivalvia), Schnecken (Gastropoda) sowie Krebstiere (Crustacea) näher untersucht. Die Intensität der Interaktion ist maßgeblich von der Aufnahme von MP durch die verschiedenen Arten abhängig. Anhand von zahlreichen Aufnahmestudien mit verschiedenen limnischen Arten, darunter den Muschelarten Dreissena polymorpha, Sinanodonta woodiana und Anodonta anatina, der Lungenschnecke Lymnaea stagnalis sowie der Amphipodenart Gammarus pulex, wurde nachgewiesen, dass die MP-Aufnahme von den Eigenschaften der exponierten Arten bzw. Individuen, den MP-Charakteristika sowie den Expositionsbedingungen abhängt. Die Experimente mit Muscheln verdeutlichten die rasche Aufnahme, aber auch Exkretion von MP-Partikeln innerhalb weniger Stunden. In allen drei Artgruppen war die Aufnahme konzentrationsabhängig mit zunehmender Aufnahme bei steigenden MP-Konzentrationen. Die Muschelexperimente zeigten jedoch auch, dass eine gleichzeitige Exposition mit anderen Partikeln (z.B. Nahrung) zu einer reduzierten Aufnahme führt. Auch die Größe der Testorganismen beeinflusste die Aufnahme: So nahmen im Fall der Muscheln und Krebse kleinere Individuen (bzw. im Fall der Muscheln auch Arten) relativ pro Körpermasse mehr MP-Partikel auf als größere Individuen bzw. Arten. Für alle untersuchten Arten wurde darüber hinaus gezeigt, dass die MP-Größe relevanten Einfluss auf die Menge an aufgenommenen Partikeln hat.
Ein Vergleich zwischen den Artgruppen zeigte, dass Muscheln als filtrierende Organismen in den Laboruntersuchungen bei gleicher Expositionskonzentration mehr MP aufnahmen als Krebse (Zerkleinerer) und Schnecken (Weidegänger). Im Gegensatz zu Muscheln nutzen Krebstiere und Schnecken allerdings die Grenzschicht zwischen Wasser- und Sedimentphase als Suchraum für ihre Nahrung und sind in der Umwelt (auf Grund des höheren MP-Vorkommens in Sedimenten) somit möglicherweise gegenüber höheren MP-Konzentrationen exponiert als Muscheln. Die Extrapolation der gewonnenen Labordaten sowie der Vergleich mit publizierten Umweltdaten legen allerdings nahe, dass das MP-Vorkommen in Individuen aller drei Artgruppen bisher auf einige wenige MP-Partikel begrenzt ist. Ausgeprägte Unterschiede zwischen den Artgruppen sind bisher nicht erkennbar.
MP-Toxizitätsstudien mit G. pulex, L. stagnalis sowie D. polymorpha konnten trotz der Berücksichtigung einer Vielzahl an Endpunkten (Mortalität, Reproduktion, Nahrungsaufnahme, oxidativer Stress, Energiereserven, Immunzellaktivität) und trotz des Einsatzes zum Teil sehr hoher MP-Konzentrationen weit oberhalb aktueller Umweltkonzentrationen nur sehr wenige MP-induzierte Effekte nachweisen, darunter eine Steigerung der Filtrationsaktivität (D. polymorpha) bzw. Veränderung der Immunfunktion von Hämolymphzellen (L. stagnalis).
Zur weiteren Risikoabschätzung wurden diese Studienergebnisse mit publizierten Daten für marine und limnische Muschel- und Krebsarten in Artenempfindlichkeitsverteilungen (Species Sensitivity Distributions, SSD) zusammengeführt und jeweils eine SSD für Muscheln und Krebstiere erstellt. Die Erstellung einer SSD nur für limnische Arten ist zum jetzigen Zeitpunkt auf Grund der geringen Datenlage noch nicht möglich.
...
Human GLUTs represent a family of specialized transporters that facilitate the diffusion of hexoses through membranes along a concentration gradient. The 14 isoforms share high sequence identity but differ in substrate specificity and affinity, and tissue distribution. According to their structure similarity, GLUTs are divided into three classes, with class 1 comprising the most intensively studied isoforms GLUTs1 4. An abnormal function of different GLUT members has been related to the pathogenesis of various diseases, including cancer and diabetes. Hence, GLUTs are the subject of intensive research, and efforts concentrate on identifying GLUT-selective ligands for putative medical purposes and their application in studies aiming to further unravel the metabolic roles of these transporters.
The hexose transporter deficient (hxt0) yeast strain EBY.VW4000 is devoid of all its endogenous hexose transporters and unable to grow on glucose or related hexoses. This strain has proven to be a valuable platform to investigate heterologous transporters due to its easy handling, increased robustness, and versatile applications. However, the functional expression of GLUTs in yeast requires certain modifications. Single point mutations of GLUT1 and GLUT5 led to their functional expression in EBY.VW4000, whereas the native GLUT1 was actively expressed in EBY.S7, a hxt0 strain carrying the fgy1 mutation that putatively reduces the phosphatidylinositol-4-phosphate (PI4P) content in the plasma membrane. GLUT4 was only actively expressed in the hxt0 strain SDY.022, which also contains the fgy1 mutation and in which ERG4 is additionally deleted. Erg4 is one of the late enzymes in the ergosterol pathway, and therefore SDY.022 probably has an altered sterol composition in its membrane.
The goal of this thesis was to actively express GLUT2 and GLUT3 in a hxt0 yeast strain, providing a convenient system for their ligand screening. A PCR-derived amino acid exchange in the sequence of GLUT3 enabled its functional expression in EBY.VW4000 and the unmodified GLUT3 protein was active in EBY.S7. Functional expression of GLUT2 was achieved by rational design. The extracellular loop between the transmembrane regions 1 and 2 is significantly larger in GLUT2 than in other class 1 GLUTs. By truncating this loop by 34 amino acids and exchanging an alanine for a serine, a GLUT3-like loop was implemented. The resulting construct GLUT2∆loopS was functional in EBY.S7. With an additional point mutation in the transmembrane region 11, GLUT2∆loopS_Q455R was also actively expressed in EBY.VW4000. Inhibition studies with the known GLUT inhibitors phloretin and quercetin showed a reduced transporter activity for GLUT2 and GLUT3 in uptake assays and growth tests when inhibitors were present, demonstrating that both systems are amenable for ligand screening experiments.
The newly established GLUT2 yeast system was then used to screen a library of compounds pre-selected by in silico screening. Thereby, eleven identified GLUT2 inhibitors exhibited strong potencies with IC50 values ranging from 0.61 to 19.3 µM. By employing the other yeast systems, these compounds were tested for their effects on GLUT1, and GLUTs3-5, revealing that nine of the identified ligands were GLUT2-selective. In contrast, one was a pan-class 1 inhibitor (inhibiting GLUTs1-4), and one affected GLUT2 and GLUT5, the two fructose transporting isoforms. These compounds will serve as useful tools for investigations on the role of GLUT2 in metabolic diseases and might even evolve into pharmaceutical agents targeting GLUT2-associated diseases.
Due to the beneficial effect of the putatively changed sterol composition in SDY.022 (by ERG4 deletion) on the functional expression of GLUT4, it was hypothesized that the presence of the human sterol cholesterol, or cholesterol-like sterols, might have a beneficial effect on GLUT expression, too. Thus, it was attempted to generate hxt0 strains that synthesize these sterols by genetic modifications targeting the ergosterol pathway. In the scope of these experiments, several strains with different sterol compositions were generated. Drop tests on glucose medium with the different strains expressing GLUT1 or GLUT4 revealed that the deletion of ERG6 is clearly advantageous for a functional expression of GLUT1 (but not GLUT4). This indicates that the methyl group at the ergosterol side chain (introduced by Erg6 and reduced by Erg4) negatively influences GLUT1 activity. However, this effect on GLUT1 activity was less pronounced than the putative altered PI4P content in EBY.S7.
Additionally, in this thesis, a new tool to measure glucose transport rates of transporters expressed in the hxt0 yeast system was developed to facilitate their kinetic characterization. For this, the pH-sensitive GFP variant pHluorin was employed as a biosensor for the cytosolic pH (pHcyt) by measuring the ratio (R390/470) of emission intensities at 512 nm from two different excitation wavelengths (390 and 470 nm). Sugar-starved cells exhibit a slightly acidic pHcyt because ATP production is depleted, reducing the activity of ATP-dependent proton pumps.
...
The development of photosynthesis was a highlight in the progression of bacteria. In addition to the photosystems with their structural proteins, the photosynthesis apparatus consists of different cofactors including essential carotenoids. Thus, the evolution of the carotenoid pathways in relation to the functionality of the resulting structures in photosynthesis is the focus of this review. Analysis of carotenoid pathway genes indicates early evolutionary roots in prokaryotes. The pathway complexity leading to a multitude of structures is a result of gene acquisition, including their functional modifications, emergence of novel genes and gene exchange between species. Along with the progression of photosynthesis, carotenoid pathways coevolved with photosynthesis according to their advancing functionality. Cyanobacteria, with their oxygenic photosynthesis, became a landmark for evolutionary events including carotenogenesis. Concurrent with endosymbiosis, the cyanobacterial carotenoid pathways were inherited into algal plastids. In the lineage leading to Chlorophyta and plants, carotenoids evolved to their prominent role in protection and regulation of light energy input as constituents of a highly efficient light-harvesting complex.
Rolf van Dick und Holger Horz haben in ihrem UniReport-Essay »Bestmögliche Bildung und Ausbildung für alle« gefordert und die Kritik von Hans Peter Klein und Julian Nida-Rümelin am aktuellen Akademisierungswahn scharf zurückgewiesen. Nida-Rümelin und Klein antworten nun im Gespräch gemeinsam auf die zentralen Thesen von van Dick und Horz.
Anfang Februar veröffentlichte die Pressestelle der Goethe-Universität die Meldung „In der Stadt bauen Kaninchen dichter: Große Bauten für die ländliche Großfamilie, kleine Bauten für das städtische Pärchen.“ Die Meldung beruhte auf einem Fachartikel der Arbeitsgruppe Ökologie und Evolution im Journal of Zoology. Rasend schnell verbreitete sich die Meldung in den Medien – wir haben Madlen Ziege, Doktorandin in der Arbeitsgruppe Ökologie und Evolution einmal danach befragt, wie die Forschung mit populären oder besser: popularisierten Meldungen umgeht.