Design and application of cyclic di-GMP biosensor.

Abstract

c-di-GMP, a ubiquitous second messenger in bacterial domain, regulates various bacterial phenotypes including biofilm formation. Biofilm formation causes severe problems in medical and industry field. Hence, a genetically encoded FRET-based biosensor was designed to visualize the c-di-GMP level. VCA0042 and MrKH biosensors flanked with fluorescent proteins (mCerulean and mVenus) were constructed due to their distinct ranges of sensitization towards the change of c-di- GMP level. Their function and feasibility were verified in vitro and in vivo. In vitro, fluorescence titration was performed in which various concentration of c-di-GMP were given. In vivo, biosensors were expressed in E. coli BL-21 (DE3) and different stress inducers (aminoglycosides and 3-indolylacetonitrile) were given to manipulate the c-di-GMP level within the cell to validate the FRET response of biosensors. It was shown that FRET response of both biosensors reacts inversely with the c-di- GMP level. When c-di-GMP levels elevated, the FRET efficiency decreased due to the conformational change of the PilZ domain protein (VCA0042 and MrKH) sandwiching between the fluorescent proteins. It was noted that these biosensors would be a useful tool in visualizing cytosolic level of c-di-GMP, which confer phenotypes among the bacteria assisting in design optimization of antibiotics and antimicrobial agent.