The conjugative system encoded by the integrative conjugative element ICEclc in Pseudomonas putida

Integrative and conjugative elements (ICE) are a widespread class of mobile genetic elements, which play an important role for bacterial evolution and adaptation to new niches. ICE transfer between cells requires a multi-subunit protein complex known as type IV secretion system (T4SS) which is usually self-encoded. Although several T4SS have been discovered and characterized in the years, they mostly belong to mobile plasmids and ICE-encoded T4SS remain severely understudied. Here we focused on the T4SS encoded by the ICEclc element first found in Pseudomonas knackmussii B13 in two identical copies. The core region of ICEclc, which includes the genetic locus encoding for the T4SS, is conserved among several β- and γ-proteobacteria, thus understanding peculiarities about this conjugation system would be representative for a large number of mobile elements. In this work we mainly focused on the extensive characterization of the ICEclc T4SS genetic locus with a combination of ‘in silico’, molecular biology and microscopy approaches. The first chapter introduces the theme of horizontal gene transfer with a closer look at integrative and conjugative elements characteristics, evolutionary importance and DNA conjugation mechanism with in depth description of the up to date knowledge in T4SS classification and structural composition. In the second chapter we present the general characterization of the 24 genes of the ICEclc T4SS locus. With bioinformatics analysis we inferred homologies between ICE encoded and known T4SS components. Single gene knockouts of 22 out of the 24 open reading frames were used to understand their essentiality for ICE transfer. To better understand T4SS localization at single cell level, we fused nine predicted T4SS subunits to fluorescent proteins and studied their cellular localization. By coupling fluorescent labelling and gene deletions we showed possible interactions between several T4SS subunits and we proposed a dynamic model of T4SS assembly. The third chapter focuses on the characterization of the conjugative pilus, one of the four major protein subassemblies of T4SS. With protein 3D structure prediction, we identified the gene encoding the pilin in ICEclc, being orf66625. With a microscopy technique based on chemical labelling of cysteine residues we could observe the conjugative pilus in vivo. In addition, we used cryo-CLEM to gain detailed morphological information on this cellular appendage. The fourth chapter presents a study on the dynamic behaviour of two ICEclc T4SS subunits. Here we focused on IceB7 and IceB4 located in the outer and inner-membrane respectively. With high resolution confocal microscopy we followed the localization of the two labelled subunits overtime both in presence and absence of recipient cells and we inferred their dynamics with custom made image analysis pipelines. Our results suggest a highly stable IceB7, which is one of the core members of the outer membrane assembly and a highly dynamic IceB4, suggesting that the ATPase of the T4SS are not constitutively docked to the conjugative machinery and that they might do so only when ICE DNA is about to be transferred. Finally, we conclude with a general discussion on the outcomes and relevance of this work and the perspective for future studies. -- Les éléments intégratifs et conjugatifs (ICE) constituent une classe répandue d'éléments génétiques mobiles qui jouent un rôle important dans l'évolution bactérienne et l'adaptation à de nouvelles niches. Le transfert des ICE nécessite le contact entre deux bactéries et la mise en place d’un complexe protéique reliant les deux cellules appelé système de sécrétion de type IV (T4SS). Bien que plusieurs T4SS appartenant à des familles distinctes aient été découverts et caractérisés au cours des dernières décennies, la plupart appartiennent à des plasmides mobiles et les T4SS codés par les ICE restent gravement sous-étudiés. Dans cette étude, nous nous sommes concentrés sur le T4SS codé par l'élément ICEclc, initialement découvert en deux copies identiques intégrées dans le chromosome de Pseudomonas knackmussii B13. La région centrale d’ICEclc, qui inclut le locus génétique codant le T4SS, est conservée chez plusieurs éléments apparentés présents dans le génome de protéobactéries des groupes β et γ. Ainsi, la compréhension des particularités de ce système de conjugaison serait représentatif d'un grand nombre d'éléments mobiles. Dans ce travail, nous avons focalisé notre attention sur la caractérisation approfondie du locus génétique du T4SS d’ICEclc en utilisant une combinaison d'approches bioinformatiques, génétiques, et microscopiques. Le premier chapitre introduit le thème du transfert horizontal de gènes en se penchant sur les caractéristiques des éléments intégratifs et conjugatifs, leur importance évolutive et le mécanisme de transfert d'ADN par conjugaison, avec une description approfondie des connaissances actuelles sur la classification et la composition structurelle du T4SS. Dans le deuxième chapitre, nous présentons la caractérisation générale des 24 gènes du locus T4SS d'ICEclc. Grâce à une analyse bioinformatique des homologies entre les composants du T4SS codés par ICEclc et d’autres déjà connus, nous avons inféré des fonctions aux produits de la majorité des gènes du locus T4SS. 22 variants d’ICEclc délétés pour 1 des 24 gènes codant des sous-unités putatives du T4SS ont été utilisés pour comprendre la nécessité de chaque sous-unité pour le transfert de l'ICE. Afin de mieux comprendre la dynamique de mise en place et la localisation des différents parties du T4SS au niveau de la cellule individuelle, nous avons fusionné neuf sous-unités prédites du T4SS à des protéines fluorescentes, et étudié leur localisation cellulaire par microscopie à épifluorescence. En associant le marquage fluorescent et les délétions géniques, nous avons montré des interactions possibles entre plusieurs sous-unités du T4SS et avons proposé un modèle dynamique de l'assemblage du T4SS. Le troisième chapitre se concentre sur la caractérisation du pilus de conjugaison, l'un des quatre sous-ensembles protéiques du T4SS. Une analyse bioinformatique reposant sur la prédiction de la structure tridimensionnelle des protéines a permis d’identifier le gène orf66625 codant la piline (sous unités protéique majeure du pilus) chez ICEclc. Grâce à une technique de microscopie basée sur le marquage chimique des cystéines, nous avons pu observer le pilus conjugatif in vivo. De plus, nous avons obtenu des informations morphologiques détaillées sur cet appendice cellulaire grâce à une technique spécifique de microscopie électronique, appelée cryo-CLEM. Le quatrième chapitre présente une étude sur la dynamique d’IceB7 et IceB4, deux sous-unités du T4SS d'ICEclc situées dans la membrane externe et la membrane interne respectivement. Grace à la microscopie confocale à haute résolution, nous avons suivi la localisation des deux sous-unités au fil du temps, en présence et en absence de cellules receveuses, et analysé leur dynamique à l'aide de « pipelines » d'analyse d'images développés par nos soins. D’une part, nos résultats suggèrent qu’IceB7 est hautement stable, et est l'un des membres centraux de l'assemblage du complexe dans la membrane externe. D’autre part, IceB4 est hautement dynamique, ce qui suggère que cette protéine n'est pas constitutivement ancrée à la machinerie de transfert, et pourrait l’être uniquement lorsque l'ADN de l'ICE est sur le point d'être transféré. Enfin, ce manuscrit se termine par une discussion générale sur les résultats et la pertinence de ce travail, ainsi que les perspectives pour des études futures

The IT revolution across the U.S. states

This paper presents evidence on the geographical dimension of the IT revolution in the U.S. economy. BEA and Census data show that, although neither IT diffusion nor the productivity revival was geographically narrow, the matching of the two across the U.S. states has been far from perfect. The late 1990s productivity acceleration mostly occurred in those states specialized in the production of IT goods & services as well as of non-IT durable goods. When those states are excluded from the sample, the remaining states do not exhibit any significant acceleration in productivity. In particular, the association between productivity gains and IT use is at best weak at the state level. This contrasts with previous aggregate and sector evidence, where the importance of both IT production and use was stressed.

THE IT REVOLUTION ACROSS THE UNITED STATES

In this paper we study the geography of the IT revolution in the U.S. economy. By relating the intensity of IT production and diffusion to labor productivity growth for the United States, we find three main results. First, states with above-average production intensity of IT manufacturing show more growth acceleration than other states. Second, the same applies to states with above-average IT diffusion. Yet, while the result for IT-producing states is strong, the result for IT-using states is somewhat smaller and less robust across specifications. Third, we also reconcile our state-wide pieces of evidence with previous industry and aggregate evidence. Accelerating productivity growth in IT-producing states stems from both IT-producing and IT-using industries in those states and is not a manifestation of the exclusive importance of IT production. Moreover, the less robust evidence for IT-using states is due to lower growth contributions from IT-producing and other industries in these states, not a symptom of a missing effect of IT usage. Copyright � 2006 The Authors; Journal compilation � International Association for Research in Income and Wealth 2006.

Effectiveness of Different Modalities of Remote Online Training in Young Healthy Males

Since 2020 there has been an increase in demand for home workouts. Therefore, different ways of delivering distance training have been proposed to promote “stay active at home.” This study aimed to compare the effectiveness of three different training programs consisting of a total of 15 workouts (three sessions per week): supervised livestreaming (LS), unsupervised following a video recording (VR), and unsupervised following a written program (WP). Changes in anthropometric and cardiovascular variables, muscle fitness, and physical activity levels were evaluated. To provide a meaningful analysis for significant comparisons between small groups, mean differences (∆), 95% confidence interval (95% C.I.), and Cohen’s effect sizes (E.S.) were also calculated. The three training modalities increased physical activity levels, with an adherence rate of LS = 93.3%, VR = 86%, and WP = 74%. Although there was no reduction in body weight, waist circumference decreased by 1.3 cm (95% C.I. = −2.1, −0.5; E.S. = 0.170; p < 0.004). Furthermore, where LS, VR, and WP resulted in improvements in muscle fitness, only LS showed changes in cardiovascular variables, such as resting heart rate (∆ = −7.3 bpm; 95% C.I. = −11.9, −2.7; E.S. = 1.296; p < 0.001) and Ruffier’s index (∆ = −2.1bpm; 95% C.I. = −3.5, −0.8; E.S. 1.099; p < 0.001). Remote online training proved its effectiveness over a short period of time. However, supervised training proved to be the most effective, highlighting the importance of an experienced trainer

Fuori dalla Crisi

Uno sguardo alle esperienze di altri paesi proponendo soluzioni concrete per superare la spirale negativa tra il riequilibrio dei conti pubblici e l\u2019adozione di politiche fiscali recessive. La conclusione \ue8 che non c\u2019\ue8 alternativa alla crescita per innovazione, come insegnano le esperienze di successo di Silicon Valley, Finlandia e Israele. Ecco allora un vero e proprio programma di riforme per far ripartire il credito, dare autonomia alle scuole, digitalizzare la pubblica amministrazione e non solo. Un piano con cui dovrebbe confrontarsi chi \ue8 oggi al governo del Paese

EC aid to associated countries: distribution and determinants

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