Sensitive and Broadly Compatible Transcription Factor-Based Biosensor for Monitoring c-di-GMP Dynamics in Biofilms.

Biofilms are ubiquitous and have many negative effects, for example, in infections or biocorrosion. Given the critical role of the second messenger cyclic di-GMP (c-di-GMP) in biofilm formation, targeting a reduction in intracellular concentrations of c-di-GMP is believed to be a key aspect in the development of biofilm mitigation strategies. To facilitate this effort, here, we developed a transcription factor (TF)-based biosensor that integrates the TF FleQ from with a P-P tandem promoter. The dynamic range of the biosensor was optimized by fine-tuning the TF expression. The biosensor exhibited broad compatibility and effectiveness in detecting decreases in c-di-GMP levels across various biofilm model organisms, including strains lacking FleQ or its homologues, such as , , , and , as well as containing FleQ. Additionally, we monitored c-di-GMP levels in biofilms formed by and through a ratiometric, image-based quantification method. The methodology used the green fluorescence protein (GFP) as a reporter for c-di-GMP levels and 4',6-diamidino-2-phenylindole (DAPI) or the monomeric red fluorescence protein (mRFP) as the indicator for biofilm biomass. The GFP/DAPI or GFP/mRFP ratio gives effective c-di-GMP per unit of biomass. This TF-based biosensor provides an important tool to study c-di-GMP dynamics, which facilitates efforts in developing biofilm control strategies and understanding regulatory networks for biofilm development.