| Pseudomonas putida KT2440 is a model strain widely used in industrial production.Therefore,clarifying the environmental tolerance mechanism of Pseudomonas putida KT2440 so that it can withstand the pressure of industrial production is of great significance to guide the production practice of KT2440.Quorum sensing(QS)is widely involved in the regulation of bacterial environmental adaptability.Autoinducer-2(AI-2),a signal molecule that mediates intraspecific and interspecific communication,can regulate a variety of physiological processes of bacteria,including biofilm formation,virulence and drug resistance.At present,three types of AI-2 receptors have been identified,including Lux P,Lsr B and transmembrane signal transduction proteins containing d CACHE domain.Among them,AI-2 receptors containing d CACHE domain widely exist in bacteria.However,it has not been reported whether AI-2 receptor exists in Pseudomonas putida KT2440 and whether AI-2 is involved in regulating the physiological function of KT2440.This study explored the chemotactic receptors that mediate the chemotactic response of Pseudomonas putida KT2440 to AI-2,and to explore the regulatory effect of AI-2 signal on the chemotactic movement and biofilm formation of Pseudomonas putida KT2440 through chemotactic receptors.The specific research results are as follows:1.Pseudomonas putida KT2440 has chemotaxis to AI-2The chemotaxis of Pseudomonas putida KT2440 to AI-2 was detected by capillary quantitative analysis and soft agar plate gradient assays.Both results showed that Pseudomonas putida KT2440 had chemotaxis to AI-2.This suggests that the receptor containing AI-2 in Pseudomonas putida KT2440 can mediate its chemotaxis to AI-2.2.Identification of AI-2 receptor in Pseudomonas putida KT2440In order to dig out the AI-2 receptor in Pseudomonas putida KT2440,this study used the amino acid sequence of the known AI-2 receptor Tlp Q in Pseudomonas aeruginosa to search the blastp in the genome of Pseudomonas putida KT2440,and retrieved the protein with the highest homology,methylated chemoattractant receptor protein Mcp U.Then,the bioinformatics analysis and molecular docking experiment of Mcp U showed that Mcp U had the structural basis of combining with AI-2.Then,the ligand binding domain(LBD)of Mcp U was heterologously expressed and purified.The interaction between Mcp U-LBD and AI-2 was analyzed by bioluminescence experiment of Vibrio Harvey and isothermal titration calorimetry(ITC).The results showed that there was a high affinity interaction between Mcp U-LBD and AI-2,which proved that Mcp U was the receptor of AI-2.3.The chemotactic response of Pseudomonas putida KT2440 to AI-2 depends on methylated chemoreceptor Mcp UThe chemotaxis of wild-type strain,mcp U gene deleted mutant and mcp U gene complementary strain to AI-2 were detected by soft agar plate gradient assays and capillary quantitative analysis.The results showed that the wild-type strain had chemotaxis to AI-2,while the mcp U gene deleted mutant did not show chemotaxis to AI-2,but the mcp U gene complementary strain showed obvious chemotaxis to AI-2.Therefore,the chemotactic response of Pseudomonas putida KT2440 to AI-2 depends on the methylation chemotactic receptor Mcp U.4.AI-2 enhanced the biofilm forming ability of Pseudomonas putida KT2440 through Mcp UThis study further determined the effect of AI-2 on biofilm formation of Pseudomonas putida KT2440 and Δmcp U on biofilm formation.The results showed that exogenous addition of AI-2 significantly increased the biofilm formation of strain KT2440,while AI-2had no significant effect on the biofilm formation ability of strain Δmcp U.In order to eliminate the effect of strain growth on biofilm formation,the effect of AI-2 on the growth of KT2440 and Δmcp U was measured in LB liquid medium.The results showed that the growth rate of Δmcp U and KT2440 was basically the same when AI-2 was added.The above studies showed that AI-2 enhanced the biofilm forming ability of Pseudomonas putida KT2440 through Mcp U.In conclusion,this study found the receptor methylation chemotactic receptor Mcp U of quorum sensing signal molecule AI-2 in Pseudomonas putida KT2440,and proved that AI-2 regulates the chemotaxis and biofilm formation of Pseudomonas putida KT2440 through this receptor.These results suggest that Pseudomonas putida KT2440 can sense AI-2 signals produced by other bacteria in the environment by its chemoreceptor Mcp U,and enhance its biofilm formation ability,so as to improve its environmental adaptability.Therefore,enhancing the biofilm forming ability of Pseudomonas putida KT2440 by synthesizing AI-2 signal or co culturing it with other AI-2 producing strains can be used as a new strategy to improve the environmental adaptation of Pseudomonas putida KT2440. |
