Study On The Mechanism Of Polyphosphate Metabolism And Stress Response Of Deinococcus Radiodurans

The extremophilic bacterium Deinococcus radiodurans（DR）,known for its high resistance to oxidative stress and excellent DNA damage repair,relies on a variety of defense systems including its polyphosphate（polyP）metabolic products and unique cellular envelope structures.In DR cells,polyP synthesis is mediated by polyphosphate kinase（DrPPK）,and polyP degradation is mediated by exopolyphosphatase（DrPPX）into monophosphates.However,the molecular mechanisms of polyP metabolism in DR and its response to environmental stress remain unclear.This study employed biochemical and molecular biology,biophysics,and computational biology techniques to investigate the molecular basis of DrPPX catalytic function and structural adaptive evolution mechanism of PPX homologous proteins,and explore liquid-liquid phase separation formation of intracellular polyP and its related cellular stress response.This study also investigated the interaction between DR and environmental pollutant-nanoplastics.The conclusions were obtained as follows:1)Molecular mechanism of DrPPX cleaving its substrate polyP was clarified.Protein complex structure analysis of DrPPX revealed that the aspartic acid residue D7 formed a coordination bond with divalent metal ions and water molecules,while E114,D136,and E143 polarized the conserved water molecule to launch a nucleophilic attack on the terminal phosphate group of polyP,leading to the breakage of high-energy phosphate bonds between the terminal phosphates of polyP and release of a phosphate ion（Pi）.When small-molecule metabolites such as PO₄^3-,P3（polyP with three Pi units）occupied the active site,they can competitively inhibit and regulate the polyP hydrolytic activity of DrPPX.Meanwhile,the signaling molecule ppGpp might also act as an inhibitor on DrPPX.2)By comparing the differences in stuctures of PPX homologs,we found a negative correlation between the length ofα-helical interdomain linker（α-linker）connecting the N-and C-terminal domains of PPX and the overall structural stability as well as enzymatic activity of PPX homologs.PPXs with a shorterα-linker exhibited stronger domain interaction and enzymatic activity.We extended this rule to the PPX homologous proteins from the extreme bacteria of Deinococus Thermus phylum.Evolutionary analysis and biochemical experiments indicate that PPXs from bacteria such as Thermus,Meiothermus,and Vulcaniibacterium,which can tolerate high-temperature environments such as deep-sea hydrothermal vents and volcanoes（where often contain high polyP concentrations）,had shorterα-linker and stronger polyP degradation activity and thermal stability.These findings demonstrate the adaptive evolutionary characteristics of PPX molecular structure and provide a reference for the rational design of polyP-degrading enzymes with high efficiency.3)The existence and dynamic changes of coacervate droplets of intracellular polyP in DR were discovered.DrPPK and DrPPX were involved in the formation and dynamic process of coacervate droplets mediated by liquid-liquid phase separation（LLPS）of intracellular polyP.The key binding sites for electrostatic attraction of the DrPPK with negatively charged polyP were the conserved positively charged residues R404,R434,K462,R644,R649.On the other hand,DrPPX protein interacted with polyP droplets,leading to droplet dissolution.PolyP coacervate droplets underwent dynamic changes under oxidative stress and might participate in the regulation of stress-response proteins in the cell.Proteomic analysis identified potential interacting proteins in the DrPPK-polyP coacervate droplets,which were involved in various pathways.Among these proteins,DrMFD and DrRnpA participated in nucleotide excision repair and tRNA maturation,respectively.In vivo and in vitro experiments confirmed the interaction between DrMFD,DrRnpA and DrPPK-polyP coacervate droplets.It is proposed that DR cells may shield these important proteins from environmental stress damage in the polyP droplets formed by LLPS,and then use DrPPX to disassemble the droplets and release the proteins to participate in cell recovery from damage when the stress is relieved.These results explored the dynamic regulation function of polyP and oxidative stress response of DR from the perspective of LLPS.4)We also investigated the interaction between biological cells including DR and environmental pollutant-nanoplastics.The absorption capacity of D.radiodurans,E.coli,and Bacillus subtilis for polystyrene nanoparticles（PS nanoparticles）was studied.The results showed that the entry of nanoparticles into bacteria was mainly depending on physical characteristics such as surface charge and particle size,as well as cell envelope structure of bacteria.Positively charged PS nanoparticles（PS-NH₂,80 nm）could effectively penetrate and accumulate inside the cells by forming strong electrostatic interactions with the negatively charged cell membrane.PS-NH₂ caused the disruption of the cell membrane structure,resulting in cell growth inhibition and death.Neutral（PS）and negatively charged PS nanoparticles（PS-COOH）could hardly enter the cells.Compared to E.coli and B.subtilis,D.radiodurans,which has a thicker and more complicated cell envelope structure,absorbed and accumulated fewer nanoparticles.Further studies using fluorescence labeling and molecular dynamics simulations revealed the action characteristics and transmembrane uptake mechanism of nanoparticles with different charge on bacterial cell membranes.Moreover,we showed that nanoparticles could enter crops and accumulate in the fruits,indicating that nanoplastic pollutants may affect ecosystem and food chain safety.In summary,this study elucidated the mechanism and molecular basis of polyP degradation by the exopolyphosphatase DrPPX in DR,as well as the structural differences and adaptive evolution of different PPXs.The study also revealed the existence and dynamic process of polyP coacervate droplet,and demonstrated the functional involvement of polyP metabolism in oxidative stress response of DR.Additionally,the study investigated the interaction between cells including DR and nanoplastics,providing a reference for evaluating the relevant risks of nanoplastics to ecosystem.These findings enriched the understanding of polyP metabolism and provide new ideas for the directed evolution and rational design of efficient polyP degrading enzymes,and provided new insights into the stress-response mechanisms of DR.