| Lactic acid bacteria(LAB),as important microorganisms in the food industry and human intestine,is vulnerable to environmental changes,and the presence of biofilm can effectively improve the resistance of LAB.However,the mechanism by which the quorum sensing(QS)system regulates their biofilm formation is not clear.In this study,we screened and identified beneficial LAB with high biofilm-forming properties,also analyzed their tolerance to the adverse environment and their abilities to produce the signal molecule AI-2 based on different growth stages of the strain,and explored the relationship between strain resistance and the signal molecule AI-2.Then we knocked down the key gene luxS of the signal molecule AI-2 based on genome-wide data analysis.The mechanism of the LuxS/AI-2 QS system on biofilm formation in LAB was clarified using transcriptomic and proteomic techniques.The main findings of this study are as follows:The 241 strains of LAB isolated from yak dairy products in Tibet and sour horse milk dairy products in Ximeng were used for the study.The ability of the strains to tolerate pH 3.0 and low concentration of bile salt(0.05 %)was used as the evaluation index for the initial screening,moreover 64 strains of LAB with strong tolerance and good vitality to environments were screened.The high biofilm-forming ability of 64 strains of LAB was measured by the crystalline violet staining method,and 25 strains of high biofilm-producing strains were screened out.The results of the artificial gastrointestinal fluid tolerance test showed that 17 strains had a relatively high survival rate after 3 h incubation in simulated gastrointestinal fluid,among which strains TG5-1-4,TZ3-1-8 and RM1-1-7 could be grown in simulated intestinal fluid.The results of harmful metabolites test showed that 15 strains had negative effects for harmful metabolites.The secretion capacity of the signal molecule AI-2 was determined for 25 strains with high biofilm-producing LAB,only 4 strains were high in the production of signal molecule AI-2,and the 4 strains were identified as Lactobacillus plantarum RM3-1-3,Enterococcus faecium TZ2-1-1,Lactobacillus fermentum 733 and Pediococcus acidilactici 132.The L.plantarum RM3-1-3,E.faecium TZ2-1-1,L.fermentum 733 and P.acidilactici 132 with acid resistance,bile salt resistance,strong ability to produce signal molecule AI-2 and high biofilm-forming properties were selected as the research objects.By preparing planktonic and biofilm strains,their tolerance to different stress environments was studied respectively,and the ability of the strains to produce signal molecule AI-2 under stress was determined.The results showed that the viable number of the strains was suppressed under different stress conditions,but the biofilm strains showed better activity,and the environmental stress had a greater effect on the activity of the signal molecule AI-2.The changes of the signal molecule AI-2 activity were different in acid stress,but when the pH was 4.5,the secretion of the signal molecule AI-2 of E.faecium TZ2-1-1,L.fermentum 733,and P.acidilactici 132 in the biofilm state were reached the maximum,except for L.plantarum RM3-1-3.During bile salt stress,the activity of signal molecule AI-2 tended to decrease with increasing concentration of bile salt,but most of the planktonic strains exhibited higher AI-2 activity of the signaling molecule than the biofilm state in high bile salt concentration environment.In temperature stress,four strains produced almost no signal molecule AI-2 when the incubation temperature was 4,10,55,and 70 ℃,when the culture temperature was 45 ℃,except for L.fermentum 733,the secretion of signal molecule AI-2 was lower than that of the control(37 ℃)and the planktonic strains was more capable of secreting signal molecules.The AI-2 activity of the signal molecule decreased with the increase of Na Cl concentration during salt stress,but the AI-2 secretion was severely inhibited when the Na Cl concentration was higher than 4.5 %.There was strain specificity in the effect of freezing stress on the signal molecule AI-2 activity of LAB.The beneficial L.fermentum 733 with high biofilm-forming properties with good resistance to adverse environments and high activity of the signal molecule AI-2 was selected as the research object.Through the analysis of its whole genome sequencing data,orf01877,a key gene regulating QS,was located in which is a key gene encoding LuxS protein synthesisand.Therefore,the key gene of AI-2 synthesis of L.fermentum 733,luxS,was knocked out.The λRed recombinant technology was used to successfully construct a luxS gene-deficient strain,and the conditions of L.fermentum 733 knockouts were optimized.The best electrotransformation conditions were obtained: the strain was cultured with 0.7 mol/L Na Cl until the middle of logarithm to prepare the sensory state,the highest transformation efficiency was achieved by electrotransformation with plasmid addition of 2 μL,a voltage of 1.8 KV,an antibiotic concentration of 2.5 μg/mL,and an L-arabinose concentration of 100 mmol/L.Based on FTIR and cell physiological assays,it was concluded that knockdown of the luxS gene significantly reduced the biofilm production of L.fermentum 733,as well as the resistance to acid,bile salt,high temperature,and hyperosmotic environment.The reduction in biofilm production was mainly influenced by the deletion of the luxS gene,which led to a significant reduction in the content of unsaturated fatty acids,EPS,protein,and e DNA in biofilm and biofilm fluidity.The correlation analysis concluded that protein was the most important factor affecting biofilm formation before and after a knockdown.Combined the transcriptome with the proteome,the effects of luxS gene deletion on biofilm formation of L.fermentum 733 were analyzed,whose results showed that the deletion of the luxS gene affected the synthesis of the signal molecule AI-2,leading to the down-regulated expression of mRNA and protein-encoding by wecB and reducing the amount of EPS synthesis.It would also restrict the amount of L-homocysteine synthesis and affect L-cysteine synthesis,leading to a decrease in the expression of the mRNA and protein-encoding by metC gene,which in turn limits the catabolism of L-cysteine to pyruvate.And the reduction of pyruvate production affects biofilm formation mainly through the following three pathways: Firstly,when pyruvate reduces lactate,a large amount of ATP is produced,and the down-regulation of luxS gene significantly down-regulates the expression of mRNA and protein encoded by nrdD gene,which reduces the amount of ATP synthesis that in turn reduces the synthesis of dATP and dGTP,leading to the reduction of e DNA synthesis.Secondly,the reduction of ATP synthesis resulted in down-regulation of the expression of mRNA and protein encoded by the ATP-dependent gene idhl,resulting in reduced accumulation of the downstream product ketoglutarate.Because the lack of synthetic raw material affects the synthesis of arginine,which makes the expression of Arg family mRNA and proteins significantly down-regulated,thus hindering the synthesis of extracellular proteins.Finally,The decrease in the amount of pyruvate synthesis leads to the blockage of Acetyl-CoA synthesis,which affects the synthesis and metabolism of fatty acids in biofilm,while the down-regulation of mRNA and protein expression of the Acc and Fab families inhibits the elongation of fatty acid carbon chains and the synthesis of unsaturated fatty acids,resulting in poor biofilm fluidity.In summary,the LuxS/AI-2 QS system influences the biofilm formation of L.fermentum 733 through the regulation of the content of major components in the biofilm by the signal molecule AI-2,which in turn affects the resistance of the strain to adverse environments.This finding provides a theoretical basis for later targeted regulation of lactic acid bacteria biofilm formation and modification of its resistance. |
PDF Full Text Request