Cyanobacteria exhibit a distinctive lifestyle as photoautotrophic bacteria, necessitating rapid responses to environmental shifts that jeopardize their carbon/nitrogen (C/N) balance. To maintain homeostasis, cyanobacteria employ cyclic nucleotide second messengers, such as cyclic di-adenosine monophosphate (c-di-AMP)¹. In cyanobacteria, c-di-AMP has now been implicated in a broader range of functions, including C/N balance regulation², day-night acclimation³, and natural competence⁴. While mechanistic aspects of C-regulation have been elucidated³, N-regulation remains unexplored. In the search for novel c-di-AMP targets, we identified the glutamine (Gln) transporter BgtAB as a c-di-AMP target. Gln was reported to be toxic for cyanobacteria⁵, but its mode of action remained elusive. Surprisingly, the c-di-AMP-free mutant (ΔdacA) was able to grow on toxic Gln concentrations and use it as sole N-source, while wildtype (WT) died. We found that ΔdacA exhibited impaired Gln uptake compared to WT. Here, targeted metabolomics unveiled an overaccumulation of arginine and its intermediates in WT cells growing on Gln. In contrast, Gln metabolization was severely hampered in ΔdacA, highlighting a role for c-di-AMP in controlling N-metabolism. We further show that the high Gln uptake in WT induces dacA gene expression and c-di-AMP overproduction, which is known to be toxic for bacteria⁶. Overall, our findings suggest that c-di-AMP positively regulates BgtAB, and the resulting Gln uptake triggers c-di-AMP overproduction, establishing a lethal feedback loop. This work presents the first mechanistic explanation of Gln toxicity in cyanobacteria due to c-di-AMP signalling and showcases the role of c-di-AMP in N-metabolism.

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