(556f) Mapping the Sequence-Function Landscape for Quorum Sensing Specificity: Mitigating Signal Crosstalk of the Lasr Quorum Sensor for Programmable Bacterial Consortia

Programmable intercellular communication using quorum sensing components allows for coordinated and dynamic responses to be engineered in synthetic microbial consortia for biomanufacturing and other applications. Homoserine lactone-mediated quorum sensors are well-studied and widely applied, yet they commonly exhibit some degree of crosstalk with other LuxR-type quorum sensors natively. This signal interference is often detrimental to applications where specific intercellular signaling allows for more tightly controlled cell-cell communication. In this project, we apply targeted protein engineering strategies to elucidate the sequence-function relationship for a LuxR-type quorum sensing regulator and aim to identify variants with improved signaling specificity across a set of commonly used quorum sensors.

Here we target the LasR quorum sensor, which is among the highest level of crosstalk observed and for which the protein-ligand interactions and protein structure have been well studied. Mutations of ligand binding residues were generated for LasR using site directed mutagenesis. Among other mutations, a LasR mutation V76T has been identified that confers selectivity towards its cognate 3-oxo-C12-HSL signal relative to induction with the set of noncognate signals. A combinatorial pooled saturation mutagenesis library of LasR variants (9,486 mutants) was designed by mutating a ligand binding pocket region of LasR (L125 – L130) by custom scripts and then constructed using an oligo pool to build the LasR sensor library. LasR variants were designed to contain single, double, and triple mutations with all amino acid substitutions. Sequencing of the pooled library confirmed all 9,486 LasR protein variants (100% design coverage) present in the pool. Pooled sort-seq assays using the cognate and noncognate homoserine lactone (HSL) were performed to determine the activation of each LasR variant for both HSL ligands. Based on this dataset, analyses identified sensor variants with improved specificity and positions to maintain the function of the LasR sensor (e.g. G126). The elucidated sequence-function relationship reveals that single mutations can confer LasR with improved specificity, while double or triple mutations can give rise to variants with interesting properties, such as reversed signal specificity. This work informs how the LasR quorum sensor can be engineered to mitigate signal crosstalk and elucidates the tradeoff of sensitivity and specificity for this sensor. This approach can be used for further high-throughput engineering of orthogonal HSL-mediated quorum sensors necessary for programming bacterial consortia.