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 Pseudomonas aeruginosa with a P R -P pel 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 Escherichia coli, Shewanella oneidensis, Comamonas testosteroni, and Acinetobacter baumannii, as well as P. aeruginosa containing FleQ. Additionally, we monitored c-di-GMP levels in biofilms formed by P. aeruginosa and S. oneidensis 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.