Inhibitory Mechanism Of D-tryptophan Based On PpGpp Regulation On Spoilage Of Shewanella Baltica

Shewanella baltica is a dominant spoilage bacterium in seafood because of its capability of growing at low temperature and under high osmotic stress.Previous studies have shown that the exogenous addition of D-tryptophan(D-Trp)can effectively inhibit some pathogenic bacteria in a high-salt environment,companied with the up-regulation of intracellular second messenger guanosine tetraphosphate(ppGpp).However,the ultimate reason of its antibacterial mechanism is still unknown.In this work,we used S.baltica as the research object and tried to investigate the molecular mechanism of the inhibitory effectiveness of D-Trp on S.baltica.We hypothesized that D-Trp alter the osmotolerance of S.baltica by regulating ppGpp signaling pathway.The main research results are as follows:(1)Inhibitory effect of D-Trp on the growth and osmotolerance of S.baltica.By measuring the growth of bacteria at different concentrations of Na Cl and D-Trp,we found that 40 m M D-Trp combined with 3% Na Cl could completely inhibit the growth of S.baltica.The transcriptomic analysis and LC/MS revealed that D-Trp could affect the growth of S.baltica through amino acid metabolism.On this basis,we found that D-Trp effectively inhibited quorum-sensing signaling molecules DKPs(cyclo-(Lphe-L-pro),cyclo-(L-pro-L-Leu),cyclo-(L-Leu-L-Leu)by LC/MS,companied by the reduction of bacterial adhesion,biofilm formation,and drug resistance.The sterile salmon fillet experiment showed that exogenous addition of 40 m M D-Trp significantly reduced the TVC in the fish pieces and delayed the generation of TVB-N,indicating that D-Trp wound reduce the spoilage potential of S.baltica.(2)The regulation mechanism of D-Trp on bacterial intracellular second messenger ppGpp was explored.The transcriptomics revealed that the ppGpp synthesis domain was significantly up-regulated,whereas the ppGpp decomposition gene(Spo T)was significantly down-regulated under D-Trp treatment.High performance liquid chromatography(HPLC)analysis revealed that intercellular level of ppGpp was significantly upregulated after D-Trp treatment.Molecular docking further showed that D-Trp interacted with the active sites of ppGpp synthetic protein Rel A and degraded protein Spo T by van der Waals force,pi-pi bond,and traditional hydrogen bond,which affected the physiological function of the protein.Molecular dynamics analysis also suggest that D-Trp squeezes and stretches the loose protein structure of Rel A and Spo T.q PCR experiments verified the regulation of D-Trp on the synthesis of ppGpp gene in S.baltica.By constructing Rel A,Spo T knockout strains and double knockout strain,we found that the antibacterial activity of D-Trp was reduced,supporting by the fact that the ppGpp knockout strains recovered growth under the inhibitory conditions of D-Trp.These results indicates that D-Trp inhibit bacterial growth by up-regulating the content of ppGpp in bacterial cells.(3)Mechanisms of ppGpp-mediated spoilage in S.baltica.The typical spoilage indicators(p H,TVC,TVB-N,extracellular protease activity)in S.baltica-inoculated sterile salmon fillets were investigated.The results showed that TVC,TVB-N,and extracellular protease activity of ppGpp deletion strain(Δrel A)and ppGpp knockout strain(ΔRΔS)were significantly lower than those of wild strain and knockout strainΔSpo T.The motility experiments indicated that the swimming ability of the strains decreased after ppGpp knockout.Furthermore,bacterial adhesion and biofilm formation are enhanced after ppGpp knockout.CLSM showed that the biofilm of the double deletion strain was thicker and more complete in structure.The extracellular polysaccharide contents were determined by Congo red and phenol-concentrated sulfuric acid methods,and the results showed that the ΔRΔS had significantly higher extracellular polysaccharide production than the wild strain and the single knockout strains.Through transcriptomic analysis and q PCR verification,we found that ppGpp deletion affected flagellar motor genes,trimethylamine synthesis genes,protease activity genes,lipid metabolism genes,exopolysaccharide regulatory genes,biofilm regulatory genes,and amino acid metabolism genes.Our studies could provide a novel preservative for the inhibiting of specific spoilage bacteria in seafood.We also revealed the bacterial inhibition mechanism of D-Trp was partly regulated by ppGpp,which may provide a certain theoretical basis and practical significance for the preservation of aquatic products.