50 research outputs found

    Etude de la transformation plasmidique naturelle d'Escherichia coli et de ses relations éventuelles avec la compétence programmée pour la transformation génétique et la compétence dite nutritionnelle

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    Bien que la bactérie Escherichia coli ne soit pas connue pour être naturellement transformable, mon travail de thèse montre que l'on peut obtenir des transformants plasmidiques spontanément sur boîte. Cette transformation n'est pas induite par les cations divalents au contraire de la transformation ‘artificielle chimique’ (Sun et al., FEMS Microbiol. Lett. 2006. 265: 249–255). Les bactéries transformables utilisent une machinerie protéique transmembranaire, évolutivement conservée, pour internaliser l'ADN exogène sous forme simple brin. E.coli possède l'ensemble des gènes codant pour cette machinerie. J'ai inactivé les gènes clés de cette machinerie, dont hofQ (canal transmembranaire externe) et ycaI (canal transmembranaire interne), et observé qu'aucun de ces mutants n'est affecté pour la transformation sur boîte. L'ADN plasmidique ne pénètre donc pas via la machinerie de transformation, mais plutôt sous forme double brin ce que suggèrent les courbes de réponse à la concentration d'ADN (Sun et al., J. Bacteriol. 2009. 191: 713-719). Le troisième volet de ma thèse a consisté à tenter de mieux caractériser un phénomène appelé 'compétence nutritionnelle', appellation qui désigne la capacité d'utiliser l'ADN comme source de carbone. Pour établir si différents gènes de la machinerie de transformation étaient impliqués, j'ai cherché à reproduire les expériences publiées de croissance de la souche ZK126 sur milieu minimum M63 contenant de l'ADN. Malgré de nombreuses tentatives et contrôles, je n'ai pas pu reproduire ces expériences, ce qui m'a amené à clore mon mémoire de thèse par une discussion critique des données publiées relatives à la compétence nutritionnelle de E. coli.While Escherichia coli is not considered to belong to naturally transformable species, I established a transformation system allowing spontaneous plasmid transformation on plate (Sun et al., FEMS Microbiol. Lett. 2006. 265: 249–255). Transformation is not induced by divalent cations in contrast to chemically-induced 'artificial transformation' (Sun et al., J. Bacteriol. 2009. 191: 713-719). As DNA uptake in naturally transformable bacteria relies on a conserved multiprotein machinery and the E. coli genome contains all genes encoding this machinery, I investigated whether key genes are required for plasmid transformation. None of the mutants I constructed, including hofQ and ycaI which encode putative outer and inner membrane channel proteins were affected, indicating that plasmid DNA is not taken up via the transformation machinery. We proposed that plasmid DNA instead enters the cytoplasm as double stranded material as suggested by response curves to DNA concentration (Sun et al., J. Bacteriol. 2009. Ibid.). In the last part of my thesis, I reinvestigated so-called ‘nutritional competence’ of E. coli. Previously work reported that E. coli cells are able to use DNA as the sole carbon source. I wished to establish whether this phenomenon relies on the above-mentioned DNA uptake machinery. I therefore tried to reproduce the published growth experiments of E. coli ZK126 on M63 minimal medium with DNA. Despite numerous attempts and controls, I could not observe any growth. This failure to reproduce published observations led me to conclude my thesis by an in-depth discussion of the three articles dedicated to so-called nutritional competence of E. coli

    Modeling of wireless remote shape control for beams using nonlinear photostrictive actuators

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    AbstractPhotostrictive materials produce mechanical strain when irradiated by ultraviolet light, thus may be used in wireless remote control of smart microstructures. This paper presents an investigation into modelling and static shape control of beams with nonlinear photostrictive actuators. Governing equations of beams bonded with photostrictive actuator patches are derived to study the interaction between the photostrictive actuators and the host beams. An analytical solution method is presented to solve the governing equations of the beams with discretely distributed photostrictive actuators. An iterative procedure is developed to find optimal light intensities in photostrictive actuators that best match the actuated shape to the desired one. An example is given to illustrate the model and shape control of a beam with PLZT actuators

    Pull in and Push Out: Mechanisms of Horizontal Gene Transfer in Bacteria

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    Horizontal gene transfer (HGT) plays an important role in bacterial evolution. It is well accepted that DNA is pulled/pushed into recipient cells by conserved membrane-associated DNA transport systems, which allow the entry of only single-stranded DNA (ssDNA). However, recent studies have uncovered a new type of natural bacterial transformation in which double-stranded DNA (dsDNA) is taken up into the cytoplasm, thus complementing the existing methods of DNA transfer among bacteria. Regulated by the stationary-phase regulators RpoS and cAMP receptor protein (CRP), Escherichia coli establishes competence for natural transformation with dsDNA, which occurs in agar plates. To pass across the outer membrane, a putative channel, which may compete for the substrate with the porin OmpA, may mediate the transfer of exogenous dsDNA into the cell. To pass across the inner membrane, dsDNA may be bound to the periplasmic protein YdcS, which delivers it into the inner membrane channel formed by YdcV. The discovery of cell-to-cell contact-dependent plasmid transformation implies the presence of additional mechanism(s) of transformation. This review will summarize the current knowledge about mechanisms of HGT with an emphasis on recent progresses regarding non-canonical mechanisms of natural transformation. Fully understanding the mechanisms of HGT will provide a foundation for monitoring and controlling multidrug resistance

    Horizontal gene transfer mediated bacterial antibiotic resistance

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    Bacterial antibiotic resistance, especially multidrug resistance (MDR), has become a global challenge, threatening human and animal health, food and environment safety. The number of human deaths accounted for by multidrug resistance (MDR) was estimated to increase to 10 million by 2050, exceeding the number of deaths arising from cancer (WHO, 2014). Although a bacterium is able to establish antibiotic resistance through spontaneous mutation (Salverda et al., 2017), development of MDR in the bacterium would take a long time if it only relies on self-adaptive mutation. Horizontal gene transfer (HGT) allows bacteria to exchange their genetic materials (including antibiotic resistance genes, ARGs) among diverse species (Le Roux and Blokesch, 2018), greatly fostering collaboration among bacterial population in MDR development. Recent studies reveal emergence of ‘superbugs’ that carry a number of HGT-transferred ARGs on plasmids and tolerate almost all antibiotics (Mathers et al., 2015; Wang and Sun, 2015; Malhotra-Kumar et al., 2016). These MDR plasmids are able to be further transferred to different bacterial species, creating new ‘superbugs’ that grow in different environments. Global emergence of ‘superbugs’ carrying MDR plasmids (e. g. NDM-1 and MCR-1) in various environmental niches (e. g. patients, animals and soil) indicates rapid propagation of MDR among bacterial populations. Although HGT and MDR were found to be tightly linked in ‘superbugs’, as revealed by surveillance studies, our knowledge about how and to what extend HGT propels development of MDR under different environmental conditions remain inadequate. The 22 publications collected in the topic “Horizontal Gene Transfer Mediated Bacterial Antibiotic Resistance” show new discoveries and recent advances concerning this issue in a wide range of fields, providing a basis for collaboratively controlling MDR in the future

    A Three-Phase Interleaved Floating Output Boost Converter

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    High step-up dc-dc converter is an essential part in several renewable energy systems. In this paper, a new topology of step-up dc-dc converter based on interleaved structure is proposed. The proposed converter uses three energy storing capacitors to achieve a high voltage gain. Besides the high voltage gain feature, the proposed converter also reduces the voltage stress across the semiconductor switches. This helps in using low rating switching devices which can reduce the overall size and cost of the converter. The operating principle of the proposed converter is discussed in detail and its principle waveforms are analyzed. An experiment is carried out on a 20 V input, 130 V output, and 21 W power prototype of the proposed converter in the laboratory to verify the performance of the proposed converter. An efficiency of 91.3% is achieved at the rated load

    RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli

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    Spontaneous plasmid transformation of Escherichia coli is independent of the DNA uptake machinery for single-stranded DNA (ssDNA) entry. The one-hit kinetic pattern of plasmid transformation indicates that double-stranded DNA (dsDNA) enters E. coli cells on agar plates. However, DNA uptake and transformation regulation remain unclear in this new type of plasmid transformation. In this study, we developed our previous plasmid transformation system and induced competence at early stationary phase. Despite of inoculum size, the development of competence was determined by optical cell density. DNase I interruption experiment showed that DNA was taken up exponentially within the initial 2 minutes and most transforming DNA entered E. coli cells within 10 minutes on LB-agar plates. A half-order kinetics between recipient cells and transformants was identified when cell density was high on plates. To determine whether the stationary phase master regulator RpoS plays roles in plasmid transformation, we investigated the effects of inactivating and over-expressing its encoding gene rpoS on plasmid transformation. The inactivation of rpoS systematically reduced transformation frequency, while over-expressing rpoS increased plasmid transformation. Normally, RpoS recognizes promoters by its lysine 173 (K173). We found that the K173E mutation caused RpoS unable to promote plasmid transformation, further confirming a role of RpoS in regulating plasmid transformation. In classical transformation, DNA was transferred across membranes by DNA uptake proteins and integrated by DNA processing proteins. At stationary growth phase, RpoS regulates some genes encoding membrane/periplasmic proteins and DNA processing proteins. We quantified transcription of 22 of them and found that transcription of only 4 genes (osmC, yqjC, ygiW and ugpC) encoding membrane/periplasmic proteins showed significant differential expression when wildtype RpoS and RpoSK173E mutant were expressed. Further investigation showed that inactivation of any one of these genes did not significantly reduce transformation, suggesting that RpoS may regulate plasmid transformation through other/multiple target genes

    Robust estimation of bacterial cell count from optical density

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    Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data
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