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Herein we report the use of an environmental multimetal(loid)-resistant strain, MF05, to biosynthesize single- or multi-element nanostructures under anaerobic conditions. Inorganic nanostructure synthesis typically requires methodologies and conditions that are harsh and environmentally hazardous. Thus, green/eco-friendly procedures are desirable, where the use of microorganisms and their extracts as bionanofactories is a reliable strategy. First, MF05 was entirely sequenced and identified as an Escherichia coli -related strain with some genetic differences from the traditional BW25113. Secondly, we compared the CdS nanostructure biosynthesis by whole-cell in a design defined minimal culture medium containing sulfite as the only sulfur source to obtain sulfide reduction from a low-cost chalcogen reactant. Under anaerobic conditions, this process was greatly favored, and irregular CdS (ex. 370 nm; em. 520–530 nm) was obtained. When other chalcogenites were tested (selenite and tellurite), only spherical Se 0 and elongated Te 0 nanostructures were observed by TEM and analyzed by SEM-EDX. In addition, enzymatic-mediated chalcogenite (sulfite, selenite, and tellurite) reduction was assessed by using MF05 crude extracts in anaerobiosis; similar results for nanostructures were obtained; however Se 0 and Te 0 formation were more regular in shape and cleaner (with less background). Finally, the in vitro nanostructure biosynthesis was assessed with salts of Ag, Au, Cd, and Li alone or in combination with chalcogenites. Several single or binary nanostructures were detected. Our results showed that MF05 is a versatile anaerobic bionanofactory for different types of inorganic NS. synthesis.

The tellurium oxyanion tellurite induces oxidative stress in most microorganisms. In Escherichia coli, tellurite exposure results in high levels of oxidized proteins and membrane lipid peroxides, inactivation of oxidation-sensitive enzymes and reduced glutathione content. In this work, we show that tellurite-exposed E. coli exhibits transcriptional activation of the zwf gene, encoding glucose 6-phosphate dehydrogenase (G6PDH), which in turn results in augmented synthesis of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Increased zwf transcription under tellurite stress results mainly from reactive oxygen species (ROS) generation and not from a depletion of cellular glutathione. In addition, the observed increase of G6PDH activity was paralleled by accumulation of glucose-6-phosphate (G6P), suggesting a metabolic flux shift toward the pentose phosphate shunt. Upon zwf overexpression, bacterial cells also show increased levels of antioxidant molecules (NADPH, GSH), better-protected oxidation-sensitive enzymes and decreased amounts of oxidized proteins and membrane lipids. These results suggest that by increasing NADPH content, G6PDH plays an important role in E. coli survival under tellurite stress.

D-tagatose is a high-value, low-calorie sweetener that can be produced from dairy lactose via a two-step enzymatic route: lactose hydrolysis to galactose followed by galactose isomerization to tagatose. Here, we combined genomics, in silico structural analysis, and biochemical assays to evaluate the lactose-to-tagatose conversion potential of an Antarctic isolate, L47, identified as Ewingella americana (NCBI accession SAMN54554459). Genome mining revealed one L-arabinose isomerase gene (araA) and three β-galactosidase genes (bgaA, bglY, lacZ), an uncommon combination in a single bacterium. Recombinant AraA was produced in Escherichia coli and biochemically characterized, showing Mn2+ dependence and measurable D-galactose isomerization, reaching ~18% tagatose from 100 mM galactose after 48 h under the tested conditions. In contrast, the β-galactosidases were predominantly recovered as insoluble aggregates in E. coli; therefore, β-galactosidase activity was assessed using washed inclusion-body preparations. Under these conditions, BgaA displayed the most consistent o-NPG hydrolyzing activity, whereas BglY and LacZ did not yield reproducible activity. Overall, our results identify BgaA as the most tractable lactose-hydrolyzing candidate from L47 in the current workflow and indicate that AraA performance is the principal bottleneck toward an efficient lactose-to-tagatose process, motivating future optimization at the enzyme and process levels.

Biochemical, genetic, enzymatic and molecular approaches were used to demonstrate, for the first time, that tellurite (TeO(3) (2-)) toxicity in E. coli involves superoxide formation. This radical is derived, at least in part, from enzymatic TeO(3) (2-) reduction. This conclusion is supported by the following observations made in K(2)TeO(3)-treated E. coli BW25113: i) induction of the ibpA gene encoding for the small heat shock protein IbpA, which has been associated with resistance to superoxide, ii) increase of cytoplasmic reactive oxygen species (ROS) as determined with ROS-specific probe 2'7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA), iii) increase of carbonyl content in cellular proteins, iv) increase in the generation of thiobarbituric acid-reactive substances (TBARs), v) inactivation of oxidative stress-sensitive [Fe-S] enzymes such as aconitase, vi) increase of superoxide dismutase (SOD) activity, vii) increase of sodA, sodB and soxS mRNA transcription, and viii) generation of superoxide radical during in vitro enzymatic reduction of potassium tellurite.

The metalloid tellurite is highly toxic to microorganisms. Several mechanisms of action have been proposed, including thiol depletion and generation of hydrogen peroxide and superoxide, but none of them can fully explain its toxicity. Here we use a combination of directed evolution and chemical and biochemical approaches to demonstrate that tellurite inhibits heme biosynthesis, leading to the accumulation of intermediates of this pathway and hydroxyl radical. Unexpectedly, the development of tellurite resistance is accompanied by increased susceptibility to hydrogen peroxide. Furthermore, we show that the heme precursor 5-aminolevulinic acid, which is used as an antimicrobial agent in photodynamic therapy, potentiates tellurite toxicity. Our results define a mechanism of tellurite toxicity and warrant further research on the potential use of the combination of tellurite and 5-aminolevulinic acid in antimicrobial therapy. The mechanisms of action of the antibacterial metalloid tellurite are unclear. Here, the authors show that tellurite induces an accumulation of hydroxyl radical and intermediates of heme biosynthesis in E. coli , and that the heme precursor 5-aminolevulinic acid potentiates tellurite toxicity.

In this study, a versatile synthesis of silver nanoparticles of well-defined size by using hydrogels as a template and stabilizer of nanoparticle size is reported. The prepared hydrogels are based on polyvinyl alcohol and maleic acid as crosslinker agents. Three hydrogels with the same nature were synthesized, however, the crosslinking degree was varied. The silver nanoparticles were synthesized into each prepared hydrogel matrix achieving three significant, different-sized nanoparticles that were spherical in shape with a narrow size distribution. It is likely that the polymer network stabilized the nanoparticles. It was determined that the hydrogel network structure can control the size and shape of the nanoparticles. The hydrogel/silver nanohybrids were characterized by swelling degree, Thermal Gravimetric Analysis (TGA), Fourier Transform Infrared (FT-IR), Scanning Electron Microscopy (SEM) and Transmission Electron Microscope (TEM). Antibacterial activity against Staphylococcus aureus was evaluated, confirming antimicrobial action of the encapsulated silver nanoparticles into the hydrogels.

Tellurite (TeO32−) is a highly soluble and toxic oxyanion that inhibits the growth of Escherichia coli at concentrations as low as ~1 µg/mL. This toxicity has been primarily attributed to the generation of reactive oxygen species (ROS) during its intracellular reduction by thiol-containing molecules and NAD(P)H-dependent enzymes. However, under anaerobic conditions, E. coli exhibits significantly increased tellurite tolerance—up to 100-fold in minimal media—suggesting the involvement of additional, ROS-independent mechanisms. In this study, we combined chemical-genomic screening, untargeted metabolomics, and targeted biochemical assays to investigate the effects of tellurite under both aerobic and anaerobic conditions. Our findings reveal that tellurite perturbs amino acid and nucleotide metabolism, leading to intracellular imbalances that impair protein synthesis. Additionally, tellurite induces notable changes in membrane lipid composition, particularly in phosphatidylethanolamine derivatives, which may influence biophysical properties of the membrane, such as fluidity or curvature. This membrane remodeling could contribute to the increased resistance observed under anaerobic conditions, although direct evidence of altered membrane fluidity remains to be established. Overall, these results demonstrate that tellurite toxicity extends beyond oxidative stress, impacting central metabolic pathways and membrane-associated functions regardless of oxygen availability.

Iron is essential for bacterial survival, yet its excess can be harmful due to its role in increasing oxidative stress. Enterococcus faecalis , a common member of the human gut microbiota, must carefully balance its iron levels to survive in changing environments. Here, we investigate how E. faecalis compensates for the reduced availability of glutathione, a key antioxidant, when exposed to high iron concentrations. We discovered that E. faecalis lowers its intracellular iron levels when glutathione biosynthesis is disrupted and reprograms its metabolism to prioritize energy production, potentially to fuel stress response mechanisms under iron-induced oxidative conditions. These findings enhance our understanding of bacterial adaptation under oxidative stress and suggest that interfering with arginine metabolic pathways could represent novel strategies to combat E. faecalis infections.

Escherichia coli glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) are key enzymes of the pentose phosphate pathway, responsible for the NADPH production in cells. We investigated modification of both enzymes mediated by peroxyl radicals (ROO · ) to determine their respective susceptibilities to and mechanisms of oxidation. G6PDH and 6PGDH were incubated with AAPH (2,2′-azobis(2-methylpropionamidine)dihydrochloride), which was employed as ROO · source. The enzymatic activities of both enzymes were determined by NADPH release, with oxidative modifications examined by electrophoresis and liquid chromatography (LC) with fluorescence and mass (MS) detection. The activity of G6PDH decreased up to 62.0 ± 15.0% after 180 min incubation with 100 mM AAPH, whilst almost total inactivation of 6PGDH was determined under the same conditions. Although both proteins contain abundant Tyr (particularly 6PGDH), these residues were minimally affected by ROO · , with Trp and Met being major targets. LC–MS and in silico analysis showed that the modification sites of G6PDH are distant to the active site, consistent with a dispersed distribution of modifications, and inactivation resulting from oxidation of multiple Trp and Met residues. In contrast, the sites of oxidation detected on 6PGDH are located close to its catalytic site indicating a more localized oxidation, and a consequent high susceptibility to ROO · -mediated inactivation.

Tellurium nanoparticles (TeNPs) are emerging as valuable materials in various technological and biomedical applications due to their unique physicochemical properties. In general, TeNPs are prepared using chemical methods based on a redox reaction in which strong reducing agents are employed which are often toxic and harmful to the environment. Biological biosynthesis provides a green strategy for substituting the commonly used reducing chemical agents with microorganisms or enzymes. Among the enzymes noted as key players in microbial tellurite reduction, glutathione reductase (GR) has been identified; however, its specific role in enhancing TeNP biosynthesis has yet to be fully elucidated. In this study, we aimed to evaluate the impact of GR overexpression on TeNP production in Escherichia coli (E. coli). For this purpose, four GR enzymes from different microorganisms identified as tellurite resistant were heterogeneously expressed and purified from E. coli. The kinetic parameters for NADPH and oxidized glutathione (GSSG), the native substrates of GR, were determined to evaluate their TR activity under saturated NADPH concentrations. The GR from Pseudomonas lini strain BNF22 presented the highest catalytic efficiency for NADPH and exhibited greater TR activity. This enzyme was overexpressed in E. coli MG1655 (DE3) and cells overexpressing GR increased the yield of TeNPs in those cells, presenting an increased elemental cell tellurium composition. Our results provide valuable insights for the development of engineered E. coli as a platform for TeNP biosynthesis. Using microorganisms as a green strategy for TeNP production, the results of this study highlight the enzymatic mechanisms underlying the role of GR in the biosynthesis of TeNPs.