Mol Plant Microbe Interact. 2013 Jun 7

Carbonell A, Maliogka VI, Pérez JD, Salvador Esteban B, San León D, Garcia JA, Simón-Mateo C.

PPV-D and PPV-R are two isolates from strain D of Plum pox virus (PPV) that differ in host specificity. Previous analyses of chimeras originating from PPV-R and PPV-D suggested that the N-terminus of the coat protein (CP) includes host specific pathogenicity determinants.

Here, these determinants were mapped precisely by analyzing the infectivity in herbaceous and woody species of chimeras containing a fragment of the 3’ region of PPV-D –including the region coding for the CP– in a PPV-R backbone. These chimeras were not infectious in Prunus persica, but systemically infected Nicotiana clevelandii and Nicotiana benthamiana when specific amino acids (aa) were modified or deleted in a short 30-aa region of the N-terminus of the CP. Most of these mutations did not reduce PPV fitness in Prunus although others impaired systemic infection in this host.

We propose a model in which the N-terminus of the CP, highly relevant for virus systemic movement, is targeted by a host defense mechanism in Nicotiana. Mutations in this short region allow PPV to overcome the defense response in this host but can compromise the efficiency of PPV systemic movement in other hosts such as Prunus.

Environ Microbiol Rep. 2012 Jun;4(3):335-41

Tamames J, de la Peña S, de Lorenzo V.

In any metagenomic project, the coverage obtained for each particular species depends on its abundance. This makes it difficult to determine a priori the amount of DNA sequencing necessary to obtain a high coverage for the dominant genomes in an environment.

To aid the design of metagenomic sequencing projects, we have developed COVER, a web-based tool that allows the estimation of the coverage achieved for each species in an environmental sample. COVER uses a set of 16S rRNA sequences to produce an estimate of the number of operational taxonomic units (OTUs) in the sample, provides a taxonomic assignment for them, estimates their genome sizes and, most critically, corrects for the number of unobserved OTUs. COVER then calculates the amount of sequencing needed to achieve a given goal. Our tests and simulations indicate that the results obtained through COVER are in very good agreement with the experimental results.

Environ Microbiol Rep. 2013 Apr;5(2):291-300

Sevilla E, Silva-Jiménez H, Duque E, Krell T, Rojo F.

Sensor kinases play a key role in sensing and responding to environmental and physiological signals in bacteria. In this study we characterized a previously unknown orphan hybrid sensor kinase from Pseudomonas putida, which is conserved in several Pseudomonads.

Inactivation of the gene coding for this sensor kinase, which we have named HskA, modified the expression of at least 85 genes in cells growing in a complete medium. HskA showed a strong influence on the composition of the electron transport chain. In cells growing exponentially in a complete medium, the absence of HskA led to a significant reduction in the expression of the genes coding for the bc1 complex and for the CIO and Cbb3-1 terminal oxidases. In stationary phase cells, however, lack of HskA caused a higher expression of the Cyo terminal oxidase and a lower expression of the Aa3 terminal oxidase. The HskA polypeptide shows two PAS (signal-sensing) domains, a transmitter domain containing the invariant phosphorylatable histidine and an ATP binding site, and a receiver domain containing the conserved aspartate capable of transphosphorylation, but lacks an Hpt module. It is therefore a hybrid sensor kinase. Phosphorylation assays showed that purified HskA undergoes autophosphorylation in the presence of ATP.

Plant Cell. 2013 Jun 14

Sauer M, Delgadillo MO, Zouhar J, Reynolds GD, Pennington JG, Jiang L, Liljegren SJ, Stierhof YD, De Jaeger G, Otegui MS, Bednarek SY, Rojo E.

Many soluble proteins transit through the trans-Golgi network (TGN) and the prevacuolar compartment (PVC) en route to the vacuole, but our mechanistic understanding of this vectorial trafficking step in plants is limited. In particular, it is unknown whether clathrin-coated vesicles (CCVs) participate in this transport step.

Through a screen for modified transport to the vacuole (mtv) mutants that secrete the vacuolar protein VAC2, we identified MTV1, which encodes an EPSIN N-TERMINAL HOMOLOGY protein, and MTV4, which encodes the ADP ribosylation factor GTPase-activating protein NEVERSHED/AGD5. MTV1 and NEV/AGD5 have overlapping expression patterns and interact genetically to transport vacuolar cargo and promote plant growth, but they have no apparent roles in protein secretion or endocytosis. MTV1 and NEV/AGD5 colocalize with clathrin at the TGN and are incorporated into CCVs. Importantly, mtv1 nev/agd5 double mutants show altered subcellular distribution of CCV cargo exported from the TGN. Moreover, MTV1 binds clathrin in vitro, and NEV/AGD5 associates in vivo with clathrin, directly linking these proteins to CCV formation.

These results indicate that MTV1 and NEV/AGD5 are key effectors for CCV-mediated trafficking of vacuolar proteins from the TGN to the PVC in plants.

PLoS One. 2013 Jun 20;8(6):e65986

Goñi-Moreno A, Amos M, de la Cruz F.

Recent efforts in synthetic biology have focussed on the implementation of logical functions within living cells. One aim is to facilitate both internal "re-programming" and external control of cells, with potential applications in a wide range of domains. However, fundamental limitations on the degree to which single cells may be re-engineered have led to a growth of interest in multicellular systems, in which a "computation" is distributed over a number of different cell types, in a manner analogous to modern computer networks. Within this model, individual cell type perform specific sub-tasks, the results of which are then communicated to other cell types for further processing. The manner in which outputs are communicated is therefore of great significance to the overall success of such a scheme. Previous experiments in distributed cellular computation have used global communication schemes, such as quorum sensing (QS), to implement the "wiring" between cell types. While useful, this method lacks specificity, and limits the amount of information that may be transferred at any one time. We propose an alternative scheme, based on specific cell-cell conjugation. This mechanism allows for the direct transfer of genetic information between bacteria, via circular DNA strands known as plasmids.

We design a multi-cellular population that is able to compute, in a distributed fashion, a Boolean XOR function. Through this, we describe a general scheme for distributed logic that works by mixing different strains in a single population; this constitutes an important advantage of our novel approach. Importantly, the amount of genetic information exchanged through conjugation is significantly higher than the amount possible through QS-based communication. We provide full computational modelling and simulation results, using deterministic, stochastic and spatially-explicit methods. These simulations explore the behaviour of one possible conjugation-wired cellular computing system under different conditions, and provide baseline information for future laboratory implementations.

Immunity. 2013 Jun 27;38(6):1176-86

Del Fresno C, Soulat D, Roth S, Blazek K, Udalova I, Sancho D, Ruland J, Ardavín C.

Type I interferon (IFN) is crucial during infection through its antiviral properties and by coordinating the immunocompetent cells involved in antiviral or antibacterial immunity. Type I IFN (IFN-α and IFN-β) is produced after virus or bacteria recognition by cytosolic receptors or membrane-bound TLR receptors following the activation of the transcription factors IRF3 or IRF7. IFN-β production after fungal infection was recently reported, although the underlying mechanism remains controversial.

Here we describe that IFN-β production by dendritic cells (DCs) induced by Candida albicans is largely dependent on Dectin-1- and Dectin-2-mediated signaling. Dectin-1-induced IFN-β production required the tyrosine kinase Syk and the transcription factor IRF5. Type I IFN receptor-deficient mice had a lower survival after C. albicans infection, paralleled by defective renal neutrophil infiltration. IFN-β production by renal infiltrating leukocytes was severely reduced in C. albicans-infected mice with Syk-deficient DCs. These data indicate that Dectin-induced IFN-β production by renal DCs is crucial for defense against C. albicans infection.

Cell Rep. 2013 Jul 2. pii: S2211-1247(13)00292-1

Gatchalian J, Fütterer A, Rothbart SB, Tong Q, Rincon-Arano H, Sánchez de Diego A, Groudine M, Strahl BD, Martínez-A C, van Wely KH, Kutateladze TG.

Death Inducer Obliterator 3 (Dido3) is implicated in the maintenance of stem cell genomic stability and tumorigenesis. Here, we show that Dido3 regulates the expression of stemness genes in embryonic stem cells through its plant homeodomain (PHD) finger.

Binding of Dido3 PHD to histone H3K4me3 is disrupted by threonine phosphorylation that triggers Dido3 translocation from chromatin to the mitotic spindle. The crystal structure of Dido3 PHD in complex with H3K4me3 reveals an atypical aromatic-cage-like binding site that contains a histidine residue. Biochemical, structural, and mutational analyses of the binding mechanism identified the determinants of specificity and affinity and explained the inability of homologous PHF3 to bind H3K4me3. Together, our findings reveal a link between the transcriptional control in embryonic development and regulation of cell division.

PLoS One. 2013 Jun 27;8(6):e66894

García-Arriaza J, Arnáez P, Gómez CE, Sorzano CO, Esteban M.

Poxvirus vector Modified Vaccinia Virus Ankara (MVA) expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (termed MVA-B) is a promising HIV/AIDS vaccine candidate, as confirmed from results obtained in a prophylactic phase I clinical trial in humans. To improve the immunogenicity elicited by MVA-B, we have generated and characterized the innate immune sensing and the in vivo immunogenicity profile of a vector with a double deletion in two vaccinia virus (VACV) genes (C6L and K7R) coding for inhibitors of interferon (IFN) signaling pathways. The innate immune signals elicited by MVA-B deletion mutants (MVA-B ΔC6L and MVA-B ΔC6L/K7R) in human macrophages and monocyte-derived dendritic cells (moDCs) showed an up-regulation of the expression of IFN-β, IFN-α/β-inducible genes, TNF-α, and other cytokines and chemokines. A DNA prime/MVA boost immunization protocol in mice revealed that these MVA-B deletion mutants were able to improve the magnitude and quality of HIV-1-specific CD4+ and CD8+ T cell adaptive and memory immune responses, which were mostly mediated by CD8+ T cells of an effector phenotype, with MVA-B ΔC6L/K7R being the most immunogenic virus recombinant. CD4+ T cell responses were mainly directed against Env, while GPN-specific CD8+ T cell responses were induced preferentially by the MVA-B deletion mutants. Furthermore, antibody levels to Env in the memory phase were slightly enhanced by the MVA-B deletion mutants compared to the parental MVA-B.

These findings revealed that double deletion of VACV genes that act blocking intracellularly the IFN signaling pathway confers an immunological benefit, inducing innate immune responses and increases in the magnitude, quality and durability of the HIV-1-specific T cell immune responses. Our observations highlighted the immunomodulatory role of the VACV genes C6L and K7R, and that targeting common pathways, like IRF3/IFN-β signaling, could be a general strategy to improve the immunogenicity of poxvirus-based vaccine candidates.

PLoS One. 2013 Jun 14;8(6):e65526

Sanz-Sánchez L, Risco C.

Inside cells, viruses build specialized compartments for replication and morphogenesis. We observed that virus release associates with specific structures found on the surface of mammalian cells. Cultured adherent cells were infected with a bunyavirus and processed for oriented sectioning and transmission electron microscopy. Imaging of cell basal regions showed sophisticated multilamellar structures (MLS) and extracellular filament bundles with attached viruses.

Correlative light and electron microscopy confirmed that both MLS and filaments proliferated during the maximum egress of new viruses. MLS dimensions and structure were reminiscent of those reported for the nanostructures on gecko fingertips, which are responsible for the extraordinary attachment capacity of these lizards. As infected cells with MLS were more resistant to detachment than control cells, we propose an adhesive function for these structures, which would compensate for the loss of adherence during release of new virus progeny.

Produciéndose cerca de 10.000 alteraciones diarias en el genoma de cada célula, el trabajo que hacen proteínas reparadoras del ADN como las que estudia en su laboratorio del Centro Nacional de Biotecnología del CSIC (CNB) el grupo dirigido por el biofísico Fernando Moreno-Herrero es tremendamente importante.

Junto a bioquímicos de la Universidad de Bristol, han publicado en la revista PNAS el mecanismo de exploración o escaneo que utiliza la proteína AddAB en el proceso de reparación de un corte de la doble hélice de ADN. Como nos explica Moreno-Herrero, reparar los daños en esencial ya que su acumulación puede acabar provocando "la muerte celular o incluso degenerar en una célula cancerígena".

Con anterioridad, el grupo del CNB había demostrado que para que la proteína AddAB recorriera el cromosoma separando las dos hebras necesita estar unida a unas secuencias del genoma llamadas Chi. Ahora además, utilizando la técnica de pinzas magnéticas, han sido capaces de estudiar molécula a molécula cómo recorre el ADN, a qué velocidad y hasta las pausas que hacen. Sus resultados muestran que las características de la translocación a lo largo del ADN dependen del contenido en citosina y guanina y de la presencia de las secuencias Chi.

Desde el CNB, la investigadora postdoctoral Carolina Carrasco nos explica que en su estudio han podido ver "que cuando la proteína encuentra una verdadera secuencia Chi, realiza una pausa en esta posición después de la cual reinicia nuevamente su trayectoria". Y es que cuando AddAB se encuentra con secuencias parecidas a Chi, también se detiene en un intento fallido de reconocer estas pseudo-Chi, siendo la duración de las pausas en estos casos menores.

Basándose en la capacidad de dicha proteína de reconocer específicamente las secuencias Chi, los investigadores han propuesto un modelo mecánico para explicar el origen de ambas pausas. La forma de la pausa y el aparente cambio de velocidad después de la misma actúan cómo un filtro selectivo cuando se encuentra ante una secuencia Chi genuina.

• Carrasco C, Gilhooly NS, Dillingham MS, Moreno-Herrero F. On the mechanism of recombination hotspot scanning during double-stranded DNA break resection. PNAS June 24, 2013.