Sulfane Sulfur Act As A Common Signal To Regulate MarR Family Transcriptional Factors

H2S is generally considered to be the third type gas signal molecule behind NO and CO,regulating important physiological process in mammals.However,there are few studies with microorganisms,and they are mainly related to sulfur metabolism.Whether H2S is involved in other physiological activities remains to be explored.Sulfane sulfur refers to sulfur-containing molecules with redox activity that can oxidize or reduce biological molecules under physiological conditions,in which the valence state of sulfur atom is usually 0 or-1.H2S and sulfane sulfur are inherent components in cells.They have similar functions and can be transformed into each other through a variety of enzymatic and redox reactions.Therefore,which is the real signal molecule has been a research hot topic in recent years.Recently,more and more evidence show that H2S is only act as signal precursor,transmitting between cells.Sulfane sulfur is the specific form to play the signal function.The previous results also showed that sulfane sulfur could affect the drug resistance and pigment secretion of P.aeruginosa by regulating MarR family transcription factors MexR and OspR,respectively.However,further evidence is needed to prove whether sulfane sulfur is a common signal molecule.Whether sulfane sulfur has a general regulatory effect on MarR family proteins and affects more physiological activities are also worth further exploration.The thesis describes that sulfane sulfur functions as an inducer of the three transcription regulators of the MarR family,Escherichia coli MarR,Staphylococcus aureus MgrA,and Pseudomonas aeruginosa OhrR.EMSA experiments showed that sulfane sulfur could reduce the affinity of MarR、MgrA、OhrR to their cognate DNA probes,indicating that these transcriptional factors could respond to sulfane sulfur in vitro.The cysteine residues of these transcriptional factors were then mutated to serine.It was found that the mutant protein MarR-5XS,which contains a single Cys80 only,could still sense sulfane sulfur,indicating that Cys80 played a key role in that process.The mutant protein MgrA-C12S was found to lose its sensitivity to sulfane sulfur,indicating that Cys12 was important for sensing sulfane sulfur.As for OhrR,the single mutantion of three cysteine didn’t affect the ability to respond to sulfane,but double mutation did,indicating all these three cysteines were responsible for sensing sulfane sulfur.The results of the constructed fluorescence reporters were consistent with the in vitro experiments,MarR、MgrA and OhrR responded to sulfane sulfur in vivo,and caused the increasing of fluorescence level in a concentration-dependent manner.Further,it was found that the mutation of Cys80 of MarR significantly reduced the fluorescence level induced by sulfane sulfur compared with the wild type strain,indicating Cys80 was important.The mutation of Cys12 also led to a significant decrease in the fluorescence level induced by sulfane sulfur,emphasizing the key role of Cys12 of MgrA in the progress of responding to sulfane sulfur.Size-exclusion chromatography was used to analyze the aggregation of transcriptional factors.Sulfane sulfur promoted MarR to form tetramer and higher-multimers.Similarly,sulfane sulfur caused the formation of MgrA tetramer.These results indicated that sulfane sulfur changed the conformation of MarR and MgrA.Subsequently,Liquid Chromatography-Mass Spectrometry(LC-MS)comfirmed the formation of disulfide and trisulfide bond between Cys80 in two MarR dimmers after sulfane sulfur treatmenty.The formation of disulfide bond between Cys12 of MgrA dimers and Cys’2 of persulfide and trisulfide were also detected after MgrA was treated by sulfane sulfur.As for OhrR treated by sulfane sulfur,the formation of disulfide bond was detected between cys9 and cys19,cys9 and cys121,cys19 and cys121.The homologous modeling analysis confirmd that the intramolecular disulfide bond was likely formed between cys9 and cys19,and the intermolecular disulfide bond was likely formed between cys9 and cys121,cys19 and cys121.Further exploration was carried out by non-reducing SDS-PAGE,it was found that sulfane sulfur induced the formation of MarR and MgrA dimers from monomers,but couldn’t promote MarR-C80S and MgrA-C12S to form dimers.In addition,sulfane sulfur could also promote OhrR、OhrR-C9S and OhrR-C19S instead of OhrR-C121S to form dimer.These results showed that the mechanism of MarR,MgrA and OhrR sensing sulfane sulfur was to utilize cysteine residues to form disulfide,which caused conformational changes and reduced the affinity to cognate DNA probe,leading to the releasing of the repression of downstream genes.However,the specific mechanism was slightly different.Sulfane sulfur caused the formation of disulfide bond and trisulfide bond between Cys80 of two MarR dimers.Similarly,sulfane sulfur also induced the formation of disulfide bond between Cys12 of the two MgrA dimers.The difference was that the persulfide and trisulfide modification of Cys12 was also comfirmed.As for OhrR,sulfane sulfur promoted the formation of intramolecular disulfide bond between cys9 and cys19,and intermolecular disulfide bonds between cys9 and cys121,cys19 and cys121 respectively.PDO,a key enzyme to degrade sulfane sulfur,was expressed in the constructed fluorescence reporter system strains.And the growth and fluorescence curves were detected.It was found that the introduction of PDO could significantly reduce the overall fluorescence level compared with the reporter strains without PDO,indicating that these transcriptional factors could respond to endogenous sulfane sulfur.In summary,this study comfirmed the common regulation of sulfane sulfur to MarR family transcriptional factors and revealed the specific mechanism of regulation,providing new evidence for the signal function of sulfane sulfur.In addition,it was also proved that sulfane sulfur could regulate transcriptional factors related to other physiological activities rather than sulfur metabolism,providing theoretical basis for further investigation of new physiological function of sulfane sulfur.