Functional Characterization Of Chemoreceptor Encoding Genes Atu0514 And Atu2173 In Agrobacterium Fabrum C58

Bacterial chemotaxis to plant root secretory nutrients permits bacteria to congregate around plant roots and hence interact with root tissue.As a result,bacterial chemotaxis is thought to be a crucial step in the formation of bacterial-plant relationships.As a plant pathogen in soil,Agrobacterium fabrum may invade both the interrhizosphere and root tissues of plants.The chemoreceptor(also known as methyl-accepting chemotaxis protein,MCP)is the major signaling protein in the bacterial chemotaxis signaling pathway.MCP specifically senses and binds chemo-effectors and transmits the sensed signal to the downstream proteins of the chemotaixs signaling system.The genome of Agrobacterium fabrum C58 contains 20 MCP-encoding genes,but lacking studies about the functions of these 20 chemoreceptors.As a result,studying the function of Agrobacterium fabrum chemotactic receptors can help us learn more about the bacteria's chemotactic signaling pathways.atu0514 is the only chemoreceptor gene located in the che operon of Agrobacterium fabrum.Hence,the identification of the exact function of atu0514-encoding chemoreceptor(here,named as MCP514)will be very important for us to understand more deeply the chemotaxis signal transduction mechanism of A.fabrum.Chemotaxis phenotyping tests revealed that deleting atu0514 dramatically reduced Agrobacterium fabrum chemotactic response to four distinct chemicals:sucrose,valine,citric acid,and acetosyringone(AS).MCP514 localizes at cell pole through interactions between its hairpin substructure domain and other transmembrane chemotactic receptor hairpin substructure domains,according to data from a series of EGFP-MCP514 fusion protein fluorescence subcellular localization assays.Mutations in MCP514 N336 and V353 sites impact Agrobacterium fabrum C58 chemotaxis,indicating that MCP514 is involved in Agrobacterium fabrum C58 signaling to a range of chemicals via interactions with other chemotactic receptors.MCP2173 is a chemotactic receptor with a dCache₁ ligand binding domain that is encoded by atu2173,a chemotactic receptor gene found on the circular chromosome of Agrobacterium fabrum C58.Differential fluorescence scanning(DSF)was used to evaluate 17 amino acid ligands for binding to MCP2173 in vitro.In vivo capillary chemotaxis assay revealed that deletion of atu2173 impacted the chemotactic response of Agrobacterium fabrum C58 to these 17 amino acids in a distinct way,demonstrating that MCP2173 is the primary amino acid chemotactic receptor in Agrobacterium fabrum C58.Mutations at four sites,R127,Y145,D147,and D174,caused MCP2173 to lose its chemotactic response to amino acids,indicating that four sites,R127,Y145,D147,and D174,are critical for amino acid recognition by Agrobacterium fabrum C58.The four sites,R127,Y145,D147,and D174,are important for Agrobacterium fabrum C58 to recognize amino acids.The deletion of MCP2173 diminished the amount of biofilm production in Agrobacterium fabrum C58,showing that the chemoreceptor MCP2173 is important in the formation of biofilm in Agrobacterium fabrum C58.In conclusion,MCP514 affects the chemotaxis of Agrobacterium fabrum C58 to different substances through interactions with other chemoreceptors,while MCP2173 is the main amino acid chemoreceptor of Agrobacterium fabrum C58 and is involved in biofilm formation.The results of this study will promote a better understanding of the molecular mechanisms and physiological functions of chemotaxis in Agrobacterium fabrum C58,which will help to further improve the utilization of Agrobacterium fabrum C58.