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Depending on the biochemical and biotechnical approach, the aim of this work was to understand the mechanism of protein-glucan interactions in regulation and control of starch degradation. Although starch degradation starts with the phosphorylation process, the mechanisms by which this process is controlling and adjusting starch degradation are not yet fully understood. Phosphorylation is a major process performed by the two dikinases enzymes α-glucan, water dikinase (GWD) and phosphoglucan water dikinase (PWD). GWD and PWD enzymes phosphorylate the starch granule surface; thereby stimulate starch degradation by hydrolytic enzymes. Despite these important roles for GWD and PWD, so far the biochemical processes by which these enzymes are able to regulate and adjust the rate of phosphate incorporation into starch during the degradation process haven‘t been understood. Recently, some proteins were found associated with the starch granule. Two of these proteins are named Early Starvation Protein 1 (ESV1) and its homologue Like-Early Starvation Protein 1 (LESV). It was supposed that both are involved in the control of starch degradation, but their function has not been clearly known until now. To understand how ESV1 and LESV-glucan interactions are regulated and affect the starch breakdown, it was analyzed the influence of ESV1 and LESV proteins on the phosphorylating enzyme GWD and PWD and hydrolysing enzymes ISA, BAM, and AMY. However, the analysis determined the location of LESV and ESV1 in the chloroplast stroma of Arabidopsis. Mass spectrometry data predicted ESV1and LESV proteins as a product of the At1g42430 and At3g55760 genes with a predicted mass of ~50 kDa and ~66 kDa, respectively. The ChloroP program predicted that ESV1 lacks the chloroplast transit peptide, but it predicted the first 56 amino acids N-terminal region as a chloroplast transit peptide for LESV. Usually, the transit peptide is processed during transport of the proteins into plastids. Given that this processing is critical, two forms of each ESV1 and LESV were generated and purified, a full-length form and a truncated form that lacks the transit peptide, namely, (ESV1and tESV1) and (LESV and tLESV), respectively. Both protein forms were included in the analysis assays, but only slight differences in glucan binding and protein action between ESV1 and tESV1 were observed, while no differences in the glucan binding and effect on the GWD and PWD action were observed between LESV and tLESV. The results revealed that the presence of the N-terminal is not massively altering the action of ESV1 or LESV. Therefore, it was only used the ESV1 and tLESV forms data to explain the function of both proteins.
However, the analysis of the results revealed that LESV and ESV1 proteins bind strongly at the starch granule surface. Furthermore, not all of both proteins were released after their incubation with starches after washing the granules with 2% [w/v] SDS indicates to their binding to the deeper layers of the granule surface. Supporting of this finding comes after the binding of both proteins to starches after removing the free glucans chains from the surface by the action of ISA and BAM. Although both proteins are capable of binding to the starch structure, only LESV showed binding to amylose, while in ESV1, binding was not observed. The alteration of glucan structures at the starch granule surface is essential for the incorporation of phosphate into starch granule while the phosphorylation of starch by GWD and PWD increased after removing the free glucan chains by ISA. Furthermore, PWD showed the possibility of starch phosphorylation without prephosphorylation by GWD.
Biochemical studies on protein-glucan interactions between LESV or ESV1 with different types of starch showed a potentially important mechanism of regulating and adjusting the phosphorylation process while the binding of LESV and ESV1 leads to altering the glucan structures of starches, hence, render the effect of the action of dikinases enzymes (GWD and PWD) more able to control the rate of starch degradation. Despite the presence of ESV1 which revealed an antagonistic effect on the PWD action as the PWD action was decreased without prephosphorylation by GWD and increased after prephosphorylation by GWD (Chapter 4), PWD showed a significant reduction in its action with or without prephosphorylation by GWD in the presence of ESV1 whether separately or together with LESV (Chapter 5). However, the presence of LESV and ESV1 together revealed the same effect compared to the effect of each one alone on the phosphorylation process, therefore it is difficult to distinguish the specific function between them. However, non-interactions were detected between LESV and ESV1 or between each of them with GWD and PWD or between GWD and PWD indicating the independent work for these proteins. It was also observed that the alteration of the starch structure by LESV and ESV1 plays a role in adjusting starch degradation rates not only by affecting the dikinases but also by affecting some of the hydrolysing enzymes since it was found that the presence of LESV and ESV1leads to the reduction of the action of BAM, but does not abolish it.
Bacteria are one of the most widespread kinds of microorganisms that play essential roles in many biological and ecological processes. Bacteria live either as independent individuals or in organized communities. At the level of single cells, interactions between bacteria, their neighbors, and the surrounding physical and chemical environment are the foundations of microbial processes. Modern microscopy imaging techniques provide attractive and promising means to study the impact of these interactions on the dynamics of bacteria. The aim of this dissertation is to deepen our understanding four fundamental bacterial processes – single-cell motility, chemotaxis, bacterial interactions with environmental constraints, and their communication with neighbors – through a live cell imaging technique. By exploring these processes, we expanded our knowledge on so far unexplained mechanisms of bacterial interactions.
Firstly, we studied the motility of the soil bacterium Pseudomonas putida (P. putida), which swims through flagella propulsion, and has a complex, multi-mode swimming tactic. It was recently reported that P. putida exhibits several distinct swimming modes – the flagella can push and pull the cell body or wrap around it. Using a new combined phase-contrast and fluorescence imaging set-up, the swimming mode (push, pull, or wrapped) of each run phase was automatically recorded, which provided the full swimming statistics of the multi-mode swimmer. Furthermore, the investigation of cell interactions with a solid boundary illustrated an asymmetry for the different swimming modes; in contrast to the push and pull modes, the curvature of runs in wrapped mode was not affected by the solid boundary. This finding suggested that having a multi-mode swimming strategy may provide further versatility to react to environmental constraints.
Then we determined how P. putida navigates toward chemoattractants, i.e. its chemotaxis strategies. We found that individual run modes show distinct chemotactic responses in nutrition gradients. In particular, P. putida cells exhibited an asymmetry in their chemotactic responsiveness; the wrapped mode (slow swimming mode) was affected by the chemoattractant, whereas the push mode (fast swimming mode) was not. These results can be seen as a starting point to understand more complex chemotaxis strategies of multi-mode swimmers going beyond the well-known paradigm of Escherichia coli, that exhibits only one swimming mode.
Finally we considered the cell dynamics in a dense population. Besides physical interactions with their neighbors, cells communicate their activities and orchestrate their population behaviors via quorum-sensing. Molecules that are secreted to the surrounding by the bacterial cells, act as signals and regulate the cell population behaviour. We studied P. putida’s motility in a dense population by exposing the cells to environments with different concentrations of chemical signals. We found that higher amounts of chemical signals in the surrounding influenced the single-cell behaviourr, suggesting that cell-cell communications may also affect the flagellar dynamics.
In summary, this dissertation studies the dynamics of a bacterium with a multi-mode swimming tactic and how it is affected by the surrounding environment using microscopy imaging. The detailed description of the bacterial motility in fundamental bacterial processes can provide new insights into the ecology of microorganisms.
Under an ecological speciation scenario, the radiation of African weakly electric fish (genus Campylomormyrus) is caused by an adaptation to different food sources, associated with diversification of the electric organ discharge (EOD). This study experimentally investigates a phenotype-environment correlation to further support this scenario. Our behavioural experiments showed that three sympatric Campylomormyrus species with significantly divergent snout morphology differentially react to variation in substrate structure. While the short snout species (C. tamandua) exhibits preference to sandy substrate, the long snout species (C. rhynchophorus) significantly prefers a stone substrate for feeding. A third species with intermediate snout size (C. compressirostris) does not exhibit any substrate preference. This preference is matched with the observation that long-snouted specimens probe deeper into the stone substrate, presumably enabling them to reach prey more distant to the substrate surface. These findings suggest that the diverse feeding apparatus in the genus Campylomormyrus may have evolved in adaptation to specific microhabitats, i.e., substrate structures where these fish forage. Whether the parallel divergence in EOD is functionally related to this adaptation or solely serves as a prezygotic isolation mechanism remains to be elucidated.
Potato is the 4th most important food crop in the world. Especially in tropical and sub-tropical potato production, drought is a yield limiting factor. Potato is sensitive to water stress. Potato yield loss under water stress could be reduced by using tolerant varieties and adjusted agronomic practices. Direct selection for yield under water-stressed conditions requires long selection cycles. Thus, identification of markers for marker-assisted selection may speed up breeding. The objective of this thesis is to identify morphological markers for drought tolerance by continuously monitoring plant growth and canopy temperature with an automatic phenotyping system.
The phenotyping was performed in drought-stress experiments that were conducted in population A with 64 genotypes and population B with 21 genotypes in the screenhouse in 2015 and 2016 (population A) and in 2017 and 2018 (population B). Drought tolerance was quantified as deviation of the relative tuber starch yield from the experimental median (DRYM) and parent median (DRYMp). Relative tuber starch yield is starch yield under drought stress relative to the average starch yield of the respective cultivar under control conditions in the same experiment. The specific DRYM value was calculated based on the yield data of the same experiment or the global DRYM that was calculated from yield data derived from data combined over yeas of respective population or across multiple experiments including VALDIS and TROST experiments (2011-2016).
Analysis of variance found a significant effect of genotype on DRYM indicating that the tolerance variation required for marker identification was given in both populations.
Canopy growth was monitored continuously six times a day over five to ten weeks by a laser scanner system and yielded information on leaf area, plant height and leaf angle for population A and additionally on leaf inclination and light penetration depth for population B. Canopy temperature was measured 48 times a day over six to seven weeks by infrared thermometry in population B. From the continuous IRT surface temperature data set, the canopy temperature for each plant was selected by matching the time stamp of the IRT data with laser scanner data.
Mean, maximum, range and growth rate values were calculated from continuous laser scanner measurements of respective canopy parameters. Among the canopy parameters, the maximum and mean values in long-term stress conditions showed better correlation with DRYM values calculated in the same experiment than growth rate and diurnal range values. Therefore, drought tolerance index prediction was done from maximum and mean values of canopy parameters.
The tolerance index in specific experiment condition was linearly predicted by simple regression model from different single canopy parameters under long-term stress condition in population A (2016) and population B (2017 and 2018). Among the canopy parameters maximum light penetration depth (2017), mean leaf angle (2017, 2018, and 2016), mean leaf inclination or mean canopy temperature depression (2017 and 2018), maximum plant height (2017) were selected as tolerance predictors. However, no single parameters were sufficient to predict DRYM. Therefore, several independent parameters were integrated in a multiple regression model.
In multiple regression model, specific experiment DRYM values in population A was predicted from mean leaf angle (2016). In population B, specific tolerance could be predicted from maximum light penetration depth and mean leaf inclination (2017) and mean leaf inclination (2018) or mean canopy temperature depression and mean leaf angle (2018).
In data combined over season of population A, the multiple linear regression model selected maximum plant height and mean leaf angle as tolerance predictor. In Population B, mean leaf inclination was selected as tolerance predictor. However, in population A, the variation explained by the final model was too low.
Furthermore, the average tolerances respective to parent median (2011-2018) across FGH plants or all plants (FGH and field) were predicted from maximum plant height (population A) and maximum plant height and mean leaf inclination (population B). Altogether, canopy parameters could be used as markers for drought tolerance. Therefore, water stress breeding in potato could be speed up through using leaf inclination, light penetration depth, plant height and canopy temperature depression as markers for drought tolerance, especially in long-term stress conditions.
Glycosylphosphatidylinositols (GPIs) are highly complex glycolipids that serve as membrane anchors to a large variety of eukaryotic proteins. These are covalently attached to a group of peripheral proteins called GPI-anchored proteins (GPI-APs) through a post-translational modification in the endoplasmic reticulum. The GPI anchor is a unique structure composed of a glycan, with phospholipid tail at one end and a phosphoethanolamine linker at the other where the protein attaches. The glycan part of the GPI comprises a conserved pseudopentasaccharide core that could branch out to carry additional glycosyl or phosphoethanolamine units. GPI-APs are involved in a diverse range of cellular processes, few of which are signal transduction, protein trafficking, pathogenesis by protozoan parasites like the malaria- causing parasite Plasmodium falciparum. GPIs can also exist freely on the membrane surface without an attached protein such as those found in parasites like Toxoplasma gondii, the causative agent of Toxoplasmosis. These molecules are both structurally and functionally diverse, however, their structure-function relationship is still poorly understood. This is mainly because no clear picture exists regarding how the protein and the glycan arrange with respect to the lipid layer. Direct experimental evidence is rather scarce, due to which inconclusive pictures have emerged, especially regarding the orientation of GPIs and GPI-APs on membrane surfaces and the role of GPIs in membrane organization. It appears that computational modelling through molecular dynamics simulations would be a useful method to make progress. In this thesis, we attempt to explore characteristics of GPI anchors and GPI-APs embedded in lipid bilayers by constructing molecular models at two different resolutions – all-atom and coarse-grained.
First, we show how to construct a modular molecular model of GPIs and GPI-anchored proteins that can be readily extended to a broad variety of systems, addressing the micro-heterogeneity of GPIs. We do so by creating a hybrid link to which GPIs of diverse branching and lipid tails of varying saturation with their optimized force fields, GLYCAM06 and Lipid14 respectively, can be attached. Using microsecond simulations, we demonstrate that GPI prefers to “flop-down” on the membrane, thereby, strongly interacting with the lipid heads, over standing upright like a “lollipop”. Secondly, we extend the model of the GPI core to carry out a systematic study of the structural aspects of GPIs carrying different side chains (parasitic and human GPI variants) inserted in lipid bilayers. Our results demonstrate the importance of the side branch residues as these are the most accessible, and thereby, recognizable epitopes. This finding qualitatively agrees with experimental observations that highlight the role of the side branches in immunogenicity of GPIs and the specificity thereof. The overall flop-down orientation of the GPIs with respect to the bilayer surface presents the side chain residues to face the solvent. Upon attaching the green fluorescent protein (GFP) to the GPI, it is seen to lie in close proximity to the bilayer, interacting both with the lipid heads and glycan part of the GPI. However the orientation of GFP is sensitive to the type of GPI it is attached to. Finally, we construct a coarse-grained model of the GPI and GPI-anchored GFP using a modified version of the MARTINI force-field, using which the timescale is enhanced by at least an order of magnitude compared to the atomistic system.
This study provides a theoretical perspective on the conformational behavior of the GPI core and some of its branched variations in presence of lipid bilayers, as well as draws comparisons with experimental observations. Our modular atomistic model of GPI can be further employed to study GPIs of variable branching, and thereby, aid in designing future experiments especially in the area of vaccines and drug therapies. Our coarse-grained model can be used to study dynamic aspects of GPIs and GPI-APs w.r.t plasma membrane organization. Furthermore, the backmapping technique of converting coarse-grained trajectory back to the atomistic model would enable in-depth structural analysis with ample conformational sampling.
Coarse-grained molecular model for the Glycosylphosphatidylinositol anchor with and without protein
(2020)
Glycosylphosphatidylinositol (GPI) anchors are a unique class of complex glycolipids that anchor a great variety of proteins to the extracellular leaflet of plasma membranes of eukaryotic cells. These anchors can exist either with or without an attached protein called GPI-anchored protein (GPI-AP) both in vitro and in vivo. Although GPIs are known to participate in a broad range of cellular functions, it is to a large extent unknown how these are related to GPI structure and composition. Their conformational flexibility and microheterogeneity make it difficult to study them experimentally. Simplified atomistic models are amenable to all-atom computer simulations in small lipid bilayer patches but not suitable for studying their partitioning and trafficking in complex and heterogeneous membranes. Here, we present a coarse-grained model of the GPI anchor constructed with a modified version of the MARTINI force field that is suited for modeling carbohydrates, proteins, and lipids in an aqueous environment using MARTINI's polarizable water. The nonbonded interactions for sugars were reparametrized by calculating their partitioning free energies between polar and apolar phases. In addition, sugar-sugar interactions were optimized by adjusting the second virial coefficients of osmotic pressures for solutions of glucose, sucrose, and trehalose to match with experimental data. With respect to the conformational dynamics of GPI-anchored green fluorescent protein, the accessible time scales are now at least an order of magnitude larger than for the all-atom system. This is particularly important for fine-tuning the mutual interactions of lipids, carbohydrates, and amino acids when comparing to experimental results. We discuss the prospective use of the coarse-grained GPI model for studying protein-sorting and trafficking in membrane models.
Consensify
(2020)
A standard practise in palaeogenome analysis is the conversion of mapped short read data into pseudohaploid sequences, frequently by selecting a single high-quality nucleotide at random from the stack of mapped reads. This controls for biases due to differential sequencing coverage, but it does not control for differential rates and types of sequencing error, which are frequently large and variable in datasets obtained from ancient samples. These errors have the potential to distort phylogenetic and population clustering analyses, and to mislead tests of admixture using D statistics. We introduce Consensify, a method for generating pseudohaploid sequences, which controls for biases resulting from differential sequencing coverage while greatly reducing error rates. The error correction is derived directly from the data itself, without the requirement for additional genomic resources or simplifying assumptions such as contemporaneous sampling. For phylogenetic and population clustering analysis, we find that Consensify is less affected by artefacts than methods based on single read sampling. For D statistics, Consensify is more resistant to false positives and appears to be less affected by biases resulting from different laboratory protocols than other frequently used methods. Although Consensify is developed with palaeogenomic data in mind, it is applicable for any low to medium coverage short read datasets. We predict that Consensify will be a useful tool for future studies of palaeogenomes.
Despite the increasing number of species invasions, the factors driving invasiveness are still under debate. This is particularly the case for “invisible” invasions by aquatic microbial species. Since in many cases only a few individuals or propagules enter a new habitat, their genetic variation is low and might limit their invasion success, known as the genetic bottleneck. Thus, a key question is, how genetic identity and diversity of invading species influences their invasion success and, subsequently, affect the resident community. We conducted invader-addition experiments using genetically different strains of the globally invasive, aquatic cyanobacterium Raphidiopsis raciborskii (formerly: Cylindrospermopsis raciborskii) to determine the role of invader identity and genetic diversity (strain richness) at four levels of herbivory. We tested the invasion success of solitary single strain invasions against the invader genetic diversity, which was experimentally increased up to ten strains (multi-strain populations). By using amplicon sequencing we determined the strain-specific invasion success in the multi-strain treatments and compared those with the success of these strains in the single-strain treatments. Furthermore, we tested for the invasion success under different herbivore pressures. We showed that high grazing pressure by a generalist herbivore prevented invasion, whereas a specialist herbivore enabled coexistence of consumer and invader. We found a weak effect of diversity on invasion success only under highly competitive conditions. When invasions were successful, the magnitude of this success was strain-specific and consistent among invasions performed with single-strain or multi-strain populations. A strain-specific effect was also observed on the resident phytoplankton community composition, highlighting the strong role of invader genetic identity. Our results point to a strong effect of the genetic identity on the invasion success under low predation pressure. The genetic diversity of the invader population, however, had little effect on invasion success in our study, in contrast to most previous findings. Instead, it is the interaction between the consumer abundance and type together with the strain identity of the invader that defined invasion success. This study underlines the importance of strain choice in invasion research and in ecological studies in general.
Starch and Glycogen Analyses
(2020)
For complex carbohydrates, such as glycogen and starch, various analytical methods and techniques exist allowing the detailed characterization of these storage carbohydrates. In this article, we give a brief overview of the most frequently used methods, techniques, and results. Furthermore, we give insights in the isolation, purification, and fragmentation of both starch and glycogen. An overview of the different structural levels of the glucans is given and the corresponding analytical techniques are discussed. Moreover, future perspectives of the analytical needs and the challenges of the currently developing scientific questions are included
In nature, plants are often subjected to periods of recurrent environmental stress that can strongly affect their development and productivity. To cope with these conditions, plants can remember a previous stress, which allows them to respond more efficiently to a subsequent stress, a phenomenon known as priming. This ability can be maintained at the somatic level for a few days or weeks after the stress is perceived, suggesting that plants can store information of a past stress during this recovery phase. While the immediate responses to a single stress event have been extensively studied, knowledge on priming effects and how stress memory is stored is still scarce. At the molecular level, memory of a past condition often involves changes in chromatin structure and organization, which may be maintained independently from transcription. In this review, we will summarize the most recent developments in the field and discuss how different levels of chromatin regulation contribute to priming and plant abiotic stress memory.
Pollen records from Siberia are mostly absent in global or Northern Hemisphere synthesis works. Here we present a taxonomically harmonized and temporally standardized pollen dataset that was synthesized using 173 palynological records from Siberia and adjacent areas (northeastern Asia, 42-75 degrees N, 50-180 degrees E). Pollen data were taxonomically harmonized, i.e. the original 437 taxa were assigned to 106 combined pollen taxa. Age-depth models for all records were revised by applying a constant Bayesian age-depth modelling routine. The pollen dataset is available as count data and percentage data in a table format (taxa vs. samples), with age information for each sample. The dataset has relatively few sites covering the last glacial period between 40 and 11.5 ka (calibrated thousands of years before 1950 CE) particularly from the central and western part of the study area. In the Holocene period, the dataset has many sites from most of the area, with the exception of the central part of Siberia. Of the 173 pollen records, 81 % of pollen counts were downloaded from open databases (GPD, EPD, PANGAEA) and 10 % were contributions by the original data gatherers, while a few were digitized from publications. Most of the pollen records originate from peatlands (48 %) and lake sediments (33 %). Most of the records (83 %) have >= 3 dates, allowing the establishment of reliable chronologies. The dataset can be used for various purposes, including pollen data mapping (example maps for Larix at selected time slices are shown) as well as quantitative climate and vegetation reconstructions. The datasets for pollen counts and pollen percentages are available at https://doi.org/10.1594/PANGAEA.898616 (Cao et al., 2019a), also including the site information, data source, original publication, dating data, and the plant functional type for each pollen taxa.
What Colin Reynolds could tell us about nutrient limitation, N:P ratios and eutrophication control
(2020)
Colin Reynolds exquisitely consolidated our understanding of driving forces shaping phytoplankton communities and those setting the upper limit to biomass yield, with limitation typically shifting from light in winter to phosphorus in spring. Nonetheless, co-limitation is frequently postulated from enhanced growth responses to enrichments with both N and P or from N:P ranging around the Redfield ratio, concluding a need to reduce both N and P in order to mitigate eutrophication. Here, we review the current understanding of limitation through N and P and of co-limitation. We conclude that Reynolds is still correct: (i) Liebig's law of the minimum holds and reducing P is sufficient, provided concentrations achieved are low enough; (ii) analyses of nutrient limitation need to exclude evidently non-limiting situations, i.e. where soluble P exceeds 3-10 mu g/l, dissolved N exceeds 100-130 mu g/l and total P and N support high biomass levels with self-shading causing light limitation; (iii) additionally decreasing N to limiting concentrations may be useful in specific situations (e.g. shallow waterbodies with high internal P and pronounced denitrification); (iv) management decisions require local, situation-specific assessments. The value of research on stoichiometry and co-limitation lies in promoting our understanding of phytoplankton ecophysiology and community ecology.
Using individual-based modeling to understand grassland diversity and resilience in the Anthropocene
(2020)
The world’s grassland systems are increasingly threatened by anthropogenic change. Susceptible to a variety of different stressors, from land-use intensification to climate change, understanding the mechanisms driving the maintenance of these systems’ biodiversity and stability, and how these mechanisms may shift under human-mediated disturbance, is thus critical for successfully navigating the next century. Within this dissertation, I use an individual-based and spatially-explicit model of grassland community assembly (IBC-grass) to examine several processes, thought key to understanding their biodiversity and stability and how it changes under stress. In the first chapter of my thesis, I examine the conditions under which intraspecific trait variation influences the diversity of simulated grassland communities. In the second and third chapters of my thesis, I shift focus towards understanding how belowground herbivores influence the stability of these grassland systems to either a disturbance that results in increased, stochastic, plant mortality, or eutrophication.
Intraspecific trait variation (ITV), or variation in trait values between individuals of the same species, is fundamental to the structure of ecological communities. However, because it has historically been difficult to incorporate into theoretical and statistical models, it has remained largely overlooked in community-level analyses. This reality is quickly shifting, however, as a consensus of research suggests that it may compose a sizeable proportion of the total variation within an ecological community and that it may play a critical role in determining if species coexist. Despite this increasing awareness that ITV matters, there is little consensus of the magnitude and direction of its influence. Therefore, to better understand how ITV changes the assembly of grassland communities, in the first chapter of my thesis, I incorporate it into an established, individual-based grassland community model, simulating both pairwise invasion experiments as well as the assembly of communities with varying initial diversities. By varying the amount of ITV in these species’ functional traits, I examine the magnitude and direction of ITV’s influence on pairwise invasibility and community coexistence. During pairwise invasion, ITV enables the weakest species to more frequently invade the competitively superior species, however, this influence does not generally scale to the community level. Indeed, unless the community has low alpha- and beta- diversity, there will be little effect of ITV in bolstering diversity. In these situations, since the trait axis is sparsely filled, the competitively inferior may suffer less competition and therefore ITV may buffer the persistence and abundance of these species for some time.
In the second and third chapters of my thesis, I model how one of the most ubiquitous trophic interactions within grasslands, herbivory belowground, influences their diversity and stability. Until recently, the fundamental difficulty in studying a process within the soil has left belowground herbivory “out of sight, out of mind.” This dilemma presents an opportunity for simulation models to explore how this understudied process may alter community dynamics. In the second chapter of my thesis, I implement belowground herbivory – represented by the weekly removal of plant biomass – into IBC-grass. Then, by introducing a pulse disturbance, modelled as the stochastic mortality of some percentage of the plant community, I observe how the presence of belowground herbivores influences the resistance and recovery of Shannon diversity in these communities. I find that high resource, low diversity, communities are significantly more destabilized by the presence of belowground herbivores after disturbance. Depending on the timing of the disturbance and whether the grassland’s seed bank persists for more than one season, the impact of the disturbance – and subsequently the influence of the herbivores – can be greatly reduced. However, because human-mediated eutrophication increases the amount of resources in the soil, thus pressuring grassland systems, our results suggest that the influence of these herbivores may become more important over time.
In the third chapter of my thesis, I delve further into understanding the mechanistic underpinnings of belowground herbivores on the diversity of grasslands by replicating an empirical mesocosm experiment that crosses the presence of herbivores above- and below-ground with eutrophication. I show that while aboveground herbivory, as predicted by theory and frequently observed in experiments, mitigates the impact of eutrophication on species diversity, belowground herbivores counterintuitively reduce biodiversity. Indeed, this influence positively interacts with the eutrophication process, amplifying its negative impact on diversity. I discovered the mechanism underlying this surprising pattern to be that, as the herbivores consume roots, they increase the proportion of root resources to root biomass. Because root competition is often symmetric, herbivory fails to mitigate any asymmetries in the plants’ competitive dynamics. However, since the remaining roots have more abundant access to resources, the plants’ competition shifts aboveground, towards asymmetric competition for light. This leads the community towards a low-diversity state, composed of mostly high-performance, large plant species. We further argue that this pattern will emerge unless the plants’ root competition is asymmetric, in which case, like its counterpart aboveground, belowground herbivory may buffer diversity by reducing this asymmetry between the competitively superior and inferior plants.
I conclude my dissertation by discussing the implications of my research on the state of the art in intraspecific trait variation and belowground herbivory, with emphasis on the necessity of more diverse theory development in the study of these fundamental interactions. My results suggest that the influence of these processes on the biodiversity and stability of grassland systems is underappreciated and multidimensional, and must be thoroughly explored if researchers wish to predict how the world’s grasslands will respond to anthropogenic change. Further, should researchers myopically focus on understanding central ecological interactions through only mathematically tractable analyses, they may miss entire suites of potential coexistence mechanisms that can increase the coviability of species, potentially leading to coexistence over ecologically-significant timespans. Individual-based modelling, therefore, with its focus on individual interactions, will prove a critical tool in the coming decades for understanding how local interactions scale to larger contexts, and how these interactions shape ecological communities and further predicting how these systems will change under human-mediated stress.
Adverse environmental conditions are detrimental to plant growth and development. Acclimation to abiotic stress conditions involves activation of signaling pathways which often results in changes in gene expression via networks of transcription factors (TFs). Mediator is a highly conserved co-regulator complex and an essential component of the transcriptional machinery in eukaryotes. Some Mediator subunits have been implicated in stress-responsive signaling pathways; however, much remains unknown regarding the role of plant Mediator in abiotic stress responses. Here, we use RNA-seq to analyze the transcriptional response of Arabidopsis thaliana to heat, cold and salt stress conditions. We identify a set of common abiotic stress regulons and describe the sequential and combinatorial nature of TFs involved in their transcriptional regulation. Furthermore, we identify stress-specific roles for the Mediator subunits MED9, MED16, MED18 and CDK8, and putative TFs connecting them to different stress signaling pathways. Our data also indicate different modes of action for subunits or modules of Mediator at the same gene loci, including a co-repressor function for MED16 prior to stress. These results illuminate a poorly understood but important player in the transcriptional response of plants to abiotic stress and identify target genes and mechanisms as a prelude to further biochemical characterization.
Of city and village mice
(2020)
A fundamental question of current ecological research concerns the drives and limits of species responses to human-induced rapid environmental change (HIREC). Behavioural responses to HIREC are a key component because behaviour links individual responses to population and community changes. Ongoing fast urbanization provides an ideal setting to test the functional role of behaviour for responses to HIREC. Consistent behavioural differences between conspecifics (animal personality) may be important determinants or constraints of animals’ adaptation to urban habitats. We tested whether urban and rural populations of small mammals differ in mean trait expression, flexibility and repeatability of behaviours associated to risk-taking and exploratory tendencies. Using a standardized behavioural test in the field, we quantified spatial exploration and boldness of striped field mice (Apodemus agrarius, n = 96) from nine sub-populations, presenting different levels of urbanisation and anthropogenic disturbance. The level of urbanisation positively correlated with boldness, spatial exploration and behavioural flexibility, with urban dwellers being bolder, more explorative and more flexible in some traits than rural conspecifics. Thus, individuals seem to distribute in a non-random way in response to human disturbance based on their behavioural characteristics. Animal personality might therefore play a key role in successful coping with the challenges of HIREC.
Since the golden era of antibiotics natural products are of ever growing interest to both basic research and applied sciences as they are the main source of new bioactive compounds delivering lead structures for new pharmaceuticals with potent antibiotic, anti-inflammatory or anti-cancer activities. Alongside the technological advances in high-throughput genome sequencing and the better understanding of the general organization of those modular biosynthetic assembly lines of secondary metabolites, there was also a shift from wet-lab screening of active cell extracts towards algorithm-based in silico screening for new natural product biosynthesis gene clusters (BGCs). Although the increasing availability of full genome sequences revealed that such non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS) and ribosomally synthesized and post-translationally modified peptides (RiPPs) can be found in all three kingdoms of life, certain phyla like actinobacteria and cyanobacteria show a very high density of these secondary metabolite BGCs.
The facultative symbiotic, N2-fixing model organism N. punctiforme PCC73102 is a terrestrial type IV cyanobacterium that not only dedicates are very large fraction of its genome to secondary metabolite production but is also amenable to genetic modification. AntiSMASH analysis of the genome showed that there are sixteen potential secondary metabolite BGCs encoded in N. punctiforme, but until now there were only two compounds assigned to their respective BGC leaving the remaining fourteen orphan. This makes the organism a perfect subject for the establishment of a novel combinatorial genomic mining approach for the detection of new natural products.
In the course of this study a combinatorial approach of genomic mining, independent monitoring techniques and alteration of cultivation conditions lead to new insights in cyanobacterial natural product biosynthesis and ultimately to the description of a novel compound produced by N. punctiforme. With the generation and investigation of a reporter strain library consisting of CFP-producing transcriptional reporter mutants for every predicted secondary metabolite BGC of N. punctiforme, it could be shown that natural product expression is in fact not silent for all those BGCs where no compound can be detected. Instead several distinct expression patterns could be described highlighting that secondary metabolite production is under tight regulation and only a minor fraction of these BGCs is in fact silent under standard laboratory conditions. Furthermore, increasing light intensity and carbon dioxide availability and cultivating N. punctiforme to very high cell densities had a tremendous impact on the overall metabolic activity of the organism. Investigation of high density cultivated cell extracts ultimately lead to the detection of a so far undescribed set of microviridins with unusual extended peptide sequences named Microviridin N3 – N9. Both cultivation of the transcriptional reporter mutants as well as RTqPCR-based detection of secondary metabolite BGC transcription levels revealed that in fact 50% of N. punctiforme’s natural product BGCs are upregulated under high cell density conditions. In contrast to this very broad response, co-cultivation of N. punctiforme in chemical or physical contact with a N-deprived host plant (Blasia pusilla) lead to a very specific upregulation of two natural product BGCs, namely RIPP3 and RIPP4. Although this response could be confirmed by various independent monitoring techniques and heavy analytical efforts were spent, no compound could be assigned to either of these BGCs.
This study is the first in-depth systematic investigation of a cyanobacterial secondary metabolome by a combinatorial approach of genome mining and independent monitoring techniques that can serve as a new strategic approach to gain further insight into natural product synthesis of various organisms. Although there are single well described examples of secondary metabolites like the cell differentiation factor PatS in Anabaena sp. strain PCC 7120, the level and extent of regulation observed in this study is unprecedented and understanding of these mechanisms might be the key to streamline natural product discovery. However, the results of this study also highlight that induction of secondary metabolite BGCs is not the real challenge. Instead the new insights point towards analytical issues being a severe hurdle and finding reliable strategies to overcome these problems might as well drive natural product discovery.
Background
Parasitoid wasps have fascinating life cycles and play an important role in trophic networks, yet little is known about their genome content and function. Parasitoids that infect aphids are an important group with the potential for biological control. Their success depends on adapting to develop inside aphids and overcoming both host aphid defenses and their protective endosymbionts.
Results
We present the de novo genome assemblies, detailed annotation, and comparative analysis of two closely related parasitoid wasps that target pest aphids: Aphidius ervi and Lysiphlebus fabarum (Hymenoptera: Braconidae: Aphidiinae). The genomes are small (139 and 141 Mbp) and the most AT-rich reported thus far for any arthropod (GC content: 25.8 and 23.8%). This nucleotide bias is accompanied by skewed codon usage and is stronger in genes with adult-biased expression. AT-richness may be the consequence of reduced genome size, a near absence of DNA methylation, and energy efficiency. We identify missing desaturase genes, whose absence may underlie mimicry in the cuticular hydrocarbon profile of L. fabarum. We highlight key gene groups including those underlying venom composition, chemosensory perception, and sex determination, as well as potential losses in immune pathway genes.
Conclusions
These findings are of fundamental interest for insect evolution and biological control applications. They provide a strong foundation for further functional studies into coevolution between parasitoids and their hosts. Both genomes are available at https://bipaa.genouest.org.
The transfer of Microcystis aeruginosa from freshwater to estuaries has been described worldwide and salinity is reported as the main factor controlling the expansion of M. aeruginosa to coastal environments. Analyzing the expression levels of targeted genes and employing both targeted and non-targeted metabolomic approaches, this study investigated the effect of a sudden salt increase on the physiological and metabolic responses of two toxic M. aeruginosa strains separately isolated from fresh and brackish waters, respectively, PCC 7820 and 7806. Supported by differences in gene expressions and metabolic profiles, salt tolerance was found to be strain specific. An increase in salinity decreased the growth of M. aeruginosa with a lesser impact on the brackish strain. The production of intracellular microcystin variants in response to salt stress correlated well to the growth rate for both strains. Furthermore, the release of microcystins into the surrounding medium only occurred at the highest salinity treatment when cell lysis occurred. This study suggests that the physiological responses of M. aeruginosa involve the accumulation of common metabolites but that the intraspecific salt tolerance is based on the accumulation of specific metabolites. While one of these was determined to be sucrose, many others remain to be identified. Taken together, these results provide evidence that M. aeruginosa is relatively salt tolerant in the mesohaline zone and microcystin (MC) release only occurs when the capacity of the cells to deal with salt increase is exceeded.
Invasive species frequently differentiate phenotypically in novel environments within a few generations, often even with limited genetic variation. For the invasive plants Solidago canadensis and S. gigantea, we tested whether such differentiation might have occurred through heritable epigenetic changes in cytosine methylation. In a 2-year common-garden experiment, we grew plants from seeds collected along a latitudinal gradient in their non-native Central European range to test for trait differentiation and whether differentiation disappeared when seeds were treated with the demethylation agent zebularine. Microsatellite markers revealed no population structure along the latitudinal gradient in S. canadensis, but three genetic clusters in S. gigantea. Solidago canadensis showed latitudinal clines in flowering phenology and growth. In S. gigantea, the number of clonal offspring decreased with latitude. Although zebularine had a significant effect on early growth, probably through effects on cytosine methylation, latitudinal clines remained (or even got stronger) in plants raised from seeds treated with zebularine. Thus, our experiment provides no evidence that epigenetic mechanisms by selective cytosine methylation contribute to the observed phenotypic differentiation in invasive goldenrods in Central Europe.
The shape of a defense-growth trade-off governs seasonal trait dynamics in natural phytoplankton
(2020)
Theory predicts that trade-offs, quantifying costs of functional trait adjustments, crucially affect community trait adaptation to altered environmental conditions, but empirical verification is scarce. We evaluated trait dynamics (antipredator defense, maximum growth rate, and phosphate affinity) of a lake phytoplankton community in a seasonally changing environment, using literature trait data and 21 years of species-resolved high-frequency biomass measurements. The trait data indicated a concave defense-growth trade-off, promoting fast-growing species with intermediate defense. With seasonally increasing grazing pressure, the community shifted toward higher defense levels at the cost of lower growth rates along the trade-off curve, while phosphate affinity explained some deviations from it. We discuss how low fitness differences of species, inferred from model simulations, in concert with stabilizing mechanisms, e.g., arising from further trait dimensions, may lead to the observed phytoplankton diversity. In conclusion, quantifying trade-offs is key for predictions of community trait adaptation and biodiversity under environmental change.
Trehalose 6-phosphate (Tre6P) is a sucrose signalling metabolite that has been implicated in regulation of shoot branching, but its precise role is not understood.
We expressed tagged forms of TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1) to determine where Tre6P is synthesized in arabidopsis (Arabidopsis thaliana), and investigated the impact of localized changes in Tre6P levels, in axillary buds or vascular tissues, on shoot branching in wild-type and branching mutant backgrounds.
TPS1 is expressed in axillary buds and the subtending vasculature, as well as in the leaf and stem vasculature. Expression of a heterologous Tre6P phosphatase (TPP) to lower Tre6P in axillary buds strongly delayed bud outgrowth in long days and inhibited branching in short days. TPP expression in the vasculature also delayed lateral bud outgrowth and decreased branching. Increased Tre6P in the vasculature enhanced branching and was accompanied by higher expression of FLOWERING LOCUS T (FT) and upregulation of sucrose transporters. Increased vascular Tre6P levels enhanced branching in branched1 but not in ft mutant backgrounds.
These results provide direct genetic evidence of a local role for Tre6P in regulation of axillary bud outgrowth within the buds themselves, and also connect Tre6P with systemic regulation of shoot branching via FT.
Tre6P synthesis by TPS1 is essential for embryogenesis and postembryonic growth in Arabidopsis, and appropriate Suc signaling by Tre6P is dependent on the noncatalytic domains of TPS1. In Arabidopsis (Arabidopsis thaliana), TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1) catalyzes the synthesis of the sucrose-signaling metabolite trehalose 6-phosphate (Tre6P) and is essential for embryogenesis and normal postembryonic growth and development. To understand its molecular functions, we transformed the embryo-lethal tps1-1 null mutant with various forms of TPS1 and with a heterologous TPS (OtsA) from Escherichia coli, under the control of the TPS1 promoter, and tested for complementation. TPS1 protein localized predominantly in the phloem-loading zone and guard cells in leaves, root vasculature, and shoot apical meristem, implicating it in both local and systemic signaling of Suc status. The protein is targeted mainly to the nucleus. Restoring Tre6P synthesis was both necessary and sufficient to rescue the tps1-1 mutant through embryogenesis. However, postembryonic growth and the sucrose-Tre6P relationship were disrupted in some complementation lines. A point mutation (A119W) in the catalytic domain or truncating the C-terminal domain of TPS1 severely compromised growth. Despite having high Tre6P levels, these plants never flowered, possibly because Tre6P signaling was disrupted by two unidentified disaccharide-monophosphates that appeared in these plants. The noncatalytic domains of TPS1 ensure its targeting to the correct subcellular compartment and its catalytic fidelity and are required for appropriate signaling of Suc status by Tre6P.
Understanding how organisms adapt to their local environment is a major focus of evolutionary biology. Local adaptation occurs when the forces of divergent natural selection are strong enough compared to the action of other evolutionary forces. An improved understanding of the genetic basis of local adaptation can inform about the evolutionary processes in populations and is of major importance because of its relevance to altered selection pressures due to climate change. So far, most insights have been gained by studying model organisms, but our understanding about the genetic basis of local adaptation in wild populations of species with little genomic resources is still limited.
With the work presented in this thesis I therefore set out to provide insights into the genetic basis of local adaptation in populations of two voles species: the common vole (Microtus arvalis) and the bank vole (Myodes glareolus). Both voles species are small mammals, they have a high evolutionary potential compared to their dispersal capabilities and are thus likely to show genetic responses to local conditions, moreover, they have a wide distribution in which they experience a broad range of different environmental conditions, this makes them an ideal species to study local adaptation.
The first study focused on producing a novel mitochondrial genome to facilitate further research in M. arvalis. To this end, I generated the first mitochondrial genome of M. arvalis using shotgun sequencing and an iterative mapping approach. This was subsequently used in a phylogenetic analysis that produced novel insights into the phylogenetic relationships of the Arvicolinae.
The following two studies then focused on the genetic basis of local adaptation using ddRAD-sequencing data and genome scan methods. The first of these involved sequencing the genomic DNA of individuals from three low-altitude and three high-altitude M. arvalis study sites in the Swiss Alps. High-altitude environments with their low temperatures and low levels of oxygen (hypoxia) pose considerable challenges for small mammals. With their small body size and proportional large body surface they have to sustain high rates of aerobic metabolism to support thermogenesis and locomotion, which can be restricted with only limited levels of oxygen available. To generate insights into high-altitude adaptation I identified a large number of single nucleotide polymorphisms (SNPs). These data were first used to identify high levels of differentiation between study sites and a clear pattern of population structure, in line with a signal of isolation by distance. Using genome scan methods, I then identified signals of selection associated with differences in altitude in genes with functions related to oxygen transport into tissue and genes related to aerobic metabolic pathways. This indicates that hypoxia is an important selection pressure driving local adaptation at high altitude in M. arvalis. A number of these genes were linked with high-altitude adaptation in other species before, which lead to the suggestion that high-altitude populations of several species have evolved in a similar manner as a response to the unique conditions at high altitude
The next study also involved the genetic basis of local adaptation, here I provided insights into climate-related adaptation in M. glareolus across its European distribution. Climate is an important environmental factor affecting the physiology of all organisms. In this study I identified a large number of SNPs in individuals from twelve M. glareolus populations distributed across Europe. I used these, to first establish that populations are highly differentiated and found a strong pattern of population structure with signal of isolation by distance. I then employed genome scan methods to identify candidate loci showing signals of selection associated with climate, with a particular emphasis on polygenic loci. A multivariate analysis was used to determine that temperature was the most important climate variable responsible for adaptive genetic variation among all variables tested. By using novel methods and genome annotation of related species I identified the function of genes of candidate loci. This showed that genes under selection have functions related to energy homeostasis and immune processes. Suggesting that M. glareolus populations have evolved in response to local temperature and specific local pathogenic selection pressures.
The studies presented in this thesis provide evidence for the genetic basis of local adaptation in two vole species across different environmental gradients, suggesting that the identified genes are involved in local adaptation. This demonstrates that with the help of novel methods the study of wild populations, which often have little genomic resources available, can provide unique insights into evolutionary processes.
Cardiac valves are essential for the continuous and unidirectional flow of blood throughout the body. During embryonic development, their formation is strictly connected to the mechanical forces exerted by blood flow. The endocardium that lines the interior of the heart is a specialized endothelial tissue and is highly sensitive to fluid shear stress. Endocardial cells harbor a signal transduction machinery required for the translation of these forces into biochemical signaling, which strongly impacts cardiac morphogenesis and physiology. To date, we lack a solid understanding on the mechanisms by which endocardial cells sense the dynamic mechanical stimuli and how they trigger different cellular responses. In the zebrafish embryo, endocardial cells at the atrioventricular canal respond to blood flow by rearranging from a monolayer to a double-layer, composed of a luminal cell population subjected to blood flow and an abluminal one that is not exposed to it. These early morphological changes lead to the formation of an immature valve leaflet. While previous studies mainly focused on genes that are positively regulated by shear stress, the mechanisms regulating cell behaviors and fates in cells that lack the stimulus of blood flow are largely unknown. One key discovery of my work is that the flow-sensitive Notch receptor and Krüppel-like factor (Klf) 2, one of the best characterized flow-regulated transcriptional factors, are activated by shear stress but that they function in two parallel signal transduction pathways. Each of these two pathways is essential for the rearrangement of atrioventricular cells into an immature double-layered valve leaflets. A second key discovery of my study is the finding that both Notch and Klf2 signaling negatively regulate the expression of the angiogenesis receptor Vegfr3/Flt4, which becomes restricted to abluminal endocardial cells of the valve leaflet. Within these cells, Flt4 downregulates the expressions of the cell adhesion proteins Alcam and VE-cadherin. A loss of Flt4 causes abluminal endocardial cells to ectopically express Notch, which is normally restricted to luminal cells, and impairs valve morphology. My study suggests that abluminal endocardial cells that do not experience mechanical stimuli loose Notch expression and this triggers expression of Flt4. In turn, Flt4 negatively regulates Notch on the abluminal side of the valve leaflet. These antagonistic signaling activities and fine-tuned gene regulatory mechanisms ultimately shape cardiac valve leaflets by inducing unique differences in the fates of endocardial cells.
After endosymbiosis, chloroplasts lost most of their genome. Many former endosymbiotic genes are now nucleus-encoded and the products are re-imported post-translationally. Consequently, photosynthetic complexes are built of nucleus- and plastid-encoded subunits in a well-defined stoichiometry. In Chlamydomonas, the translation of chloroplast-encoded photosynthetic core subunits is feedback-regulated by the assembly state of the complexes they reside in. This process is called Control by Epistasy of Synthesis (CES) and enables the efficient production of photosynthetic core subunits in stoichiometric amounts. In chloroplasts of embryophytes, only Rubisco subunits have been shown to be feedback-regulated. That opens the question if there is additional CES regulation in embryophytes. I analyzed chloroplast gene expression in tobacco and Arabidopsis mutants with assembly defects for each photosynthetic complex to broadly answer this question. My results (i) confirmed CES within Rubisco and hint to potential translational feedback regulation in the synthesis of (ii) cytochrome b6f (Cyt b6f) and (iii) photosystem II (PSII) subunits. This work suggests a CES network in PSII that links psbD, psbA, psbB, psbE, and potentially psbH expression by a feedback mechanism that at least partially differs from that described in Chlamydomonas. Intriguingly, in the Cyt b6f complex, a positive feedback regulation that coordinates the synthesis of PetA and PetB was observed, which was not previously reported in Chlamydomonas. No evidence for CES interactions was found in the expression of NDH and ATP synthase subunits of embryophytes. Altogether, this work provides solid evidence for novel assembly-dependent feedback regulation mechanisms controlling the expression of photosynthetic genes in chloroplasts of embryophytes.
In order to obtain a comprehensive inventory of the rbcL and psbA RNA-binding proteomes (including factors that regulate their expression, especially factors involved in CES), an aptamer based affinity purification method was adapted and refined for the specific purification these transcripts from tobacco chloroplasts. To this end, three different aptamers (MS2, Sephadex ,and streptavidin binding) were stably introduced into the 3’ UTRs of psbA and rbcL by chloroplast transformation. RNA aptamer based purification and subsequent chip analysis (RAP Chip) demonstrated a strong enrichment of psbA and rbcL transcripts and currently, ongoing mass spectrometry analyses shall reveal potential regulatory factors. Furthermore, the suborganellar localization of MS2 tagged psbA and rbcL transcripts was analyzed by a combined affinity, immunology, and electron microscopy approach and demonstrated the potential of aptamer tags for the examination of the spatial distribution of chloroplast transcripts.
We expressed Dictyostelium lamin (NE81) lacking both a functional nuclear localization signal and a CAAX-box for C-terminal lipid modification. This lamin mutant assembled into supramolecular, three-dimensional clusters in the cytosol that disassembled at the onset of mitosis and re-assembled in late telophase, thus mimicking the behavior of the endogenous protein. As disassembly is regulated by CDK1-mediated phosphorylation at serine 122, we generated a phosphomimetic S122E mutant called GFP-NE81-S122E-∆NLS∆CLIM. Surprisingly, during imaging, the fusion protein assembled into cytosolic clusters, similar to the protein lacking the phosphomimetic mutation. Clusters disassembled again in the darkness. Assembly could be induced with blue but not green or near ultraviolet light, and it was independent of the fusion tag. Assembly similarly occurred upon cell flattening. Earlier reports and own observations suggested that both blue light and cell flattening could result in a decrease of intracellular pH. Indeed, keeping the cells at low pH also reversibly induced cluster formation. Our results indicate that lamin assembly can be induced by various stress factors and that these are transduced via intracellular acidification. Although these effects have been shown in a phosphomimetic CDK1 mutant of the Dictyostelium lamin, they are likely relevant also for wild-type lamin.
New Methods, New Concepts
(2020)
Microbial interactions play an essential role in aquatic ecosystems and are of the great interest for both marine and freshwater ecologists. Recent development of new technologies and methods allowed to reveal many functional mechanisms and create new concepts. Yet, many fundamental aspects of microbial interactions have been almost exclusively studied for marine pelagic and benthic ecosystems. These studies resulted in a formulation of the Black Queen Hypothesis, a development of the phycosphere concept for pelagic communities, and a realization of microbial communication as a key mechanism for microbial interactions. In freshwater ecosystems, especially for periphyton communities, studies focus mainly on physiology, biodiversity, biological indication, and assessment, but the many aspects of microbial interactions are neglected to a large extent. Since periphyton plays a great role for aquatic nutrient cycling, provides the basis for water purification, and can be regarded as a hotspot of microbial biodiversity, we highlight that more in-depth studies on microbial interactions in periphyton are needed to improve our understanding on functioning of freshwater ecosystems. In this paper we first present an overview on recent concepts (e.g., the “Black Queen Hypothesis”) derived from state-of-the-art OMICS methods including metagenomics, metatranscriptomics, and metabolomics. We then point to the avenues how these methods can be applied for future studies on biodiversity and the ecological role of freshwater periphyton, a yet largely neglected component of many freshwater ecosystems.
Objective
Plant carnivory is distributed across the tree of life and has evolved at least six times independently, but sequenced and annotated nuclear genomes of carnivorous plants are currently lacking. We have sequenced and structurally annotated the nuclear genome of the carnivorous Roridula gorgonias and that of a non-carnivorous relative, Madeira’s lily-of-the-valley-tree, Clethra arborea, both within the Ericales. This data adds an important resource to study the evolutionary genetics of plant carnivory across angiosperm lineages and also for functional and systematic aspects of plants within the Ericales.
Results
Our assemblies have total lengths of 284 Mbp (R. gorgonias) and 511 Mbp (C. arborea) and show high BUSCO scores of 84.2% and 89.5%, respectively. We used their predicted genes together with publicly available data from other Ericales’ genomes and transcriptomes to assemble a phylogenomic data set for the inference of a species tree. However, groups of orthologs showed a marked absence of species represented by a transcriptome. We discuss possible reasons and caution against combining predicted genes from genome- and transriptome-based assemblies.
Engineering biotechnological microorganisms to use methanol as a feedstock for bioproduction is a major goal for the synthetic metabolism community. Here, we aim to redesign the natural serine cycle for implementation in E. coli. We propose the homoserine cycle, relying on two promiscuous formaldehyde aldolase reactions, as a superior pathway design. The homoserine cycle is expected to outperform the serine cycle and its variants with respect to biomass yield, thermodynamic favorability, and integration with host endogenous metabolism. Even as compared to the RuMP cycle, the most efficient naturally occurring methanol assimilation route, the homoserine cycle is expected to support higher yields of a wide array of products. We test the in vivo feasibility of the homoserine cycle by constructing several E. coli gene deletion strains whose growth is coupled to the activity of different pathway segments. Using this approach, we demonstrate that all required promiscuous enzymes are active enough to enable growth of the auxotrophic strains. Our findings thus identify a novel metabolic solution that opens the way to an optimized methylotrophic platform.
Formaldehyde is a highly reactive compound that participates in multiple spontaneous reactions, but these are mostly deleterious and damage cellular components. In contrast, the spontaneous condensation of formaldehyde with tetrahydrofolate (THF) has been proposed to contribute to the assimilation of this intermediate during growth on C1 carbon sources such as methanol. However, the in vivo rate of this condensation reaction is unknown and its possible contribution to growth remains elusive. Here, we used microbial platforms to assess the rate of this condensation in the cellular environment. We constructed Escherichia coli strains lacking the enzymes that naturally produce 5,10-methylene-THF. These strains were able to grow on minimal medium only when equipped with a sarcosine (N-methyl-glycine) oxidation pathway that sustained a high cellular concentration of formaldehyde, which spontaneously reacts with THF to produce 5,10-methylene-THF. We used flux balance analysis to derive the rate of the spontaneous condensation from the observed growth rate. According to this, we calculated that a microorganism obtaining its entire biomass via the spontaneous condensation of formaldehyde with THF would have a doubling time of more than three weeks. Hence, this spontaneous reaction is unlikely to serve as an effective route for formaldehyde assimilation.
Forage availability has been suggested as one driver of the observed decline in honey bees. However, little is known about the effects of its spatiotemporal variation on colony success. We present a modeling framework for assessing honey bee colony viability in cropping systems. Based on two real farmland structures, we developed a landscape generator to design cropping systems varying in crop species identity, diversity, and relative abundance. The landscape scenarios generated were evaluated using the existing honey bee colony model BEEHAVE, which links foraging to in-hive dynamics. We thereby explored how different cropping systems determine spatiotemporal forage availability and, in turn, honey bee colony viability (e.g., time to extinction, TTE) and resilience (indicated by, e.g., brood mortality). To assess overall colony viability, we developed metrics,P(H)andP(P,)which quantified how much nectar and pollen provided by a cropping system per year was converted into a colony's adult worker population. Both crop species identity and diversity determined the temporal continuity in nectar and pollen supply and thus colony viability. Overall farmland structure and relative crop abundance were less important, but details mattered. For monocultures and for four-crop species systems composed of cereals, oilseed rape, maize, and sunflower,P(H)andP(P)were below the viability threshold. Such cropping systems showed frequent, badly timed, and prolonged forage gaps leading to detrimental cascading effects on life stages and in-hive work force, which critically reduced colony resilience. Four-crop systems composed of rye-grass-dandelion pasture, trefoil-grass pasture, sunflower, and phacelia ensured continuous nectar and pollen supply resulting in TTE > 5 yr, andP(H)(269.5 kg) andP(P)(108 kg) being above viability thresholds for 5 yr. Overall, trefoil-grass pasture, oilseed rape, buckwheat, and phacelia improved the temporal continuity in forage supply and colony's viability. Our results are hypothetical as they are obtained from simplified landscape settings, but they nevertheless match empirical observations, in particular the viability threshold. Our framework can be used to assess the effects of cropping systems on honey bee viability and to develop land-use strategies that help maintain pollination services by avoiding prolonged and badly timed forage gaps.
In nature, bacteria are found to reside in multicellular communities encased in self-produced extracellular matrices. Indeed, biofilms are the default lifestyle of the bacteria which cause persistent infections in humans. The biofilm assembly protects bacterial cells from desiccation and limits the effectiveness of antimicrobial treatments. A myriad of biomolecules in the extracellular matrix, including proteins, exopolysaccharides, lipids, extracellular DNA and other, form a dense and viscoelastic three dimensional network. Many studies emphasized that a destabilization of the mechanical integrity of biofilm architectures potentially eliminates the protective shield and renders bacteria more susceptible to the immune system and antibiotics. Pantoea stewartii is a plant pathogen which infects monocotyledons such as maize and sweet corn. These bacteria produce dense biofilms in the xylem of infected plants which cause wilting of plants and crops. Stewartan is an exopolysaccharide which is produced by Pantoea stewartii and secreted as the major component to the extracellular matrix. It consists of heptasaccharide repeating units with a high degree of polymerization (2-4 MDa). In this work, the physicochemical properties of stewartan were investigated to understand the contributions of this exopolysaccharide to the mechanical integrity and cohesiveness of Pantoea stewartii biofilms. Therefore, a coarse-grained model of stewartan was developed with computational techniques to obtain a model for its three dimensional structural features. Here, coarse-grained molecular dynamic simulations revealed that the exopolysaccharide forms a hydrogel in which the exopolysaccharide chains arrange into a three dimensional mesh-like network. Simulations at different concentrations were used to investigate the influence of the water content on the network formation. Stewartan was further purified from 72 h grown Pantoea stewartii biofilms and the diffusion of bacteriophage and differently-sized nanoparticles (which ranged from 1.1 to 193 nm diameter) was analyzed in reconstituted stewartan solutions. Fluorescence correlation spectroscopy and single-particle tracking revealed that the stewartan network impeded the mobility of a set of differently-sized fluorescent particles in a size-dependent manner. Diffusion of these particles became more anomalous, as characterized by fitting the diffusion data to an anomalous diffusion model, with increasing stewartan concentrations. Further bulk and microrheological experiments were used to analyze the transitions in stewartan fluid behavior and stewartan chain entanglements were described. Moreover, it was noticed, that a small fraction of bacteriophage particles was trapped in small-sized pores deviating from classical random walks which highlighted the structural heterogeneity of the stewartan network. Additionally, the mobility of fluorescent particles
also depended on the charge of the stewartan exopolysaccharide and a model of a molecular sieve for the stewartan network was proposed. The here reported structural features of the stewartan polymers were used to provide a detailed description of the mechanical properties of typically glycan-based biofilms such as the one from Pantoea stewartii.
In addition, the mechanical properties of the biofilm architecture are permanently sensed by the embedded bacteria and enzymatic modifications of the extracellular matrix take place to address environmental cues. Hence, in this work the influence of enzymatic degradation of the stewartan exopolysaccharides on the overall exopolysaccharide network structure was analyzed to describe relevant physiological processes in Pantoea stewartii biofilms. Here, the stewartan hydrolysis kinetics of the tailspike protein from the ΦEa1h bacteriophage, which is naturally found to infect Pantoea stewartii cells, was compared to WceF. The latter protein is expressed from the Pantoea stewartii stewartan biosynthesis gene cluster wce I-III. The degradation of stewartan by the ΦEa1h tailspike protein was shown to be much faster than the hydrolysis kinetics of WceF, although both enzymes cleaved the β D GalIII(1→3)-α-D-GalI glycosidic linkage from the stewartan backbone. Oligosaccharide fragments which were produced during the stewartan cleavage, were analyzed in size-exclusion chromatography and capillary electrophoresis. Bioinformatic studies and the analysis of a WceF crystal structure revealed a remarkably high structural similarity of both proteins thus unveiling WceF as a bacterial tailspike-like protein. As a consequence, WceF might play a role in stewartan chain length control in Pantoea stewartii biofilms.
The aim of this study was to assess the ability of the FFQ to describe reliable and valid dietary pattern (DP) scores. In a total of 134 participants of the European Prospective Investigation into Cancer and Nutrition-Potsdam study aged 35-67 years, the FFQ was applied twice (baseline and after 1 year) to assess its reliability. Between November 1995 and March 1997, twelve 24-h dietary recalls (24HDR) as reference instrument were applied to assess the validity of the FFQ. Exploratory DP were derived by principal component analyses. Investigated predefined DP were the Alternative Healthy Eating Index (AHEI) and two Mediterranean diet indices. From dietary data of each FFQ, two exploratory DP were retained, but differed in highly loading food groups, resulting in moderate correlations (r 0 center dot 45-0 center dot 58). The predefined indices showed higher correlations between the FFQ (r(AHEI) 0 center dot 62, r(Mediterranean Diet Pyramid Index (MedPyr)) 0 center dot 62 and r(traditional Mediterranean Diet Score (tMDS)) 0 center dot 51). From 24HDR dietary data, one exploratory DP retained differed in composition to the first FFQ-based DP, but showed similarities to the second DP, reflected by a good correlation (r 0 center dot 70). The predefined DP correlated moderately (r 0 center dot 40-0 center dot 60). To conclude, long-term analyses on exploratory DP should be interpreted with caution, due to only moderate reliability. The validity differed extensively for the two exploratory DP. The investigated predefined DP showed a better reliability and a moderate validity, comparable to other studies. Within the two Mediterranean diet indices, the MedPyr performed better than the tMDs in this middle-aged, semi-urban German study population.
Differentially-charged liposomes interact with alphaherpesviruses and interfere with virus entry
(2020)
Exposure of phosphatidylserine (PS) in the outer leaflet of the plasma membrane is induced by infection with several members of the Alphaherpesvirinae subfamily. There is evidence that PS is used by the equine herpesvirus type 1 (EHV-1) during entry, but the exact role of PS and other phospholipids in the entry process remains unknown. Here, we investigated the interaction of differently charged phospholipids with virus particles and determined their influence on infection. Our data show that liposomes containing negatively charged PS or positively charged DOTAP (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium) inhibited EHV-1 infection, while neutral phosphatidylcholine (PC) had no effect. Inhibition of infection with PS was transient, decreased with time, and was dose dependent. Our findings indicate that both cationic and anionic phospholipids can interact with the virus and reduce infectivity, while, presumably, acting through different mechanisms. Charged phospholipids were found to have antiviral effects and may be used to inhibit EHV-1 infection.
Broad and unspecific use of antibiotics accelerates spread of resistances. Sensitive and robust pathogen detection is thus important for a more targeted application. Bacteriophages contain a large repertoire of pathogen-binding proteins. These tailspike proteins (TSP) often bind surface glycans and represent a promising design platform for specific pathogen sensors. We analysed bacteriophage Sf6 TSP that recognizes the O-polysaccharide of dysentery-causing Shigella flexneri to develop variants with increased sensitivity for sensor applications. Ligand polyrhamnose backbone conformations were obtained from 2D H-1,H-1-trNOESY NMR utilizing methine-methine and methine-methyl correlations. They agreed well with conformations obtained from molecular dynamics (MD), validating the method for further predictions. In a set of mutants, MD predicted ligand flexibilities that were in good correlation with binding strength as confirmed on immobilized S. flexneri O-polysaccharide (PS) with surface plasmon resonance. In silico approaches combined with rapid screening on PS surfaces hence provide valuable strategies for TSP-based pathogen sensor design.
Island disharmony refers to the biased representation of higher taxa on islands compared to their mainland source regions and represents a central concept in island biology. Here, we develop a generalizable framework for approximating these source regions and conduct the first global assessment of island disharmony and its underlying drivers. We compiled vascular plant species lists for 178 oceanic islands and 735 mainland regions. Using mainland data only, we modelled species turnover as a function of environmental and geographic distance and predicted the proportion of shared species between each island and mainland region. We then quantified the over- or under-representation of families on individual islands (representational disharmony) by contrasting the observed number of species against a null model of random colonization from the mainland source pool, and analysed the effects of six family-level functional traits on the resulting measure. Furthermore, we aggregated the values of representational disharmony per island to characterize overall taxonomic bias of a given flora (compositional disharmony), and analysed this second measure as a function of four island biogeographical variables. Our results indicate considerable variation in representational disharmony both within and among plant families. Examples of generally over-represented families include Urticaceae, Convolvulaceae and almost all pteridophyte families. Other families such as Asteraceae and Orchidaceae were generally under-represented, with local peaks of over-representation in known radiation hotspots. Abiotic pollination and a lack of dispersal specialization were most strongly associated with an insular over-representation of families, whereas other family-level traits showed minor effects. With respect to compositional disharmony, large, high-elevation islands tended to have the most disharmonic floras. Our results provide important insights into the taxon- and island-specific drivers of disharmony. The proposed framework allows overcoming the limitations of previous approaches and provides a quantitative basis for incorporating functional and phylogenetic approaches into future studies of island disharmony.
The biosynthesis of the molybdenum cofactor (Moco) is highly conserved among all kingdoms of life. In all molybdoenzymes containing Moco, the molybdenum atom is coordinated to a dithiolene group present in the pterin-based 6-alkyl side chain of molybdopterin (MPT). In general, the biosynthesis of Moco can be divided into four steps in in bacteria: (i) the starting point is the formation of the cyclic pyranopterin monophosphate (cPMP) from 5 '-GTP, (ii) in the second step the two sulfur atoms are inserted into cPMP leading to the formation of MPT, (iii) in the third step the molybdenum atom is inserted into MPT to form Moco and (iv) in the fourth step bis-Mo-MPT is formed and an additional modification of Moco is possible with the attachment of a nucleotide (CMP or GMP) to the phosphate group of MPT, forming the dinucleotide variants of Moco. This review presents an update on the well-characterized Moco biosynthesis in the model organism Escherichia coli including novel discoveries from the recent years.
Role of diversification rates and evolutionary history as a driver of plant naturalization success
(2020)
Human introductions of species beyond their natural ranges and their subsequent establishment are defining features of global environmental change. However, naturalized plants are not uniformly distributed across phylogenetic lineages, with some families contributing disproportionately more to the global alien species pool than others. Additionally, lineages differ in diversification rates, and high diversification rates have been associated with characteristics that increase species naturalization success. Here, we investigate the role of diversification rates in explaining the naturalization success of angiosperm plant families.
We use five global data sets that include native and alien plant species distribution, horticultural use of plants, and a time-calibrated angiosperm phylogeny. Using phylogenetic generalized linear mixed models, we analysed the effect of diversification rate, different geographical range measures, and horticultural use on the naturalization success of plant families.
We show that a family's naturalization success is positively associated with its evolutionary history, native range size, and economic use. Investigating interactive effects of these predictors shows that native range size and geographic distribution additionally affect naturalization success. High diversification rates and large ranges increase naturalization success, especially of temperate families.
We suggest this may result from lower ecological specialization in temperate families with large ranges, compared with tropical families with smaller ranges.
The Cheb Basin (CZ) is a shallow Neogene intracontinental basin located in the western Eger Rift. The Cheb Basin is characterized by active seismicity and diffuse degassing of mantle-derived CO2 in mofette fields. Within the Cheb Basin, the Hartoušov mofette field shows a daily CO2 flux of 23–97 tons. More than 99% of CO2 released over an area of 0.35 km2. Seismic active periods have been observed in 2000 and 2014 in the Hartoušov mofette field. Due to the active geodynamic processes, the Cheb Basin is considered to be an ideal region for the continental deep biosphere research focussing on the interaction of biological processes with geological processes.
To study the influence of CO2 degassing on microbial community in the surface and subsurface environments, two 3-m shallow drillings and a 108.5-m deep scientific drilling were conducted in 2015 and 2016 respectively. Additionally, the fluid retrieved from the deep drilling borehole was also recovered. The different ecosystems were compared regarding their geochemical properties, microbial abundances, and microbial community structures. The geochemistry of the mofette is characterized by low pH, high TOC, and sulfate contents while the subsurface environment shows a neutral pH, and various TOC and sulfate contents in different lithological settings. Striking differences in the microbial community highlight the substantial impact of elevated CO2 concentrations and high saline groundwater on microbial processes. In general, the microorganisms had low abundance in the deep subsurface sediment compared with the shallow mofette. However, within the mofette and the deep subsurface sediment, the abundance of microbes does not show a typical decrease with depth, indicating that the uprising CO2-rich groundwater has a strong influence on the microbial communities via providing sufficient substrate for anaerobic chemolithoautotrophic microorganisms. Illumina MiSeq sequencing of the 16S rRNA genes and multivariate statistics reveals that the pH strongly influences the microbial community composition in the mofette, while the subsurface microbial community is significantly influenced by the groundwater which motivated by the degassing CO2. Acidophilic microorganisms show a much higher relative abundance in the mofette. Meanwhile, the OTUs assigned to family Comamonadaceae are the dominant taxa which characterize the subsurface communities. Additionally, taxa involved in sulfur cycling characterizing the microbial communities in both mofette and CO2 dominated subsurface environments.
Another investigated important geo–bio interaction is the influence of the seismic activity. During seismic events, released H2 may serve as the electron donor for microbial hydrogenotrophic processes, such as methanogenesis. To determine whether the seismic events can potentially trigger methanogenesis by the elevated geogenic H2 concentration, we performed laboratory simulation experiments with sediments retrieved from the drillings. The simulation results indicate that after the addition of hydrogen, substantial amounts of methane were produced in incubated mofette sediments and deep subsurface sediments. The methanogenic hydrogenotrophic genera Methanobacterium was highly enriched during the incubation. The modeling of the in-situ observation of the earthquake swarm period in 2000 at the Novy Kostel focal area/Czech Republic and our laboratory simulation experiments reveals a close relation between seismic activities and microbial methane production via earthquake-induced H2 release. We thus conclude that H2 – which is released during seismic activity – can potentially trigger methanogenic activity in the deep subsurface. Based on this conclusion, we further hypothesize that the hydrogenotrophic early life on Earth was boosted by the Late Heavy Bombardment induced seismic activity in approximately 4.2 to 3.8 Ga.
In a changing world, phytoplankton communities face a large variety of challenges including altered light regimes. These alterations are caused by more pronounced stratification due to rising temperatures, enhanced eutrophication, and browning of lakes. Community responses toward these effects can emerge as alterations in physiology, biomass, biochemical composition, or diversity. In this study, we addressed the combined effects of changes in light and nutrient conditions on community responses. In particular, we investigated how light intensity and variability under two nutrient conditions influence (1) fast responses such as adjustments in photosynthesis, (2) intermediate responses such as pigment adaptation and (3) slow responses such as changes in community biomass and species composition. Therefore, we exposed communities consisting of five phytoplankton species belonging to different taxonomic groups to two constant and two variable light intensity treatments combined with two levels of phosphorus supply. The tested phytoplankton communities exhibited increased fast reactions of photosynthetic processes to light variability and light intensity. The adjustment of their light harvesting mechanisms via community pigment composition was not affected by light intensity, variability, or nutrient supply. However, pigment specific effects of light intensity, light variability, and nutrient supply on the proportion of the respective pigments were detected. Biomass was positively affected by higher light intensity and nutrient concentrations while the direction of the effect of variability was modulated by light intensity. Light variability had a negative impact on biomass at low, but a positive impact at high light intensity. The effects on community composition were species specific. Generally, the proportion of green algae was higher under high light intensity, whereas the cyanobacterium performed better under low light conditions. In addition to that, the diatom and the cryptophyte performed better with high nutrient supply while the green algae as well as the cyanobacterium performed better at low nutrient conditions. This shows that light intensity, light variability, and nutrient supply interactively affect communities. Furthermore, the responses are highly species and pigment specific, thus to clarify the effects of climate change a deeper understanding of the effects of light variability and species interactions within communities is important.
Studies on the diversity, distribution and ecological role of Saprolegniales (Oomycota) in freshwater ecosystems are currently receiving attention due to a greater understanding of their role in carbon cycling in various aquatic ecosystems. In this study, we characterized several Saprolegniales species isolated from Anzali lagoon, Gilan province, Iran, using morphological and molecular methods. Four species of Saprolegnia were identified, including S. anisospora and S. diclina as first reports for Iran, as well as Achlya strains, which were closely related to A. bisexualis, A. debaryana and A. intricata. Evaluation of the ligno-, cellulo- and chitinolytic activities was performed using plate assay methods. Most of the Saprolegniales isolates were obtained in autumn, and nearly 50% of the strains showed chitinolytic and cellulolytic activities. However, only a few Saprolegniales strains showed lignolytic activities. This study has important implications for better understanding the ecological niche of oomycetes, and to differentiate them from morphologically similar, but functionally different aquatic fungi in freshwater ecosystems.
The Arctic region is especially impacted by global warming as temperatures in high latitude regions have increased and are predicted to further rise at levels above the global average. This is crucial to Arctic soils and the shallow shelves of the Arctic Ocean as they are underlain by permafrost. Perennially frozen ground is a habitat for a large number and great diversity of viable microorganisms, which can remain active even under freezing conditions. Warming and thawing of permafrost makes trapped soil organic carbon more accessible to microorganisms. They can transform it to the greenhouse gases carbon dioxide, methane and nitrous oxide. On the other hand, it is assumed that thawing of the frozen ground stimulates microbial activity and carbon turnover. This can lead to a positive feedback loop of warming and greenhouse gas release.
Submarine permafrost covers most areas of the Siberian Arctic Shelf and contains a large though unquantified carbon pool. However, submarine permafrost is not only affected by changes in the thermal regime but by drastic changes in the geochemical composition as it formed under terrestrial conditions and was inundated by Holocene sea level rise and coastal erosion. Seawater infiltration into permafrost sediments resulted in an increase of the pore water salinity and, thus, in thawing of permafrost in the upper sediment layers even at subzero temperatures. The permafrost below, which was not affected by seawater, remained ice-bonded, but warmed through seawater heat fluxes.
The objective of this thesis was to study microbial communities in submarine permafrost with a focus on their response to seawater influence and long-term warming using a combined approach of molecular biological and physicochemical analyses. The microbial abundance, community composition and structure as well as the diversity were investigated in drill cores from two locations in the Laptev Sea, which were subjected to submarine conditions for centuries to millennia. The microbial abundance was measured through total cell counts and copy numbers of the 16S rRNA gene and of functional genes. The latter comprised genes which are indicative for methane production (mcrA) and sulfate reduction (dsrB). The microbial community was characterized by high-throughput-sequencing of the 16S rRNA gene. Physicochemical analyses included the determination of the pore water geochemical and stable isotopic composition, which were used to describe the degree of seawater influence. One major outcome of the thesis is that the submarine permafrost stratified into different so-called pore water units centuries as well as millennia after inundation: (i) sediments that were mixed with seafloor sediments, (ii) sediments that were infiltrated with seawater, and (iii) sediments that were unaffected by seawater. This stratification was reflected in the submarine permafrost microbial community composition only millennia after inundation but not on time-scales of centuries.
Changes in the community composition as well as abundance were used as a measure for microbial activity and the microbial response to changing thermal and geochemical conditions. The results were discussed in the context of permafrost temperature, pore water composition, paleo-climatic proxies and sediment age. The combination of permafrost warming and increasing salinity as well as permafrost warming alone resulted in a disturbance of the microbial communities at least on time-scales of centuries. This was expressed by a loss of microbial abundance and bacterial diversity. At the same time, the bacterial community of seawater unaffected but warmed permafrost was mainly determined by environmental and climatic conditions at the time of sediment deposition. A stimulating effect of warming was observed only in seawater unaffected permafrost after millennia-scale inundation, visible through increased microbial abundance and reduced amounts of substrate.
Despite submarine exposure for centuries to millennia, the community of bacteria in submarine permafrost still generally resembled the community of terrestrial permafrost. It was dominated by phyla like Actinobacteria, Chloroflexi, Firmicutes, Gemmatimonadetes and Proteobacteria, which can be active under freezing conditions.
Moreover, the archaeal communities of both study sites were found to harbor high abundances of marine and terrestrial anaerobic methane oxidizing archaea (ANME). Results also suggested ANME populations to be active under in situ conditions at subzero temperatures. Modeling showed that potential anaerobic oxidation of methane (AOM) could mitigate the release of almost all stored or microbially produced methane from thawing submarine permafrost.
Based on the findings presented in this thesis, permafrost warming and thawing under submarine conditions as well as permafrost warming without thaw are supposed to have marginal effects on the microbial abundance and community composition, and therefore likely also on carbon mobilization and the formation of methane. Thawing under submarine conditions even stimulates AOM and thus mitigates the release of methane.
Monocytes and macrophages are key players in maintaining immune homeostasis. Identifying strategies to manipulate their functions via gene delivery is thus of great interest for immunological research and biomedical applications. We set out to establish conditions for mRNA transfection in hard-to-transfect primary human monocytes and monocyte-derived macrophages due to the great potential of gene expression from in vitro transcribed mRNA for modulating cell phenotypes. mRNA doses, nucleotide modifications, and different carriers were systematically explored in order to optimize high mRNA transfer rates while minimizing cell stress and immune activation. We selected three commercially available mRNA transfection reagents including liposome and polymer-based formulations, covering different application spectra. Our results demonstrate that liposomal reagents can particularly combine high gene transfer rates with only moderate immune cell activation. For the latter, use of specific nucleotide modifications proved essential. In addition to improving efficacy of gene transfer, our findings address discrete aspects of innate immune activation using cytokine and surface marker expression, as well as cell viability as key readouts to judge overall transfection efficiency. The impact of this study goes beyond optimizing transfection conditions for immune cells, by providing a framework for assessing new gene carrier systems for monocyte and macrophage, tailored to specific applications.
In June 2019, more than a hundred plant researchers met in Cologne, Germany, for the 6th European Workshop on Plant Chromatin (EWPC). This conference brought together a highly dynamic community of researchers with the common aim to understand how chromatin organization controls gene expression, development, and plant responses to the environment. New evidence showing how epigenetic states are set, perpetuated, and inherited were presented, and novel data related to the three-dimensional organization of chromatin within the nucleus were discussed. At the level of the nucleosome, its composition by different histone variants and their specialized histone deposition complexes were addressed as well as the mechanisms involved in histone post-translational modifications and their role in gene expression. The keynote lecture on plant DNA methylation by Julie Law (SALK Institute) and the tribute session to Lars Hennig, honoring the memory of one of the founders of the EWPC who contributed to promote the plant chromatin and epigenetic field in Europe, added a very special note to this gathering. In this perspective article we summarize some of the most outstanding data and advances on plant chromatin research presented at this workshop.
Accurately quantifying species' area requirements is a prerequisite for effective area-based conservation. This typically involves collecting tracking data on species of interest and then conducting home-range analyses. Problematically, autocorrelation in tracking data can result in space needs being severely underestimated. Based on the previous work, we hypothesized the magnitude of underestimation varies with body mass, a relationship that could have serious conservation implications. To evaluate this hypothesis for terrestrial mammals, we estimated home-range areas with global positioning system (GPS) locations from 757 individuals across 61 globally distributed mammalian species with body masses ranging from 0.4 to 4000 kg. We then applied block cross-validation to quantify bias in empirical home-range estimates. Area requirements of mammals <10 kg were underestimated by a mean approximately15%, and species weighing approximately100 kg were underestimated by approximately50% on average. Thus, we found area estimation was subject to autocorrelation-induced bias that was worse for large species. Combined with the fact that extinction risk increases as body mass increases, the allometric scaling of bias we observed suggests the most threatened species are also likely to be those with the least accurate home-range estimates. As a correction, we tested whether data thinning or autocorrelation-informed home-range estimation minimized the scaling effect of autocorrelation on area estimates. Data thinning required an approximately93% data loss to achieve statistical independence with 95% confidence and was, therefore, not a viable solution. In contrast, autocorrelation-informed home-range estimation resulted in consistently accurate estimates irrespective of mass. When relating body mass to home range size, we detected that correcting for autocorrelation resulted in a scaling exponent significantly >1, meaning the scaling of the relationship changed substantially at the upper end of the mass spectrum.
Metagenomic sequencing has revolutionised our knowledge of virus diversity, with new virus sequences being reported faster than ever before. However, virus discovery from metagenomic sequencing usually depends on detectable homology: without a sufficiently close relative, so-called ‘dark’ virus sequences remain unrecognisable. An alternative approach is to use virus-identification methods that do not depend on detecting homology, such as virus recognition by host antiviral immunity. For example, virus-derived small RNAs have previously been used to propose ‘dark’ virus sequences associated with the Drosophilidae (Diptera). Here, we combine published Drosophila data with a comprehensive search of transcriptomic sequences and selected meta-transcriptomic datasets to identify a completely new lineage of segmented positive-sense single-stranded RNA viruses that we provisionally refer to as the Quenyaviruses. Each of the five segments contains a single open reading frame, with most encoding proteins showing no detectable similarity to characterised viruses, and one sharing a small number of residues with the RNA-dependent RNA polymerases of single- and double-stranded RNA viruses. Using these sequences, we identify close relatives in approximately 20 arthropods, including insects, crustaceans, spiders, and a myriapod. Using a more conserved sequence from the putative polymerase, we further identify relatives in meta-transcriptomic datasets from gut, gill, and lung tissues of vertebrates, reflecting infections of vertebrates or of their associated parasites. Our data illustrate the utility of small RNAs to detect viruses with limited sequence conservation, and provide robust evidence for a new deeply divergent and phylogenetically distinct RNA virus lineage.
Utilising a reconstructed ancestral mitochondrial genome of a clade to design hybridisation capture baits can provide the opportunity for recovering mitochondrial sequences from all its descendent and even sister lineages. This approach is useful for taxa with no extant close relatives, as is often the case for rare or extinct species, and is a viable approach for the analysis of historical museum specimens. Asiatic linsangs (genus Prionodon) exemplify this situation, being rare Southeast Asian carnivores for which little molecular data is available. Using ancestral capture we recover partial mitochondrial genome sequences for seven banded linsangs (P. linsang) from historical specimens, representing the first intraspecific genetic dataset for this species. We additionally assemble a high quality mitogenome for the banded linsang using shotgun sequencing for time-calibrated phylogenetic analysis. This reveals a deep divergence between the two Asiatic linsang species (P. linsang, P. pardicolor), with an estimated divergence of ~12 million years (Ma). Although our sample size precludes any robust interpretation of the population structure of the banded linsang, we recover two distinct matrilines with an estimated tMRCA of ~1 Ma. Our results can be used as a basis for further investigation of the Asiatic linsangs, and further demonstrate the utility of ancestral capture for studying divergent taxa without close relatives.
Thermal stress response is an essential physiological trait that determines occurrence and temporal succession in nature, including response to climate change. We compared temperature-related demography in closely related heat-tolerant and heat-sensitive Brachionus rotifer species. We found significant differences in heat response, with the heat-sensitive species adopting a strategy of long survival and low population growth, while the heat-tolerant followed the opposite strategy. In both species, we examined the genetic basis of physiological variation by comparing gene expression across increasing temperatures. Comparative transcriptomic analyses identified shared and opposing responses to heat. Interestingly, expression of heat shock proteins (hsps) was strikingly different in the two species and mirrored differences in population growth rates, showing that hsp genes are likely a key component of a species’ adaptation to different temperatures. Temperature induction caused opposing patterns of expression in further functional categories including energy, carbohydrate and lipid metabolism, and in genes related to ribosomal proteins. In the heat-sensitive species, elevated temperatures caused up-regulation of genes related to meiosis induction and post-translational histone modifications. This work demonstrates the sweeping reorganizations of biological functions that accompany temperature adaptation in these two species and reveals potential molecular mechanisms that might be activated for adaptation to global warming.
Identifying the entirety of gene regulatory interactions in a biological system offers the possibility to determine the key molecular factors that affect important traits on the level of cells, tissues, and whole organisms. Despite the development of experimental approaches and technologies for identification of direct binding of transcription factors (TFs) to promoter regions of downstream target genes, computational approaches that utilize large compendia of transcriptomics data are still the predominant methods used to predict direct downstream targets of TFs, and thus reconstruct genome-wide gene-regulatory networks (GRNs). These approaches can broadly be categorized into unsupervised and supervised, based on whether data about known, experimentally verified gene-regulatory interactions are used in the process of reconstructing the underlying GRN. Here, we first describe the generic steps of supervised approaches for GRN reconstruction, since they have been recently shown to result in improved accuracy of the resulting networks? We also illustrate how they can be used with data from model organisms to obtain more accurate prediction of gene regulatory interactions.
Plants located adjacent to agricultural fields are important for maintaining biodiversity in semi-natural landscapes. To avoid undesired impacts on these plants due to herbicide application on the arable fields, regulatory risk assessments are conducted prior to registration to ensure proposed uses of plant protection products do not present an unacceptable risk. The current risk assessment approach for these non-target terrestrial plants (NTTPs) examines impacts at the individual-level as a surrogate approach for protecting the plant community due to the inherent difficulties of directly assessing population or community level impacts. However, modelling approaches are suitable higher tier tools to upscale individual-level effects to community level. IBC-grass is a sophisticated plant community model, which has already been applied in several studies. However, as it is a console application software, it was not deemed sufficiently user-friendly for risk managers and assessors to be conveniently operated without prior expertise in ecological models. Here, we present a user-friendly and open source graphical user interface (GUI) for the application of IBC-grass in regulatory herbicide risk assessment. It facilitates the use of the plant community model for predicting long-term impacts of herbicide applications on NTTP communities. The GUI offers two options to integrate herbicide impacts: (1) dose responses based on current standard experiments (acc. to testing guidelines) and (2) based on specific effect intensities. Both options represent suitable higher tier options for future risk assessments of NTTPs as well as for research on the ecological relevance of effects.