Cyclic-di-GMP binds to histidine kinase RavS to control RavS-RavR phosphotransfer and regulates the bacterial lifestyle transition between virulence and swimming

The two-component signalling system (TCS) comprising a histidine kinase (HK) and a response regulator (RR) is the predominant bacterial sense-and-response machinery. Because bacterial cells usually encode a number of TCSs to adapt to various ecological niches, the specificity of a TCS is in the centre of regulation. Specificity of TCS is defined by the capability and velocity of phosphoryl transfer between a cognate HK and a RR. Here, we provide genetic, enzymology and structural data demonstrating that the second messenger cyclic-di-GMP physically and specifically binds to RavS, a HK of the phytopathogenic, gramnegative bacterium Xanthomonas campestris pv. campestris. The [c-di-GMP]-RavS interaction substantially promotes specificity between RavS and RavR, a GGDEF-EAL domaincontaining RR, by reinforcing the kinetic preference of RavS to phosphorylate RavR. [c-diGMP]- RavS binding effectively decreases the phosphorylation level of RavS and negatively regulates bacterial swimming. Intriguingly, the EAL domain of RavR counteracts the above regulation by degrading c-di-GMP and then increasing the level of phosphorylated RavS. Therefore, RavR acts as a bifunctional phosphate sink that finely controls the level of phosphorylated RavS. These biochemical processes interactively modulate the phosphoryl flux between RavS-RavR and bacterial lifestyle transition. Our results revealed that c-di-GMP acts as an allosteric effector to dynamically modulate specificity between HK and RR.