The Catalase-Peroxidase KatG Is Required for Virulence of Xanthomonas campestris pv. campestris in a Host Plant by Providing Protection against Low Levels of H2O2▿

Xanthomonas campestris pv. campestris katG encodes a catalase-peroxidase that has a role in protecting the bacterium against micromolar concentrations of H2O2. A knockout mutation in katG that causes loss of catalase-peroxidase activity correlates with increased susceptibility to H2O2 and a superoxide generator and is avirulent in a plant model system. katG expression is induced by oxidants in an OxyR-dependent manner

Overproduction of the cyanobacterial hydrogenase and selection of a mutant thriving on urea, as a possible step towards the future production of hydrogen coupled with water treatment.

Using a combination of various types of genetic manipulations (promoter replacement and gene cloning in replicating plasmid expression vector), we have overproduced the complex hydrogenase enzyme in the model cyanobacterium Synechocystis PCC6803. This new strain overproduces all twelve following proteins: HoxEFUYH (hydrogen production), HoxW (maturation of the HoxH subunit of hydrogenase) and HypABCDEF (assembly of the [NiFe] redox center of HoxHY hydrogenase). This strain when grown in the presence of a suitable quantities of nickel and iron used here exhibits a strong (25-fold) increase in hydrogenase activity, as compared to the WT strain growing in the standard medium. Hence, this strain can be very useful for future analyses of the cyanobacterial [NiFe] hydrogenase to determine its structure and, in turn, improve its tolerance to oxygen with the future goal of increasing hydrogen production. We also report the counterintuitive notion that lowering the activity of the Synechocystis urease can increase the photoproduction of biomass from urea-polluted waters, without decreasing hydrogenase activity. Such cyanobacterial factories with high hydrogenase activity and a healthy growth on urea constitute an important step towards the future development of an economical industrial processes coupling H2 production from solar energy and CO2, with wastewater treatment (urea depollution)

Inhibition of protein kinase C promotes dengue virus replication

International audienceBACKGROUND:Dengue virus (DENV) is a member of the Flaviviridae family, transmitted to human via mosquito. DENV infection is common in tropical areas and occasionally causes life-threatening symptoms. DENV contains a relatively short positive-stranded RNA genome, which encodes ten viral proteins. Thus, the viral life cycle is necessarily rely on or regulated by host factors.METHODS:In silico analyses in conjunction with in vitro kinase assay were used to study kinases that potentially phosphorylate DENV NS5. Potential kinase was inhibited or activated by a specific inhibitor (or siRNA), or an activator. Results of the inhibition and activation on viral entry/replication and host cell survival were examined.RESULTS:Our in silico analyses indicated that the non-structural protein 5 (NS5), especially the RNA-dependent RNA polymerase (RdRp) domain, contains conserved phosphorylation sites for protein kinase C (PKC). Phosphorylation of NS5 RdRp was further verified by PKC in vitro kinase assay. Inhibitions of PKC by a PKC-specific chemical inhibitor or siRNA suppressed NS5 phosphorylation in vivo, increased viral replication and reduced viability of the DENV-infected cells. In contrary, activation of PKC effectively suppressed intracellular viral number.CONCLUSIONS:These results indicated that PKC may act as a restricting mechanism that modulates the DENV replication and represses the viral outburst in the host cells

Exopolysaccharides protect Synechocystis against the deleterious effects of Titanium dioxide nanoparticles in natural and artificial waters

International audienceWe have studied the effect of TiO2 nanoparticles (NPs) on the model cyanobacteria Synechocystis PCC6803. We used well-characterized NPs suspensions in artificial and natural (Seine River, France) waters. We report that NPs trigger direct (cell killing) and indirect (cell sedimentation precluding the capture of light, which is crucial to photosynthesis) deleterious effects. Both toxic effects increase with NPs concentration and are exacerbated by the presence of UVAs that increase the production of Reactive Oxygen Species (hydroxyl and superoxide radicals) by TiO2 NPs. Furthermore, we compared the responses of the wild-type strain of Synechocystis, which possesses abundant exopolysaccharides surrounding the cells, to that of an EPS-depleted mutant. We show, for the first time, that the exopolysaccharides play a crucial role in Synechocystis protection against cell killing caused by TiO2 NP

Schematic representation of the <i>Synechocystis</i> strains constructed in this study.

<p><i>Synechocystis</i> cells are represented by oval shapes showing their chromosome attached to the cell membrane. The pCE-<i>hypABCDEF</i> replicating plasmid is represented by circles. The <i>hoxEFUYHW</i> and <i>hypABCDEF</i> genes and the antibiotic resistance markers are shown by large arrows pointing towards the direction of their transcription. The triangle represents the strong lambda phage pR promoter (λ<i>p</i>_R); CE for strong <u>c</u>onstitutive <u>e</u>xpression.</p

The abundance of total EPS influences the zeta potential of <i>Synechocystis</i>.

<p>(<b>A</b>) Zeta values for the WT and mutant cells harbouring either a single or a double deletion of the genes <i>sll0923</i>, <i>sll5052</i>, <i>slr1875</i> and <i>sll1581</i>, as indicated. (<b>B</b>) Histogram plots of the total amounts of EPS (CPS+RPS) of each strain. All results are expressed as means ± standard deviation of the data obtained after three biological repetitions of every assay.</p

Comparative analysis of the strains over-expressing the genes <i>hoxEFUYH</i> (CE1) or <i>hoxEFUYHW</i> (CE4; CE4u) alone, or together with the <i>hypABCDEF</i> genes (CE5; CE5u).

<p>All experiments were performed at least three times. (<b>A</b>) Typical growth of the wild type (WT; squares), CE-<i>hoxEFUYH</i> (CE1; white triangles) and CE-<i>hoxEFUYHW</i> (CE4; black squares) cells incubated under standard conditions. (<b>B</b>) Histogram plot representation of transcript abundance (measured by Real-time quantitative PCR) of the <i>hoxEFUYHW</i> genes in strains WT (small light-grey bars), CE1 (grey rectangles) and CE4 (hatched bars) mutants. (<b>C</b>) Western blot analysis of the abundance of the HoxF and HoxH proteins in WT, CE1, CE4 and CE5 cells growing in MM* medium (MM + 17 μM Fe). (<b>D</b>) Histogram plot representation of the transcript abundance (RT-qPCR) of the <i>hypABC-F</i> genes in the strains WT (light-grey rectangles), CE1 (grey) and CE5 (arrow-filled bars). (<b>E</b>) Histograms representation of the hydrogenase activities of WT (light grey), CE1 (grey), CE2 (dark grey) CE4 (light grey-hatched bars), CE5 (white arrow-filled bars), CE4u (dark grey-hatched bars) and CE5u cells (grey arrow-filled bars) growing in standard medium (MM) or MM* (MM + 17 μM Fe) supplemented with 2.5 μM NiSO₄.</p

Influence of urea on the growth of <i>Synechocystis</i> WT and mutants overexpressing the <i>hoxEFUYHW</i> genes alone (CE4) or in combination with the <i>hypABCDEF</i> genes (CE5).

<p>(<b>A</b>) Typical growth of WT cells cultivated on medium containing Ni (1 μM) and urea (2.5–20 mM) as the sole nitrogen source. (<b>B</b>) Typical growth on urea (5 mM as the sole nitrogen source) and Ni (2.5 μM) of the WT strain, and the CE4 and CE5 strain without or with (CE4u and CE5u) a mutation in <i>ureG</i>. Influence of prolonged growth on urea (5 mM as the sole nitrogen source) and Ni (2.5 μM) on the cell appearance <b>C</b>) and urease activity (<b>D</b>) of the studied strains. All experiments were performed at least three times.</p