IT’S THE LITTLE THINGS: AN EXPLORATION OF SMALL RNAS AND SELFISH GENETIC ELEMENTS OF THE HUMAN BACTERIAL PATHOGENS COXIELLA BURNETII AND BARTONELLA BACILLIFORMIS

Coxiella burnetii is a Gram-negative gammaproteobacterium and zoonotic agent of Q fever in humans. Previous work in our lab has demonstrated that C. burnetii codes for several small RNAs (sRNAs) that are differentially expressed between in vivo and in vitro growth conditions. sRNAs serve as post-transcriptional regulatory effectors involved in the control of nearly all biological processes. We demonstrated that several of the identified sRNAs, namely Coxiella burnetii small RNA 3 (CbsR3), Cbsr13, and CbsR16, represent members of two novel families of miniature inverted-repeat transposable elements (MITEs), termed QMITE1 and QMITE2. Furthermore, we have characterized a highly expressed, infection-specific sRNA, CbsR12, and have determined that it is necessary for expansion of the C. burnetii intracellular niche in a human monocyte-derived alveolar macrophage cell line. We have determined that CbsR12 may participate in broad gene regulation by acting as an RNA sponge for the global regulatory RNA-binding protein CsrA. Additionally, CbsR12 is a trans-acting sRNA that targets transcripts of the carA, metK, and cvpD genes in vitro and in vivo. Bartonella bacilliformis is a Gram-negative alphaproteobacterium and the etiological agent of Carrión\u27s disease in humans. B. bacilliformis is spread between humans through the bite of female phlebotomine sand flies. As a result, the pathogen encounters significant environmental shifts during its life cycle, including changes in pH and temperature. Bacterial sRNAs can serve as a means of rapid regulation under shifting environmental conditions. We therefore performed total RNA-sequencing analyses on B. bacilliformis grown in vitro then shifted to one of ten distinct conditions that simulate various environments encountered by the pathogen during its life cycle. From this, we identified 160 sRNAs significantly expressed under at least one of the conditions tested. Northern blot analysis was used to confirm the expression of eight novel sRNAs. We also characterized a Bartonella bacilliformis group I intron (BbgpI) that disrupts an un-annotated tRNACCUArg gene and determined that the intron splices in vivo and self-splices in vitro. Furthermore, we verified the predicted molecular targeting of a sand fly-specific sRNA, Bartonella bacilliformis small RNA 9 (BbsR9), to transcripts of the ftsH, nuoF, and gcvT genes, in vitro

Whole-genome resequencing of Escherichia coli K-12 MG1655 undergoing short-term laboratory evolution in lactate minimal media reveals flexible selection of adaptive mutations

Background Short-term laboratory evolution of bacteria followed by genomic sequencing provides insight into the mechanism of adaptive evolution, such as the number of mutations needed for adaptation, genotype-phenotype relationships, and the reproducibility of adaptive outcomes. Results In the present study, we describe the genome sequencing of 11 endpoints of Escherichia coli that underwent 60-day laboratory adaptive evolution under growth rate selection pressure in lactate minimal media. Two to eight mutations were identified per endpoint. Generally, each endpoint acquired mutations to different genes. The most notable exception was an 82 base-pair deletion in the rph-pyrE operon that appeared in 7 of the 11 adapted strains. This mutation conferred an approximately 15% increase to the growth rate when experimentally introduced to the wild-type background and resulted in an approximately 30% increase to growth rate when introduced to a background already harboring two adaptive mutations. Additionally, most endpoints had a mutation in a regulatory gene (crp or relA, for example) or the RNA polymerase. Conclusions The 82 base-pair deletion found in the rph-pyrE operon of many endpoints may function to relieve a pyrimidine biosynthesis defect present in MG1655. In contrast, a variety of regulators acquire mutations in the different endpoints, suggesting flexibility in overcoming regulatory challenges in the adaptation

Similarity-based gene detection: using COGs to find evolutionarily-conserved ORFs

BACKGROUND: Experimental verification of gene products has not kept pace with the rapid growth of microbial sequence information. However, existing annotations of gene locations contain sufficient information to screen for probable errors. Furthermore, comparisons among genomes become more informative as more genomes are examined. We studied all open reading frames (ORFs) of at least 30 codons from the genomes of 27 sequenced bacterial strains. We grouped the potential peptide sequences encoded from the ORFs by forming Clusters of Orthologous Groups (COGs). We used this grouping in order to find homologous relationships that would not be distinguishable from noise when using simple BLAST searches. Although COG analysis was initially developed to group annotated genes, we applied it to the task of grouping anonymous DNA sequences that may encode proteins. RESULTS: "Mixed COGs" of ORFs (clusters in which some sequences correspond to annotated genes and some do not) are attractive targets when seeking errors of gene predicion. Examination of mixed COGs reveals some situations in which genes appear to have been missed in current annotations and a smaller number of regions that appear to have been annotated as gene loci erroneously. This technique can also be used to detect potential pseudogenes or sequencing errors. Our method uses an adjustable parameter for degree of conservation among the studied genomes (stringency). We detail results for one level of stringency at which we found 83 potential genes which had not previously been identified, 60 potential pseudogenes, and 7 sequences with existing gene annotations that are probably incorrect. CONCLUSION: Systematic study of sequence conservation offers a way to improve existing annotations by identifying potentially homologous regions where the annotation of the presence or absence of a gene is inconsistent among genomes

Codon usage adaptation in prokaryotic genomes

La tesi esta basada en l'adaptació de l'ús de codons a genomes procariotes, especialment l'adaptació de l'ús de codons a una alta expressió. Hi ha un grup de genomes procariotes, els quals estan sota una selecció traduccional, que tenen un grup de gens amb un ús de codons esbiaixat de la resta de gens del genoma i adaptats a l'abundància dels tRNA. Hem desenvolupat un nou algoritme per a avaluar si un genoma esta sota selecció traduccional i predir els gens altament expressat de tots els genomes sota selecció traduccional. Aquestes prediccions són públiques a la base de dades HEG-DB (http://genomes.urv.cat/HEG-DB), la qual s'ha publicat a la revista Nucleic Acids Research. Les prediccions de gens altament expressats s'han fet servir com a filtre en les prediccions de gens adquirits per transferència horitzontal, ja que els gens altament expressats molts cops son predits com a falsos positius en la predicció de gens adquirits. Amb les dades de la predicció de gens altament expressats, també hem desenvolupat una nova eina Bioinformàtica, anomenada OPTIMIZER (http://genomes.urv.cat/OPTIMIZER) i publicada al Nucleic Acids Research, per tal d'optimitzar l'ús de codons d'un gen per a incrementar la seva expressió en experiments d'expressió heteròloga de proteïnes. També hem estudiat un cas particular d'adaptació de l'ús de codons. El cas de l' 'amelioration', que és l'adaptació de l'ús de codons que pateix un gen inserit en un genoma hoste. Aquest cas l'hem estudiat amb els gens mitocondrials que varen saltar al genoma nuclear i varen haver d'adaptar el seu us de codons mitocondrial a l'ús de codons del genoma nuclear. Per tal d'estudiar l''amelioration', hem desenvolupat un nou índex anomenat CAI esperat (eCAI) i una nova eina Bioinformàtica anomenada CAIcal (http://genomes.urv.cat/CAIcal), que està en procés de revisió a la revista BMC Bioinformatics. Analitzant l'anàlisi de l'ús de codons dels genomes completament sequenciats vàrem realitzar una troballa que s'aparta una mica del tema central de la tesi. Vàrem veure que els genomes que estan adaptats a la (hiper)termofília tenen un patró de l'ús de codons i d'aminoàcids diferent a la resta de genomes (mesòfils). Aquest fet ens ha permès descobrir casos de guany i pèrdua (recents i antics) de la capacitat d'adaptació termofílica en genomes procariotes. Aquests resultats han donat lloc a una publicació a la revista Trends in Genetics. Durant la tesi he realitzat una estada de 4 mesos (Febrer - Juny, 2006) en el laboratori de bioinformàtica del departament de biologia de la universitat nacional d'Irlanda a Maynooth sota la supervisió del Dr James McInerney on vaig desenvolupar un nou programa per a la comparació d'arbres filogenètics anomenat TOPD/FMTS (http://genomes.urv.cat/topd) el qual està publicat a la revista Bioinformatics.This thesis is based in codon usage adaptation in prokaryotic genomes, especially the codon usage adaptation to a high expression. In genomes under translational selection, the group of highly expressed genes has a codon usage adapted to the most abundant tRNA species. We have developed a new iterative algorithm which predicts a group of highly expressed genes in genomes under translational selection by using the Codon Adaptation Index and the group of ribosomal protein genes as a seed. We have developed a new genomic database, called HEG-DB, to store genes that are predicted as highly expressed in prokaryotic complete genomes under strong translational selection. The database is freely available at http://genomes.urv.cat/HEG-DB and it has been published in Nucleic Acids Research. The predicted highly expressed genes are used as an initial filter to reduce the number of false positives of the Horizontal Gene Transfer Database, due to highly expressed genes are usually false positive in predictions of acquired genes. We have developed a new web sever, called OPTIMIZER (http://genomes.urv.cat/OPTIMIZER), which has been published in Nucleic Acids Research, to optimize the codon usage of DNA or RNA sequences. This new web server can be used to predict and optimize the level expression of a gene in heterologous gene expression or to express new genes that confer new metabolic capabilities in a given species. We have also analyzed an especial case of codon usage adaptation, which is called 'amelioration'. The 'amelioration' is the adaptation of foreign genes to a new genome. This is the case of mitochondrial genes encoded in the human nuclear genome and originally encoded in the proto-mitochondria. To test the 'amelioration' process we have developed an expected value of CAI (eCAI) to find out whether the differences in the CAI are statistically significant or whether they are the product of biased nucleotide and/or amino acid composition and a new bioinformatics tool called CAIcal (http://genomes.urv.cat/CAIcal). We have also analyzed the evolution of thermophilic adaptation in prokaryotes and we suggest that the amino acid composition signature in thermophilic organisms is a consequence of or an adaptation to living at high temperatures, not its cause. Our findings suggest that there have been several cases where the capacity for thermophilic adaptation has been gained or lost throughout the evolution of prokaryotes. These results have been published in Trends in Genetics. During my thesis I have worked for four months in the Bioinformatics Laboratory of the Biology Department at the National University of Ireland under the supervision of Dr James O. McInerney where I developed a new software program to compare phylogenetic trees called TOPD/FMTS (http://genomes.urv.cat/topd), that has been published in Bioinformatics

Host cell engineering for the production of methacrylate esters

Microbial bioprocess serves as an alternative route for the sustainable production of a variety of chemicals. Recent bioprocess development efforts has allowed its application for the commercial production of certain industrially relevant chemicals. However, most are still in the exploratory or precommercialization stage due to a variety of bottlenecks that needs to be addressed prior to commercialization. This includes the bioprocess route being developed by Lucite International for the production of butyl methacrylate, which could be part of an integrated process for the production of methacrylate esters. In this bioprocess, commercial viability is attainable with a butyl methacrylate titre of 10-20% v/v. One of the bottlenecks in this proposed bioprocess is the toxicity of the bioproduct towards the production strain, which could limit the attainable product titre. A previous study on its toxicity led to the isolation of E. coli strains that can grow vial cultures with BMA at 20% v/v. However, these strains were unable to demonstrate tolerance in a well-mixed environment. Thus, there is still a need to develop a robust host strain that can tolerate butyl methacrylate at the desired product titre. E. coli BW25113 was explored as the potential host strain. Adaptive evolution via sequential batch and chemostat cultures were used to generate E. coli strains with tolerance for butyl methacrylate at 20% v/v. Genome shuffling was also used to further improve growth of E. coli with butyl methacrylate at 20% v/v. The possible mechanisms of tolerance for butyl methacrylate were determined with the use of genomic DNA and RNA sequencing of the evolved strains. The ability of the evolved strains to produce BMA was also tested by introduction of the heterologous pathway. Adaptive evolution, through sequential batch and chemostat cultures, was successful in generating various E. coli strains with improved growth in the presence of BMA up to 20% v/v. Each of the evolved strains acquired various mutations that include an acrR mutation along with either a marR, soxR, and rob. The mutations acquired allowed increased expression in acrAB, which suggests that the AcrAB-TolC efflux pump might play an important part in the tolerance for butyl methacrylate. Exposure of the evolved strain to butyl methacrylate stimulated the activation of genes that belong to the oxidative stress, heat shock, phage shock, and acid stress response systems and membrane modifying, energy generating, and essential building block synthesizing enzymes. It also resulted in the repression of the genes related to DNA replication and protein synthesis. The use of the evolved strains as host cell for production did not show an improvement in butyl methacrylate titre in comparison to the parental strain. However, butyl methacrylate production seems to be limited by factors other than toxicity. Thus, there is a need for further investigation and improvement of the production pathway

The Role of Genomic Context in Bacterial Growth Homeostasis

The growth of bacteria is a complex but well-orchestrated dance involving the repetitive and reproducible production of their diverse cellular components in order to divide. A lot can go astray and therefore the cell has developed several strategies in order to ensure everything remains synchronized. This problem is only further complicated as the cells adjust their growth rate to their living conditions resulting in ripple effects throughout the cell physiology. One notable change is that as nutrient availability and quality increases so too does the average size and the concentration of ribosomes in the cell. The latter enables the production of the largest macromolecule faction in the cell (proteins) including the production of more ribosomes required to maintain the protein synthesis requirements. With the increase in volume of the cell comes a required increase in surface area, and a disbalance between these two would result in untenable levels of internal pressure. How then do bacteria ensure that volume growth is synchronized with the production of cell envelope components so that cell homeostasis is maintained, especially in the face of fluctuating growth rate? Genomic context is known to assist in co-regulation of genes thereby synchronizing them to respond to different cellular stimuli. As the bacterial genome is highly fluid, the existence of conserved genomic contexts suggests important loci of co-regulation. Could it be in these gene clusters that a possible link between growth and surface expansion is found? To answer this question this thesis undertook three missions, firstly we established a genome comparison tool (www.GenCoDB.org) that will take advantage of the ever-growing availability of bacterial genomes to assist us in the analysis, comparison, and quantification of genome contexts. This will rely on novel strategies in order to: accommodate the breadth of genome data available in a computationally efficient manner, reduce the effect of sampling bias that plague most bacterial datasets and ensure candidates are considered significant for their evolutionary context. The availability of GenCoDB is sure to facilitate genomic context research in the microbiology community and improve accessibility to non-bioinformatics to this wellspring of important biological data. With the swath of genomic neighbourhood data, we then sought to understand and analyse the evolution of conserved gene clusters in order to narrow down possible volume-surface regulating candidates. By tracking the evolution of gene clusters throughout the Bacteria kingdom we found that co-orientation is strongly conserved, however, this does not influence the subsequent context around the cluster nor the expansion of the cluster. We found that vertical transmission and not horizontal gene transfer was found to be the driving factor of gene cluster occurrence in chromosomes and that the origin and terminus are hotspots for cluster maintenance. Finally, we found that despite the apparent frequency of operon organization in gene clusters, gene clusters appear to be maintained due to other selective pressures such as within-cluster protein-protein interactions and the essential status of their genes. We suggest that operons are a consequence and not a cause co-localization over evolutionary time. We identified a single gene cluster candidate that met all the requirements we believe are required for cell growth homeostasis of synchronized surface and volume expansion. These requirements were a broad conservation within Bacteria, and a connection between ribosome-associated proteins (growth) with cell envelope synthesizes. In agreement with our evolution studies we found that whilst the cluster was co-regulated this did not appear to be the selective pressure that brought these different processes together. Instead we found a potential role of genomic channelling, linking the production of pyrimidines with the synthesis of the cell envelope which is reliant on the co-localization of this cluster. Together, this work will forward the understanding of chromosome evolution in Bacteria and the potential implications of genomic context in metabolite utilization. It challenges the roles that operons and horizontal gene transfer play in the long-term evolution of gene order and it provides a new quantitative and statistical resource providing access to over 1.9 million gene neighbourhoods

Specific and global networks of gene regulation in Streptomyces coelicolor

Streptomycetes produce a plethora of secondary metabolites, including antibiotics, and undergo a complex developmental cycle. The GC-rich species, Streptomyces coelicolor is used by many laboratories as a model for studying gene regulation, morphological development, cellular physiology and microbial signalling. A genome-wide view of many factors that control S. coelicolor gene expression at the level of transcription initiation and beyond was obtained successfully from a combination of RNA-sequencing approaches. For instance, sites of transcription initiation, vegetative promoters, leaderless mRNAs, sites involved in the processing and degradation of rRNA, tRNA and mRNA, and small RNAs, including those that may be involved in attenuation-like switching mechanisms, were successfully detected. Many of the small RNAs identified in this study are novel. Overall, our approaches show the ability to identify new layers of transcriptional complexity associated with several key regulators of secondary metabolism and morphological development in S. coelicolor. Here we were able to show that AtrA activates the transcription of ssgR, the gene product of which is in turn required for the transcription of ssgA, the best-studied SALP, which has a crucial role in septation and the morphology aerial hyphae. AtrA also binds to the promoter region of leuA2, which encodes α-isopropylmalate that directly utilise acetyl-CoA. Interestingly, crude extract from M1146 strain (Δact, Δred, Δcpk, and Δcda) was found to inhibit the DNA-binding activity of AtrA; however, the specificity of the small molecule(s) interaction with AtrA should be investigated. Addition of the 3 x FLAG tag™ to the N-terminus of AtrA does not hinder its ability to substitute functionally for untagged AtrA in S. coelicolor and can be used for the mapping of AtrA binding sites by ChIP-sequencing. Taken together the above suggest that AtrA has a direct role in morphological development and coordinating the utilisation of acetyl-CoA for primary and secondary metabolism

Genome evolution and systems biology in bacterial endosymbionts of insects

Gene loss is the most important event in the process of genome reduction that appears associated with bacterial endosymbionts of insects. These small genomes were derived features evolved from ancestral prokaryotes with larger genome sizes, consequence of a massive process of genome reduction due to drastic changes in the ecological conditions and evolutionary pressures acting on these prokaryotic lineages during their ecological transition to host-dependent lifestyle. In the present thesis, the process of genome reduction is studied from different perspectives. In the first chapter, genome rearrangements have been studied in a set of 31 complete γ-proteobacterial genomes that includes five genomes of bacterial endosymbionts of insects. This is carried out by comparing the order of a subset of 244 single-copy orthologous genes presents in all the genomes and calculating the number of inversions and breakpoints between each genome pair. This reveals that inversions were the main rearrangement event in γ-proteobacteria evolution, with a progressive increase in the number of rearrangements with increased evolutionary distance. However, significant heterogeneity in different γ-proteobacterial lineages was also detected, with a significant acceleration in the rates of genome rearrangements in bacterial endosymbionts of insects at initial stages of the association. In the second chapter, the structure and functional capabilities of Sodalis glossinidius has been studied. S. glossinidius is the secondary endosymbiont of tsetse flies, and it´s at very initial stages of genome reduction process. It´s genome is experiencing a massive process of gene inactivation, with 972 pseudogenes (inactivated genes) that were described but not annotated in the original annotation of the genome. In this chapter, a complete functional re-annotation of this genome was carried out, that includes the characterization of 1501 pseudogenes though analysis of S. glossinidius intergenic regions. A massive presence of CDSs related with mobile genetic elements and surface proteins were detected, being also the functional classes most affected by pseudogenization. The reconstruction of the metabolic map of S. glossinidius revealed a functional profile very similar to that of free-living enterics, with inactivation of L-arginine biosynthesis pathway, whereas the comparison with Wigglesworthia glossinidia (tsetse primary endosymbiont) reveals possible cases of metabolic complementation between both tsetse endosymbionts at thiamine, coenzyme A and tetrahydrofolate biosynthesis level. Finally, in the third chapter of the thesis, the complete reductive evolution process associated with S. glossinidius was studied from a systems biology perspective through the reconstruction of their genome-scale metabolic networks at different stages of this process and the prediction of their internal reaction fluxes under different external conditions through Flux Balance Analysis. This revealed the decisive role of the pseudogenization of genes involved in L-arginine and glycogen biosynthesis and specially the pseudogenization of the key anaplerotic enzyme phosphoenolpyruvate carboxylase in the ecological transition to a host-dependent lifestyle experienced by S. glossinidius. A progressive decrease in network robustness to gene deletion events and to changes in particular reaction fluxes were detected. Finally, reductive evolution simulations over the functional network of S. glossinidius under different external conditions revealed a higher plasticity in minimal networks evolved in a nutrient-rich environment, and allow defining different sets of essential and disposable genes based on their presence or absence in minimal metabolic networks. These essential genes had more optimized patterns of codon usage and more restricted patterns of sequence evolution than disposable genes that could be lost without affecting the functionality of the network. However, lineage-specific estimates of dN and dS in S. glossinidius and Escherichia coli revealed that common features of ancient bacterial endosymbionts like acceleration in the rates of sequence evolution and the loss of adaptative codon usage were starting to affect S. glossinidius evolution.En esta tesis doctoral, el proceso de reducción genómica característico de bacterias endosimbiontes de insectos ha sido estudiado utilizando diferentes aproximaciones computacionales basadas en la genómica comparada y la biología de sistemas. Por un lado, las dinámicas de reordenaciones genómicas han sido estudiadas en un subconjunto de 31 genomas completos de γ-proteobacterias que incluyen 5 genomas completos de endosimbiontes bacterianos de insectos, revelando una aceleración significativa de las tasas de reordenaciones en estos genomas en etapas iniciales del proceso de reducción. Posteriormente, el genoma de Sodalis glossinidius, el endosimbionte secundario de la mosca tsétsé, fue re-anotado con el objetivo de evaluar el impacto de los procesos de inactivación génica y proliferación de elementos genéticos móviles en etapas tempranas del proceso de reducción, asi como su impacto sobre las capacidades funcionales de la bacteria en el contexto ecológico de su coexistencia con el endosimbionte primario ancestral Wigglesworthia glossinidia. Finalmente, el proceso completo de reducción genómica en S. glossinidius ha sido estudiado a través de la reconstrucción de su red metabólica a diferentes etapas de este proceso y su análisis funcional mediante Análisis de Balance de Flujos, evaluando la robustez de las redes frente a sucesos de deleción asi como las dinámicas evolutivas de genes esenciales y no esenciales en base a su presencia en redes mínimas evolucionadas a partir de la red funcional. Este análisis permitió identificar sucesos de inactivación génica con efectos drásticos sobre las capacidades funcionales del sistema como los genes implicados en la biosíntesis de arginina y glicógeno, y especialmente la inactivación de la enzima fosfoenolpiruvato carboxilasa, asi como una disminución progresiva de la robustez de las redes frente a diferentes sucesos mutacionales asociada al proceso de pérdida génica. Finalmente, simulaciones de evolución reductiva sobre la red funcional bajo diferentes condiciones de entorno ha permitido definir conjuntos de genes esenciales y delecionables en base a su presencia o ausencia en las redes mínimas producto de las simulaciones, revelando una mayor conservación a nivel de secuencia y un uso de codones más optimizado en genes esenciales frente a genes cuya pérdida no afecta a la funcionalidad del sistema

RNA-basierte Kontrolle der Expression des Typ III Sekretionssystems von Yersinia pseudotuberculosis

Expression of Ysc-Yop type III secretion system requires a tight regulation, which is conferred by two RNA-binding proteins YopD and CsrA. The underlying study demonstrated that expression of the transcriptional master regulator lcrF of the Ysc-Yop T3SS is controlled in intertwined feedback loops by these RNA-binding proteins. CsrA is a global acting post-transcriptional regulator that coordinates metabolic and virulence gene expression. This study demonstrated that expression of csrA is precisely controlled in an autoregulatory feedback loop, indirectly on the transcriptional and directly on the post-transcriptional level. CsrA was found to directly bind to two sites in the 5'-UTR of its transcript, from which one site is located within the ribosomal binding site. By this mechanism, CsrA was shown to block translational initiation and restrict CsrA synthesis when a certain CsrA amount is reached. Under non-secretion conditions, the RNA-binding protein YopD was shown to bind to the csrA transcript thereby masking a CsrA binding site and preventing the direct autoinhibition of csrA. Analysis of the role of CsrA and YopD in the lcrF regulation demonstrated a direct interaction of both RNA-binding proteins with the yscW-lcrF transcript. The 5'-UTR of lcrF contains an RNA-thermometer structure, which prevents ribosomal access through a base-pairing mechanism. Furthermore, this study demonstrated, CsrA interacts with the ribosomal binding site of lcrF and modulates the opening state of the RNA thermometer. Both regulators modulate the stability of the lcrF transcript. CsrA stabilizes and YopD destabilizes the lcrF mRNA. This is caused by the YopD- and CsrA-dependent control of the RNA degradosome. Furthermore, new putative CsrA targets could be identified by an interactome analysis with FLAG-tagged CsrA protein. At body host temperature, CsrA specifically interacts with tRNAs and transcripts of tRNA processing proteins. In summary, this study revealed the complex interplay of YopD and CsrA in controlling the expression of the T3SS master regulator lcrF. Both regulators are in involved in the post-transcriptional control of lcrF expression under non-secretion conditions and confer an antagonistic regulation.Die Expression des Ysc-Yop Typ III Sekretionssystem setzt eine präzise Regulation voraus. Diese erfolgt durch die zwei RNA-bindenden Proteine YopD und CsrA. Die vorliegende Studie zeigte, dass die Expression des transkriptionellen Hauptregulators des Ysc-Yop T3SS lcrF in einer ineinander verschlungenen Rückkopplung durch diese RNA-bindenden Proteine kontrolliert wird. CsrA ist ein globaler posttranskriptioneller Regulator, der sowohl die Expression von metabolischen als auch Virulenzgenen koordiniert. Diese Studie zeigte, dass die Expression von csrA präzise in einer autoregulatorischen Rückkopplung kontrolliert wird - indirekt auf transkriptioneller und direkt auf posttranskriptioneller Ebene. Es konnte gezeigt werden, dass CsrA das eigene Transkript an zwei Stellen bindet, von der eine mit der ribosomalen Bindestelle überlappt. Durch diesen Mechanismus, blockt CsrA die Translation seines Transkripts und begrenzt die Synthese von weiteren CsrA Molekülen. Unter Nicht-Sekretionsbedingungen bindet das RNA-bindende Protein YopD das csrA-Transkript und maskiert somit eine der beiden CsrA Bindestelle. Dadurch wird die direkte Autoinhibition von csrA behindert. Die Analyse der Rolle von YopD und CsrA in der lcrF Regulation zeigte die direkte Interaktion beider RNA-Bindeproteine mit dem yscW-lcrF Transkript. Die 5'-UTR von lcrF enthält ein RNA-Thermometer, welches den ribosomalen Zugang durch einen Basenpaarungsmechanismus einschränkt. Desweiteren konnte in dieser Studie gezeigt werden, dass CsrA mit der ribosomalen Bindestelle von lcrF interagiert und den Öffnungsgrad des RNA-Thermometers moduliert. Die lcrF-RNA-Stabilität wird durch beide Regulatoren moduliert. CsrA stabilisiert und YopD destabilisiert das lcrF-Transkript. Dies wird durch die CsrA- und YopD-bedingte Kontrolle des RNA Degradosoms verursacht. Außerdem konnten neue mögliche CsrA-Ziel-Transkripte durch eine Interaktionsanalyse mit FLAG-markiertem CsrA-Protein aufgedeckt werden. Bei 37 °C interagiert CsrA spezifisch mit tRNAs sowie den Transkripten von tRNA-modifizierenden Proteinen. Zusammengefasst beleuchtete diese Studie das komplexe Zusammenspiel von YopD und CsrA in der Kontrolle des T3SS-Hauptregulators lcrF. Beide Regulatoren sind an der posttranskriptionellen Kontrolle der lcrF-Expression beteiligt, haben aber einen entgegengesetzten Effekt

NMR-Untersuchungen zu dynamischen Umfaltungsprozessen in RNA-Molekülen

The following thesis is concerned with the elucidation of structural changes of RNA molecules during the time course of dynamic processes that are commonly denoted as folding reactions. In contrast to the field of protein folding, the concept of RNA folding comprises not only folding reactions itself but also refolding- or conformational switching- and assembly processes (see chapter III). The method in this thesis to monitor these diverse processes is high resolution liquid-state NMR spectroscopy. To understand the reactions is of considerable interest, because most biological active RNA molecules function by changing their conformation. This can be either an intrinsic property of their respective sequence or may happen in response to a cellular signal such as small molecular ligand binding (like in the aptamer and riboswitch case), protein or metal binding. The first part of the thesis (chapters II & III) provides a general overview over the field of RNA structure and RNA folding. The two chapters aim at introducing the reader into the current status of research in the field. Chapters II is structured such that primary structure is first described then secondary and tertiary structure elements of RNA structure. A special emphasis is given to bistable RNA systems that are functionally important and represent models to understand fundamental questions of RNA conformational switching. RNA folding in vitro as well as in vivo situations is discussed in Chapter III. The following chapters IV and V also belong to the introduction part and review critically the NMR methods that were used to understand the nature and the dynamics of the conformational/structural transitions in RNA. A general overview of NMR methods quantifying dynamics of biomolecules is provided in chapter IV. A detailed discussion of solvent exchange rates and time-resolved NMR, as the two major techniques used, follows. In the final chapter V of the first part the NMR parameters used in structure calculation and structure calculation itself are conferred. The second part of the thesis, which is the cumulative part, encompasses the conducted original work. Chapter VI reviews the general NMR techniques applied and explains their applicability in the field of RNA structural and biochemical studies in several model cases. Chapter VII describes the achievement of a complete resonance assignment of an RNA model molecule (14mer cUUCGg tetral-loop RNA) and introduces a new technique to assign quaternary carbon resonances of the nucleobases. Furthermore, it reports on a conformational analysis of the sugar backbone in this RNA hairpin molecule in conjunction with a parameterization of 1J scalar couplings. Achievements: • Establishment of two new NMR pulse-sequences facilitating the assignment of quaternary carbons in RNA nucleobases • First complete (99.5%) NMR resonance assignment of an RNA molecule (14mer) including 1H, 13C, 15N, 31P resonances • Description of RNA backbone conformation by a complete set of NMR parameters • Description of the backbone conformational dependence in RNA of new NMR parameters (1J scalar couplings) Chapters VII & VIII summarize the real-NMR studies that were conducted to elucidate the conformational switching events of several RNA systems. Chapter VIII gives an overview on the experiments that were accomplished on three different bistable RNAs. These molecules where chosen to be good model systems for RNA refolding reactions and so consequently served as reporters of conformational switching events of RNA secondary structure elements. Achievements: • First kinetic studies of RNA refolding reactions with atomic resolution by NMR • Application of [new] RT-NMR techniques either regarding the photolytic initiation of the reaction or regarding the readout of the reaction • Discovery of different RNA refolding mechanisms for different RNA molecules Deciphering of a general rule for RNA refolding methodology to conformational switching processes of RNA tertiary structure elements. The models for these processes were a) the guanine-dependent riboswitch RNA and b) the minimal hammerhead ribozyme. Achievements: • NMR spectroscopic assignment of imino-resonances of the hypoxanthine bound guanine-dependent riboswitch RNA • Application of RT-NMR techniques to monitor the ligand induced conformational switch of the aptamer domain of the guanine-dependent riboswitch RNA at atomic resolution • Translation of kinetic information into structural information • Deciphering a folding mechanism for the guanine riboswitch aptamer domain • Application of RT-NMR techniques to monitor the reaction of the catalytically active mHHR RNA at atomic resolution In the appendices the new NMR pulse-sequences and the experimental parameters are described, which are not explicitly treated in the respective manuscripts.Die vorliegende Doktorarbeit beschäftigt sich mit den strukturellen Änderungen in RNA Molekülen während dynamischer konformationeller Änderungen, die gemeinhin als RNA-Faltung bezeichnet werden. Im Gegensatz zur Proteinfaltung sind RNA-Faltungsprozesse nicht exklusiv als die Faltung einer definierten Konformation aus einem Ensemble an ungefalteten, d.h. ausgehend von unstrukturierten Molekülen, zu verstehen. RNA-Faltung beinhaltet vielmehr die strukturelle Umwandlung verschiedener stabiler Konformationen (die als RNA-Umfaltung benannt wird) und den Aufbau von molekularen Komplexen aus mehreren Molekülen (siehe Kapitel III). Die experimentelle Technik, die hier zur Untersuchung dieser Prozesse genutzt wurde, ist die hochauflösende Flüssig-NMR-Spektroskopie. Das Verständnis der strukturellen und biophysikalischen Grundlagen solcher Umfaltungsreaktionen von RNA ist essentiell, da solche konformationellen Änderungen die biologische Funktion der Moleküle modulieren. Dabei ist zu bemerken, dass eine Umfaltungsreaktion eine intrinsische Eigenschaft einer gegebenen RNA-Sequenz sein kann oder die Antwort auf ein externes zelluläres Signal, wie die Bindung eines niedermolekularen Liganden (z.B. in Aptameren und in Riboswitch RNAs), eines Proteins oder eines Metall-Ions. Der erste Teil dieser Doktorarbeit (Kapitel I & II) hält einen Überblick über die Themengebiete RNA-Struktur und RNA-Faltung bereit. Beide Kapitel führen in den derzeitigen Stand der Forschung ein. Kapitel II führt dabei entlang der hierarchischen Ordnung von RNA Molekülen und diskutiert die Eigenschaften von Primär-, Sekundär- und Tertiär-Strukturelementen. Ein besonderes Augenmerk wird dabei auf bistabile RNA Systeme gelegt; ihre wichtige biologische Funktionalität wird dargestellt, ebenso wird das Potential ausgeleuchtet, diese funktionale Klasse von RNA Molekülen als Modellsysteme zu nutzen, um fundamentale Fragen zu konformationellen Übergängen in RNA zu beantworten. In Kapitel III folgt sodann die Diskussion über RNA-Faltung in in vitro Experimenten als auch im zellulären Kontext (in vivo). Die Kapitel IV und V besprechen die NMR-spektroskopischen Techniken, die genutzt werden, um die Art und die dynamischen Eigenschaften von konformationellen/strukturellen Umwandlungen in RNA zu untersuchen. Hierbei wird der Schwerpunkt auf die verwendeten Techniken des Wasseraustauschs an labilen Protonen und der zeitaufgelösten NMR-Spektroskopie gelegt. Der zweite Teil der Doktorarbeit fasst kumulativ die durchgeführten Studien zusammen. Kapitel VI bespricht hierbei die grundlegenden NMR Techniken, die zur Strukturaufklärung von RNA Molekülen angewendet werden und zeigt deren Anwendungsmöglichkeiten an unterschiedlichen Beispielen von strukturellen und biochemischen Studien. Das folgende Kapitel VII beschreibt die komplette Resonanzzuordnung eines RNA Modell-Moleküls (14mer cUUCGg tetra-loop RNA) und stellt eine neue Pulstechnik vor, die zur Zuordnung der Resonanzen von quatären Kohlenstoffen in Purinbasen benützt werden kann. Weiterhin schließt sich ein Report an, wie die Konformation des Zuckerrückgrates in RNA-Molekülen bestimmt wird und schlägt mittels einer an oben genanntem Modellsystem durchgeführte Parametrisierung 1J skalare Kopplungen als neue Strukturparameter vor. Kapitel VII & VIII fassen die hierzu durchgeführten RT-NMR Studien zusammen. Kapitel VIII gibt hierbei einen Überblick über die Untersuchungen an drei bistabilen RNA-Systemen. Diese Moleküle wurden ausgewählt, da sie als Modelle für RNA-Umfaltungsreakionen dienen. Das finale Kapitel IX behandelt die Anwendung der oben ausgeführten neuen Methodologie auf konformationelle Umwandlungen von RNA Tertiär-Strukturelementen: a) Guanin-abhängige Riboswitch RNA (GSW) und b) Minimales "hammerhead" Ribozym (mHHR)