Domain-Swapped LuxR-Type Quorum Sensing Receptors Reveal Divergent Ligand-Response Mechanisms among Homologues.

LuxI/R quorum sensing controls diverse cell density-dependent behaviors in gram-negative bacteria, yet the molecular basis of LuxR-type receptor response to ligands remains poorly defined. This gap limits both mechanistic understanding of signaling and the rational design of synthetic LuxR modulators. LuxR homologues exhibit two response modes: associative receptors require N-acyl l-homoserine lactone (AHL) signal binding to enable DNA binding and transcriptional activation, whereas dissociative receptors are active without ligand and inhibited upon AHL binding. Herein, we dissect determinants of ligand-response type using domain swapping and mutagenesis across four archetypical receptors: the associative receptors LasR (Pseudomonas aeruginosa) and MrtR (Mesorhizobium tianshanense), and the dissociative receptors EsaR (Pantoea stewartii) and ExpR2 (Pectobacterium versatile). Analyses of domain-swapped receptors revealed that the ligand-binding domain largely dictates associative versus dissociative behavior in response to native AHL agonists. Consistently, non-native AHL-derived antagonists retained their activity when DNA-binding domains were interchanged, underscoring the primacy of the ligand-binding module. We also found that the extended interdomain linker characteristic of dissociative receptors does not determine response mechanism. Instead, our data implicate receptor-specific interdomain interactions in activation. Notably, deletion of a single residue in EsaR converted this dissociative receptor into an associative one, representing, to our knowledge, the first example of such a mechanistic inversion in a LuxR-type protein. Together, these findings define key structural features governing ligand response and reveal unexpected mechanistic plasticity, providing a foundation for more informed design of next-generation quorum sensing modulators with enhanced specificity, potency, and predictable activity across diverse receptors.