Autoinducer-fluorophore conjugates enable FRET in LuxR proteins in vitro and in cells.

Cell-to-cell signaling, or quorum sensing (QS), in many Gram-negative bacteria is governed by small molecule signals (N-acyl-L-homoserine lactones, AHLs) and their cognate receptors (LuxR-type proteins). The mechanistic underpinnings of QS in these bacteria are severely limited due to the challenges of isolating and manipulating most LuxR-type proteins. Reports of quantitative direct-binding experiments on LuxR-type proteins are scarce, and robust and generalizable methods that provide such data are largely nonexistent. We report herein a Förster resonance energy transfer (FRET) assay that leverages (1) conserved tryptophans located in the LuxR-type protein ligand-binding site and synthetic fluorophore-AHL conjugates, and (2) isolation of the proteins bound to weak agonists. The FRET assay permits straightforward measurement of ligand-binding affinities with receptor-either in vitro or in cells-and was shown to be compatible with six LuxR-type proteins. These methods will advance fundamental investigations of LuxR-type protein mechanism and the development of small molecule QS modulators.

Methods for the directed evolution of biomolecular interactions.

Noncovalent interactions between biomolecules such as proteins and nucleic acids coordinate all cellular processes through changes in proximity. Tools that perturb these interactions are and will continue to be highly valuable for basic and translational scientific endeavors. By taking cues from natural systems, such as the adaptive immune system, we can design directed evolution platforms that can generate proteins that bind to biomolecules of interest. In recent years, the platforms used to direct the evolution of biomolecular binders have greatly expanded the range of types of interactions one can evolve. Herein, we review recent advances in methods to evolve protein-protein, protein-RNA, and protein-DNA interactions.

Chemical Control of Quorum Sensing in E. coli: Identification of Small Molecule Modulators of SdiA and Mechanistic Characterization of a Covalent Inhibitor.

Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of severe diarrheal disease in humans. Cattle are the natural reservoir of EHEC, and approximately 75% of EHEC infections in humans stem from bovine products. Many common bacterial pathogens, including EHEC, rely on chemical communication systems, such as quorum sensing (QS), to regulate virulence and facilitate host colonization. EHEC uses SdiA from E. coli (SdiAEC), an orphan LuxR-type receptor, to sense N-acyl l-homoserine lactone (AHL) QS signals produced by other members of the bovine enteric microbiome. SdiAEC regulates two phenotypes critical for colonizing cattle: acid resistance and the formation of attaching and effacing lesions. Despite the importance of SdiAEC, there is very little known about its selectivity for different AHL signals, and no chemical inhibitors that act specifically on SdiAEC have been reported. Such compounds would represent valuable tools to study the roles of QS in EHEC virulence. To identify chemical modulators of SdiAEC and delineate the structure-activity relationships (SARs) for AHL activity in this receptor, we report herein the screening of a focused library composed largely of AHLs and AHL analogues in an SdiAEC reporter assay. We describe the identity and SARs of potent modulators of SdiAEC activity, examine the promiscuity of SdiAEC, characterize the mechanism of a covalent inhibitor, and provide phenotypic assay data to support that these compounds can control SdiAEC-dependent acid resistance in E. coli. These SdiAEC modulators could be used to advance the study of LuxR-type receptor/ligand interactions, the biological roles of orphan LuxR-type receptors, and potential QS-based therapeutic approaches.

Omics Technologies to Understand Activation of a Biosynthetic Gene Cluster in Micromonospora sp. WMMB235: Deciphering Keyicin Biosynthesis.

DNA sequencing of a large collection of bacterial genomes reveals a wealth of orphan biosynthetic gene clusters (BGCs) with no identifiable products. BGC silencing, for those orphan clusters that are truly silent, rather than those whose products have simply evaded detection and cluster correlation, is postulated to result from transcriptional inactivation of these clusters under standard laboratory conditions. Here, we employ a multi-omics approach to demonstrate how interspecies interactions modulate the keyicin producing kyc cluster at the transcriptome level in cocultures of kyc-bearing Micromonospora sp. and a Rhodococcus sp. We further correlate coculture dependent changes in keyicin production to changes in transcriptomic and proteomic profiles and show that these changes are attributable to small molecule signaling consistent with a quorum sensing pathway. In piecing together the various elements underlying keyicin production in coculture, this study highlights how omics technologies can expedite future efforts to understand and exploit silent BGCs.

Non-native autoinducer analogs capable of modulating the SdiA quorum sensing receptor in Salmonella enterica serovar Typhimurium.

Quorum sensing (QS) allows many common bacterial pathogens to coordinate group behaviors such as virulence factor production, host colonization, and biofilm formation at high population densities. This cell-cell signaling process is regulated by N -acyl L-homoserine lactone (AHL) signals, or autoinducers, and LuxR-type receptors in Gram-negative bacteria. SdiA is an orphan LuxR-type receptor found in Escherichia, Salmonella, Klebsiella, and Enterobacter genera that responds to AHL signals produced by other species and regulates genes involved in several aspects of host colonization. The inhibition of QS using non-native small molecules that target LuxR-type receptors offers a non-biocidal approach for studying, and potentially controlling, virulence in these bacteria. To date, few studies have characterized the features of AHLs and other small molecules capable of SdiA agonism, and no SdiA antagonists have been reported. Herein, we report the screening of a set of AHL analogs to both uncover agonists and antagonists of SdiA and to start to delineate structure-activity relationships (SARs) for SdiA:AHL interactions. Using a cell-based reporter of SdiA in Salmonella enterica serovar Typhimurium, several non-natural SdiA agonists and the first set of SdiA antagonists were identified and characterized. These compounds represent new chemical probes for exploring the mechanisms by which SdiA functions during infection and its role in interspecies interactions. Moreover, as SdiA is highly stable when produced in vitro, these compounds could advance fundamental studies of LuxR-type receptor:ligand interactions that engender both agonism and antagonism.