ExsA is an AraC-family transcriptional regulator that controls expression of T3SS genes in P. aeruginosa. ExsA binds to DNA at T3SS promoters and activates transcription. In the work presented here I examine the stoichiometry, ligand-interaction properties, and transcriptional activation mechanism of ExsA. I determined that ExsA is largely monomeric in solution. ExsA binds T3SS promoter DNA with high affinity resulting in two ExsA-DNA complexes. Whereas the lower molecular weight complex represents a single molecule of ExsA bound to DNA, the higher molecular weight complex represents two molecules of ExsA bound to adjacent sites at T3SS promoters. I next analyzed the mechanism by which ExsD negatively effects ExsA function. Chromatin Immuno-Precipitation Assays (ChIP) demonstrate that ExsD inhibits the DNA-binding activity of ExsA in vivo. Finally, I characterized the mechanism of transcriptional activation by ExsA. ExsA-dependent promoters contain regions that resemble consensus σ70 -35 and -10 recognition hexamers. The spacing between these regions, however, is increased 4-5 bp compared to the σ70 consensus. Nevertheless, I demonstrate that T3SS promoters are dependent on σ70-RNA polymerase (RNAP). Using the abortive initiation assay I discovered that ExsA recruits RNA polymerase to the PexsC and PexsD promoters. Potassium permanganate footprints indicate that following recruitment, RNAP facilitates unwinding of DNA at the -10 hexamer of T3SS promoters. Transcriptional activators generally recruit RNAP by contacting the α or σ70 subunits (or both). I have found that ExsA recruits RNAP to the PexsC and PexsD promoters by contacting region 4.2 of σ70. Although I have established the role of the -10 hexamer, the function of a near-consensus, putative -35 remains puzzling. in vitro transcription assays with mutations in the PexsC -35 hexamer reveals that this region is dispensable for ExsA-independent transcription. This data may suggest that what was thought to be a -35 hexamer is really just an ExsA binding site. Consistent with this hypothesis, I provide evidence that suggests an extended -10 element at PexsC may function to compensate for the lack of a -35 hexamer.

The ability of bacteria to detect their surroundings and enact an appropriate response is critical for survival. Translation of external signals into a coherent response requires specific control over the transcription of DNA into RNA. Much of the regulation at this step is accomplished by transcriptional regulators, proteins that bind to DNA and alter gene expression. A wide-spread variety of regulators in bacteria is the AraC-family. These regulators are divided into two conserved domains and respond to a variety of compounds owing to different N-terminal domains. A subfamily of these regulators, GATase1-like AraC-family transcriptional regulators (GATRs), is described. These proteins contain an N-terminal domain with structural characteristics similar to enzymes that synthesize amine-containing compounds. Members of this subfamily of transcriptional regulators are found in a wide range of bacteria, however, few are characterized. A relatively high number of GATRs are encoded in the Burkholderia thailandensis genome. Therefore, we utilized this bacterium as a model to explore the function and diversity of these regulators. GATRs in B. thailandensis divided into two groups based on bioinformatics analysis. The first group includes three members which we identified that contribute to the positive regulation of glycine betaine (GB) catabolism. GB can be utilized as a nutrient source or as a potent osmoprotectant. The regulation of this pathway in B. thailandensis differs from previously established models due to the interplay of these regulators. Homologs of two other GATRs in this group were identified that regulate carnitine and arginine catabolism. The second group of GATRs contains uncharacterized members with no known functions. A genetic strategy for engineering constitutive GATRs was developed and employed to investigate the transcriptional regulons of these GATRs. This approach yielded the identification of a novel GATR that represses expression of an operon producing a formaldehyde detoxification system, and is the first example of a GATR that functions as a repressor.

Transcription of the Pseudomonas aeruginosa type III secretion system is controlled by ExsA, a member of the AraC/XylS family of regulators. ExsA is comprised of an amino terminal domain that is involved in self-association and regulatory functions, and a carboxy-terminal domain that contains two helix-turn helix (HTH) DNA-binding motifs which contact promoter DNA. Previous work from our lab determined the function of the two independent ExsA domains and found that each ExsA-dependent promoter contains two adjacent binding sites for monomeric ExsA. The promoter-proximal site (binding site 1) consists of highly conserved GnC and TGnnA sequences that are individually recognized by the two HTH DNA-binding motifs of an ExsA monomer. Nevertheless, the details of how ExsA recognizes and binds to ExsA-dependent promoters were still unknown. In chapter II I show that the two ExsA monomers bind to promoter regions in a head-to-tail orientation and identify residues in the first HTH of ExsA that contact the GnC sequence. Likewise, residues located in the second HTH motif, which contribute to the recognition of the TGnnA sequence, were also identified. While the GnC and TGnnA sequences are important for binding to site 1, the promoter-distal binding sites (site 2) lack obvious similarity among themselves or with binding site 1. Site 2 in the PexsC promoter region contains a GnC sequence that is functionally equivalent to the GnC in site 1 and recognized by the first HTH motif of an ExsA monomer and the second HTH interacts with an adenine residue in binding site 2. A comparison of hybrid promoters composed of binding site 2 from one promoter fused to binding site 1 derived from another promoter indicates that ExsA-binding affinity, promoter strength, and the degree of promoter bending are properties that are largely determined by binding site 2. Through the course of the ExsA studies I observed that the amino acids that comprise the HTH motifs of ExsA are nearly identical to those in LcrF/VirF, the activators of T3SS gene expression in the pathogenic yersiniae. In chapter III I tested the hypothesis that ExsA/LcrF/VirF recognize a common nucleotide sequence. Here I report that Yersinia pestis LcrF binds to and activates transcription of ExsA-dependent promoters in P. aeruginosa, and that plasmid expressed ExsA complements a Y. pestis lcrF mutant for T3SS gene expression. Mutations that disrupt the ExsA consensus-binding sites in both P. aeruginosa and Y. pestis T3SS promoters prevent activation by ExsA and LcrF. All of the data combined demonstrate that ExsA and LcrF recognize a common nucleotide sequence. Nevertheless, the DNA binding properties of ExsA and LcrF are distinct. Whereas two ExsA monomers are sequentially recruited to the promoter region, LcrF binds to promoter DNA as a preformed dimer and has a higher capacity to bend DNA. An LcrF mutant defective for dimerization bound promoter DNA with properties similar to ExsA. Finally, I demonstrate that the activators of T3SS gene expression from Photorhabdus luminescens, Aeromonas hydrophila, and Vibrio parahaemolyticus are also sensitive to mutations that disrupt the ExsA-consensus binding site. Taken together, this work shows that ExsA binding and activation at T3SS gene promoters serves as a model system by which the DNA binding properties of other AraC family transcriptional activators can be predicted.

Pathogenesis of enterotoxigenic Escherichia coli (ETEC) and Shigella flexneri relies predominantly on members of the AraC/XylS family of transcriptional regulators, Rns (or its homolog, CfaD) and MxiE, respectively. Rns/CfaD regulate the expression of pili, which allow the bacteria to attach to the intestinal epithelium. Better understanding of the role Rns plays in virulence was attained by expanding our knowledge of the Rns regulon, revealing that it functions as an activator of cexE, a previously uncharacterized gene. By in vitro DNase I footprinting two Rns-binding sites were identified upstream of cexEp, both of which are required for full activation of cexE. The amino terminus of CexE also contains a secretory signal peptide that is removed during translocation to the periplasm. Though the function of CexE remains unknown, these studies suggest that CexE is a novel ETEC virulence factor since it is regulated by Rns/CfaD. In Shigella flexneri, the expression of a subset of virulence genes (including, ipaH9.8 and ospE2) is dependent upon the activator MxiE and a cytoplasmic chaperone IpgC. To define the molecular mechanism of transcriptional activation by this chaperone-activator pair, an in vitro pull down assay was performed revealing that MxiE specifically interacts with IpgC in a complex. Additionally, IpgC recognizes three polypeptide regions in MxiE: within MxiE(1-46), MxiE(46-110) and MxiE(196-216). Furthermore, it seems that MxiE and IpgC regulate transcription of ipaH9.8 and ospE2 promoters differently. In the bacterium, the formation of the MxiE-IpgC complex is initially prevented because IpgC is sequestered in individual complexes with effector proteins, IpaB and IpaC. Upon contact with an eukaryotic host cell the effector proteins are secreted, thereby freeing IpgC to form a complex with MxiE and activate the expression of virulence genes. This new characterization of the role of Rns and MxiE in virulence gene regulation in ETEC and S. flexneri, respectively will give new insights into the pathogenesis of the regulators.

Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2015-08-07T14:08:44Z No. of bitstreams: 0 / Made available in DSpace on 2015-08-07T14:08:44Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

L'expression des gènes codant pour le Système de Sécrétion de Type III (SST3), facteur de virulence majeur de Pseudomonas aeruginosa, est activée par ExsA. Ce facteur de transcription appartient à la famille des régulateurs de type AraC/XylS caractérisés par un domaine de fixation à l'ADN comportant deux motifs hélice-tour-hélice. L'activité de ces protéines est généralement régulée par la fixation d'un ligand sur un domaine supplémentaire non conservé. Ce ligand peut être de nature protéique dans le cas de certains régulateurs contrôlant la synthèse de SST3. Ainsi, l'activité transcriptionnelle de ExsA est inhibée par l'anti-activateur ExsD. L'étude de la fonctionnalité de ExsA et de ses domaines par des approches in vitro et in vivo a révélé que le domaine C-terminal de ExsA est bien le domaine de fixation à l'ADN et que deux monomères se fixent sur le promoteur de l'opéron des gènes de régulation du SST3 (pC). Ce domaine isolé possède une affinité pour pC et une activité transcriptionelle inférieure à celle de ExsA. En effet, la fixation efficace de ExsA sur le promoteur pC requiert sa dimérisation à travers son domaine N-terminal. La dernière hélice  de ce domaine N-ter semble jouer un rôle majeur dans la dimérisation de ExsA. Le deuxième objectif de ma thèse était de comprendre l'interaction entre ExsA et ExsD et d'identifier le mécanisme grâce auquel l'inhibiteur empêche ExsA d'activer la transcription des gènes du SST3. Après co-production des deux protéines, le complexe ExsA/ExsD a été purifié puis caractérisé. ExsA et ExsD forment un complexe hétérodimérique au sein duquel l'inhibiteur empêche le facteur de transcription de se fixer à l'ADN.

Seules deux structures du système nerveux central adulte, la zone sous-granulaire et la zone sous-ventriculaire, produisent continuellement de nouveaux neurones et sont considérées comme neurogènes. En dehors de ces zones délimitées, le tissu nerveux ne possède pas de telles facultés. Leurs influences anti-neurogènes peuvent cependant être mises entre parenthèses dans certaines conditions. Cela se produit après neurectomie vestibulaire unilatérale (NVU) chez le chat adulte ; en effet, des études immunohistochimiques et comportementales nous ont permis de découvrir l'existence d'une neurogenèse réactionnelle de type GABAergique dans les noyaux vestibulaires désafférentés situés dans le tronc cérébral. Nos résultats témoignent de l'implication fonctionnelle de cette prolifération cellulaire dans la restauration des fonctions posturo-locomotrices suite à une NVU. Nous avons également mis en évidence que les caractéristiques et l'intensité de la désafférentation vestibulaire déterminent, non seulement, le décours temporel de la restauration des fonctions vestibulaires, mais aussi les différents mécanismes cellulaires post-lésionnels et le potentiel neurogène des noyaux vestibulaires. En outre, nous avons démontré que l'activation ou le blocage des récepteurs GABA de type A influence d'une part, les différentes étapes de la neurogenèse réactionnelle dans les noyaux vestibulaires, et détermine d'autre part le décours de la récupération comportementale des animaux. Ainsi, le système GABAergique joue-t-il un rôle important dans la régulation de la neurogenèse induite après NVU et de sa fonctionnalité. / Only two structures of the adult central nervous system: the subgranular zone and the subventricular zone, produce continuously new neurons and are considered as neurogenic. Outside these two delimited areas, nervous tissue does not have such faculties. The anti-neurogenic influences can however be removed under specific conditions. That is what happens after unilateral vestibular neurectomy (UVN) in the adult cat: behavioral and immunohistochemical studies have demonstrated the existence of a reactive GABAergic neurogenesis in the deafferented vestibular nuclei located in the brainstem. Our results demonstrate the functional role of the vestibular cell proliferation in the postural locomotor function recovery after UVN. We also demonstrated that characteristics and intensity of the vestibular lesion, not only determine the time course of recovery of vestibular function, but also the post-lesional cellular mechanisms and the neurogenic potential occurring in the vestibular nuclei. In addition, we showed that activation or blockade of GABA type A receptors influences the different steps of neurogenesis in the vestibular nuclei, and also determines the time course of behavioral recovery. Thus, the GABAergic system influences reactive neurogenesis that is benefic for vestibular compensation process. Finally, the results of a recent study demonstrated that vestibular-hippocampal relations exist, and that stress induced by vestibular deafferentation can modulate adult neurogenesis in both the vestibular nuclei and the dentate gyrus in the adult cat.

No description available.