Vibrio cholerae, the causative agent of cholera, has been a lethal enteric pathogen to humans for most of recorded history. Even though it is well studied, it still kills many people every year due to rapid and severe dehydrations from diarrhea. Part of what makes V. cholerae such an effective pathogen is its ability to control virulence factors depending on its environment. ToxR is a major virulence protein that has upstream control of most of the virulence genes that are turned on when in a human host. Two of the most critical virulence factors, toxin coregulated pilus and cholera toxin are controlled by ToxR. CsrA is a protein that regulates many cellular functions in V. cholerae, including glycogen synthesis, motility, and biofilm production. Preliminary data suggests a link between CsrA and the regulation of ToxR. In order to study CsrA as it relates to ToxR regulation, a csrA mutant must be generated in V. cholerae. CsrA plays such an important role in glycogen metabolism that a csrA mutant is not viable due to excessive glycogen levels. In order to make a viable csrA mutant, glycogen synthesis has to be turned off. In this research, I attempt to make a viable V. cholerae csrA mutant by deleting csrA in a strain that is deficient for glycogen synthesis (glg). Normally without CsrA, glycogen in the cell would increase to a detrimental level. Since a glg⁻ csrA⁻ mutant lacks the ability to make glycogen, the levels never reach a lethal level, allowing the mutant to survive without functional CsrA. Such a glg- csrA- double mutant's ToxR regulation can be studied by growth in various media by measuring OmpU and OmpT expression. Using PCR, restriction enzymes, and DNA ligase, a suicide plasmid was created containing sequences that flank the csrA gene but instead of the csrA gene, a chloramphenicol resistance cassette was inserted. Through bacterial conjugation this plasmid was introduced into three V. cholerae glg- strains. Allelic exchange was carried out utilizing the homology between the DNA flanking wild type csrA and the csrA deletion with chloramphenicol cassette. This first crossover event was initiated with the requirement of the [pi] protein for the plasmid to replicate. Without the pir gene to create [pi] protein, selection for antibiotic resistance required that the plasmid integrate into the genome. This was selected based on the plasmid encoded ampicillin resistance. After the second crossover event, there were two possible outcomes of excision: reverting to wild type csrA or retention of the csrA mutation. The csrA mutant was selected based on its sucrose and chloramphenicol resistance and ampicillin sensitivity. / text

The quorum-sensing response of Vibrio fischeri involves a complex network of genes (encoding regulatory proteins as well as sRNAs), that govern host-association and production of bioluminescence. A key regulator of this system is LuxR, which is the transcriptional activator of the lux operon as well as several other genes in. LuxR also autoregulates its own transcription, which we have shown causes bistability and hysteresis in the quorum-sensing response. This behavior allows the system to maintain a stable and robust response in the face of environmental fluctuation or decreases in external autoinducer concentration caused by other sources. There are many factors that are known to regulate luxR expression, including the ArcA redox-responsive regulator, the cAMP-CRP secondary metabolism regulator, and components of the quorum-sensing pathway like LitR. Because of this, LuxR levels are critical in both the timing of quorum-sensing induction, as well as the maintenance of the response over time. This makes it a potential target for multiple levels of regulation in response to factors such as environmental and metabolic conditions, as well as other components of the quorum-sensing network. Another important global regulatory protein in V. fischeri (and most other species of Gram-negative proteobacteria) is the post-transcriptional regulator CsrA. CsrA controls processes involved in carbon storage and utilization, as well as the transition from exponential to stationary phase growth. We have demonstrated that CsrA is regulated by two sRNAs (CsrB1 and CsrB2) in V. fischeri. Because CsrA regulates changes in cell behavior and is an important metabolic regulator, there is a good possibility that it has some interactions with the quorum-sensing regulon, whose endproduct, bioluminescence, creates a large metabolic demand from the cell. In an effort to determine at which point in the quorum-sensing regulatory network CsrA regulation is important, epistasis experiments were designed using factorial design, which is a subset of statistical analysis of variance (ANOVA). This method was used to generate a high degree of confidence in the data, so that even minor interactions in the regulatory networks could be established. By altering the levels of CsrA expression in various mutant strains of V. fischeri, we have demonstrated that CsrA acts by an unknown mechanism to increase the transcription of luxR when the quorum-sensing regulator LitR is absent. Our results also demonstrated that CsrA mediates this effect through repression of ArcA activity, which is known to act directly on the luxR and luxI intergenic region as a repressor. This indicates that CsrA may bypass the upstream parts of the quorum-sensing regulatory cascade that lead to litR activation, so that LitR and LuxR may be regulated differently in response to certain conditions. This work has shown that the interactions between global regulons can coordinately control the amount of quorum-sensing induction by affecting the level of LuxR in the cell. The balance of these regulatory networks allows the cell to tightly regulate the quorum-sensing response. Thus, LuxR serves as a critical regulatory hub in the cell, at which multiple signals can be integrated in order to generate the appropriate cellular response. / Ph. D.

Vibrio parahaemolyticus is considered to be an emerging, yet understudied, human pathogen. The V. parahaemolyticus BB22OP genome was sequenced to allow for a comparative analysis between the genome of BB22OP and another previously sequenced, pathogenic strain of V. parahaemolyticus, RIMD2210633. V. parahaemolyticus BB22OP is interesting because it exhibits a spontaneous phenotypic switch in colony morphology due to the loss of a functional OpaR; this also influences virulence. OpaR is the major quorum-sensing regulator in V. parahaemolyticus homologous to LuxR from V. harveyi. When opaR is removed from the RIMD2210633 genome, the same phenotypic switch is not seen indicating a difference between the quorum-sensing systems in these two strains. Understanding the regulatory variation in these two strains has the potential to provide key insights into the control of pathogenesis in this organism. Initially, the BB22OP genome sequencing results aligned into 125 contigs. The genome has now been assembled into two distinct chromosomes with only two gaps remaining to be filled. These gaps are located in the integron region, which is difficult to assemble due to its structure. The integron is a series of gene cassettes separated by inverted repeats that facilitate recombination events that build the integron. The integron region is further evidence of genetic differences between the two strains. The integron in the RIMD2210633 strain is comprised of 69 gene cassettes, while the BB22OP integron contains at least 86 gene cassettes. There are 313 genes novel to the BB22OP genome, which could result in the phenotypic differences seen in these two strains. Additionally five of the 313 genes are predicted to be transcriptional regulators indicating the potential for differential gene regulation. Further comparative analysis will likely reveal more phenotypic divergence between the physiology of RIMD2210633 and BB22OP. Additionally, the CsrA regulatory network was explored in RIMD2210633. CsrA was first characterized in E. coli as a global regulator of carbon storage and metabolism. RIMD2210633 contains a CsrA homolog and was predicted to contain four CsrA-regulating sRNAs (CsrB1-3 and CsrC), and this work confirmed that these sRNAs regulate CsrA in the same manner as in E. coli. CsrA and the same CsrA-regulating sRNAs were found in the BB22OP genome as well. Since CsrA is known to regulate glycogen production, a qualitative iodine-staining plate assay and a quantitative glycogen assay were used to indirectly measure CsrA activity in the presence and absence of individual regulatory sRNAs. The RIMD2210633 CsrA, CsrB1, CsrB2, CsrB3 and CsrC were shown to have the predicted physiological role in recombinant E. coli, with higher glycogen levels observed when CsrA was active and lower levels when each of the sRNAs was overexpressed. CsrA is also known to regulate biofilm production and virulence factors. In an attempt to develop a screening method for potential CsrA targets, a transcriptional/translational fusion system was developed. Transcriptional and translational fusions to β-galactosidase were created to PdksA, PglgC1 and PtoxR from RIMD2210633. CsrA or CsrB2 was overexpressed in recombinant E. coli containing each of the fusion constructs in order to see what happens to the gene expression from these promoters at low and high CsrA activity levels. Surprisingly, changing the activity levels of CsrA impacted both transcriptional and translational levels making the results of the assay difficult to interpret. Collectively these efforts have enhanced our understanding of V. parahaemolyticus. In particular, the sequencing of BB22OP has allowed for a comparative analysis between the BB22OP and RIMD2210633 strains. These strains have remarkably conserved genomes despite the phenotypic differences they exhibit. It appears there is variation in the quorum-sensing systems of these two strains. Further analysis will reveal how the quorum-sensing regulons differ and how this impacts the virulence of these two pathogenic V. parahaemolyticus strains. / Master of Science

Pseudomonas aeruginosa RsmA belongs to the CsrA family of RNA binding proteins. CsrA family members are post-transcriptional regulators of global gene expression and usually function to inhibit translation of target genes, but in some cases can also exert positive regulatory effects. Previous work from our lab determined that RsmA is required for maximal T3SS gene expression in P. aeruginosa strain PA103. Nevertheless, the molecular mechanism underlying the RsmA-mediated control of T3SS gene expression was unknown. Expression of the T3SS is under the direct control of ExsA, a transcriptional activator. Previous microarray analyses showed that exsA transcript levels were reduced two-fold in an rsmA mutant. In chapter II I examine the role of RsmA in regulating ExsA expression. I demonstrate that expression of a ExsA-LacZ translational fusion was reduced two-fold in an rsmA mutant suggesting a specific effect of RsmA on ExsA expression. The effect of RsmA on ExsA expression occurs at a post-transcriptional level and is independent of mRNA and protein stabilization mechanisms. RsmA directly interacts with the exsCEBA transcript at multiple sites. Truncation analyses indicate that the -37 to +85 region (relative to the ATG start codon) is necessary and sufficient for RsmA-dependent control. I identified two binding sites, BS1 (-25 bp) and BS2 (+84), involved in the interaction of RsmA with the exsA transcript using sequence analysis, site-directed mutagenesis, EMSA assays, RNase footprints, and RNaseH cleavage assays. Mutagenesis of both binding sites results in an RsmA-independent phenotype. I further demonstrate that RsmA is able to activate ExsA expression. I propose a model wherein RsmA relieves a block on ExsA translation. Collectively, this work shows that RsmA directly binds and activates ExsA expression at the post-transcriptional level. Most Pseudomonas species carry at least two homologs of CsrA on the chromosome, but only one copy had been identified in P. aeruginosa. Through the course of other projects in the lab, we observed several phenotypes that could not be accounted for by a single copy of RsmA. In collaboration with the Wolfgang lab, we identified a second CsrA homolog, RsmF in P. aeruginosa. RsmF is dimeric in solution. The structure of RsmF differs substantially from other CsrA homologs by having alpha-helices located between the beta-2 and beta-3 strands. In chapter III I examine the role of RsmF in regulating RsmA-controlled processes associated with acute (T3SS) and chronic (T6SS and biofilm formation) infection. I discovered that while an rsmF mutant alone does not exhibit a phenotype, simultaneous deletion of both rsmA and rsmF significantly accentuates the phenotypes exhibited by an rsmA mutant alone. I show that RsmA directly binds and represses RsmF translation and that the small regulatory RNAs RsmZ and RsmY do not significantly modulate RsmF activity. Site-directed mutagenesis revealed that Arg 62, located in the beta-1 and beta-5 fold, is essential for biological activity in vivo and RNA-binding in vitro suggesting a conserved mechanism of RNA recognition maintained across all CsrA family members. Finally, I show that RsmF binds to only a subset of RsmA targets and is not involved in the regulation of all RsmA-controlled processes. In chapter IV I identified high-affinity RNA ligands from a chemically synthesized oligonucleotide library using systematic evolution of ligands by exponential enrichment (SELEX) and high-througput sequencing. From preliminary analyses of high-throughput sequencing data, the RsmF-binding consensus was determined as 5'-RUACARGGAC-3', with the ARGGA motif being 95% conserved. Collectively, this work shows that Rsm homologs play important roles in regulating virulence gene expression in P. aeruginosa.

RÉSUMÉCsrA est un régulateur post-transcriptionnel contrôlant l’expression et/ou la stabilité des ARNm auxquels il se lie. Il appartient à la famille des régulateurs globaux et contrôle une grande variété de fonctions apparemment non liées. Dans le cas de CsrA, il s’agit principalement de fonctions métaboliques et de fonctions liées aux comportements sociaux des bactéries. Cependant, les limites de la régulation exercée par CsrA sur la physiologie cellulaire sont floues car ses cibles directes semblent abondantes mais difficiles à identifier et, parmi celles-ci, d’autres régulateurs important étendent indirectement l’influence de CsrA.Au cours de cette thèse, nous avons étudié les effets de la délétion du gène csrA à l’échelle de la population bactérienne (1), de la cellule bactérienne (2) et au niveau génétique (3) dans une souche d’Escherichia coli uropathogène. Les infections urinaires font parties des infections bactériennes les plus courantes, présentent un mécanisme de chronicité faisant intervenir la formation de biofilms et E. coli est le principal agent responsable de ces infections. (1) Nous avons montré que l’architecture des biofilms formés par la souche uropathogène d’E. coli était différente de celle décrite pour la souche de laboratoire et que la délétion de csrA affectait fortement cette architecture. (2) Nous avons également montré que le gène csrA n’était pas essentiel mais que sa délétion entraînait un défaut de croissance ainsi qu’une perte d’homéostasie de l’enveloppe. (3) Finalement, nous avons étudié des mutants compensatoires obtenus au travers d’une expérience d’évolution expérimentale partant du mutant ΔcsrA et montré que les différents phénotypes testés étaient restaurés dans ces mutants compensatoires sans qu’aucun changement génétique ne soit identifié.SUMMARYCsrA is a global post-transcriptional regulator controlling the expression/stability of its mRNA targets. It regulates a wide variety of apparently unrelated functions mainly related to metabolism and social behaviors. However, the limits of the regulation mediated by CsrA are not clear as its regulon is large and contains many other regulators extending indirectly its influence.In this thesis, we study the consequences of the deletion of csrA at the population level (1), cellular level (2) and genetic level (3) in an uropathogenic strain of Escherichia coli. Urinary tract infections are among the most frequent bacterial infections, present chronicity mechanism involving biofilms formation and are most of the time caused by E. coli. (1) We showed that the architecture of biofilms formed by the uropathogenic strain is different from that of the lab-strain of E. coli and that CsrA is necessary to generate this particular architecture. (2) We also showed that csrA gene is not essential and that its deletion provokes a growth defect and a loss of the envelope homeostasis. (3) Finally, we studied compensatory mutants selected through experimental evolution and showed that tested phenotypes are restored in these mutants without any genetic change being identified. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

CsrA is a global post-transcriptional regulatory protein that has a great impact on many physiological pathways in a cell. It regulates central carbon metabolism, motility and biofilm formation as well as virulence, pathogenesis, quorum sensing and the oxidative stress response. However, CsrA is in turn also regulated by CsrB and CsrC sRNAs, among other factors. In this study, we explore its regulatory effect on three different genes/operons, cstA, glgCAP and flhDC. We performed beta-galactosidase assays on wild type (wt) and RNase P temperature sensitive (ts) cells to determine the dependence on RNaseP for the CsrA, CsrB and CsrC regulatory cascade on these three genes. Our results showed a clear decrease in cstA and glgCAP activity during CsrA activation, suggesting a regulatory cascade in which inhibition of RNase P leads to an inactivation of CsrB and CsrC. This in turn leads to an activation of CsrA and an inhibition of cstA and glgCAP. The effect on flhDC, however, was not as clear and needs further investigation.

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