RNA silencing is a major regulator of gene expression in plants, controlling from development to transposable element silencing and stress responses. As part of the silencing machinery, micro (mi)RNAs orchestrate silencing of their targets, either directly or through cascades of secondary small interfering (si)RNAs. To investigate the role of RNA silencing in plant immunity, I chose to focus on the miR482/2118 family, because of its diversity and presence in many plant species since the appearance of seed plants, with most genomes containing several copies, and because its members target sequences conserved in a family of disease resistance genes known Nucleotide biding site leucine-rich repeat (NLR) genes. In this dissertation, I wanted to address the extent to which the miRNA family and its derived phasiRNAs regulate expression of defence genes as well as contribute to quantitative resistance in crops. I explore the structural differences of miR482/2118 members in Solanum lycopersicum and show that they are functionally significant and affect their target preferences. My approach was based on small RNA sequencing and degradome data to characterize targets of these miRNAs, including the recently discovered tomato TAS5 locus. I also generated transgenic tomatoes constitutively expressing target mimic RNAs that sequester different miR482/2118 members. These tomato mimic RNA lines were less susceptible than their non-transgenic precursors to pathogens Phytophthora infestans and Pseudomonas syringae. Additionally, I investigated the role of small RNAs and their effector proteins during vegetative and reproductive development in tomato. I employed transcript and small RNA sequencing and CRISPR-Cas9 techniques of gene editing to investigate the impact of these factors in gamete viability and transposable element silencing in vegetative meristems. The results presented here provide new evidence about the extent that RNA silencing contributes to the regulation of vital processes in plants. My study primarily explores the extent to which structural differences between the members of the miR482/2118 family affect their range of action, and the use of target mimics against these miRNAs as biotechnological approach for enhancing disease resistance in highly bred cultivars.

abstract: Biomass synthesis is a competing factor in biological systems geared towards generation of commodity and specialty chemicals, ultimately limiting maximum titer and yield; in this thesis, a widely generalizable, modular approach focused on decoupling biomass synthesis from the production of the phenylalanine in a genetically modified strain of E. coli BW25113 was explored with the use of synthetic trans-encoded small RNA (sRNA) to achieve greater efficiency. The naturally occurring sRNA MicC was used as a scaffold, and combined on a plasmid with a promoter for anhydrous tetracycline (aTc) and a T1/TE terminator. The coding sequence corresponding to the target binding site for fourteen potentially growth-essential gene targets as well as non-essential lacZ was placed in the seed region of the of the sRNA scaffold and transformed into BW25113, effectively generating a unique strain for each gene target. The BW25113 strain corresponding to each gene target was screened in M9 minimal media; decreased optical density and elongated cell morphology changes were observed and quantified in all induced sRNA cases where growth-essential genes were targeted. Six of the strains targeting different aspects of cell division that effectively suppressed growth and resulted in increased cell size were then screened for viability and metabolic activity in a scaled-up shaker flask experiment; all six strains were shown to be viable during stationary phase, and a metabolite analysis showed increased specific glucose consumption rates in induced strains, with unaffected specific glucose consumption rates in uninduced strains. The growth suppression, morphology and metabolic activity of the induced strains in BW25113 was compared to the bacteriostatic additives chloramphenicol, tetracycline, and streptomycin. At this same scale, the sRNA plasmid targeting the gene murA was transformed into BW25113 pINT-GA, a phenylalanine overproducer with the feedback resistant genes aroG and pheA overexpressed. Two induction times were explored during exponential phase, and while the optimal induction time was found to increase titer and yield amongst the BW25113 pINT-GA murA sRNA variant, overall this did not have as great a titer or yield as the BW25113 pINT-GA strain without the sRNA plasmid; this may be a result of the cell filamentation. / Dissertation/Thesis / Masters Thesis Chemical Engineering 2016

Staphylococcus aureus is a highly problematic human pathogen due to its ability to cause devastating infections, paired with a capacity to withstand the action of a large fraction of available antibiotics. Both pathogenicity and antibiotic resistance are encoded by numerous genomic elements, though the expression of these factors is energetically costly and not always beneficial for cellular survival. Therefore, S. aureus has developed sophisticated regulatory networks to integrate a multitude of signals, enabling it to navigate the delicate balance between its pathogenic lifestyle and baseline needs for cellular energy homeostasis. It is thus imperative to study S. aureus behavior and its underlying regulatory circuits not as isolated entities, but rather holistically as part of an optimized, highly interconnected system. To do so, we must seek to achieve a comprehensive understanding of all encoded regulators, that is, not only historically well defined elements like transcription factors, two-component systems and σ factors, but also the lesser studied ’non-classical’ regulators like small regulatory RNAs and regulatory subunits of RNA-dependent RNA polymerase (RNAP). To this end, we describe here the identification of numerous, previously unidentified sRNAs and their incorporation into a new standardized cataloging and annotation system. The identification and annotation procedures are based on a number of RNAseq experiments performed in three different S. aureus backgrounds (MRSA252, NCTC 8325, and USA300). We then apply RNAseq to evaluate the expression patterns of these elements when grown in human serum, thus probing for possible connections between sRNAs and S. aureus pathogenicity. In addition, we characterize the role of two small RNAP subunits, δ and ω, for S. aureus RNAP function. δ is of particular interest, as it is unique to Gram-positive bacteria; deletion of the subunit results in a loss of transcriptional stringency and decreased expression of numerous virulence determinants. These alterations are accompanied by impaired survival of the δ mutant in whole human blood, increased phagocytosis by human leukocytes, and decreased survival in a murine model septicemia when compared to its parental strain. In contrast, there is no indication of direct and gene-specific transcriptional functions for ω. Rather, we describe a role for ω in the structural integrity of the RNAP complex, where its loss leads to a structural disturbance in the RNAP complex that causes altered affinities for (alternative) σ factors and the δ subunit. Overall, the findings presented here contribute to a better understanding of the intricate regulatory systems that guide the lifestyle of an organism that presents an immense burden to patients and our health care system alike.

Serratia sp. ATCC 39006 (S39006) is a Gram-negative bacterium that is virulent in plant (potato) and animal (Caenorhabditis elegans) models. It produces two secondary metabolite antibiotics, prodigiosin and a carbapenem, and the plant cell wall degrading exoenzymes, pectate lyase and cellulase. A complex regulatory network controls production of prodigiosin, including a quorum sensing (QS) system, and the role of post-transcriptional regulation was investigated. It was hypothesized that Hfq-dependent small regulatory RNAs (sRNAs) might also play a role. Hfq is an RNA chaperone involved in post-transcriptional regulation that plays a key role in stress response and virulence in other bacterial species. An S39006 ∆hfq mutant was constructed and in the mutants production of prodigiosin and carbapenem was abolished, while production of the QS molecule, butanoyl homoserine lactone (BHL), was unaffected. Using transcriptional fusions, it was found that Hfq regulated the QS response regulators, SmaR and CarR. Additionally, exoenzyme production and swimming motility were decreased in the ∆hfq mutant, and virulence was attenuated in potato and C. elegans. It was also shown that the phenotype of an hfq mutant is independent of its role in regulating the stationary phase sigma factor, rpoS. In order to define the complete regulon of Hfq and identify relevant potential sRNAs, deep sequencing of strand-specific cDNAs (RNA-seq) was used to analyse the whole transcriptome of S39006 WT and the ∆hfq mutant. The regulon of another post-transcriptional regulator, RsmA, also involved in regulating prodigiosin production, was investigated by performing RNA-seq on an rsmA mutant. Moreover, global changes in the proteome of the hfq mutant was analysed using an LC-MS/MS approach with isobaric tags for relative and absolute quantification (iTRAQ). This study confirms a role for Hfq in pathogenesis and the regulation of antibiotic production in S39006, and begins to provide a systems-level understanding of Hfq and RsmA regulation using a combination of transcriptomics and proteomics.

Les petits ARN (ARNS) sont impliqués dans la régulation post-transcriptionnelle des voies métaboliques et dans les réponses aux stress et la virulence. Chez Staphyloccus Aureus, RNAIII, l’effecteur principal du système AGR, agit comme un ARN antisens pour réguler l’expression de protéines de parois cellulaires et de nombreux cytotoxines en fin de phase de croissance exponentielle. Une série de petits ARN-RSA, ont été découverts par notre équipe en 2009. Les objectifs de cette étude sont : 1) Construire des outils génétiques pour étudier leurs fonctions, II) Analyser leur expression dans diverses conditions in Vitro et in Vivo. Ainsi, nous avons déterminé le niveau d’expression de 4 ARNS (RSAA, RSAE, RASG et RSAH) et ARNIII, lorsque la bactérie a été exposée à divers antibiotiques ou en contact avec d’autres bactéries comme Pseudomonas. Des concentrations sub-inhibitrices (1/16 et 1/8 MIC) de la lévofloxacine augmentent l’expression de RSAH dans RN6390, mas pas dans HG001. Le niveau d’expression de RSAG et RSAH ont été augmentés en présence de P. Aeruginosa, mais le mécanisme de cet effet n’a pas été élucidé. Les données obtenues à partir d’échantillons humains ont montré que l’expression globale des cinq petits ARN a été extrêmement variable dans les abcès, plus homogène dans les crachats des patients atteints de mucoviscidose, et très homogène dans les échantillons de colonisation nasale. Le modèle d’expression particulier des ARNS associé à la colonisation nasale pourrait refléter le commensalisme de S. Aureus dans cette niche. / Small RNAs (sRNAs) are involved in the post-transcriptional regulation of metabolic pathways and in responses to stress and virulence. In Staphylococcus aureus, RNAIII, the main effector of the Agr system, acts as an antisense RNA to regulate the expression of many cell wall proteins and cytotoxins in late-exponential growth phase. A series of novel sRNAs – Rsa were discovered by our team in 2009. The objective of this study is i) to construct genetic tools to study their functions and ii) to analyze their expression in various conditions in vitro and in vivo. Thus, we determined the expression level of 4 sRNAs (RsaA, RsaE, RsaG and RsaH) and RNAIII, when the bacterium was exposed to various antibiotics or in contact with other bacteria such as Pseudomonas. Subinhibitory concentrations (1/16 and 1/8 MIC) of levofloxacin could enhance the RsaH expression in RN6390, not in HG001. RsaG and RsaH level were increased in the presence of P. aeruginosa, however the mechanism of this effect has not been elucidated. Data obtained from human samples showed that the global expression of the five sRNAs was extremely variable in the abscesses, more homogeneous in the sputa of cystic fibrosis patients, and highly uniform in the nasal carrier samples. The unique expression pattern of sRNA associated with nasal colonization might reflect the commensalism of S. aureus in this niche.

No description available.

Ancestral environmental conditions can instruct offspring development, although the mechanism(s) underlying such transgenerational epigenetic inheritance is unclear. In murine models focused on paternal dietary effects, we and others have identified tRNA fragments (tRFs) in mature sperm as potential carriers of epigenetic information. In our search for molecular targets of specific tRFs, we observed that altering the level of 5’-tRF Glycine-GCC (tRF-GG) in mouse embryonic stem cells (mESCs) and preimplantation embryos modulates the expression of the endogenous retrovirus MERV-L and genes regulated by MERV-L. Intriguingly, transient derepression of MERV-L is associated with totipotency of two-cell stage embryos and a subset of two-cell-like mESCs. Here, I reveal the mechanistic basis for tRF-GG regulation of MERV-L. I show that tRF-GG supports the production of numerous small nuclear RNAs associated with the Cajal body, in mouse and human embryonic stem cells. In particular, tRF-GG modulates the levels of U7 snRNA to ensure an adequate supply of histone proteins. This in turn safeguards heterochromatin-mediated transcriptional repression of MERV-L elements. Importantly, tRF-GG effects on histone mRNA levels, activity of a histone 3’UTR reporter, and expression of MERV-L associated transcripts can all be suppressed by appropriate manipulation of U7 RNA levels. I also show that hnRNPF and H bind directly to tRF-GG, and display a stark overlap of in vivo functions to tRF-GG. Together, this data uncovers a conserved mechanism for a 5’ tRNA fragment in the fine-tuning of a regulatory cascade to modulate global chromatin organization during pre-implantation development.

Enteropathogenic bacteria, such as Shigella and Salmonella, are associated with diarrheal diseases, which remain a significant cause of infant mortality worldwide. The secretion of protein effectors by the type III secretion apparatus (T3SA) is used by these pathogens to invade human cells and modulate host cell functions. First, we used RNA-Seq to analyze the differential transcriptome of Shigella flexneri when the T3SA is active or inactive. This allowed us to identify two uncharacterized genes that were temporarily named gem1 and gem3 and whose expression was regulated by MxiE and IpgC as other late substrates of the T3SA. Finally, we pursued the characterization of gem1 and gem3 at the protein level and renamed them icaT and icaR, respectively, when we found their protein products were secreted by the T3SA. Furthermore, we find homologs of icaT and icaR with a conserved MxiE box in several E. coli phylogroups. We also demonstrated that these homologous genes could be reactivated when both MxiE and IpgC were introduced in these strains. This discovery paved a new perspective on the evolution of pathogenesis into the E. coli lineage as both commensal and pathogenic strains harbored these genes. Treating infections caused by Enterobacteriaceae is becoming more challenging due to growing antibiotic resistance and no vaccines are widely available. Accordingly, the World Health Organization (WHO) recognized that we entered the "post-antibiotic era," where new antibiotics or antivirulence drugs are urgently needed, including for Shigella. The T3SA is an attractive target for antivirulence drugs, which may become alternative to classical antibiotics. Through screening 3,000 compounds, we found two novel inhibitors of the T3SA. Our data suggested that one of these candidate inhibitors, a dipyridyl-containing compound, reduces the virulence of Shigella at the transcriptional level. Indeed, the virulence inhibition occurs via the repression of the transcriptional activator VirB by the small chromosomal RNA RyhB, which is upregulated by this compound through an unknown mechanism involving the pyridyl groups. The repression of VirB induced by this molecule reduce the expression of several genes encoding parts of the T3SA. In comparison, the second compound is a quinone that seems to affect the assembly of the T3SA.

In this work, dRNA-seq (differential RNA sequencing) and RNAtag-seq were applied to first define the global transcriptome architecture of C. difficile, followed by Hfq RIP-seq (RNA immunoprecipitation followed by RNA-seq) and RIL-seq (RNA interaction by ligation and sequencing) to characterize the Hfq-mediated sRNA interactome on a transcriptome-wide scale. These approaches resulted in the annotation of > 60 novel sRNAs. Notably, it not only revealed 50 Hfq-bound sRNAs, but also > 1000 mRNA-sRNA interactions, confirming Hfq as a global RNA matchmaker in C. difficile. Similar to its function in Gram-negative species, deletion of Hfq resulted in decreased sRNA half-lives, providing evidence that Hfq affects sRNA stability in C. difficile. Finally, several sRNAs and their function in various infection relevant conditions were characterized. The sRNA nc085 directly interacts with the two-component response regulator eutV, resulting in regulation of ethanolamine utilization, an abundant intestinal carbon and nitrogen source known to impact C. difficile pathogenicity. Meanwhile, SpoY and SpoX regulate translation of the master regulator of sporulation spo0A in vivo, thereby affecting sporulation initiation. Furthermore, SpoY and SpoX deletion significantly impacts C. difficile gut colonization and spore burden in a mouse model of C. difficile infection. / Der anaerobe Gram-positive humanpathogene Erreger Clostridioides difficile (C. difficile) gilt als Hauptursache für nosokomiale Antibiotika-assoziierte Diarrhöe. Verschiedene Virulenzfaktoren und -eigenschaften beeinflussen das Fortschreiten und den Schweregrad der Krankheit, darunter Toxinexpression und Sporenbildung. Kleine regulatorische RNAs (sRNAs) sind bekannte post- transkriptionelle Regulatoren von Virulenz- und Stress-assoziierten Stoffwechselwegen in vielen pathogenen Bakterien. In Gram-negativen Arten wird sRNA-abhängige post-transkriptionelle Regulierung häufig durch das RNA-Chaperon Hfq vermittelt, welches die sRNA-mRNA- Basenpaarung erleichtert. Trotz ihrer Bedeutung in Gram-negativen Bakterien ist vergleichsweise wenig über die verschiedenen Aspekte der post-transkriptionellen Regulation in Gram-positiven Arten bekannt. Erste Daten deuten auf eine wichtige Funktion von Hfq bei der Regulierung verschiedener infektionsassoziierter Signalwege in C. difficile hin, sowie auf die Existenz eines umfangreichen post-transkriptionellen Netzwerks. Eine globale Identifizierung von Hfq- assoziierten RNAs und deren Einfluss auf die Virulenz von und Kolonisierung durch C. difficile ist jedoch bisher noch nicht erfolgt. In dieser Arbeit wurde dRNA-seq (differentielle RNA-Sequenzierung) und RNAtag-seq angewandt, um zunächst die globale Transkriptom-Architektur von C. difficile zu definieren. Anschließend wurde Hfq RIP-seq (RNA-Immunpräzipitation gefolgt von RNA-seq) und RIL-seq (RNA-Interaktion durch Ligation und Sequenzierung) durchgeführt, um das Hfq-vermittelte sRNA-Interaktom auf globaler Ebene zu charakterisieren. Diese Ansätze führten zur Annotation von > 60 neuen sRNAs. Darüber hinaus wurden 50 Hfq-gebundene sRNAs, sowie > 1000 mRNA- sRNA-Interaktionen identifiziert, wodurch Hfq als globaler RNA-Matchmaker in C. difficile bestätigt wurde. Analog zu seiner Funktion in Gram-negativen Arten, führte die Deletion von Hfq zu verringerten sRNA-Halbwertszeiten, was darauf hindeutet, dass Hfq die sRNA-Stabilität in C. difficile beeinflusst. Schließlich wurden mehrere sRNAs und ihre Funktion unter verschiedenen infektionsrelevanten Bedingungen charakterisiert. Die sRNA nc085 interagiert direkt mit dem Zweikomponenten-Regulator eutV, was zu einer Regulierung der Ethanolaminverwertung führt. Als häufig vorkommenden Kohlenstoff- und Stickstoffquelle im Darm, kann Ethanolamin die Pathogenität von C. difficile beeinflussen. SpoY und SpoX regulieren dagegen die Translation des Hauptregulators der Sporulation spo0A in vivo und damit die Sporulationsinitiation. Darüber hinaus hat die Deletion von SpoY und SpoX signifikante Auswirkungen auf die Besiedlung des Darms mit C. difficile sowie die Sporenbelastung in einem Mausmodell der C. difficile-Infektion. Insgesamt liefert diese Arbeit Beweise für eine umfassende Hfq-abhängige post-transkriptionelle Regulierung, die die Physiologie und Virulenz eines Gram-positiven Erregers beeinflusst. Auch wenn mit dieser Arbeit die Charakterisierung der sRNA-vermittelten Regulation in C. difficile gerade erst begonnen hat, können die RIL-seq-Daten als Grundlage für zukünftige mechanistische Studien der RNA-basierten Genregulation in C. difficile herangezogen werden.

No description available.