Characterization of cell response induced by different stresses in Escherichia coli

La prolifération bactérienne requiert la coordination entre les grands processus du cycle cellulaire qui sont la réplication et la ségrégation de l’ADN, l’élongation et la division cellulaire. Durant leur vie, les bactéries sont exposées à différents stress endogènes ou exogènes (antibiotiques, pH, manque de nutriments…) qui peuvent perturber le cycle cellulaire. Ces conditions défavorables activent alors une réponse cellulaire qui vise à améliorer la survie aux stress. Chez E. coli, la formation de cassures sur le chromosome induit la réponse SOS qui inhibe la division des cellules. Dans ce contexte, la bactérie continue à s’allonger ce qui aboutit à la formation d’une cellule filamenteuse. La filamentation a longtemps été considérée comme un symptôme de mort cellulaire, mais des études récentes suggèrent qu’il s’agirait plutôt d’un changement de morphologie transitoire qui améliorerait la survie en conditions défavorables. L’objectif principal de cette thèse est de caractériser le processus de filamentation et surtout le redémarrage de la division du filament, permettant un retour à une croissance normale de la bactérie. J’ai pour cela mis en place une approche combinant la microscopie en cellules vivantes en chambre microfluidique, la cytométrie en flux, la microbiologie traditionnelle et la génétique bactérienne. L’association de ces techniques constitue une approche globale permettant de caractériser l’effet d’un stress sur la viabilité, la morphologie et le contenu en ADN des bactéries, et ce de la cellule unique à l’échelle de la population. Cette approche a permis de décrire comment les cellules filamenteuses se divisent rapidement en cellules viables et de comprendre comment cet état de différenciation cellulaire transitoire et réversible constitue une stratégie efficace de survie aux stress. Par ailleurs, l’expertise développée au cours de ces travaux m’a permis d’être impliqué dans l ‘étude du transfert de gène de résistance à la tétracycline par conjugaison bactérienne. Ces mêmes expertisent ont aussi permis la caractérisation de l’effet de biocides induisant la réponse aux stress de l’enveloppe et la visualisation de l’effet de la production chez E. coli de deux toxines prédites pour être impliquées dans un système d’inhibition de croissance contact-dépendant chez A. baumannii.Bacterial growth requires coordination between the main cell cycle processes that are DNA replication and segregation, elongation and cell division. During their life, bacteria are exposed to different endogenous or exogenous stresses (antibiotics, pH, nutrients starvation…) that can disturb the bacteria cell cycle. Those hostile life conditions trigger a cellular response aiming at improving survival in stress conditions. In E. coli, DNA breaks induce the SOS response that inhibits cell division while the bacteria continue to elongate, resulting in the formation of a filamentous cell. Filamentation has long been considered as a symptom of cell death, however recent studies suggest that this phenotype could instead be a transient morphology change improving the survival in hostile environments. The main objective of this thesis is to characterize the filamentation process, especially the restart of the filament division allowing to resume normal bacterial growth. To do so, I developped an approach combining live-cell microscopy in microfluidic chamber, flow cytometry, traditional microbiology technics and bacterial genetics. Association of those techniques constitutes a global approach allowing characterization of the stress effect on bacterial viability, morphology and DNA content, from the single cell to the population level. This experimental framework allowed to describe how filamentous cells quickly divide into viable cells, thus understanding how this transient and reversible cellular differentiation state constitute an efficient stress-survival strategy. Furthermore, the expertise I developed during this ph.D. project allowed me to be involved into the study of drug-resistance acquisition by gene transfer through bacterial DNA conjugation. Besides, I contributed to the characterization of the effects of biocides inducing envelop stress response and to the characterize the impact on E. coli of the production of Acinetobacter baumannii toxins predicted to be involved in contact-dependant growth inhibitio

Caractérisation de la réponse cellulaire associée à différents stress chez la bactérie Escherichia coli

Bacterial growth requires coordination between the main cell cycle processes that are DNA replication and segregation, elongation and cell division. During their life, bacteria are exposed to different endogenous or exogenous stresses (antibiotics, pH, nutrients starvation…) that can disturb the bacteria cell cycle. Those hostile life conditions trigger a cellular response aiming at improving survival in stress conditions. In E. coli, DNA breaks induce the SOS response that inhibits cell division while the bacteria continue to elongate, resulting in the formation of a filamentous cell. Filamentation has long been considered as a symptom of cell death, however recent studies suggest that this phenotype could instead be a transient morphology change improving the survival in hostile environments. The main objective of this thesis is to characterize the filamentation process, especially the restart of the filament division allowing to resume normal bacterial growth. To do so, I developped an approach combining live-cell microscopy in microfluidic chamber, flow cytometry, traditional microbiology technics and bacterial genetics. Association of those techniques constitutes a global approach allowing characterization of the stress effect on bacterial viability, morphology and DNA content, from the single cell to the population level. This experimental framework allowed to describe how filamentous cells quickly divide into viable cells, thus understanding how this transient and reversible cellular differentiation state constitute an efficient stress-survival strategy. Furthermore, the expertise I developed during this ph.D. project allowed me to be involved into the study of drug-resistance acquisition by gene transfer through bacterial DNA conjugation. Besides, I contributed to the characterization of the effects of biocides inducing envelop stress response and to the characterize the impact on E. coli of the production of Acinetobacter baumannii toxins predicted to be involved in contact-dependant growth inhibitionLa prolifération bactérienne requiert la coordination entre les grands processus du cycle cellulaire qui sont la réplication et la ségrégation de l’ADN, l’élongation et la division cellulaire. Durant leur vie, les bactéries sont exposées à différents stress endogènes ou exogènes (antibiotiques, pH, manque de nutriments…) qui peuvent perturber le cycle cellulaire. Ces conditions défavorables activent alors une réponse cellulaire qui vise à améliorer la survie aux stress. Chez E. coli, la formation de cassures sur le chromosome induit la réponse SOS qui inhibe la division des cellules. Dans ce contexte, la bactérie continue à s’allonger ce qui aboutit à la formation d’une cellule filamenteuse. La filamentation a longtemps été considérée comme un symptôme de mort cellulaire, mais des études récentes suggèrent qu’il s’agirait plutôt d’un changement de morphologie transitoire qui améliorerait la survie en conditions défavorables. L’objectif principal de cette thèse est de caractériser le processus de filamentation et surtout le redémarrage de la division du filament, permettant un retour à une croissance normale de la bactérie. J’ai pour cela mis en place une approche combinant la microscopie en cellules vivantes en chambre microfluidique, la cytométrie en flux, la microbiologie traditionnelle et la génétique bactérienne. L’association de ces techniques constitue une approche globale permettant de caractériser l’effet d’un stress sur la viabilité, la morphologie et le contenu en ADN des bactéries, et ce de la cellule unique à l’échelle de la population. Cette approche a permis de décrire comment les cellules filamenteuses se divisent rapidement en cellules viables et de comprendre comment cet état de différenciation cellulaire transitoire et réversible constitue une stratégie efficace de survie aux stress. Par ailleurs, l’expertise développée au cours de ces travaux m’a permis d’être impliqué dans l ‘étude du transfert de gène de résistance à la tétracycline par conjugaison bactérienne. Ces mêmes expertisent ont aussi permis la caractérisation de l’effet de biocides induisant la réponse aux stress de l’enveloppe et la visualisation de l’effet de la production chez E. coli de deux toxines prédites pour être impliquées dans un système d’inhibition de croissance contact-dépendant chez A. baumannii

Original sequence divergence among Pseudomonas putida CadRs drive specificity

International audienceBacteria, especially those living in soils, are in constant contact with metals. Transition metals like Fe or Zn, are required for proper growth. Some other metals like Cd or Hg are only toxic. Several systems exist to detoxify cells when these metals are present in concentrations harmful to biological systems. The expression of these systems is under control of specialized regulatory proteins able to detect metals and to regulate cognate detoxifying systems. In this work we report on the characterisation of the metallo-regulator CadR from P. putida KT2440. By using gene reporter assays, we investigated the repertoire of metals detected by CadR. We show that CadR is much more responsive to Hg than to Cd, as compared to CadR from P. putida 06909. CadR from P. putida KT2440 differs in only 3 amino-acids in its metal-binding domain with respect to CadR from P. putida 06909. We show that these residues are important determinants of metal selectivity by engineering a modified CadR

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli.

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon bactericidal antibiotic treatment, the bulk of the bacterial population is rapidly killed. This first rapid phase of killing is followed by a substantial decrease in the rate of killing as the persister cells remain viable. Classically, persistence is determined at the population level by time/kill assays performed with high doses of antibiotics and for defined exposure times. While this method provides information about the level of persister cells and the killing kinetics, it fails to reflect the intrinsic cell-to-cell heterogeneity underlying the persistence phenomenon. The protocol described here combines classical time/kill assays with single-cell analysis using real-time fluorescence microscopy. By using appropriate fluorescent reporters, the microscopy imaging of live cells can provide information regarding the effects of the antibiotic on cellular processes, such as chromosome replication and segregation, cell elongation, and cell division. Combining population and single-cell analysis allows for the molecular and cellular characterization of the persistence phenotype.info:eu-repo/semantics/publishe

Micro-addition of Fe in highly alloyed Cu-Ti alloys to improve both formability and strength

Cu-Be alloys provide excellent electrical and mechanical properties, but present serious health hazards during manufacturing. Among alternative alloys, the Cu-Ti system has the highest yield strength; however, Ti cannot be easily solutionized at concentrations above 4 wt%, resulting in a relatively low formability. In this study, Cu-xTi-yFe (x = 3, 5, 6 wt% and y = 0, 0.3 wt%) alloys were studied after both solution-annealing and age-hardening through mechanical testing and microstructure analysis. Micro-additions of Fe kept high concentration of Ti in solid solution (up to 6 wt%) after water quenching and suppressed the classical “wave-like” early-stage precipitation. Instead, a new dispersion of nano precipitates was observed. This behavior results in doubling the ductility in the solution annealed state (up to 48% elongation), together with maintaining a very high strength after ageing (up to 975 MPa) from precipitation of metastable nano α-Cu4Ti. This study shows that Fe micro-additions, when combined with a higher amounts of Ti (6 wt%), enables the production of Cu-based alloys combining high formability and strength, providing an excellent alternative to Cu-Be in mechanical applications

Role of AcrAB-TolC multidrug efflux pump in drug-resistance acquisition by plasmid transfer

International audienceDrug-resistance dissemination by horizontal gene transfer remains poorly understood at the cellular scale. Using live-cell microscopy, we reveal the dynamics of resistance acquisition by transfer of the Escherichia coli fertility factor–conjugation plasmid encoding the tetracycline-efflux pump TetA. The entry of the single-stranded DNA plasmid into the recipient cell is rapidly followed by complementary-strand synthesis, plasmid-gene expression, and production of TetA. In the presence of translation-inhibiting antibiotics, resistance acquisition depends on the AcrAB-TolC multidrug efflux pump, because it reduces tetracycline concentrations in the cell. Protein synthesis can thus persist and TetA expression can be initiated immediately after plasmid acquisition. AcrAB-TolC efflux activity can also preserve resistance acquisition by plasmid transfer in the presence of antibiotics with other modes of actio

The Two-Component System ZraPSR Is a Novel ESR that Contributes to Intrinsic Antibiotic Tolerance in Escherichia coli

International audienceDuring their lifecycle, bacteria are exposed to continuous changes in their environment, some of which are stressful and can be harmful. The cell envelope is the first line of defense against a hostile environment, but it is also the first target for damage. To deal with this problem, bacteria have evolved systems collectively called "envelope stress response," or ESR, dedicated to the detection and repair of damaged components. Here we decided to investigate whether the atypical two-component system ZraP-SR is a novel ESR. Based on the screening of more than 240 drugs using the Biolog technology, we show that the deletion of zraP or zraR confers increased susceptibility to five classes of antibiotics and to some environmental stress targeting the envelope. Using a microscopy approach, we also establish that ZraP and ZraR are required to maintain envelope integrity. So far, the ZraR regulator was only known to activate the transcription of zraP and zraSR. Using chromatin immunoprecipitation followed by sequencing and RT-qPCR, we have now identified 25 additional genes regulated by ZraR, the majority of which are involved in the response against stress. Taken together, our results demonstrate that ZraP-SR is a novel ESR

Pushing the limits of nickel detection to nanomolar range using a set of engineered bioluminescent Escherichia coli

International audienceThe detection of nickel in water is of great importance due to its harmfulness for living organism. A way to detect Ni is the use of whole-cell biosensors. The aim of the present work was to build a light-emitting bacterial biosensor for the detection of Ni with high specificity and low detection limit properties. For that purpose, the regulatory circuit implemented relied on the RcnR Ni/Co metallo-regulator and its rcnA natural target promoter fused to the lux reporter genes. To convert RcnR to specifically detect Ni, several mutations were tested and the C35A retained. Deleting the Ni efflux pump rcnA and introducing genes encoding several Ni-uptake systems lowered the detection thresholds. When these constructs were assayed in several Escherichia coli strains, it appeared that the detection thresholds were highly variable. The TD2158 wild-type E. coli gave rise to a biosensor ten times more active and sensitive than its W3110 E. coli K12 equivalent. This biosensor was able to confidently detect Ni concentrations as little as 80 nM (4.7 μg l −1), which makes its use compatible with the norms governing the drinking water quality

Effect of Aneurysm and Patient Characteristics on Intracranial Aneurysm Wall Thickness

Background: The circle of Willis is a network of arteries allowing blood supply to the brain. Bulging of these arteries leads to formation of intracranial aneurysm (IA). Subarachnoid hemorrhage (SAH) due to IA rupture is among the leading causes of disability in the western world. The formation and rupture of IAs is a complex pathological process not completely understood. In the present study, we have precisely measured aneurysmal wall thickness and its uniformity on histological sections and investigated for associations between IA wall thickness/uniformity and commonly admitted risk factors for IA rupture. Methods: Fifty-five aneurysm domes were obtained at the Geneva University Hospitals during microsurgery after clipping of the IA neck. Samples were embedded in paraffin, sectioned and stained with hematoxylin-eosin to measure IA wall thickness. The mean, minimum, and maximum wall thickness as well as thickness uniformity was measured for each IA. Clinical data related to IA characteristics (ruptured or unruptured, vascular location, maximum dome diameter, neck size, bottleneck factor, aspect and morphology), and patient characteristics [age, smoking, hypertension, sex, ethnicity, previous SAH, positive family history for IA/SAH, presence of multiple IAs and diagnosis of polycystic kidney disease (PKD)] were collected. Results: We found positive correlations between maximum dome diameter or neck size and IA wall thickness and thickness uniformity. PKD patients had thinner IA walls. No associations were found between smoking, hypertension, sex, IA multiplicity, rupture status or vascular location, and IA wall thickness. No correlation was found between patient age and IA wall thickness. The group of IAs with non-uniform wall thickness contained more ruptured IAs, women and patients harboring multiple IAs. Finally, PHASES and ELAPSS scores were positively correlated with higher IA wall heterogeneity. Conclusion: Among our patient and aneurysm characteristics of interest, maximum dome diameter, neck size and PKD were the three factors having the most significant impact on IA wall thickness and thickness uniformity. Moreover, wall thickness heterogeneity was more observed in ruptured IAs, in women and in patients with multiple IAs. Advanced medical imaging allowing in vivo measurement of IA wall thickness would certainly improve personalized management of the disease and patient care