A Cytosolic Reductase Pathway is Required for Efficient N-Glycosylation of an STT3B-Dependent Acceptor Site

N-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here we use an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could also be mimicked by the addition of a membrane impermeable reducing agent. The identified hypoglycosylated acceptor site is adjacent to a cysteine involved in a short range disulfide, which has been shown to be dependent on the STT3B-containing oligosaccharyl transferase. We also show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A and STT3B-dependent glycosylation. Our results provide further insight into the important role of the ER redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.

Transcriptional Profiling of Myceliophthora thermophila on Galactose and Metabolic Engineering for Improved Galactose Utilization

Efficient biological conversion of all sugars from lignocellulosic biomass is necessary for the cost-effective production of biofuels and commodity chemicals. Galactose is one of the most abundant sugar in many hemicelluloses, and it will be important to capture this carbon for an efficient bioconversion process of plant biomass. Thermophilic fungus Myceliophthora thermophila has been used as a cell factory to produce biochemicals directly from renewable polysaccharides. In this study, we draw out the two native galactose utilization pathways, including the Leloir pathway and oxido-reductive pathway, and identify the significance and contribution of them, through transcriptional profiling analysis of M. thermophila and its mutants on galactose. We find that galactokinase was necessary for galactose transporter expression, and disruption of galK resulted in decreased galactose utilization. Through metabolic engineering, both galactokinase deletion and galactose transporter overexpression can activate internal the oxido-reductive pathway and improve the consumption rate of galactose. Finally, the heterologous galactose-degradation pathway, De Ley–Doudoroff (DLD) pathway, was successfully integrated into M. thermophila, and the consumption rate of galactose in the engineered strain was increased by 57%. Our study focuses on metabolic engineering for accelerating galactose utilization in a thermophilic fungus that will be beneficial for the rational design of fungal strains to produce biofuels and biochemicals from a variety of feedstocks with abundant galactose.

Role of the high-affinity reductive iron acquisition pathway of Candida albicans in prostaglandin E2 production, virulence, and interaction with Pseudomonas aeruginosa

Components of the iron reductive pathway of Candida albicans have been implicated in the production of prostaglandin E2 (PGE2) and virulence. However, it is unknown whether other components of this pathway influence PGE2. We investigated the role of the iron reductive pathway of C. albicans in biofilm formation, PGE2 production, and virulence in Caenorhabditis elegans. Additionally, as the co-occurrence of C. albicans and Pseudomonas aeruginosa in host tissues is frequent and involves competition for host-associated iron, we examined the effects of this interaction. Deletion of multicopper oxidase gene, FET99, and iron permease genes, FTH1 and FTH2, affected biofilm metabolic activity, and for the FTH2 mutant, also biofilm morphology. Deletion of CCC1 (vacuolar iron transporter) and CCC2 (P-type ATPase copper importer) also influenced biofilm morphology. For PGE2 production, deletion of FET99, FTH1, FTH2, CCC1, and CCC2 caused a significant reduction by monomicrobial biofilms, while FTH2deletion caused the highest reduction in polymicrobial biofilms. URA3 positive mutants of FET99 and FTH2 demonstrated attenuated virulence in C. elegans, potentially due to the inability of mutants to form hyphae in vivo. Deductively, the role of the iron reductive pathway in PGE2 synthesis is indirect, possibly due to their role in iron homeostasis. Lay Summary Iron uptake is vital for disease-causing microbes like Candida albicans. Using strains deficient in some iron-uptake genes, we show that iron-uptake genes, especially FET99 and FTH2, play a role in biofilm formation, prostaglandin production, and virulence in the nematode infection model.

Photocatalytic reductive defluorination of perfluorooctanoic acid in water under visible light irradiation: the role of electron donor

Visible light-driven defluorination of PFOA was achieved via a photo-reductive pathway by using Pt–Bi2O4 as a photocatalyst and KI as an electron donor.

Development of Ratiometric Fluorescence Sensors Based on CdSe/ZnS Quantum Dots for the Detection of Hydrogen Peroxide

In this study, carboxyl group functionalized-CdSe/ZnS quantum dots (QDs) and aminofluorescein (AF)-encapsulated polymer particles were synthesized and immobilized to a sol–gel mixture of glycidoxypropyl trimethoxysilane (GPTMS) and aminopropyl trimethoxysilane (APTMS) for the fabrication of a hydrogen peroxide-sensing membrane. CdSe/ZnS QDs were used for the redox reaction of hydrogen peroxide (H2O2) via a reductive pathway by transferring electrons to the acceptor that led to fluorescence quenching of QDs, while AF was used as a reference dye. Herein, the ratiometric fluorescence intensity of CdSe/ZnS QDs and AF was proportional to the concentration of hydrogen peroxide. The fluorescence membrane (i.e., QD–AF membrane) could detect hydrogen peroxide in linear detection ranges from 0.1 to 1.0 mM with a detection limit (LOD) of 0.016 mM and from 1.0 to 10 mM with an LOD of 0.058 mM. The sensitivity of the QD–AF membrane was increased by immobilizing horseradish peroxidase (HRP) over the surface of the QD–AF membrane (i.e., HRP–QD–AF membrane). The HRP–QD–AF membrane had an LOD of 0.011 mM for 0.1–1 mM H2O2 and an LOD of 0.068 mM for 1–10 mM H2O2. It showed higher sensitivity than the QD–AF membrane only, although both membranes had good selectivity. The HRP–QD–AF membrane could be applied to determine the concentration of hydrogen peroxide in wastewater, while the QD–AF membrane could be employed for the detection of α-ketobutyrate.

The Mammalian Cytosolic Thioredoxin Reductase Pathway Acts via a Membrane Protein to Reduce ER-localised Proteins

SummaryFolding of proteins entering the mammalian secretory pathway requires the insertion of the correct disulfide bonds. Disulfide formation involves both an oxidative pathway for their insertion and a reductive pathway to remove incorrectly formed disulfides. Reduction of these disulfides is critical for correct folding and degradation of misfolded proteins. Previously, we showed that the reductive pathway is driven by NADPH generated in the cytosol. Here, by reconstituting the pathway using purified proteins and ER microsomal membranes, we demonstrate that the thioredoxin reductase system provides the minimal cytosolic components required for reducing proteins within the ER lumen. In particular, saturation of the pathway and its protease sensitivity demonstrates the requirement for a membrane protein to shuttle electrons from the cytosol to the ER lumen. These results provide compelling evidence for the critical role of the cytosol in regulating ER redox homeostasis to ensure correct protein folding and to facilitate the degradation of misfolded ER proteins.

Complete Genome Sequence of Escherichia coli BE104, an MC4100 Derivative Lacking the Methionine Reductive Pathway

In this announcement, we present the complete annotated genome sequence of an Escherichia coli MC4100 mutant strain, BE104. This strain has several methionine sulfoxide reductase gene deletions, making it ideal for studying enzymes that alter the redox state of methionine.

Biodegradation of 3,5-dinitrosalicylic acid by Phanerochaete chrysosporium

Despite intensive efforts put on prevention of environment pollution by nitroaromatic compounds, these xenobiotics have not been eliminated from the biosphere. The physicochemical properties make nitroaromatics extremely recalcitrant to biodegradation. Therefore, microbial degraders of these pollutants are sought after. This paper reports preliminary results of the study on degradation of 3,5-dinitrosalicylic acid (DNS) by a basidiomycetous fungus Phanerochaete chrysosporium under stationary conditions in a culture medium containing 0.05–0.5% v/v of DNS. The results obtained suggest that the fungus degrades DNS through the reductive pathway.

Aerobic Transformation of 2,4-Dinitrotoluene byEscherichia coliand Its Implications for the Detection of Trace Explosives

ABSTRACTDNT (2,4-dinitrotoluene), a volatile impurity in military-grade 2,4,6-trinitrotoluene (TNT)-based explosives, is a potential tracer for the detection of buried landmines and other explosive devices. We have previously described anEscherichia colibioreporter strain engineered to detect traces of DNT and have demonstrated that theyqjFgene promoter, the sensing element of this bioreporter, is induced not by DNT but by at least one of its transformation products. In the present study, we have characterized the initial stages of DNT biotransformation inE. coli, have identified the key metabolic products in this reductive pathway, and demonstrate that the main DNT metabolite that inducesyqjFis 2,4,5-trihydroxytoluene. We further show thatE. colicannot utilize DNT as a sole carbon or nitrogen source and propose that this compound is metabolized in order to neutralize its toxicity to the cells.IMPORTANCEThe information provided in this article sheds new light both on the microbial biodegradability of nitroaromatic compounds and on the metabolic capabilities ofE. coli. By doing so, it also clarifies the pathway leading to the previously unexplained induction of theE. coli yqjFgene by 2,4-dinitrotoluene, an impurity that accompanies 2,4,6-trinitrotoluene (TNT)-based explosives. Our improved understanding of these processes will serve to molecularly enhance the performance of a previously described microbial bioreporter of buried landmines and other explosive devices, in which theyqjFgene promoter serves as the sensing element.