Bacteriophage GC1, a novel tectivirus infecting gluconobacter cerinus, an acetic acid bacterium associated with wine-making

The Gluconobacter phage GC1 is a novel member of the Tectiviridae family isolated from a juice sample collected during dry white wine making. The bacteriophage infects Gluconobacter cerinus, an acetic acid bacterium which represents a spoilage microorganism during wine making, mainly because it is able to produce ethyl alcohol and transform it into acetic acid. Transmission electron microscopy revealed tail-less icosahedral particles with a diameter of similar to 78 nm. The linear double-stranded DNA genome of GC1 (16,523 base pairs) contains terminal inverted repeats and carries 36 open reading frames, only a handful of which could be functionally annotated. These encode for the key proteins involved in DNA replication (protein-primed family B DNA polymerase) as well as in virion structure and assembly (major capsid protein, genome packaging ATPase (adenosine triphosphatase) and several minor capsid proteins). GC1 is the first tectivirus infecting an alphaproteobacterial host and is thus far the only temperate tectivirus of gram-negative bacteria. Based on distinctive sequence and life-style features, we propose that GC1 represents a new genus within the Tectiviridae, which we tentatively named Gammatectivirus. Furthermore, GC1 helps to bridge the gap in the sequence space between alphatectiviruses and betatectiviruses

Effect of microwave treatment for wine microbial stabilisation: Potential use of a Weibullian mathematical model

The aim of this research was to investigate the efficiency of microwaves (MW) for wine microbiological stabilization and thereby assess the potential application of a Weibullian mathematical model in wine industry. The study focused on (i) the influence of treatment time (come-up time and temperature maintenance) and temperature on a simple wine-like matrix, as well as (ii) the specific resistance of yeast and bacteria selected according to their representativeness in red wines (A. aceti ATCC_15973, S. cerevisiae FX10, B. bruxellensis CRBO_L0619 and AWRI_1499), and finally (iii) the potential usefulness of a new mathematical model adapted to heat treatment to evaluate strain sensitivity. This research aimed to show that microwave treatment decreases the main wine-associated microorganism populations and that the reduction in viable counts mainly depends on the MW treatment temperature. Complete microbiological stabilisation was obtained under a temperature of 50 °C a very short treatment time, except in the case of A. aceti ATCC_15973. Furthermore, the Weibull parameters analyses showed that heat inactivation a wine microorganism studied did not follow first-order kinetics, indicating that we should change our way of studying thermal death curves. The findings from this study suggest that MW could be useful for the wine industry as a complement or an alternative to sulphite utilisation

Int J Food Microbiol.

Oenophages have so far been mostly isolated from red wines under malolactic fermentation (MLF), and correspond to temperate or ex-temperate phages of Oenococcus oeni. Their genomes are clustered into 4 integrase gene sequence groups, which are also related to the chromosomal integration site. Our aims were to survey the occurrence of oenophages in a broader and more diverse collection of samples than those previously explored. Active phages were isolated from 33 out of 166 samples, which mostly originated from must and MLF. Seventy one phage lysates were produced and 30% were assigned to a novel group with unusual genomic characteristics, called unk. All unk members produced similar RAPD and DNA restriction patterns, were negative by PCR for the signature sequences previously identified in the integrase and endolysin genes of oenophages, and lacked any BamHI restriction site in their genome. The data support that development of additional and novel signature genes for assessing oenophage diversity is now required

Physical, anatomical, and biochemical composition of skins cell walls from two grapevine cultivars (Vitis vinifera) of Champagne region related to their susceptibility to Botrytis cinerea during ripening

International audienceThis work investigated the structural, biochemical, and molecular characteristics of grape skin cell wall during ripening, related to susceptibility to Botrytis cinerea. The comparative study between the two main grape cultivars in Champagne region, Pinot noir and Chardonnay, quantified: (1) the maturity and physical profile of grape skin; (2) the morphological characteristics; (3) soluble pectic polysaccharides located in grape skin cell walls; and (4) the gene expression of the two main degrading enzymes (VvPME1 and VvPG1) and PME activity. During the maturation period, the grape skins of the two cultivars appear different in their structure and composition. Chardonnay is characterized by higher relative humidity (RH) and level of VvPG1 expression, lower disease incidence and penetrometry values, and thicker cell walls than Pinot noir skins. Thus, the cell wall composition is sufficiently different between grape varieties from the same area to allow their discrimination and could be used to better manage the harvest date

Relationship Between Hg Speciation and Hg Methylation/Demethylation Processes in the Sulfate-Reducing Bacterium Pseudodesulfovibrio hydrargyri: Evidences From HERFD-XANES and Nano-XRF

International audienceMicroorganisms are key players in the transformation of mercury into neurotoxic methylmercury (MeHg). Nevertheless, this mechanism and the opposite MeHg demethylation remain poorly understood. Here, we explored the impact of inorganic mercury (IHg) and MeHg concentrations from 0.05 to 50 µM on the production and degradation of MeHg in two sulfate-reducing bacteria, Pseudodesulfovibrio hydrargyri BerOc1 able to methylate and demethylate mercury and Desulfovibrio desulfuricans G200 only able to demethylate MeHg. MeHg produced by BerOc1 increased with increasing IHg concentration with a maximum attained for 5 µM, and suggested a saturation of the process. MeHg was mainly found in the supernatant suggesting its export from the cell. Hg L 3-edge High-Energy-Resolution-Fluorescence-Detected-X-ray-Absorption-Near-Edge-Structure spectroscopy (HERFD-XANES) identified MeHg produced by BerOc1 as MeHg-cysteine 2 form. A dominant tetracoordinated βHgS form was detected for BerOc1 exposed to the lowest IHg concentrations where methylation was detected. In contrast, at the highest exposure (50 µM) where Hg methylation was abolished, Hg species drastically changed suggesting a role of Hg speciation in the production of MeHg. The tetracoordinated βHgS was likely present as nano-particles as suggested by transmission electron microscopy combined to X-ray energy dispersive spectroscopy (TEM-X-EDS) and nano-X ray fluorescence (nano-XRF). When exposed to MeHg, the production of IHg, on the contrary, increased with the increase of MeHg Frontiers in Microbiology | www.frontiersin.org

Design, synthesis, and biological evaluation of a multifunctional neuropeptide-Y conjugate for selective nuclear delivery of radiolanthanides

International audienceBackground Targeting G protein-coupled receptors on the surface of cancer cells with peptide ligands is a promising concept for the selective tumor delivery of therapeutically active cargos, including radiometals for targeted radionuclide therapy (TRT). Recently, the radiolanthanide terbium-161 (Tb-161) gained significant interest for TRT application, since it decays with medium-energy beta-radiation but also emits a significant amount of conversion and Auger electrons with short tissue penetration range. The therapeutic efficiency of radiometals emitting Auger electrons, like Tb-161, can therefore be highly boosted by an additional subcellular delivery into the nucleus, in order to facilitate maximum dose deposition to the DNA. In this study, we describe the design of a multifunctional, radiolabeled neuropeptide-Y (NPY) conjugate, to address radiolanthanides to the nucleus of cells naturally overexpressing the human Y-1 receptor (hY(1)R). By using solid-phase peptide synthesis, the hY(1)R-preferring [F-7,P-34]-NPY was modified with a fatty acid, a cathepsin B-cleavable linker, followed by a nuclear localization sequence (NLS), and a DOTA chelator (compound pb12). In this proof-of-concept study, labeling was performed with either native terbium-159 (Tb-nat), as surrogate for Tb-161, or with indium-111 (In-111). Results [Tb-nat]Tb-pb12 showed a preserved high binding affinity to endogenous hY(1)R on MCF-7 cells and was able to induce receptor activation and internalization similar to the hY(1)R-preferring [F-7,P-34]-NPY. Specific internalization of the In-111-labeled conjugate into MCF-7 cells was observed, and importantly, time-dependent nuclear uptake of In-111 was demonstrated. Study of metabolic stability showed that the peptide is insufficiently stable in human plasma. This was confirmed by injection of [In-111]In-pb12 in nude mice bearing MCF-7 xenograft which showed specific uptake only at very early time point. Conclusion The multifunctional NPY conjugate with a releasable DOTA-NLS unit represents a promising concept for enhanced TRT with Auger electron-emitting radiolanthanides. Our research is now focusing on improving the reported concept with respect to the poor plasmatic stability of this promising radiopeptide

Wine phenolic compounds differently affect the host-killing activity of two lytic bacteriophages infecting the lactic acid bacterium oenococcus oeni

International audienceTo provide insights into phage-host interactions during winemaking, we assessed whether phenolic compounds modulate the phage predation of Oenococcus oeni. Centrifugal partition chromatography was used to fractionate the phenolic compounds of a model red wine. The ability of lytic oenophage OE33PA to kill its host was reduced in the presence of two collected fractions in which we identified five compounds. Three, namely, quercetin, myricetin and p-coumaric acid, significantly reduced the phage predation of O. oeni when provided as individual pure molecules, as also did other structurally related compounds such as cinnamic acid. Their presence was correlated with a reduced adsorption rate of phage OE33PA on its host. Strikingly, none of the identified compounds affected the killing activity of the distantly related lytic phage Vinitor162. OE33PA and Vinitor162 were shown to exhibit different entry mechanisms to penetrate into bacterial cells. We propose that ligand-receptor interactions that mediate phage adsorption to the cell surface are diverse in O. oeni and are subject to differential interference by phenolic compounds. Their presence did not induce any modifications in the cell surface as visualized by TEM. Interestingly, docking analyses suggest that quercetin and cinnamic acid may interact with the tail of OE33PA and compete with host recognition

Synaptic and supra-synaptic organisation of the dopaminergic projection to the striatum

Dopamine transmission is a monoaminergic system involved in reward processing and motor control. Volume transmission is thought to be the main mechanism by which monoamines modulate effector transmission though synaptic structures are scarcely described. Here, we applied a fluorescence activated synaptosome sorting workflow to dopaminergic projections to the striatum and explored cellular and molecular features of the dopaminergic synaptome. This demonstrated that dopaminergic varicosities adhere to post-synaptic membrane baring cognate receptors. We further identified a specific bond of varicosities to glutamatergic or GABAergic synapses in structures we named dopaminergic “hub synapses”. Finally, we showed that the synaptic adhesion protein SynCAM 2 is strongly expressed at dopaminergic hub synapses. Our data strongly suggest that neuromodulation frequently operates from hub-synapses on local receptors, presumably in conjunction with extra-synaptic volume transmission. We provide a new framework for the molecular exploration of dopaminergic synapses and more generally on discrete synapse populations ex-vivo

HDAC inhibition induces expression of scaffolding proteins critical for tumor progression in pediatric glioma: focus on EBP50 and IRSp53

International audienceBackground. Diffuse midline glioma (DMG) is a pediatric malignancy with poor prognosis. Most children die less than one year after diagnosis. Recently, mutations in histone H3 have been identified and are believed to be oncogenic drivers. Targeting this epigenetic abnormality using histone deacetylase (HDAC) inhibitors such as panobinostat (PS) is therefore a novel therapeutic option currently evaluated in clinical trials. Methods. BH3 profiling revealed engagement in an irreversible apoptotic process of glioma cells exposed to PS confirmed by annexin-V/propidium iodide staining. Using proteomic analysis of 3 DMG cell lines, we identified 2 proteins deregulated after PS treatment. We investigated biological effects of their downregulation by silencing RNA but also combinatory effects with PS treatment in vitro and in vivo using a chick embryo DMG model. Electron microscopy was used to validate protein localization. Results. Scaffolding proteins EBP50 and IRSp53 were upregulated by PS treatment. Reduction of these proteins in DMG cell lines leads to blockade of proliferation and migration, invasion, and an increase of apoptosis. EBP50 was found to be expressed in cytoplasm and nucleus in DMG cells, confirming known oncogenic locations of the protein. Treatment of glioma cells with PS together with genetic or chemical inhibition of EBP50 leads to more effective reduction of cell growth in vitro and in vivo. Conclusion. Our data reveal a specific relation between HDAC inhibitors and scaffolding protein deregulation which might have a potential for therapeutic intervention for cancer treatment

Characterization of a new virulent phage infecting the lactic acid bacterium Oenococcus oeni

International audiencePhi OE33PA is a new virulent siphophage infecting Oenococcus oeni that was isolated from a red wine collected in Pauillac (France). Although the phage could not lysogenize its host, a conserved sequence within the integrase genes harbored by oenophages could be detected, and corresponded to a B-type integrase. The phage host range encompassed ten out of the 38 O. oeni strains tested. One-step growth kinetics revealed latent and burst periods of 4 and 5 h, respectively, with a burst size of about 45 plaque-forming units per infected cell. The phage had a distinctive restriction profile when compared with Phi 10 MC, another B-type oenophage previously isolated in our laboratory. Incubation in wine could inactivate high-titer suspensions of FOE33PA in a short time at room temperature. However, encapsidated phage DNA could still be detected by real time PCR, and these non-infectious viruses dominated the wine samples showing that direct enumeration of phages in wine samples using the double-layer agar technique only informs about the quantity of residual infectious phages. Kinetics studies throughout the fermentation using both qPCR as well as plating should now provide reliable understanding of the phage dynamics during wine making