Abstract
<jats:p> Common bacteria coordinate group behaviors in a cell-density–dependent manner through quorum sensing. In Gram-negative bacteria, quorum sensing is typically mediated by LuxI/LuxR systems, where LuxI-type synthases produce <jats:italic toggle="yes">N</jats:italic> -acyl L-homoserine lactone (AHL) signals that are detected by LuxR-type receptors to regulate gene expression. Although AHLs share a conserved Lhomoserine lactone headgroup, receptor selectivity is encoded by the acyl tail, which varies in length and oxidation state. Here, we systematically interrogate the chemical determinants of AHL activity by synthesizing a set of 37 analogues spanning all acyl chain lengths from C1 to C20 with and without 3-oxo substitution. Evaluation of these compounds across seven LuxR-type receptors using cell-based reporter assays revealed detailed structure–activity relationships linking acyl tail architecture to receptor modulation. We identified distinct chain-length optima for agonism and antagonism and demonstrated that both parameters are strongly receptor-dependent. Interestingly, many of the antagonists displayed non-classical partial agonism behavior, with both antagonistic and agonistic activity on a receptor depending on concentration. Together, these results define several key chemical features governing LuxR:ligand recognition and establish generalizable principles for tuning AHL activity across some of the most studied LuxR-type receptors. This work also provides a framework for understanding how structural variation in native AHLs influences signaling specificity and for building hypotheses about the potential roles of LuxI/LuxR quorum sensing on interspecies interactions in complex mixed-microbial environments. </jats:p>