Substrate dependent homeostatic control of c-di-AMP synthase (MsDisA) and hydrolase (MsPDE) from Mycobacterium smegmatis

Abstract

AbstractIn bacteria, cyclic-di-nucleotide based second messengers regulate various physiological processes including the stress response. For the past few decades, cyclic diadenosine monophosphate (c-di-AMP) is emerging as a crucial second messenger in bacterial world. It is an essential molecule and has been implicated in fatty acid metabolism, antibiotic resistance, biofilm formation, virulence and activates the cytosolic pathway of innate immunity in host cell. The level of c-di-AMP is maintained within the cell by the action of two opposing enzymes, namely diadenylate cyclases and phosphodiesterases. However, such kind of c-di-AMP modulation remains to be explored in Mycobacterium smegmatis. Here, we systematically investigate the c-di-AMP synthase (MsDisA) and a hydrolase (MsPDE) from M. smegmatis at different pHs and osmolytic conditions. Our biochemical assays showed that the MsDisA activity is enhanced during the alkaline stress and c-di-AMP is readily produced without any intermediates. At pH 9.4, the MsDisA promoter activity increases significantly, further strengthening this observation. However, under physiological conditions, the activity of MsDisA is moderate with the formation of intermediates. We also observe that the size of MsDisA is significantly increased upon incubation with substrate. To further get deep insights into the structural characteristics, we report a 3.8 Å cryo-EM structure of the MsDisA protein, distinct from the earlier reported structure of DisA from Thermotoga maritima. The domain mutant experiments prove that the N-terminal minimal region can form a functional octamer. Thus, our results reveal how mycobacterial c-di-AMP is biochemically and structurally regulated in response to different environment.