A dual-threshold system relying on multiple c-di-GMP metabolic enzymes controls cell fate of a cyanobacterium.

Cyclic-di-GMP (c-di-GMP) is a ubiquitous second messenger in bacteria and regulates a variety of cell activities. Many bacteria contain multiple enzymes involved in c-di-GMP synthesis or degradation; however, how they coordinate with each other to orchestrate c-di-GMP homeostasis remains unclear. Here, using the cyanobacterium Anabaena PCC 7120 as a model, we created cdG0 and cdGmax strains by deleting all 8 and 14 genes, respectively, that encode enzymes with c-di-GMP degradation and synthesis domains, alongside a collection of mutants with various numbers of these genes deleted. Our findings demonstrate that c-di-GMP in Anabaena not only modulates cell size but is also indispensable for cell viability. Quantitative analysis established two critical physiological thresholds in vivo: a minimal c-di-GMP level required for cell size maintenance and a lower, lethal threshold essential for survival. We show that the 16 enzymes involved in c-di-GMP turnover in Anabaena function as an electromechanical-like dual relay to control c-di-GMP dynamics, with different modules contributing to c-di-GMP homeostasis or responding as an SOS alarm when the c-di-GMP concentration drops below the lethal threshold. Both effects of c-di-GMP on cell size reduction and cell viability are mediated by the cyclic-di-GMP receptor (CdgR), depending on the amount of the c-di-GMP-free form of CdgR available because of titration by c-di-GMP in the cells. The system, with the two concentration thresholds of c-di-GMP that dictate cell size and viability, respectively, enables dynamic cellular adaptation while preventing lethal effects.