Structures and failure mechanisms analysis of Turtle Mountain

The 30 M m3 rockslide that occurred on the east face of Turtle Mountain in the Crowsnest Pass area (Alberta) in 1903 is one of the most famous landslides in the world. In this paper, the structural features of the South part of Turtle Mountain are investigated in order to understand the present-day scar morphology and to identify the most important failure mechanisms. The structural features were mapped using a high resolution digital elevation model (DEM) in order to have a large overview of the relevant structural features. At the same time, a field survey was carried out and small scale fractures were analyzed in different parts of southern Turtle Mountain in order to confirm the DEM analysis. Results allow to identify six main discontinuity sets that influence the Turtle Mountain morphology. These discontinuity sets were then used to identify the potential failure mechanisms affecting Third Peak and South Peak area.

Two alternate mechanisms contribute to the persistence of interdependent lineages in Pogonomyrmex harvester ants.

Some populations of Pogonomyrmex harvester ants comprise pairs of highly differentiated lineages with queens mating at random with several males of their own and of the alternate lineage. These queens produce two types of diploid offspring, those fertilized by males of the queens' lineage which develop into new queens and those fertilized by males of the other lineage which mostly develop into functionally sterile workers. This unusual mode of genetic caste determination has been found in 26 populations and a total of four lineage pairs (F(1)-F(2), G(1)-G(2), H(1)-H(2) and J(1)-J(2)) have been described in these populations. Despite the fact that a few interlineage queens are produced, previous studies revealed that there is a complete lack of genetic introgression between lineages. Here we quantify the proportion of interlineage queens produced in each of the four lineage pairs and determine the fate of these queens. In the F(1)-F(2), G(1)-G(2) and H(1)-H(2) lineage pairs, interlineage queens were produced by a minority of colonies. These colonies exclusively produced interlineage queens and workers, suggesting that interlineage eggs can develop into queens in these three pairs of lineages in the absence of competition with pure-lineage brood. An analysis of three key stages of the colony life cycle revealed that colonies headed by interlineage queens failed to grow sufficiently to produce reproductive individuals. In laboratory comparisons, interlineage queens produced fewer viable eggs, with the effect that they raised fewer workers and lost more weight per worker produced than pure-lineage queens. In the J(1)-J(2) lineage pair, we did not find a single interlineage queen, raising the possibility that interlineage eggs have completely lost the ability to develop into queens in this lineage pair. Hence, two distinct mechanisms seem to account for the complete lack of between-lineage gene flow in the F(1)-F(2), G(1)-G(2), H(1)-H(2) and J(1)-J(2) lineage pairs.

Switching between apparently redundant iron-uptake mechanisms benefits bacteria in changeable environments.

Bacteria often possess multiple siderophore-based iron uptake systems for scavenging this vital resource from their environment. However, some siderophores seem redundant, because they have limited iron-binding efficiency and are seldom expressed under iron limitation. Here, we investigate the conundrum of why selection does not eliminate this apparent redundancy. We focus on Pseudomonas aeruginosa, a bacterium that can produce two siderophores-the highly efficient but metabolically expensive pyoverdine, and the inefficient but metabolically cheap pyochelin. We found that the bacteria possess molecular mechanisms to phenotypically switch from mainly producing pyoverdine under severe iron limitation to mainly producing pyochelin when iron is only moderately limited. We further show that strains exclusively producing pyochelin grew significantly better than strains exclusively producing pyoverdine under moderate iron limitation, whereas the inverse was seen under severe iron limitation. This suggests that pyochelin is not redundant, but that switching between siderophore strategies might be beneficial to trade off efficiencies versus costs of siderophores. Indeed, simulations parameterized from our data confirmed that strains retaining the capacity to switch between siderophores significantly outcompeted strains defective for one or the other siderophore under fluctuating iron availabilities. Finally, we discuss how siderophore switching can be viewed as a form of collective decision-making, whereby a coordinated shift in behaviour at the group level emerges as a result of positive and negative feedback loops operating among individuals at the local scale.

The murine model of infection with Leishmania major and its importance for the deciphering of mechanisms underlying differences in Th cell differentiation in mice from different genetic backgrounds.

Mice from the majority of inbred strains are resistant to infection by Leishmania major, an obligate intracellular protozoan parasite of macrophages in the mammalian host. In contrast, mice from BALB strains are unable to control infection and develop progressive disease. In this model of infection, genetically determined resistance and susceptibility have been clearly shown to result from the appearance of parasite-specific CD4+ T helper 1 or T helper 2 cells, respectively. This murine model of infection is considered as one of the best experimental systems for the study of the mechanisms operating in vivo at the initiation of polarised T helper 1 and T helper 2 cell maturation. Among the several factors influencing Th cell development, cytokines themselves critically regulate this process. The results accumulated during the last years have clarified some aspects of the role played by cytokines in Th cell differentiation. They are providing critical information that may ultimately lead to the rational devise of means by which to tailor immune responses to the effector functions that are most efficient in preventing and/or controlling infections with pathogens.

Molecular mechanisms of drug resistance in clinical Candida species isolated from tunisian hospitals.

Antifungal resistance of Candida species is a clinical problem in the management of diseases caused by these pathogens. In this study we identified from a collection of 423 clinical samples taken from Tunisian hospitals two clinical Candida species (Candida albicans JEY355 and Candida tropicalis JEY162) with decreased susceptibility to azoles and polyenes. For JEY355, the fluconazole (FLC) MIC was 8 μg/ml. Azole resistance in C. albicans JEY355 was mainly caused by overexpression of a multidrug efflux pump of the major facilitator superfamily, Mdr1. The regulator of Mdr1, MRR1, contained a yet-unknown gain-of-function mutation (V877F) causing MDR1 overexpression. The C. tropicalis JEY162 isolate demonstrated cross-resistance between FLC (MIC > 128 μg/ml), voriconazole (MIC > 16 μg/ml), and amphotericin B (MIC > 32 μg/ml). Sterol analysis using gas chromatography-mass spectrometry revealed that ergosterol was undetectable in JEY162 and that it accumulated 14α-methyl fecosterol, thus indicating a perturbation in the function of at least two main ergosterol biosynthesis proteins (Erg11 and Erg3). Sequence analyses of C. tropicalis ERG11 (CtERG11) and CtERG3 from JEY162 revealed a deletion of 132 nucleotides and a single amino acid substitution (S258F), respectively. These two alleles were demonstrated to be nonfunctional and thus are consistent with previous studies showing that ERG11 mutants can only survive in combination with other ERG3 mutations. CtERG3 and CtERG11 wild-type alleles were replaced by the defective genes in a wild-type C. tropicalis strain, resulting in a drug resistance phenotype identical to that of JEY162. This genetic evidence demonstrated that CtERG3 and CtERG11 mutations participated in drug resistance. During reconstitution of the drug resistance in C. tropicalis, a strain was obtained harboring only defective Cterg11 allele and containing as a major sterol the toxic metabolite 14α-methyl-ergosta-8,24(28)-dien-3α,6β-diol, suggesting that ERG3 was still functional. This strain therefore challenged the current belief that ERG11 mutations cannot be viable unless accompanied by compensatory mutations. In conclusion, this study, in addition to identifying a novel MRR1 mutation in C. albicans, constitutes the first report on a clinical C. tropicalis with defective activity of sterol 14α-demethylase and sterol Δ(5,6)-desaturase leading to azole-polyene cross-resistance.

IL-2- and CD25-dependent immunoregulatory mechanisms in the homeostasis of T-cell subsets.

IL-2 plays a pivotal role in regulating the adaptive immune system by controlling the survival and proliferation of regulatory T (Treg) cells, which are required for the maintenance of immune tolerance. Moreover, IL-2 is implicated in the differentiation and homeostasis of effector T-cell subsets, including T(H)1, T(H)2, T(H)17, and memory CD8+ T cells. The IL-2 receptor is composed of 3 distinct subunits, namely the alpha (CD25), beta (CD122), and gamma (gammac) chains. Of crucial importance for the delivery of IL-2 signals to Treg cells is the expression of CD25, which, along with CD122 and gammac, confers high affinity binding to IL-2. Notably, recent findings suggest a novel role for CD25, whereby CD25 molecules on Treg cells and possibly other cells are capable of influencing T-cell homeostasis by means of IL-2 deprivation. This review explores these findings and integrates them into our current understanding of T-cell homeostasis.

Peroxynitrite is a key mediator of the cardioprotection afforded by ischemic postconditioning in vivo.

Myocardial ischemic postconditioning (PosC) describes an acquired resistance to lethal ischemia-reperfusion (I/R) injury afforded by brief episodes of I/R applied immediately after the ischemic insult. Cardioprotection is conveyed by parallel signaling pathways converging to prevent mitochondria permeability transition. Recent observations indicated that PostC is associated with free radicals generation, including nitric oxide (NO(.)) and superoxide (O2 (.-)), and that cardioprotection is abrogated by antioxidants. Since NO. And O2 (. -) react to form peroxynitrite, we hypothesized that postC might trigger the formation of peroxyntrite to promote cardioprotection in vivo. Rats were exposed to 45 min of myocardial ischemia followed by 3h reperfusion. PostC (3 cycles of 30 seconds ischemia/30 seconds reperfusion) was applied at the end of index ischemia. In a subgroup of rats, the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrinato iron (FeTPPS) was given intravenously (10 mg/kg(-1)) 5 minutes before PostC. Myocardial nitrotyrosine was determined as an index of peroxynitrite formation. Infarct size (colorimetric technique and plasma creatine kinase-CK-levels) and left ventricle (LV) function (micro-tip pressure transducer), were determined. A significant generation of 3-nitrotyrosine was detected just after the PostC manoeuvre. PostC resulted in a marked reduction of infarct size, CK release and LV systolic dysfunction. Treatment with FeTPPS before PostC abrogated the beneficial effects of PostC on myocardial infarct size and LV function. Thus, peroxynitrite formed in the myocardium during PostC induces cardioprotective mechanisms improving both structural and functional integrity of the left ventricle exposed to ischemia and reperfusion in vivo.

Multiple expression control mechanisms of peroxisome proliferator-activated receptors and their target genes.

The peroxisome proliferator-activated receptors (PPAR) alpha, beta/delta and gamma belong to the nuclear hormone receptor superfamily. As ligand-activated receptors, they form a functional transcriptional unit upon heterodimerization with retinoid X receptors (RXRs). PPARs are activated by fatty acids and their derivatives, whereas RXR is activated by 9-cis retinoic acid. This heterodimer binds to peroxisome proliferator response elements (PPRE) residing in target genes and stimulates their expression. Recent reports now indicate that PPARs and RXRs can function independently, in the absence of a hetero-partner, to modulate gene expression. Of importance, these non-canonical mechanisms underscore the impact of both cofactors and DNA on gene expression. Furthermore, these different mechanisms reveal the increasing repertoire of PPAR 'target' genes that now encompasses non-PPREs containing genes. It is also becoming apparent that understanding the regulation of PPAR expression and activity, can itself have a significant influence on how the expression of subgroups of target genes is studied and integrated in current knowledge.

The pollutant diethylhexyl phthalate regulates hepatic energy metabolism via species-specific PPARalpha-dependent mechanisms.

Background: The modulation of energetic homeostasis by pollutants has recently emerged as a potential contributor to the onset of metabolic disorders. Diethylhexyl phthalate (DEHP) is a widely used industrial plasticizer to which humans are widely exposed. Phthalates can activate the three peroxisome proliferatoractivated receptor (PPAR) isotypes on cellular models and induce peroxisome proliferation in rodents.Objectives: In this study, we aimed to evaluate the systemic and metabolic consequences of DEHP exposure that have remained so far unexplored and to characterize the underlying molecular mechanisms of action.Methods: As a proof of concept and mechanism, genetically engineered mouse models of PPARs were exposed to high doses of DEHP, followed by metabolic and molecular analyses.Results: DEHP-treated mice were protected from diet-induced obesity via PPARalpha-dependent activation of hepatic fatty acid catabolism, whereas the activity of neither PPARbeta nor PPARgamma was affected. However, the lean phenotype observed in response to DEHP in wild-type mice was surprisingly abolished in PPARalpha-humanized mice. These species differences are associated with a different pattern of coregulator recruitment.Conclusion: These results demonstrate that DEHP exerts species-specific metabolic actions that rely to a large extent on PPARalpha signaling and highlight the metabolic importance of the species-specific activation of PPARalpha by xenobiotic compounds. Editor's SummaryDiethylhexyl phthalate (DEHP) is an industrial plasticizer used in cosmetics, medical devices, food packaging, and other applications. Evidence that DEHP metabolites can activate peroxisome proliferatoractivated receptors (PPARs) involved in fatty acid oxidation (PPARalpha and PPARbeta) and adiposite function and insulin resistance (PPARgamma) has raised concerns about potential effects of DEHP on metabolic homeostasis. In rodents, PPARalpha activation also induces hepatic peroxisome proliferation, but this response to PPARalpha activation is not observed in humans. Feige et al. (p. 234) evaluated systemic and metabolic consequences of high-dose oral DEHP in combination with a high-fat diet in wild-type mice and genetically engineered mouse PPAR models. The authors report that mice exposed to DEHP gained less weight than controls, without modifying their feeding behavior; they also exhibited lower triglyceride levels, smaller adipocytes, and improved glucose tolerance compared with controls. These effects, which were observed in mice fed both high-fat and standard diets, appeared to be mediated by PPARalpha-dependent activation of hepatic fatty acid catabolism without apparent involvement of PPARbeta or PPARgamma. However, mouse models that expressed human (versus mouse) PPARalpha tended to gain more weight on a high-fat diet than their DHEP-unexposed counterparts. The authors conclude that findings support species-specific metabolic effects of DEHP mediated by PPARalpha activation.

Beyond splitting: observer-rated defense mechanisms in borderline personality disorder

Defense mechanism is a key concept in the psychoanalytic psychopathology of borderline personality disorder (BPD). Theoretical and empirical elaborations on this question are briefly reviewed and discussed with respect to process assessment of defense mechanisms; we put forward observer-rater methodology as an accurate means of assessing unconscious in-session processes. A sample of 25 patients presenting with BPD were interviewed, as were subjects from a matched control group without psychiatric symptoms (n = 25), using a psychodynamic interview paradigm. These interviews were transcribed and rated using the Defense Mechanisms Rating Scales. The results indicate that, compared to controls, patients with BPD used higher percentages of a action, borderline, disavowal, narcissistic, and hysteric defenses, along with lower levels of mature and obsessional defenses. Overall defensive functioning was significantly lower in the patients with BPD, compared to controls. Narcissistic defenses were related with symptom level. These results are discussed in light of previous studies on defensive functioning of BPD and the literature on psychoanalytic psychopathology. These results have several important clinical implications.

Mechanisms of skin adherence and invasion by dermatophytes.

Dermatophytes are keratinophilic fungi that can be pathogenic for humans and animals by infecting the stratum corneum, nails, claws or hair. The first infection step consists of adherence of arthroconidia to the stratum corneum. The mechanisms and the kinetics of adherence have been investigated using different in vitro and ex vivo experimental models, most notably showing the role of a secreted serine protease from Microsporum canis in fungal adherence to feline corneocytes. After germination of the arthroconidia, dermatophytes invade keratinised structures that have to be digested into short peptides and amino acids to be assimilated. Although many proteases, including keratinolytic ones, have been characterised, the understanding of dermatophyte invasion mechanisms remains speculative. To date, research on mechanisms of dermatophyte infection focused mainly on both secreted endoproteases and exoproteases, but their precise role in both fungal adherence and skin invasion should be further explored.

Mechanisms of postprandial hyperglycemia in liver transplant recipients: comparison of liver transplant patients with kidney transplant patients and healthy controls.

Impaired glucose tolerance or diabetes mellitus are frequent complications after organ transplantation, and are usually attributed to glucocorticoid and immunosuppressive treatments. Liver transplantation results in total hepatic denervation which may also affect glucoregulation. We therefore evaluated postprandial glucose metabolism in a group of patients with liver cirrhosis before and after orthotopic liver transplantation. Seven patients with liver cirrhosis of various etiologies, 6 patients having received a kidney transplant, and 6 healthy subjects were studied. Their glucose metabolism was evaluated in the basal state and over 4 hours after ingestion of a glucose load with 6.6 (2) H glucose dilution analysis. The patients with liver cirrhosis were studied before, and again 4 weeks (range 2-6) and 38 weeks (range 20-76, n=6) after orthotopic liver transplantation. Basal glucose metabolism was similar in liver and kidney transplant recipients. Impaired glucose tolerance was present in both groups, but postprandial hyperglycemia was exaggerated and lasted longer in liver transplant patients. Postprandial insulinemia was lower in liver transplant recipients, while C-peptide concentrations were comparable to those of kidney transplant recipients, indicating increased insulin clearance. Glucose turnover was not altered in both groups of patients during the initial 3 hours after glucose ingestion, but was higher in liver transplant early after transplantation during the fourth hour. Postprandial hyperglycemia remained unchanged in liver transplant recipients 38 weeks after liver transplantation, despite substantial reduction of immunosuppressive and glucocorticoid doses. We conclude that liver transplant recipients have severe postprandial hyperglycemia which can be attributed to insulinopenia (secondary, at least in part, to increased insulin clearance) and a late increased glucose turnover. These changes may be secondary to hepatic denervation.

Dibenzofuran degradation by Sphingomonas wittichii RW1 under environmental stresses

Sphingomonas wittichii is a gram-negative Alpha-proteobacterium, capable of degrading xenobiotic compounds such as dibenzofuran (DBF), dibenzo-p-dioxin, carbazole, 2-hydroxybiphenyl or nitro diphenyl ether herbicides. The metabolism of strain RW1 has been the subject of previous studies and a number of genes involved in DBF degradation have been characterized. It is known that RW1 posseses a unique initial DBF dioxygenase (encoded by the dxnAl gene) that catalyzes the first step in the degradation pathway. None of the organisms known to be able to degrade DBF have a similar dioxygenase, the closest match being the DBF dioxygenase from Rhodococcus sp. with an overall amino acid similarity of 45%. Genes participating in the conversion of the metabolite salicylate via the ortho-cleavage pathway to TCA cycle intermediates were identified as well. Apart from this scarce information, however, there is a lack of global knowledge on the genes that are involved in DBF degradation by strain RW1 and the influence of environmental stresses on DBF-dependent global gene expression. A global analysis is necessary, because it may help to better understand the behaviour of the strain under field conditions and suggest improvements for the current bioaugmentation practice. Chapter 2 describes the results of whole-genome analysis to characterize the genes involved in DBF degradation by RW1. Micro-array analysis allowed us to detect differences in gene transcription when strain RW1 was exposed to DBF. This was complemented by ultra-high throughput sequencing of mutants no longer capable of growing on salicylate and DBF. Some of the genes of the ortho-cleavage pathway were induced 2 to 4 times in the presence of DBF, as well as the initial DBF dioxygenase. However two gene clusters, named 4925 and 5102 were induced up to 19 times in response to DBF induction. The cluster 4925 is putatively participating in a meta-cleavage pathway while the cluster 5102 might be part of a gentisate pathway. The three pathways, ortho-cleavage, meta-cleavage and gentisate pathway seem to be active in parallel when strain RW1 is exposed to DBF, presenting evidence for a redundancy of genes for DBF degradation in the genome of RW1. Chapter 3 focuses on exploiting genetic tools to construct bioreporters representative for DBF degradation in RW1. A set of basic tools for genetic manipulation in Sphingomonas wittichii RW1 was tested and optimized. Both plasmids and mini-transposons were evaluated for their ability to be maintained in RW1 with or without antibiotic selection pressure, and for their ability to lead to fluorescent protein expression in strain RW1 from a constitutive promoter. Putative promoter regions of three of the previously found DBF-induced genes (Swit_4925, Swit_5102 and Swit_4897-dxnAl) were then used to construct eg/^-bioreporters in RW1. Chapter 4 describes the use of the constructed RW1-based bioreporter strains for examining the expression of the DBF degradation pathway genes under microcosm conditions. The bioreporter strains were first exposed to different carbon sources in liquid culture to calibrate the egfp induction. Contrary to our expectations from micro-array analysis only the construct with the promoter from gene cluster 4925 responded to DBF, whereas the other two constructs did not show specific induction with DBF. The response from the bioreporters was subsequently tested for sensitivity to water stress, given that this could have an important impact in soils. Exposure to liquid cultures with decreasing water potential, achieved by NaCl or PEG addition to the growth media, showed that eGFP expression in RW1 from the promoter regions 4925 and 5102 was not directly influenced by water stress, but only through an overall reduction in growth rate. In contrast, expression of eGFP from the dxnAl or an uspA promoter was also directly dependent on the extent of water stress. The RW1 with the 4925 construct was subsequently used in soil microcosms to evaluate DBF bioavailability to the cells in presence or absence of native microbiota or other contaminated material. We found that RW1 could grow on DBF added to soil, but bioreporter expression suggested that competition with native microbiota for DBF intermediates may limit its ability to proliferate to a maximum. Chapter 5 describes the results from the experiments carried out to more specifically detect genes of RW1 that might be implicated in water stress resistance. Hereto we created transposon mutagenesis libraries in RW1, either with a classical mini-Tn5 or with a variant that would express egfp when the transposon would insert in a gene induced under water stress. Classical mutant libraries were screened by replica plating under high and low water stress conditions (achieved by adding NaCl to the agar medium). In addition, we screened for smaller microcolonies formed by mutants in agarose beads that could be analized with flow cytometry. A number of mutants impaired to grow on NaCl-supplemented media were recovered and the transposon insertion sites sequenced. In a second procedure we screened by flow cytometry for mutants with a higher eGFP production after exposure to growth medium with higher NaCl concentrations. Mutants from both libraries rarely overlapped. Discovered gene functions of the transposon insertions pointed to compatible solute synthesis (glutamate and proline), cell membrane synthesis and modification of cell membrane composition. The results obtained in the present study give us a more complete picture of the mechanisms of DBF degradation by S. wittichii RW1, how it reacts to different DBF availability and how the DBF catabolic activity may be affected by the conditions found in contaminated environments. - Sphingomonas wittichii est une alpha-protéobactérie gram-négative, capable de dégrader des composés xénobiotiques tels que le dibenzofurane (DBF), la dibenzo-p-dioxine, le carbazole, le 2-hydroxybiphényle ou les herbicides dérivés du nitro-diphényléther. Le métabolisme de la souche RW1 a fait l'objet d'études antérieures et un certain nombre de gènes impliqués dans la dégradation du DBF ont été caractérisés. Il est connu que RW1 possède une unique dioxygénase DBF initiale (codée par le gène dxnAl) qui catalyse la première étape de la voie de dégradation. Aucun des organismes connus pour être capables de dégrader le DBF n'a de dioxygénase similaire. L'enzyme la plus proche étant la DBF dioxygénase de Rhodococcus sp. avec 45% d'acides aminés conservés. Les gènes qui participent à la transformation du salicylate en métabolites intermédiaires du cycle de Krebs par la voie ort/io-cleavage ont aussi été identifiés. Outre ces informations lacunaires, il y a un manque de connaissances sur l'ensemble des gènes impliqués dans la dégradation du DBF par la souche RW1 ainsi que l'effet des stress environnementaux sur l'expression génétique globale, en présence du DBF. Une analyse globale est nécessaire, car elle peut aider à mieux comprendre le comportement de la souche dans les conditions de terrain et de proposer des améliorations pour l'utilisation de la bio-augmentation comme technique de bio-remédiation. Le chapitre 2 décrit les résultats de l'analyse du génome pour caractériser les gènes impliqués dans la dégradation du DBF par RW1. Une analyse de micro-arrays nous a permis de détecter des différences dans la transcription des gènes lorsque la souche RW1 a été exposée au DBF. L'analyse a été complétée par le criblage à ultra-haut débit de mutants qui n'étaient plus capables de croître avec le salicylate ou le DBF comme seule source de carbone. Certains des gènes de la voie ortho-cleavage, dont la DBF dioxygénase initiale, ont xî été induits 2 à 4 fois, en présence du DBF. Cependant, deux groupes de gènes, nommés 4925 et 5102 ont été induits jusqu'à 19 fois en réponse au DBF. Le cluster 4925 participe probablement dans une voie de meta-cleavage tandis que le cluster 5102 pourrait faire partie d'une voie du gentisate. Les trois voies, ortho-cleavage, meta-cleavage et la voie du gentisate semblent être activées en parallèle lorsque la souche RW1 est exposée au DBF, ce qui représente une redondance de voies pour la dégradation du DBF dans le génome de RW1. Le chapitre 3 se concentre sur l'exploitation des outils génétiques pour la construction de biorapporteurs de la dégradation du DBF par RW1. Un ensemble d'outils de base pour la manipulation génétique dans Sphingomonas wittichii RW1 a été testé et optimisé. Deux plasmides et mini-transposons ont été évalués pour leur capacité à être maintenu dans RW1 avec ou sans pression de sélection par des antibiotiques, et pour leur capacité à exprimer la protéine fluorescente verte (eGFP) dans la souche RW1. Les trois promoteurs des gènes Swit_4925, Swit_5102 et Swit_4897 (dxnAl), induits en réponse au DBF, ont ensuite été utilisés pour construire des biorapporteurs dans RW1. Le chapitre 4 décrit l'utilisation des souches biorapportrices construites pour l'analyse de l'expression des gènes de la voie de dégradation du DBF dans des microcosmes avec différents types de sols. Les souches biorapportrices ont d'abord été exposées à différentes sources de carbone en cultures liquides afin de calibrer l'induction de la eGFP. La construction avec le promoteur du gène 4925 a permis une réponse au DBF. Mais contrairement à nos attentes, basées sur les résultats de l'analyse des micro-arrays, les deux autres constructions n'ont pas montré d'induction spécifique au DBF. La réponse des biorapporteurs a ensuite été testée pour la sensibilité au stress hydrique, étant donné que cela pourrait avoir un impact important dans les microcosmes. La diminution du potentiel hydrique en culture liquide est obtenue par addition de NaCl ou de PEG au milieu de croissance. Nous avons montré que l'expression de la eGFP contrôlée par les promoteurs 4925 et 5102 n'était pas directement influencée par le stress hydrique, mais seulement par une réduction globale des taux de croissance. En revanche, l'expression de la eGFP dépendante des promoteurs dxnAl et uspA était aussi directement dépendante de l'ampleur du stress hydrique. La souche avec la construction 4925 a été utilisée par la suite dans des microcosmes avec différents types de sols pour évaluer la biodisponibilité du DBF en présence ou absence des microbes indigènes et d'autres composés contaminants. Nous avons constaté que RW1 pouvait se développer si le DBF a été ajouté au sol, mais l'expression de la eGFP par le biorapporteur suggère que la compétition avec la microbiota indigène pour les métabolites intermédiaires du DBF peut limiter sa capacité à proliférer de manière optimale. Le chapitre 5 décrit les résultats des expériences réalisées afin de détecter spécifiquement les gènes de RW1 qui pourraient être impliquées dans la résistance au stress hydrique. Ici on a crée des bibliothèques de mutants de RW1 par transposon, soit avec un mini-Tn5 classique ou avec une variante qui exprime la eGFP lorsque le transposon s'insère dans un gène induit par le stress hydrique. Les bibliothèques de mutants ont été criblées par la méthode classique de repiquage sur boîtes, dans des conditions de stress hydrique élevé (obtenu par l'addition de NaCl dans les boîtes). En outre, nous avons criblé des micro¬colonies dans des billes d'agarose qui ont pu être analysées par cytométrie de flux. Un certain nombre de mutants déficients à croître sur des milieux supplémentés avec du NaCl ont été isolés et les sites d'insertion du transposon séquencés. Dans une deuxième procédure nous avons criblé par cytométrie de flux des mutants avec une production de eGFP supérieure, après exposition à un milieu de croissance avec une concentration élevée de NaCl. Les mutants obtenus dans les deux bibliothèques n'étaient pas similaires. Les fonctions des gènes où se trouvent les insertions de transposons sont impliqués dans la synthèse de solutés compatibles (glutamate et de la proline), dans la synthèse de la membrane cellulaire et dans la modification de la composition de la membrane cellulaire. Les résultats obtenus dans la présente étude nous donnent une image plus complète des mécanismes de dégradation du DBF par S. wittichii RW1, comment cette souche réagit à la disponibilité du DBF et comment l'activité catabolique peut être affectée par les conditions rencontrées dans des environnements contaminés.