Hunting polymers-transforming microbes from mangrove soils
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PROVIDER: PRJEB72453 | ENA |
REPOSITORIES: ENA
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Project description:Background: Circulating polymers of alpha1-antitrypsin (α1AT) are chemo-attractant for neutrophils and contribute to inflammation in pulmonary, vascular and adipose tissues. Cellular factors affecting the intracellular itinerary of mutant polymerogenic α1AT remain obscure. Methods: Here, we report on an unbiased genome-wide CRISPR/Cas9 screen for regulators of trafficking of the polymerogenic α1AT-H334D variant. Single guide RNAs targeting genes whose inactivation enhanced accumulation of polymeric α1AT were enriched by iterative construction of CRISPR libraries based on genomic DNA from fixed cells selected for high polymer content by fluorescence-activated cell sorting. This approach bypassed the limitation to conventional enrichment schemes imposed by cell fixation. Results: Our screen identified 121 genes involved in polymer retention at false discovery rate < 0.1. From that set of genes, the pathway ‘cargo loading into COPII-coated vesicles’ was overrepresented with 16 significant genes, including two transmembrane cargo receptors, LMAN1 (ERGIG-53) and SURF4. LMAN1 and SURF4-disrupted cells displayed a secretion defect extended beyond α1AT monomers to polymers, whose low-level secretion was especially dependent on SURF4 and correlated with SURF4-α1AT-H334D physical interaction and with enhanced co-localisation of polymeric α1AT-H334D with the endoplasmic reticulum (ER). Conclusions: These findings suggest that ER cargo receptors co-ordinate intracellular progression of α1AT out of the ER and modulate the accumulation of polymeric α1AT not only by controlling the concentration of precursor monomers but also through a previously-unrecognised role in secretion of the polymers themselves.
2021-06-02 | GSE158574 | GEO
Project description:The serpinopathies are human pathologies caused by mutations that promote polymerisation and intracellular deposition of proteins of the serpin superfamily, leading to a poorly understood cell toxicity. The dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) is caused by polymerisation of the neuronal serpin neuroserpin (NS) within the endoplasmic reticulum (ER) of neurons. We have generated transgenic neural progenitor cell (NPC) cultures from mouse embryonic cerebral cortex, stably expressing the control protein GFP (green fluorescent protein), or human wild type, G392E or deltaNS. We have characterised these cells in the proliferative state and after differentiation to neurons. Our results show that G392E NS formed polymers that were mostly retained within the ER, while wild type NS was correctly secreted as a monomeric protein into the culture medium. DeltaNS was absent at steady state due to its rapid degradation, but it was easily detected upon proteasomal block. Looking at their intracellular distribution, wild type NS was found in partial co-localisation with ER and Golgi markers, while G392E NS was localised within the ER only. Furthermore, polymers of NS were detected by ELISA and immunofluorescence in neurons expressing the mutant but not the wild type protein. We used our model system to investigate which cellular pathways were activated by intracellular polymers of G392E NS by performing RNA sequencing of differentiated cells expressing G392E NS or the negative control protein GFP, and identified 747 genes with a significant upregulation (623) or downregulation (124) in G392E NS-expressing cells. We focused our attention on genes involved in the defence against oxidative stress that were up-regulated in cells expressing G392E NS. Inhibition of these defences by specific pharmacological reagents uncovered the damaging effects of NS polymers. Our results support a role for oxidative stress in the cellular toxicity underlying the neurodegenerative dementia FENIB.
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Project description:Producing the fuels and chemicals from renewable plant biomass has been thought as a feasible way for global sustainable development. However, the economical efficiency of biorefinery remains challenges. Here a cellulolytic thermophilic fungus, Myceliophthora thermophila, was constructed into a platform through metabolic engineering, which can efficiently convert lignocellulose to important bulk chemicals for polymers, four carbon 1, 4-diacids (malic and succinic acid), directly from lignocellulose without any extra enzymes addition or complicated pretreatment, with titer of over 200 g/L on cellulose and 110 g/L on plant biomass (corncob) during fed-batch fermentation. Our study represents a milestone of consolidated bioprocessing technology (CBP) and offers a new promising system for cost-effectively production of biomass-based chemicals and potentially fuels.
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Project description:Microbial community diversity and composition are important for the maintenance of mangrove ecosystem. Bacterial and archaeal community composition of the Bamenwan Mangrove Wetland soil in Hainan, China, was determined using pyrosequencing technique. Bacterial community composition presented differences among the five soil samples. Rhizobiales with higher abundance were observed in inner mangrove forest samples, while Desulfobacterales were in the seaward edge samples, and Frankiales, Gaiellales and Rhodospirillales in the landedge sample. For archaea, Crenarchaeota and Euryarchaeota dominated in five samples, but the proportion in each samples were different. Dominant archaeal community composition at the order level was similar in the seaward edge samples. The dominant archaeal clusters in the two inner mangrove forest samples were different, with Soil Crenarchaeotic Group (SCG) and Halobacteriales in sample inside of Bruguiera sexangula forest and SCG, Methanosarcinales and Marine Benthic Group B (MBGB) in sample inside of Xylocarpus mekongensis forest. The dominant archaeal clusters in land sample were unique, with Terrestrial Group and South African Gold Mine Group 1. The metabolic pathways including metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems and human diseases were all detected for bacterial and archaeal functional profiles, but metabolic potentials among five samples were different.
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Project description:Nitrate reduction is considered to be a minor microbial pathway in the oxidation of mangrove-derived organic matter due to a limited supply of nitrate in mangrove soils. At a limited availability of this electron acceptor compared to the supply of degradable carbon, nitrate ammonification is thought to be the preferential pathway of nitrate reduction. Mangrove forest mutually differ in their productivity, which may lead to different available carbon to nitrate ratios in their soil. Hence, nitrate ammonification is expected to be of more importance in high- compared to low-productive forests. The hypothesis was tested in flow-through reactors that contain undisturbed mangrove soils from high-productive Avicennia germinans and Rhizophora mangle forests in Florida and low-productive Avicennia marina forests in Saudi Arabia. Nitrate was undetectable in the soils from both regions. It was assumed that a legacy of nitrate ammonification would be reflected by a higher ammonium production from these soils upon the addition of nitrate. Unexpectedly, the soils from the low-productive forests in Saudi Arabia produced considerably more ammonium than the soils from the high-productive forests in Florida. Hence, other environmental factors than productivity must govern the selection of nitrate ammonification or denitrification. A rather intriguing observation was the 1:1 production of nitrite and ammonium during the consumption of nitrate, more or less independent from sampling region, location, sampling depth, mangrove species and from the absence or presence of additional degradable carbon. This 1:1 ratio points to a coupled production of ammonium and nitrite by one group of nitrate-reducing microorganisms. Such a production of nitrite will be hidden by the presence of active nitrite-reducing microorganisms under the nitrate-limited conditions of most mangrove forest soils.
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Project description:Dibutyl phthalate (DBP) is well known as a high-priority pollutant. This study explored the impacts of DBP on the metabolic pathways of microbes in black soils in the short term (20 days). The results showed that the microbial communities were changed in black soils with DBP. In nitrogen cycling, the abundances of the genes were elevated by DBP. DBP contamination facilitated 3'-phosphoadenosine-5'-phosphosulfate (PAPS) formation, and the gene flux of sulfate metabolism was increased. The total abundances of ABC transporters and the gene abundances of the monosaccharide-transporting ATPases MalK and MsmK were increased by DBP. The total abundance of two-component system (TCS) genes and the gene abundances of malate dehydrogenase, histidine kinase and citryl-CoA lyase were increased after DBP contamination. The total abundance of phosphotransferase system (PTS) genes and the gene abundances of phosphotransferase, Crr and BglF were raised by DBP. The increased gene abundances of ABC transporters, TCS and PTS could be the reasons for the acceleration of nitrogen, carbon and sulfate metabolism. The degrading-genes of DBP were increased markedly in soil exposed to DBP. In summary, DBP contamination altered the microbial community and enhanced the gene abundances of the carbon, nitrogen and sulfur metabolism in black soils in the short term.
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