The Mechanism Of Sulfane Sulfur Regulating Secondary Metabolism And Differentiation In Streptomyces Coelicolor

Hydrogen sulfide(H2S)is a new signaling molecule that plays an important role in many physiological processes.H2S and sulfane sulfur usually coexist in the cytoplasm.Recent studies suggest that H2S plays a signaling role via sulfane sulfur that has stronger nucleophilicity and higher ability to modify sulfhydryl groups than H2S.Sulfane sulfur includes inorganic polysulfides(HS,H,n≥2),organic polysulfides(RSSnH and RSSR,n≥2),and organic persulfides(RSSH),and they are common cellular components in mammalian cells.It plays an important role in antioxidation,neural transmission,anti-inflammation,and regulation functions.Sulfane sulfur is also widely present in microorganisms and has important physiological functions.For example,in Staphylococcus aureus,sulfane sulfur is involved in the regulation of bacterial pathogenicity.In Pseudomonas aeruginosa PAO1,the level of sulfane sulfur is related to bacterial pathogenicity and drug resistance.In Synechococcus sp.PCC7002,the level of sulfane sulfur is related to bacterial photosynthesis.Actinomycetes are an important source of natural products.About 70%of antibiotics and anticancer drugs on the market are derived from natural products of Actinomycetes.Most of the products are polyketides.Streptomyces coelicolor M145 is a typical actinomycete,and its growth and development are complex.During the process,various secondary metabolites are produced,including several polyketide antibiotics.Most researchers focus on mining new metabolic pathways of these synthetic gene clusters,while less research has been conducted on the regulation of these synthetic gene clusters.The purpose of this project is to explore the regulation of sulfane sulfur on secondary metabolites biosynthesis and development process of S.coelicolor M145.The main research contents of this dissertation are as follows:1.Sulfane sulfur activates ACT production and sporulation in S.coelicolor M145.A gene cluster controlling intracellular sulfane sulfur levels is identified.Genetically modifying this gene cluster can change sulfane sulfur levels,thereby altering the actinorhodin production and sporulation.Exogenously adding sodium hydrosulfide(NaHS)and sublimed sulfur(S8)to the solid medium promoted the production of secondary metabolite ACT and sporulation of the wild type strain.Persulfide dioxygenase(PDO)oxidizes sulfane sulfur and reduces intracellular sulfane sulfur content.In the ΔScpdo strain,the endogenous sulfane sulfur content was increased.The ΔScpdo strain produced high ACT and increased sporulation.The effects of exogenous and endogenous sulfane sulfur proved that it acts as a signal molecule to affect the synthesis of secondary metabolites,and development of Streptomyces.This bacterial species carries a natural gene circuit containing four genes that encode a CsoR-like transcription factor(ScCsoR),persulfide dioxygenase(ScPDO),rhodanese and a sulfite transporter,which were shown to be responsible for sensing and removal of excessive sulfane sulfur.RT-qPCR and EMSA results showed that ScCsoR directly bound to the Scpdo promoter region to negatively regulate its mRNA level,and also directly inhibited the transcription of its own and Scrhod.In addition,RT-PCR indicated that Scpdo and SctauE were co-transcribed.Therefore,ScCsoR regulated the expression of Scpdo,SctauE,SccsoR and Scrhod and the encoded proteins were involved in sulfane sulfur oxidation.The regulatory factor ScCsoR was identified to specifically sense polysulfides.Mass spectrometry analysis revealed that Cys37 residues of ScCsoR could be modified with sulfane sulfur to form a persulfidation form,and the modified ScCSoR did not bind to the promoters,thereby activating the transcription of ScPDO.The deletion of ScCsoR decreased cellular sulfane sulfur,while the deletion of ScPDO increased its levels.The increased sulfane sulfur promoted actinorhodin production and sporulation.This study unveiled a natural gene circuit for maintaining sulfane sulfur homeostasis in bacteria and identified the trigger effect of sulfane sulfur on actinorhodin production,presenting a new approach for activating polyketide gene clusters in actinomycetes.2.Sulfane sulfur as a signal molecule regulates the expression level of AdpA,and the specific pathway by which sulfane sulfur affects the synthesis of ACT is clarified.However,ScCsoR cannot directly regulate ACT synthesis,and the molecular mechanism of sulfane sulfur affecting ACT needs to be further analyzed.So why is AdpA the next subject of study?First,we found that the expression of AdpA was the largest when the content of sulfane sulfur was the highest;second,AdpA directly regulated ACT synthesis(actⅡ-4,a specific activating gene for ACT synthesis)and morphological differentiation(wblA and ssgA,etc.);third,It contains 4 cysteines,which provides the possibility to sense sulfane sulfur.By analyzing the phenotypes of wt,ΔadpA and ΔadpA::adpA,the results showed that exogenous cysteine and thiosulfate no longer induced ΔadpA to produce high ACT.When the wild type and ΔadpA were induced with 400 μM HSnH and S8,the mRNA level of actⅡ-4,a specific activating regulator responsible for ACT synthesis,in the wild type were significantly increased,but the mRNA level of actⅡ-4 in ΔadpA not be induced.These results suggest that sulfane sulfur promotes wt high production of ACT dependent on AdpA.A fluorescent reporter system constructed in Streptomyces also verified this conclusion.To explore the effect of sulfane sulfur on AdpA and its target genes,we carried out a series of experiments.EMSA results indicated that sulfane sulfur affects its interaction with itself,actⅡ-4 and wblA promoter DNA.RT-qPCR detected increased mRNA level in the non-knockout portion(adpA-wt)of ΔadpA,suggesting that AdpA is an auto-repressor.In addition,an increase in the mRNA level of adpA-wt was detected in the ΔScpdo strain that contained a relatively high content of sulfane sulfur,further demonstrating that sulfane sulfur induces the transcription of adpA.The results of FP(Fluorescence Polarization)experiment showed that sulfane sulfur weakened the affinities of AdpA to adpA,actⅡ-4 and wblA promoters,further suggesting that sulfane sulfur is an inducer of AdpA.Mathematical modeling was used to help understand the mechanism of how sulfane sulfur affecting AdpA and other genes expression.When the intracellular sulfane sulfur content is high in the early growth stage,the content of AdpA modified by sulfane sulfur increased.At this time,AdpA-S is the main form,which has low activation effect on ActⅡ-4 expression.When sulfane sulfur content decreases,the content of AdpA-S also decrease,resulting in the major form of AdpA-apo,which has high activation effect on ActⅡ-4 expression.Based on this model,it speculated that sulfane sulfur can temporarily break the auto-inhibitory regulation in AdpA expression,making AdpA accumulate to a higher level which lasts for a longer time,eventually leads to more ActⅡ-4 expression and increased ACT production.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.To further analyze the mechanism of AdpA sensing sulfane sulfur,we single-mutated the four Cys of AdpA to Ser and complemented it into ΔadpA.After phenotypic analysis,the sporulation phenotype of the ΔadpA::adpAC62S strain was not restored,suggesting that Cys62 are very important for the normal biological function of the strain.In addition,the AdpAC62S protein bound to its target DNA abnormally and partially lost sulfane sulfur sensing function.FP experiments also demonstrated that the ability of AdpAC62S binding to DNA was affected.The LC-MS/MS data showed that sulfane sulfur formed a Cys62-SSH modification at Cys62-SH.These results suggest that sulfane sulfur influenced the activity of AdpA via a persulfidation on its Cys62.To further reveal the mechanism,we used AlphaFold2 to predict the complete 3D structure of AdpA.The structure shows that Cys62 are located in the shallow region of the protein surface,from the perspective of conservation,this part has high variability,and sulfane sulfur is likely to be formed Cys62-SSH at Cys62-SH,provides theoretical support for the conclusions of the LC-MS/MS.In addition,the highly conserved Cys126 residues are found to be located on the sidewall that resembles a small molecule binding pocket,which is likely to contain ligand small molecules.The conservation of different domains of AdpA protein was further analyzed,and both ends of the protein were highly flexible and variable,which provided theoretical support for explaining the various DNA binding models of AdpA.Bioinformatics analysis showed that the four Cys residues on AdpA were conserved in different Streptomyces,suggesting that the signaling effect of sulfane sulfur by modifying regulatory proteins may be a general mechanism.The expression of AdpA is regulated in several ways.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.3.AdpA affects its content by directly regulating sulfane sulfur oxidation and reduction pathways,and is an important factor in Streptomyces coping with sulfur stress and survival.In addition to regulating the synthesis of secondary metabolites and differentiation,AdpA can also regulate the content of sulfane sulfur.It was found that the deletion of adpA resulted in enhanced sensitivity of the strain to HSnH and Sg.Sequence analysis and EMSA results showed that the promoters of genes related to the PDO sulfane sulfur oxidation pathway and the Trx sulfane sulfur reduction system both contained AdpA recognition sequences,and AdpA directly regulated these two pathways.The sulfane sulfur oxidation and reduction pathways were able to respond to HSnH and S8 in the wt strain,while their responses were significantly attenuated in ΔadpA:especially under anaerobic conditions,this effect was more pronounced.It was also found that the absence of adpA resulted in the strain being unable to survive under anaerobic conditions.Therefore,the content of this chapter revealed that AdpA is an important factor to cope with sulfur stress and survival in Streptomyces,and discovered the regulator AdpA that regulates both sulfane sulfur oxidation and reduction pathways.The closed regulatory loop of the content has deepened the researchers’ understanding of the regulation of microbial sulfur metabolism.In conclusion,the physiological function of sulfane sulfur was explored in S.coelicolor M145,and the specific pathway of its effect on the synthesis of antibiotic ACT was revealed.It can regulate the content of sulfane sulfur,and provide a theoretical basis for further revealing the role of sulfane sulfur in signal regulation in vivo.This study provides a new opportunity for activating silent gene clusters and increasing the production of polyketides.