Desulfovibrio vulgaris Hildenborough (DvH) is a sulfate-reducing bacterium that has the potential to be an agent of bioremediation by aiding in the precipitation of heavy metals and radionuclides from contaminated environments via its natural metabolism. Because such environments are commonly associated with a variety of bacterial stressors, it is important to understand how Desulfovibrio regulates its response to changing conditions for successful environmental application. Very few types of regulation have been characterized in Desulfovibrio and many regulatory networks remain unknown. The importance of regulation by both cis and trans acting RNA molecules has become increasingly evident over the last decade. Both small RNAs and riboswitches have been shown in other bacteria to play key roles in sensing cellular conditions and eliciting the appropriate responses. A previous study in this laboratory identified several small regulatory RNAs, but their biological roles have remained largely unknown. Additionally, a few uncharacterized riboswitches have been predicted in DvH based on genome sequence analysis. This dissertation aimed to characterize the most conserved DvH sRNA and to investigate its potential dual role as a S-adenosylmethionine (SAM) sensing riboswitch. Expression of the sRNA, Dv SIC19, was confirmed during normal growth in lactate/sulfate medium via Northern blot analysis. Sequence and expression analyses also indicated that Dv SIC19 is located upstream of a small hypothetical protein DVU1170 and that the two genes are co-transcribed. Molecular techniques and computational analysis were also employed to determine a role for Dv SIC19 and to identify potential targets. Stress analysis using qRT-PCR suggested a potential role for Dv SIC19 under normal growth conditions as opposed to being involved in a specific stress response. While characterization of Dv SIC19 was ongoing, re-annotation of the DvH genome indicated that Dv SIC19 shared both sequence and structural similarity to the SAM-I class of riboswitches. Multiple techniques, both in vitro and in vivo were used to verify the riboswitch activity of Dv SIC19 and its response to SAM. Determining that Dv SIC19 played some role in the methionine biosynthesis pathway lead to the identification of a mRNA target encoding SahR, a predicted transcriptional regulator of methionine biosynthesis genes. Subsequent electrophoretic mobility shift assays confirmed the ability of Dv SIC19 to bind the sahR transcript and qRT-PCR analysis of a Dv SIC19 deletion strain suggested a negative regulatory role. This study presented the first regulatory role for a newly discovered sRNA in Desulfovibrio. Additionally, this study verified that Dv SIC19 acts not only as a trans regulatory molecule, but also as a cis regulatory element in the methionine biosynthesis pathway of DvH.

Desulfovibrio vulgaris strain Hildenborough is a key organism in the bioremediation of environments contaminated with anthropogenic sources of heavy metals and radionuclides. In order for D. vulgaris to be optimized for remediation of such sites in the environment, it is necessary to understand the organism's response to key stressors that are found in conjunction with both heavy metals and radionuclides. Few regulators have been discovered in this bacterium and even less is known about its genetic regulatory response to changing conditions in its environment. This dissertation project aimed to use computational, molecular, and culture-based methods to identify novel, small, regulatory RNAs (sRNAs)--an important class of cellular regulators that have yet to be studied in D. vulgaris--and to determine their effect on cellular processes within the organism. Computational and transcriptomic high-throughput sequencing resulted in over 200 high-quality putative sRNAs with the expression of sixteen of these sRNAs verified by Northern blot analysis. Eight of the confirmed sRNAs were selected and further characterized within this study resulting in three trans--or intergenic--encoded sRNA, three cis--or antisense--encoded sRNAs, and two sRNAs that may encode small proteins. These eight sRNAs included growth phase-specific sRNAs, differentially expressed sRNAs, as well as sRNAs processed into multiple forms. Molecular techniques such as quantitative reverse-transcriptase PCR (qRT-PCR) indicated differential expression of the confirmed sRNAs under a variety of stress conditions. Culture-based methods confirmed growth deficiencies in deletion and over-expression mutants of the sRNAs tested under stressors suggested during qRT-PCR analysis. Both of these tests suggested that the sRNAs discovered in this study participate within the cellular response of the bacterium to environmental stress. Computational methods were employed to reveal likely putative mRNA targets for the confirmed sRNAs in order to determine interactions and function within the regulatory networks. This study presents the first evidence of the existence and expression of sRNAs within D. vulgaris Hildenborough. Furthermore, the information gathered about the sRNAs analyzed in this work indicates that sRNAs are an important part of the regulatory response mechanism of D. vulgaris. Further research into target identification and interaction can close the loop of these regulatory networks and solidify the role of sRNAs in D. vulgaris.

The goal of this paper was to determine rate of presence of whipworms of genus Trichuris in bodies of selected ruminants (sheep, roe deer) in certain areas and to morphologically state different species of whipworms using molecular revision and professional literature on samples found during helmitological dissections of selected ruminants. Two hypotheses were stated: H1: species that are found in highest volume in case of roe deer and sheep are whipworms Trichuris discolor and Trichuris ovis H2: these whipworms can not be positively distinguished when using morphometrical methods. Material needed for the study, i.e. the intestines of examined ruminants, was recovered in different areas of Czech Republic. Later were the intestines dissected in a laboratory using standardized procedure and hereby collected samples were analysed. Based on selected methods it was determined that in roe deer the rate of occurence of Trichuris discolor is much higher compared to that of Trichuris ovis. With sheep the difference between rates of presence is smaller. These results confirm the first hypothesis by showing high rate of presence of whipworms in these hosts. Collected females of genus Trichurids were morphometrically differentiated by their sex and in 4 morphotypes. Following this differentiation, the most present were the females of morphotype M2, those with a vulval opening without an everted vagina. The second hypothesis was also confirmed. Multihosting species Trichuris discolor and Trichuris ovis are prevalent in the bodies of roe deer and sheep. Thus we can say the roe deer are a potential source of whipworm contamination to sheep breeding. It can not be excluded that sheep are infected by roe deer and vice versa. Molecular determination is a necessary tool for correct assessment of whipworm species, considering the fact that morphological methods may lead to incorrect results.

Francisella tularensis is the causative agent of the disease tularemia and a potential bioterrorism agent. Few regulators have been identified in this organism and little is known about its genetic regulatory networks. In this dissertation project, culture-based and molecular methods were used to both determine the role of the RNA chaperone protein Hfq and identify potential novel small RNAs in F. tularensis subsp. novicida strain U112. The Hfq protein is recognized as an important regulatory factor in a variety of cellular processes, including stress resistance and pathogenesis, and has been shown in several bacteria to interact with small RNAs as a post-transcriptional regulator of mRNA stability and translation. Molecular methods were employed to determine that hfq is potentially transcribed in an operon with both the immediate up- and downstream genes. Phenotypic analysis of two transposon insertions within the hfq ORF revealed that the N-terminal region of the Hfq protein is more important for stress tolerance than the C-terminal end. Complete deletion of hfq resulted in a variety of growth defects under certain stress conditions such as heat-shock, low pH, and oxidative stress. Gene expression of hfq under several of these conditions changed significantly, further suggesting a role for the protein during stress tolerance. Because Hfq likely functions as a global regulator, the expression of several genes in the hfq mutant strain were compared to wild-type and some were significantly altered in particular growth backgrounds. The hfq mutant also exhibited a delayed entry into stationary phase and increased biofilm formation under certain conditions. Shotgun cloning and high-throughput sequencing were used to generate a list of potential sRNAs, an important class of regulators that had yet to be studied in F. novicida. Three candidates were selected and their expression verified using Northern blot analysis and self-ligating RACE. The sRNA transcript designated CISC-1 appears important for certain aspects of cell growth and is differently expressed under several stress conditions. ISC-2 is a transcript that has a minor effect on cell growth during exponential phase, but is upregulated during stationary phase. The third sRNA, ISC-16, is highly conserved among Francisella species and is potentially important for the biosynthesis of bacterial fatty acids. These sRNAs represent an important group of regulators that, along with the Hfq protein, could be important for controlling global gene expression in Francisella.

B. anthracis alters its virulence gene expression profile in response to a number of environmental signals, including levels of bicarbonate and CO2. Virulence plasmid pXO1 is important to Bacillus anthracis pathogenicity as it carries the genes encoding the anthrax toxin and virulence regulatory factors. Induction of toxin and other virulence genes requires the pXO1-encoded AtxA regulatory protein. The cytochrome c maturation system influences the expression of virulence factors in Bacillus anthracis. B. anthracis carries two copies of the ccdA gene, encoding predicted thiol-disulfide oxidoreductases that contribute to cytochrome c maturation. Loss of both ccdA genes results in a reduction of cytochrome c production, an increase in virulence factor expression, and a reduction in sporulation efficiency. pXO1 also carries a gene encoding an Hfq-like protein, pXO1-137. Loss of pXO1-137 results in significant growth defects and reductions in toxin gene expression only when grown under toxin inducing conditions. Similarly, loss of a small RNA on pXO1, sRNA-1, results in similar growth defects and reductions in toxin gene production. Both increased and decreased expression of pXO1-137 and sRNA-1 result in growth defects suggesting narrow functional set points for Hfq and sRNA levels.

Regulation of gene expression by RNA polymerase (RNAP) is an essential ability of living organisms, required for their adaption to a changing environment and ultimately enabling their survival. Interaction of RNAP with ribonucleic acids (DNA or RNA) is crucial for transcription and its regulation. This Doctoral Thesis contains two projects addressing interactions of RNAP with nucleic acids: (i) Transcription of modified DNA templates and (ii) Ms1, a small RNA (sRNA) from M. smegmatis. (i) We investigated the influence of modifications in the major groove of DNA on bacterial transcription in vitro. We found out that transcription of modified DNA templates is influenced on the transcription initiation level and that the promoter sequence is important for the effect of the modifications. Furthermore, we successfully performed transcription switch ON and OFF in vitro by bioorthogonal reactions. This regulation of transcription by artificial DNA modifications has a future in biotechnologies and/or medical therapy. (ii) Regulators of transcription are also small non-coding RNAs. These molecules have an important role in gene expression regulation among prokaryotes and eukaryotes. Ms1 is an sRNA found in mycobacteria. It makes a complex with the RNAP core and it is abundant in stationary phase (in amounts...

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Microorganisms are exposed to constantly changing environments, and consequently have evolved mechanisms to rapidly adapt their physiology upon stress imposition. These adaptive responses are coordinated through the rewiring of gene expression via complex networks that control the transcriptional program and the activity of post-transcriptional regulators. Although transcription factors primarily determine which genes are expressed, post-transcriptional regulation has a major role in fine-tuning the dynamics of gene expression. Post-transcriptional control is exerted by RNA-binding proteins and small regulatory RNAs (sRNAs) that bind to mRNA targets and modulate their synthesis, degradation and translation efficiency. In Escherichia coli, sRNAs associated with an RNA chaperone, Hfq, are key post-transcriptional regulators, yet the functions of most of these sRNAs are still unknown. The first step in understanding the roles of sRNAs in regulating gene expression is to identify their targets. To generate transcriptome-wide maps of Hfq-mediated sRNA-mRNA binding, we applied CLASH (cross-linking, ligation and sequencing of hybrids), a method that combines in vivo capture of RNA-RNA interactions, high-throughput sequencing and computational analyses, in E. coli. We uncovered thousands of dynamic growth-stage dependent association of Hfq to sRNAs and mRNAs. The latter confirmed known sRNA-target pairs and identified additional targets for known sRNAs, as well as novel sRNAs in various genomic features along with their targets. These data significantly expand our knowledge of the sRNA-target interaction networks in E.coli. In particular, the Hfq CLASH data indicated 3'-UTRs of mRNAs as major reservoirs of sRNAs, and the utilization of these may be more common than anticipated. Our findings also provide mechanistic insights that ensue from the identification of tens of sRNA-sRNA interactions that point to extensive sponging activity among regulatory RNAs: many sRNAs appear to be able to interact and repress the functions of other base-pairing sRNAs. We validated and highlighted the biological significance of some of the CLASH results by characterizing a 3'-UTR derived sRNA, MdoR (mal-dependent OMP repressor). This sRNA emerges by processing of the last transcript of malEFG polycistron, encoding components of maltose transport system. We found MdoR directly downregulates several major porins, whilst derepressing the maltose-specific porin LamB via destabilization of its inhibitor, MicA, likely by a sponging mechanism. Physiologically, MdoR contributes to the remodelling of envelope composition and links nutrient sensing to envelope stress responses during maltose assimilation. MdoR is a clear example of how cells integrate circuitry through multiple networks as part of their adaptive responses and how the CLASH methodology can help expand our understanding of sRNA-based regulation.

Listeriosis is a potentially lethal disease caused by the Gram-positive facultative intracellular pathogen Listeria monocytogenes (L.m.). L.m. is found ubiquitously in the environment and infects humans via ingestion of contaminated food. Contaminated products are usually derived from ruminants and involve dairy products and different kinds of processed meat. Listeriosis is a potential lifethreatening disease with a total mortality rate of 20-30 %. The development of listeriosis may lead to meningitis and septicemia or other invasive diseases. Pregnant women are of increased risk of developing listeriosis and a materno-fetal infection commonly lead to spontaneous abortion or still-birth. Regulation of gene expression, and specifically virulence gene expression, is essential for pathogenic bacteria to be equipped for handling counteractions from the host as well as thriving in the often hostile environment. In pathogenic Listeria, virulence gene expression is under the control of the global virulence gene regulator PrfA. The expression of prfA is highly regulated at the transcriptional, post-transcriptional and post- translational level. We have identified a novel type of post-transcriptional regulation of prfA-mRNA by a trans-acting riboswitch element (SreA). By binding to the leader region of prfA-mRNA, SreA negatively regulates the expression of prfA. To our knowledge, this is the first description of a cis-acting riboswitch capable of functioning as a small RNA in trans, regulating targets on distant sites. To date, there have been around 100 sRNAs identified in Listeria monocytogenes, but experimental data is still limited. We have characterized a blood induced sRNA, Rli38, which is important for full virulence during oral infection of mice. Our data suggest that Rli38 regulates the expression of at least two proteins; OppD (Oligopeptide transport protein) and IsdG (heme degrading monooxygenase). Both of these proteins have been implicated in the infectious cycle of L.m. We speculate that the virulence phenotype of an ∆rli38 mutant is possibly mediated through the effect of these proteins. L.m. is a complex pathogen, able to infect and replicate in a variety of organs and cause several distinctive forms of disease. These qualities of L.m. generate difficulties in simulating human listeriosis in animal models, as entailed by the multitude of models used in the field. In this work, we have evaluated the use of an alternative animal model in studying listeriosis. Our results describe the differentiated virulence potential of wildtype bacteria and a ∆prfA mutant strain in the chicken embryo by live/death screening and organ colonization. Large differences in mean time to death were found between wild-type and the ∆prfA strain and ∆prfA cells displayed a considerable defect in colonization of the embryonal liver. The results presented in this thesis show that the chicken embryo infection model is a valuable and convenient tool in studying end-outcome and organ colonization of Listeria monocytogenes. Taken together, this thesis describes the characterization of two previously unknown sRNAs in the human pathogen Listeria monocytogenes and the use of an alternative infection model for simulating listeriosis.

The Gram-negative pathogen Vibrio cholerae uses variety of regulatory molecules to modulate expression of virulence factors. One important regulatory element of microorganisms is small non-coding RNAs (sRNAs), which control various cell functions such as expression of cell membrane proteins, mRNA decay and riboswitches. In this thesis studies, we demonstrated the roles of the sRNAs VrrA in regulation of outer membrane protein expression, biofilm formation and expression of ribosome binding proteins. In addition, we showed that VrrB, a newly discovered sRNA, played a role in amino acid dependent starvation survival of V. cholerae and might functioned as a riboswitch. VrrA, a 140-nt sRNAs in V. cholerae, was controlled by the alternative sigma factor σE. The outer membrane protein, OmpT is known to be regulated by environmental signals such as pH and temperature via the ToxR regulon and carbon source signals via the cAMP–CRP complex. Our studies provide new insight into the regulation of OmpT by signals received via the σE regulon through VrrA. We demonstrated that VrrA down-regulate ompT translation by base-pairing with the 5′ region of the ompT mRNA in a Hfq (RNA chaperone protein) dependent manner. V. cholerae biofilms contain three matrix proteins—RbmA, RbmC and Bap1—and exopolysaccharide. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. In our studies, we demonstrated that VrrA negatively regulated rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation was not stringently dependent on Hfq. In V. cholerae, VC0706 (Vrp) and VC2530 proteins are homologous to ribosome-associated inhibitor A (RaiA) and hibernation promoting factor (HPF) of Escherichia coli, respectively. HPF facilitates stationary phase survival through ribosome hibernation. We showed that VrrA repressed Vrp protein expression by base-pairing to the 5´ region of vrp mRNA and that this regulation required Hfq. We also showed that Vrp was highly expressed during stationary phase growth and associated with the ribosomes of V. cholerae. We further demonstrated that Vrp and VC2530 were important for V. cholerae starvation survival under nutrient-deficient conditions. While VC2530 was down-regulated in bacterial cells lacking vrrA, mutation of vrp resulted in increased expression of VC2530. Riboswitches are an important class of regulators in bacteria, which are most often located in the 5' untranslated region (5´ UTR) of bacterial mRNA. In this study, we discovered the novel non-coding sRNA, VrrB located at the 5´ UTR of a downstream gene encoding Vibrio auxotropic factor A (VafA) for phenylalanine. In V. cholerae, reduced production of VafA was observed in the presence of phenylalanine and phenylpyruvate in the culture media. Some analogs of phenylalanine and phenylpyruvate could also modulate the expression of VafA. Furthermore, bacterial cells lacking the vrrB gene exhibited high production of VafA, suggesting that VrrB might function as a riboswitch that controls VafA expression.