The Investigation Of The Formation,transfer,and Inhibitory Effect Of Elemental Sulfur In Microorganisms

Sulfur is the fifth most abundant element on Earth and one of the main elements in biogeochemical cycles.Sulfur is important and indispensable for all living organisms.Sulfur is commonly found in nature in the form of sulfide,sulfate and elemental sulfur.Hydrogen sulfide(H2S)is an acute and highly toxic substance,but recent studies have shown that H2S is the third type of signaling molecule after carbon monoxide and nitric oxide,and is involved in various physiological processes in living organisms.Many heterotrophic bacteria can use sulfide:quinone reductase(SQR)and persulfide dioxygenase(PDO)to oxidize H2S,referred to as SQR-PDO pathway.In addition,sulfur transferase(ST)can also be involved.In the SQR-PDO pathway,SQR first oxidizes sulfide to polysulfide(H2Sn,n≥2),H2Sn reacts with glutathione(GSH)spontaneously or with the help of ST to generate glutathione persulfide(GSSH),GSSH is oxidized by PDO to sulfite,sulfite and GSSH generate thiosulfate spontaneously or with the help of ST.H2Sn is often considered to be the final product of H2S oxidation by SQR.It is unclear whether elemental sulfur(S8),the more stable form of sulfane sulfur,is a product of SQR.If S8 is the product of SQR,there are several questions surrounding the generation of S8:How is S8 formed during H2S oxidation by SQR?Is S8 accumulated inside or outside of the bacterial cell?Can S8 produced by heterotrophic bacteria form sulfur globules?These questions should be studied.S8 can enter cells,but there is currently no clear understanding on how S8 is transferred.S8 is often used as fungicides,but the antifungal mechanism is unclear.GSH is the main reducing thiol in fungi and plays an important role in antioxidation.GSH can react with S8 to generate H2S and glutathione disulfide(GSSG).Are H2S and GSSG related to the toxic effect of S8?In order to answer the questions,the research contents of this thesis are as follows:1.Two SQR containing heterotrophic bacteria:Ec(Cpsqr)(Escherichia coli MG 1655 expressing the sqr gene from Cupriavidus pinatubonensis JMP134)and Corynebacterium vitaeruminis DSM20294 that contains an sqr gene were selected to detect their products of sulfide oxidation.The two bacteria without PDO do not further oxidize H2Sn and GSSH.They oxidized H2S to mainly S8.Using transmission electron microscopy and energy spectrum scanning,it was found that S8 accumulated in the cytoplasm in the form of sulfur globules.The effect of intracellular sulfur globules on the physiology of heterotrophic bacteria was further investigated.The production of sulfur globules resulted in a reduction in celllular thiols with the accumulation of GSSG.The process of sulfide oxidation by Ec(Cpsqr)cell membrane fractions and the reaction of GSH and S8 were studied in vitro,and H2Sn(n≥2),GSnH(n≥2)and GSnG(n≥2)were generated during S8 formation.The S8 formation was a complex process with the dynamic changes of intermediate products.On the bassi of the results,a model of S8 formation by SQR oxidizing sulfide in heterotrophic bacteria was proposed.S8 may be formed in three ways:(1)SQR oxidizes sulfide to generate short chain H2Sn,whic reacts with intracellular GSH to generate short chain GSnH,The short chain GSnH will further react with GSH to generate GSnG and H2S,the generated H2S will be oxidized by SQR again,and finally the intracellular GSH will be consumed,and the short chain H2Sn will elongate to H2S9,before spontaneously forming S8.(2)GSnH generated during SQR oxidation of sulfide may react with another GSnH to generate H2Sn and GSnG,and H2Sn elongate to H2S9 to spontaneously form S8.(3)GSnH may react with H2Sn to generate GS9H,which spontaneously forming S8 and GSH.H2Sn is a key intermediate in the three pathways of S8 formation.These three pathways may occur simultaneously,but because the intracellular GSH content is limited,the first pathway is the main source of S8 formation.After S8 accumulates in heterotrophic bacteria,it will form sulfur globules2.Different bacteria were studied,and the results show that two S8 transfer phenomena,S8 transmission between bacteria and S8 transmission from extracellular to intracellular,are very common in nature.Using filter to separate S8(bacteria with S8 accumulation or added S8)and the recipient bacteria,the sulfur transfer stopped.The results indicate that S8 transmission requires direct contact with the recipient bacteria.It ruled out the possibility of S8 being converted into soluble substance to enter the recipient bacteria.Using gene knockout and preparation of GSH liposomes,the possibilities of S8 transmission via known protein-assisted mechanisms were ruled out.Further,S8 was found to accumulate in the cell membrane,as S8 was soluble in phospholipid bilayer.When S8 was mixed with bacterial cells,S8 was immediately adsorbed around bacterial cells,as shown by centrifugation through a sucrose gradient and electron microscopy.Further analysis showed that S8 could also be dissolved into lipopolysaccharide and peptidoglycan,the main component of cell envelope.The mechanism of S8 transmission was proposed.When the bacteria with S8 accumulation are in direct contact with the recipient bacteria,or when S8 in the environment is adsorbed on the recipient bacteria S8 can enter the cell by dissolving in the cell membrane,and the whole process does not require any protein assistance.3.The effects of S8 on three microorganisms:E.coli,Staphylococcus aureus and Saccharomyces cerevisiae were tested by measuring growth curve,minimum inhibitory concentration,and viability.S.cerevisiae was the most sensitive to S8.S8 rapidly entered S.cerevisiae cells,and inside S8 reacts with intracellular GSH to produce H2S and GSSG.The effects of H2S and GSSG on S.cerevisiae were investigated separately.H2S inhibited the oxygen consumption rate of S.cerevisiae,but the inhibition could be relieved by removing H2S.The H2S inhibition was identify on the electron transport chain of S.cerevisiae.GSSG had a different effect,increasing disulfide stress and oxidative stress.Thus,the mechanism of antifungal effect of S8 is via its function as a strong oxidant.When S8 enters the fungal cell,it will oxidize the intracellular GSH to GSSG,and produce H2S.H2S can inhibit the electron transport chain to reduce ATP synthesis and glucose metabolism.Accumulation of GSSG causes fungal cells to experience oxidative stress.Both H2S and GSSG inhibited fungal growth.In summary,this thesis focuses on the process of SQR containing heterotrophic bacteria oxidizing H2S to form S8 intracellularly under aerobic conditions,the mechanism of S8 transfer through dissolution in the cell membrane,and the mechanism of S8 as a strong oxidant inhibiting fungal growth.These studies contribute to a better understanding of the role of microorganisms in the sulfur cycle in nature,and have implications for the use of S8 to treat fungal infections in plants and animals.