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.