Elucidating the role of c-di-AMP in Mycobacterium smegmatis: Phenotypic characterization and functional analysis

Cyclic-di-AMP (c-di-AMP) is an important secondary messenger molecule that plays a critical role in monitoring several important cellular processes, especially in several Gram-positive bacteria. In this study, we seek to unravel the physiological significance of the molecule c-di-AMP in Mycobacterium smegmatis under different conditions, using strains with altered c-di-AMP levels: c-di-AMP null mutant (ΔdisA) and a c-di-AMP over-expression mutant (Δpde). Our thorough analysis of the mutants revealed that the intracellular concentration of c-di-AMP could determine many basic phenotypes such as colony architecture, cell shape, cell size, membrane permeability etc. Additionally, it was shown to play a significant role in multiple stress adaptation pathways in the case of different DNA and membrane stresses. Our study also revealed how the biofilm phenotypes of M. smegmatis cells are altered with high intracellular c-di-AMP concentration. Next, we checked how c-di-AMP contributes to antibiotic resistance or susceptibility characteristics of M. smegmatis, which was followed by a detailed transcriptome profile analysis to reveal key genes and pathways such as translation, arginine biosynthesis, cell wall and plasma membrane are regulated by c-di-AMP in mycobacteria.