Fig. 2: Widespread occurrence of GAPES1 domain-containing YeaJ homologs capable of sensing AI-2 in the order Enterobacterales.

a Schematic illustrating the predicted domain organization of YeaJ homologs. Protein sequences were analyzed using hmmscan program against the Pfam 34.0 database. TM transmembrane domain, ECD extracytoplasmic domain, CD cytoplasmic domain. b GAPES1 domains from YeaJ homologs in bacterial species belonging to the Enterobacteriaceae, Pectobacteriaceae, and Hafniaceae families are capable of retaining AI-2. GAPES1 domains from YeaJ homologs were expressed and purified as His₆ fusion proteins in a luxS⁺ or luxS^- E. coli strain. Bioluminescence in V. harveyi strain MM32 was measured following the addition of a buffer control or ligands released from the purified proteins upon denaturing by heating. YeaJ-LBD was used as a positive control. AI-2 activity is presented as mean ± s.e.m. of three independent experiments. The NCBI accession numbers for YeaJ homologs are provided and the bacterial species to which they belong are given in parentheses. c AI-2-binding GAPES1 domains of YeaJ homologs harbor two conserved residues corresponding to Y210 and D239 of YeaJ. GAPES1 domains of YeaJ and its homologs that have been tested for AI-2 binding activity were aligned using ClustalW embedded in MEGA7 software. Two positions corresponding to Y210 and D239 of YeaJ that may be involved in AI-2 binding are labeled with red arrows. The conserved residues in the two positions are highlighted in yellow and non-conserved residues are highlighted in purple. The NCBI accession number for each YeaJ homolog is included. d Isotherms representing binding of two mutants of YeaJ-LBD (Y210A or D239A) with DPD/AI-2. The binding affinity was determined using ITC. The data shown were one representative of three independent experiments with similar results. The K_d and complex stoichiometry (n) are presented as mean ± s.d. of three independent experiments. Source data are provided as a Source Data file.