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Xenorhabdus and Photorhabdus are bacterial genera that live in symbiosis with entomopathogenic nematodes of the genera Steinernema and Heterorhabditis, respectively. These nematodes infect insect larvae through the trachea and then enter the hemocoel. Once inside the hemocoel, the nematodes release the bacteria through their intestine. Thereafter, the bacteria become active and kill the larvae within 48 h. During this process, the immune system of the insect host is compromised by molecules produced and secreted by the bacteria. This illustrates that the bacteria possess not only a large arsenal of biological weaponry such as antibiotics and fungicides but also lipases, proteases, etc. Therefore, they are not only able to kill the insect but also protect the cadaver from other food competitors.
During the past decades, a large number of natural products have been identified from Xenorhabdus and Photorhabdus. However, the targets and functions for many of these biological molecules are still unknown. Therefore, the goal of the doctoral thesis is to elucidate the modes of action of these natural products from Xenorhabdus and Photorhabdus with the main focus on non-ribosomal peptides (NRPs). The work can be divided into two parts. Initially, it starts with the synthesis of natural compounds and various chemically modified derivatives. Besides that, a number of peptides were synthesized for other projects to either verify their structures or quantify the amount produced by the bacteria. Then, secondary analysis methods are applied and provide additional insight into the modes of action of these compounds.
During the thesis, I carried out peptide synthesis either manually or with an automatic synthesizer system from Biotage. Here, the Fmoc-protecting group strategy was preferred in most cases. Natural products, such as silathride, xenoautoxin, phenylethylamide, tryptamide, rhabdopeptide, 3-hydroxyoctanoic acid, and PAX, were produced during this process. Furthermore, new peptide derivatives derived from synthetic NRPS approaches using the XU concept or SYNZIP were generated as standards.
Most of these natural compounds were experimentally verified by MIC tests (broth microdilution, plate diffusion) to be biologically active. For example, silathride, phenylethylamide, and tryptamide showed quorum quenching effects when tested against Chromobacterium violaceum. Initial results from collaborators (PD Dr. Nadja Hellmann/Mainz) showed that tryptamide and phenylethylamide interact with membrane or membrane proteins.
(R)-3-hydroxyoctanoic acid was synthesized to verify the molecule structure of phototemtide A, a cyclic lipopeptide with antiprotozoal activity. The rhabdopeptides are another class, which showed remarkable antiprotozoal effects. However, their mode of action was unknown. These compounds are relatively short peptide sequences, which contain hydrophobic residues, such as valine, leucine, or phenylalanine. Moreover, they possess N methylation, resulting in a rod-shaped highly hydrophobic structure. In this work, I synthesized eight new derivatives of rhabdopeptides for photo-affinity labeling (PAL). These molecules should react covalently under UV-light irradiation with the biological target of the peptides. In addition, these derivatives can be enriched in a pull-down assay using click chemistry. Afterward, analytic methods such as mass detection (proteome analysis) can be applied to elucidate the protein targets.
The PAX peptides derivatives are well-known to have anti-microbial activities and believed to be secreted into the environment by the producing bacteria. However, I found that the majority of these peptides are located in the cell pellet fraction and not in the supernatant. This has been shown through quantification using HPLC MS. New PAX derivatives were synthesized, which carry a moiety suitable for covalent modification using click-chemistry, therefore being functionalizable with a fluorescence dye. In collaboration with Dr. Christoph Spahn (Prof. Dr. Mike Heilemann group), we used confocal, as well as super-resolution microscopy, in particular, single-molecule localization microscopy (SMLM) to investigate the spatial distribution of clickable PAX molecules and revealed that they localize at the bacterial membrane. Furthermore, bioactivity assays revealed that the promotor exchanged X. doucetiae PAX mutants, which do not produce PAX molecules without chemical induction (hereby termed as pax-), were more susceptible to several insect AMPs tested. Based on these findings, a new dual mechanism of action for PAX was proposed. Besides the previously shown antimicrobial activity, these molecules with a positive net charge of +5 (pH = 7) would bind to the negatively charged bacterial surface. Hereby, the surface charge (typically negative) would be inversed resulting in a protective effect for Xenorhabdus against other positively charged AMPs. Furthermore, PAX was investigated as AMP against E. coli to study its antimicrobial mechanism of action. Here, the results show that PAX can disrupt the E. coli membrane at higher concentrations (> 30 µg/ml), enter the cytosol, and lead to reorganization of subcellular structures, such as the nucleoid during this process.
Another aspect of secondary analysis is the application of proteomic analysis. Therefore, I induced X. nematophila, X. szentirmaii, and P. luminescens with insect lysate. These samples were analyzed using HPLC-MS/MS (Q Exactive) together with a database approach (Maxquant/Andromeda). The results showed that in all strains the lipid degradation and the glyoxylate pathway were induced. This is in line with the given insect lysate diet, which mostly contained lipids. Moreover, several interesting unknown peptides and proteins were also upregulated and might get into the focus of future research.
The genus Giraffa likely evolved around seven million years ago in Indo-Asia and spread over the Arabian-African land bridge into Eastern Africa. The oldest fossil of the African lineage was found in Kenya and dated to 7-5.4 Mya. Beside modern giraffe, four additional African species have likely existed (G. gracilis, G. pygmaea, G. stillei, and G. jumae). Based on their morphological similarities, G. gracilis is often considered to be the closest relative of the modern giraffe. Nevertheless, the phylogeny within the genus Giraffa is largely unresolved.
Modern giraffe (Giraffa sp.) have been neglected by the scientific community for a long time and still very little is known about their biology. Traditionally, present-day giraffe have been considered a single species (G. camelopardalis) which is divided into six to eleven subspecies, with nine subspecies being the most accepted classification. This classification was based on morphological differences and geographic ranges. However, recent genetic analyses found hidden diversity within Giraffa and proposed four genetically distinct giraffe species (G. camelopardalis, G. reticulata, G. tippelskirchi, G. giraffa) with presumably little gene flow among them.
Gene flow on a population level is the exchange of genetic information among populations facilitated by the migration of individuals between populations. Additionally, it is an important criterion to delineate species, because many species concepts, especially the Biological Species Concept, rely on the concept of reproductive isolation. Yet, new genetic methods are identifying an increasing number of species that show signs of introgressive hybridization or gene flow among them. Therefore, strict reproductive isolation cannot always be applied to delineate species, especially in young, probably still diverging, species such as giraffe.
Therefore, giraffe are ideal study organisms to investigate the level of gene flow in recently diverged species with adjacent or potentially overlapping ranges. Furthermore, their recent classification as “Vulnerable” by the IUCN and their unreliable distribution maps require the genetic evaluation of their population structure, distribution and conservation status.
In Publication 1 (Winter et al. (2018a), Ecological Genetics and Genomics, 7–8, 1–5), I studied the distribution and matrilineal population structure of Angolan giraffe (G. giraffa angolensis) using sequences from the cytochrome b gene (1,140 bp) and the mitochondrial control region for individuals from across their known range and beyond, and additionally including individuals from all known giraffe species and subspecies. The reconstruction of a phylogenetic tree and a mitochondrial haplotype network allowed to identify the most easterly known natural population of Angolan giraffe, a population that was previously assigned to their sister-subspecies South African giraffe (G. giraffa giraffa), indicating the limit of classification by morphology and geography. Furthermore, the analyses show that Namibia’s iconic desert-dwelling giraffe population is genetically distinct, even from the nearest population at Etosha National Park, suggesting very limited, if any, natural exchange of matrilines. Yet, no geographic barriers are known for this region that would prevent genetic exchange. Therefore, the two populations are likely on different evolutionary trajectories. Limited individuals with an Etosha haplotype further suggest that translocation of Etosha giraffe into the desert population had only a minor impact on the local population. Two separate haplogroups within Etosha National Park suggest an “out of Etosha” radiation of Angolan giraffe to the East followed by a later back-migration.
In Publication 2 (Winter et al. (2018b), Ecology and Evolution, 8(20), 10156–10165), I investigated the genetic population structure of giraffe across their range (n = 137) with focus on the amount of gene flow among the proposed giraffe species with a 3-fold increased set of nuclear introns (n = 21). Limited gene flow of less than one effective migrant per generation, even between the closely related northern (G. camelopardalis) and reticulated giraffe (G. reticulata) further supports the existence of four giraffe species by a different methodology, gene flow. This is significant because most species concepts build on reproductive isolation. Furthermore, this result is corroborated by four distinct major clades in a phylogenetic tree analysis, and distinct clusters in Principal Component Analysis and STRUCTURE analysis. All these analyses suggest a low level of genetic exchange among the four giraffe species and, therefore, a high degree of reproductive isolation in accordance with the Biological Species Concept (BSC). In Addition, only a single individual in 137 was identified as being potential of natural hybrid origin, which promotes the four-species concept further. ...
The Mediterranean realm, comprising the Mediterranean and Macaronesian regions, has long been recognized as one of the world’s biodiversity hotspots, owing to its remarkable species richness and endemism. Several hypotheses on biotic and abiotic drivers of species diversification in the region have been often proposed but rarely tested in an explicit phylogenetic framework. Here, we investigate the impact of both species-intrinsic and -extrinsic factors on diversification in the species-rich, cosmopolitan Limonium, an angiosperm genus with center of diversity in the Mediterranean. First, we infer and time-calibrate the largest Limonium phylogeny to date. We then estimate ancestral ranges and diversification dynamics at both global and regional scales. At the global scale, we test whether the identified shifts in diversification rates are linked to specific geological and/or climatic events in the Mediterranean area and/or asexual reproduction (apomixis). Our results support a late Paleogene origin in the proto-Mediterranean area for Limonium, followed by extensive in situ diversification in the Mediterranean region during the late Miocene, Pliocene, and Pleistocene. We found significant increases of diversification rates in the “Mediterranean lineage” associated with the Messinian Salinity Crisis, onset of Mediterranean climate, Plio-Pleistocene sea-level fluctuations, and apomixis. Additionally, the Euro-Mediterranean area acted as the major source of species dispersals to the surrounding areas. At the regional scale, we infer the biogeographic origins of insular endemics in the oceanic archipelagos of Macaronesia, and test whether woodiness in the Canarian Nobiles clade is a derived trait linked to insular life and a biotic driver of diversification. We find that Limonium species diversity on the Canary Islands and Cape Verde archipelagos is the product of multiple colonization events followed by in situ diversification, and that woodiness of the Canarian endemics is indeed a derived trait but is not associated with a significant shift to higher diversification rates. Our study expands knowledge on how the interaction between abiotic and biotic drivers shape the uneven distribution of species diversity across taxonomic and geographical scales.
To support future research based on natural sciences collection data, DiSSCo (Distributed System of Scientific Collections) – the European Research Infrastructure for Natural Science Collections – adopts Digital Object Architecture as the basis for its planned data infrastructure. Using the outputs of one Research Data Alliance (RDA) interest group (IG) and five working groups (WGs) we show how RDA recommendations and supporting documents have been applied to the various stages of the DiSSCo data lifecycle.
Currently one of the biggest challenges for society is to combat global warming. A solution to this global threat is the implementation of a CO2-based bioeconomy and a H2-based bioenergy economy. Anaerobic lithotrophic bacteria such as the acetogenic bacteria are key players in the global carbon and H2 cycle and thus prime candidates as driving forces in a H2- and CO2-bioeconomy. Naturally, they convert two molecules of CO2 via the Wood-Ljungdahl pathway (WLP) to one molecule of acetyl-CoA which can be converted to different C2-products (acetate or ethanol) or elongated to C4 (butyrate) or C5-products (caproate). Since there is no net ATP generation from acetate formation, an electron-transport phosphorylation (ETP) module is hooked up to the WLP. ETP provides the cell with additional ATP, but the ATP gain is very low, only a fraction of an ATP per mol of acetate. Since acetogens live at the thermodynamic edge of life, metabolic engineering to obtain high-value products is currently limited by the low energy status of the cells that allows for the production of only a few compounds with rather low specificity. To set the stage for acetogens as production platforms for a wide range of bioproducts from CO2, the energetic barriers have to be overcome. This review summarizes the pathway, the energetics of the pathway and describes ways to overcome energetic barriers in acetogenic C1 conversion.
Cell fate clusters in ICM organoids arise from cell fate heredity and division: a modelling approach
(2020)
During the mammalian preimplantation phase, cells undergo two subsequent cell fate decisions. During the first decision, the trophectoderm and the inner cell mass are formed. Subsequently, the inner cell mass segregates into the epiblast and the primitive endoderm. Inner cell mass organoids represent an experimental model system, mimicking the second cell fate decision. It has been shown that cells of the same fate tend to cluster stronger than expected for random cell fate decisions. Three major processes are hypothesised to contribute to the cell fate arrangements: (1) chemical signalling; (2) cell sorting; and (3) cell proliferation. In order to quantify the influence of cell proliferation on the observed cell lineage type clustering, we developed an agent-based model accounting for mechanical cell–cell interaction, i.e. adhesion and repulsion, cell division, stochastic cell fate decision and cell fate heredity. The model supports the hypothesis that initial cell fate acquisition is a stochastically driven process, taking place in the early development of inner cell mass organoids. Further, we show that the observed neighbourhood structures can emerge solely due to cell fate heredity during cell division.
Reprogramming of tomato leaf metabolome by the activity of heat stress transcription factor HsfB1
(2020)
Plants respond to high temperatures with global changes of the transcriptome, proteome, and metabolome. Heat stress transcription factors (Hsfs) are the core regulators of transcriptome responses as they control the reprogramming of expression of hundreds of genes. The thermotolerance-related function of Hsfs is mainly based on the regulation of many heat shock proteins (HSPs). Instead, the Hsf-dependent reprogramming of metabolic pathways and their contribution to thermotolerance are not well described. In tomato (Solanum lycopersicum), manipulation of HsfB1, either by suppression or overexpression (OE) leads to enhanced thermotolerance and coincides with distinct profile of metabolic routes based on a metabolome profiling of wild-type (WT) and HsfB1 transgenic plants. Leaves of HsfB1 knock-down plants show an accumulation of metabolites with a positive effect on thermotolerance such as the sugars sucrose and glucose and the polyamine putrescine. OE of HsfB1 leads to the accumulation of products of the phenylpropanoid and flavonoid pathways, including several caffeoyl quinic acid isomers. The latter is due to the enhanced transcription of genes coding key enzymes in both pathways, in some cases in both non-stressed and stressed plants. Our results show that beyond the control of the expression of Hsfs and HSPs, HsfB1 has a wider activity range by regulating important metabolic pathways providing an important link between stress response and physiological tomato development.
Mutualistic interactions between plants and animals can affect both plant and animal communities, and potentially leave imprints on plant demography. Yet, no study has simultaneously tested how trait variation in plant resources shapes the diversity of animal consumers, and how these interactions influence seedling recruitment. Here, we analyzed whether (i) phylogenetic diversity and functional diversity of fruiting plants were correlated with the corresponding diversity of frugivorous birds, and (ii) whether phylogenetic diversity and functional identity of plant and bird communities influenced the corresponding diversity and identity of seedling communities. We recorded mutualistic interactions between fleshy-fruited plants and frugivorous birds and seedling communities in 10 plots along an elevational gradient in the Colombian Andes. We built a phylogeny for plants/seedlings and birds and measured relevant morphological plant and bird traits that influence plant-bird interactions and seedling recruitment. We found that phylogenetic diversity and functional diversity of frugivorous birds were positively associated with the corresponding diversities of fruiting plants, consistent with a bottom-up effect of plants on birds. Moreover, the phylogenetic diversity of seedlings was related to the phylogenetic diversity of plants, but was unrelated to the phylogenetic diversity of frugivorous birds, suggesting that top-down effects of animals on seedlings were weak. Mean seed mass of seedling communities was positively associated with the mean fruit mass of plants, but was not associated with the mean avian body mass in the frugivore communities. Our study shows that variation in the traits of fleshy-fruited plants was associated with the diversity of frugivorous birds and affected the future trajectory of seedling recruitment, whereas the morphological traits of animal seed dispersers were unrelated to the phylogenetic and functional structure of seedling communities. These findings suggest that bottom-up effects are more important than top-down effects for seed-dispersal interactions and seedling recruitment in diverse tropical communities.
In biological systems (cell culture media, cells, body fluids), drugs/toxicants are usually not freely dissolved but partially bound to biomolecules; only a fraction of the chemical is free/unbound (fu). To predict pharmacological effects and toxicity, it is important that the fu of the drug is known. As the differences between free and nominal concentrations are determined by test system parameters (e.g., the protein and lipid content, and the type of surface material), comparison of nominal concentrations between two different new approach methods (NAM) may lead to faulty conclusions. The same problem exists when in vitro concentrations are compared to those in human subjects. Therefore, the respective fu of a chemical in a test system needs to be determined for in vitro-to-in vivo extrapolations (IVIVE). Besides direct measurements, prediction models can help to obtain fu. Here we describe a simplified approach to approximate fu and provide background information on the underlying assumptions. Comparative predictions and measurements of fu of various drugs are shown to exemplify the approach. Basic input data, like protein and lipid concentrations, are also provided. Beyond such test systems data, the only required chemical-specific inputs are the lipophilicity of the candidate drug and its ionization state, as determined by the dissociation constants of its acidic or basic groups. This overview is intended to be used by any lab scientist without specific toxicokinetics training to obtain an estimate of fu in a given cell culture medium.