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.

The pathway of recombining short homologous ends in Escherichia coli revealed by the genetic study.

The recombination of short homologous ends in Escherichia coli has been known for 30 years, and it is often used for both site-directed mutagenesis and in vivo cloning. For cloning, a plasmid and target DNA fragments were converted into linear DNA fragments with short homologous ends, which are joined via recombination inside E. coli after transformation. Here this mechanism of joining homologous ends in E. coli was determined by a linearized plasmid with short homologous ends. Two 3'-5' exonucleases ExoIII and ExoX with nonprocessive activity digested linear dsDNA to generate 5' single-strand overhangs, which annealed with each other. The polymerase activity of DNA polymerase I (Pol I) was exclusively employed to fill in the gaps. The strand displacement activity and the 5'-3' exonuclease activity of Pol I were also required, likely to generate 5' phosphate termini for subsequent ligation. Ligase A (LigA) joined the nicks to finish the process. The model involving 5' single-stranded overhangs is different from established recombination pathways that all generate 3' single-stranded overhangs. This recombination is likely common in bacteria since the involved enzymes are ubiquitous.

The Pathway of Sulfide Oxidation to Octasulfur Globules in the Cytoplasm of Aerobic Bacteria.

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 GSnH (n ≥ 2) and H2Sn before producing octasulfur (S8), the main component of elemental sulfur. GSnH 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 S8. S8 aggregated into sulfur globules in the cytoplasm. The results highlight the process of sulfide oxidation to S8 globules in the bacterial cytoplasm and demonstrate the potential of using heterotrophic bacteria with SQR to convert toxic H2S into relatively benign S8 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.

Sulfane Sulfur is an intrinsic signal activating MexR-regulated antibiotic resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO1, an opportunistic human pathogen, deploys several strategies to resist antibiotics. It uses multidrug efflux pumps, including the MexAB-OprM pump, for antibiotic resistance, and it also produces hydrogen sulfide (H2 S) that provides some defense against antibiotics. MexR functions as a transcriptional repressor of the mexAB-oprM operon. MexR responds to oxidative stresses caused by antibiotic exposure, and it also displays a growth phase-dependent derepression of the mexAB-oprM operon. However, the intrinsic inducer has not been identified. Here, we report that P. aeruginosa PAO1 produced sulfane sulfur, including glutathione persulfide and inorganic polysulfide, produced from either H2 S oxidation or from L-cysteine metabolism. Sulfane sulfur directly reacted with MexR, forming di- and trisulfide cross-links between two Cys residues, to derepress the mexAB-oprM operon. Levels of cellular sulfane sulfur and mexAB-oprM expression varied during growth, and both reached the maximum during the stationary phase of growth. Thus, self-produced H2 S and sulfane sulfur may facilitate antibiotic resistance via inducing the expression of antibiotic resistance genes.

The Heterotrophic Bacterium Cupriavidus pinatubonensis JMP134 Oxidizes Sulfide to Sulfate with Thiosulfate as a Key Intermediate.

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 gene-deletion 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.

Sensitive Method for Reliable Quantification of Sulfane Sulfur in Biological Samples.

Sulfane sulfur has been recognized as a common cellular component, participating in regulating enzyme activities and signaling pathways. However, the quantification of total sulfane sulfur in biological samples is still a challenge. Here, we developed a method to address the need. All tested sulfane sulfur reacted with sulfite and quantitatively converted to thiosulfate when heated at 95 °C in a solution of pH 9.5 for 10 min. The assay condition was also sufficient to convert total sulfane sulfur in biological samples to thiosulfate for further derivatization and quantification. We applied the method to detect sulfane sulfur contents at different growth phases of bacteria, yeast, mammalian cells, and zebrafish. Total sulfane sulfur contents in all of them increased in the early stage, kept at a steady state for a period, and declined sharply in the late stage of the growth. Sulfane sulfur contents varied in different species. For Escherichia coli, growth media also affected the sulfane sulfur contents. Total sulfane sulfur contents from different organs of mouse and shrimp were also detected, varying from 1 to 10 nmol/(mg of protein). Thus, the new method is suitable for the quantification of total sulfane sulfur in biological samples.

Developing Polysulfide-Sensitive GFPs for Real-Time Analysis of Polysulfides in Live Cells and Subcellular Organelles.

Polysulfides are newly discovered cellular contents, and they are involved in multiple intracellular processes, including redox homeostasis and protein sulfhydration. The dynamic changes of polysulfides inside the cell are directly related to these processes. To monitor the intracellular dynamics and subcellular levels of polysulfides, we developed green-fluorescent-protein (GFP)-based probes that are polysulfide-specific. A pair of cysteine residues was introduced near the GFP chromophore with the spatial distance between the cysteine residues designed to allow the formation of internal -S n- ( n ≥ 3) bonds but not -S2- (disulfide) bonds. We tested these probes in model microorganisms and found that they displayed ratiometric changes to intracellular polysulfides that had clear variations associated with the growth phases. The distribution of polysulfides in subcellular organelles is heterogeneous, suggesting that polysulfides have multiple origins and functions in cells. These probes provided long-desired tools for polysulfide in vivo studies.

FisR activates σ54 -dependent transcription of sulfide-oxidizing genes in Cupriavidus pinatubonensis JMP134.

Some heterotrophic bacteria are able to oxidize sulfide (H2 S, HS- and S2- ) to sulfite and thiosulfate via polysulfide. The genes coding for the oxidation enzymes in Cupriavidus pinatubonensis JMP134 have recently been identified; however, their regulation is unknown. A regulator gene is adjacent to the operon of the sulfide-oxidizing genes, encoding a σ54 -dependent transcription factor (FisR) with three domains: an R domain, an AAA+ domain and a DNA-binding domain. Here it is reported that the regulator responds to the presence of sulfide and activates the sulfide-oxidizing genes. FisR binds to its cognate operator at -114 to -135 bp of the transcription start of the operon. When polysulfide reacts with the R domain of FisR through the three conserved cysteine residues (C53, C64 and C71), FisR activates the expression of the operon. FisR is highly sensitive to polysulfide, activating σ54 -dependent transcription of sulfide-oxidizing genes for sulfide removal. Further, sequence analysis indicates that FisR-type regulators are relatively common for controlling sulfide-oxidizing genes under sulfide stress in the Proteobacteria.

The Complete Pathway for Thiosulfate Utilization in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is known to grow with thiosulfate as a sulfur source, and it produces more ethanol when using thiosulfate than using sulfate. Here, we report how it assimilates thiosulfate. S. cerevisiae absorbed thiosulfate into the cell through two sulfate permeases, Sul1 and Sul2. Two rhodaneses, Rdl1 and Rdl2, converted thiosulfate to a persulfide and sulfite. The persulfide was reduced by cellular thiols to H2S, and sulfite was reduced by sulfite reductase to H2S. Cysteine synthase incorporated H2S into O-acetyl-l-homoserine to produce l-homocysteine, which is the precursor for cysteine and methionine in S. cerevisiae Several other rhodaneses replaced Rdl1 and Rdl2 for thiosulfate utilization in the yeast. Thus, any organisms with the sulfate assimilation system potentially could use thiosulfate as a sulfur source, since rhodaneses are common in most organisms.IMPORTANCE The complete pathway of thiosulfate assimilation in baker's yeast is determined. The finding reveals the extensive overlap between sulfate and thiosulfate assimilation. Rhodanese is the only additional enzyme for thiosulfate utilization. The common presence of rhodanese in most organisms, including Bacteria, Archaea, and Eukarya, suggests that most organisms with the sulfate assimilation system also use thiosulfate. Since it takes less energy to reduce thiosulfate than sulfate for assimilation, thiosulfate has the potential to become a choice of sulfur in optimized media for industrial fermentation.

Everyone loves an underdog: metabolic engineering of the xylose oxidative pathway in recombinant microorganisms.

The D-xylose oxidative pathway (XOP) has recently been employed in several recombinant microorganisms for growth or for the production of several valuable compounds. The XOP is initiated by D-xylose oxidation to D-xylonolactone, which is then hydrolyzed into D-xylonic acid. D-Xylonic acid is then dehydrated to form 2-keto-3-deoxy-D-xylonic acid, which may be further dehydrated then oxidized into α-ketoglutarate or undergo aldol cleavage to form pyruvate and glycolaldehyde. This review introduces a brief discussion about XOP and its discovery in bacteria and archaea, such as Caulobacter crescentus and Haloferax volcanii. Furthermore, the current advances in the metabolic engineering of recombinant strains employing the XOP are discussed. This includes utilization of XOP for the production of diols, triols, and short-chain organic acids in Escherichia coli, Saccharomyces cerevisiae, and Corynebacterium glutamicum. Improving the D-xylose uptake, growth yields, and product titer through several metabolic engineering techniques bring some of these recombinant strains close to industrial viability. However, more developments are still needed to optimize the XOP pathway in the host strains, particularly in the minimization of by-product formation.

Integrated, systems metabolic picture of acetone-butanol-ethanol fermentation by Clostridium acetobutylicum.

Microbial metabolism involves complex, system-level processes implemented via the orchestration of metabolic reactions, gene regulation, and environmental cues. One canonical example of such processes is acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum, during which cells convert carbon sources to organic acids that are later reassimilated to produce solvents as a strategy for cellular survival. The complexity and systems nature of the process have been largely underappreciated, rendering challenges in understanding and optimizing solvent production. Here, we present a system-level computational framework for ABE fermentation that combines metabolic reactions, gene regulation, and environmental cues. We developed the framework by decomposing the entire system into three modules, building each module separately, and then assembling them back into an integrated system. During the model construction, a bottom-up approach was used to link molecular events at the single-cell level into the events at the population level. The integrated model was able to successfully reproduce ABE fermentations of the WT C. acetobutylicum (ATCC 824), as well as its mutants, using data obtained from our own experiments and from literature. Furthermore, the model confers successful predictions of the fermentations with various network perturbations across metabolic, genetic, and environmental aspects. From foundation to applications, the framework advances our understanding of complex clostridial metabolism and physiology and also facilitates the development of systems engineering strategies for the production of advanced biofuels.

H2S biotreatment with sulfide-oxidizing heterotrophic bacteria.

Many industrial activities produce H2S, which is toxic at high levels and odorous at even very low levels. Chemolithotrophic sulfur-oxidizing bacteria are often used in its remediation. Recently, we have reported that many heterotrophic bacteria can use sulfide:quinone oxidoreductase and persulfide dioxygenase to oxidize H2S to thiosulfate and sulfite. These bacteria may also potentially be used in H2S biotreatment. Here we report how various heterotrophic bacteria with these enzymes were cultured with organic compounds and the cells were able to rapidly oxidize H2S to zero-valence sulfur and thiosulfate, causing no apparent acidification. Some also converted the produced thiosulfate to tetrathionate. The rates of sulfide oxidation by some of the tested bacteria in suspension, ranging from 8 to 50 µmol min-1 g-1 of cell dry weight at pH 7.4, sufficient for H2S biotreatment. The immobilized bacteria removed H2S as efficiently as the bacteria in suspension, and the inclusion of Fe3O4 nanoparticles during immobilization resulted in increased efficiency for sulfide removal, in part due to chemical oxidation H2S by Fe3O4. Thus, heterotrophic bacteria may be used for H2S biotreatment under aerobic conditions.

Recombinant Escherichia coli with sulfide:quinone oxidoreductase and persulfide dioxygenase rapidly oxidises sulfide to sulfite and thiosulfate via a new pathway.

Many heterotrophic bacteria contain sulfide:quinone oxidoreductase (SQR) and persulfide dioxygenase (PDO) genes. It is unclear how these enzymes cooperate to oxidise sulfide in bacteria. Cupriavidus pinatubonensis JMP134 contains a gene cluster of sqr and pdo, and their functions were analysed in Escherichia coli. Recombinant E. coli cells with SQR and PDO rapidly oxidised sulfide to thiosulfate and sulfite. The SQR also contains a DUF442 domain that was shown to have rhodanese activities. E. coli cells with PDO and SQR-C94S, an active site mutant of the rhodanese domain, oxidised sulfide to thiosulfate with transitory accumulation of polysulfides. Cellular and enzymatic evidence showed that DUF442 speeds up the reaction of polysulfides with glutathione to produce glutathione persulfide (GSSH). Thus, SQR oxidises sulfide to polysulfides; rhodanese enhances the reaction of polysulfides with glutathione to produce GSSH; PDO oxidises GSSH to sulfite; sulfite spontaneously reacts with polysulfides to generate thiosulfate. The pathway is different from the proposed mitochondrial pathway because it has polysulfides, that is, disulfide and trisulfide, as intermediates. The data demonstrated that heterotrophic bacteria with SQR and PDO can rapidly oxidise sulfide to thiosulfate and sulfite, providing the foundation for using heterotrophic bacteria with SQR and PDO for sulfide bioremediation.

Autonomous production of 1,4-butanediol via a de novo biosynthesis pathway in engineered Escherichia coli.

1,4-Butanediol (BD) is an important chemical that is widely used in industry with an annual demand of one million metric tons. Here we report a modular development of engineered bacteria for successful BD bio-production. Using a systems engineering concept, we partitioned our development into two parts: namely BD biosynthesis and production control. The former was implemented through a de novo pathway that functions as an enzymatic reactor, while the latter was accomplished via synthetic circuits serving as genetic controllers. To facilitate development, the carbon utilizations were also partitioned into two routes. d-xylose was exclusively designated for BD production with other carbon sources utilized for cellular growth. Additionally, a quorum-sensing mechanism was exploited for production control, and the resulting strain was capable of autonomous production of BD. This study represents an example of the synergy between synthetic biology and metabolic engineering, affirming the need for deeper integration of the two fields.

Identification of aldehyde reductase catalyzing the terminal step for conversion of xylose to butanetriol in engineered Escherichia coli.

Biosynthetic pathways for the production of biofuels often rely on inherent aldehyde reductases (ALRs) of the microbial host. These native ALRs play vital roles in the success of the microbial production of 1,3-propanediol, 1,4-butanediol, and isobutanol. In the present study, the main ALR for 1,2,4-butanetriol (BT) production in Escherichia coli was identified. Results of real-time PCR analysis for ALRs in EWBT305 revealed the increased expression of adhP, fucO, adhE, and yqhD genes during BT production. The highest increase of expression was observed up to four times in yqhD. Singular deletion of adhP, fucO, or adhE gene showed marginal differences in BT production compared to that of the parent strain, EWBT305. Remarkably, yqhD gene deletion (KBTA4 strain) almost completely abolished BT production while its re-introduction (wild-type gene with its native promoter) on a low copy plasmid restored 75 % of BT production (KBTA4-2 strain). This suggests that yqhD gene is the main ALR of the BT pathway. In addition, KBTA4 showed almost no NADPH-dependent ALR activity, but was also restored upon re-introduction of the yqhD gene (KBTA4-2 strain). Therefore, the required ALR activity to complete the BT pathway was mainly contributed by YqhD. Increased gene expression and promiscuity of YqhD were both found essential factors to render YqhD as the key ALR for the BT pathway.

MEP pathway-mediated isopentenol production in metabolically engineered Escherichia coli.

BACKGROUND: Isopentenols, such as prenol and isoprenol, are promising advanced biofuels because of their higher energy densities and better combustion efficiencies compared with ethanol. Microbial production of isopentenols has been developed recently via metabolically engineered E. coli. However, current yields remain low and the underlying pathways require systematic optimization. RESULTS: In this study, we targeted the E. coli native 2-methyl-(D)-erythritol-4-phosphate (MEP) pathway and its upstream glycolysis pathway for the optimization of isopentenol production. Two codon optimized genes, nudF and yhfR from Bacillus subtilis, were synthesized and expressed in E. coli W3110 to confer the isopentenol production of the strain. Two key enzymes (IspG and Dxs) were then overexpressed to optimize the E. coli native MEP pathway, which led to a significant increase (3.3-fold) in isopentenol production. Subsequently, the glycolysis pathway was tuned to enhance the precursor and NADPH supplies for the MEP pathway by activating the pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (ED), which resulted in additional 1.9 folds of increase in isopentenol production. A 5 L-scale batch cultivation experiment was finally implemented, showing a total of 61.9 mg L(-1) isopentenol production from 20 g L(-1) of glucose. CONCLUSION: The isopentenol production was successfully increased through multi-step optimization of the MEP and its upstream glycolysis pathways. It demonstrated that the total fluxes and their balance of the precursors of the MEP pathway are of critical importance in isopentenol production. In the future, an elucidation of the contribution of PPP and ED to MEP is needed for further optimization of isopentenol production.

Metabolic engineering of Escherichia coli for biosynthesis of D-galactonate.

D-galactose is an attractive substrate for bioconversion. Herein, Escherichia coli was metabolically engineered to convert D-galactose into D-galactonate, a valuable compound in the polymer and cosmetic industries. D-galactonate productions by engineered E. coli strains were observed in shake flask cultivations containing 2 g L(-1) D-galactose. Engineered E. coli expressing gld coding for galactose dehydrogenase from Pseudomonas syringae was able to produce 0.17 g L(-1) D-galactonate. Inherent metabolic pathways for assimilating both D-galactose and D-galactonate were blocked to enhance the production of D-galactonate. This approach finally led to a 7.3-fold increase with D-galactonate concentration of 1.24 g L(-1) and yield of 62.0 %. Batch fermentation in 20 g L(-1) D-galactose of E. coli ∆galK∆dgoK mutant expressing the gld resulted in 17.6 g L(-1) of D-galactonate accumulation and highest yield of 88.1 %. Metabolic engineering strategy developed in this study could be useful for industrial production of D-galactonate.

Combining De Ley-Doudoroff and methylerythritol phosphate pathways for enhanced isoprene biosynthesis from D-galactose.

An engineered Escherichia coli strain was developed for enhanced isoprene production using D-galactose as substrate. Isoprene is a valuable compound that can be biosynthetically produced from pyruvate and glyceraldehyde-3-phosphate (G3P) through the methylerythritol phosphate pathway (MEP). The Leloir and De Ley-Doudoroff (DD) pathways are known existing routes in E. coli that can supply the MEP precursors from D-galactose. The DD pathway was selected as it is capable of supplying equimolar amounts of pyruvate and G3P simultaneously. To exclusively direct D-galactose toward the DD pathway, an E. coli ΔgalK strain with blocked Leloir pathway was used as the host. To obtain a fully functional DD pathway, a dehydrogenase encoding gene (gld) was recruited from Pseudomonas syringae to catalyze D-galactose conversion to D-galactonate. Overexpressions of endogenous genes known as MEP bottlenecks, and a heterologous gene, were conducted to enhance and enable isoprene production, respectively. Growth test confirmed a functional DD pathway concomitant with equimolar generation of pyruvate and G3P, in contrast to the wild-type strain where G3P was limiting. Finally, the engineered strain with combined DD-MEP pathway exhibited the highest isoprene production. This suggests that the equimolar pyruvate and G3P pools resulted in a more efficient carbon flux toward isoprene production. This strategy provides a new platform for developing improved isoprenoid producing strains through the combined DD-MEP pathway.

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.

The Activity of YCA1 Metacaspase Is Regulated by Reactive Sulfane Sulfur via Persulfidation in Saccharomyces cerevisiae.

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.