Characteristics Of Biofilm Formation And Spoilage Of Psychrotrophic Pseudomonas At Low Temperature Based On Whole Genome And Transcriptome

Cold chain at low temperature can effectively retard the microbial activities of in the process of production,storage and transportation,maintaining the quality and safety of agricultural products.However,psychrotrophic Pseudomonas remains proliferation and metabolism at low temperature,which contributed to the quality deterioration of cold chain animal-derived foods stored aerobically.Metabolic diversity makes it well adapted to the low temperature environment and competition from other psychrotrophic microorganisms,leading to becoming the dominant spoilage bacteria.Psychrotrophic Pseudomonas has a strong ability to form biofilm,which makes it easy to attach to the surface of food and utensils to survive and spread,increasing persistent contamination.In view of this,three psychrotrophic Pseudomonas strains isolated from fish and beef were investigated the characteristics of biofilm formation and spoilage at low temperature based on whole genome and transcriptome.The regulatory mechanism of quorum sensing(QS)on Pseudomonas biofilm and spoilage was further explored.The main results are as follows:(1)The genomic sequences of Pseudomonas fluorescens PF07,Pseudomonas lundensis PL28 and Pseudomonas psychrophile PP26 were obtained by whole genome sequencing,with the length of 6.13,4.83 and 4.67 Mb,respectively.Pan-genome analysis revealed 304 core genes,mainly involved in translation,ribosomal structure and biosynthesis,amino acid transport and metabolism,and energy production and conversion.Functional annotations showed that the three strains contained a large number of genes involved in amino acid metabolism,carbohydrate synthesis and decomposition and transmembrane transport.Comparative genomic analysis indicated that three Pseudomonas contained most of the biofilmrelated genes,the biofilm forming genes are mainly associated with quorum sensing,c AMP/Vfr,c-di-GMP and Gac/ RSM pathways(lux,crp,wsp,gac),EPS(psl,alg),adhesion(yfc,pga),motility(pil,flg,fli,mot),chemotaxis(che)and dispersion(bdl A,dks A).Spoilage potential was closely related to various families encoding alkaline metalloproteinase apr,amino acid decarboxylase spe and polyamine transporter pot,encoding ED pathway glucose hydrolase glk,zwf,edd,encoding lipase synthesis lip and other gene families.In addition,temperature stressrelated cold shock protein coding genes csp,tap B and heat shock protein cap,dna were identified,and multiple σ factors such as rpo S,rpo N,and rpo D were also distributed.(2)Biofilm formation characteristics of Pseudomonas spoilers at 25 ℃,15 ℃ and 4 ℃were compared,as well as the resistance of mature biofilm to chemical and heat treatment.The results showed that the biofilm formed slowly at 4 ℃ and reached the OD peak of 3.69～3.91 at120～144 h,but was significantly higher than the peak at 15 and 25 ℃.The secretion of extracellular polymeric substances(EPS)increased significantly at low temperature,of which extracellular proteins and extracellular polysaccharides accounted for 71.03% ～ 77.44% and21.39% ～ 27.71%,respectively.Thicker spatial structure of mature biofilm was observed at4 ℃,with the thickness ranging from 42.7 ～ 54.6 μm.Biofilm transferred from hydrophobicity to moderate hydrophobicity,and the motility was significantly inhibited at 4 ℃.The resistance of mature biofilm formed at 4 ℃ to Na Cl O and heat treatment were significantly increased(P< 0.05),suggesting that the stress resistance of biofilm at different temperatures was significantly related to the secretion of EPS matrix.In addition,PF07 maintained a higher amount of biofilm within 192 h at low temperature,and EPS content,motility and biomass residue of 0.06% Na Cl O treatment were higher than those of PL28 and PP26.(3)The spoilage characteristics of PF07 and PL28 at 25 ℃,15 ℃ and 4 ℃ were compared,and the biomarkers produced in the process of spoilage were screened.It was found that the two Pseudomonas had higher growth density and higher protease activity at 15 ℃ than at 25 ℃and 4 ℃.PF07 and PL28 showed high protease activity and produced TVB-N content of 18.76～ 29.32 mg N/m L in fish and beef juice for 7 days.The two Pseudomonas in fish juice produced several key volatile organic compounds(VOCs)showed that butanoic acid,ethyl ester and hexanoic acid,ethyl ester could be used as potential biomarkers in the spoilage of Pseudomonas.Transcriptome analysis further showed that the differentially expressed genes(DEGs)of PF07 at 0 ～ 3 and 3 ～ 7 days were 1423 and 1117,respectively,which were mainly involved in twocomponent system,ABC transport,sulfur metabolism,ribosome and aminoacyl t RNA biosynthesis.Moreover,51 and 24 genes were continuously up-regulated or down-regulated,respectively,during the refrigerated storage.Lipid metabolism genes encoding 3-hydroxyacylCo A dehydrogenase and acyl-Co A dehydrogenase promoted the decomposition and utilization of fatty acids and carbohydrate.Metabolic genes,such as amino acid dehydrogenase,branched chain amino acid transporter permease and sulfate ABC transporter,promoted the decomposition from proteins to amines production for two Pseudomonas strain.(4)The regulation of QS on the biofilm and spoilage potential of PF07 and PL28 were further explored based on spoilage phenotypic and transcriptomics.HPLC-MS/MS identified that PF07 produced four kinds of AHLs signal molecules,while PL28 had almost no AHLs activities.Exogenous C4-HSL apparently increased the biofilm formation,EPS production and spoilage characteristics of PF07 and PL28,such as protease activity and siderophore production.The artificial contamination results of refrigerated fish indicated that the exogenous C4-HSL could significantly accelerated the accumulation of soluble peptides,TVB-N and p H,and reduce the color difference.Transcriptome analysis found that exogenous C4-HSL resulted in273 and 297 DEGs in PF07 and PL28,respectively,among which the ribosome and spoilagerelated metabolic pathways were significantly regulated.The catabolism of amino acids,carbohydrates and lipids in the substrate of PF07 and PL28 was promoted by C4-HSL,as well as the secretion of hydrolase,resulting in the enhancement of the spoilage ability.Compared to the up-regulation of genes related to iron uptake involving in production of siderophore for PF07,the genes associated with iron and sulfur metabolism for PL28 were up-regulated.Additionally,more genes being sensitive environmental stress to C4-HSL in PL28,with increased signal transduction and stress resistance,and decreased motility.To sum up,the genomes of psychrotrophic Pseudomonas PF07,PL28 and PP26 contained a large number of genes of biofilm formation,amino acid metabolism and carbohydrate synthesis and decomposition.These Pseudomonas spoilers secreted a large number of extracellular matrix and promoted the development of thicker mature biofilms,which increased their resistance against disinfection at low temperature.Meanwhile,Pseudomonas secreted protease at low temperature,which closely related to high spoilage-related metabolites.C4-HSL-mediated QS promoted Pseudomonas biofilm formation and its spoilage potential related to iron transport.