| Sulfur plays important roles in individual organisms as an indispensable element and participates in active biogeochemical cycling.The valence of sulfur ranges from-2 to+6.Hydrogen sulfide with-2 valence has long been considered as the third gastrotransmitter after NO and CO,and plays an important role in cell signaling and maintenance of redox homeostasis.Excessive hydrogen sulfide is also toxic.It can inhibit the activity of cytochrome c oxidase,disrupting respiratory electron transport chain for respiration.Therefore,the removal of hydrogen sulfide is very important to organisms.Polysulfide is one of the oxidation metabolites of hydrogen sulfide.It is a kind of important sulfur compounds in geochemical and microbiological processes.It participates in many important environmental processes such as sulfidization of organic matters,formation of pyrite and metal chelation.At the same time,polysulfide is common component inside cells,and participates in many important intracellular physiological processes including signaling,maintenance of redox state,metabolic regulation,biosynthesis and intracellular oxidation resistance,and more and more evidences show that polysulfides,as one of sulfane sulfur,are actually playing the role of signaling molecules in the cell.Therefore,whether in the environment or in cells,the maintenance of polysulfide homeostasis is crucial.Our laboratory has reported a complete sulfides oxidation pathway in an environmental heterotrophic bacteria Cupriavidus pinatubonensis JMP134.It is mainly composed of sulfide:quinone oxidoreductase(SQR),persulfide dioxygenase(PDO)and the sulfur transferase(rhodanese),which work together to complete the oxidation of sulfide to thiosulfate or sulfite.What's more.SQR and PDO exist simultaneously in many bacterias and work synergistically.However,some bacterias only have sqr or pdo gene alone,resulting they can only oxidize hydrogen sulfide to polysulfide or oxidize polysulfide to the sulfite.There is a question about the transportation of polysulfides inside and outside cells as well as among the cells which is unknown.We found that the operon of pdo genes in many bacteria are co-transcribed with two or three yedE genes,encoding membrane proteins which may related to the transportation of sulfur compounds.Bioinformatics analysis indicate that YedEs have structural and functional relationship with many proteins involved in sulfur metabolism.At the same time,a family proteins of transcriptional regulators YedR commonly found upstream of the yedE genes.Therefore,we speculate that the proteins encoded by yedEs and their upstream regulatory protein may play a role in maintaining intracellular polysulfide homeostasis in conjunction with PDOIn C.pinatubonesis JMP134,YedEl,YedE2 and YedE3 are three small membrane proteins,each containing transmembrane domains.Our physiological and biochemical experiments by heterologous expression of three proteins indicated that YedE1E2 synergistically accelerates the utilization of polysulfides by promoting the transport of polysulfides into the cells,but there is no significant effect on the efflux of polysulfides.YedE3 has no significant effect on polysulfide transport.Sequence analysis indicated that each YedEl and YedE2 contain a conserved Cys.The mutation of conserved Cys sites and a series of physiological and biochemical experiments revealed that they all participate in the function of YedEs in the transporting of polysulfide.Phylogenetic analysis indicated that the yedEl and yedE2 genes are mainly present in the ProteobacteriaIn this paper,the possible transcriptional regulator gene yedR,upstream of yedE1E2 in C.pinatubonensis JMP134 was knocked out.The results of real-time PCR showed that the transcription level of the downstream yedE1E2 gene was significantly increased after knockout.We purified the YedR protein in vitro,and the EMSA results indicated that it can specifically bind to the region near the promoter upstream of the gene.Thus,we determined that the YedR protein acts as a transcriptional repressor of the downstream yedE1E2 gene and regulates the transcription level of them.In addition,the 5' RACE experiment identified the transcription start site of the operon.We constructed the promoter fluorescence reporter system to further validate the transcriptional regulation of the YedR protein for downstream genes.At the same time.it was proved by using different sulfides to induce the reporting system that the YedR protein can specifically sense the sulfane sulfur and is concentration-dependent.The EMSA experiments in vitro also showed that the sulfane sulfur can well deactivate the specific binding of the YedR protein to the targeted DNA sequence,once again confirming the above results.There are four Cys sites in the YedR protein sequence,and two conserved Cys sites have been confirmed to be involved in the process of YedR protein sensing sulfane sulfur.The function of the other two Cyss are still unclear.The results of homology modeling indicated that the mechanism of which the YedR protein senses the sulfane sulfur and thus relieve the suppression may be related to the conformational change caused by the change of protein redox state.Through a series of experiments,the YedE1 E2 proteins were revealed as a possible polysulfide transporter.We confirmed that the YedR protein.a transcriptional repressor.could regulate the downstream genes by sensing the sulfane sulfur,and preliminarily proposed the mechanism that YedE12 protein and YedR protein may cooperate with PDO protein to maintain intracellular polysulfide homeostasis indicating the direction for exploring sulfur metabolism in heterotrophic microorganisms. |
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