Exploring the evolutionary divergence of cyclic di-nucleotide signaling in diverse mycobacterial species.

Cyclic dinucleotides (CDNs), such as cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP), are key second messengers that regulate fundamental bacterial processes, including cell wall synthesis, biofilm formation, antibiotic resistance, stress response, and virulence. These pathways are particularly relevant in major pathogens like Mycobacterium tuberculosis. Increasing evidence highlights the pathogenic potential of non-tuberculous mycobacteria (NTM), originally environmental species that are emerging as significant human pathogens. Understanding the evolution of CDN signaling may therefore provide critical insights into this transition. Our comparative genomic analysis revealed that the c-di-AMP synthase disA is present as a single copy in nearly all mycobacterial genomes, except within the genus Mycolicibacter.The corresponding phosphodiesterases, pde and ataC, are variably distributed, with pathogenic mycobacteria showing a preference for pde over ataC. In contrast to the relatively conserved c-di-AMP system, the c-di-GMP pathway comprising diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), exhibits remarkable variation in gene presence/absence and domain architecture across the Mycobacteriaceae family. This diversity suggests multiple independent gene gain and loss events throughout evolution, often accompanied by the acquisition of accessory domains. Evolutionary analyses reveal a clear dichotomy between the two CDN signaling systems. The c-di-AMP pathway, governed by disA, which is under strong purifying selection similar to core housekeeping genes, and pde, which shows low genetic variability, underscores its conserved and essential role in maintaining core cellular physiology. In contrast, the c-di-GMP system is markedly more heterogeneous, consistent with its function in environmental sensing and adaptation. Together, these findings highlight a sharp evolutionary split in CDN signaling within the Mycobacteriaceae family, c-di-AMP serves as an indispensable regulator of core physiological processes, whereas c-di-GMP confers flexibility for niche-specific adaptation and survival.