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Bacteria are highly organized organisms which are able to adapt to and propagate under a multitude of environmental conditions. Propagation hereby requires reliable chromosome replication and segregation which has to occur cooperatively with other cellular processes such as transcription, translation or signaling. Several mechanisms were proposed for segregation of the Escherichia coli (E. coli) chromosome, for example a mitotic-like active segregation model or entropy-based passive chromosome segregation. Another segregation model suggests coupled transcription, translation and insertion of membrane proteins (termed "transertion"), which links the replicating chromosome (nucleoid) to the growing cell cylinder.
Fluorescence microscopy was widely used to provide evidence for a distinct segregation model. However, the dynamic nature of bacterial chromosomes, the small bacterial size and the optical resolution limit of ~ 200-300 nm impair unveiling the underlying mechanisms. With the emergence of super-resolution fluorescence microscopy techniques and advanced labeling methods, a new toolbox became available enabling scientists to visualize biomolecules and cellular processes in unprecedented detail. Single-molecule localization microscopy (SMLM) represents a set of super-resolution microscopy techniques which relies on the temporal separation of the fluorescence signal and detection of single fluorophores. Separation can be achieved using photoactivatable or -convertible fluorescent proteins (FPs) in photoactivated localization microscopy (PALM), photoswitchable organic dyes in direct stochastic optical reconstruction microscopy (dSTORM) or dynamically binding fluorescent probes in point accumulation for imaging in nanoscale topography (PAINT). In all these techniques, the fluorescence emission pattern of single fluorophores is spatially localized with nanometer-precision. An artificial image is finally reconstructed from the coordinates of all single fluorophores detected. This provides a spatial resolution of ~ 20 nm, which is perfectly suited to investigate cellular processes in bacteria. In this thesis, different SMLM techniques were applied to study fundamental processes in E. coli. This includes determination of protein copy numbers and distributions as well as the nanoscale organization of nucleic acids and lipids.
A novel labeling approach was applied and used for super-resolution imaging of the E. coli nucleoid. It is based on the incorporation of the modified thymidine analogue 5-ethynyl-2’- deoxyuridine (EdU) into the replicating chromosome. Azide-functionalized organic fluorophores can be covalently attached to the ethynyl group of incorporated EdU bases using a copper-catalyzed "click chemistry" reaction. Under the investigated growth condition, E. coli cells exhibited overlapping replication cycles, which is commonly referred to as multi-fork replication and enables cells to divide faster than they can replicate the entire chromosome. dSTORM imaging of such labeled nucleoids revealed chromosome features with diameters of 50 - 200 nm, representing highly condensed DNA filaments. Sorting single E. coli cells by length allowed visualizing structural changes of the nucleoid throughout the cell cycle. Replicating nucleoids segregated and expanded along the bacterial long axis, while constantly covering the entire width of the cell. Measuring cell and nucleoid length revealed a relative nucleoid expansion rate of 78 ± 6 %. At the same time, nucleoids populated 63 ± 8 % of the cell length, almost exclusively being localized to the cylindrical part of the cell. This value was hence normalized to the cylindrical fraction of the cell, yielding a value of 79 ± 10 % (nucleoid-populated fraction of the cell cylinder), which is in good agreement with the observed relative nucleoid expansion rate. These results therefore support a growth-mediated segregation model, in which the chromosome is anchored to the inner membrane and passively segregated into the prospective daughter cells upon cell growth. 3-dimensional dSTORM imaging of labeled nucleoids confirmed that compacted nucleoids helically wrap along the inner membrane. Similar results were obtained by imaging orthogonally aligned E. coli cells using a holographic optical tweezer approach.
In order to visualize particular proteins together with the nucleoid, several correlative imaging workflows were established, facilitating multi-color SMLM imaging in single E. coli cells. These workflows bypass prior limitations of SMLM, including destruction of FPs by reactive oxygen species in copper-catalyzed click reactions or incompatibility of PALM imaging with dSTORM imaging buffers. A sequential SMLM imaging routine was developed which is based on postlabeling and retrieval of previously imaged cells. Optimal imaging conditions can be maintained for each fluorophore, enabling to extract quantitative information from PALM measurements while correlating the protein distribution to the nucleoid ultrastructure within the highly resolved cell envelope. Applying this workflow to an E. coli strain carrying a chromosomal rpoC - photoactivatable mCherry (PAmCh) fusion, transcribing RNA polymerase (RNAP) was found to be localized on the surface of nucleoids, where active genes are exposed towards the cytosol. During growth in nutrient-rich medium, the majority of RNAP molecules was bound to the chromosome, thus ensuring that the RNAP pool is equally distributed to the daughter cells upon cell division. This work represented the first triple-color SMLM study performed in E. coli cells. ...
Biophysical studies of the translation-regulating add adenine riboswitch from Vibrio vulnificus
(2017)
Bacterial gene expression can be regulated at mRNA level by cis-acting mRNA elements termed riboswitches. Riboswitches operate by conformational switching between a ligand-free and a ligand-bound state with different structures that either activate or inhibit gene expression. This PhD thesis contributes to the molecular level understanding of full-length purine riboswitches. It presents biophysical investigations on the ligand-dependent folding of the full-length translation-regulating add adenine riboswitch from the gram-negative human pathogenic marine bacterium Vibrio vulnificus (Asw). Asw has the typical bipartite riboswitch architecture with a 5’ ligand-sensing aptamer domain and a 3’ regulatory domain termed expression platform. According to the working hypothesis, Asw employs a unique thermodynamically-controlled 3-state conformational switching mechanism between an apoB, an apoA and a holo conformation to regulate translation initiation in a temperature-compensated manner. The two apo conformations are the putative translation-OFF states and the holo conformation is the putative translation-ON state of Asw. In the main project of this PhD thesis, an integrated nuclear magnetic resonance (NMR) and smFRET spectroscopic study of the full-length 112-nucleotide Asw (112Asw) was performed. The adenine-dependent folding of 112Asw was monitored at the level of base pairing interactions by NMR of the RNA imino protons, and at the level of three long-range intramolecular distances by smFRET of immobilized molecules. The integrated NMR and smFRET spectroscopic study of 112Asw yielded two major findings. First, NMR and smFRET both revealed that adenine binding to 112Asw impedes apoB formation by stabilizing the apoA secondary structure in the holo conformation without modulating tertiary structural interactions between the two riboswitch domains. This highlights the central role of competitive P1 and P4 helix formation at the interface of the aptamer and the expression platform for switching the accessibility of the ribosome binding site of 112Asw. Moreover, it strongly corroborates the hypothesis that purine riboswitches in general operate according to the key principle of a spatially decoupled secondary structural allosteric switch that proceeds without ligand-induced tertiary structural interactions between the aptamer domain and the expression platform. Second, it was uncovered by smFRET that the apoA and the holo conformation of 112Asw do not adopt a single folding state at near-physiological Mg2+ concentration. Instead, apoA and holo exhibit a persistent dynamic equilibrium between substates with an undocked (U), a short-lived docked (D1; ~s) and a Mg2+-bound long-lived docked (D2; ~10 s) aptamer kissing loop motif. In the holo conformation, the fractional population of the long-lived docked substate is ~2-fold increased compared to the apoA conformation, but undocked and docked substates are still comparably stable. The here described multiple folding states of the apoA and the holo conformation might have regulatory properties that are in between the apoB translation-OFF state and the holo-D2 translation-ON state. Additonally, an integrated NMR and smFRET analysis of 127-nucleotide Asw (127Asw) is presented. Compared to 112Asw, 127Asw is 3’-elongated by 15 nucleotides of the adenosine deaminase encoding sequence of the add gene from Vibrio vulnificus. 127Asw was chosen as mRNA template for future investigations of the interaction between Asw and the 30S ribosomal subunit. The NMR spectra of 127Asw demonstrated that 127Asw has the same overall secondary structure as 112Asw. Like for 112Asw, the combined NMR and smFRET analysis of 127Asw showed that adenine binding impedes apoB formation and stabilizes a long-lived docked aptamer kissing loop fold. However, compared to 112Asw, 127Asw has a destabilized aptamer kissing loop motif and a stabilized P4 helix in the expression platform. Finally, ligand-observed studies of the transient encounter complex between Asw and the near-cognate ligand hypoxanthine are described. By competition binding WaterLOGSY NMR experiments with hypoxanthine and the adenine analogue 2,6-diaminopurine, it could be shown that hypoxanthine binds to the same binding site of 112Asw as the cognate ligand adenine. The hypoxanthine binding constant measured with the WaterLOGSY method is in the low mM range (1.8 mM) and substantially exceeds the physiological hypoxanthine concentration in E. coli (~0.3 mM), thus ruling out that hypoxanthine binding can significantly impact the translational regulation of Asw in vivo. Also, preliminary FTIR difference spectra of 13C,15N-labelled and unlabelled hypoxanthine in complex with the pbuE adenine riboswitch aptamer and the xpt guanine riboswitch aptamer are discussed. These spectra showed a pattern of multiple IR bands that appeared to be characteristic for the respective complex.
The fact that the interaction of oligonucleotides follows strict rules has been utilized to create two- or three-dimensional objects made of DNA. With computer-assisted design of DNA sequences, any arbitrary structure on the nanometer- to micrometer-scale can be generated just by hybridization of the needed strands. As astonishing these structures are, without any modification of the DNA strands involved no function can be assigned to them. Many different ways of functionalizing DNA-nanostructures have been developed with light-responsive nanostructures having a rather subordinated role. Almost all light responsive DNA-nanostructures involve the acyclic azobenzene-linking system tAzo based on D-threoninol which is known to work best at elevated temperatures to ensure optimal switching. As the structure of DNA-constructs is mainly maintained by hydrogen-bonding, variation of the temperature should be avoided in order to keep the structure intact.
To develop a light-responsive nanostructure model system with low-temperature operating azobenzene C-nucleosides, DNA-minicircles have been utilized. Those minicircles bear a lariat-like protrusion with a 10 base long single-stranded overhang, which is responsible for the dimerization with a ring bearing a complementary binding region. DNA-minicircles have been produced in a sequential manner by building and purifying the single stranded minicircle first by splint ligation and prepratative PAGE or RP-HPLC, followed by annealing it to the outer ring and subsequent purification by molecular-weight cut-off. Imaging of DNA-minicircles by atomic force microscopy (AFM) was possible with several methods of sample preparation leading to images of varying quality. With the help of AFM, qualitative analysis of the minicircles was possible. It could be shown, that theoretical and empirical size dimensions of the rings and their interactions were in great accordance. Designing the interaction site of the minicircles proved to be the main task in this project. The amount of C-nucleosidic modifications was identified by screening, followed by a screening of their optimal position and binding partners in the counterstrand. Two azobenzene C-nucleosides in a 10mer binding region and abasic sites opposing them appeared to give the best compromise between absolute dimerization ratio and photocontrolled change of it, as identified by native PAGE. In the following, the dimerization ratios of minicircles containing azobenzene C-nucleosides were compared with minicircles containing tAzo and unmodified minicircles. It could be shown, that the tAzo-modification leads to an elevated binding affinity compared to the unmodified minicircles, but the change upon irradiation is relatively humble compared to the C-nucleosides. For the C-nucleosidic modifications dimerization ratios reached a maximum of 40% in favored trans-state, but could be almost completely turned-off when switching into cis-state. In addition, arylazopyrazole-modified C-nucleosides could be switched into trans-state by irradiating at 530 nm, which is an improvement compared to standard azobenzene, as it shifts irradiation wavelength closer to the phototherapeutic window.
The utilization of DNA-analogous C-nucleosides bring two drawbacks with them: the ribose units include the flexibility of the sugar conformation and it is reasonable to think, that upon isomerization of the azobenzene, part of the steric stress generated is compensated by the sugar reconfiguration, which is lost for duplex
destabilization. In addition, the combination of the ribosidic linker end the end-to-end distance of trans-azobenzene causes the chromophore to penetrate deep into the base stack of the opposing strand, causing a serious destabilization even in favored trans-state. The goal was to find a linker system, that combines the benefits of the azobenzene C-nucleoside without the possibility to change sugar conformation and the strong destabilization in the trans-state. For this reason locked azobenzene C-nucleosides in analogy to LNA nucleosides have been synthesized. The synthesis of LNA analogous azobenzene C-nucleosides (LNAzo) was possible over a 16-step synthesis, with the critical step being the addition of in situ lithiated azobenzene to protected sugar aldehyde. Both anomers of LNAzo and mAzo as reference where incorporated into different oligonucleotide test systems by solid phase synthesis for thorough evaluation. It could be shown, that LNAzo β has a similar performance to mAzo in DNA with overall slightly increased TM- and ΔTM-values. Performance of LNAzo β was similar to mAzo even if steric stress is reduced by using abasic sites in the counterstrand opposing the azobenzene. Only in a RNA context, the true potential of LNAzo β could be observed. In a DNA/RNA duplex, photocontrol could be improved by almost 50%, in a RNA/RNA duplex even by over 100%. Although the primary goal was the improvement of the azobenzene C-nucleoside for a DNA-nanostructure context, LNAzo β proved not to give a sufficient improvement in regard to the cost-value ratio. Never the less, the invention of the locked azobenzene C-nucleoside was a huge success for reversible photoregulation of RNA hybridization. With this, a new way to regulate RNA hybridization has been found, which could be used to create RNA therapeutics in an antisense-approach.
As LNAzo β improved duplex stability only in a limited amount in DNA, further improvements on the backbone have been declared futile and focus shifted onto optimization of the chromophore. First, the azobenzene as it is installed on the ribosidic linker decreases duplex stability by forcing its distal aromat deep into opposing base stacking region. It would be an improvement, if in favored trans-state the distal aromat would be positioned in the less confined space of either major or minor groove and only upon isomerization would shift into base pairing region. Second, the azobenzene itself is not able to contribute to attractive interactions aside from relatively weak π-interactions to adjacent nucleobases, which could be improved, if it could partake in hydrogen bonding. For those apparent reasons, 2-phenyldiazenyl-modified purines have been selected as targets. They combine the ability to contribute to hydrogen bonding of nucleobases with the photochomicity of azobenzenes. Both 2’-deoxyadenosine- and 2’-deoxyguanosine-analogue photoswitches dAAzo and dGAzo have been synthesized and incorporated into 10mer DNA test systems by solid phase synthesis. It could be shown, that duplex stability could be increased compared to established azobenzene C-nucleoside. The improvement was stronger for dAAzo than for dGAzo as in the case for guanosine the amino function on the C2-position had to be replaced by the phenyldiazenyl function, reducing its ability to form hydrogen bonds. Unfortunately, photocontrol of duplex stability caused by 2-phenyldiazenyl purines was rather limited. A reason for this could be the positioning of the distal aromat within the duplex, which can be close to the opposing nucleobase (endo-helical) or in greater distance (exo-helical). The exo-helical conformation of the trans-isomer can only switch to the exo-P-cis-conformation, which relocates the distal aromat in the minor groove, without significant impact on duplex stability.
Physical Biology is a field of life sciences dealing with the extraction of quantitative data from biophysical or molecular biological experiments with different levels of complexity. Such data are further used as parameters for mathematical models of the biological system. These models allow to predict reactions on external stimuli by describing the relevant molecular interactions and are therefore used for example to generate a deeper comprehension of complex human diseases. An essential technique in biophysical research on human diseases is fluorescence microscopy. This is a constantly developed toolbox comprising a large number of specific labeling strategies, as well as a broad spectrum of fluorescent probes. It is further minimal invasive and therefore suitable for measurements in living cells or organisms. The sensitivity of modern photo-detectors even allows for the detection of a single fluorescent probe with an accuracy of approximately 10 nm.
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The model-prediction was further verified by two color SMLM experiments. In this work the development and application of imaging-systems are described which provide quantitative data with single-molecule resolution for systems biological model approaches with a low degree of abstractness. In the near future, the impact of mathematical models in the research field of complex human diseases will increase. The predictions of these models will be more exact, the more detailed and accurate the input parameters will become. This work gives an impression of how quantitative data obtained by SMLM may serve as input parameters for mathematical models at the single-cell level.