Naphthalene Degradation and Incorporation of Naphthalene-Derived Carbon into Biomass by the Thermophile Bacillus thermoleovorans

The thermophilic aerobic bacterium Bacillus thermoleovorans Hamburg 2 grows at 60°C on naphthalene as the sole source of carbon and energy. In batch cultures, an effective substrate degradation was observed. The carbon balance, including naphthalene, metabolites, biomass, and CO₂, was determined by the application of [1-¹³C]naphthalene. The incorporation of naphthalene-derived carbon into the bulk biomass as well as into specified biomass fractions such as fatty acids and amino acids was confirmed by coupled gas chromatography-mass spectrometry (GC-MS) and isotope analyses. Metabolites were characterized by GC-MS; the established structures allow tracing the degradation pathway under thermophilic conditions. Apart from typical metabolites of naphthalene degradation known from mesophiles, intermediates such as 2,3-dihydroxynaphthalene, 2-carboxycinnamic acid, and phthalic and benzoic acid were identified for the pathway of this bacterium. These compounds indicate that naphthalene degradation by the thermophilic B. thermoleovorans differs from the known pathways found for mesophilic bacteria.     

Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells

1. Download : Download high-res image (192KB)
2. Download : Download full-size image

     

Expression of the Dominant-Negative Tail of Myosin Va Enhances Exocytosis of Large Dense Core Vesicles in Neurons

Regulated exocytosis of secretory vesicles is a fundamental process in neurotransmission and the release of hormones and growth factors. The F-actin-binding motor protein myosin Va was recently shown to be involved in exocytosis of peptide-containing large dense core vesicles of neuroendocrine cells. It has not previously been discussed whether it plays a similar role in neurons. We performed live-cell imaging of cultured hippocampal neurons to measure the exocytosis of large dense core vesicles containing fluorescently labelled neuropeptide Y. To address the role of myosin Va in this process, neurons were transfected with the dominant-negative tail domain of myosin Va (myosinVa-tail). Under control conditions, about 0.75% of the labelled large dense core vesicles underwent exocytosis during 5 min of stimulation. This value was doubled to 1.80% of the vesicles when myosinVa-tail was expressed. Depolymerization of F-actin using latrunculin B resulted in a similar increase in exocytosis in both control and myosinVa-tail expressing cells. Interestingly, the increase in exocytosis caused by myosinVa-tail expression was completely abolished in the presence of KN-62, an inhibitor of calcium–calmodulin-dependent kinase II. We suggest that myosinVa-tail causes the liberation of large dense core vesicles from the actin cytoskeleton, leading to an increase in exocytosis in the cultured hippocampal neurons. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Beobachtungen über das Befalls-“decline„-Phänomen beim Auftreten von Fußkrankheiten in Monokulturen

In comparative experiments for detecting apple chlorotic leaf spot virus (ApCLSV) in apple cultivars and wild apple varieties using graft indexing the procedure of applying the woody indicator clone Malus pumila MILL. Comparing R 12740 7A and the DAS‐ELISA technique, the reliability of the ELISA in ApCLSV detection in fruit‐bearing cultivars proved to be slightly better than that of R 12740 7A. In certain taxa of wild apple varieties, however, the reliability of the graft indexing procedure in detecting ApCLSV proved to be much better than that of ELISA. The investigation was carried out with 864 trees belonging to 16 apple cultivars and 24 wild apple varieties, respectively.     

Novel clades of soil biphenyl degraders revealed by integrating isotope probing,multi-omics,and single-cell analyses

Most microorganisms in the biosphere remain uncultured and poorly characterized. Although the surge in genome sequences has enabled insights into the genetic and metabolic properties of uncultured microorganisms, their physiology and ecological roles cannot be determined without direct probing of their activities in natural habitats. Here we employed an experimental framework coupling genome reconstruction and activity assays to characterize the largely uncultured microorganisms responsible for aerobic biodegradation of biphenyl as a proxy for a large class of environmental pollutants, polychlorinated biphenyls. We used ¹³C-labeled biphenyl in contaminated soils and traced the flow of pollutant-derived carbon into active cells using single-cell analyses and protein–stable isotope probing. The detection of ¹³C-enriched proteins linked biphenyl biodegradation to the uncultured Alphaproteobacteria clade {"type":"entrez-protein","attrs":{"text":"UBA11222","term_id":"2096922005"}}UBA11222, which we found to host a distinctive biphenyl dioxygenase gene widely retrieved from contaminated environments. The same approach indicated the capacity of Azoarcus species to oxidize biphenyl and suggested similar metabolic abilities for species of Rugosibacter. Biphenyl oxidation would thus represent formerly unrecognized ecological functions of both genera. The quantitative role of these microorganisms in pollutant degradation was resolved using single-cell-based uptake measurements. Our strategy advances our understanding of microbially mediated biodegradation processes and has general application potential for elucidating the ecological roles of uncultured microorganisms in their natural habitats.Subject terms: Microbial ecology, Biogeochemistry, Biogeochemistry, Soil microbiology, Biogeochemistry     

Genome and proteome analyses show the gaseous alkane degrader Desulfosarcina sp. strain BuS5 as an extreme metabolic specialist

The metabolic potential of the sulfate-reducing bacterium Desulfosarcina sp. strain BuS5, currently the only pure culture able to oxidize the volatile alkanes propane and butane without oxygen, was investigated via genomics, proteomics and physiology assays. Complete genome sequencing revealed that strain BuS5 encodes a single alkyl-succinate synthase, an enzyme which apparently initiates oxidation of both propane and butane. The formed alkyl-succinates are oxidized to CO₂ via beta oxidation and the oxidative Wood–Ljungdahl pathways as shown by proteogenomics analyses. Strain BuS5 conserves energy via the canonical sulfate reduction pathway and electron bifurcation. An ability to utilize long-chain fatty acids, mannose and oligopeptides, suggested by automated annotation pipelines, was not supported by physiology assays and in-depth analyses of the corresponding genetic systems. Consistently, comparative genomics revealed a streamlined BuS5 genome with a remarkable paucity of catabolic modules. These results establish strain BuS5 as an exceptional metabolic specialist, able to grow only with propane and butane, for which we propose the name Desulfosarcina aeriophaga BuS5. This highly restrictive lifestyle, most likely the result of habitat-driven evolutionary gene loss, may provide D. aeriophaga BuS5 a competitive edge in sediments impacted by natural gas seeps. Etymology: Desulfosarcina aeriophaga, aério (Greek): gas; phágos (Greek): eater; D. aeriophaga: a gas eating or gas feeding Desulfosarcina.     

Assimilation of benzene carbon through multiple trophic levels traced by different stable isotope probing methodologies

The flow of benzene carbon along a food chain consisting of bacteria and eukaryotes, including larvae (Diptera: Chironomidae), was evaluated by total lipid fatty acids (TLFAs)-, amino acid- and protein-stable isotope probing (SIP). A coconut-fibre textile, colonized by a benzene-degrading biofilm, was sampled in a system established for the remediation of benzene, toluene, ethylbenzene and xylenes (BTEX)-polluted groundwater and incubated with (12)C- and [(13)C(6)]-benzene (>99 at.%) in a batch-scale experiment for 2-8 days. After 8 days, Chironomus sp. larvae were added to study carbon flow to higher trophic levels. Gas chromatography-combustion-isotope ratio monitoring mass spectrometry of TLFA showed increased isotope ratios in the (13)C-benzene-incubated biofilm. A higher (13)C-enrichment was observed in TLFAs, indicative of Gram-negative bacteria than for Gram-positive. Fatty acid indicators of eukaryotes showed significant (13)C-incorporation, but to a lower extent than bacterial indicators. Fatty acids extracted from larvae feeding on (13)C-biofilm reached an isotopic ratio of 1.55 at.%, illustrating that the larvae feed, to some extent, on labelled biomass. No (13)C-incorporation was detectable in larval proteins after their separation by sodium-dodecyl sulphate-polyacrylamide gel electrophoresis and analysis by nano-liquid-chromatography-mass spectrometry. The flow of benzene-derived carbon could be traced in a food web consisting of bacteria and eukaryotes.     

Multi-level communication of human retinal pigment epithelial cells via tunneling nanotubes

Background

Tunneling nanotubes (TNTs) may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE) cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis.

Methodology and Principal Findings

Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca²⁺ imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells.

Conclusions and Significance

Our observations indicate that human RPE cell line ARPE-19 cells communicate by tunneling nanotubes and can support different types of intercellular traffic.     

Formation of bound residues during microbial degradation of [14C]anthracene in soil

Carbon partitioning and residue formation during microbial degradation of polycyclic aromatic hydrocarbons (PAH) in soil and soil-compost mixtures were examined by using [14C]anthracenes labeled at different positions. In native soil 43.8% of [9-14C]anthracene was mineralized by the autochthonous microflora and 45.4% was transformed into bound residues within 176 days. Addition of compost increased the metabolism (67.2% of the anthracene was mineralized) and decreased the residue formation (20. 7% of the anthracene was transformed). Thus, the higher organic carbon content after compost was added did not increase the level of residue formation. [14C]anthracene labeled at position 1,2,3,4,4a,5a was metabolized more rapidly and resulted in formation of higher levels of residues (28.5%) by the soil-compost mixture than [14C]anthracene radiolabeled at position C-9 (20.7%). Two phases of residue formation were observed in the experiments. In the first phase the original compound was sequestered in the soil, as indicated by its limited extractability. In the second phase metabolites were incorporated into humic substances after microbial degradation of the PAH (biogenic residue formation). PAH metabolites undergo oxidative coupling to phenolic compounds to form nonhydrolyzable humic substance-like macromolecules. We found indications that monomeric educts are coupled by C-C- or either bonds. Hydrolyzable ester bonds or sorption of the parent compounds plays a minor role in residue formation. Moreover, experiments performed with 14CO2 revealed that residues may arise from CO2 in the soil in amounts typical for anthracene biodegradation. The extent of residue formation depends on the metabolic capacity of the soil microflora and the characteristics of the soil. The position of the 14C label is another important factor which controls mineralization and residue formation from metabolized compounds.