Studies On The Thiosulfate Oxidation Pathway In Sulfur-reducing Shewanella Oneidensis

Thiosulfate,an important form of inorganic sulfur components,contains two sulfur atoms both at intermediate but different chemical valences（0 and+4）,thus a myriad of diverse microorganisms is able to obtain energy required for cell growth with thiosulfate as either an electron donor or acceptor.Shewanella oneidensis,a Gram-negative facultative anaerobicγ-proteobacterium renowned for its extraordinary respiratory versatility,has been extensively studied as a research model in the field of bacterial respiration.Under anaerobic conditions,S.oneidensis is able to reduce thiosulfate or elemental sulfur to sulfide,yet whether it is able to oxidize thiosulfate remains elusive.Aiming to fill this gap,this study investigates thiosulfate oxidation in S.oneidensis.The complex electron-transfer network made by multiple cytochrome c（cyt c）proteins located in the cytoplasmic membrane,the periplasm and the outer membrane of S.oneidensis is the basis of its respiratory versatility.In the known thiosulfate oxidation systems like Sox and S₄I,several critical enzymes are also cyt c proteins.Therefore,in this study,we used BLASTp with established thiosulfate-oxidizing enzymes as target proteins to search against the S.oneidensis proteome for homologous proteins.The results showed that S.oneidensis lacks homologues of typical Sox enzymes but encodes two homologues of a thiosulfate dehydrogenase in the S₄I pathway,SO＿4047 and SO＿4048,both of which are diheme cyt c proteins located in the periplasm.Based on amino acid sequence alignment and 3D structure prediction,SO＿4047 and SO＿4048 are homologous to the C-terminal and the N-terminal regions of Mp Tsd respectively,a thiosulfate dehydrogenase of the photosynthetic purple sulfur bacterium Marichromatium purpuratum,with an overall amino acid sequence identity of 39%and well-conserved residues in the critical active sites.In-frame deletion mutation and complementation experiments confirmed that SO＿4047 and SO＿4048exhibited thiosulfate-oxidizing activity and was the only enzyme for the reaction in S.oneidensis.Based on all of these,SO＿4047 and SO＿4048 were named Tsd A and Tsd B,which are subunits of thiosulfate dehydrogenase directly bound to substrates and the electron-accepting partner,respectively.In S.oneidensis,dissimilatory thiosulfate reduction depends on Psr ABC complex,catalyzing a reaction converting thiosulfate to sulfide and sulfite.Further exploration uncovered that S.oneidensis preferentially oxidized rather than reduced thiosulfate under aerobic conditions,and a small fraction of thiosulfate could be reduced only when the Tsd system was absent.By measuring the promoter activity,it was found that the promoter activity of psr under aerobic conditions was much lower not only than that of tsd,but also than itself under anaerobic conditions.Therefore,the low transcription level of psr under aerobic conditions appears to be,at least partially,responsible for the preferential oxidation of thiosulfate.Surprisingly,thiosulfate oxidation reduced the overall cyt c content,which is related to lowered intracellular c AMP levels.As a global regulatory factor,c AMP-Crp direcetly regulates expression of many cyt c genes,hence the reduced content of c AMP-Crp could result in decreased expression of the downstream genes,including those encoding cyt c proteins.The genome of S.oneidensis encodes two functional terminal oxidases to support aerobic growth,heme-copper oxidase（HCO）cyt cbb₃ and quinol oxidase cyt bd.In M.purpuratum,electrons generated from thiosulfate oxidation are suggested to be ultimately delivered onto its cyt cbb₃ oxidase to facilitate aerobic respiration.However,in this study,we found that neither cyt cbb₃ nor cyt bd could serve as the direct electron acceptor for the Tsd system in S.oneidensis.In addition,when S.oneidensis cells consume thiosulfate by oxidation,the terminal oxidase that supports cell growth is cyt bd.Finally,through genomic and phylogenetic analysis,we found that a large number of and diverse bacterial species encode either thiosulfate reductase or oxidase system in their genome,but those,like S.oneidensis,possess both thiosulfate reductase and dehydrogenase are rather limited,which is another important finding of this study.In conclusion,by focusing on the dissimilatory metabolism of thiosulfate in S.oneidensis,this study reveals the physiological functions of two previously unknown cyt c proteins and illustrates its thiosulfate oxidation characteristics and related molecular mechanisms.By doing so,the research gap in the field of bacterial thiosulfate oxidation is filled.