High-Throughput Screening to Identify Chemoreceptor Ligands.

Area of Science:

• Microbiology and Molecular Biology
• Bacterial Chemotaxis and Signal Transduction

Background:

• A significant number of bacterial chemoreceptors lack functional annotation, hindering our understanding of bacterial chemotaxis evolution.
• Knowledge of chemoreceptor function is crucial for deciphering bacterial adaptation strategies across diverse lifestyles.
• Previous experimental methods focused on individual, genetically engineered ligand binding domains (LBDs) have advanced functional annotation.

Purpose of the Study:

• To establish a robust protocol for the functional annotation of bacterial chemoreceptors.
• To enable efficient identification and characterization of ligands that bind to recombinant chemoreceptor LBDs.

Main Methods:

• Utilized a combined approach of high-throughput ligand screening via Differential Scanning Fluorimetry (DSF).
• Employed Isothermal Titration Calorimetry (ITC) for detailed characterization of ligand- LBD interactions.
• Focused on recombinant chemoreceptor ligand binding domains (LBDs) to assess ligand specificity and binding affinity.

Main Results:

• Demonstrated the efficacy of the combined DSF and ITC method for identifying ligands of chemoreceptor LBDs.
• Confirmed that major classes of LBDs can be produced recombinantly and retain ligand-binding activity.
• The protocol proved highly efficient in determining the function of previously uncharacterized chemoreceptors.

Conclusions:

• The developed protocol offers a powerful and efficient strategy for annotating bacterial chemoreceptor functions.
• This method facilitates a deeper understanding of bacterial chemotaxis systems and their evolutionary trajectories.
• The approach is applicable to a wide range of bacterial species and chemoreceptor families.