title: Intracellular co-localization of the Escherichia coli enterobactin biosynthetic enzymes EntA, EntB, and EntE.
abstract: Bacteria utilize small-molecule iron chelators called siderophores to support growth in low-iron environments. The Escherichia coli catecholate siderophore enterobactin is synthesized in the cytoplasm upon iron starvation. Seven enzymes are required for enterobactin biosynthesis: EntA-F, H. Given that EntB-EntE and EntA-EntE interactions have been reported, we investigated a possible EntA-EntB-EntE interaction in E. coli cells. We subcloned the E. coli entA and entB genes into bacterial adenylate cylase two-hybrid (BACTH) vectors allowing for co-expression of EntA and EntB with N-terminal fusions to the adenylate cyclase fragments T18 or T25. BACTH constructs were functionally validated using the CAS assay and growth studies. Co-transformants expressing T18/T25-EntA and T25/T18-EntB exhibited positive two-hybrid signals indicative of an intracellular EntA-EntB interaction. To gain further insights into the interaction interface, we performed computational docking in which an experimentally validated EntA-EntE model was docked to the EntB crystal structure. The resulting model of the EntA-EntB-EntE ternary complex predicted that the IC domain of EntB forms direct contacts with both EntA and EntE. BACTH constructs that expressed the isolated EntB IC domain fused to T18/T25 were prepared in order to investigate interactions with T25/T18-EntA and T25/T18-EntE. CAS assays and growth studies demonstrated that T25-IC co-expressed with the EntB ArCP domain could complement the E. coli entB(-) phenotype. In agreement with the ternary complex model, BACTH assays demonstrated that the EntB IC domain interacts with both EntA and EntE.
authors.affiliation: Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St., W., Montreal, H4B 1R6, Quebec, Canada.
authors.forename: Paknoosh
authors.initials: P
authors.lastname: Pakarian
authors.affiliation: Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St., W., Montreal, H4B 1R6, Quebec, Canada; Groupe de Recherche Axé sur la Structure des Protéines (GRASP), Canada. Electronic address: peter.pawelek@concordia.ca.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Pawelek
mesh_terms: D004758:Enterobactin / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000378:metabolism*; D006867:Hydrolases / Q000032:analysis / Q000378:metabolism*; D008025:Ligases / Q000032:analysis / Q000378:metabolism*; D062105:Molecular Docking Simulation; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000032:analysis / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D060066:Protein Interaction Maps
chemical_list: D029968:Escherichia coli Proteins; D004758:Enterobactin; D044925:Oxidoreductases Acting on CH-CH Group Donors; C061899:entA protein, E coli; D006867:Hydrolases; C064012:isochorismatase; D008025:Ligases; C061746:2,3-dihydroxybenzoate-AMP ligase, E coli
keywords: Bacterial two-hybrid; CAS assay; Enterobactin; Protein-protein interactions; Siderophore