Kaiserslautern - Fachbereich Biologie
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The ability to sense and respond to different environmental conditions allows living organisms to adapt quickly to their surroundings. In order to use light as a source of information, plants, fungi, and bacteria employ phytochromes. With their ability to detect far-red and red light, phytochromes constitute a major photoreceptor family. Bacterial phytochromes (BphPs) are composed of an apo-phytochrome and an open-chain tetrapyrrole, the chromophore biliverdin IXα, which mediates the photosensory properties. Depending on the photoexcitation and the quality of the incident light, phytochromes interconvert between two photoconvertible parental states: the red light-absorbing Pr-form and the far-red light-absorbing Pfr-form. In contrast to prototypical phytochromes, with a thermal stable Pr ground state, there is a group of bacterial phytochromes that exhibit dark reversion from the Pr- to the Pfr-form. These special proteins are classified as bathy phytochromes and range across different classes of bacteria. Moreover, the majority of BphPs act as sensor histidine kinases in two-component regulatory systems. The light-triggered conformational change results in the autophosphorylation of the histidine kinase domain and the transphosphorylation of an associated response regulator, inducing a cellular response. Spectroscopic analysis utilizing homologously produced protein identified PaBphP, the histidine kinase of the human opportunistic pathogen Pseudomonas aeruginosa, as a bathy phytochrome. Intensive research on PaBphP revealed evidence that the interconversion between its physiological active and inactive states is influenced by light and darkness rather than far-red and red light. In order to conduct a comprehensive systematic analysis, further bacterial phytochromes were investigated regarding their biochemical and spectroscopic behavior, as well as their autokinase activity. In addition to PaBphP, this work employs the bathy phytochromes AtBphP2, AvBphP2, XccBphP from the non-photosynthetic plant pathogens Agrobacterium tumefaciens, Allorhizobium vitis, Xanthomonas campestris, as well as RtBphP2 from the soil bacterium Ramlibacter tataouinensis. All investigated BphPs displayed a bathy-typical behavior by developing a distinct Pr-form under far-red light conditions and undergoing dark reversion to their Pfr-form. Different Pr/Pfr-fractions can be identified among the BphP populations in varying natural light conditions, including red or blue light. The Pr-form is considered as the active form due to autophosphorylation activity in the heterologously produced phytochromes when exposed to light. In the absence of light, associated with the development of the Pfr-form, the phytochromes exhibited disabled or strongly reduced autokinase activity. Additionally, light-triggered phosphorylation was observed for the response regulator PaAlgB, which is linked to the phytochrome of P. aeruginosa. This study presents the first comparative investigation of numerous bathy phytochromes under identical conditions. The work addressed a gap in the literature by providing quantitative correlation between kinase activity and calculated Pr/Pfr-fractions obtained from spectroscopic measurements. The biological role of PaBphP was partially elucidated through phenotypic characterization employing P. aeruginosa mutant and overexpression strains. The generation of a functional model was possible by considering the postulated functions of the other phytochromes found in the literature. In summary, bathy BphPs are hypothesized to modulate bacterial virulence according to the circadian day/night rhythm of their hosts. The pathogens are believed to reduce their virulence during daylight hours to evade immune and defense reactions, while increasing their virulence during the evening and night, enabling more effective infections.
Climate change will have severe consequences on Eastern Boundary Upwelling Systems (EBUS). They host the largest fisheries in the world supporting the life of millions of people due to their tremendous primary production. Therefore, it is of utmost importance to better understand predicted impacts like alternating upwelling intensities and light impediment on the structure and the trophic role of protistan plankton communities as they form the basis of the food web. Numerical models estimate the intensification of the frequency in eddy formation. These ocean features are of particular importance due to their influence on the distribution and diversity of plankton communities and the access to resources, which are still not well understood even to the present day. My PhD thesis entails two subjects conducted during large-scaled cooperation projects REEBUS (Role of Eddies in Eastern Boundary Upwelling Systems) and CUSCO (Coastal Upwelling System in a Changing Ocean).
Subject I of my study was conducted within the multidisciplinary framework REEBUS to investigate the influence of eddies on the biological carbon pump in the Canary Current System (CanCS). More specifically, the aim was to find out how mesoscale cyclonic eddies affect the regional diversity, structure, and trophic role of protistan plankton communities in a subtropical oligotrophic oceanic offshore region.
Samples were taken during the M156 and M160 cruises in the Atlantic Ocean around Cape Verde during July and December 2019, respectively. Three eddies with varying ages of emergence and three water layers (deep chlorophyll maximum DCM, right beneath the DCM and oxygen minimum zone OMZ) were sampled. Additional stations without eddy perturbation were analyzed as references. The effect of oceanic mesoscale cyclonic eddies on protistan plankton communities was analyzed by implementing three approaches. (i) V9 18S rRNA gene amplicons were examined to analyze the diversity and structure of the plankton communities and to infer their role in the biological carbon pump. (ii) By assigning functional traits to taxonomically assigned eDNA sequences, functional richness and ecological strategies (ES) were determined. (iii) Grazing experiments were conducted to assess abundance and carbon transfer from prokaryotes to phagotrophic protists.
All three eddies examined in this study differed in their ASV abundance, diversity, and taxonomic composition with the most pronounced differences in the DCM. Dinoflagellates were the most abundant taxa in all three depth layers. Other dominating taxa were radiolarians, Discoba and haptophytes. The trait-approach could only assign ~15% of all ASVs and revealed in general a relatively high functional richness. But no unique ES was determined within a specific eddy. This indicates pronounced functional redundancy, which is recognized to be correlated with ecosystem resilience and robustness by providing a degree of buffering capacity in the face of biodiversity loss. Elevated microbial abundances as well as bacterivory were clearly associated to mesoscale eddy features, albeit with remarkable seasonal fluctuations. Since eddy activity is expected to increase on a global scale in future climate change scenarios, cyclonic eddies could counteract climate change by enhancing carbon sequestration to abyssal depths. The findings demonstrate that cyclonic eddies are unique, heterogeneous, and abundant ecosystems with trapped water masses in which characteristic protistan plankton develop as the eddies age and migrate westward into subtropical oligotrophic offshore waters. Therefore, eddies influence regional protistan plankton diversity qualitatively and quantitatively.
Subject II of my PhD project contributed to the CUSCO field campaign to identify the influence of varying upwelling intensities in combination with distinct light treatments on the whole food web structure and carbon pump in the Humboldt Current System (HCS) off Peru. To accomplish such a task, eight offshore-mesocosms were deployed and two light scenarios (low light, LL; high light, HL) were created by darkening half of the mesocosms. Upwelling was simulated by injecting distinct proportions (0%, 15%, 30% and 45%) of collected deep-water (DW) into each of the moored mesocosms. My aim was to examine the changes in diversity, structure, and trophic role of protistan plankton communities for the induced manipulations by analyzing the V9 18S rRNA gene amplicons and performing short-term grazing experiments.
The upwelling simulations induced a significant increase in alpha diversity under both light conditions. In austral summer, reflected by HL conditions, a generally higher alpha diversity was recorded compared to the austral winter simulation, instigated by LL treatment. Significant alterations of the protistan plankton community structure could likewise be observed. Diatoms were associated to increased levels of DW addition in the mimicked austral winter situation. Under nutrient depletion, chlorophytes exhibited high relative abundances in the simulated austral winter scenario. Dinoflagellates dominated the austral summer condition in all upwelling simulations. Tendencies of reduced unicellular eukaryotes and increased prokaryotic abundances were determined under light impediment. Protistan-mediated mortality of prokaryotes also decreased by ~30% in the mimicked austral winter scenario.
The findings indicate that the microbial loop is a more relevant factor in the structure of the food web in austral summer and is more focused on the utilization of diatoms in austral winter in the HCS off Peru. It was evident that distinct light intensities coupled with multiple upwelling scenarios could lead to alterations in biochemical cycles, trophic interactions, and ecosystem services. Considering the threat of climate change, the predicted relocation of EBUS could limit primary production and lengthen the food web structure with severe socio-economic consequences.
Living systems incessantly engage in the regulation of their cellular processes to fulfill their biological functions. Beyond development-related adjustments or cell cycle oscillations, environmental fluctuations compel the system to reorganize metabolic pathways, structural components, or molecular repair and reconstitution mechanisms. These responses manifest across diverse temporal scales, necessitating an intricate regulatory orchestration. Time series experiments have become increasingly popular for charting the chronological order and elucidating the underlying mechanisms. In the era of high-throughput technologies, the majority of cellular molecules can be analyzed in one fell swoop, generating a comprehensive snapshot of the status quo of most present molecules. Methodological advancements also permit the monitoring not only of molecular abundances but also the functional status of transcripts and proteins. However, due to the still high efforts associated with such experiments, the number of measured time points and the replication of measurements remains limited. Resulting datasets contain signals from thousands of molecules, yet they are sparse in temporal resolution and are often imprecise due to biological variability and technical measurement inaccuracies.
This thesis explores the complexities arising from the examination of short time series data and introduces pioneering tools that offer fresh insights into the realm of biological time series analysis. The broad spectrum of analytic possibilities ranges from a molecule-centric investigation of individual time courses to a holistic aggregation of the system’s response to its main characteristics. By creating a modeling framework that applies domain-specific constraints, time-course signals can be transformed from a series of discrete data points into a continuous curve. These curves align with current biological conjectures about molecule kinetics being smooth and devoid of superfluous oscillations. Noise present at individual time points is judiciously accounted for during curve fitting, mitigating the impact of time points with high variance on the curve. Subsequent classification is based on the features of these curves (extreme points and inflection points) and ensures a reduction in data amount and complexity. Succinct labels assigned to each molecule's kinetics encapsulate the signal's most notable features. Besides this modeling approach, an innovative enrichment strategy is introduced, that is independent of prior data partitioning and capable of segregating the temporal response into its thermodynamically relevant components. This approach allows for a continuous assessment of each molecule's contribution to these components, obviating the need for exclusive allocation. The application of various analytical approaches to heat acclimation experiments in Chlamydomonas highlights the relevance and potential of time series experiments and specifically tailored analysis techniques. The integration of different system levels has led to the identification of regulatory peculiarities, such as an increased correlation between transcripts and corresponding proteins during acclimation responses. These and other insights may herald new avenues of research that could ultimately enhance plant robustness in the face of increasing environmental perturbations.
The growing popularity of time series experiments necessitates dedicated analytical approaches that empower researchers and analysts to decipher patterns, discern trends, and unravel the underlying structures within the data, facilitating predictions and the derivation of meaningful conclusions that could potentially build bridges between the interweaved systems levels.
Regulation of sucrose transport between source and sink tissues is critical for plant development and properties. In cells, the dynamic vacuolar sugar homeostasis is maintained by the controlled regulation of the activities of sugar importers and exporters residing in the tonoplast. We show here that the EARLY RESPONSE TO DEHYDRATION6-LIKE4 protein, being the closest homolog to the proton/glucose symporter ERDL6, resides in the vacuolar membrane. We raised both, molecular expression and data deriving from non-aqueous fractionation studies indicating that ERDL4 was involved in glucose and fructose allocation across the tonoplast. Surprisingly, overexpression of ERDL4 increased total sugar levels in leaves, which is due to a concomitantly induced stimulation of TST2 expression, coding for the major vacuolar sugar loader. This conclusion is supported by the notion that tst1-2 knockout lines overexpressing ERDL4 lack increased cellular sugar levels. That ERDL4 activity contributes to the coordination of cellular sugar homeostasis is further indicated by two observations. Firstly, ERDL4 and TST genes exhibit an opposite regulation during a diurnal rhythm, secondly, the ERDL4 gene is markedly expressed during cold acclimation representing a situation in which TST activity needs to be upregulated. Moreover, ERDL4-overexpressing plants show larger size of rosettes and roots, a delayed flowering and increased total seed yield. In summary, we identified a novel factor influencing source to sink transfer of sucrose and by this governing plant organ development.
Chromosomal aberrations are manifold changes in the configuration of the DNA. Each cell in a tumor
may accumulate different karyotype changes, making it challenging to determine the causes and
consequences of this instability. Therefore, model systems have been developed in the past to
generate and study specific genome alterations. In this thesis, I present the results of my studies on
three types of chromosomal aberrations, all of which may contribute to tumor development or
progression.
Chromothripsis is a phenomenon that describes a one-off massive chromosomal disruption and
reassembly, perhaps arising via DNA damage micronuclei (MN). MN are small DNA-packed nuclear
envelopes. I tested potential causes of DNA damage in MN and found that the rupture of the MN
envelope and the entry of cytosolic fractions increase DNA damage in MN. Furthermore, I addressed
the question of what physiological consequences cell lines with an additional rearranged chromosome
have compared to those with an intact extra chromosome. Strikingly, the cells with more
rearrangements showed a functional advantage resulting in an improved fitness potential.
However, the engineering of polysomic cell lines with fully intact additional chromosomes increases
various cellular stress responses and reduces the proliferation capacity. To investigate how cancer cells
overcome the detrimental consequences of aneuploidy, I explored physiological adaptations of model
cells with a defined additional chromosome that underwent in vivo and in vitro evolution. Interestingly,
unfavorable phenotypes of aneuploid cells, such as the replication stress, were mitigated upon
evolution. Furthermore, I examined the replication on single molecule resolution, showing alteration
after evolution that might underlie the replication stress bypass or tolerance.
In contrast to these unbalanced forms of genomic aberrations, whole genome doubling (WGD) leads
to a full doubled chromosome set, which was shown to evolve into aneuploid karyotypes by
chromosomal instability (CIN), frequently by losing chromosomes. Cells that underwent WGD
accumulate DNA damage in the S phase. I performed a single molecule analysis on the DNA during the
first cell cycle after WGD to elucidate how the DNA damage arises and found that the number of active
origins is not sufficient to replicate the doubled amount of DNA in the first S phase after WGD faithfully.
This starts a genome-destabilizing cascade that eventually promotes tumorigenesis, metastasis, and
poor patient outcome.
Taken together, these studies provide insights into the causes and consequences of three types of
genomic aberrations: chromothripsis, polysomy, and WGD. However different these phenomena may
be, they share one common feature – they contribute to tumor development and progression.
Therefore, elucidating the aberrant cell functions caused by genomic aberrations contributes to a
better understanding of a cancer cell's nature and will perhaps help to find new cancer therapy targets.
Phycobilisomes (PBS) are the major light-harvesting complexes for the majority of cyanobacteria
and allow these organisms to absorb in the so-called green gap. They consist of smaller units called
phycobiliproteins (PBPs), which are composed of an α- and a β-subunit with covalently bound
linear tetrapyrroles (phycobilins). The latter are attached to the apo-PBPs by phycobiliprotein
lyases. Interestingly, cyanobacteria of the genus Prochlorococcus lack complete PBS and instead
use prochlorophyte chlorophyll-binding proteins (Pcbs), which effectively utilize the energy of the
blue light region. The low-light-adapted (LL) strain Prochlorococcus marinus SS120 has a single
PBP, phycoerythrin-III (PE-III). It has been postulated that PE-III is chromophorylated with the
phycobilins phycourobilin (PUB) and phycoerythrobilin (PEB) in a 3:1 ratio. Thereby, the function
of PE-III remains unclear so far, so that light-gathering function and also photoreceptor function
are discussed.
The main goal of this work was to characterize the assembly of PE-III and thus the function of the
six putative phycobiliprotein lyases of P. marinus SS120. Previous work found that the individual
lyases could not be produced in soluble form, so we switched to a dual pDuet™ plasmid system in
E. coli, which was successfully established. Investigation of the binding of PEB to Apo-PE
revealed that the CpeS lyase specifically chromophorylated Cys82 with 3Z-PEB. Unfortunately,
additional chromophorylation could not be observed using the pDuet system. Therefore, in a
second part of the work, the entire PE gene cluster from P. marinus SS120 was to be introduced
into E. coli and expressed. Although the gene cluster was successfully transcribed within E. coli,
no translation was observed, possibly due to incompatible translation initiation between
Prochlorococcus and E. coli. The introduction of a mini PE cluster (CpeAB) into the
cyanobacterium Synechococcus sp. PCC 7002 was also successfully performed, in which case
production of CpeB but not CpeA from Prochlorococcus was detected. Recombinant CpeB was
also detected together with intrinsic PBP in Synechococcussp. 7002, indicating structural similarity
and incorporation into PBS in Synechococcus sp. 7002. Overall, the obtained results suggest that a
cyanobacterial host is a good option for the studies on the assembly of PE-III from P. marinus and,
based on this, future work could aim at generating an artificial operon using synthetic biology to
achieve efficient translation of all genes.
The vast majority of all mitochondrial proteins are synthesized in the cytosol. These proteins carry characteristic targeting motifs within their sequence, which allows for the binding of chaperones, that in turn usher precursors to the mitochondrial surface for import and assembly. Though, our understanding of these early reactions is still lacking, recent efforts have shown that the ER surface can facilitate the import of mitochondrial proteins (ER-SURF) with the help of the J-protein Djp1. Close cooperation of organelles in form of membrane contact sites is crucial for cellular function. The aim of my work was to investigate whether ER-mitochondria contact sites are critical for the transfer of proteins from the ER to mitochondria.
Several contact sites have been characterized between ER and mitochondria in S. cerevisiae. One contact site is called the ER mitochondria encounter structure (ERMES) and another is partly formed by Tom70. Owing to the high propensity of suppressor mutations in ERMES, I employed a knockdown approach to deplete this contact site. Using an inducible CRISPR interference (CRISPRi) system, I could rapidly and efficiently deplete Mdm34, which is a part of ERMES. I could show that depletion of Mdm34 had a synthetic negative effect in combination with a deletion of TOM70. Loss of both contact sites led to a strong decrease of many mitochondrial proteins in the whole cell proteome. Using affinity purification of ER and mitochondria in conjunction with mass spectrometry I could demonstrate that a specific set of mitochondrial proteins are enriched on the ER upon loss of Mdm34 and Tom70, which mainly were proteins of the inner membrane e.g., Oxa1 and Cox5A. Moreover, I was able to validate that the import of these proteins was hampered upon loss of both contact sites. Also, in vivo the biogenesis of Oxa1 was impeded upon single loss of Mdm34 or Tom70 and strongly impaired if both were lost. Analysis of the maximum hydrophobicity of inner membrane proteins in the ER-SURF set revealed on average a significantly higher peak compared to other inner membrane proteins. I could show that deleting or swapping the transmembrane domain of Cox5A would make it contact site independent or reliant on contact sites respectively, as revealed by an in vitro import assay.
In this study I was able to demonstrate the involvement of membrane contact sites in ER-SURF and identify a list of putative clients. Furthermore, I could show that hydrophobicity of the transmembrane segment of inner membrane proteins is one determinant for ER-SURF dependence.
Drought is a significant environmental factor that can impair plant growth and development, leading to reduced crop productivity or even plant death. Maintaining sugar distribution from source to sink is crucial for increasing crop production under water limitation conditions. Numerous studies have suggested that nutrition fertilization, especially potassium (K), can enhance plant growth and yield production. To investigate the mechanism of K in sugar long-distance transportation under drought stress, we established a soil-grow system and a hydroponic-grow system with varying amounts of potassium supplementation and analyzed the biochemical and molecular responses in Arabidopsis and potato plants under drought stress conditions. Our findings showed that excess potassium fertilization limited sucrose metabolism, leading to lower drought tolerance in Arabidopsis in both grow systems. However, higher potassium supplementation altered sugar relocation and potassium movement, resulting in an increase in starch yield production in both potato plants with different sink strength capacities. We also proposed that a low amount of sodium increases Arabidopsis drought tolerance under low potassium conditions since a low amount of sodium can improve the control of osmotic potential, leading to more water being retained in plant cells.
Silicon (Si) has received considerable attention recently for its potential in mitigating drought stress, although the effects vary among different plant species. To investigate the mechanism of Si in drought stress tolerance, we applied monosilicic acid in hydroponic media and then applied PEG8000 to simulate drought stress. Our findings revealed that Si-dependent drought mitigation occurred more in the shoot than in the root of Arabidopsis, and we observed silicon accumulation in the shoot of Arabidopsis. In Si-treated plants, more glucose was accumulated in the vacuole, leading to better osmotic potential control under drought stress. RNA sequencing analysis showed that Si altered the activity of sugar transporters and the sugar metabolism process, and increased photosynthesis. However, Si-dependent regulation in sugar transporter showed different responses in potato. Understanding the mechanism of Si in potato requires further studies. Overall, our dissertation provides important information for clarifying the mechanism of Si in drought stress, which forms the basis for further investigation.
The amyloid precursor protein (APP) is a key molecular component of Alzheimer’s disease (AD) pathogenesis. Proteolytic APP processing generates various cleavage products, including extracellular amyloid beta (Aβ) and the cytoplasmic APP intracellular domain (AICD). Although the role of AICD in the activation of kinase signaling pathways is well established in the context
of full-length APP, little is known about intracellular effects of the AICD fragment, particularly within discrete neuronal compartments. Deficits in fast axonal transport (FAT) and axonopathy documented in AD-affected neurons prompted us to evaluate potential axon-autonomous effects of the AICD fragment for the first time. Vesicle motility assays using the isolated squid axoplasm
preparation revealed inhibition of FAT by AICD. Biochemical experiments linked this effect to aberrant activation of selected axonal kinases and heightened phosphorylation of the anterograde motor protein conventional kinesin, consistent with precedents showing phosphorylation-dependent regulation of motors proteins powering FAT. Pharmacological inhibitors of these kinases alleviated the AICD inhibitory effect on FAT. Deletion experiments indicated this effect requires a sequence encompassing the NPTY motif in AICD and interacting axonal proteins containing a phosphotyrosinebinding domain. Collectively, these results provide a proof of principle for axon-specific effects of AICD, further suggesting a potential mechanistic framework linking alterations in APP processing, FAT deficits, and axonal pathology in AD.
Like many other bacteria, the opportunistic pathogen P. aeruginosa encodes a broad network of enzymes that regulate the intracellular concentration of the second messenger c-di-GMP. One of these enzymes is the phosphodiesterase NbdA that consists of three domains: a membrane anchored, putative sensory MHYT domain, a non-functional diguanylate cyclase domain with degenerated GGDEF motif and an active PDE domain with EAL motif. Analysis of the nbdA open reading frame by 5’-RACE PCR revealed an erroneous annotation of nbdA in the Pseudomonas database with the ORF 170 bp shorter than previously predicted. The newly defined promoter region of nbdA contains recognition sites for the alternative sigma-factor RpoS as well as the transcription factor AmrZ. Promoter analysis within PAO1 wt as well as rpoS and amrZ mutant strains utilizing transcriptional fusions of the nbdA promoter to the reporter gene lacZ revealed transcriptional activation of nbdA by RpoS in stationary growth phase and transcriptional repression by AmrZ. Additionally, no influence of nitrite and neither exogenous nor endogenous NO on nbdA transcription could be shown in this study. However, deletion of the nitrite reductase gene nirS led to a strong increase of nbdA promoter activity which needs to be characterized further. Predicted secondary structures of the 5’-UTR of the nbdA mRNA indicated either an RNA thermometer function of the mRNA or post-transcriptional regulation of nbdA by the RNA binding proteins RsmA and RsmF. Nevertheless, translational studies using fusions of the 5’ UTR of nbdA to the reporter gene bgaB did not verify either of these hypotheses. In general, nbdA translational levels were very low and neither the production of the reporter BgaB nor genomically encoded NbdA could be detected on a western blot. Overproduction of NbdA variants induced many phenotypic changes in motility and biofilm formation. But strains overproducing variants containing the MHYT domain revealed greatly elongated cells and were impaired in surface growth, indicating a misbalance in the membrane protein homeostasis. Therefore, these phenotypes have to be interpreted very critically. Microscopic studies with fluorescently tagged NbdA revealed either a diffuse fluorescent signal of NbdA or the formation of fluorescent foci which were located mainly at the cell poles. Co-localization studies with the polar flagellum and the chemotaxis protein CheA showed that NbdA is not generally localizing to the flagellated cell pole. NbdA localization indicates the control of a specific local c-di-GMP pool in the cell which is most likely involved in MapZ mediated chemotactic flagellar motor switching.