Cyanide and sulfite are environmentally relevant pollutants that require reliable monitoring because of their toxicity, industrial use, and potential co-occurrence in contaminated waters. However, conventional methods often involve laborious pretreatment, expensive instrumentation, and matrix interference, limiting rapid environmental analysis. Here, we report a rhodanese-coupled nanoporous gold (NPG) electrochemical platform for dual detection of cyanide and sulfite using an NPG-modified glassy carbon electrode (NPG/GCE). The NPG interface enabled sensitive sulfite oxidation with a distinct peak at +0.14 V, whereas cyanide was indirectly quantified through the Rdl2-catalyzed stoichiometric reaction between cyanide and thiosulfate. This reaction produces thiocyanate and sulfite in a 1:1 ratio, and the generated sulfite, rather than thiocyanate, was selected as the electroactive reporter. This signal-conversion strategy converted poorly electroactive cyanide into a readily detectable species. The platform showed a linear range of 0.1-2000 μM for direct sulfite detection and 0.1-1000 μM for indirect Rdl2-assisted cyanide detection based on the generated sulfite oxidation signal, with corresponding detection limits of 50.44 nM and 48.42 nM, respectively. It also exhibited good operational and Rdl2 storage stability, acceptable anti-interference performance, and satisfactory spike-recovery results in environmental water samples, with recoveries of 97.63%-110.09% for sulfite and 96.90%-108.20% for cyanide. This system integrates Rdl2 specificity with NPG-based signal amplification, offering a simple enzymatic signal-conversion strategy for electrochemical monitoring of poorly electroactive pollutants.

Thiosulfate serves as a critical intermediate in the sulfur biogeochemical cycle and is frequently oxidized by the sulfur oxidation (Sox) system to sulfate in bacteria. Cupriavidus pinatubonensis JMP134 employs a complete Sox system to oxidize S2O32- to SO42-, yet the regulatory mechanisms underlying this process remain unclear. A regulator gene located adjacent to the sox genes cluster encodes an ArsR-type transcriptional regulator (SoxR). Here we report the regulation of the sox genes by SoxR in C. pinatubonensis JMP134. The sox genes are transcribed as an operon from a promoter in the front of soxR. SoxR binds to DNA 23-bp downstream of the transcription start site, blocking the sox transcription. We present evidence that SoxR does not directly respond to S2O32- but instead reacts with reactive sulfane sulfur via Cys43 and Cys98, and this interaction is contingent upon a functional Sox system. These findings will deepen our understanding of the regulation of the Sox system in bacteria.

Iron-sulfur clusters are versatile protein cofactors involved in diverse biological processes, but their role in hydrogen sulfide/hydrosulfide (H2S/HS-) sensing remains largely unexplored. Here, we report that the Bacillus licheniformis sensor kinase NreB contains an unusual [2Fe-2S] cluster within its PAS domain. A 1.52-Å crystal structure reveals a distinct coordination geometry where three conserved cysteine residues and a non-cysteinyl sulfur ligand stabilize the cluster. Biochemical and native mass spectrometry support assignment of the predominant ligand state as -SH and show enrichment of -SSH/SSOH-like state upon sulfide exposure in the presence of O2, correlating with increased NreB kinase activity. Electron paramagnetic resonance spectroscopy shows that the cluster retains its oxidized [2Fe-2S]2+ state during sulfide-sensing. Molecular dynamics simulations further reveal transient solvent and HS- accessibility to the buried cluster, providing a physical basis for ligand entry. Here, we show that bacteria sense sulfide via a three-cysteine-coordinated Fe-S cluster with a labile sulfur ligand.

Natural bioactive compounds (NBC) have been research targets for decades due to their various usages in agriculture, industry, and more importantly, pharmaceuticals [...]

The results of protein thiols reacting with oxidants may be different in the presence or absence of glutathione (GSH). Upon exposure to oxidants, such as Cu2+ and polysulfide, the multiple drug resistant regulator MarR dimer in Escherichia coli is believed to form tetramers linked by disulfide bonds between its Cys80 thiols. We confirmed this observation in the absence of GSH; however, the MarR-Cys80 thiol was primarily glutathionylated in the presence of GSH after MarR was treated with various oxidants, including octasulfur (S8), Cu2+, H2O2, ClO-, and a NO donor. When using S8 as the oxidizing agent, we identified four pathways to induce MarR-Cys80 glutathionylation. Since E. coli contains high concentrations of GSH, MarR is likely glutathionylated instead of forming tetramers inside the cells. When E. coli was exposed to S8, cellular levels of protein glutathionylation were increased and most MarR was glutathionylated, as shown by Western blot and LC-MS analyses. The glutathionylated MarR displayed reduced affinity to its cognate operator, resulting in the expression of its repressed genes. The results highlight the need to consider the wide presence of GSH when investigating protein thiol modification.

Monitoring etoposide is important due to its wide usage in anti-tumor therapy; however, the commonly used HPLC method is expensive and often requires complicated extraction and detection procedures. Electrochemical analysis has great application prospects because of its rapid response and high specificity, sensitivity, and efficiency with low cost and high convenience. In this study, we constructed a nanoporous gold (NPG)-modified GCE for the detection of etoposide. The electrochemical oxidation of etoposide by NPG caused a sensitive current peak at +0.27 V with good reproductivity in 50 mM of phosphate buffer (pH 7.4). The relationship between etoposide concentration and peak current was linear in the range between 0.1 and 20 μM and between 20 and 150 μM, with a detection sensitivity of 681.8 μA mM−1 cm−2 and 197.2 μA mM−1 cm−2, respectively, and a limit of detection (LOD) reaching 20 nM. The electrode had a good anti-interference ability to several common anions and cations. Spiked recovery tests in serum, urine, and fermentation broth verified the excellent performance of the sensor in terms of sensitivity, reproducibility, and specificity. This may provide a promising tool for the detection of etoposide in biological samples.

Podophyllotoxin (PTOX) is naturally produced by the plant Podophyllum species. Some of its derivatives are anticancer drugs, which are produced mainly by using chemical semi-synthesis methods. Recombinant bacteria have great potential in large-scale production of the derivatives of PTOX. In addition to introducing the correct enzymes, the transportation of PTOX into the cells is an important factor, which limits its modification in the bacteria. Here, we improved the cellular uptake of PTOX into Escherichia coli with the help of the zero-valent sulfur transporter YedE1E2 in the presence of cetyltrimethylammonium bromide (CTAB). CTAB promoted the uptake of PTOX, but induced the production of reactive oxygen species. A protein complex (YedE1E2) of YedE1 and YedE2 enabled E. coli cells to resist CTAB by reducing reactive oxygen species, and YedE1E2 was a hypothetical transporter. Further investigation showed that YedE1E2 facilitated the uptake of extracellular zero-valent sulfur across the cytoplasmic membrane and the formation of glutathione persulfide (GSSH) inside the cells. The increased GSSH minimized oxidative stress. Our results indicate that YedE1E2 is a zero-valent sulfur transporter and it also facilitates CTAB-assisted uptake of PTOX by recombinant bacteria.

YCA1, the only metacaspase in Saccharomyces cerevisiae, plays important roles in the regulation of chronological lifespan, apoptosis, and cytokinesis. YCA1 has protein hydrolase activity and functions by cleaving itself and target proteins. However, there are few reports about the regulation of YCA1 activity. In this study, we observed that reactive sulfane sulfur (RSS) can inhibit the activity of YCA1. In vitro experiments demonstrated that RSS reacted with the Cys276 of YCA1, the residue central to its protein hydrolase activity, to form a persulfidation modification (protein-SSH). This modification inhibited both its self-cleavage and the cleavage of its substrate protein, BIR1. To investigate further, we constructed a low-endogenous-RSS mutant of S. cerevisiae, BY4742 Δcys3, in which the RSS-producing enzyme cystathionine-γ-lyase (CYS3) was knocked out. The activity of YCA1 was significantly increased by the deletion of CYS3. Moreover, increased YCA1 activity led to reduced chronological lifespan (CLS) and CLS-driven apoptosis. This study unveils the first endogenous factor that regulates YCA1 activity, introduces a novel mechanism of how yeast cells regulate chronological lifespan, and broadens our understanding of the multifaceted roles played by RSS.

Horizontal gene transfer has been demonstrated to be an important driver for the emergency of multidrug-resistant pathogens. Recently, a transferable gene cluster tmexCD1-toprJ1 of the resistance-nodulation-division (RND) superfamily was identified in the plasmids of animal-derived Klebsiella pneumoniae strains, with a higher efflux capacity for various drugs than the Escherichia coli AcrAB-TolC homolog system. In this study, we focused on the differences in the inner membrane pump of these two systems and identified some key residues that contribute to the robust efflux activity of the TMexCD1 system. With the aid of homologous modeling and molecular docking, eight residues from the proximal binding pocket (PBP) and nine from the distal binding pocket (DBP) were selected and subjected to site-directed mutagenesis. Several of them, such as S134, I139, D181, and A290, were shown to be important for substrate binding in the DBP region, and all residues in PBP and DBP showed certain substrate preferences. Apart from the conservative switch loop (L613-623(TMexD1)) previously identified in the E. coli AcrB (EcAcrB), a relatively unconservative loop (L665-675T(MexD1)) at the bottom of PBP was proposed as a critical element for the robust activity of TMexD1, due to variations at sites E669, G670, N673, and S674 compared to EcAcrAB, and the significantly altered efflux activity due to their mutations. The conservation and flexibility of these key factors can contribute to the evolution of the RND efflux pumps and thus serve as potential targets for developing inhibitors to block the widespread of the TMexCD1 system.

Abstract Asparagus, a vital economic contributor, is a well‐liked vegetable grown around the globe, and some secondary metabolites in its spear are beneficial to human health. Asparagus spears possess a significant quantity of nutrients and phytochemicals; however, the difference in these chemical compositions among various varieties has not been sufficiently studied. This work aimed to detect the chemical compositions of 30 varieties of asparagus and to assess them by principal component analysis (PCA). The results showed that the contents of these chemical compositions varied in varieties. Selenium (Se, 1.12–2.9 μg/100 g dry‐weight [DW]) was abundant in asparagus, with an average dry matter content of 8.25%. Free amino acids (5.60–9.98 g/100 g DW) and polyphenols (6.34–8.67 mg/g DW) were both present in high amounts, along with flavonoids (4.218–8.22 mg/g DW) and protodioscin (0.44–1.96 mg/g DW). Correlation analysis, PCA, and hierarchical cluster analysis were used to conduct a comprehensive evaluation of asparagus. Atlas, Appolo, Jinggang 111, Jingke 2, and WS‐1 were the top five varieties with comprehensive scores. This study provided valuable data for the breeding, quality improvement, processing, and utilization of asparagus varieties in the future.

Zero-valent sulfur, commonly utilized as a fertilizer or fungicide, is prevalent in various environmental contexts. Its most stable and predominant form, octasulfur (S8), plays a crucial role in microbial sulfur metabolism, either through oxidation or reduction. However, the mechanism underlying its cellular uptake remains elusive. We presented evidence that zero-valent sulfur was adsorbed to the cell surface and then dissolved into the membrane lipid layer as lipid-soluble S8 molecules, which reacted with cellular low-molecular thiols to form persulfide, e.g., glutathione persulfide (GSSH), in the cytoplasm. The process brought extracellular zero-valent sulfur into the cells. When persulfide dioxygenase is present in the cells, GSSH will be oxidized. Otherwise, GSSH will react with another glutathione (GSH) to produce glutathione disulfide (GSSG) and hydrogen sulfide (H2S). The mechanism is different from simple diffusion, as insoluble S8 becomes soluble GSSH after crossing the cytoplasmic membrane. The uptake process is limited by physical contact of insoluble zero-valent sulfur with microbial cells and the regeneration of cellular thiols. Our findings elucidate the cellular uptake mechanism of zero-valent sulfur, which provides critical information for its application in agricultural practices and the bioremediation of sulfur contaminants and heavy metals.

Hydrogen sulfide (H2S) and its oxidation product zero-valent sulfur (S0) play important roles in animals, plants, and bacteria. Inside cells, S0 exists in various forms, including polysulfide and persulfide, which are collectively referred to as sulfane sulfur. Due to the known health benefits, the donors of H2S and sulfane sulfur have been developed and tested. Among them, thiosulfate is a known H2S and sulfane sulfur donor. We have previously reported that thiosulfate is an effective sulfane sulfur donor in Escherichia coli; however, it is unclear how it converts thiosulfate to cellular sulfane sulfur. In this study, we showed that one of the various rhodaneses, PspE, in E. coli was responsible for the conversion. After the thiosulfate addition, the ΔpspE mutant did not increase cellular sulfane sulfur, but the wild type and the complemented strain ΔpspE::pspE increased cellular sulfane sulfur from about 92 μM to 220 μM and 355 μM, respectively. LC-MS analysis revealed a significant increase in glutathione persulfide (GSSH) in the wild type and the ΔpspE::pspE strain. The kinetic analysis supported that PspE was the most effective rhodanese in E. coli in converting thiosulfate to glutathione persulfide. The increased cellular sulfane sulfur alleviated the toxicity of hydrogen peroxide during E. coli growth. Although cellular thiols might reduce the increased cellular sulfane sulfur to H2S, increased H2S was not detected in the wild type. The finding that rhodanese is required to convert thiosulfate to cellular sulfane sulfur in E. coli may guide the use of thiosulfate as the donor of H2S and sulfane sulfur in human and animal tests.

Bacillus licheniformis MW3 degrades bird feathers. Feather keratin is rich in cysteine, which is metabolized to produce hazardous sulfide and sulfane sulfur. A challenge to B. licheniformis MW3 growing on feathers is to detoxify them. Here we identified a gene cluster in B. licheniformis MW3 to deal with these toxicity. The cluster contains 11 genes: the first gene yrkD encodes a repressor, the 8(th) and 9(th) genes nreB and nreC encode a two-component regulatory system, and the 10th and 11th genes encode sulfide: quinone reductase (SQR) and persulfide oxygenase (PDO). SQR and PDO collectively oxidize sulfide and sulfane sulfur to sulfite. YrkD sensed sulfane sulfur to derepress the 11 genes. The NreBC system sensed sulfide and further amplified the transcription of sqr and pdo. The two regulatory systems synergistically controlled the expression of the gene cluster, which was required for the bacterium to grow on feather. The findings highlight the necessity of removing sulfide and sulfane sulfur during feather degradation and may help with bioremediation of feather waste and sulfide pollution.

Zero-valent sulfur is accumulated in the cytoplasm of certain sulfur-oxidizing or reducing microorganisms. When these microorganisms are unable to metabolize zero-valent sulfur, they produce sulfur globules that mainly consist of octasulfur (S8), a common species of elemental sulfur. The intracellular zero-valent sulfur was easily transferred to other bacteria and the yeast Saccharomyces cerevisiae for metabolism. After eliminating all known potential mechanisms of zero-valent sulfur transfer between cells, we hypothesized and tested whether S8 was directly transferred. S8 was shown to be soluble and enriched in membrane lipids. The transfer of S8 molecules occurred between live cells, inactivated cells, and liposomes via physical contact. Low-molecular thiols, such as glutathione, reacted with S8 in the cell membranes to produce glutathione persulfide that was soluble in the cytoplasm. In the recipient cells, glutathione persulfide was either metabolized by enzymes or spontaneously reacted with another glutathione to produce hydrogen sulfide and glutathione disulfide. The new mechanism of zero-valent sulfur transfer as membrane lipid-soluble S8 molecules is common among tested microorganisms and may also occur in the environment for microorganisms to share and use zero-valent sulfur.

Despite being present in minimal amounts, vitamin B2 (VB2), vitamin C (VC), and vitamin B6 (VB6) each play indispensable roles in human metabolisms. Given that VB2, VC, and VB6 cannot be synthesized by the human body, detections of these three vitamins both in fermentation liquid where vitamins are industrially manufactured and in human serum where vitamin concentrations could be clinically controlled are of significant importance. Here, a nanoporous gold (NPAu) modified screen-printed electrode (NPAu/SPE) was fabricated to detect VB2, VC, and VB6 based on NPAu’s electro-oxidation towards vitamins. Owing to the wide separation of peak potentials among VB2, VC, and VB6, the simultaneous detection of these three vitamins was achieved by the NPAu/SPE within a potential range from −0.8 V to 0.8 V. The achieved limits of detection (LOD) for VB2, VC, and VB6 were 0.46, 6.44, and 1.92 μM, with sensitivities of 68.58, 4.77, and 15.94 μA/μM, respectively. Subsequent reliability experiments suggested that the NPAu/SPE exhibited solid anti-interference capability and repeatability. Additionally, the real-sample detection of the NPAu/SPE towards VB2, VC, and VB6 was achieved both in human serum and in fermentation liquid with comparable accuracy (the recovery rates were from 89.8% to 111.7%) as high-performance liquid chromatography (HPLC). Moreover, the portable NPAu/SPE showed comparable performance in terms of the LOD and linear dynamic range when compared to glassy carbon electrodes (GCE) limited to laboratory detection. The proposed NPAu/SPE possesses various advantageous properties including portability, easy fabrication, high sensitivity, and cost-efficiency, making it a potential candidate for clinical and industrial multi-vitamins analysis.

Sulfane sulfur, including organic persulfide and polysulfide, is a normal cellular component, and its level varies during growth. It is emerging as a signaling molecule in bacteria, regulating the gene regulator MarR in Escherichia coli, MexR in Pseudomonas aeruginosa, and MgrA of Staphylococcus aureus. They are MarR-family regulators and are often repressors for multiple antibiotic resistance genes. Here, we report that another MarR-type regulator OhrR that represses the expression of itself and a thiol peroxidase gene ohr in P. aeruginosa PAO1 also responded to sulfane sulfur. PaOhrR formed disulfide bonds between three Cys residues within a dimer after polysulfide treatment. The modification reduced its affinity to its cognate DNA binding site. An Escherichia coli reporter system, in which mKate was under the repression of OhrR, showed that PaOhrR derepressed its controlled gene when polysulfide was added, whereas the mutant PaOhrR with two Cys residues changed to Ser residues did not respond to polysulfide. The expression of the PaOhrR-repressed mKate was significantly increased when the cells enter the late log phase when cellular sulfane sulfur reached a maximum, but the mKate expression under the control of the PaOhrR-C9SC19S double mutant was not increased. Furthermore, the expression levels of ohrR and ohr in P. aeruginosa PAO1 were significantly increased when cellular sulfane sulfur was high. Thus, PaOhrR senses both exogenous and intrinsic sulfane sulfur to derepress its controlled genes. The finding also suggests that sulfane sulfur may be a common inducer of the MarR-type regulators, which may confer the bacteria to resist certain stresses without being exposed to the stresses.

Elemental sulfur is a common fungicide, but its inhibition mechanism is unclear. Here, we investigated the effects of elemental sulfur on the single-celled fungus Saccharomyces cerevisiae and showed that the inhibition was due to its function as a strong oxidant. It rapidly entered S. cerevisiae. Inside the cytoplasm, it reacted with glutathione to generate glutathione persulfide that then reacted with another glutathione to produce H2S and glutathione disulfide. H2S reversibly inhibited the oxygen consumption by the mitochondrial electron transport chain, and the accumulation of glutathione disulfide caused disulfide stress and increased reactive oxygen species in S. cerevisiae. Elemental sulfur inhibited the growth of S. cerevisiae; however, it did not kill the yeast for up to 2 h exposure. The combined action of elemental sulfur and hosts’ immune responses may lead to the demise of fungal pathogens.

Sulfur-oxidizing bacteria can oxidize hydrogen sulfide (H2S) to produce sulfur globules. Although the process is common, the pathway is unclear. In recombinant Escherichia coli and wild-type Corynebacterium vitaeruminis DSM 20294 with sulfide:quinone oxidoreductase (SQR) but no enzymes to oxidize zero valence sulfur, SQR oxidized H2S into short-chain inorganic polysulfide (H2Sn, n >= 2) and organic polysulfide (RSnH, n >= 2), which reacted with each other to form long-chain GS(n)H (n >= 2) and H2Sn before producing octasulfur (S-8), the main component of elemental sulfur. GS(n)H also reacted with glutathione (GSH) to form GSnG (n >= 2) and H2S; H2S was again oxidized by SQR. After GSH was depleted, SQR simply oxidized H2S to H2Sn, which spontaneously generated S-8. S-8 aggregated into sulfur globules in the cytoplasm. The results highlight the process of sulfide oxidation to S-8 globules in the bacterial cytoplasm and demonstrate the potential of using heterotrophic bacteria with SQR to convert toxic H2S into relatively benign S-8 globules. IMPORTANCE Our results provide evidence of H2S oxidation producing octasulfur globules via sulfide:quinone oxidoreductase (SQR) catalysis and spontaneous reactions in the bacterial cytoplasm. Since the process is an important event in geochemical cycling, a better understanding facilitates further studies and provides theoretical support for using heterotrophic bacteria with SQR to oxidize toxic H2S into sulfur globules for recovery.

Elemental sulfur and sulfite have been used to inhibit the growth of yeasts, but thiosulfate has not been reported to be toxic to yeasts. We observed that thiosulfate was more inhibitory than sulfite to Saccharomyces cerevisiae growing in a common yeast medium. At pH < 4, thiosulfate was a source of elemental sulfur and sulfurous acid, and both were highly toxic to the yeast. At pH 6, thiosulfate directly inhibited the electron transport chain in yeast mitochondria, leading to reductions in oxygen consumption, mitochondrial membrane potential and cellular ATP. Although thiosulfate was converted to sulfite and H2S by the mitochondrial rhodanese Rdl1, its toxicity was not due to H2S as the rdl1-deletion mutant that produced significantly less H2S was more sensitive to thiosulfate than the wild type. Evidence suggests that thiosulfate inhibits cytochrome c oxidase of the electron transport chain in yeast mitochondria. Thus, thiosulfate is a potential agent against yeasts.

Heterotrophic bacteria actively participate in the biogeochemical cycle of sulfur on Earth. The heterotrophic bacterium Cupriavidus pinatubonensis JMP134 contains several enzymes involved in sulfur oxidation, but how these enzymes work together to oxidize sulfide in the bacterium has not been studied. Using genedeletion and whole-cell assays, we determined that the bacterium uses sulfide:quinone oxidoreductase to oxidize sulfide to polysulfide, which is further oxidized to sulfite by persulfide dioxygenase. Sulfite spontaneously reacts with polysulfide to produce thiosulfate. The sulfur-oxidizing (Sox) system oxidizes thiosulfate to sulfate. Flavocytochrome c sulfide dehydrogenase enhances thiosulfate oxidation by the Sox system but couples with the Sox system for sulfide oxidation to sulfate in the absence of sulfide:quinone oxidoreductase. Thus, C. pinatubonensis JMP134 contains a main pathway and a contingent pathway for sulfide oxidation. IMPORTANCE We establish a new pathway of sulfide oxidation with thiosulfate as a key intermediate in Cupriavidus pinatubonensis JMP134. The bacterium mainly oxidizes sulfide by using sulfide:quinone oxidoreductase, persulfide dioxygenase, and the Sox system with thiosulfate as a key intermediate. Although the purified and reconstituted Sox system oxidizes sulfide, its rate of sulfide oxidation in C pinatubonensis JMP134 is too low to be physiologically relevant. The findings reveal how these sulfur-oxidizing enzymes participate in sulfide oxidation in a single bacterium.