Regulation Of RpoS/RpoN Protein In Shewanella And Pseudomonas Isolated From The Fish

Microbial activity is by far the major factor affecting the quality of fresh aquatic product.Shewanella baltica and Pseudomonas fluorescens are widely known to be responsible for specific spoilage organisms（SSO）in refrigerated marine fish,due to the high amine metabolism,the strong proteolytic ability and biofilm forming ability respectively.Those specific spoilage bacteria could encounter a series of stresses in the various unfavorable environmental condition during the processing and storage of food,which is closely related to the bacteria stress response factor Sigma.The RpoS and RpoN in bacterium,as main proteins of Sigma factor 70 family and 54 family,respectively,playing important regulatory roles in the metabolic processes,such as stresses condition,swimming and virulence factor secretion.However,few study about sigma factor in food spoilage bacteria was reported.To explore the survival and metabolism of spoilage bacteria under various stresses condition,the regulation of RpoS/RpoN protein in Shewanella baltica and Pseudomonas fluorescens were investigated.The survival and biofilm formation of two fish spoilage bacteria in three kinds of stress condition were assessed.Then bacterial growth,biofilm biomass,motility,stress resistance and spoilage potential were compared between wild type（WT）and mutants of rpoS and rpoN constructed by suicide plasmid and homologous recombination.Additionally,the differences in gene transcription levels and related metabolic pathways in RpoN mutant of S.baltica were analyzed.The research will provide a new theoretical basis for revealing the mechanism of food microbial spoilage,and lays a solid foundation for the healthy development of the aquatic industry.The main results are as follows:S.baltica and P.fluorescens showed significant changes in growth,biofilm biomass and protease activity under different culture temperature,nutrient and oxidative stress conditions.At 10℃,20℃,and 30℃ the biofilm was easily formed,and was slower at4℃.Four bacteria did not grow at 37℃.The amount of biofilm biomass decreased sharply at 1/5LB concentration,and the biofilm biomass increased in the addition of glucose at 2%or higher concentration.However,stress cultures suppletmented 1%³%NaCl inhibited biofilm.The concentration of metal ions(Fe³⁺,Cu²⁺,Mg²⁺,Ca²⁺)greatly affected the growth and biofilm biomass of two spoilage bacteria,in which 10mmol/L Ca²⁺,1mmol/L Mg²⁺,0.3mmol/L Cu²⁺,0.3mmol/LFe³⁺promotes biofilm biomass.In the presence of 0¹mg/mL acetic acid and 0⁰.2%sodium hypochlorite,the biofilm biomass and adhesion of S.baltica and P.fluorescens decreased.The adhesion ability of S.baltica did not affect significantly supplemented by 3%NaCl and 3%Glucose,while the adhesion of P.fluorescens decreased under 3%NaCl,but increased under 3%Glucose.The protease activity decreased in the four strains under 3%NaCl,and was promoted in 3%Glucose.The RpoS protein similarity between S.baltica and P.fluorescens was 86.16%.Two mutants of rpoS gene（ΔrpoS）in S.baltica and P.fluorescens were constructed.The differences in bacteria growth,biofilm biomass,stress resistance and spoilage potential of wild-type strains（WT）andΔrpoS strains were constructed and compared.It were found that the deletion of rpoS gene did not affect the growth of two spoilage potential and the biofilm biomass of S.baltica.P.fluorescens（ΔrpoS）was significantly higher than wild-type strain in high glucose,high sodium chloride and acidic environment.S.baltica（ΔrpoS）has reduced motility and flagellum,but rpoS deficiency does not affect the motility of P.fluorescens.In the sterilized fish juice,the content of TMA of the S.baltica（ΔrpoS）was decreased,the EPS production was increased,protease activity and TVB-N was not affected.The protease activity and EPS of P.fluorescens（ΔrpoS）was increased.The survival rate of the wild-type strain of S.baltica in 30%NaCl,10%ethanol,45℃,10mmol/L hydrogen peroxide,0.001%sodium hypochlorite,150μg/mL crystal violet stress were 1.25,2.42,4.55,1.76,8.32 and 3.62 times ofΔrpoS,respectively.P.fluorescens wild-type strain were 1.01,9.00,1.11,8.80,1.26 and 1.31 times ofΔrpoS strain.RpoS protein significantly enhances the ability of S.baltica and P.fluorescens to resist stress environment（P<0.05）.The similarity of RpoN protein between S.baltica and P.fluorescens was 86.24%.Two mutants of rpoN gene（ΔrpoN）in S.baltica and P.fluorescens constructed,and the strain growth,biofilm biomass,stress resistance,drug resistance,spoilage potential and quorum sensing of WT andΔrpoN strains were compared.Results showed that twoΔrpoN delayed the growth rate,but did not affect the cell number.TheΔrpoN strains retarded the biofilm development of spoilage bacteria,and theΔrpoN formed higher biofilm biomass than the wild-type for 36h at 30℃.High sodium chloride and acidic stresses inhibited the biofilm formation,especiallyΔrpoN strains.Compared with WT,two mutants basically lost swimming and had no flagellum structure observed by transmission electron microscopy.Moreover,except 30%NaCl and 10mmol/L hydrogen peroxide,the survival rate inΔrpoN of S.baltica treatd by 10%ethanol,45℃,0.001%sodium hypochlorite and 150μg/mL crystal violet apparently reduced,while the survival rate inΔrpoN of P.fluorescens only decreased in the treatment of hydrogen peroxide and sodium hypochlorite.Furthermore,P.fluorescens has more severe antibiotics resistant compared with S.baltica.Among the 19 antibiotics tested,ΔrpoN of S.baltica weakened resistance to 18 antibiotics,whileΔrpoN of P.fluorescens attenuates15 antibiotic resistance.Additionally,the deletion of rpoN gene in two spoilage strains greatly affected quorum sensing activity.TheΔrpoN strain significantly reduced two DKPs in S.baltica,including cyclo-（L-Pro-L-Leu）and cyclo-（L-Pro-L-Phe）involving in the regulation of spoilage enzymes,whileΔrpoN decreased the AHLs activity in P.fluorescen,especially C4-HSL,which caused to interfer with quorum sensing regulatroy function.Transcriptomics analysis explored the regulation of RpoN protein in S.baltica.Fourteen flagellum assembly genes was down-regulated,which was consistance with the decrease of swimming inΔrpoN.A large number of genes involving in carbon and nitrogen metabolism pathways were down-regulated,particularly for the amines metabolism related spoilage metabolites in S.baltica.The genes associated with biofilm formation,stresses,drug resistance,protease and quorum sensing were down-regulated inΔrpoN,which were consistant with the above phenotypic changes.The reliability of transcriptomics in S.baltica were verified by RT-PCR.Five genes expression related biofilm,flagellum,protease,and LuxR in Pseudomonas phenotypes were also found to decrease,similarly to effects of phenotypes.In summary,RpoS and RpoN in S.baltica and P.fluorescens affected the biofilm biomass,survive in stresses,and spoilage potential.However,the regulation differed slightly for two sigma factors.RpoS protein mainly regulated the response to oxidative and acidic stresses in spoilage bacteria,partially affect spoilage and motility,and did not influence growth and biofilm formation.RpoN protein in two spoilage strains are mainly responsible for regulation of carbon and nitrogen metabolism,flagellum assembly and biofilm formation and partially affected the resistence to stresses and antibiotics.Furthermore,RpoS and RpoN regulate the physiological functions of S.baltica and P.fluorescens,and RpoN is mainly involved in cell biofilm synthesis,oxidative phosphorylase synthesis transport,flagellar growth,etc.RpoS is mainly responsible for the anabolism of enzymes involved in stress processes,maintaining normal intracellular osmotic balance.