Background

Cyclic di-adenosine monophosphate (c-di-AMP) is an essential secondary messenger that plays a pivotal role in regulating various physiological processes, including osmoregulation, DNA integrity maintenance, sporulation, cell wall homeostasis, ion-channel regulation, antibiotic resistance, virulence gene expression, acid resistance, and carbon metabolism [1,2]. In the bacterial kingdom, c-di-AMP synthesis is catalyzed by di-adenylate cyclase (DAC) domain–containing proteins, which convert two ATP molecules into c-di-AMP in the presence of metal ions such as Mg and Mn. To date, five classes of DAC domain–containing proteins have been identified in bacteria: DNA integrity scanning protein A (DisA), c-di-AMP synthase A (CdaA), c-di-AMP synthase sporulation-specific (CdaS), c-di-AMP synthase N-terminal TM segment (CdaM), and c-di-AMP synthase Z (CdaZ) [3,4]. DisA, an octameric protein, has dual functions: it can bind to Holliday junction DNA or synthesize c-di-AMP [1]. Mycobacterium smegmatis is a non-pathogenic, rapidly growing bacterium from the genus Mycobacterium. It serves as a widely used model organism for studying mycobacteria, including pathogenic species like Mycobacterium tuberculosis and non-tuberculous mycobacteria (NTM) pathogens, such as Mycobacterium abscessus and Mycobacterium avium [5]. In M. smegmatis, the gene MSMEG_6080 encodes the c-di-AMP synthase DisA, which catalyzes c-di-AMP synthesis in the presence of Mg ions. DisA contains three main domains: the N-terminal DAC domain-1, the C-terminal DNA binding domain-3, and a linker domain (domain-2) that connects domain 1 and domain 2 [4,6]. The enzyme's active site is located at the interface of the DAC domains [1]. The cellular concentration of c-di-AMP is regulated by phosphodiesterases (PDEs), which are located in different operons and contain DHH-DHHA1 or HD (His-Asp) domains [7–9]. PDEs hydrolyze c-di-AMP into pApA or AMP. These enzymes typically feature a unique structural alignment, consisting of an N-terminal linked to a degenerate GGDEF domain and a C-terminal DHH-DHHA1 module, essential for c-di-AMP hydrolysis [9]. In Mycobacterium tuberculosis, the PDE (CnpB) has a core DHH-DHHA1 domain that hydrolyzes c-di-AMP first to linear 5'-pApA and then to two 5'-AMP molecules [8]. Similarly, the PDE in M. smegmatis (MSMEG_2630) contains a DHH-DHHA1 domain without additional regulatory domains found in the GdpP protein family of Bacillus subtilis. All types of PDEs require specific divalent metal ions (Mg, Mn, Co) for their activity [1,2,7]. The opposing activities of these enzymes maintain the homeostasis of c-di-AMP within bacterial cells. Herein, we describe an HPLC-based protocol to study the c-di-AMP synthesis by DisA protein from M. smegmatis. This protocol outlines a straightforward method for quantifying c-di-AMP synthesized by mycobacterial DisA. The method is adapted from protocols by Bai et al., Christen et al., and Ryjenkov et al., with necessary modifications [10–13]. This HPLC-based method allows quick, easy, and quantitative estimation of synthesized c-di-AMP by DisA, avoiding the utilization of potentially hazardous radioactive substrates and products.