Study On The Function Of ECP Fimbriae In Colonization Of Pine Wood Nematodes By Enterobacter Ludwigii AA4

Enterobacter ludwigii AA4 is a gram-negative biocontrol bacterium belonging to Enterobacter,which is isolated from plant rhizosphere.It can colonize pine wood nematode effectively in the process of interaction with pine wood nematode.ECP fimbriae are ubiquitous in symbiotic strains of animals and plants and pathogenic Escherichia coli,and play the role of colonization factors by promoting the interaction between bacteria and host cells and the interaction between bacteria in the biofilm community.In this study,the ECP fimbriae gene cluster was identified and its function was studied on the plant rhizosphere growth-promoting bacteria Enterobacter ludwigii AA4.The main results are as follows:(1)Bioinformatics analysis confirmed that there were five core proteins for ECP fimbriae assembly in strain AA4,including EcpA,the main subunit of fimbriae,EcpD,which is responsible for binding to the receptor,chaperone proteins EcpB,EcpE and guide protein EcpC.(2)The transcription of ECP component in AA4 of Enterobacter Ludwig was detected.It was found that ECP component ecpA,ecpB,ecpC,ecpD and ecpE could be transcribed and located on the same operon.(3)The single gene mutants of five components of ECP,AA4ΔecpA,AA4ΔecpB,AA4ΔecpC,AA4ΔecpD,AA4ΔecpE and ECP gene cluster mutants AA4ΔecpBCDE and complementary strains AA4ΔecpA-C and AA4ΔecpE-C were constructed by homologous recombination technique.There was no significant difference in growth between the mutant strain and the wild type AA4 strain.(4)The fimbriae assembly of ECP component mutants in strain AA4 was observed by transmission electron microscope.It was found that the fimbriae assembly ability of ecpA,ecpE and ecpABCDE knockout mutants decreased,so the key components in the assembly process of ECP fimbriae in strain AA4 were the main subunit EcpA and chaperone protein EcpE.(5)It was found that the deletion of ecpA,ecpE gene and ecpABCDE gene cluster weakened the colonization ability of strain AA4 on pine wood nematode.Through functional study,it was further found that the deletion of ecpE gene and ecpABCDE gene cluster weakened the biofilm formation ability of strain AA4,and the deletion of ecpA,ecpE gene cluster and ecpABCDE gene cluster also weakened the movement ability of strain AA4,but the deletion of ecpABCDE gene cluster could not change the antioxidant ability of strain AA4.To sum up,the ecpA,ecpE and ecpABCDE gene clusters of strain AA4 participate in bacterial colonization on nematodes by affecting bacterial movement ability and biofilm formation.The above results will lay a theoretical foundation for revealing the colonization mechanism of strain AA4 in pine wood nematode and provide scientific basis for the research and development and efficient application of microbial preparation for the control of pine wood nematode.