| Sulfur is one of the important elements that make up living organisms.At the beginning of life on Earth,sulfur,especially reduced sulfur,has been ubiquitous in the environment and played a significant role in the origin of life.Today,with the changes of the ecological environment,although reduced sulfur is not abundant in most environments,only in a few special environments,such as hot springs and volcanic vents,it still maintains an important physiological function in organisms.Reactive sulfane sulfur species are normal cellular components in organisms.Reactive sulfane sulfur species include hydrogen polysulfides(H2Sn,n?2),organic polysulfides(RSSnH,RSSnR,n?2),and organic persulfides(RSSH).They possess both nucleophilic and electrophilic characteristics,while thiols(cysteine,GSH,etc.)are generally nucleophilic.As nucleophiles,they are better reductants than thiols;as electrophiles,they can transfer electrophilic sulfane sulfur(S0)to protein thiols to generate protein-SSH,affecting certain enzyme activities and protecting protein thiols from irreversible oxidation.Owing to the dual-reactivities,reactive sulfane sulfur is involved in many cellular processes,such as redox homeostasis,virulence in pathogenic bacteria,and biogenesis of mitochondria.Sulfane sulfur also functions as antioxidants inside cells.Albeit the good roles,sulfane sulfur may be toxic at high concentrations.Indeed,elemental sulfur has been used as an antimicrobial agent for ages.Although its toxicity mechanism is unclear,a recent study suggested that sulfur is transported into the cell in the form of hydrogen polysulfide,inducing protein persulfidation as a possible toxic mechanism.Organosulfur compounds can be used to treat antibiotic-resistant bacteria,and they are converted to hydrogen polysulfide for the toxicity.Both bacteria and fungi display reduced viability being exposed to sulfane sulfur in excess.Therefore,intracellular sulfane sulfur is likely to be maintained within a range for microorganisms under normal conditions.Multiple pathways for sulfane sulfur generation have been discovered.3-mercaptopyruvate sulfurtransferase and cysteinyl-tRNA synthetase produce sulfane sulfur from cysteine;Cystathionine ?-synthase and cystathionine ?-lyase catalyze cystine to produce sulfane sulfur,but their major product is sulfane sulfur or H2S is controversial in vivo because of the low concentration cystine in reduced cellular milieu;In addition.sulfide:quinone oxidoreductase and superoxide dismutase(SOD)produce sulfane sulfur from H2S.Most microorganisms possess several of these pathways.Two sulfane sulfur removal pathways have been found.Aerobic microorganisms may apply persulfide dioxygenase to remove excessive sulfane sulfur,and the persulfide dioxygenase expression can be induced by sulfane sulfur via sulfane sulfur-sensing transcription factors.Reactive sulfane sulfur can be also reduced to H2S.Glutaredoxin and thioredoxin were previously thought to reduce the disulfide bonds formed by oxidative stress,and recent studies have shown that they also catalyze the reduction of sulfane sulfur to Glutaredoxin and thioredoxin are more effective in reducing sulfane sulfur than GSH.However,it is unclear whether or not sulfane sulfur induces the expression of glutaredoxin and thioredoxin.Escherichia coli does not contain sulfane sulfur oxidation systems,but contains thioredoxins(Trx)and glutaredoxins(Grx),and the expression of TrxC and GrxA is regulated by a global transcriptional regulator OxyR.OxyR was initially identified as a regulator responding to reactive oxygen species(ROS).ROS triggers the formation of intra disulfide bond between Cys19 and Cys-208 or oxidizes Cys199 to C199-SOH.The exact mechanism is still in debate.In this thesis,E.coli was used as the research object to study the changes of intracellular sulfane sulfur level in E.coli,the mechanism of OxyR senses and regulates intracellular sulfane sulfur and exogenous reactive sulfane sulfur stress.In addition,the potential of application of heterotrophic bacteria containing sulfide:quinone oxidoreductase and persulfide dioxygenase in H2S biotreatment was also studied.The main contents are as follows:1.The accumulation and reduction of endogenous sulfane sulfur in E.coliThe intracellular sulfane sulfur of E.coli was reduced to H2S by reducing agent,and then the concentration of sulfane sulfur in E.coli was determined by the amount of H2S.Using this method,the intracellular sulfane sulfur concentration of E.coli grown to stable stage in LB medium was measured to be about 800 ?M.SSP4 is a fluorescent probe for the specific detection of sulfane sulfur.The SSP4 is used to characterize the change of intracellular sulfane sulfur concentration in E.coli.The intracellular sulfane sulfur started to accumulate at middle-log phase and reached the maximum at early stationary phase.When the stationary-phased cells were transferred into fresh medium(OD600=1),their intracellular sulfane sulfur decreased quickly with concomitant release of H2S.This phenomenon suggests that sulfane sulfur may be reduced to H2S by enzymes,such as thioredoxin and glutaredoxin.E.coli contains several thioredoxins and glutaredoxins.When glutaredoxins was over expressed in vivo,they could partially decrease the sulfane sulfur accumulation.When co-expression of thioredoxin with thioredoxin reductase prevented the increase of sulfane sulfur during the log phase of growth.These results confirmed that thioredoxins and glutaredoxins are involved in the reduction of endogenous sulfane sulfur in E.coli.The transcriptional regulator OxyR controls the expression of partial thioredoxin and glutaredoxin(TrxC and GrxA),which responds oxidative stress and then activates TrxC and GrxA the transcription of TrxC and GrxA.Meanwhile,the peroxidase KatG is also induced by OxyR.TrxC,GrxA and KatG have long been thought of in dealing with oxidative stress.TrxC and GrxA reduce the disulfide bond caused by oxidative stress.KatG directly catalyze the disproportionation of hydrogen peroxide to produce water and oxygen.But recent studies have shown that these three enzymes can also act on the reactive sulfane sulfur.TrxC and GrxA can reduce inorganic polysulfides and persulfides,while KatG has the activity of oxidizing inorganic polysulfides.Thus,OxyR may play a role in regulating intracellular sulfane sulfur.Based on the above assumptions,we deleted oxyR gene in E.coli and observed that the mutant had higher intracellular sulfane sulfur than wt did at log-phase.When E.coli ?oxyR cells at the stationary phase were transferred into fresh LB medium,the decrease of intracellular sulfane sulfur and the release of H2S were slower than that of the cells.This result confirms our assumptions.The sulfane sulfur stress test showed that ?oxyR more sensitive to exogenously added H2Sn under both aerobic and anaerobic conditions.After complementing oxyR into E.coli ?oxyR,the strain regained the tolerance to H2Sn.The results indicated that OxyR plays an important role in dealing with the exogenous H2Sn stress.The combined results suggested that OxyR regulates the production of thioredoxin and glutaredoxin that reduce sulfane sulfur to H2S by sensing sulfane sulfur.2.The mechanism of OxyR regulating sulfane sulfur levelsIn order to verify the above hypothesis,we constructed three reporting plasmids with an mKate gene under the control of the trxC,grxA,or katG promoter.These plasmids were transformed into E.coli wt and AoxyR.The results indicate that exogenous addition of H2Sn can induce the expression of TrxC,GrxA and KatG in E.coli wt.However,in E.coli ?oxyR,the three promoters led to constantly low expressions of mKate with or without added H2Sn,and H2Sn loses the induction effect.Complementation of oxyR restored the mutant's response to H2Sn.This suggests that it is through OxyR that H2Sn induces the expression of trxC,grxA and katG,three sulfane sulfur removal genes.This OxyR-dependent induction effect is present in both aerobic and anaerobic conditions.We also proved that sulfide did not induce the expression of related genes in wt,excluding the signal function of sulfide.When we used E.coli cells harboring a sulfide:quinone oxidoreductase of C.pinatubonensis JMP134.the added sulfide was oxidized to H2Sn19,which induced the expression of trxC,grxA and katG.The regulatory domin of OxyR contains two cysteine residues that can sense peroxides,Cys199 and Cys208.In order to study the mechanism of OxyR sensing sulfane sulfur,we mutated Cys199 and Cys208 on OxyR separately or simultaneously to serine(Ser)in plasmids.OxyRc199S,OxyRC208S,and OxyRc199S.C208S all lost the induction effect.The results indicated that CyS199 and Cys208 are involved in H2Sn sensing.In vitro transcription-translation results showed that the OxyR reacted with H2Sn had more than 5 times the transcriptional activity compared with reduced OxyR,confirming that OxvR could be activated directly by reaction with H2Sn,Meanwhile.in vitro transcription-translation results confirmed OxyRC199S,OxyRC208S and OxyRC199S mutant proteins cannot be activated by H2Sn and loses its regulatory effect,which again indicates that Cys199 and Cys208 play an indispensable role in sensing H2Sn.Mass spectrometry analysis was performed to analyze the H2Sn-treated OxyR.The results showed that Cys199 on OxyR was modified by H2Sn to form persulfide modification(OxyR-SSH),while Cys208 was not modified.These data collectively indicated that OxyR senses H2Sn via persulfidation on Cys199,other than forming disulfide or-Sn-(n?3)bond between Cys199 and Cys208.As results of mutation suggest that Cys208 also plays an indispensable role,we speculate that it may play a role in stabilizing the persulfidated Cysl99.3.OxyR-related proteome,transcriptome and bioinformatics analysisThe signal function of reactive sulfane sulfur is to modify thiol of proteins by persulfidation,which affects the activity of proteins.Knockout OxyR lead to intracellular sulfane sulfur levels rise,so we compared the amount and species of persulfidated proteins in ?oxyR and wt using a biotin labeling method.Results showed that the ?oxyR had higher amount of persulfidated protein than wt;In terms of species,66 persulfidated modified proteins were detected in wt,while ?oxyR contained 118 persulfidated proteins;51 proteins were shared between them;15 proteins were wt-specific,and 67 were only detectable at ?oxyR and was not present in wt.Through the gene ontology analysis to persulfidated proteins of wt and ?oxvR revealed that the biological process,cellular component and molecular function all contained persulfidated proteins,especially those related to energy metabolism.The difference in the types of persulfidated proteins between wt and ?oxyR may have a broad effect on the physiological processes of cells.OxyR is a global transcriptional regulator that was first discovered to regulate oxidative stress,and we found it can also sense sulfane sulfur.Therefore,we used the RNA Seq method to study the global transcriptional response of E.coli confront H2Sn stress or H2O2 stress.At the global level,there were similarities and differences.For the genes that OxyR related,the response of E.coli has difference in the degree ofup-regulation between the two stresses.It may reflects the different allosteric regulation of OxyR cause multi-level transcriptional responses.Bioinformatics analysis showed that OxyR is widely distributed in bacteria.More than 50%of the bacteria contain OxyR.It was also found to be present in bacteria that inhabit environments with high concentrations of reactive sulfane sulfur and in a number of obligate anaerobic bacteria.For these bacteria.OxyR is likely used to deal with reactive sulfane sulfur stress.On the geological evolution timescale,the evolutionary age or the earliest appearance time of some OxyR-containing bacteria is in a period when the Earth's oxygen content is very low and the reactive sulfane sulfur content is high.OxyR is an ancient protein whose role at that time may be mainly to respond to reactive sulfane sulfur stress and to regulate intracellular sulfane sulfur levels.In conclusion,we discovered that E.coli uses OxyR to sense reactive sulfane sulfur and activates the genes for its removal.The persulfidation of OxyR is the fifth type of modification for OxyR activation.Since OxyR is widely distributed in both aerobic and anaerobic bacteria,the OxyR-regulated network may represent a conserved mechanism that bacteria can resort to when confronting endogenous and/or exogenous sulfane sulfur stress.4.Application of H2S oxidation system of heterotrophic bacteria in H2S biotreatmentIn addition to thioredoxins and glutaredoxins that reduce sulfane sulfur to H2S in bacteria,suifide:quinone oxidoreductase(SQR)and persulfide dioxygenase(PDO)can also oxidize H2S and sulfane sulfur to sulfite and thiosulfate.Thus,these bacteria have the potential to be applied to the H2S biotreatment.H2S is produced by various industrial and natural activities,such as petroleum refining,methane-containing biogas production and wastewater treatment.H2S,however,is malodorous and toxic that needs to be treated.Biofiltration is often used in H2S removal from waste gas or waste water because of its effectiveness,low energy consumption and minimal by-product generation compared to chemical and physical treatments.Researchers have tried different reactor designs,various packing materials and nutrients for biofiltration to achieve stable efficiency of H2S elimination.However,biofiltration is limited to the use of a few of chemolithotrophic sulfur-oxidizing bacteria,such as Thiobacillus spp.,Acidithiobacillus spp.and green sulfur bacterium.A problem of using these sulfur-oxidizing bacteria is the production of sulfuric acid,leading to acidification of the liquid phase.To control this problem,the ratio of H2S and O2 must be strictly controlled,bringing more costs.Here we made a thorough investigation on the efficiency of H2S oxidation of these bacteria containing sqr and pdo.Compared with several widely used autotrophic bacteria,some heterotrophic bacteria were found to have the same oxidation efficiency as reported autotrophic bacteria.We showed heterotrophic sulfide oxidizing bacteria oxidize sulfide without causing acidification,probably due to the lack of sulfuric acid production.Based on the above studies,we developed a method to immobilize cells with Fe3O4 nanoparticles for the efficient and recyclable removal of H2S. |
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