Nuevos varsovianos, nuevos madrileños. Visiones literarias de los ‘exiliados’ en la Varsovia y el Madrid de posguerra: el caso de Zły de Leopold Tyrmand y La colmena de Camilo José Cela

El artículo tiene por objetivo identificar las similitudes y diferencias en el tratamiento de los nuevos varsovianos y nuevos madrileños en las novelas Zły de Leopold Tyrmand y La colmena de Camilo José Cela. Estas novelas fueron escritas en unos contextos paralelos: tras la segunda guerra mundial y la guerra civil española, en el ámbito del régimen ‘comunista’ polaco y del Estado franquista, respectivamente. El análisis presta una especial atención a la distribución espacial de los nuevos varsovianos y nuevos madrileños –es decir, los personajes ‘exiliados’ en la Varsovia y el Madrid de posguerra– dentro de ambos cuerpos novelescos, incluyendo cuestiones de identidad, e intenta verificar el grado de veracidad representado por sendas novelas

Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity.

Published versio

The role of the conserved phenylalanine in the σ54-interacting GAFTGA motif of bacterial enhancer binding proteins

σ54-dependent transcription requires activation by bacterial enhancer binding proteins (bEBPs). bEBPs are members of the AAA+ (ATPases associated with various cellular activities) protein family and typically form hexameric structures that are crucial for their ATPase activity. The precise mechanism by which the energy derived from ATP hydrolysis is coupled to biological output has several unknowns. Here we use Escherichia coli PspF, a model bEBP involved in the transcription of stress response genes (psp operon), to study determinants of its contact features with the closed promoter complex. We demonstrate that substitution of a highly conserved phenylalanine (F85) residue within the L1 loop GAFTGA motif affects (i) the ATP hydrolysis rate of PspF, demonstrating the link between L1 and the nucleotide binding pocket; (ii) the internal organization of the hexameric ring; and (iii) σ54 interactions. Importantly, we provide evidence for a close relationship between F85 and the −12 DNA fork junction structure, which may contribute to key interactions during the energy coupling step and the subsequent remodelling of the Eσ54 closed complex. The functionality of F85 is distinct from that of other GAFTGA residues, especially T86 where in contrast to F85 a clean uncoupling phenotype is observed

Functional roles of the pre-sensor I insertion sequence in an AAA+ bacterial enhancer binding protein

Molecular machines belonging to the AAA+ superfamily of ATPases use NTP hydrolysis to remodel their versatile substrates. The presence of an insertion sequence defines the major phylogenetic pre-sensor I insertion (pre-SIi) AAA+ superclade. In the bacterial σ54-dependent enhancer binding protein phage shock protein F (PspF) the pre-SIi loop adopts different conformations depending on the nucleotide-bound state. Single amino acid substitutions within the dynamic pre-SIi loop of PspF drastically change the ATP hydrolysis parameters, indicating a structural link to the distant hydrolysis site. We used a site-specific protein–DNA proximity assay to measure the contribution of the pre-SIi loop in σ54-dependent transcription and demonstrate that the pre-SIi loop is a major structural feature mediating nucleotide state-dependent differential engagement with Eσ54. We suggest that much, if not all, of the action of the pre-SIi loop is mediated through the L1 loop and relies on a conserved molecular switch, identified in a crystal structure of one pre-SIi variant and in accordance with the high covariance between some pre-SIi residues and distinct residues outside the pre-SIi sequence

Essential roles of three enhancer sites in σ54-dependent transcription by the nitric oxide sensing regulatory protein NorR

The bacterial activator protein NorR binds to enhancer-like elements, upstream of the promoter site, and activates σ54-dependent transcription of genes that encode nitric oxide detoxifying enzymes (NorVW), in response to NO stress. Unique to the norVW promoter in Escherichia coli is the presence of three enhancer sites associated with a binding site for σ54-RNA polymerase. Here we show that all three sites are required for NorR-dependent catalysis of open complex formation by σ54-RNAP holoenzyme (Eσ54). We demonstrate that this is essentially due to the need for all three enhancers for maximal ATPase activity of NorR, energy from which is used to remodel the closed Eσ54 complex and allow melting of the promoter DNA. We also find that site-specific DNA binding per se promotes oligomerisation but the DNA flanking the three sites is needed to further stabilise the functional higher order oligomer of NorR at the enhancers

Essential roles of three enhancer sites in σ54-dependent transcription by the nitric oxide sensing regulatory protein NorR

The bacterial activator protein NorR binds to enhancer-like elements, upstream of the promoter site, and activates σ54-dependent transcription of genes that encode nitric oxide detoxifying enzymes (NorVW), in response to NO stress. Unique to the norVW promoter in Escherichia coli is the presence of three enhancer sites associated with a binding site for σ54-RNA polymerase. Here we show that all three sites are required for NorR-dependent catalysis of open complex formation by σ54-RNAP holoenzyme (Eσ54). We demonstrate that this is essentially due to the need for all three enhancers for maximal ATPase activity of NorR, energy from which is used to remodel the closed Eσ54 complex and allow melting of the promoter DNA. We also find that site-specific DNA binding per se promotes oligomerisation but the DNA flanking the three sites is needed to further stabilise the functional higher order oligomer of NorR at the enhancers

Broad Spectrum Pro-Quorum-Sensing Molecules as Inhibitors of Virulence in Vibrios

Quorum sensing (QS) is a bacterial cell-cell communication process that relies on the production and detection of extracellular signal molecules called autoinducers. QS allows bacteria to perform collective activities. Vibrio cholerae, a pathogen that causes an acute disease, uses QS to repress virulence factor production and biofilm formation. Thus, molecules that activate QS in V. cholerae have the potential to control pathogenicity in this globally important bacterium. Using a whole-cell high-throughput screen, we identified eleven molecules that activate V. cholerae QS: eight molecules are receptor agonists and three molecules are antagonists of LuxO, the central NtrC-type response regulator that controls the global V. cholerae QS cascade. The LuxO inhibitors act by an uncompetitive mechanism by binding to the pre-formed LuxO-ATP complex to inhibit ATP hydrolysis. Genetic analyses suggest that the inhibitors bind in close proximity to the Walker B motif. The inhibitors display broad-spectrum capability in activation of QS in Vibrio species that employ LuxO. To the best of our knowledge, these are the first molecules identified that inhibit the ATPase activity of a NtrC-type response regulator. Our discovery supports the idea that exploiting pro-QS molecules is a promising strategy for the development of novel anti-infectives

Promoter and regulon analysis of nitrogen assimilation factor, σ54, reveal alternative strategy for E. coli MG1655 flagellar biosynthesis

Bacteria core RNA polymerase (RNAP) must associate with a σ factor to recognize promoter sequences. Promoters recognized by the σ54 (or σN) associated RNA polymerase are unique in having conserved positions around −24 and −12 nucleotides upstream from the transcriptional start site. Using DNA microarrays representing the entire Escherichia coli genome and promoter validation approaches, we identify 40 in vivo targets of σ54, the nitrogen assimilation σ factor, and estimate that there are 70 σ54 promoters in total. Immunoprecipitation assays have been performed to further evaluate the efficiency of our approaches. In addition, promoter consensus binding search and primer extension assay helped us to identify a new σ54 promoter carried by insB-5 in the upstream of flhDC operon. The involvement of σ54 in flagellar biosynthesis in sequenced E. coli strain MG1655 indicates a fluid gene regulation phenomenon carried by some mobile elements in bacteria genome