Domain-swapped LuxR-type quorum sensing receptors reveal divergent ligand-response mechanisms among homologs

Many common bacteria use quorum sensing to regulate cell density-dependent phenotypes, including luminescence, biofilm formation, virulence, and symbiosis. The LuxI/R system is the best-characterized quorum sensing pathway in Gram-negative bacteria and consists of a LuxI-type synthase that produces an N-acyl L-homoserine lactone (AHL) autoinducer and a LuxR-type transcription factor that is regulated by AHL binding. Binding of native AHL signal promotes DNA binding and transcriptional regulation in some LuxR homologs (associative-type), while other homologs regulate transcription in the absence of ligand and are inactivated by native signal binding (dissociative-type). To better characterize what features determine ligand-response type, we generated structural mutants of two associative receptors (LasR of Pseudomonas aeruginosa and MrtR of Mesorhizobium tianshanense) and two dissociative receptors (EsaR of Pantoea stewartii and ExpR2 of Pectobacterium versatile). Swapping domains between these receptors revealed that the ligand-binding domain primarily determines associative vs. dissociative activity in response to native AHL agonists. Further, non-native AHL-derived antagonists maintained their activity profiles in receptors with interchanged DNA-binding domains. We also found that the extended linker between domains observed in the dissociative receptors does not determine mechanism of ligand response, and that inter-domain interactions may play an important role in activation for some receptors but not others. Notably, deletion of just one residue from the dissociative receptor EsaR produced a mutant with associative activity, the first time such mechanism switching has been reported for a LuxR-type receptor. These findings illuminate features essential for ligand response and highlight the mechanistic diversity of the LuxR family. IMPORTANCELuxI/R quorum sensing regulates various cell density-dependent phenotypes in Gram-negative bacteria. Prior research has developed small molecule modulators of LuxR-type receptors, with potential applications in anti-virulence, anti-biofouling, and bioengineering. Competitive antagonists have been reported for receptors active in the presence of native ligand but not for receptors active in its absence. A lack of knowledge about the molecular mechanisms of receptor response to ligand limits both our fundamental understanding of the LuxI/R quorum sensing process and the rational design of chemical modulators with superior activity profiles. We used a structural mutagenesis strategy with four LuxR-type receptors that operate via two distinct mechanisms to begin to dissect the structural features that drive differences in ligand response between receptors. These insights could aid in efforts to characterize novel LuxR homologs, understand potential interspecies communication via quorum sensing, and develop improved chemical probes to alter LuxR-type receptor activity.