Effects Of Flagellar Key Regulatory Genes On The Biological Characteristics In Pseudomonas Putida KT2442

Flagellum is a main bacterial motive organelle that protrudes from the surface of the cell.Its synthesis and assembly involve a variety of proteins.The defects of flagellum might influence the formation of bacterial biofilm,the secretion of proteins or other biological processes.Therefore,it is of theoretical significance to study the effect of flagella gene mutants on the biological characteristics of the strain and the regulation mechanism of the related biological process.In this study,the mutant strains of flagella defect were constructed by knocking out the flagellum synthesis-related genes in Pseudomonas putida chromosome.The flagella synthesis and regulatory mechanism of Type VI Secretion System(T6SS)of each strain was explored by phenotypic analysis and transcriptional analysis.The results are listed below:(1)Based on homologous recombination,the flagella-related genes fleQ,fli A,rpoN,c-di-GMP degradation-related gene bifA and T6 SS gene hcp1 were deleted from the P.putida KT2442 chromosome,respectively.?fleQ,?fliA,?rpoN,?bifA,?hcp1 and the corresponding complemented strains were constructed.(2)By swimming motility analysis,secretion protein SDS-PAGE analysis,RT-qPCR and transmission electron microscopy,we confirmed that fleQ,fliA,rpoN are required for the biosynthesis of flagella,while the mutation of bifA and hcp1 have no significant effect on the flagella swimming motility.Among them,the mutation of rpoN would influence the growth of the strain.?rpoN cells grow slower than the wild-type KT2442.(3)Transcriptome analysis revealed that in early exponential phase ?fleQ,T6 SS related genes including hcp1 were significantly up-regulated.In late exponential phase KT2442,these genes were similarly up-regulated.However,some other T6 SS genes were differentially regulated.It indicated that FleQ was involved in part of the T6 SS regulation.In early exponential phase ?fleQ transcriptome,several genes relevant to c-di-GMP biosynthesis,cellulose synthesis and surface adhesion proteins were regulated.FleQ represses the expression of cellulose synthesis whereas activates the expression of surface adhesion proteins.(4)Based on SDS-PAGE and RT-qPCR,we found that wild-type KT2442 could secrete large amounts of Hcp1 protein in late exponential phase,which cannot be observed in early exponential phase.In early exponential phase ?fleQ,?fliA,?rpoN,the secretion of Hcp1 was increased in varied levels and easily visualized on SDS-PAGE compared to wild-type KT2442.Combined with RT-qPCR results,it shows FleQ and RpoN repress the expression of T6 SS genes.(5)?hcp1 were constructed to investigate the function of T6 SS and antibacterial activity analysis shows the T6 SS activity of P.putida can inhibit the growth of other bacteria.In this study,FleQ was involved in several biological processes including flagella biosynthesis,T6 SS,cellulose synthesis and surface adhesion proteins expression,which would beneficial to study and reconstruction of the P.putida regulation network.