Montanlandschaft Oberhalbstein – prähistorische Kupferproduktion in Graubünden

Seit Jahrzehnten als prähistorisches Bergbaugebiet bekannt, war das Oberhalbstein in den vergangenen Jahren erstmals Gegenstand systematischer montanarchäologischer Forschungen. Dabei wurden nicht nur Dutzende neue Fundstellen entdeckt, sondern erstmals auch zentrale Verhüttungsbefunde wie Öfen und Röstbette ausgegraben. Zudem konnten gleich mehrere Kupferbergwerke aus der Bronze- und Eisenzeit untersucht werden – eine schweizweite Premiere! Dank ausführlicher Geländearbeiten, Materialauswertungen und Laboranalysen kann nun der gesamte «Oberhalbstein-Prozess» vom Erz bis zum Metall rekonstruiert werden

Ght Protein of Neisseria meningitidis Is Involved in the Regulation of Lipopolysaccharide Biosynthesis

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and is responsible for the barrier function of this membrane. A ght mutant of Neisseria meningitidis that showed increased sensitivity to hydrophobic toxic compounds, suggesting a breach in this permeability barrier, was previously described. Here, we assessed whether this phenotype was possibly caused by a defect in LPS transport or synthesis. The total amount of LPS appeared to be drastically reduced in a ght mutant, but the residual LPS was still detected at the cell surface, suggesting that LPS transport was not impaired. The ght mutant was rapidly overgrown by pseudorevertants that produced normal levels of LPS. Genetic analysis of these pseudorevertants revealed that the lpxC gene, which encodes a key enzyme in LPS synthesis, was fused to the promoter of the upstream-located pilE gene, resulting in severe lpxC overexpression. Analysis of phoA and lacZ gene fusions indicated that Ght is an inner membrane protein with an N-terminal membrane anchor and its bulk located in the cytoplasm, where it could potentially interact with LpxC. Cell fractionation experiments indeed indicated that Ght tethers LpxC to the membrane. We suggest that Ght regulates LPS biosynthesis by affecting the activity of LpxC. Possibly, this mechanism acts in the previously observed feedback inhibition of LPS synthesis that occurs when LPS transport is hampered

Ght Protein of Neisseria meningitidis Is Involved in the Regulation of Lipopolysaccharide Biosynthesis

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and is responsible for the barrier function of this membrane. A ght mutant of Neisseria meningitidis that showed increased sensitivity to hydrophobic toxic compounds, suggesting a breach in this permeability barrier, was previously described. Here, we assessed whether this phenotype was possibly caused by a defect in LPS transport or synthesis. The total amount of LPS appeared to be drastically reduced in a ght mutant, but the residual LPS was still detected at the cell surface, suggesting that LPS transport was not impaired. The ght mutant was rapidly overgrown by pseudorevertants that produced normal levels of LPS. Genetic analysis of these pseudorevertants revealed that the lpxC gene, which encodes a key enzyme in LPS synthesis, was fused to the promoter of the upstream-located pilE gene, resulting in severe lpxC overexpression. Analysis of phoA and lacZ gene fusions indicated that Ght is an inner membrane protein with an N-terminal membrane anchor and its bulk located in the cytoplasm, where it could potentially interact with LpxC. Cell fractionation experiments indeed indicated that Ght tethers LpxC to the membrane. We suggest that Ght regulates LPS biosynthesis by affecting the activity of LpxC. Possibly, this mechanism acts in the previously observed feedback inhibition of LPS synthesis that occurs when LPS transport is hampered

Genes Activated by Vibrio cholerae upon Exposure to Caenorhabditis elegans Reveal the Mannose-Sensitive Hemagglutinin To Be Essential for Colonization

The waterborne diarrheal disease cholera is caused by the bacterium Vibrio cholerae. The facultative human pathogen persists as a natural inhabitant in the aquatic ecosystem between outbreaks. In contrast to the human host, V. cholerae requires a different set of genes to survive in this hostile environment. For example, predatory micrograzers are commonly found in the aquatic environment and use bacteria as a nutrient source, but knowledge of the interaction between bacterivorous grazers and V. cholerae is limited. In this study, we successfully adapted a genetic reporter technology and identified more than 100 genes activated by V. cholerae upon exposure to the bacterium-grazing nematode Caenorhabditis elegans. This screen provides a first glimpse into responses and adaptational strategies of the bacterial pathogen against such natural predators. Subsequent phenotypic characterization revealed the mannose-sensitive hemagglutinin to be crucial for colonization of the worm, which causes developmental delay and growth retardation.During its life cycle, the facultative human pathogen Vibrio cholerae, which is the causative agent of the diarrheal disease cholera, needs to adapt to a variety of different conditions, such as the human host or the aquatic environment. Importantly, cholera infections originate from the aquatic reservoir where V. cholerae persists between the outbreaks. In the aquatic environment, bacteria are constantly threatened by predatory protozoa and nematodes, but our knowledge of the response pathways and adaptation strategies of V. cholerae to such stressors is limited. Using a temporally controlled reporter system of transcription, we identified more than 100 genes of V. cholerae induced upon exposure to the nematode Caenorhabditis elegans, which emerged recently as a valuable model for environmental predation during the aquatic lifestyle of V. cholerae. Besides others, we identified and validated the genes encoding the mannose-sensitive hemagglutinin (MSHA) type IV pilus to be significantly induced upon exposure to the nematode. Subsequent analyses demonstrated that the mannose-sensitive hemagglutinin is crucial for attachment of V. cholerae in the pharynx of the worm and initiation of colonization, which results in growth retardation and developmental delay of C. elegans. Thus, the surface adhesion factor MSHA could be linked to a fitness advantage of V. cholerae upon contact with bacterium-grazing nematodes

Visualization of NET formation by <i>V. cholerae</i>.

<p>Human neutrophils were stimulated for 6 h either with PMA, <i>V. cholerae</i> WT or Δ<i>dns</i>Δ<i>xds</i> mutant (MOI 40) or left untreated (unstimulated). <b>A</b>. Shown are representative immunofluorescent micrographs of DNA (DAPI, blue) and neutrophil elastase (NE, Cy3 conjugated, red) stained samples as well as <i>V. cholerae</i> expressing GFP (green). <b>B</b>. Microscopic evaluation of NET formation by human neutrophils stimulated with PMA, <i>V. cholerae</i> WT or Δ<i>dns</i>Δ<i>xds</i> mutant or left untreated (unstim). The percentage of cells undergoing NET formation was determined using ImageJ as explained in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003614#s4" target="_blank">Materials and Methods</a> section. Shown are medians with the interquartile range. At least nine images from two independent donors were analyzed for each data set. Significant differences between the data sets are marked by asterisks (<i>P</i><0.05; Kruskal-Wallis test followed by post-hoc Dunn's multiple comparisons).</p

The two extracellular nucleases of <i>V. cholerae</i> are able to degrade NETs.

<p><b>A</b>. DNA release of neutrophils was stimulated with PMA (indicated by the arrow “PMA”) and followed by incubation with the respective <i>V. cholerae</i> strain (MOI 40, time point of addition is indicated by the arrow “Vch”). Staining of DNA by the cell impermeant fluorescent DNA dye Sytox green was measured in 10 min intervals. Values are presented as percentage of DNA fluorescence compared with the Triton ×100 lysis control (100%) indicating NET formation, respectively. Shown are medians of at least six measurements out of three independent donors. <b>B</b>. Human neutrophils were stimulated with <i>V. cholerae</i> WT or Δ<i>dns</i>Δ<i>xds</i> mutant (MOI 4) in presence of the cell impermeant fluorescent DNA dye Sytox green and monitored by live cell imaging. Shown are images of the indicated time points. The complete movies are available as <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003614#ppat.1003614.s005" target="_blank">movie S1</a> (<i>V. cholerae</i> WT) and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003614#ppat.1003614.s006" target="_blank">S2</a> (Δ<i>dns</i>Δ<i>xds</i> mutant) in the supporting information.</p

<i>V. cholerae</i> stimulates ROS production and NET formation in human neutrophils.

<p><b>A–B</b>. ROS production by human neutrophils incubated with the indicated <i>V. cholerae</i> strains and MOI was measured by a luminometric assay. The y-axis shows the area under the curve representing the ROS production over 6 h. Shown are medians of at least six measurements out of three independent donors. The error bars represent the interquartile range. The ROS dynamics are available as supporting <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003614#ppat.1003614.s002" target="_blank">figure S2B and C. </a><b>C–H</b>. DNA release of neutrophils incubated with PMA or the respective <i>V. cholerae</i> strain and MOI. Untreated neutrophils served as an unstimulated control. Staining of DNA by the cell impermeant fluorescent DNA dye Sytox green was measured in 10 min intervals. Values are presented as percentage of DNA fluorescence compared with the Triton ×100 lysis control (100%) indicating NET formation, respectively. DNAse I was added after 6 h for the indicated data sets (+DNAse I). Shown are medians of at least six measurements out of three (C–F) or two (G and H) independent donors.</p

Reconstitution of isotopically labeled ribosomal protein L29 in the 50S large ribosomal subunit for solution-state and solid-state NMR.

Solid-state nuclear magnetic resonance (NMR) has recently emerged as a method of choice to study structural and dynamic properties of large biomolecular complexes at atomic resolution. Indeed, recent technological and methodological developments have enabled the study of ever more complex systems in the solid-state. However, to explore multicomponent protein complexes by NMR, specific labeling schemes need to be developed that are dependent on the biological question to be answered. We show here how to reconstitute an isotopically labeled protein within the unlabeled 50S or 70S ribosomal subunit. In particular, we focus on the 63-residue ribosomal protein L29 (~7&nbsp;kDa), which is located at the exit of the tunnel of the large 50S ribosomal subunit (~1.5&nbsp;MDa). The aim of this work is the preparation of a suitable sample to investigate allosteric conformational changes in a ribosomal protein that are induced by the nascent polypeptide chain and that trigger the interaction with different chaperones (e.g., trigger factor or SRP)

Vibrio cholerae evades neutrophil extracellular traps by the activity of two extracellular nucleases

The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation