Study On The Function Of Quorum Sensing LuxS Protein And Cyclo-(L-Pro-L-Leu) In Shewanella Baltica

In recent years,more and more scholars have been interested in the QS system of food spoilage bacteria.Shewanella baltica with strong spoilage ability is a specific spoilage organism(SSO)in various seafoods.Previous studies have shown that AI-2 and DKPs signal molecules were detected in S.baltica,and the supplement of cyclo-(L-Pro-L-Leu)could enhance the spoilage potential of S.baltica.However,the receptor LuxR protein was not found,and molecular information and function of autoinducers in the spoilage of S.baltica are rare.In this study,the LuxS and LuxR protein in S.baltica was analyzed by bioinformatics.luxS gene-deleted strains was obtained and phenotypes of wild strain and mutant were compared.The molecular mechanism of cyclo-(L-Pro-L-Leu)on S.baltica was analyzed based on the transcriptomics.The main results are as follows:The signal molecules in S.baltica were detected by GC,HPLC-MS/MS,Chromobacterium violaceum C.V026 and V.harveyi BB170 biosensor.AI-2 and cyclo-(L-Pro-L-Leu),cyclo-(L-Pro-L-Phe)were found in S.baltica while AHLs signaling molecules were not.The bioinformatics analysis including the basic information and phylogenetic tree analysis showed that eleven luxR family homologous genes were found in the S.baltica genome,while luxI homologous genes were not.In addtion,eight luxR proteins were related to the two-component system.The protein sequences of AEG 10146.1 and AEG11800.1 were observed to be possible LuxR solo proteins in NCBI based on the phylogenetic tree and conserved amino acid analysis of the LuxR protein of known QS receptors.Two corresponding luxR genes were amplified by PCR in S.baltica SB 11.Meanwhile,the S.baltica SB 11 luxS gene was amplified and ORF of luxS gene was 510 bp.The BLASTP analysis showed that the LuxS protein of S.baltica shared the high sequence similarity(80%)with Shewanella putrefaciens,Vibrio vulnificus and Vibrio parahaemolyticus.Phylogenetic tree and secondary structure of protein analysis revealed that LuxS was a conserved protein and possessed His-54,His-58,Glu-57 and Cys-127,which formed the binding sites for divalent zinc ions.In addition,Ser-6,His-11 and Arg-39 and Glu-93 in LuxS protein played important roles in activity.Three-dimensional structure simulation showed that LuxS protein was composed of four ?-helices and five?-sheets,and the surface of each subunit had a zinc ion binding site.S.baltica SB 11 luxS gene mutant strain was constructed,verified by PCR amplification and AI-2 activity.The growth,biofilm formation,spoilage phenotypes and protein expression were compared between wild SB 11 and mutant at 4? and 28?.The growth of wild strain and luxS-deficient strain had no significant difference,however,the biofilm formation in deficient strain sharply reduced.Compared to wild strain,biofilm in the luxs-deficient strain decreased 20%after incubation for 96 h at 4?,and decreased 28%after incubation at 28? for 24 h.Similarly,the amount of extracellular polysaccharide in wild and mutant strains were 0.060 mg/mL and 0.051 mg/mL for 120 h at 4?,and 0.064 mg/mL and 0.056 mg/mL for 24 h at 28 ? respectively.The cell adhesion levels of the wild type strain and luxS gene-deleted was 7.25 and 6.78 logioCFU/cm2 at 4? for 72 h,and reached to 6.85 and 6.40 Log10CFU/cm2 at 28 ? for 24 h,respectively.Fluorescence microscopic observation showed that SB 11 adhered rapidly to the coverslip surface and accumulated a large number of biofilms,while mutant strain seemed to form a flat sparse biofilm,and the bacteria do not aggregate into clusters.Meanwhile,the absence of luxS.gene promoted the ability of bacterial migration.After 120 h,the diffusion diameter of wild strain was only 80%compared to the diffusion diameter of mutant strain at 4?.After 48 h incubation at 28 ?,the diffusion diameter of wild strain and mutant strain were 71 and 53 mm,respectively.However,the protease activity and production of trimethylamine,putrescine and volatile base nitrogen between wild and mutant was no great difference.It was indicated that luxS gene was involving in biofilm formation,adhesion,swimming,extracellular polysaccharide formation,however,was not assciated with the regulation of spoilage potential in S.baltica SB11.SDS-PAGE also showed that the different protein expression ranging 28 from 44.3 KDa was observed between the mutant and wild-type strain,suggest that the different proteins might participated in the biofilm formation and swimming ability.Exogenous addition of cyclo-(L-Pro-L-Leu)enhanced the biofilm formation,swimming,adhesion,lipase activity,and production of trimethylamineand volatile basic nitrogen formation,while it didn't affect the growth of S.baltica SB11.Transcriptional analysis showed that exogenous addition of cyclo-(L-Pro-L-Leu)could result in 130 and 89 significant differentially expressed genes,in which 122 and 65 genes up-regulated at 4 h and 12 h incubation respectively.According to the GO and KEGG pathways analysis,it was found that ribosome,carbon metabolism,fatty acid metabolism,two-component system,flagellum assembly,bacterial chemotaxis and other metabolic pathways in S.baltica differed significantly.Various adhesion-related genes were upregulated,such as transcriptional regulatory gene regulating CsgAB operon of bacterial extracellular protein fiber(Curli),pilW gene of the stable protein of the Type ? pili,and the homologous gene of Pseudomonas fluorescens lap A,which may promote the adhesion and colonization of S.baltica SB11.Upregulation of multiple flagellum and chemotaxis-related genes caused the increase of bacterial motility,such as fliM,FlgD,FliA,methylation receptor chemokine gene.Futhennore,the gene of c-di-GMP molecule synthetase also upregulated,which enhances the biofilm-forming.After spoilage gene-related genes such as torF,torA,ODC,up-regulated after 12h.In addition,the gene expression level of AEG 11800 significantly increased when exogenous cyclo-(L-Pro-L-Leu)was added,suggesting that it is probably LuxR solo protein.RT-PCR showed that the increasing expression levels of fliA,tor A and ODC genes were consistent with that of the transcriptome group in 4 h and 12 h in the presence of cyclo-(L-Pro-L-Leu).In summary,signal molecules such as AI-2 and cyclo-(L-Pro-L-Leu),cyclo-(L-Pro-L-Phe)were detected in S.balticca.The LuxS protein was conserved,as a functional protein of LuxS protein family.A LuxR family protein in S.baltica was identified,which may be QS LuxR solo.cyclo-(L-Pro-L-Leu)could enhance the spoilage ability of S.baltica.The addition of cyclo-(L-Pro-L-Leu)upregulated the expression of adhesion,motility and corruption related genes in S.baltica by the transcriptional analysis.