Rapid Detection of the Anti-Tumor Drug Etoposide in Biological Samples by Using a Nanoporous-Gold-Based Electrochemical Sensor.

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.

Microorganisms uptake zero-valent sulfur via membrane lipid dissolution of octasulfur and intracellular solubilization as persulfide.

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.

Using the sulfide-oxidizing bacterium Geobacillus thermodenitrificans to restrict H2S release during chicken manure composting.

Hydrogen sulfide (H2S) is a toxic gas massively released during chicken manure composting. Diminishing its release requires efficient and low cost methods. In recent years, heterotrophic bacteria capable of rapid H2S oxidation have been discovered but their applications in environmental improvement are rarely reported. Herein, we investigated H2S oxidation activity of a heterotrophic thermophilic bacterium Geobacillus thermodenitrificans DSM465, which contains a H2S oxidation pathway composed by sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO). This strain rapidly oxidized H2S to sulfane sulfur and thiosulfate. The oxidation rate reached 5.73 µmol min-1·g-1 of cell dry weight. We used G. thermodenitrificans DSM465 to restrict H2S release during chicken manure composting. The H2S emission during composting process reduced by 27.5% and sulfate content in the final compost increased by 34.4%. In addition, this strain prolonged the high temperature phase by 7 days. Thus, using G. thermodenitrificans DSM465 to control H2S release was an efficient and economic method. This study provided a new strategy for making waste composting environmental friendly and shed light on perspective applications of heterotrophic H2S oxidation bacteria in environmental improvements.

Rhodobacteraceae methanethiol oxidases catalyze methanethiol degradation to produce sulfane sulfur other than hydrogen sulfide.

Methanethiol (MT) is a sulfur-containing compound produced during dimethylsulfoniopropionate (DMSP) degradation by marine bacteria. The C-S bond of MT can be cleaved by methanethiol oxidases (MTOs) to release a sulfur atom. However, the cleaving process remains unclear, and the species of sulfur product is uncertain. It has long been assumed that MTOs produce hydrogen sulfide (H2S) from MT. Herein, we studied the MTOs in the Rhodobacteraceae family-whose members are important DMSP degraders ubiquitous in marine environments. We identified 57 MTOs from 1,904 Rhodobacteraceae genomes. These MTOs were grouped into two major clusters. Cluster 1 members share three conserved cysteine residues, while cluster 2 members contain one conserved cysteine residue. We examined the products of three representative MTOs both in vitro and in vivo. All of them produced sulfane sulfur other than H2S from MT. Their conserved cysteines are substrate-binding sites in which the MTO-S-S-CH3 complex is formed. This finding clarified the sulfur product of MTOs and enlightened the MTO-catalyzing process. Moreover, this study connected DMSP degradation with sulfane sulfur metabolism, filling a critical gap in the DMSP degradation pathway and representing new knowledge in the marine sulfur cycle field. IMPORTANCE: This study overthrows a long-time assumption that methanethiol oxidases (MTOs) cleave the C-S bond of methanethiol to produce both H2S and H2O2-the former is a strong reductant and the latter is a strong oxidant. From a chemistry viewpoint, this reaction is difficult to happen. Investigations on three representative MTOs indicated that sulfane sulfur (S0) was the direct product, and no H2O2 was produced. Finally, the products of MTOs were corrected to be S0 and H2O. This finding connected dimethylsulfoniopropionate (DMSP) degradation with sulfane sulfur metabolism, filling a critical gap in the DMSP degradation pathway and representing new knowledge in the marine sulfur cycle field.

A common mechanism for rapid transfer of zero-valent sulfur between microbial cells.

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.

The Rhodanese PspE Converts Thiosulfate to Cellular Sulfane Sulfur in Escherichia coli.

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.

Investigating the debrominations of a subset of brominated flame retardants by biogenic reactive sulfur species.

Brominated flame retardants (BFRs) are persistent organic pollutants. Many bacteria are able to debrominate BFRs, but the underlying mechanism is unclear. Herein, we discovered that reactive sulfur species (RSS), which have strong reductive activity and are commonly present in bacteria, might be one of the reasons leading to such ability. Experiments performed with RSS (H2S and HSSH) and BFRs indicated that RSS can debrominate BFRs via two different mechanisms simultaneously: the substitutive debromination that generates thiol-BFRs and the reductive debromination that generates hydrogenated BFRs. Debromination reactions rapidly happened under neutral pH and ambient temperature, and the debromination degree was around 30% - 55% in one hour. Two Pseudomonas strains, Pseudomonas sp. C27 and Pseudomonas putida B6-2 both produced extracellular RSS and showed debromination activity. C27 debrominated HBCD, TBECH, and TBP by 5.4%, 17.7%, and 15.9% in two days. Whereas, B6-2 debrominated the three BFRs by 0.4%, 0.6%, and 0.3% in two days. The two bacteria produced different amounts and species of RSS, which were likely responsible for the contrasted degrees of the debromination. Our finding unveiled a novel, non-enzymatic debromination mechanism that many bacteria may possess. RSS producing bacteria have potentials to contribute to bioremediation of BFRs-polluted environments.

The Pleiotropic Regulator AdpA Regulates the Removal of Excessive Sulfane Sulfur in Streptomyces coelicolor.

Reactive sulfane sulfur (RSS), including persulfide, polysulfide, and elemental sulfur (S8), has important physiological functions, such as resisting antibiotics in Pseudomonas aeruginosa and Escherichia coli and regulating secondary metabolites production in Streptomyces spp. However, at excessive levels it is toxic. Streptomyces cells may use known enzymes to remove extra sulfane sulfur, and an unknown regulator is involved in the regulation of these enzymes. AdpA is a multi-functional transcriptional regulator universally present in Streptomyces spp. Herein, we report that AdpA was essential for Streptomyces coelicolor survival when facing external RSS stress. AdpA deletion also resulted in intracellular RSS accumulation. Thioredoxins and thioredoxin reductases were responsible for anti-RSS stress via reducing RSS to gaseous hydrogen sulfide (H2S). AdpA directly activated the expression of these enzymes at the presence of excess RSS. Since AdpA and thioredoxin systems are widely present in Streptomyces, this finding unveiled a new mechanism of anti-RSS stress by these bacteria.

A sulfide-sensor and a sulfane sulfur-sensor collectively regulate sulfur-oxidation for feather degradation by Bacillus licheniformis.

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 8th and 9th 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.

A Zero-Valent Sulfur Transporter Helps Podophyllotoxin Uptake into Bacterial Cells in the Presence of CTAB.

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.

An Optimized Transformation Protocol for Escherichia coli BW3KD with Supreme DNA Assembly Efficiency.

DNA cloning requires two steps: the assembly of recombinant DNA molecules and the transformation of the product into a host organism for replication. High efficiencies in both processes can increase the success rate. Recently, we developed an Escherichia coli BW3KD strain with higher transformation efficiency than commonly used cloning strains. Here, we further developed a simple method named TSS-HI (transformation storage solution optimized by Hannahan and Inoue method) for competent cell preparation, which combined the advantages of three common methods for operational simplicity and high transformation efficiency. When competent BW3KD cells were prepared using this developed method, the transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/µg DNA, which exceeded the levels of commercial chemically competent cells and homemade electrocompetent cells. BW3KD cells formed colonies within 7 h on lysogeny broth agar plates, quicker than the well-known fast-growing E. coli cloning strain Mach1. The competent cells worked effectively for the transformation of assembled DNA of 1 to 7 fragments in one step and promoted efficiencies of transformation or cloning with large plasmids. The cloning efficiency of BW3KD cells prepared by this method increased up to 828-fold over that of E. coli XL1-Blue MRF' cells prepared by a common method. Thus, competent cells are suitable for different cloning jobs and should help with the increased demand for DNA assembly in biological studies and biotechnology. IMPORTANCE DNA transformation is commonly used in cloning; however, high transformation efficiency becomes a limiting factor in many applications, such as the construction of CRISPR and DNA libraries, the assembly of multiple fragments, and the transformation of large plasmids. We developed a new competent cell preparation method with unmatched transformation efficiency. When the BW3KD strain, derived from Escherichia coli BW25113 cells, was prepared by this method, its transformation efficiency reached up to (7.21 ± 1.85) × 109 CFU/µg DNA, which broke the record for chemically prepared competent cells. Routine cloning could be completed in 1 day due to the high growth rate of this strain. The competent cells were shown to be highly efficient for transformation or cloning with large plasmids and for the assembly of multiple fragments. The results highlight the effectiveness of the new protocol and the usefulness of the BW3KD strain as the host.

Sulfane Sulfur Is an Intrinsic Signal for the Organic Peroxide Sensor OhrR of Pseudomonas aeruginosa.

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.

A Caveat When Using Alkyl Halides as Tagging Agents to Detect/Quantify Reactive Sulfur Species.

Using alkyl halides to tag reactive sulfur species (RSSs) (H2S, per/polysulfide, and protein-SSH) is an extensively applied approach. The underlying supposition is that, as with thiols, RSS reacts with alkyl halides via a nucleophilic substitution reaction. We found that this supposition is facing a challenge. RSS also initiates a reductive dehalogenation reaction, which generates the reduced unloaded tag and oxidized RSS. Therefore, RSS content in bio-samples might be underestimated, and its species might not be precisely determined when using alkyl halide agents for its analysis. To calculate to the extent of this underestimation, further studies are still required.

Synechococcus sp. PCC7002 Uses Peroxiredoxin to Cope with Reactive Sulfur Species Stress.

Cyanobacteria are a widely distributed group of microorganisms in the ocean, and they often need to cope with the stress of reactive sulfur species, such as sulfide and sulfane sulfur. Sulfane sulfur refers to the various forms of zero-valent sulfur, including persulfide, polysulfide, and element sulfur (S8). Although sulfane sulfur participates in signaling transduction and resistance to reactive oxygen species in cyanobacteria, it is toxic at high concentrations and induces sulfur stress, which has similar effects to oxidative stress. In this study, we report that Synechococcus sp. PCC7002 uses peroxiredoxin to cope with the stress of cellular sulfane sulfur. Synechococcus sp. PCC7002 contains six peroxiredoxins, and all were induced by S8. Peroxiredoxin I (PrxI) reduced S8 to H2S by forming a disulfide bond between residues Cys53 and Cys153 of the enzyme. A partial deletion strain of Synechococcus sp. PCC7002 with decreased copy numbers of the prxI gene was more sensitive to S8 than was the wild type. Thus, peroxiredoxin is involved in maintaining the homeostasis of cellular sulfane sulfur in cyanobacteria. Given that peroxiredoxin evolved before the occurrence of O2 on Earth, its original function could have been to cope with reactive sulfur species stress, and that function has been preserved. IMPORTANCE Cyanobacteria are the earliest microorganisms that perform oxygenic photosynthesis, which has played a key role in the evolution of life on Earth, and they are the most important primary producers in the modern oceans. The cyanobacterium Synechococcus sp. PCC7002 uses peroxiredoxin to reduce high levels of sulfane sulfur. That function is possibly the original role of peroxiredoxin, as the enzyme evolved before the appearance of O2 on Earth. The preservation of the reduction of sulfane sulfur by peroxiredoxin5-type peroxiredoxins may offer cyanobacteria an advantage in the complex environment of the modern oceans.

Optimization of a Method for Detecting Intracellular Sulfane Sulfur Levels and Evaluation of Reagents That Affect the Levels in Escherichia coli.

Sulfane sulfur is a class of compounds containing zero-valent sulfur. Most sulfane sulfur compounds are reactive and play important signaling roles. Key enzymes involved in the production and metabolism of sulfane sulfur have been characterized; however, little is known about how to change intracellular sulfane sulfur (iSS) levels. To accurately measure iSS, we optimized a previously reported method, in which reactive iSS reacts with sulfite to produce thiosulfate, a stable sulfane sulfur compound, before detection. With the improved method, several factors were tested to influence iSS in Escherichia coli. Temperature, pH, and osmotic pressure showed little effect. At commonly used concentrations, most tested oxidants, including hydrogen peroxide, tert-butyl hydroperoxide, hypochlorous acid, and diamide, did not affect iSS, but carbonyl cyanide m-chlorophenyl hydrazone increased iSS. For reductants, 10 mM dithiothreitol significantly decreased iSS, but tris(2-carboxyethyl)phosphine did not. Among different sulfur-bearing compounds, NaHS, cysteine, S2O32- and diallyl disulfide increased iSS, of which only S2O32- did not inhibit E. coli growth at 10 mM or less. Thus, with the improved method, we have identified reagents that may be used to change iSS in E. coli and other organisms, providing tools to further study the physiological functions of iSS.

Rhodaneses minimize the accumulation of cellular sulfane sulfur to avoid disulfide stress during sulfide oxidation in bacteria.

Heterotrophic bacteria and human mitochondria often use sulfide: quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO) to oxidize sulfide to sulfite and thiosulfate. Bioinformatic analysis showed that the genes encoding RHOD domains were widely presented in annotated sqr-pdo operons and grouped into three types: fused with an SQR domain, fused with a PDO domain, and dissociated proteins. Biochemical evidence suggests that RHODs facilitate the formation of thiosulfate and promote the reaction between inorganic polysulfide and glutathione to produce glutathione polysulfide. However, the physiological roles of RHODs during sulfide oxidation by SQR and PDO could only be tested in an RHOD-free host. To test this, 8 genes encoding RHOD domains in Escherichia coli MG1655 were deleted to produce E. coli RHOD-8K. The sqrCp and pdoCp genes from Cupriavidus pinatubonensis JMP134 were cloned into E. coli RHOD-8K. SQRCp contains a fused RHOD domain at the N-terminus. When the fused RHOD domain of SQRCp was inactivated, the cells oxidized sulfide into increased thiosulfate with the accumulation of cellular sulfane sulfur in comparison with cells containing the intact sqrCp and pdoCp. The complementation of dissociated DUF442 minimized the accumulation of cellular sulfane sulfur and reduced the production of thiosulfate. Further analysis showed that the fused DUF442 domain modulated the activity of SQRCp and prevented it from directly passing the produced sulfane sulfur to GSH. Whereas, the dissociated DUF442 enhanced the PDOCp activity by several folds. Both DUF442 forms minimized the accumulation of cellular sulfane sulfur, which spontaneously reacted with GSH to produce GSSG, causing disulfide stress during sulfide oxidation. Thus, RHODs may play multiple roles during sulfide oxidation.

Engineered Escherichia coli Nissle 1917 with urate oxidase and an oxygen-recycling system for hyperuricemia treatment.

Hyperuricemia is the second most prevalent metabolic disease to human health after diabetes. Only a few clinical drugs are available, and most of them have serious side effects. The human body does not have urate oxidase, and uric acid is secreted via the kidney or the intestine. Reduction through kidney secretion is often the cause of hyperuricemia. We hypothesized that the intestine secretion could be enhanced when a recombinant urate-degrading bacterium was introduced into the gut. We engineered an Escherichia coli Nissle 1917 strain with a plasmid containing a gene cassette that encoded two proteins PucL and PucM for urate metabolism from Bacillus subtilis, the urate importer YgfU and catalase KatG from E. coli, and the bacterial hemoglobin Vhb from Vitreoscilla sp. The recombinant E. coli strain effectively degraded uric acid under hypoxic conditions. A new method to induce hyperuricemia in mice was developed by intravenously injecting uric acid. The engineered Escherichia coli strain significantly lowered the serum uric acid when introduced into the gut or directly injected into the blood vessel. The results support the use of urate-degrading bacteria in the gut to treat hyperuricemia. Direct injecting bacteria into blood vessels to treat metabolic diseases is proof of concept, and it has been tried to treat solid tumors.

Sulfane Sulfur Posttranslationally Modifies the Global Regulator AdpA to Influence Actinorhodin Production and Morphological Differentiation of Streptomyces coelicolor.

The transcription factor AdpA is a key regulator controlling both secondary metabolism and morphological differentiation in Streptomyces. Due to its critical functions, its expression undergoes multilevel regulations at transcriptional, posttranscriptional, and translational levels, yet no posttranslational regulation has been reported. Sulfane sulfur, such as hydro polysulfide (HSnH, n ≥ 2) and organic polysulfide (RSnH, n ≥ 2), is common inside microorganisms, but its physiological functions are largely unclear. Here, we discovered that sulfane sulfur posttranslationally modifies AdpA in Streptomyces coelicolor via specifically reacting with Cys62 of AdpA to form a persulfide (Cys62-SSH). This modification decreases the affinity of AdpA to its self-promoter PadpA, allowing increased expression of adpA, further promoting the expression of its target genes actII-4 and wblA. ActII-4 activates actinorhodin biosynthesis, and WblA regulates morphological development. Bioinformatics analyses indicated that AdpA-Cys62 is highly conserved in Streptomyces, suggesting the prevalence of such modification in this genus. Thus, our study unveils a new type of regulation on the AdpA activity and sheds a light on how sulfane sulfur stimulates the production of antibiotics in Streptomyces. IMPORTANCEStreptomyces species produce a myriad of natural products with (potential) clinical applications. While the database of biosynthetic gene clusters is quickly expanding, their regulation mechanisms are rarely known. Sulfane sulfur species are commonly present in microorganisms with unclear functions. Here, we discovered that sulfane sulfur increases actinorhodin (ACT) production in S. coelicolor. The underlying mechanism is that sulfane sulfur specifically reacts with AdpA, a global transcription factor controlling both ACT gene cluster and morphological differentiation-related genes, to form sulfhydrated AdpA. This modification changes the dynamics of AdpA-controlled gene networks and leads to high expression of ACT biosynthetic genes. Given the wide prevalence of AdpA and sulfane sulfur in Streptomyces, this mechanism may represent a common regulating pattern of all AdpA-controlled biosynthetic pathways. Thus, this finding provides a new strategy for mining and activating valuable biosynthetic gene clusters.

Escherichia coli BW25113 Competent Cells Prepared Using a Simple Chemical Method Have Unmatched Transformation and Cloning Efficiencies.

Escherichia coli recA - strains are usually used for cloning to prevent insert instability via RecA-dependent recombination. Here, we report that E. coli BW25113 (recA +) competent cells prepared by using a previously reported transformation and storage solution (TSS) had 100-fold or higher transformation efficiency than the commonly used E. coli cloning strains, including XL1-Blue MRF'. The cloning success rates with E. coli BW25113 were 440 to 1,267-fold higher than those with E. coli XL1-Blue MRF' when several inserts were assembled into four vectors by using a simple DNA assembly method. The difference was in part due to RecA, as the recA deletion in E. coli BW25113 reduced the transformation efficiency by 16 folds and cloning success rate by about 10 folds. However, the transformation efficiency and the cloning success rate of the recA deletion mutant of E. coli BW25113 are still 12- and >48-fold higher than those of E. coli XL1-Blue MRF', which is a commonly used cloning strain. The cloned inserts with different lengths of homologous sequences were assembled into four vectors and transformed into E. coli BW25113, and they were stably maintained in BW25113. Thus, we recommend using E. coli BW25113 for efficient cloning and DNA assembly.

Elemental Sulfur Inhibits Yeast Growth via Producing Toxic Sulfide and Causing Disulfide Stress.

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.