Identification of cyclic di-GMP protein receptors: high-throughput screening strategies and experimental verification / 生物工程学报

ABSTRACT

cyclic di-GMP (c-di-GMP) is a universal second messenger in bacterial cells. It regulates various biological processes such as biofilm development, pathogenicity, motility, exopolysaccharide (EPS) production and cell cycle. The second messenger exerts its function by binding to effectors, such as riboswitches and proteins. However, due to the diverse conformations of c-di-GMP, its effectors are hardly to be predicted by homology search. Identification of c-di-GMP effectors is the initial step to investigate its regulatory function in bacterial signal transduction, however, it remains to be a technically difficult task. Here we reviewed the mechanism of biofilm development controlled by c-di-GMP through binding to various types of protein effectors, and summarized the screening strategies, including genetics analysis, protein pull-down combined with LC/MS/MS identification, DRaCALA systematic screening and molecular docking-based prediction. We also summarized experimental methods for verifying protein-c-di-GMP interaction, including isothermal titration calorimetry, surface plasmon resonance, microscale thermophoresis etc. In addition, we discussed the advantages and disadvantages of these strategies and methods. The present review aims to facilitate the future investigations that are focused on regulatory role of novel c-di-GMP effectors.