Initial Step of Selenite Reduction via Thioredoxin for Bacterial Selenoprotein Biosynthesis

Many organisms reductively assimilate selenite to synthesize selenoprotein. Although the thioredoxin system, consisting of thioredoxin 1 (TrxA) and thioredoxin reductase with NADPH, can reduce selenite and is considered to facilitate selenite assimilation, the detailed mechanism remains obscure. Here, we show that selenite was reduced by the thioredoxin system from Pseudomonas stutzeri only in the presence of the TrxA (PsTrxA), and this system was specific to selenite among the oxyanions examined. Mutational analysis revealed that Cys33 and Cys36 residues in PsTrxA are important for selenite reduction. Free thiol-labeling assays suggested that Cys33 is more reactive than Cys36. Mass spectrometry analysis suggested that PsTrxA reduces selenite via PsTrxA-SeO intermediate formation. Furthermore, an in vivo formate dehydrogenase activity assay in Escherichia coli with a gene disruption suggested that TrxA is important for selenoprotein biosynthesis. The introduction of PsTrxA complemented the effects of TrxA disruption in E. coli cells, only when PsTrxA contained Cys33 and Cys36. Based on these results, we proposed the early steps of the link between selenite and selenoprotein biosynthesis via the formation of TrxA–selenium complexes.

Untargeted Metabolomics Investigation on Selenite Reduction to Elemental Selenium by Bacillus mycoides SeITE01

Bacillus mycoides SeITE01 is an environmental isolate that transforms the oxyanion selenite (SeO32−) into the less bioavailable elemental selenium (Se0) forming biogenic selenium nanoparticles (Bio-SeNPs). In the present study, the reduction of sodium selenite (Na2SeO3) by SeITE01 strain and the effect of SeO32− exposure on the bacterial cells was examined through untargeted metabolomics. A time-course approach was used to monitor both cell pellet and cell free spent medium (referred as intracellular and extracellular, respectively) metabolites in SeITE01 cells treated or not with SeO32−. The results show substantial biochemical changes in SeITE01 cells when exposed to SeO32−. The initial uptake of SeO32− by SeITE01 cells (3h after inoculation) shows both an increase in intracellular levels of 4-hydroxybenzoate and indole-3-acetic acid, and an extracellular accumulation of guanosine, which are metabolites involved in general stress response adapting strategies. Proactive and defensive mechanisms against SeO32− are observed between the end of lag (12h) and beginning of exponential (18h) phases. Glutathione and N-acetyl-L-cysteine are thiol compounds that would be mainly involved in Painter-type reaction for the reduction and detoxification of SeO32− to Se0. In these growth stages, thiol metabolites perform a dual role, both acting against the toxic and harmful presence of the oxyanion and as substrate or reducing sources to scavenge ROS production. Moreover, detection of the amino acids L-threonine and ornithine suggests changes in membrane lipids. Starting from stationary phase (24 and 48h), metabolites related to the formation and release of SeNPs in the extracellular environment begin to be observed. 5-hydroxyindole acetate, D-[+]-glucosamine, 4-methyl-2-oxo pentanoic acid, and ethanolamine phosphate may represent signaling strategies following SeNPs release from the cytoplasmic compartment, with consequent damage to SeITE01 cell membranes. This is also accompanied by intracellular accumulation of trans-4-hydroxyproline and L-proline, which likely represent osmoprotectant activity. The identification of these metabolites suggests the activation of signaling strategies that would protect the bacterial cells from SeO32− toxicity while it is converting into SeNPs.

Raman spectroscopic characterization of selenite reduction by the bacterium Azospirillum thiophilum in the presence of an increased concentration of sulphate

In the biomass of A. thiophilum BV-S grown in the presence of 7 mM Na2SO4, Raman spectroscopy showed a peak at 348 cm–1 (Se–S bond) in addition to a peak at 250 cm–1 (amorphous modification of Se).

Metabolic transformation of selenium (IV) by bacteria of the genus Azospirillum

Possible mechanisms of selenite reduction by bacteria of the genus Azospirillum are studied. A method is proposed for producing extracellular Se nanoparticles homogeneous by size which have been characterised by various methods.

Development of Selenium Nanoparticle Based Agriculture Sensor for Heavy Metal Toxicity Detection

The presence of heavy metals in increased concentrations in the environment has become a global environmental concern. This rapid increase in heavy metals in the environment is attributed to enhanced industrial and mining activities. Metal ions possess a lengthy half-life and property to bioaccumulate, are non-biodegradable and, thus, are a threat to the human health. A number of conventional spectroscopic and chromatographic techniques are being used for the detection of heavy metals, but these suffer from various limitations. Nano-based sensors have emerged as potential candidates for the sensitive and selective detection of heavy metals. Thus, the present study was focused on the synthesis of selenium nanoparticles (SeNPs) by using selenite-reducing bacteria in the development of a heavy metal toxicity biosensor. During the biosynthesis of selenium nanoparticles, supernatants of the overnight-grown culture were treated with Na2SeO32− and incubated for 24 h at 37 °C. The as-synthesized nanoparticles were characterized by UV–Vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) and transmission electron microscopy (TEM) analyses. XRD and TEM results confirmed the formation of SeNPs in sizes ranging from 35 to 40 nm, with face-centered cubic (FCC) structures. The bioreduction process and validation of the formation of SeNPs was further confirmed by FTIR studies. The reduction in the biosynthesis of SeNPs using bacterial metabolite due to heavy metal cytotoxicity was analyzed by the colorimetric bioassay (SE Assay). The inhibition of selenite reduction and loss of red color in the presence of heavy metals may serve as a biosensor for heavy metal toxicity analysis. Thus, this biosensor development is aimed at improving the sensitivity and specificity of analytic detection.

NAD(P)H-dependent thioredoxin-disulfide reductase TrxR is essential for tellurite and selenite reduction and resistance in Bacillus sp. Y3

ABSTRACT Microbial reduction of selenite [Se(IV)] and tellurite [Te(IV)] to Se(0) and Te(0) can function as a detoxification mechanism and serve in energy conservation. In this study, Bacillus sp. Y3 was isolated and demonstrated to have an ability of simultaneous reduction of Se(IV) and Te(IV) during aerobic cultivation, with reduction efficiencies of 100% and 90%, respectively. Proteomics analysis revealed that the putative thioredoxin disulfide reductase (TrxR) and sulfate and energy metabolic pathway proteins were significantly upregulated after the addition of Se(IV) and Te(IV). qRT-PCR also showed an increased trxR transcription level in the presence of Se(IV) and Te(IV). Compared with a wild-type Escherichia coli strain, the TrxR-overexpressed E. coli strain showed higher Se(IV) and Te(IV) resistance levels and reduction efficiencies. Additionally, the TrxR showed in vitro Se(IV) and Te(IV) reduction activities when NADPH or NADH were present. When NADPH was used as the electron donor, the optimum conditions for enzyme activities were pH 8.0 and 37°C. The Km values of Te(IV) and Se(IV) were 16.31 and 2.91 mM, and the Vmax values of Te(IV) and Se(IV) were 12.23 and 11.20 µM min−1 mg−1, respectively. The discovery of the new reductive enzyme TrxR enriches the repertoire of the bacterial Se(IV) and Te(IV) resistance and reduction mechanisms. Bacillus sp. Y3 can efficiently reduce Se(IV) and Te(IV) simultaneously. Strain Y3 provides potential applications for selenite and tellurite bioremediation. The TrxR enzyme shows high catalytic activity for reducing Se(IV) and Te(IV). The discovery of TrxR enriches the bacterial Se(IV) and Te(IV) reduction mechanisms.