Search Results (9)

Representatives of the colorless sulfur bacteria of the genus Beggiatoa use reduced sulfur compounds in the processes of lithotrophic growth, which is accompanied by the storage of intracellular sulfur. However, it is still unknown how the transformation of intracellular sulfur occurs in Beggiatoa representatives. Annotation of the genome of Beggiatoa leptomitoformis D-402 did not identify any genes for the oxidation or reduction of elemental sulfur. By searching BLASTP, two putative persulfide dioxygenase (PDO) homologs were found in the genome of B. leptomitoformis. In some heterotrophic prokaryotes, PDO is involved in the oxidation of sulfane sulfur. According to HPLC-MS/MS, the revealed protein was reliably detected in a culture sample grown only in the presence of endogenous sulfur and CO₂. The recombinant protein from B. leptomitoformis was active in the presence of glutathione persulfide. The crystal structure of recombinant PDO exhibited consistency with known structures of type I PDO. Thus, it was shown that B. leptomitoformis uses PDO to oxidize endogenous sulfur. Additionally, on the basis of HPLC-MS/MS, RT-qPCR, and the study of PDO reaction products, we predicted the interrelation of PDO and Sox-system function in the oxidation of endogenous sulfur in B. leptomitoformis and the connection of this process with energy metabolism. Full article

Cyanobacteria can perform both anoxygenic and oxygenic photosynthesis, a characteristic which ensured that these organisms were crucial in the evolution of the early Earth and the biosphere. Reactive oxygen species (ROS) produced in oxygenic photosynthesis and reactive sulfur species (RSS) produced in anoxygenic photosynthesis are closely related to intracellular redox equilibrium. ROS comprise superoxide anion (O₂^●−), hydrogen peroxide (H₂O₂), and hydroxyl radicals (^●OH). RSS comprise H₂S and sulfane sulfur (persulfide, polysulfide, and S₈). Although the sensing mechanism for ROS in cyanobacteria has been explored, that of RSS has not been elucidated. Here, we studied the function of the transcriptional repressor PerR in RSS sensing in Synechococcus sp. PCC7002 (PCC7002). PerR was previously reported to sense ROS; however, our results revealed that it also participated in RSS sensing. PerR repressed the expression of prxI and downregulated the tolerance of PCC7002 to polysulfide (H₂S_n). The reporter system indicated that PerR sensed H₂S_n. Cys¹²¹ of the Cys4:Zn²⁺ site, which contains four cysteines (Cys¹²¹, Cys¹²⁴, Cys¹⁶⁰, and Cys¹⁶³) bound to one zinc atom, could be modified by H₂S_n to Cys¹²¹-SSH, as a result of which the zinc atom was released from the site. Moreover, Cys¹⁹ could also be modified by polysulfide to Cys¹⁹-SSH. Thus, our results reveal that PerR, a representative of the Cys₄ zinc finger proteins, senses H₂S_n. Our findings provide a new perspective to explore the adaptation strategy of cyanobacteria in Proterozoic and contemporary sulfurization oceans. Full article

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. Full article

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. Full article

The studies concerned the expression of sulfurtransferases and cystathionine beta-synthase in six human leukemia cell lines: B cell acute lymphoblastic leukemia-B-ALL (REH cells), T cell acute lymphoblastic leukemia-T-ALL (DND-41 and MOLT-4 cells), acute myeloid leukemia—AML (MV4-11 and MOLM-14 cells), and chronic myeloid leukemia—CML (K562 cells). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were performed to determine the expression of thiosulfate sulfurtransferase, 3-mercaptopyruvate sulfurtransferase, gamma-cystathionase, and cystathionine beta-synthase on the mRNA and protein level. Interestingly, we found significant differences in the mRNA and protein levels of sulfurtransferases and cystathionine beta-synthase in the studied leukemia cells. The obtained results may contribute to elucidating the significance of the differences between the studied cells in the field of sulfur compound metabolism and finding new promising ways to inhibit the proliferation of various types of leukemic cells by modulating the activity of sulfurtransferases, cystathionine beta-synthase, and, consequently, the change of intracellular level of sulfane sulfur as well as H₂S and reactive oxygen species production. Full article

Transforming growth factor-β₁ (TGF-β₁) occurs at high levels at damage sites of vascular endothelial cell layers and regulates the functions of vascular endothelial cells. Reactive sulfur species (RSS), such as cysteine persulfide, glutathione persulfide, and hydrogen persulfide, are cytoprotective factors against electrophiles such as reactive oxygen species and heavy metals. Previously, we reported that sodium trisulfide, a sulfane sulfur donor, promotes vascular endothelial cell proliferation. The objective of the present study was to clarify the regulation and significance of RSS synthesis in vascular endothelial cells after exposure to TGF-β₁. Bovine aortic endothelial cells in a culture system were treated with TGF-β₁ to assess the expression of intracellular RSS, the effect of RSS on cell proliferation in the presence of TGF-β₁, induction of RSS-producing enzymes by TGF-β₁, and intracellular signal pathways that mediate this induction. The results suggest that TGF-β₁ increased intracellular RSS levels to modulate its inhibitory effect on proliferation. The increased production of RSS, probably high-molecular-mass RSS, was due to the induction of cystathionine γ-lyase and cystathionine β-synthase, which are RSS-producing enzymes, and the induction was mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 pathways in vascular endothelial cells. TGF-β₁ regulates vascular endothelial cell functions such as proliferation and fibrinolytic activity; intracellular high-molecular-mass RSS, which are increased by TGF-β₁, may modulate the regulation activity in vascular endothelial cells. Full article

Reactive sulfane sulfur, including persulfide and polysulfide, is a type of regular cellular component, playing an antioxidant role. Its function may be organelle-dependent; however, the shortage of probes for detecting organellar reactive sulfane sulfur has hindered further investigation. Herein, we reported a red fluorescent protein (mCherry)-based probe for specifically detecting intracellular reactive sulfane sulfur. By mutating two amino acid residues of mCherry (A150 and S151) to cysteine residues, we constructed a mCherry mutant, which reacted with reactive sulfane sulfur to form an intramolecular –S_n– bond (n ≥ 3). The bond largely decreased the intensity of 610 nm emission (excitation at 587 nm) and slightly increased the intensity of 466 nm emission (excitation at 406 nm). The 466/610 nm emission ratio was used to indicate the relative abundance of reactive sulfane sulfur. We then expressed this mutant in the cytoplasm and mitochondria of Saccharomyces cerevisiae. The 466/610 nm emission ratio revealed that mitochondria had a higher level of reactive sulfane sulfur than cytoplasm. Thus, the mCherry mutant can be used as a specific probe for detecting reactive sulfane sulfur in vivo. Full article

Hydrogen sulfide (H₂S) is an endogenously produced signaling molecule. The enzymes 3-mercaptopyruvate sulfurtransferase (MST), partly localized in mitochondria, and the inner mitochondrial membrane-associated sulfide:quinone oxidoreductase (SQR), besides being respectively involved in the synthesis and catabolism of H₂S, generate sulfane sulfur species such as persulfides and polysulfides, currently recognized as mediating some of the H₂S biological effects. Reprogramming of H₂S metabolism was reported to support cellular proliferation and energy metabolism in cancer cells. As oxidative stress is a cancer hallmark and N-acetylcysteine (NAC) was recently suggested to act as an antioxidant by increasing intracellular levels of sulfane sulfur species, here we evaluated the effect of prolonged exposure to NAC on the H₂S metabolism of SW480 colon cancer cells. Cells exposed to NAC for 24 h displayed increased expression and activity of MST and SQR. Furthermore, NAC was shown to: (i) persist at detectable levels inside the cells exposed to the drug for up to 24 h and (ii) sustain H₂S synthesis by human MST more effectively than cysteine, as shown working on the isolated recombinant enzyme. We conclude that prolonged exposure of colon cancer cells to NAC stimulates H₂S metabolism and that NAC can serve as a substrate for human MST. Full article

Hydrogen sulfide (H₂S) is the most recently established gaseous vasodilator, enzymatically produced from cysteine metabolism, involved in a number of pathophysiological processes. However, its accurate detection in vivo is critical due to its volatility and tendency to form sulfane sulfur derivatives, thus limiting the data interpretation of its biological roles. We developed new applications of the simple and rapid method to measure H₂S release in cell culture systems, based on the lead acetate strip test. This test, previously prevalently used in microbiology, was compared with the agar trap method, applied, in parallel, on both cell cultures and cell-free samples. Sulfane sulfur represents the major species derived from intracellular H₂S. Various fluorescent probes are available for quantitation of H₂S derivatives intracellularly. We present here an alternative to the classic imaging method for sulfane sulfur evaluation, running on a flow cytometer, based on SSP4 probe labeling. Flow cytometry turned out to be more direct, fully quantitative and less time-consuming compared to microscopy and more precise with respect to the fluorescence multi-plate reader assay. The new application methods for H₂S determination appear to be fully suitable for the analysis of H₂S release and sulfane sulfur content in biological samples. Full article