Serratia marcescens, a member of Enterobacteriaceae family, is a causative agent of nosocomial and opportunistic infections. Numerous reports show that the multidrug resistance among S. marcescens is growing. This organism has high-level intrinsic resistance to a variety of antimicrobial agents, which makes the treatment of infections caused by this bacterium very difficult. The major mechanism for antibiotic resistance, especially to fluoroquinolones, in Gram-negative organisms is the active efflux of the antibiotic molecule mediated by efflux pumps belonging to the Resistance-Nodulation-Cell Division (RND) family. It was previously shown that the SdeAB and SdeXY multidrug efflux pumps are important for conferring the intrinsic drug resistance in S.marcescens. In Escherichia coli, the up-regulation of transcriptional activators, such as MarA, SoxS and Rob, affect transcription of acrAB, tolC and micF. Over-expression of Rob results in increased expression of the E. coli AcrAB-TolC efflux pump and decreases outer membrane permeability through up-regulation of micF, resulting in multidrug, organic solvent and heavy metal resistance. In the present study, we report the identification of a rob gene in S. marcescens which has a 70% identity at the DNA level and 71% identity at the amino acid level to that of E. coli. Moreover, the S. marcescens rob demonstrated similar properties to the E. coli rob including having an effect on expression of outer membrane protein F (OmpF) and over-expression of SdeAB and SdeXY, conferring antibiotic resistance to divergent antibacterial agents and tolerance to organic solvents. We performed rob promoter evaluations using transcriptional fusions to the Green Fluorescence Protein (GFP) in the vector pGlow-TOPO and constructed a rob knock-out using the TargeTron Gene Knockout System. Promoter activity assessment, using the pGlow-TOPO reporter plasmid, showed that rob had higher promoter activity at 37°C than 30°C. In the presence of 2,2’-dipyridyl, rob promoter activity was observed to be slightly increased in the early and mid-log phase by 1.4 and 1.1 fold, respectively. We also showed that sodium decanoate and sodium salicylate can reduce the transcription of rob at 30°C and 37°C. This reduction was observed more potently when rob was exposed to sodium decanoate at 30°C. Minimum inhibitory concentration (MIC) for various antibiotics of the S. marcescens rob knock-out demonstrated a decrease in susceptibility to nalidixic acid, tetracycline, chloramphenicol, ciprofloxacin, norfloxacin, and ofloxacin. Over-expression of rob resulted in an increased resistance by 4, 2, and 2-fold to nalidixic acid, tetracycline and chloramphenicol, respectively. In addition, rob over-production displayed 8, 4, and 4-fold increase in resistance to ciprofloxacin, norfloxacin, ofloxacin, respectively. To discover the role of rob in the efflux mechanism, we performed ethidium bromide accumulation assays on over-expressing and knock-out strains. Organic solvent tolerance assays were carried out using n-hexane to determine if rob is involved in expression of efflux pumps. We found the rob null mutant to be sensitive to n-hexane while the over-expression of rob resulted in resistance to n-hexane. RT-PCR of the rob knock-out strain showed a decrease in expression of micF, ompC, sdeXY, sdeAB and tolC, respectively, and an increase in the expression of ompF. To conclude, we identified a rob homolog in S. marcescens which contributes to resistance to multiple antibiotics and tolerance to organic solvent.

Serratia marcescens, a member of Enterobacteriaceae family, is a causative agent of nosocomial and opportunistic infections. Numerous reports show that the multidrug resistance among S. marcescens is growing. This organism has high-level intrinsic resistance to a variety of antimicrobial agents, which makes the treatment of infections caused by this bacterium very difficult. The major mechanism for antibiotic resistance, especially to fluoroquinolones, in Gram-negative organisms is the active efflux of the antibiotic molecule mediated by efflux pumps belonging to the Resistance-Nodulation-Cell Division (RND) family. It was previously shown that the SdeAB and SdeXY multidrug efflux pumps are important for conferring the intrinsic drug resistance in S.marcescens. In Escherichia coli, the up-regulation of transcriptional activators, such as MarA, SoxS and Rob, affect transcription of acrAB, tolC and micF. Over-expression of Rob results in increased expression of the E. coli AcrAB-TolC efflux pump and decreases outer membrane permeability through up-regulation of micF, resulting in multidrug, organic solvent and heavy metal resistance. In the present study, we report the identification of a rob gene in S. marcescens which has a 70% identity at the DNA level and 71% identity at the amino acid level to that of E. coli. Moreover, the S. marcescens rob demonstrated similar properties to the E. coli rob including having an effect on expression of outer membrane protein F (OmpF) and over-expression of SdeAB and SdeXY, conferring antibiotic resistance to divergent antibacterial agents and tolerance to organic solvents. We performed rob promoter evaluations using transcriptional fusions to the Green Fluorescence Protein (GFP) in the vector pGlow-TOPO and constructed a rob knock-out using the TargeTron Gene Knockout System. Promoter activity assessment, using the pGlow-TOPO reporter plasmid, showed that rob had higher promoter activity at 37°C than 30°C. In the presence of 2,2’-dipyridyl, rob promoter activity was observed to be slightly increased in the early and mid-log phase by 1.4 and 1.1 fold, respectively. We also showed that sodium decanoate and sodium salicylate can reduce the transcription of rob at 30°C and 37°C. This reduction was observed more potently when rob was exposed to sodium decanoate at 30°C. Minimum inhibitory concentration (MIC) for various antibiotics of the S. marcescens rob knock-out demonstrated a decrease in susceptibility to nalidixic acid, tetracycline, chloramphenicol, ciprofloxacin, norfloxacin, and ofloxacin. Over-expression of rob resulted in an increased resistance by 4, 2, and 2-fold to nalidixic acid, tetracycline and chloramphenicol, respectively. In addition, rob over-production displayed 8, 4, and 4-fold increase in resistance to ciprofloxacin, norfloxacin, ofloxacin, respectively. To discover the role of rob in the efflux mechanism, we performed ethidium bromide accumulation assays on over-expressing and knock-out strains. Organic solvent tolerance assays were carried out using n-hexane to determine if rob is involved in expression of efflux pumps. We found the rob null mutant to be sensitive to n-hexane while the over-expression of rob resulted in resistance to n-hexane. RT-PCR of the rob knock-out strain showed a decrease in expression of micF, ompC, sdeXY, sdeAB and tolC, respectively, and an increase in the expression of ompF. To conclude, we identified a rob homolog in S. marcescens which contributes to resistance to multiple antibiotics and tolerance to organic solvent.

Drosophila melanogaster is a widely used and efficient genetic model to study nervous system development. The conservation of many genes from Drosophila to vertebrates and a short reproduction cycle makes the fruitfly a great tool for providing insight into crucial events in nervous system formation. In studying the development of the sensory nervous system, Drosophila also provides a model for understanding the formation and function of structurally diverse cilia. Cilia are hairlike organelles present throughout our bodies and responsible for many processes such as chemo, mechano, and thermosensation, fluid movement, hearing and fertility. In Drosophila the only somatic ciliated cells are the Type I sensory neurons in which a cilium forms the sensory dendrite. There are more than two diverse subtypes of the ciliated sensory neurons and the mechanism by which this diversity is achieved remains unclear. The mechanism of ciliated sensory neuron differentiation was hereby studied on an example of a differentially expressed ciliary gene - CG6129 - a Drosophila orthologue of human Rootletin, a main protein components of ciliary rootlets. CG6129 expression is specific to the ciliated cells and exhibits so called chordotonal-enriched pattern - a strong and permanent expression in the chordotonal subtype of type I neurons and weaker and transient expression in the external sensory subtype. I have shown that CG6129 knock-down causes severe disruption of the chordotonal organs function without any obvious change in the structure of the cilium, other than the lack of ciliary rootlet. The function of the external sensory subtype was only slightly affected which further highlights the difference between the two types of ciliated sensory organs. The fact that CG6129 is differentially expressed in the two subtypes of the Drosophila ciliated sensory neurons suggests that the genes involved in the formation of various cilia are differentially regulated. I have shown that CG6129 is regulated by the two well known ciliary transcription factors - RFX and fd3F (distant homologue of Foxj1). Of the two enhancers found the early-to-late enhancer is almost entirely dependent on RFX and not on fd3F while the late enhancer is dependent on both fd3F and RFX. The fact that there is some residual CG6129 expression in the absence of both RFX and fd3F suggests involvement of another regulator that may contribute to the cilia diversity. Zmynd10 is a recently characterised ciliary gene that is involved in the axonemal dynein arms assembly. Mutations in human Zmynd10 cause primary ciliary dyskinesia (PCD) and Drosophila Zmynd10 mutants have immotile cilia that lack dynein arms. Due to the presence of specific protein domains Zmynd10 has been suggested to act as a transcriptional regulator. I have shown that the transcript levels of CG6129 and other ciliary genes are reduced in the Zmynd10 mutant. This implies that Zmynd10 may regulate ciliary genes on a transcriptional or post transcriptional level and may contribute to the regulatory network governing ciliogenesis.

No description available.

A ampliação do conhecimento sobre as formas de comunicação, controle e regulação existentes em bactérias traz luz aos avanços no combate das infecções hospitalares que são responsáveis por inúmeros prejuízos relacionados à saúde pública em todo planeta. DUMOULIN et al (2013), descreveram o regulador transcricional (RT) ElrR, que regula positivamente a transcrição do gene elrA, um fator de virulência de Enterococcus faecalis. ElrA apresenta grande similaridade com as internalinas de Listeria monocytogenes, que facilitam a invasão da bactéria ao hospedeiro. ElrR é considerada como pertencente à família Rgg-like de RT exclusivo de bactérias Gram positivas. Por vários motivos a família Rgg foi inserida à superfamília RNPP, gerando a superfamília RRNPP de RT. Os RRNPP fazem parte de um sistema de regulação por quorum sensing (QS), um sistema de comunicação célula-célula dependente de densidade celular, com função associada na ativação ou inibição da expressão de proteínas relacionadas, dentre outros, à virulência, formação de biofilme e esporulação. Para a melhor compreensão do mecanismo de como ocorre a ativação da transcrição do fator de virulência ElrA, este trabalho apresenta resultados de expressão heteróloga em E. coli e purificação das proteínas ElrR e ElrA, bem como resultados de experimentos biofísicos que caracterizam algumas propriedades estruturais e biológicas destas proteínas. Utilizando técnicas de cromatografia, espectroscopia de dicroísmo circular (CD), anisotropia de fluorescência, espalhamento dinâmico de luz (DLS), cristalografia de raios X e ressonância plasmônica de superfície (SPR), foi possível a obtenção da estrutura tridimensional de ElrR e de indícios da interação com uma região de 25bp do DNA. Realizou-se ainda, em colaboração com Dra. Pascale Serror, a tentativa de obtenção da molécula autoindutora (AI) de ElrR. São apresentados primeiros resultados da obtenção heteróloga de ElrA, sua purificação e cristalização, com importantes características que permitirão a continuação da investigação deste fator de virulência. ElrR é composta somente por alfa-hélices e apresenta-se dimérico em solução. Apesar da similaridade estrutural dos RRNPP, a identidade da sequência entre ElrR e os outros membros é extremamente baixa, o que motivou a resolução das fases cristalográficas experimentalmente. A estrutura de ElrR apresenta-se similar às homólogas, porém, com maior interface de interação entre os protômeros, que formam o dímero. O sítio de ligação do AI, em ElrR, apresenta-se mais amplo, com cavidade maior que as demais estruturas estudadas, conservando vários dos resíduos apresentados nos homólogos que realizam a estabilização do AI. Os altos fatores de temperatura dos cristais de ElrR, adicionado a anisotropia dos átomos, de uma das estruturas obtidas, apresenta a grande flexibilidade desse RT. Os indícios de interação entre ElrR e DNA aqui apresentados, obtidos por SPR e anisotropia de fluorescência, apresentam que ElrR liga especificamente ao fragmento proposto do DNA, ainda na ausência do AI. A não cristalização do complexo (ElrR-DNA), adicionada a alta flexibilidade apresentada na estrutura e a instabilidade observada na ligação ao DNA (por SPR) apontam para a obrigatoriedade da molécula de regulação (AI) para que o complexo ElrR-DNA seja estável. / The enhancing of the knowledge about communication, control and regulation in bacteria bring possibilities on the advance of hospital-acquired infections control responsible for various prejudices related to public health worldwide. DUMOULIN, et al (2013) described ElrR, a transcriptional regulator (TR), that positively regulates transcription of the elrA gene, which codifies a virulence factor of Enterococcus faecalis. ElrA shows high similarity with Listeria monocytogeneses internalins, which facilitates host invasion by these bacteria. ElrR are considered belonging to Rgg-like TR family exclusive of Gram positive bacteria. Several reasons include the Rgg family into the RNPP superfamily, generating the RRNPP superfamily of TR. The RRNPP are controlled by a quorum sensing (QS) regulation system, a cell-cell communication system based on cellular density that activates or inhibits the expression of proteins related with virulence, biofilm formation, sporulation, and others. For a better understanding of the transcription activation mechanism of ElrA, this work shows ElrR and ElrA heterologous expression in E. coli and purification of these proteins, as well as biophysics assays to characterize some structural and biological features of both proteins. Using chromatography, circular dichroism (CD), fluorescence anisotropy, dynamic light scattering (DLS), X-ray crystallography and surface plasmon resonance (SPR) technics, it was possible to obtain the tridimensional structure of ElrR, and evidences of ElrR-DNA complex formation, confirming DNA interaction site of ElrR with a 25 bp fragment. In collaboration with Dr. Pascale Serror, we attempted to achieve the ElrR auto-induction (AI) molecule. Also, results of the heterologous obtainment of ElrA are presented, as well as ElrA purification and crystallization, presenting important characteristics which will allow the further investigation of this virulence factor in near future. ElrR is composed by alpha-helices presenting dimeric fold in solution. Despite the similarity between the RRNPP members, the low identity of ElrR to the other members motivates the experimental crystallographic phases solution. ElrR structure is very similar to the homologous structures, presenting a higher interface between the protomers that compose the dimer. Its AI binding site is wider than the other structures studied, conserving several amino acid residues presented at the homologous proteins, that stabilizes the AI molecule. High temperature factors of the amino acid residues showed in all the obtained ElrR crystallographic structures plus the anisotropy of the atoms in one of those structures show the high flexibility of this TR. The evidence of the ElrR-DNA complex presented in this study, obtained by SPR and fluorescence anisotropy, indicates that ElrR binds at the proposed DNA site even in the absence of the AI molecule. The failure to obtain the ElrR-DNA complex crystals added to the high flexibility presented at some places of the structure and the observed instability at the formed complex (observed at SPR) suggest the mandatory need of the AI molecule to create a stable ElrR-DNA complex.

A ampliação do conhecimento sobre as formas de comunicação, controle e regulação existentes em bactérias traz luz aos avanços no combate das infecções hospitalares que são responsáveis por inúmeros prejuízos relacionados à saúde pública em todo planeta. DUMOULIN et al (2013), descreveram o regulador transcricional (RT) ElrR, que regula positivamente a transcrição do gene elrA, um fator de virulência de Enterococcus faecalis. ElrA apresenta grande similaridade com as internalinas de Listeria monocytogenes, que facilitam a invasão da bactéria ao hospedeiro. ElrR é considerada como pertencente à família Rgg-like de RT exclusivo de bactérias Gram positivas. Por vários motivos a família Rgg foi inserida à superfamília RNPP, gerando a superfamília RRNPP de RT. Os RRNPP fazem parte de um sistema de regulação por quorum sensing (QS), um sistema de comunicação célula-célula dependente de densidade celular, com função associada na ativação ou inibição da expressão de proteínas relacionadas, dentre outros, à virulência, formação de biofilme e esporulação. Para a melhor compreensão do mecanismo de como ocorre a ativação da transcrição do fator de virulência ElrA, este trabalho apresenta resultados de expressão heteróloga em E. coli e purificação das proteínas ElrR e ElrA, bem como resultados de experimentos biofísicos que caracterizam algumas propriedades estruturais e biológicas destas proteínas. Utilizando técnicas de cromatografia, espectroscopia de dicroísmo circular (CD), anisotropia de fluorescência, espalhamento dinâmico de luz (DLS), cristalografia de raios X e ressonância plasmônica de superfície (SPR), foi possível a obtenção da estrutura tridimensional de ElrR e de indícios da interação com uma região de 25bp do DNA. Realizou-se ainda, em colaboração com Dra. Pascale Serror, a tentativa de obtenção da molécula autoindutora (AI) de ElrR. São apresentados primeiros resultados da obtenção heteróloga de ElrA, sua purificação e cristalização, com importantes características que permitirão a continuação da investigação deste fator de virulência. ElrR é composta somente por alfa-hélices e apresenta-se dimérico em solução. Apesar da similaridade estrutural dos RRNPP, a identidade da sequência entre ElrR e os outros membros é extremamente baixa, o que motivou a resolução das fases cristalográficas experimentalmente. A estrutura de ElrR apresenta-se similar às homólogas, porém, com maior interface de interação entre os protômeros, que formam o dímero. O sítio de ligação do AI, em ElrR, apresenta-se mais amplo, com cavidade maior que as demais estruturas estudadas, conservando vários dos resíduos apresentados nos homólogos que realizam a estabilização do AI. Os altos fatores de temperatura dos cristais de ElrR, adicionado a anisotropia dos átomos, de uma das estruturas obtidas, apresenta a grande flexibilidade desse RT. Os indícios de interação entre ElrR e DNA aqui apresentados, obtidos por SPR e anisotropia de fluorescência, apresentam que ElrR liga especificamente ao fragmento proposto do DNA, ainda na ausência do AI. A não cristalização do complexo (ElrR-DNA), adicionada a alta flexibilidade apresentada na estrutura e a instabilidade observada na ligação ao DNA (por SPR) apontam para a obrigatoriedade da molécula de regulação (AI) para que o complexo ElrR-DNA seja estável. / The enhancing of the knowledge about communication, control and regulation in bacteria bring possibilities on the advance of hospital-acquired infections control responsible for various prejudices related to public health worldwide. DUMOULIN, et al (2013) described ElrR, a transcriptional regulator (TR), that positively regulates transcription of the elrA gene, which codifies a virulence factor of Enterococcus faecalis. ElrA shows high similarity with Listeria monocytogeneses internalins, which facilitates host invasion by these bacteria. ElrR are considered belonging to Rgg-like TR family exclusive of Gram positive bacteria. Several reasons include the Rgg family into the RNPP superfamily, generating the RRNPP superfamily of TR. The RRNPP are controlled by a quorum sensing (QS) regulation system, a cell-cell communication system based on cellular density that activates or inhibits the expression of proteins related with virulence, biofilm formation, sporulation, and others. For a better understanding of the transcription activation mechanism of ElrA, this work shows ElrR and ElrA heterologous expression in E. coli and purification of these proteins, as well as biophysics assays to characterize some structural and biological features of both proteins. Using chromatography, circular dichroism (CD), fluorescence anisotropy, dynamic light scattering (DLS), X-ray crystallography and surface plasmon resonance (SPR) technics, it was possible to obtain the tridimensional structure of ElrR, and evidences of ElrR-DNA complex formation, confirming DNA interaction site of ElrR with a 25 bp fragment. In collaboration with Dr. Pascale Serror, we attempted to achieve the ElrR auto-induction (AI) molecule. Also, results of the heterologous obtainment of ElrA are presented, as well as ElrA purification and crystallization, presenting important characteristics which will allow the further investigation of this virulence factor in near future. ElrR is composed by alpha-helices presenting dimeric fold in solution. Despite the similarity between the RRNPP members, the low identity of ElrR to the other members motivates the experimental crystallographic phases solution. ElrR structure is very similar to the homologous structures, presenting a higher interface between the protomers that compose the dimer. Its AI binding site is wider than the other structures studied, conserving several amino acid residues presented at the homologous proteins, that stabilizes the AI molecule. High temperature factors of the amino acid residues showed in all the obtained ElrR crystallographic structures plus the anisotropy of the atoms in one of those structures show the high flexibility of this TR. The evidence of the ElrR-DNA complex presented in this study, obtained by SPR and fluorescence anisotropy, indicates that ElrR binds at the proposed DNA site even in the absence of the AI molecule. The failure to obtain the ElrR-DNA complex crystals added to the high flexibility presented at some places of the structure and the observed instability at the formed complex (observed at SPR) suggest the mandatory need of the AI molecule to create a stable ElrR-DNA complex.

A biofilm, which is associated with 80% of chronic infections in humans, is formed when bacteria aggregate, attach to a substrate and secrete a matrix protecting the bacteria from host cell defenses and antibiotics. Bartonella henselae (B. henselae) is the causative agent of cat scratch disease, persistent bacteremia, and one of the most frequently reported causes of blood-culture negative endocarditis (BCNE) in patients. The ability of B. henselae to adhere to the heart valve, form a biofilm and vegetation to cause endocarditis increases the morbidity and mortality rate in infected patients. The presence of a trimeric autotransporter adhesin (TAA) called Bartonella adhesin A (BadA) has been linked to biofilm formation in B. henselae. BadA is a protein of 3036 amino acids and a member of the TAAs found in Bartonella and other Gram-negative bacteria. The function of BadA has been studied in vitro and is critical for agglutination, host cell adhesion and activation of a pro-angiogenic host response. However, very little is known about badA gene regulation or the molecular basis of biofilm formation. This work aims to determine whether BadA is necessary for the establishment of biofilms and how the bacteria regulate badA expression. Using genetic mutations, real-time cell adhesion assay, RT-qPCR, and microscopy, it was shown that BadA is required for biofilm formation. Using an in-frame complete deletion strain of badA, a reduced ability to form a biofilm was observed which was restored in the deletion strain complemented with a partial badA. Analysis of the B. henselae transcriptome shows nine highly transcribed, homologous RNAs, termed Bartonella regulatory transcript (Brt1-9). The Brts are short-sized (<200 >nucleotides), highly expressed, and located in an intergenic region indicative of small RNAs (sRNA). The Brts are predicted to form a stable stem and loop structure with a potential terminator/riboswitch region on the 3′ end. Located ~20 nucleotides downstream of each Brt is a poorly transcribed helix-turn-helix DNA binding protein gene termed transcriptional regulatory protein (trps 1-9). High brt transcription stops just before the start of the trp implicating the 3’ loop of the Brt as a terminating loop. Replacement of the trp with a gfp reporter gene shows that in the absence of the 3′ end of Brt1, gfp is transcribed. Also consistent with our findings, an increase in both the transcription of trp1 and badA and the formation of a biofilm in mutants of the brt1 gene was observed. Furthermore, to determine the role of the Trp in regulating badA, an electrophoretic mobility shift assay was carried out. The data confirms that Trp1 binds the promoter region of badA gene to regulate gene expression. In summary, the brt1/trp1 regulon affects badA transcription and biofilm formation in B. henselae. Understanding the mechanism and condition(s) by which the brt/trp regulatory system regulates badA is a plausible approach to the development of treatments that target the formation of biofilm-related diseases and persistent bacteremia in humans.

<p>Bacteria adapt to changes in their environment by regulating gene transcription. TetR family transcriptional regulators (TFRs) constitute one of the largest groups of bacterial transcription factors and thus, characterization of TFRs is anticipated to be crucial for a better understanding of prokaryotic physiology. Of significant importance, the majority of TFRs are predicted to respond to small-molecule signals and an emerging paradigm suggests that identifying ligands of TFRs can provide direct insight into the biochemical functions of the genes they regulate. Regulatory target genes and small-molecule ligands are unknown for all but a few TFRs and therefore, generally applicable tools for identifying these basic elements of TFRs are highly desirable. We first investigated the use of genome context as a predictive tool for identifying regulatory targets of TFRs. We find that the majority of TFRs are divergently oriented from a neighboring gene, and those with a“200 bp rule” should allow us to predict at least one regulatory target for more than half of all TFRs in the public databases. Second, we developed a biosensor mechanism amenable to high-throughput screening for identifying ligands of TFRs of unknown function. Significantly, one of our biosensors has played an integral role in characterizing the ligands of a previously uncharacterized TFR. Thus, the combined use of the tools we have developed will provide considerable benefit in understanding bacterial small-molecules responses mediated by TFRs.</p> / Doctor of Philosophy (PhD)

La région fos de la souche d’Escherichia coli pathogène aviaire BEN2908 permet le métabolisme des fructanes, des prébiotiques largement utilisés en alimentation humaine et animale. Ce métabolisme contribue à l’implantation de BEN2908 dans son réservoir, l’intestin. La région fos, située au sein de l’îlot génomique AGI-3, est composée de 6 gènes codant pour un transporteur de sucre et des enzymes impliquées dans le métabolisme des fructanes, et d’un gène transcrit de façon divergente codant pour FosR, un régulateur transcriptionnel de la famille LacI/GalR. Nous avons montré que l’expression des gènes fos est réprimée par FosR, contrôlée par la répression catabolique et induite en présence de fructanes. FosR se lie à 2 séquences opératrices de la région promotrice de l’opéron fos et cette liaison est inhibée en présence de fructanes, surtout par le 1-kestose. La région fos confère un avantage de croissance en présence de contenu cæcal et contribue à la colonisation des cæca in vivo. De plus, AGI-3 est mobile et transférable, ce qui suggère une possible diffusion du métabolisme des fructanes au sein de l’espèce E. coli. / The fos region of the avian pathogenic Escherichia coli strain BEN2908 is involved in fructan metabolism, prebiotics widely used commercially in food products for both humans and animals. This metabolism contributes to the establishment of BEN2908 in its reservoir, the intestine. The fos region, located within the genomic island AGI-3, is composed of six genes encoding a sugar transporter and enzymes involved in fructan metabolism, and of a divergently transcribed gene encoding a transcriptional regulator, FosR, belonging to the LacI/GalR family. We demonstrated that the expression of fos genes is repressed by FosR, controlled by catabolite repression and induced in the presence of fructans. FosR binds to two operator sequences of the fos operon promoter region. This binding to DNA is inhibited in the presence of fructans, especially by 1-kestose. The fos region strongly benefits growth on cecal content and colonization of the ceca in vivo. Moreover, AGI-3 is mobile and transferable, suggesting a possible dissemination of fructan metabolism within the species E. coli.

La régulation transcriptionnelle des gènes est un processus indispensable sans lequel la diversité phénotypique des cellules ainsi que l’adaptation à leur environnement serait inexistant. L’identification des éléments de régulation dans le génome est d’une importance capitale afin de comprendre les mécanismes gouvernant l’expression des gènes spécifiques à un type cellulaire donné. Ainsi, suite au pic de LH, le follicule ovarien entre dans un programme intensif de différentiation cellulaire, orchestré par des modifications majeures du profile transcriptionnel des cellules de granulosa, déclenchant ultimement l’ovulation et la lutéinisation, processus indispensables à la fertilité femelle. L’hypothèse supportée par cette étude stipule qu’une réorganisation de la structure chromatinienne survient aux régions régulatrices d’une panoplie de gènes dans les heures suivant le pic de LH et qu’en isolant et identifiant ces régions, il serait possible de retrouver des éléments essentiels aux processus d’ovulation et de lutéinisation. Ainsi, en utilisant un protocole standard de superovulation chez la souris, les éléments de régulation se modifiant 4h suivant l’administration de hCG ont été isolés et identifiés dans les cellules de granulosa en utilisant la méthode FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) combinée à un séquençage haut débit. Cette étude a démontré que suite au stimulus ovulatoire, les cellules de granulosa subissent une reprogrammation majeure des éléments de régulation, qui est corrélée avec une modification drastique de leurs fonctions biologiques. De plus, cette étude a mis en évidence une association majoritaire des éléments de régulation à des régions intergéniques distales et à des introns, indiquant que ces régions ont une importance capitale dans la régulation transcriptionnelle dans les cellules de granulosa. Cette étude a également permis d’identifier une panoplie de régulateurs transcriptionnels reconnus pour être essentiels à la fonction ovarienne, ainsi que leur sites de liaison dans le génome, démontrant que la méthode FAIRE est une méthode assez puissante pour permettre la prédiction d’événements moléculaires précis ayant un sens physiologique réel. / Identification of regulatory elements in the genome is of paramount importance to understanding the mechanisms governing the expression of specific genes in a given cell type. Following the LH surge, the ovarian peri-ovulatory follicle enters an intensive program of cellular differentiation, orchestrated by major changes in the transcriptional profile of granulosa cells, ultimately triggering ovulation and luteinization, processes essentials for fertility in females. In the mouse, several genes essential to the success of this program are induced 2 to 6 hours after the ovulatory stimulus. Using a standard protocol for superovulation in mice, the regulatory elements were isolated and identified in granulosa cells 4h after administration of hCG using the method FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) combined with next generation sequencing. The results of this analysis demonstrate that after the ovulatory stimulus, granulosa cells undergo a major reprogramming of regulatory elements, which is correlated with the extensive changes in their biological functions. In addition, this study showed that most regulatory elements were associated with distal intergenic regions and introns, indicating that these regions are important in transcriptional regulation in granulosa cells. A variety of transcriptional regulators known to be essential for ovarian function, and their binding sites were also identified in this analysis, demonstrating that the FAIRE method has the power to predict molecular events that have correlates in the known physiology of ovarian processes.