Functional Identification Of Methyl Accepting Chemotaxis Protein Encoded By Gene Atu0514 Located In The Chemotaxis Operon Of Agrobacterium Tumefaciens

Agrobacterium tumefaciens belongs to gram-negative bacteria.It can transfer a segment of T-DNA sequence from its Ti plasmid and integrate it into the plant genome,thus causing crown galls in most dicotyledons.In the natural environment,Agrobacterium tumefaciens is usually located around the rhizosphere of plants.Chemotaxis is an important first process of its interaction with the host.Only the chemotaxis response of Agrobacterium tumefaciens to the chemical signal released by the host,can the process of infection and tumorigenesis occur in the subsequent contact with the host cells of plants.Chemotaxis is an important adaptive behavior of motile bacteria.It can promote the bacteria to move towards the attractant or away from repellent.These chemical effectors are mainly perceived by methyl accepting chemotaxis protein.The genome of Agrobacterium tumefaciens C58 has 20 genes encoding MCP homologous proteins,only atu0514 is located within chemotactic operon that encodes chemotaxis system of Agrobacterium tumefaciens.Unlike most MCPs,the MCP encoded by atu0514 does not have extracellular domain structure.It consists of ligand structure domain protoglobin,HAMP and cytoplasmic domain of MA.Therefore,it is necessary to study the function and localization of the protein encoded by atu0514 geneIn this study,Agrobacterium tumefaciens wild type C5 8 was used as the experimental material to obtain the gene deletion mutant of atu0514 by the method of unlabeled reverse screening,and the complemented strain was obtained by plasmid complementation.By measuring the growth curves of wild type strain,atu0514 mutant strain and the complemented strain,it was found that the deletion of atu0514 gene did not affect the growth of Agrobacterium tumefaciens.The effect of atu0514 gene deletion on the chemotaxis response of Agrobacterium tumefaciens was determined by the swimming plate method.It was found that the chemotaxis of Agrobacterium tumefaciens was weakened by the atu0514 gene deletion,but the chemotaxis did not disappear completely.The chemotactic response of atu0514 mutant to a single substance was determined by capillary method.The results showed that wild type C58 had chemotaxis toward 10-6M glucose,10-3M valine,10'3M arginine and 10-3M citric acid,while the deletion of atu0514 gene weakened the chemotaxis response of Agrobacterium tumefaciens to these substances,but the chemotaxis did not disappear completely.This indicates that the protein encoded by atu0514 gene may interact with other MCPs in to glucose,valine,arginine,citric acid and other signaling substances.The MA domain of Atu0514 protein was verified to interact with coupling protein CheW1 and CheW2 by bacterial two-hybrid experiment and galactosidase activity assay.In order to study the localization of Atu0514,EGFP green fluorescent protein should be fused to the C terminal of Atu0514 and construct the atu0514-egfp fluorescent fusion protein.Through fluorescence microscopy,Atu0514 was located at one pole of the cell;in the CheW1 and CheW2 deficient mutants,Atu0514 remained localized at one pole,unaffected by the CheW protein.Chemical receptor protein Atu0514 has three domain,ligand domain Protoglobin,signal transduction domain HAMP and cytoplasmic domain MA,but the MA domain contains highly conserved domains HCD that can interact with CheW and methylated domains K1 and R1,therefore,we constructed a series of expression vectors containing different length truncated atu0514-.egfp based on the structural domain to preliminarily analyze the structural domains affecting the localization of Atu0514 protein.The results showed that the removal of cytoplasmic signal domain MA affected the localization of Atu0514,which was no longer located at one pole dispersed in the cell,in addition,when the methylated domain R1 and the highly conserved domain HCD in the MA domain were absent,the Atu0514 protein was not located at one pole of the cell,but dispersed in the cell,this indicates that the Atu0514 protein interacts with other transmembrane methyl accepting chemotactic proteins through the MA domain,so that the Atu0514 protein locates at one pole of the cell.Atu0514 protein has three domains,and MA domain is the cytoplasmic signal domain of most methyl accepting chemotaxis protein,the effects of atu0514 on chemotaxis were analyzed at the molecular level.We constructed a series of point-mutated strains and verified by the swimming plate that aspartic acid at the 390th position was the key amino acid site,which affected the chemotactic response of Agrobacterium tumefaciens.The study on the function and cell distribution of Atu0514 protein lays a foundation for the study of other MCPs,a deeper understanding of chemotaxis signal transduction mechanism of Agrobacterium tumefaciens,further modification Agrobacterium tumefaciens and expansion of its utilization.