| Sulfur cycling in the biosphere is tightly interwoven with the cycling of carbon and nitrogen,through various biological and geochemical processes.Marine microorganisms,due to their high abundance,diverse metabolic activities,and tremendous adaptation potential,play an essential role in the functioning of global biogeochemical cycles and linking sulfur transformation to the cycling of carbon and nitrogen.Currently many coastal regions are severely stressed by hypoxic or anoxic conditions,leading to the accumulation of toxic sulfide.A number of recent studies have demonstrated that dissimilatory sulfur oxidation by heterotrophic bacteria can protect marine ecosystems from sulfide toxicity.Polysulfides are important sulfide oxidation intermediates that have been detected in a wide array of natural systems ranging from microbial mats to hydrothermal vents,from sediments to water column.Polysulfides are a critical part of sulfur cycling and play important and diverse roles in a variety of biogeochemical processes.Polysulfides,including HSnH(n?2),RSnH(n?2)and RSnR(n?3),are newly discovered cellular components,playing multiple roles in biological systems.They are sulfide oxidation intermediates,and chains of sulfur atoms in which one sulfur atom can move to the thiosulfoxide position on one of the other sulfur atoms.The moving sulfur atom is called "zero valent sulfur","sulfane sulfur",and"sulfur-bonded sulfur".It is believed that tautomerization of persulfides exists,leading to the formation of thiosulfoxide structures.Tautomerization would generate a reactive "singlet sulfur" that would be very electrophilic and nucleophilic.Moreover,the lability of the thiosulfoxide bond provides a facile mechanism for the reversible transfer of sulfur atoms into or out of protein sulfhydryl groups and disulfides.At physiological pH,persulfides are stronger acids compared to thiols,and so thiols predominantly exist in the protonated forms but persulfides(RSSH)exist as the deprotonated anions(RSS-).The disulfide bond in persulfides weakens the RSS-H bond relative to RS-H due to the increased stability of the perthiyl radical(RSS·)compared to thiyl radical(RS·)resulting from the resonance effect.The above structural characteristics undoubtedly contribute to the regulatory functions of polysulfides in biological systems.In sulfur cycling they can be alternatively oxidized,reduced or disproportionated,and are important for a variety of environmentally relevant processes including pyrite formation,organic matter sulfidization,isotope exchange among reduced sulfur species,and metal chelation.Polysulfides also play multiple roles in a wide array of physiological processes,including induction of Ca2+influx,regulation of tumor suppressor phosphatase,and maintenance of redox homeostasis.They also serve as potent regulators of mitochondrial biogenesis and bioenergetics.More than 40%of intracellular proteins are S-sulfhydrated.Higher cellular levels of polysulfide should lead to more protein sulfhydration and confer cells with more resistance to oxidative stress.In particular,it is clear that RSS are more chemically and biochemically versatile than ROS except similarities between ROS and RSS.More and more findings suggest that RSS may be far more prevalent in intracellular signaling than previously appreciated.RSS may be more efficacious than ROS as regulators of redox-sensitive protein cysteines.Clearly,these attributes of RSS,along with their extensive involvement in evolution,suggest that they deserve considerably more attention.Although intense studies have been conducted on polysulfides in recent years,the progress is slow,partly due to the lack of suitable methods for their dynamics analysis and subcellular distribution analysis in living cells.Herein,we report a new generation fluorescent probes that enable noneinvasive,real-time,and subcellular-level monitoring of polysulfides in living cells.These probes are green fluorescent protein(GFP)-based and specific to RSSH-type polysulfides.They can be easily deployed in desired subcellular organelles by fusing with organelle-targeting peptides,as shown in the dynamic analysis of polysulfides in Escherichia coli and Saccharomyces cerevisiae.Using Ruegeria pomeroyi DSS-3 as a model,we analyzed the metabolic mechanism of inorganic and organic sulfur.In order to improve the sensitivity,some chimeric fusion proteins of proper sulfur transferases and psGFP have been explored for their use as probes.Several factors affecting the function of fusion protein probes were analyzed.1.A pair of cysteine residues was introduced near the GFP chromophore with the spatial distance between the cysteine residues designed to allow the formation of internal-Sn-(n?3)bond but not-S2-(disulfide)bond.The GFP-based,polysulfide-specific,and reaction-reversible ratiometric probe(psGFP1.1)has been constructed for the first time.The standard redox midpoint potential of the probe is-318 mV.ROS has minimal interference to the analysis because the probe does not respond to them.At pH 7.0-9.0,the 408/488 ratio(51 0408/510488)is not obviously affected by pH because of the equal increases.We tested the probe in model microorganisms and compared with previously reported probes.Our psGFP has obvious advantages,providing long-desired tools for in vivo studies of polysulfides.The dynamic changes of intracellular polysulfide levels in E.coli growth cycle were analyzed with psGFP1.1 probe and the results were consistent with SSP4 probe.Considering endogenous H2O2 cannot reach as high as 10 mM under normal physiological conditions,the ROS interference can be neglected during polysulfide analysis with psGFP probe.We found that the content of cytoplasmic polysulfides was highly correlated with growth phases in E.coli.The change from 20%to 60%psGFPox is significant,suggesting that polysulfides are likely involved in global gene regulation and enzyme activities associated with growth phases.Using S.cerevisiae BY4742 as a model,we analyzed the polysulfides in the cytoplasm,mitochondria and peroxisomes.The distribution of polysulfides in subcellular organelles is heterogeneous,suggesting polysulfides have multiple origins and functions in cells.CBS and CSE are the major polysulfides-generating enzymes localized mainly in the cytoplasm and mitochondria.3MST and cysteinyl-tRNA synthetase produce polysulfides from cysteine instead of cystine.When cystine was added into S.cerevisiae cell suspensions,the levels of intracellular polysulfides were significantly increased in the cytoplasm,mitochondria and peroxisomes.Peroxisomes had the highest increase.We treated S.cerevisiae with cysteine and found that the polysulfides did not change in peroxisomes but obviously increased in the cytoplasm and mitochondria,implying 3MST and cysteinyl-tRNA synthetase existing in the cytoplasm and mitochondria.2.In order to further improve the detection efficiency,DUF442 and Rd12 proteins with sulfur transferase activity were linked with psGFP protein by linkers and the fusion protein probes of DUF442-psGFP and Rd12-psGFP were constructed for the first time.The ability of probes to sense different sulfane sulfur has been tested in vitro experiments,and the differences in the activity of fusion proteins and unfused psGFP proteins have been compared.Rd12-psGFP1.1 probe is significantly improved and the detection sensitivity is relatively increased.Several factors affecting the activity of the fusion protein have been analyzed in detail,and suggestions for further improvement and optimization of probe are proposed.After DUF442-psGFP,Rd12-psGFP and psGFP were treated with DTT,HSSH,S8,Me-SSS-Me and sodium thiosulfate,respectively,the reaction trend of the three proteins was basically the same.All of them can be oxidized by HSSH and S8,all can slightly sense Me-SSS-Me,and none can react with sodium thiosulfate.The fusion of DUF442 or Rd12 with psGFP has little effect on the activity of psGFP protein.Compared with psGFP,the ability of Rd12-psGFP to sense HSSH and S8 is slightly improved,and the ability of DUF442-psGFP to sense Me-SSS-Me is enhanced to some extent.DUF442 and Rd12 proteins have the activity of sulfur transferase,which can transfer sulfane sulfur from a suitable sulfur donor to a new sulfur receptor.However,in the experiment,DUF442-psGFP and Rd12?psGFP fusion proteins can not sense sodium thiosulfate,and their ability to sense HSSH is not significantly improved compared with that of unfused psGFP protein.It indicates that the fused Rd12 or DUF442 with psGFPs can not transfer sulfane sulfur from sodium thiosulfate or HSSH to psGFP,and have no obvious catalytic activity.The factors affecting the fusion protein activity have been analyzed in detail,including the domain order of fusion protein,the activity of redox enzyme,the design of linker peptide and the position relationship between redox enzymes and fluorescent proteins.3.The intracellular polysulfide level of R.pomeroyi DSS-3 has been detected with psGFP 1.1 for the first time.It is confirmed that R.pomeroyi DSS-3 can produce polysulfides,and oxidize the polysulfide to thiosulfate during the transformation of organic or inorganic sulfur.This indicates that polysulfides may be an important intermediate in the conversion process between organic sulfide and reduced inorganic sulfide,and R.pomeroyi DSS-3 plays a major role in material transformation.The changes of intracellular polysulfide level and redox state in R.pomeroyi DSS-3 growth cycle have been studied.psGFP1.1 and roGFP2 probes have been successfully expressed in R.pomeroyi DSS-3.The intracellular polysulfide level and intracellular oxidation state are related to the growth stages of the Roseobacter Polysulfides are involved in the global gene regulation and enzyme activities in the growth stages,and are accompanied by the change of intracellular oxidation state.At the stable period the intracellular polysulfide level decreases and gradually stabilizes to reach a dynamic equilibrium under the action of both thioredoxin system and glutathione system.The ability of R.pomeroyi DSS-3 to transform organic or inorganic sulfur has been investigated.R.pomeroyi DSS-3 can transform cysteine,methionine,acetone sulfur(S8)and DMSP with polysulfides producing in the cell.Most of them show a trend of increasing and then decreasing,and the polysulfide level generated is the highest during transformation of acetone sulfur(S8).R.pomeroyi DSS-3 has the ability to transform organic or inorganic sulfur into polysulfides,and the intracellular redox state changes in the process of transforming organic or inorganic sulfur into polysulfides.The metabolites in the process of R.pomeroyi DSS-3 transforming organic or inorganic sulfur have been detected,and the metabolic mechanism has been analyzed.R.pomeroyi DSS-3 can produce thiosulfate by converting cysteine,sodium hydrosulfide and acetone sulfur(S8).The most is produced by converting cysteine,while thiosulfate is not produced by converting methionine and DMSP.In the experiment,R.pomeroyi DSS-3 do not accumulate sulfites in the cell.This can be explained as follows:due to the slow oxidation rate of polysulfides in the cell,and the reaction between the sulfites generated by oxidation and the accumulated polysulfide takes place rapidly to produce thiosulfate.In summary,we have constructed GFP-based polysulfide-sensitive ratiometric probes to realize noneinvasive,real-time,and subcellular-level monitoring of polysulfides in living cells.These probes can be easily deployed in desired subcellular organelles by fusing with organelle-targeting peptides,as shown in detecting the dynamics of polysulfides in E.coli and S.cerevisiae.In order to improve the sensitivity,some chimeric fusion proteins of proper sulfur transferases and psGFP have been explored for their use as probes.Several factors and suggestions for further improvement and optimization of fusion protein probes have been proposed.Using R.pomeroyi DSS-3 as a model,we have detected the x intracellular polysulfides level with psGFP1.1,proving that R.pomeroyi DSS-3 can produce polysulfides during the transformation of organic and inorganic sulfur,and oxidize the polysulfides to thiosulfate.We have analyzed the metabolic mechanism of inorganic and organic sulfur thoroughly.This work offers an effective tool for the detection of intracellular polysulfide levels.Delineating the sulfur metabolic pathways of marine bacteria not only provides an insight into the ecological roles of marine bacteria in sulfur cycling,but also helps us understand the effects of changing environmental conditions on marine sulfur cycling and reinforces the close connection between sulfur and carbon cycling in the ocean. |
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