| Pseudomonas fluorescens is a Gram-negative bacterium with high proteolytic,lipoltic and psychrotrophic abilities.It is an important spoilage organism in raw fish,meat and dairy products.However,little research has been done on the molecular information of the Puorescens QS system and its mechanisms for biofilm formation,spoilage and stress resistance.In view of this,P.fuorescens PF07 used in this study was the dominant spoilage isolated and identified from refrigerated spoiled large yellow croake.Firstly,the AHLs activity in PF07 was detected by Chromobacterium violaceum CV026 and high performance liquid chromatography-mass mass spectrometry(LC-MS/MS).The whole genome of PF07 was predicted by using gene frame sequencing technology,and LuxI and LuxR proteins were analyzed by bioinformatics.Secondly,two in-frame deletion mutants of luxl and luxR were constructed to explore their function in P.fluorescens.The growth,biofilm formation,corruption phenotype and stress resistance were compared between PF07 wild type(WT),and ?luxR,?luxI without or with C4-HSL.Finally,transcriptomics sequencing technology was used to evaluate the gene expression and metabolic pathways between PF07 and two mutants strains to deeply understand the role of LuxI/LuxR system in P.fluorescens,which can provide a theoretical support in the development of new food preservation.The main results are as the following:It was shown that the supernatant of P.fluorescens PF07 contained three AHLs signal molecules,including C4-HSL,C10-HSL and C14-HSL,in which C4-HSL had the highest content.By sequencing genomic framework,whole genome size of PF07 was predicted to be about 6.59Mb.The number of coding gene was about 5,573 and the length was about 5,352,570 bp.There are three kinds of ncRNAs predicted in PF07(tRNA,rRNA and sRNA),6 Prophages,70 CRISPRs,and 6 secondary metabolite gene clusters,among which,hserlactone(homoserine lactone)is a cluster associated with QS AHLs.In the COG cluster analysis of PF07 function,acyl-homoserine-lactone synthase GM004424(LuxI)and LuxR family transcriptional regulator protein GM004425(LuxR)were found from the genes of Signal transduction mechanism by bioinformatics analysis.It was shown that LuxI and LuxR genes in PF07 are highly identical and similar to the homologous proteins in Pseudomonas fluorescens PFuk4,and have the same conserved amino acids.Similar 3D structure of LuxI and LuxR as the six LuxI-type synthase and LuxR receptor proteins in other strains were observed-It was therefore revealed that LuxI/LuxR in PF07 is a pair of homologous quorum sensing synthetases and receptor proteins.Two in-frame deletion mutants,?luxl and AluxR,were constructed to explore their QS signaling role in P.fluorescens.It was found that C4-HSL was almost lost in?luxl,and C4-HSL was decreased by about 38.60%in ?luxR,but had no effect on C10-HSL and C14-HSL.At 30? and 4?,the loss of luxl and luxR had no effect on bacterial growth,and reached stationary phase at 18 h and 144 h,respectively.Compared with WT strain,the ?luxl and ?luxR mutants had significantly weaker ability in biofilm formation,adhesion,extracellular polysaccharide secretion(p<0.05),however,their swimming was greatly promoted(p<0.05).Biofilm biomass of ?luxl and ?lvxR decreased by 17.22%and 14.56%at 30?,and 27.51%and 22.48%at 4?compared to WT,respectively.The supplement of C4-HSL in ?luxI resulted in increase of biofilm biomass by 12.82%at 30? and 23.66%at 4?,respectively,indicating the complemention of biofilm formation.CLSM images showed that the structure of biofilm formed by ?luxI and ?luxR was thinner and looser than wild-type strain PF07 at two temperatures.Similarly,SEM observation found that AluxI and?luxR had less extracellular secretion while C4-HSL stimulated these secretions in?luxI.The deletion of luxl and luxR also reduced the extracellular protease and siderphore production of PF07(p<0.05)in sterile fish juice media stored at 4?.Compared with WT strain,protease activity decreased by 11.94%and 12.51%in?luxl and ?luxR,respectively.However,the content of TVB-N didn't affect.The exogenous addition of C4-HSL can partially restore the defection of siderphore production and protease in ?luxl,and recovery effect on protease was weaker due to the highest recovery of 9.2%.In addition,the resistance of Aluxl and AluxR in the stressing environment of 20 mM H2O2,30%(m/v)NaCl,heating treatment at 50 ?and 150 pg/mL crystal violet apparently declined in two mutants compared to WT.,especially H2O2 The survival rates WT were 18.71 and 15.31 times of the ?luxl and AluxR on the treatment of H2O2.Transcriptomic analysis revealed that the deletion of LuxI/LuxR protein in P.fluorescens PF07 affected various biofilm related genes,including down-regulation of polysaccharide-related genes,up-regulation of pst-related genes in both Aluxl and?luxR,and up-regulation of PhoB/U two-component systems in AluxR.Up-regulation of pst system and PhoB/U two-component system might be lined to the down-regulation of c-di-GMP content in the cells,thereby promoting cell motility.Meanwhile,the genes involved in extracellular protease production(aprX),ornithine monooxygenase and stress response(rpoS,OmsC,HslV)were down-regulated in the two mutants.In addition,the expression levels of other spoiled genes in Aluxl and?luxR were also down-regulated,such as genes related to lipid metabolism,sulfur compound metabolism,aromatic compound and amino acid metabolism,odor generation,etc.The expression level of protein LuxR down-regulated in AluxI.The complement of C4-HSL only restored the partial genes in Aluxl,including ornithine monooxygenase associated with ferritin synthesis,polysaccharide biosynthesis protein related to polysaccharide synthesis,pyocin R2 holin associated with pyocyanin production,and stress response related genes OmsC,HslV and so on.Finally,RT-qPCR was used to verify the expression changes of aprX,flihA,luxl,luxR,orm,pbp and rpoS genes in four groups of bacteria.The results of qPCR were consistent with the consequence of transcriptome sequencing,which indicated that the transcriptome sequencing were reliable.The above results have shown that LuxI/LuxR is a pair of homologous quorum-sensing synthetase and receptor proteins in P.fluorescens PF07,and LuxI/LuxR can up-regulate polysaccharide-related genes,down-regulate pst-related genes and PhoB/U two-component system genes to promote biofilm formation.LuxI/LuxR also up-regulate extracellular protease,siderphore,lipid metabolism and other spoilage-related genes to promote spoilage potential.Furthermore,the system up-regulate stress-related genes to enhance their stress resistance in unfavorable envirnoment.Thus,quorum sensing system LuxI/LuxR positively regulates the biofilm formation,spoilage and stress response in P.fluorescens. |
PDF Full Text Request