Understanding c-di-GMP Regulation and Dynamics in Escherichia coli

To adapt to environmental changes, bacteria rapidly transition between motile and sessile lifestyles, which are favoured under exponential growth and stationary phase, respectively. As a central regulator of this lifestyle switching, the second messenger c-di-GMP is dynamically modulated in response to growth phase transitions and flagellar activity. In Escherichia coli, this switch is globally controlled by the opposing activities of DgcE (synthase) and PdeH (hydrolase), whose expression is inversely regulated by the the stress response sigma factor (RpoS) and flagellar sigma factor (FliA), respectively. RdcA and its accessory protein RdcB act as post-translational activators of DgcE. This thesis establishes that RdcA integrates both metabolic states (GTP levels) during growth phase transitions and flagellar gene regulation (from FliA) into c-di-GMP production. Using fluorescence microscopy to monitor protein localisation and Förster resonance energy transfer (FRET) to quantify protein-protein interactions, we demonstrate that RdcA exhibits enhanced membrane localisation and interaction with DgcE at stationary phase. We propose that RdcA detects GTP depletion during exponential-to-stationary phase transitions and activates DgcE. This post-translational control enables rapid responses, complementing the slower transcriptional control mediated by the RpoS-FliA sigma factor competition, which controls DgcE and PdeH expression levels. While RdcA activates c-di-GMP production via DgcE, its own transcription is controlled by FliA, thereby bridging the antagonistic FliA-PdeH (motility) and RpoS-DgcE (sessility) regulatory programmes. Using a combination of FRET-based c-di-GMP biosensors and a FliA-dependent transcriptional reporter, we characterise these dynamics over batch culture growth phase transitions and single-cell fluctuations in a microfluidic device. Our findings suggest two key functions of this circuit: (1) it buffers against stochastic FliA fluctuations by balancing the opposing activities of RdcA and PdeH, and (2) it generates an ultrasensitive response due to the saturable stoichiometry of the RdcA-DgcE complex. As a result, the system sharpens c-di-GMP responsiveness to growth phase transitions and drives single-cell fluctuations at steady state, thereby supporting phenotypic heterogeneity while preserving robust commitment to the motile or sessile states.