Cellular Location Of Key Enzymes In Hydrogen Sulfide Oxidization Pathway And The Metabolism Of Its Product Thiosulfate In Cupriavidus Pinatubonensis JMP134

Sulfur played a significant role in the origin of life,and its varity complex and reactive compounds are essential in the geochemical cycle,meanwhile,they also have vital function in the metabolism.The oxidation of sulfur,as a critical component in the geochemical sulfur cycle,generally refers to the oxidation process of elemental sulfur or reductive sulfide by microorganism.The final product of sulfur oxidation is sulfate which affluxes into the ecosystem,with polysulfide and thiosulfate as intermediates in the oxidation process.Sulfide,especially hydrogen sulfide(H2S),has an excellent ability to penetrate the plasma membrane and causes toxic effects to various metabolic processes in living organisms.However,except its toxicity,H2S is confirmed to be a new gasotransmitter after the CO and NO at the 1990s.It is involved in many physiological processes like apoptosis,neuromodulation,cytoprotection,by acting on ion channels,transcription factors and kinases.In the environment,H2S mainly comes from the volcanoes,hot springs or the deep ocean vents and the artificial industrial processes.And it is produced mainly from cysteine and iron-sulfur proteins in various organisms under aerobic conditions There are three enzymes,cystathionine y-lyase(CSE),cystathionine ?-synthetase(CBS)and 3-mercaptopropionate sulfur transferase(3-MST)participated in this reaction.Due to the dual function of H2S,the biosome in sulfur-rich environment developed a specific H2S metabolism to maintain its concentration in a range,with excess H2S being metabolized in cells.The endogenous produced H2S could be metabolized by different oxidation pathways.The most important biological oxidation pathway(SQR-PDO pathway)consists of sulfide:quinone oxidoreductase(SQR),persulfide dioxygenase(PDO)and sulfur transferase(RHOD)and exists in some autotriphic,heterotriphic bacteria and mammalian mitochondria.SQR oxizdes H2S to polysulfides which transfer a sulfane sulfur to glutathione(GSH)automatically or sped up by RHOD to form persulfide glutathione(GSSH).GSSH was oxided to sulfite by PDO with GSH released.The sulfite could react with excess polysulfides to generate thiosulfate,spontaneously or accelerated by RHOD.The final product thiosulfate would be oxided to sulfate by sulfur oxidation system(Sox system).As the first enzyme in the SQR-PDO pathway,SQR catalyses the convertion of H2S to polysulfides and is critical for the whole sulfur metabolism.This enzyme is widely distributed in mammalian mitochondria and microorganism.According to structural and sequence analyses,SQRs are divided into six types.The type ? SQRs play a role in sulfide-dependent energy conservation for chemolithotrophic or phototrophic growth.Type ? SQRs were recently identified from animal mitochondria and bacteria,and they may play a detoxification role.To date,studies with SQR mainly focus on its biochemical functions and crystal structures.The X-ray structure of the Aquifex aeolicus SQR confirmed it is an integral monotopic membrane protein with an amphipathic motif of a helix-turn-helix inserting about 12 A into the lipid bilayer.However,it didn't show which side of the plasma membrane it's on.Only the phototrophic bacteria Rhodobacter capsulatus SQR carrying a fusion protein of alkaline phosphatase(PhoA)suggests its periplasmic localization.Hence,there is no evidence on the structure and localization of SQRs in heterotriphic bacteria.At the meantime,the periplasmic space localization of PDOs in chemolithotrophic Acidithiobacillus spp.is also controversial,due to the lack of signaling peptides.Based on this background,we studied the subcellular localization of SQR and PDO in heterotriphic bacteria Cupriavidus pinatubonensis JMP134 by sequence analysis and gene fusion techniques.C.pinatubonensis JMP134 contains an SQR(CpSQR)that is a fusion of a rhodanese domain and a type ? SQR domain.And it contains two PDOs with PDO2 has dominated catalytic activity.The results of sulfide oxidation and SDS-PAGE of whole cell lysis,membrane component and soluble proteins,and the fluorescence imaging of CpSQR-GFP fusion protein,all showed CpSQR is a membrane protein.Amino acid sequence analysis of CpSQR showed that it consists of 558 amino acids,with 1 to 128th amino acids being the N-terminal DUF442 domain,and 158 to 554th amino acids being the C-terminal SQR domain.Four segments were predicted to possibly trans the cytoplasmic membrane:amino acids(aa)147 to 175(29 aa),or 150 to 170(21 aa),213 to 229(17 aa),and 343 to 358(16 aa).There was an atypical a helix before amino acid residues 30,62,84,107,and 125.PhoA can catalyse the hydrolysis of phosphate groups only in periplasmic space,while GFP is only active in the cytoplasm,so GFP is a suitable complement to PhoA,Based on these information,we constructed fragmentx-PhoA/GFP(x=26,49,71,83,107,115,128(DUF442);157,176,230,359,450,558(CpSQR))fusion proteins,in which PhoA without the signal peptide or GFP was linked to the C-terminally truncated CpSQR at the x sites.There was no PhoA activity in all fragmentx-PhoA fusion proteins,but all the fragmentx-GFP fusion proteins expressed GFP and the DUF442 domain is a solution protein when it expressed alone in E.coli.Therefore,we determined CpSQR is a peripheral membrane protein orienting on the cytoplasmic side of the membrane.CpPDO2 is likely a cytoplasmic protein because it does not have a signal peptide for membrane translocation,uses ferrous ion(Fe2+)for catalysis and can be massive purified as soluble protein.The results of activity detection of constructed PDO-PhoA/GFP fusion proteins confirmed our hypothesis.Interestingly,we detected green fluorescence around the cell surface in E.coli or C.pinatubonensis JMP134 containing PDO-GFP fusion protein.We then constructed several GFP fusions with PDOs from other bacteria,same phenomenon was observed.The peripheral localization in cytoplasmic of CpPDO2 and other tested bacterial PDOs maybe an intrinsic property,as a significant number of cytoplasmic proteins may interact with membranes via amphipathic ?-helixes or hydrophobic patches.According to the above analysis,the location of CpSQR and CpPDO2 close to the membrane may facilitate sulfide oxidation via metabolic channeling,enabling the bacterium to rapidly detoxify sulfide to thiosulfate.Next,we detected the thiosulfate metabolism pathway--the sulfur oxidation system(Sox).Sox system consisting of periplasmic proteins SoxYZ,SoxXA,SoxB and SoxCD,is a thiosulfate-oxidizing multienzyme complex system which was representative in Paracoccus pantotrophus GB17 and oxidizes thiosulfate to sulfate.Initially,by the enzyme SoxXA,thiosulfate is conjugated to a cysteine residue in the carrier protein SoxZY to produce SoxZY-S-thiosulfonate.Next,the terminal sulfonate group is hydrolytically removed by the enzyme SoxB to form sulfate,while the newly-exposed terminal sulfane group then oxidized to the corresponding sulfonate by the enzyme SoxCD.Finally,the sulfonate group is removed by the action of SoxB.In C.pinatubonensis JMP134,there is an intact sox system encoded by soxR-soxC-soxD-Cyc-soxY-soxZ-hyp-soxA-soxX-TrxA-soxB responsible for thiosulfate oxidation at the chromosome A.We analysed the effects of Sox core protein defections on the metabolism of thiosulfate and found strains with SoxYZ,SoxXA,SoxB mutant completely lost their ability of thiosulfate oxidation,while the SoxCD mutant could still oxide thiosulfate slowly.Then,we detected the consumption of hydrogen sulfide or polysulfide by Sox system.Owing to the SQR and FCSDs(SoxEF)could oxide hydrogen sulfide to polysulfide and PDO12 could oxide poly sulfide to sulfite,we carried out experiments in different knockout strains.In the strain Cp 4K,Cp 4K?soxYZ and Cp?pdo12,Cp?pdo12?soxCD,we detected no difference on the oxidation of hydrogen sulfide and polysulfide.These indicates Sox system could not oxide hydrogen sulfide and polysulfide,and its prominent function is oxiding thiosulfate.Finally,we anslysed the regulation of Sox system.In the chromosome A of C.pinatubonensis JMP 134,a soxR gene encoded an ArsR family transcriptional regulator is upstream of soxC.SoxR only has a HTH DNA binding domain,we then constructed a soxR-deletion(Cp?soxR)mutant by knocking out this domain in C.pinatubonensis JMP134.By comparing the thiosulfate oxidation rate of various strains,we found the CpAsoxR mutant obtaining higher ability to oxidize thiosulfate.The transcription level of soxC was up-regulated,indicating SoxR could be a repressor which inhibits the express of sox genes.Reverse-transcription PCR experiments showed the transcription from soxR to soxB is in the same mRNA.5'-RACE experiments identified the transcription starting point of soxR was the guanine nucleotide(G)at-26 bp upstream of the soxR initiation codon.SoxR specifically bound to the palindrome at the downstream of transcription starting point by Electrophoretic Mobility Shift Assay(EMSA).Thiosulfide,as well as hydrogen sulfide,polysulfide,acetone sulfur and tetrathionate could increase the GFP expression of the reporter plasmid pBBR5-Pr-soxR-gfp in Cp 3K ?soxR which knocked out gene sqr/pdo1/pdo2 and soxR in C.pinatubonensis JMP134.Meanwhile,the fluorescence intensity of the GFP reporter system was increased with the concentration of these compounds and their relationship could fitted by Hill equation.Nonetheless,when test the effect of different reagents on the binding of SoxR and DNA by EMAS,we found only polysulfide and acetone sulfur which belongs to sulfane sulfur played a significant role that dissociated DNA from the SoxR protein.This result indicated it's sulfane sulfur not thiosulfates that interact with SoxR proteins directly.When the Cys43 and Cys98 in the SoxR protein were individually mutated to serine in the reporter plasmid pBBR5-Pr-soxR-gfp,the plasmid can not response to thiosulfate,implying these two cysteins have important function.Although CpSoxR has low indentity with SoxR in P.pantotrophus GB7 and P.salicylatoxidans KCT001,they all belong to a wingled HTH repressor subfamily of ArsR family without metal ion binding site,and are induced by thiosulfate but don't interact with it directly.CpSoxR only has one DNA binding site at the PsoxR promoter,while PpSoxR and PsSoxR both have two DNA binging sites at the intergenic regions of soxS-V and soxW-X.The above results suggest that the regulation of Sox system in heterotrophic bacteria C.pinatubonensis JMP134 is similar to that of autotrophic bacteria P.Pantotrophus GB17 and P.Salicylatoxidans KCT001.They are all inhibited by the ArsR family transcription factor SoxR.Thiosulfate can eliminate this inhibition,activate downstream genes expression,and accelerate the metabolism of thiosulfate.