The C-terminal domain of the Escherichia coli RNA polymerase α subunit plays a role in the CI-dependent activation of the bacteriophage λ pM promoter

The bacteriophage λ pM promoter is required for maintenance of the λ prophage in Escherichia coli, as it facilitates transcription of the cI gene, encoding the λ repressor (CI). CI levels are maintained through a transcriptional feedback mechanism whereby CI can serve as an activator or a repressor of pM. CI activates pM through cooperative binding to the OR1 and OR2 sites within the OR operator, with the OR2-bound CI dimer making contact with domain 4 of the RNA polymerase σ subunit (σ4). Here we demonstrate that the 261 and 287 determinants of the C-terminal domain of the RNA polymerase α subunit (αCTD), as well as the DNA-binding determinant, are important for CI-dependent activation of pM. We also show that the location of αCTD at the pM promoter changes in the presence of CI. Thus, in the absence of CI, one αCTD is located on the DNA at position −44 relative to the transcription start site, whereas in the presence of CI, αCTD is located at position −54, between the CI-binding sites at OR1 and OR2. These results suggest that contacts between CI and both αCTD and σ are required for efficient CI-dependent activation of pM

Etudes numériques des instabilités en ébullition avec SIMMER-V : contribution à l'évaluation d'accidents de perte de débit dans un SFR

La R&D menée dans le cadre du développement des réacteurs Gen-IV au CEA comprend les évaluations de sûreté des concepts proposés. Les analyses de sûreté se concentrent sur les accidents graves identifiés et sont simulées par des codes spécifiques. Ces analyses incluent la séquence de perte de débit non protégée pouvant conduire à la fusion du cœur, identifiée comme accident grave. L’évaluation d’une telle séquence nécessite le calcul des instabilités diphasiques pouvant apparaître dans le système lors de l’ébullition du sodium. Dans ce contexte, cette thèse cible l'impact potentiel des instabilités d'écoulement diphasiques sur le comportement à long terme des réacteurs. Le code mécaniste d'accidents graves SIMMER-V est utilisé comme outil de référence pour les calculs transitoires. Le travail a nécessité une nouvelle méthode de couplage aux frontières facilitant la représentation des systèmes concernés. Cette version du code a été validée sur l'expérience GENESIS réalisée à Delft Univ. of Technology. La validation repose sur l’évaluation du taux de décroissance pour chaque point de la carte de stabilité. La carte de stabilité et la limite calculée sont comparées aux résultats d’ATHLET (DUT). Une autre approche pour prédire la limite de stabilité par la dérivation d'un critère de stabilité analytique et sa comparaison avec les résultats de SIMMER-V, est présentée. Des simulations SIMMER-V 3D du cœur durant l’ULOF intègrent la dynamique du combustible nucléaire et vérifient les résultats généraux du modèle. Les résultats permettent de conclure sur les critères de déclenchement des oscillations en et hors phase d'un écoulement de caloporteur à travers le cœur hétérogèneThe R&D preformed in the framework of Gen-IV reactors development at the CEA includes intensive safety assessments of the proposed SFR designs. Safety analyses concentrate on identified severe accidents which are simulated by dedicated codes. Such safety analysis chains include an unprotected loss-of-flow (ULOF) event, which can potentially lead to the core melting, and is therefore identified as a severe accident. The validation of such an event requires evaluation of sodium boiling instabilities which can appear in the system. In this context, this PhD thesis targets the potential impact of two-phase flow instabilities on long-term reactor behaviour. The mechanistic severe-accident code SIMMER-V is used as a reference tool for transient calculations. A new boundary coupling method is proposed and implemented, which facilitates representing typical loop systems used in nuclear safety analyses. The developed code version is used for validation against the GENESIS experiment performed at the Delft Univ. of Technology. The validation is done in terms of decay ratio assessment for the region of interest on the stability map. The calculated stability map and boundary are benchmarked with ATHLET (DUT) results. Another approach to predicting the stability boundary is presented by derivation of an analytical stability criterion and its comparison with numerical SIMMER-V results. The full-core 3D SIMMER-V simulations during ULOF, integrate nuclear fuel dynamics and verify the general findings of the model. The results allow concluding on the onset criteria of the in- and out-phase oscillations of a coolant flow through the heterogeneous cor

Emerging roles of toxin-antitoxin modules in bacterial pathogenesis

Toxin-antitoxin (TA) cassettes are encoded widely by bacteria. The modules typically comprise a protein toxin and protein or RNA antitoxin that sequesters the toxin factor. Toxin activation in response to environmental cues or other stresses promotes a dampening of metabolism, most notably protein translation, which permits survival until conditions improve. Emerging evidence also implicates TAs in bacterial pathogenicity. Bacterial persistence involves entry into a transient semi-dormant state in which cells survive unfavorable conditions including killing by antibiotics, which is a significant clinical problem. TA complexes play a fundamental role in inducing persistence by downregulating cellular metabolism. Bacterial biofilms are important in numerous chronic inflammatory and infectious diseases and cause serious therapeutic problems due to their multidrug tolerance and resistance to host immune system actions. Multiple TAs influence biofilm formation through a network of interactions with other factors that mediate biofilm production and maintenance. Moreover, in view of their emerging contributions to bacterial virulence, TAs are potential targets for novel prophylactic and therapeutic approaches that are required urgently in an era of expanding antibiotic resistance. This review summarizes the emerging evidence that implicates TAs in the virulence profiles of a diverse range of key bacterial pathogens that trigger serious human disease

The Axe-Txe Complex of Enterococcus faecium Presents a Multilayered Mode of Toxin-Antitoxin Gene Expression Regulation

Multidrug-resistant variants of human pathogens from the genus Enterococcus represent a significant health threat as leading agents of nosocomial infections. The easy acquisition of plasmid-borne genes is intimately involved in the spread of antibiotic resistance in enterococci. Toxin-antitoxin (TA) systems play a major role in both maintenance of mobile genetic elements that specify antibiotic resistance, and in bacterial persistence and virulence. Expression of toxin and antitoxin genes must be in balance as inappropriate levels of toxin can be dangerous to the host. The controlled production of toxin and antitoxin is usually achieved by transcriptional autoregulation of TA operons. One of the most prevalent TA modules in enterococcal species is axe-txe which is detected in a majority of clinical isolates. Here, we demonstrate that the axe-txe cassette presents a complex pattern of gene expression regulation. Axe-Txe cooperatively autorepress expression from a major promoter upstream of the cassette. However, an internal promoter that drives the production of a newly discovered transcript from within axe gene combined with a possible modulation in mRNA stability play important roles in the modulation of Axe:Txe ratio to ensure controlled release of the toxin

Role of the RNA polymerase α subunits in CII-dependent activation of the bacteriophage λ p(E) promoter: identification of important residues and positioning of the α C-terminal domains

The bacteriophage λ CII protein stimulates the activity of three phage promoters, p(E), p(I) and p(aQ), upon binding to a site overlapping the –35 element at each promoter. Here we used preparations of RNA polymerase carrying a DNA cleavage reagent attached to specific residues in the C-terminal domain of the RNA polymerase α subunit (αCTD) to demonstrate that one αCTD binds near position –41 at p(E), whilst the other αCTD binds further upstream. The αCTD bound near position –41 is oriented such that its 261 determinant is in close proximity to σ(70). The location of αCTD in CII-dependent complexes at the p(E) promoter is very similar to that found at many activator-independent promoters, and represents an alternative configuration for αCTD at promoters where activators bind sites overlapping the –35 region. We also used an in vivo alanine scan analysis to show that the DNA-binding determinant of αCTD is involved in stimulation of the p(E) promoter by CII, and this was confirmed by in vitro transcription assays. We also show that whereas the K271E substitution in αCTD results in a drastic decrease in CII-dependent activation of p(E), the p(I) and p(aQ) promoters are less sensitive to this substitution, suggesting that the role of αCTD at the three lysogenic promoters may be different

The C-terminal domain of the Escherichia

coli RNA polymerase a subunit plays a role in the CI-dependent activation of the bacteriophage j pM promote

Synthesis, pharmacological and antiviral activity of 1,3-thiazepine derivatives

The preparation of new fourteen thiourea and fourteen product of their condensation with 1,4-dibromobutane, viz. 1,3-thiazepine derivatives, of 10-isopropyl-8-methyl-4-aza-tricyclo[5.2.2.02,6]undec- 8-ene-3,5-dione and 1-isopropyl-7-methyl-4-aza-tricyclo[5.2.2.02,6]undec-8-ene-3,5-dione is described. Elemental analysis, MS and 1H NMR spectra confirmed the identity of the products. The molecular structure of linear disubstituted thiourea derivative and its cyclization product was determined by an X-ray crystal structure analysis. Two of new obtained compounds (6b0 and 7a0) were tested for their pharmacological activity on animal central nervous system (CNS) in behavioral animal tests. With relatively low acute toxicity (LD50 lower than 2000 mg kg1 i.p.) they exhibited significant influence on spontaneous locomotor activity and body temperature. Additionally, compounds reduced number of the ‘‘head twitch’’ episodes after 5-hydroksytryptophan (5-HTP) administration. New compounds were evaluated in vitro against representatives of different virus classes, such as a HIV-1 (Retrovirus), a HBV (Hepadnavirus) and the single-stranded RNAþ viruses Yellow fever virus (YFV) and Bovine viral diarrhea virus (BVDV), both belonging to Flaviridae. Three of new obtained compounds showed a modest activity against HIV-1 wtIIIB, BVDV and YFV

Identification of αCTD residues important for CI-dependent activation of

<p><b>Copyright information:</b></p><p>Taken from "The C-terminal domain of the RNA polymerase α subunit plays a role in the CI-dependent activation of the bacteriophage λ promoter"</p><p></p><p>Nucleic Acids Research 2007;35(7):2311-2320.</p><p>Published online 27 Mar 2007</p><p>PMCID:PMC1874639.</p><p>© 2007 The Author(s)</p> () Strain WAM106, containing plasmids pTJSpM, pJMH1 and pACλcI, was transformed with each of a set of plasmids encoding the RNAP α subunit in which each residue of αCTD was changed individually to alanine. Cultures were grown at 37°C to OD≈0.2 in LB medium containing appropriate antibiotics, at which time IPTG was added to a final concentration of 0.1 mM. After 60 min induction of α and CI synthesis, the β-galactosidase activities were determined. The activities are presented relative to the activity of the strain harbouring plasmid pLAW2 encoding wild-type α (100% = 2300 Miller units) and are averages of at least three independent experiments. Grey bars indicate positions when alanine occurs naturally. Black bars correspond to the residues in which alanine substitution causes a decrease in activity of ≥20% compared to wild-type α. () Structure of αCTD, showing in black the residues that are important for CI-dependent activation of . Residue K271 is highlighted in grey for reference