Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability.

Secondary structure-forming DNA sequences such as CAG repeats interfere with replication and repair, provoking fork stalling, chromosome fragility, and recombination. In budding yeast, we found that expanded CAG repeats are more likely than unexpanded repeats to localize to the nuclear periphery. This positioning is transient, occurs in late S phase, requires replication, and is associated with decreased subnuclear mobility of the locus. In contrast to persistent double-stranded breaks, expanded CAG repeats at the nuclear envelope associate with pores but not with the inner nuclear membrane protein Mps3. Relocation requires Nup84 and the Slx5/8 SUMO-dependent ubiquitin ligase but not Rad51, Mec1, or Tel1. Importantly, the presence of the Nup84 pore subcomplex and Slx5/8 suppresses CAG repeat fragility and instability. Repeat instability in nup84, slx5, or slx8 mutant cells arises through aberrant homologous recombination and is distinct from instability arising from the loss of ligase 4-dependent end-joining. Genetic and physical analysis of Rad52 sumoylation and binding at the CAG tract suggests that Slx5/8 targets sumoylated Rad52 for degradation at the pore to facilitate recovery from acute replication stress by promoting replication fork restart. We thereby confirmed that the relocation of damage to nuclear pores plays an important role in a naturally occurring repair process.

Nolz1 promotes striatal neurogenesis through the regulation of retinoic acid signaling

Background: Nolz1 is a zinc finger transcription factor whose expression is enriched in the lateral ganglionic eminence (LGE), although its function is still unknown. Results: Here we analyze the role of Nolz1 during LGE development. We show that Nolz1 expression is high in proliferating neural progenitor cells (NPCs) of the LGE subventricular zone. In addition, low levels of Nolz1 are detected in the mantle zone, as well as in the adult striatum. Similarly, Nolz1 is highly expressed in proliferating LGE-derived NPC cultures, but its levels rapidly decrease upon cell differentiation, pointing to a role of Nolz1 in the control of NPC proliferation and/or differentiation. In agreement with this hypothesis, we find that Nolz1 over-expression promotes cell cycle exit of NPCs in neurosphere cultures and negatively regulates proliferation in telencephalic organotypic cultures. Within LGE primary cultures, Nolz1 over-expression promotes the acquisition of a neuronal phenotype, since it increases the number of β-III tubulin (Tuj1)- and microtubule-associated protein (MAP)2-positive neurons, and inhibits astrocyte generation and/or differentiation. Retinoic acid (RA) is one of the most important morphogens involved in striatal neurogenesis, and regulates Nolz1 expression in different systems. Here we show that Nolz1 also responds to this morphogen in E12.5 LGE-derived cell cultures. However, Nolz1 expression is not regulated by RA in E14.5 LGE-derived cell cultures, nor is it affected during LGE development in mouse models that present decreased RA levels. Interestingly, we find that Gsx2, which is necessary for normal RA signaling during LGE development, is also required for Nolz1 expression, which is lost in Gsx2 knockout mice. These findings suggest that Nolz1 might act downstream of Gsx2 to regulate RA-induced neurogenesis. Keeping with this hypothesis, we show that Nolz1 induces the selective expression of the RA receptor (RAR)β without altering RARα or RARγ. In addition, Nozl1 over-expression increases RA signaling since it stimulates the RA response element. This RA signaling is essential for Nolz1-induced neurogenesis, which is impaired in a RA-free environment or in the presence of a RAR inverse agonist. It has been proposed that Drosophila Gsx2 and Nolz1 homologues could cooperate with the transcriptional co-repressors Groucho-TLE to regulate cell proliferation. In agreement with this view, we show that Nolz1 could act in collaboration with TLE-4, as they are expressed at the same time in NPC cultures and during mouse development. Conclusions: Nolz1 promotes RA signaling in the LGE, contributing to the striatal neurogenesis during development.

Catechol-O-methyltransferase, dopamine, and sleep-wake regulation.

NlmCategory="UNASSIGNED">Sleep and sleep disorders are complex and highly variable phenotypes regulated by many genes and environment. The catechol-O-methyltransferase (COMT) gene is an interesting candidate, being one of the major mammalian enzymes involved in the catabolism of catecholamines. The activity of COMT enzyme is genetically polymorphic due to a guanine-to-adenine transition at codon 158, resulting in a valine (Val) to methionine (Met) substitution. Individuals homozygous for the Val allele show higher COMT activity, and lower dopaminergic signaling in prefrontal cortex (PFC) than subjects homozygous for the Met allele. Since COMT has a crucial role in metabolising dopamine, it was suggested that the common functional polymorphism in the COMT gene impacts on cognitive function related to PFC, sleep-wake regulation, and potentially on sleep pathologies. The COMT Val158Met polymorphism may predict inter-individual differences in brain electroencephalography (EEG) alpha oscillations and recovery processes resulting from partial sleep loss in healthy individuals. The Val158Met polymorphism also exerts a sexual dimorphism and has a strong effect on objective daytime sleepiness in patients with narcolepsy-cataplexy. Since the COMT enzyme inactivates catecholamines, it was hypothesized that the response to stimulant drugs differs between COMT genotypes. Modafinil maintained executive functioning performance and vigilant attention throughout sleep deprivation in subjects with Val/Val genotype, but less in those with Met/Met genotype. Also, homozygous Met/Met patients with narcolepsy responded to lower doses of modafinil compared to Val/Val carriers. We review here the critical role of the common functional COMT gene polymorphism, COMT enzyme activity, and the prefrontal dopamine levels in the regulation of sleep and wakefulness in normal subjects, in narcolepsy and other sleep-related disorders, and its impact on the response to psychostimulants.

Nolz1 promotes striatal neurogenesis through the regulation of retinoic acid signaling

Background: Nolz1 is a zinc finger transcription factor whose expression is enriched in the lateral ganglionic eminence (LGE), although its function is still unknown. Results: Here we analyze the role of Nolz1 during LGE development. We show that Nolz1 expression is high in proliferating neural progenitor cells (NPCs) of the LGE subventricular zone. In addition, low levels of Nolz1 are detected in the mantle zone, as well as in the adult striatum. Similarly, Nolz1 is highly expressed in proliferating LGE-derived NPC cultures, but its levels rapidly decrease upon cell differentiation, pointing to a role of Nolz1 in the control of NPC proliferation and/or differentiation. In agreement with this hypothesis, we find that Nolz1 over-expression promotes cell cycle exit of NPCs in neurosphere cultures and negatively regulates proliferation in telencephalic organotypic cultures. Within LGE primary cultures, Nolz1 over-expression promotes the acquisition of a neuronal phenotype, since it increases the number of β-III tubulin (Tuj1)- and microtubule-associated protein (MAP)2-positive neurons, and inhibits astrocyte generation and/or differentiation. Retinoic acid (RA) is one of the most important morphogens involved in striatal neurogenesis, and regulates Nolz1 expression in different systems. Here we show that Nolz1 also responds to this morphogen in E12.5 LGE-derived cell cultures. However, Nolz1 expression is not regulated by RA in E14.5 LGE-derived cell cultures, nor is it affected during LGE development in mouse models that present decreased RA levels. Interestingly, we find that Gsx2, which is necessary for normal RA signaling during LGE development, is also required for Nolz1 expression, which is lost in Gsx2 knockout mice. These findings suggest that Nolz1 might act downstream of Gsx2 to regulate RA-induced neurogenesis. Keeping with this hypothesis, we show that Nolz1 induces the selective expression of the RA receptor (RAR)β without altering RARα or RARγ. In addition, Nozl1 over-expression increases RA signaling since it stimulates the RA response element. This RA signaling is essential for Nolz1-induced neurogenesis, which is impaired in a RA-free environment or in the presence of a RAR inverse agonist. It has been proposed that Drosophila Gsx2 and Nolz1 homologues could cooperate with the transcriptional co-repressors Groucho-TLE to regulate cell proliferation. In agreement with this view, we show that Nolz1 could act in collaboration with TLE-4, as they are expressed at the same time in NPC cultures and during mouse development. Conclusions: Nolz1 promotes RA signaling in the LGE, contributing to the striatal neurogenesis during development.

Multilayered organization of jasmonate signalling in the regulation of root growth.

Physical damage can strongly affect plant growth, reducing the biomass of developing organs situated at a distance from wounds. These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling. Using a novel assay involving repetitive cotyledon wounding in Arabidopsis seedlings, we uncovered a function of JA in suppressing cell division and elongation in roots. Regulatory JA signalling components were then manipulated to delineate their relative impacts on root growth. The new transcription factor mutant myc2-322B was isolated. In vitro transcription assays and whole-plant approaches revealed that myc2-322B is a dosage-dependent gain-of-function mutant that can amplify JA growth responses. Moreover, myc2-322B displayed extreme hypersensitivity to JA that totally suppressed root elongation. The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation. Furthermore, in a JA-deficient mutant background, ninja1 myc2-322B still repressed root elongation, indicating that it is possible to generate JA-responses in the absence of JA. We show that NINJA forms a broadly expressed regulatory layer that is required to inhibit JA signalling in the apex of roots grown under basal conditions. By contrast, MYC2, MYC3 and MYC4 displayed cell layer-specific localisations and MYC3 and MYC4 were expressed in mutually exclusive regions. In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth. Our data reveal how distinct negative regulatory layers, including both NINJA-dependent and -independent mechanisms, restrain JA responses to allow normal root growth. Mechanistic insights from this work underline the importance of mapping JA signalling components to specific cell types in order to understand and potentially engineer the growth reduction that follows physical damage.

Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2.

CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis.

The down-regulation of disgust by implementation intentions: experiential and physiological concomitants

Emotion regulation plays a key role in mental health and psychopathology. Therefore, it seems important to develop effective forms of emotion regulation. Implementation intentions are if-then plans that help people attain their self-regulatory goals. Perspective-taking and response-focused implementation intentions have been shown to reduce feelings of unpleasantness and arousal, respectively, in response to briefly presented disgusting pictures. The present study addressed the open research questions whether forming these types of implementation intentions is effective in regulating affect during prolonged presentation of disgusting pictures, and whether it is associated with changes in physiological arousal. Eighty-one participants viewed disgusting, neutral, and pleasant pictures of 6 s duration under four instructions: the goal intention to not get disgusted, this goal intention furnished with a perspective-taking or a response-focused implementation intention, and no emotion regulation instructions. The dependent variables were ratings of disgust, valence, arousal, and electrodermal activity. Only perspective-taking implementation intention participants significantly reduced their disgust and unpleasantness as compared to goal-intention and control participants. Arousal and skin conductance did not significantly differ between conditions. The effectiveness of response-focused but not perspective-taking implementation intentions seems to be substantially reduced during sustained exposure duration.

Current Sheet Regulation of Solar Near-relativistic Electron Injection Histories

We present a sample of three large near-relativistic (>50 keV) electron events observed in 2001 by both the ACE and the Ulysses spacecraft, when Ulysses was at high-northern latitudes (>60◦) and close to 2 AU. Despite the large latitudinal distance between the two spacecraft, electrons injected near the Sun reached both heliospheric locations. All three events were associated with large solar flares, strong decametric type II radio bursts and accompanied by wide (>212◦) and fast (>1400 km s−1) coronal mass ejections (CMEs). We use advanced interplanetary transport simulations and make use of the directional intensities observed in situ by the spacecraft to infer the electron injection profile close to the Sun and the interplanetary transport conditions at both low and high latitudes. For the three selected events, we find similar interplanetary transport conditions at different heliolatitudes for a given event, with values of the mean free path ranging from 0.04 AU to 0.27 AU. We find differences in the injection profiles inferred for each spacecraft. We investigate the role that sector boundaries of the heliospheric current sheet (HCS) have on determining the characteristics of the electron injection profiles. Extended injection profiles, associated with coronal shocks, are found if the magnetic footpoints of the spacecraft lay in the same magnetic sector as the associated flare, while intermittent sparse injection episodes appear when the spacecraft footpoints are in the opposite sector or a wrap in the HCS bounded the CME structure.

Dual role of LldR in regulation of the lldPRD operon, involved in l-Lactate metabolism in Escherichia coli.

The lldPRD operon of Escherichia coli, involved in L-lactate metabolism, is induced by growth in this compound. We experimentally identified that this system is transcribed from a single promoter with an initiation site located 110 nucleotides upstream of the ATG start codon. On the basis of computational data, it had been proposed that LldR and its homologue PdhR act as regulators of the lldPRD operon. Nevertheless, no experimental data on the function of these regulators have been reported so far. Here we show that induction of an lldP-lacZ fusion by L-lactate is lost in an lldR mutant, indicating the role of LldR in this induction. Expression analysis of this construct in a pdhR mutant ruled out the participation of PdhR in the control of lldPRD. Gel shift experiments showed that LldR binds to two operator sites, O1 (positions 105 to 89) and O2 (positions 22 to 38), with O1 being filled at a lower concentration of LldR. L-Lactate induced a conformational change in LldR that did not modify its DNA binding activity. Mutations in O1 and O2 enhanced the basal transcriptional level. However, only mutations in O1 abolished induction by L-lactate. Mutants with a change in helical phasing between O1 and O2 behaved like O2 mutants. These results were consistent with the hypothesis that LldR has a dual role, acting as a repressor or an activator of lldPRD. We propose that in the absence of L-lactate, LldR binds to both O1 and O2, probably leading to DNA looping and the repression of transcription. Binding of L-lactate to LldR promotes a conformational change that may disrupt the DNA loop, allowing the formation of the transcription open complex.

Brain glucose sensing in homeostatic and hedonic regulation.

Glucose homeostasis as well as homeostatic and hedonic control of feeding is regulated by hormonal, neuronal, and nutrient-related cues. Glucose, besides its role as a source of metabolic energy, is an important signal controlling hormone secretion and neuronal activity, hence contributing to whole-body metabolic integration in coordination with feeding control. Brain glucose sensing plays a key, but insufficiently explored, role in these metabolic and behavioral controls, which when deregulated may contribute to the development of obesity and diabetes. The recent introduction of innovative transgenic, pharmacogenetic, and optogenetic techniques allows unprecedented analysis of the complexity of central glucose sensing at the molecular, cellular, and neuronal circuit levels, which will lead to a new understanding of the pathogenesis of metabolic diseases.

Integrated regulation of the type III secretion system and other virulence determinants in Ralstonia solanacearum

In many plant and animal bacterial pathogens, the Type III secretion system (TTSS) that directly translocates effector proteins into the eukaryotic host cells is essential for the development of disease. In all species studied, the transcription of the TTSS and most of its effector substrates is tightly regulated by a succession of consecutively activated regulators. However, the whole genetic programme driven by these regulatory cascades is still unknown, especially in bacterial plant pathogens. Here, we have characterised the programme triggered by HrpG, a host-responsive regulator of the TTSS activation cascade in the plant pathogen Ralstonia solanacearum. We show through genome-wide expression analysis that, in addition to the TTSS, HrpG controls the expression of a previously undescribed TTSS-independent pathway that includes a number of other virulence determinants and genes likely involved in adaptation to life in the host. Functional studies revealed that this second pathway co-ordinates the bacterial production of plant cell wall-degrading enzymes, exopolysaccharide, and the phytohormones ethylene and auxin. We provide experimental evidence that these activities contribute to pathogenicity. We also show that the ethylene produced by R. solanacearum is able to modulate the expression of host genes and can therefore interfere with the signalling of plant defence responses. These results provide a new, integrated view of plant bacterial pathogenicity, where a common regulator activates synchronously upon infection the TTSS, other virulence determinants and a number of adaptive functions, which act co-operatively to cause disease.

Regulation of bistable ICEclc transfer in pseudomonas knackmussii B13

L'élément génétique intégratif et conjugatif auto-transférable de 103 kb qui se trouve dans le génome de Pseudomonas knackmussii B13 (ICEc/c) confère la capacité de dégrader le 3-chlorobenzoate et le 2-aminophénol. L'élément ICE c/c peut être transféré par conjugaison de la souche B13 à diverses bêta- et gamma- protéobactéries. Seule une sous-population de 3 à 5% des cellules transfère l'élément, les cellules dites "compétentes pour le transfert". L'acquisition de la compétence pour le transfert est vraisemblablement la conséquence d'une régulation bistable, conduisant une partie des cellules au transfert de l'élément ICE c/c tandis que, dans les autres, l'élément reste quiescent et ne se transfère pas. À ce jour, les mécanismes et les acteurs moléculaires qui régulent l'activation bistable de l'élément sont restés inconnus. Mon travail de doctorat visait à identifier les éléments bistables du régulon de la compétence pour le transfert et d'analyser les fondements moléculaires de la bistabilité de l'élément ICE c/c chez P. knackmussii. Le premier chapitre introduit le thème du transfert génétique horizontal avec un accent particulier sur les éléments intégratifs et conjugatifs (ICE) et ICEcIc. L'état actuel des connaissances sur l'organisation génétique, la régulation, l'intégration et le transfert de différents modèles de ICEs est exposé en détail. En outre, je m'étends sur les phénomènes d'hétérogénéité et de bistabilité phénotyplques, qu'on peut distinguer dans une population isogénique dans des conditions de culture homogènes, et qui sont susceptibles de jouer un rôle dans le transfert de l'élément ICE c/c, dans la mesure où il ne s'active et n'est transférable que dans une très petite sous-population de cellules. Dans le chapitre 2, je présente une analyse globale des régions promotrices minimales des gènes appartenant au régulon de la compétence pour le transfert de l'élément ICE c/c. Nous avons étudié les caractéristiques d'expression des promoteurs et, s'ils s'avéraient bistables, leur activation dans le temps par comparaison avec le mutant lntB13. Pour ce faire, nous avons utilisé des fusions de promoteurs avec des gènes rapporteurs et testé l'expression bistable chez P. knackmussii par microscopie à épifluorescence. Pour six promoteurs présentant une expression bistable, nous avons employé de la microscopie temporelle pour déterminer la chronologie de leur expression par rapport à Pint et PinR. Parmi eux, nous avons identifié deux gènes exprimés précocement et trois gènes exprimés tardivement dans le processus d'acquisition de la compétence de transfert. Dans le chapitre 3, j'expose une analyse d'expression génétique pour l'un des groupes de gènes dont la transcription est la plus élevée dans la région conservée de ICE c/c, les gènes orf81655-orf68241 contenus dans une région de 14 kb. Nous montrons d'abord que cet opéron fait partie du même régulon bistable que intB13 et inrR et analysons les caractéristiques génétiques qui conduisent à une transcription élevée. Nous étudions les fonctions biologiques de ce groupe de gènes par des délétlons ciblées et montrons que certaines d'entre elles empêchent le transfert de l'élément. Nous approfondissons la caractérlsatlon de I'orf8l655 en construisant une fusion transcrlptionnelle avec le gène codant pour la protéine fluorescente verte (egfp) (en utilisant le système minl-Tn5). L'expression de Vorf81655 dans des cellules individuelles est comparée au signal mesuré par hybridation in situ en fluorescence (FISH) sur le ARN messager du gène. En utilisant FISH, des délétlons du promoteur et de l'analyse directe de transcription, nous avons localisé la région promotrice du groupe de gènes. En outre, nous avons utilisé des mutations dirigées pour comprendre la bistabilité de cette région promotrice, caractérisée par une transcription très élevée et une traduction lente de l'ARN messager.  Dans le chapitre 4, nous nous efforçons de comprendre comment la bistabilité est générée au sein du régulon te de l'élément ICE c/c. Pour ce faire, nous avons tenté de reconstituer une expression bistable, dans un hôte qui ne présente pas de bistabilité naturellement, à partir d'éléments génétiques individuels. L'hôte choisi est Pseudomonas putida dans lequel nous avons introduit une copie unique de Pint, PinR ou PaipA fusionnés à la egfp, construits qui permettent d'observer l'apparition de bistabilité. Nous avons ensuite construit différents assemblages de composants génétiques de l'élément ICE c/c, en nous concentrant sur la région parA-inrR. En effet, nous avons pu démontrer qu'une expression bistable apparaît dans P. putida grâce à ces éléments en l'absence de l'élément ICE c/c complet. À noter que la plupart des construits génétiques activent PaipA ou P|,,R, mais qu'un seul recrée la bistabilité de Pint, ce qui suggère que la région parA-inrR permet à la fois d'engendrer la bistabilité et d'opérer la transition entre les promoteurs précoces et les promoteurs tardifs du régulon de la bistabilité. Dans le chapitre 5, nous concluons sur une discussion de la pertinence de nos résultats et sur de futures perspectives de recherche. -- The 103-kb self-transmissible integrative and conjugative element (ICE) of Pseudomonas knackmussii B13 (ICEc/c) confers the capacity to degrade 3- chlorobenzoate and 2-aminophenol. ICEc/c can be conjugated from strain B13 to a variety of Beta- and Gammaproteobacteria. Interestingly, ICE c/c transfer is observed in a subpopulatlon of cells (3-5%) only, the so-called 'transfer competent' cells. The formation of transfer competence (tc) is thought to be the consequence of a 'bistable' decision, which forces those cells to follow the developmental path which leads to ICEc/c transfer, whereas in others ICE c/c remains silent and does not transfer. So far, the mechanisms and molecular partners generating this bistable transfer activation in cells of P. knackmussii B13 remain mostly unidentified. This thesis aimed at understanding the extent of the tc bistability regulon and to dissect the molecular basis of bistabillty formation of ICEc/c in P. knackmussii. The first chapter is a general Introduction on horizontal gene transfer (HGT) with particular emphasis on ICEs and ICE c/c. The emphasis is made on the current knowledge about the HGT gene organization, regulation and specific integration and transfer aspects of the different ICEs models. Furthermore, I focus on the phenomena of phenotypic heterogeneity and bistability (the property of two distinguishable phenotypes existing within an isogenic population under homogeneous conditions), which may play a particular role in ICEc/c behaviour, since ICE activation and transfer only occurs in a very small subpopulation of cells. In Chapter Two, I focus on a global analysis of the different core promoters that might belong to the ICEc/c tc pathway regulon. We studied both expression patterns of ICEc/c promoters and, once being identified as "bistable", their temporal activation compared to that of intB13. In order to do this, we used promoter reporter fusions and tested blstability expression in P. knackmussii using epifluorescence microscopy. For the 6 promoters that showed bistable expression, we used time-lapse microscopy to study the timing of promoter expression in comparison to that of P,,,t or PlnR. We could establish two "early" and 3 "late" phase promoters in the process of transfer competence. In Chapter Three, I focused my attention on analysis of gene expression of one of the most highly transcribed gene clusters in the conserved core region of ICEc/c, a 14-kb gene cluster formed by the genes orf81655-orf68241. First we showed that this operon is part of the same bistability 'regulon' as intB13 and inrR, and analysed the genetic features that lead to high transcription. We studied the potential biological function of this cluster for ICE c/c by making specific gene deletions, showing that some interrupt ICEc/c transfer. We further analysed the orfdl655 promoter by constructing transcriptional egfp fusion reporter strains using the miniTn5 delivery system. Expression of the orf81655 promoter in single cells was compared to signals measured by Fluorescence In Situ Hybridization (FISH) on orfSl655 mRNA. We localized the promoter region of the gene cluster using FISH, promoter deletions, and by direct transcript analysis. We further used site-directed mutagenesis to understand the bistability character of the promoter region and the extremely high transcription but low translation from this mRNA. In Chapter Four, we set out to understand how bistability is generated in the tc pathway of ICEc/c. For this we tried rebuilding bistable expression from ICEc/c individual gene components in a host, which normally does not display bistability. As host we used P. putida without ICEc/c but with a single copy Pint-, PlnR- or PalpA- egfp fusion that enabled us to verify bistability formation. Subsequently, we built different assemblages of ICEc/c gene components, focusing on the parA-inrR region. Indeed, we found that bistable expression can be build from those components in P. putida without ICEc/c. Interestingly, most genetic constructs activated PaipA or PlnR, but only one resulted in bistable activation of PinT. This suggests that the parA-inrR region acts as a bistability "generator", but also as a bistability "relay" from early to late promoters in the tc pathway hierarchy. In the final fifth chapter, we conclude with a discussion of the relevance of the present thesis and the resulting perspectives for future studies.