Cationic hydrous thorium dioxide colloids – a useful tool for staining negatively charged surface matrices of bacteria for use in energy-filtered transmission electron microscopy

BACKGROUND: Synthesis of cationic hydrous thorium dioxide colloids (ca. 1.0 to 1.7 nm) has been originally described by Müller [22] and Groot [11] and these have been used by Groot to stain acidic glucosaminoglycans for ultrastructure research of different tissues by conventional transmission electron microscopy. RESULTS: Synthesis of colloidal thorium dioxide has been modified and its use as a suitable stain of acidic mucopolysaccharides and other anionic biopolymers from bacteria, either as whole mount preparations or as preembedment labels, is described. The differences in stain behavior relative to commonly used rutheniumred-lysine and Alcian Blue™ electron dense acidic stains has been investigated and its use is exemplified for Pseudomonas aeruginosa adjacent cell wall biopolymers. For the first time thorificated biopolymers, i.e. bacterial outer cell wall layers, have been analysed at the ultrastructural level with electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI), leading to excellent contrast and signal strength for these extracellular biopolymers. CONCLUSION: Application of cationic hydrous ThO(2 )colloids for tracing acidic groups of the bacterial surface and/or EPS has been shown to be rather effective by transmission electron microscopy. Because of its high electron density and its good diffusibility it stains and outlines electro-negative charges within these biopolymers. In combination with ESI, based on integrated energy-filtered electron microscopy (EFTEM) Th-densities and thus negative charge densities can be discriminated from other elemental densities, especially in environmental samples, such as biofilms

The Tat system for membrane translocation of folded proteins recruits the membrane-stabilizing Psp machinery in Escherichia coli

Tat systems transport folded proteins across energized membranes of bacteria, archaea, and plant plastids. In Escherichia coli, TatBC complexes recognize the transported proteins, and TatA complexes are recruited to facilitate transport. We achieved an abstraction of TatA from membranes without use of detergents and observed a co-purification of PspA, a mem-brane- stress response protein. The N-terminal transmembrane domain of TatA was required for the interaction. Electron microscopy displayed TatA complexes in direct contact with PspA. PspB and PspC were important for the TatA-PspA contact. The activator protein PspF was not involved in the PspATatA interaction, demonstrating that basal levels of PspA already interact with TatA. Elevated TatA levels caused membrane stress that induced a strictly PspBC- and PspF-dependent up-regulation of PspA. TatA complexes were found to destabilize membranes under these conditions. At native TatA levels, PspA deficiency clearly affected anaerobic TMAO respiratory growth, suggesting that energetic costs for transport of large Tat substrates such as TMAO reductase can become growth limiting in the absence of PspA. The physiological role of PspA recruitment to TatA may therefore be the control of membrane stress at active translocons. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

Geothermometry by Raman spectroscopy of dispersed organic matter

Raman-Spektroskopie an kohligem Material (RSCM) ist eine häufig verwendete Methode, um die maximale Temperatur der Metamorphose oder die thermische Reife von Kohlen und organikreichen Sedimenten zu bestimmen. Für die Temperaturabschätzung wurden bereits mehrere Kalibrationskurven ermittelt, jedoch wird die Übertragbarkeit dieser Kalibrationen auf andere Labore durch methodische Aspekte eingeschränkt und die Vergleichbarkeit zwischen den Laboren dadurch reduziert. Die subjektive Auswertung von Spektren, das verwendete Messsystem und die Probenheterogenität bedingen die größte Streuung der Ergebnisswerte und ein Ansatz, mit dem Ziel die Vergleichbarkeit zu erhöhen, wurde formuliert. Um die Subjektivität der spektralen Auswertung zu veringern, wurde das ’IFORS’ (Iterative Fitting Of Raman Spectra) Programm geschrieben, das die automatische, Benutzer-unabhängige Auswertung von Raman-Spektren ermöglicht. Um die Streuung aufgrund des verwendeten Messsystems zu reduzieren, wurde ein Referenzprobensatz zusammengestellt, der einen Temperaturbereich von 160 °C bis 600 °C abdeckt. Während der Probenaufbereitung wurde Resonanz-Raman- Spektroskopie mit mehreren Anregungswellenlängen an dispersen Vitriniten durchgeführt, die diagenetische bis epizonale Druck- und Temperaturbedingungen erfahren hatten, um die Gleichwertigkeit der RSCM-Methode und Vitrinitreflexion zu ermitteln. Mit Hilfe des IFORS Programms wurde der ’scaled total area’ (STA) Raman Parameter ermittelt, der das Raman Spektrum von kohligem Material präzise beschreibt. Auf Grundlage der Resonanz-Raman Daten konnte gezeigt werden, dass die Methodiken der STA-Raman Spektroskopie und Vitrinitreflexion analog zueinander sind, dass die STA-RSCM Methode gegenüber der Probenaufbereitung, insbesondere dem Polieren, robust ist, und dass die Resonanz-Raman Spektren der Vitrinite eine zweistufige molekulare Entwicklung während der Inkohlung und Graphitisierung aufzeichnen. Während der ersten Stufe, die kurz nach dem Durchschreiten des Gas-Fensters endet, wachsen vor allem lineare, polyzyklische, aromatische Kohlenwasserstoffe, während in der anschließenden zweiten Stufe kondensierte Formen von polyzyklischen, aromatischen Kohlenwasserstoffen wachsen. Um die Raman Spektren von metamorphem, kohligem Material zu beschreiben, wurde die STA-RSCM Methodik erweitert und erfolgreich gegen die Temperaturinformation des Referenzprobensatzes kalibriert, so dass ein neues, überarbeitetes RSCM-Geothermometer vorgestellt werden konnte, das über einen Temperaturbereich von 160°C bis 600°C zulässig ist. Der Referenzprobensatz steht öffentlich zur Verfügung und es wird erwartet, dass der Probensatz verbessert werden kann, wenn er um Proben aus der wissenschaftilchen Gemeinschaft erweitert wird. Wenn beide Ansätze, die STA-RSCM Methodik und der Referenzprobensatz, miteinander kombiniert werden, erhöht sich die Vergleichbarkeit zwischen den Laboren und gleichzeitig steht diese geothermometrische Methode allen Laboren zur Verfügung.Raman spectroscopy of carbonaceous material (RSCM) is frequently used to determine peak metamorphic temperature or to infer the coal rank as well as the degree of organic maturation. Several temperature calibrations exist, but methodical aspects limit the portability of these calibrations among laboratories and reduce overall comparability of the method. By identifying the subjectivity of spectral evaluation, experimental setup and sample heterogeneity as major sources of bias in the method, an outline to increase comparability could be established. To reduce the subjectivity during spectral evaluation the automated, user-input independent curve-fitting software ’IFORS’ (Iterative Fitting Of Raman Spectra) has been written. To reduce the bias due to the experimental setup, a reference sample series has been collected that covers a temperature range of 160 °C to 600 °C. Multi-wavelength resonance Raman spectroscopy was performed during sample preparation on dispersed vitrinites that experienced diagenetic to epizonal pressure and temperature conditions to infer the analogy between the RSCM-method and reflectance of dispersed organic matter. The IFORS software allowed to derive the scaled total area (STA) Raman parameter which accurately characterizes Raman spectra of carbonaceous matter. Based on the resonance Raman data it could be shown that STA-RSCM method can be used in analogue to vitrinite reflectance, that this method is robust to sample preparation, especially polishing, and that the resonance Raman spectra of vitrinite reflect a two-stage molecular evolution during coalification and graphitization. During the first stage, which ends approximately after the CM passed through the gas-window, linear polycyclic aromatic structure grow, while the second stage indicates growth of condensed polycyclic aromatic structures. The STA-RSCM method has been extended to describe the Raman spectra of metamorphic CM and was successfully calibrated to the reference sample series. Thus, a revised RSCMgeothermometer valid from 160 °C to 600 °C is proposed. The sample series is available to the public and is supposed to be extended by the scientific community to further increase the quality of the reference series. When used in combination, the STA-RSCM method and the reference sample series will improve the overall comparability among laboratories and will advance the general applicability of this geothermometric method.2018-04-3

Virus-like particle production with yeast: ultrastructural and immunocytochemical insights into Pichia pastoris producing high levels of the Hepatitis B surface antigen

<p>Abstract</p> <p>Background</p> <p>A protective immune response against Hepatitis B infection can be obtained through the administration of a single viral polypeptide, the Hepatitis B surface antigen (HBsAg). Thus, the Hepatitis B vaccine is generated through the utilization of recombinant DNA technology, preferentially by using yeast-based expression systems. However, the polypeptide needs to assemble into spherical particles, so-called virus-like particles (VLPs), to elicit the required protective immune response. So far, no clear evidence has been presented showing whether HBsAg assembles in vivo inside the yeast cell into VLPs or later in vitro during down-stream processing and purification.</p> <p>Results</p> <p>High level production of HBsAg was carried out with recombinant <it>Pichia pastoris </it>using the methanol inducible <it>AOX1 </it>expression system. The recombinant vaccine was isolated in form of VLPs after several down-stream steps from detergent-treated cell lysates. Search for the intracellular localization of the antigen using electron microscopic studies in combination with immunogold labeling revealed the presence of HBsAg in an extended endoplasmic reticulum where it was found to assemble into defined multi-layered, lamellar structures. The distance between two layers was determined as ~6 nm indicating that these lamellas represent monolayers of well-ordered HBsAg subunits. We did not find any evidence for the presence of VLPs within the endoplasmic reticulum or other parts of the yeast cell.</p> <p>Conclusions</p> <p>It is concluded that high level production and intrinsic slow HBsAg VLP assembly kinetics are leading to retention and accumulation of the antigen in the endoplasmic reticulum where it assembles at least partly into defined lamellar structures. Further transport of HBsAg to the Golgi apparatus is impaired thus leading to secretory pathway disfunction and the formation of an extended endoplasmic reticulum which bulges into irregular cloud-shaped formations. As VLPs were not found within the cells it is concluded that the VLP assembly process must take place during down-stream processing after detergent-mediated disassembly of HBsAg lamellas and subsequent reassembly of HBsAg into spherical VLPs.</p

Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: Catabolic adaptation, stress responses, and autophagic processes

Background: Pichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process.Results: The shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could proceed via the ERAD pathway and through the proteasome. However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation. During the methanol fed-batch phase, induction of vacuolar proteases (e.g. strong increase of APR1) and constitutive autophagic processes were observed. Vacuolar enclosures were mainly found around peroxisomes and not close to HBsAg deposits and, thus, were most likely provoked by peroxisomal components damaged by reactive oxygen species generated by methanol oxidation.Conclusions: In the methanol fed-batch phase P. pastoris is exposed to dual stress; stress resulting from methanol degradation and stress resulting from the production of the recombinant protein leading to the induction of oxidative stress and unfolded protein response pathways, respectively. Finally, the modest increase of methanol assimilatory enzymes compared to the strong increase of methanol dissimilatory enzymes suggests here a potential to increase methanol incorporation into biomass/product through metabolic enhancement of the methanol assimilatory pathway.DBT (India)BMB

The novel extremely acidophilic, cell-wall-deficient archaeon Cuniculiplasma divulgatum gen. nov., sp. nov. represents a new family, Cuniculiplasmataceae fam. nov., of the order Thermoplasmatales

Two novel cell-wall-less, acidophilic, mesophilic, organotrophic and facultatively anaerobic archaeal strains were isolated from acidic streamers formed on the surfaces of copper-ore-containing sulfidic deposits in south-west Spain and North Wales, UK. Cells of the strains varied from 0.1 to 2 μm in size and were pleomorphic, with a tendency to form filamentous structures. The optimal pH and temperature for growth for both strains were 1.0-1.2 and 37-40 °C, with the optimal substrates for growth being beef extract (3 g l- 1) for strain S5T and beef extract with tryptone (3 and 1 g l- 1, respectively) for strain PM4. The lipid composition was dominated by intact polar lipids consisting of a glycerol dibiphytanyl glycerol tetraether (GDGT) core attached to predominantly glycosidic polar headgroups. In addition, free GDGT and small relative amounts of intact and core diether lipids were present. Strains S5T and PM4 possessed mainly menaquinones with minor fractions of thermoplasmaquinones. The DNA G+C content was 37.3 mol% in strain S5T and 37.16 mol% for strain PM4. A similarity matrix of 16S rRNA gene sequences (identical for both strains) showed their affiliation to the order Thermoplasmatales, with 73.9-86.3 % identity with sequences from members of the order with validly published names. The average nucleotide identity between genomes of the strains determined in silico was 98.75 %, suggesting, together with the 16S rRNA gene-based phylogenetic analysis, that the strains belong to the same species. A novel family, Cuniculiplasmataceae fam. nov., genus Cuniculiplasma gen. nov. and species Cuniculiplasma divulgatum sp. nov. are proposed based on the phylogenetic, chemotaxonomic analyses and physiological properties of the two isolates, S5T and PM4 ( = JCM 30641 = VKM B-2940). The type strain of Cuniculiplasma divulgatum is S5T ( = JCM 30642T = VKM B-2941T)

Biology of archaea from a novel family Cuniculiplasmataceae (Thermoplasmata) ubiquitous in hyperacidic environments

The order Thermoplasmatales (Euryarchaeota) is represented by the most acidophilic organisms known so far that are poorly amenable to cultivation. Earlier culture-independent studies in Iron Mountain (California) pointed at an abundant archaeal group, dubbed ‘G-plasma’. We examined the genomes and physiology of two cultured representatives of a Family Cuniculiplasmataceae, recently isolated from acidic (pH 1–1.5) sites in Spain and UK that are 16S rRNA gene sequence-identical with ‘G-plasma’. Organisms had largest genomes among Thermoplasmatales (1.87–1.94 Mbp), that shared 98.7–98.8% average nucleotide identities between themselves and ‘G-plasma’ and exhibited a high genome conservation even within their genomic islands, despite their remote geographical localisations. Facultatively anaerobic heterotrophs, they possess an ancestral form of A-type terminal oxygen reductase from a distinct parental clade. The lack of complete pathways for biosynthesis of histidine, valine, leucine, isoleucine, lysine and proline pre-determines the reliance on external sources of amino acids and hence the lifestyle of these organisms as scavengers of proteinaceous compounds from surrounding microbial community members. In contrast to earlier metagenomics-based assumptions, isolates were S-layer-deficient, non-motile, non-methylotrophic and devoid of iron-oxidation despite the abundance of methylotrophy substrates and ferrous iron in situ, which underlines the essentiality of experimental validation of bioinformatic predictions

Cloud Shadows in Satellite-based Solar Irradiance Estimation: Improved Correction using EUMETSAT's Cloud Top Height Data

The estimation of solar surface irradiance at high spatio-temporal resolution from geo-stationary satellite images is a well-established technique, for example by using the Heliosat method. The method has widely reduced the need for expensive ground measurements, especially in remote regions. However, the location of cloud shadows at the ground is difficult to determine and thus a significant source of errors when either the distance from the sub-satellite point or the cloud top height (CTH) increases. Although several methods have been proposed in the literature to reduce these errors, it is still an issue. We present a novel approach to correct the cloud shadow location based on the satellite-cloud-sun geometry using the CTH maps from the EUMETSAT data archive. It uses satellite viewing angles and solar position angles to determine the correct cloud shadow location for each cloudy pixel. The method is tested on cloud index (CI) maps for the months of July, August and September 2018 derived by applying the Heliosat method on the 0.6 um visible channel images from Meteosat-8 located at 41.5°E. Convective clouds with large CTHs are frequently observed over the Indian subcontinent in these three months due to the Indian summer monsoon. The global horizontal solar irradiance (GHI) obtained from the corrected CI image is validated at two BSRN stations. The normalized root mean square error (nRMSE) is reduced from 23.2% to 20.9% for the Gurgaon station and from 15.4% to 13.9% at Tiruvallur. In general, correcting the cloud shadow location on CI map improved the accuracy of the estimated GHI. Nonetheless, the method is sensitive to the accuracy of the CTH dataset and individual cases were found for which the correction reduced the accuracy