Cyclic di-GMP co-activates the two-component transcriptional regulator DevR in Mycobacterium smegmatis in response to oxidative stress

Cyclic di-GMP (c-di-GMP) is an important second messenger in bacteria, and its regulatory network has been extensively studied. However, information regarding the activation mechanisms of its receptors remains limited. In this study, we characterized the two-component regulator DevR as a new c-di-GMP receptor and further uncovered a novel co-activation mechanism for effective regulation of DevR in mycobacteria. We show that high c-di-GMP levels induce the expression of the devR operon in Mycobacterium smegmatis and increase mycobacterial survival under oxidative stress. The deletion of either DevR or its two-component kinase DevS significantly weakened the stimulating effect of c-di-GMP on oxidative-stress tolerance of mycobacteria. We also found that DevR senses the c-di-GMP signal through its C-terminal structure and that c-di-GMP alone does not directly affect the DNA-binding activity of DevR. Strikingly, c-di-GMP stimulated DevR phosphorylation by the kinase DevS, thereby activating DevR's DNA-binding affinity. In summary, our results indicated that c-di-GMP triggers a phosphorylation-dependent mechanism that co-activates DevR's transcriptional activity. Our findings suggest a novel paradigm for the cross-talk between c-di-GMP signaling and two-component regulatory systems that activates transcription of stress-response genes in bacteria.