Les petits ARN régulateurs et les systèmes à deux composants sont des régulateurs très répandus de l’expression des gènes chez les bactéries. Dans la plupart des cas, les systèmes à deux composants agissent comme des régulateurs transcriptionnels. Un grand nombre de petits ARN agissent quant à eux au niveau post-transcriptionnel en modulant la traduction et/ou la stabilité de leur(s) ARN messager(s)-cible. Des connexions entre ces deux systèmes ont récemment pu mettre en lumière des circuits de régulations complexes aux propriétés encore peu connues.Mon travail a tout d’abord porté sur la connexion entre le système à deux composants EnvZ-OmpR et les petits ARN OmrA et OmrB chez Escherichia coli. Dans un premier temps, nous avons montré qu’OmpR activait directement la transcription d’omrA et d’omrB en se fixant à leur promoteur. Cette activation permet la production des petits ARN OmrA et OmrB qui, via leur extrémité 5’ conservée, ciblent à leur tour plusieurs ARN messagers-cibles et notamment le messager ompR-envZ. En accord avec des études précédentes, le contrôle d’ompR-envZ par les Omr n’affecte pas le niveau de forme phosphorylée d’OmpR. Ce phénomène posait donc la question de l’intérêt d’une telle régulation. Nous avons ensuite pu montrer que la régulation d’omrA et d’omrB est assez unique car leurs promoteurs répondent non seulement à la forme phosphorylée mais aussi à la forme non phosphorylée d’OmpR. Ce phénomène permet à ces ARN régulateurs de limiter leur propre synthèse en ayant un effet limité sur l’expression des autres cibles d’OmpR comme les porines OmpC et OmpF.Ce travail nous a conduits à chercher à caractériser d’autres exemples de modulation des systèmes à deux composants par des petits ARN régulateurs. Nous avons notamment étudié la régulation du système NarQ-NarP. En effet, nos travaux ont montré que la synthèse de narP était contrôlée par le petit ARN RprA. Cette régulation semble affecter les cibles de NarP et en particulier l’opéron napFDAGHBC. De plus, RprA répondrait au même stimulus que le système NarQ-NarP créant ainsi un lien physiologique entre le petit ARN et sa cible.Pour finir, un autre aspect de ce travail de thèse a été de s’intéresser à la régulation d’OmrA/B dans un contexte d’infection des macrophages par une souche d’Escherichia coli pathogène, la souche LF82. En effet, des données suggéraient que ces petits ARN étaient induits au cours de l’infection. J’ai pu valider ces données et montrer que cette induction était dépendante de la présence du système EnvZ/OmpR.En conclusion, j’ai pu montrer par diverses approches que les circuits de régulation intégrant des systèmes à deux composants et des petits ARN régulateurs possédaient des propriétés assez inédites permettant à la bactérie de s’adapter à divers stress. / Small regulatory RNA (sRNAs) and two component systems (TCS) are both widespread regulators of gene expression in bacteria. While TCS are mostly transcriptional regulators, a large class of sRNAs acts as post-transcriptional regulators of gene expression by modulating translation and/or stability of target-mRNAs. Many connections have recently been unraveled between these two types of regulators, resulting in mixed regulatory circuits with poorly characterized properties.First, we have investigated in details the negative feedback circuit that exists between the EnvZ-OmpR TCS and the OmrA/B sRNAs in Escherichia coli. We have found that OmpR directly activates transcription from omrA and omrB promoters, allowing production of OmrA/B sRNAs that target multiple mRNAs through their conserved 5’ end, including the ompR-envZ mRNA. In agreement with previous reports, we have found that this control of ompR-envZ by OmrA/B sRNAs does not affect the amount of OmpR-P i.e. the presumably active form of the regulator. This phenomenon therefore raised the question of the possible interest of such a regulation. Thereafter, we found that OmrA/B regulation is really unique because they respond to the phosphorylated form but also to the unphosphorylated form of OmpR. As a result, OmrA/B limit their own synthesis while they have only a limited effect on others targets of OmpR, such as the OmpC or OmpF porins.This work led us to try to characterize other examples of modulation of two-component systems synthesis by small regulatory RNAs. In particular, we studied the regulation of the NarQ-NarP system. Indeed, our work showed that the synthesis of narP is controlled by the RprA sRNA. This regulation appears to affect NarP targets and in particular the napFDAGHBC operon. Moreover, RprA would respond to the same stimulus as the NarQ-NarP system, thus creating a physiological link between the small RNA and its target.Finally, another aspect of this work was to study the regulation of OmrA/B in a context of infection of macrophages by an Escherichia coli pathogenic strain, LF82. Indeed, data suggested that these small RNAs were induced during infection. I was able to validate these data and showed that this induction was dependent on the presence of the EnvZ-OmpR system.

The CsgD protein is the master regulator of a pathway leading to the formation of curli, in essence regulating the switch between a motile and a sessile lifestyle for bacteria. The 5’-UTR region of the csgD mRNA is a hotspot for multiple regulatory small RNAs (sRNA) involved in a complex regulatory network. Even though it is previously known how the interaction takes place it is unknown how sRNA binding affects the translational activity. In order to suggest a mode of regulation a mutational assay was performed by making changes in the csgD 5’-UTR and investigate what the translational effects were. Mutations in different regions are shown to affect the translation levels in various ways.

Cells sense the properties of the surrounding environment and convert this information into changes in gene expression. Bacteria are, in contrast to many multi-cellular eukaryotes, remarkable in their ability to cope with rapid environmental changes and to endure harsh and extreme milieus. Previously, control of gene expression was thought to be carried out exclusively by proteins. However, it is now clear that small regulatory RNAs (sRNA) also carry out gene regulatory functions. Bacteria such as E. coli harbor a large class of sRNAs that bind to mRNAs to alter translation and/or mRNA stability. By identifying mRNAs that are targeted by sRNAs, my studies have broadened the understanding of the mechanisms that underlie sRNA-dependent gene regulation, and have shed light on the impact that this type of regulation has on bacterial physiology. Control of gene expression often relies on the interplay of many regulators. This interplay is exemplified by our discovery of mutual regulation between the sRNA MicF and the globally acting transcription factor Lrp. Through double negative feedback, these two regulators respond to nutrient availability in the environment which results in reprogramming of downstream gene expression. We have also shown that both the transcription factor CsgD, and the anti-sigma factor FlgM, are repressed by the two sRNAs OmrA and OmrB, suggesting that these sRNAs are important players in the complex regulation that allow bacteria to switch between motility and sessility. Bacterial populations of genetically identical individuals show phenotypic variations when switching to the sessile state due to bistability in gene expression. While bistability has previously been demonstrated to arise from stochastic fluctuations in transcription, our results suggest that bistability possibly may arise from sRNA-dependent regulatory events also on the post-transcriptional level.