Identification of novel genetic factors that regulate c-di-AMP production in <i>Staphylococcus aureus</i> using a riboswitch-based biosensor

Nucleotide secondary messengers regulate various processes in bacteria allowing them to rapidly respond to changes in environmental conditions. c-di-AMP is an essential second messenger required for the growth of the human pathogen <i>Staphylococcus aureus</i>, regulating potassium, osmolyte uptake, and beta-lactam resistance. Cellular concentrations of c-di-AMP are regulated by the activities of two enzymes, DacA and GdpP, which synthesize and hydrolyze c-di-AMP, respectively. Besides these, only a limited number of other factors are known to regulate c-di-AMP levels. Using a c-di-AMP biosensor consisting of the <i>Bacillus subtilis</i> c-di-AMP-binding <i>kimA</i> riboswitch and <i>yfp,</i> we were able to efficiently detect differences in cellular c-di-AMP levels in <i>S. aureus</i>. To identify novel factors that regulate c-di-AMP levels, we introduced the biosensor into a library of <i>S. aureus</i> transposon mutants. In this manner, we obtained mutants with increased c-di-AMP levels that contained insertions in <i>gdpP</i> coding for the c-di-AMP hydrolase and <i>ybbR</i> (<i>cdaR</i>) coding for a c-di-AMP cyclase regulator, thus validating our screen. We also identified two high c-di-AMP mutants with insertions upstream of the <i>nrdIEF</i> operon coding for the ribonucleotide reductase enzyme. Further analysis revealed that the insertion down-regulated <i>nrdIEF</i> expression, indicating that the enzyme is a negative regulator of c-di-AMP production. This negative regulation was dependent on <i>rsh</i>, encoding for the synthase of the endogenous GdpP inhibitor (p)ppGpp. The methods established in this work can be readily adapted for use in other bacteria to uncover genetic or environmental factors regulating c-di-AMP levels.IMPORTANCEc-di-AMP is an important secondary messenger, produced by many bacterial species including the opportunistic pathogen <i>Staphylococcus aureus</i>. In this bacterium, c-di-AMP controls cell wall homeostasis, cell size, and osmotic balance. In addition, it has been shown that strains with high c-di-AMP levels exhibit increased resistance to beta-lactam antibiotics. Here, we developed a biosensor-based method for the rapid detection of c-di-AMP levels in <i>S. aureus</i>. We utilized the biosensor in a genetic screen for the identification of novel factors that impact cellular c-di-AMP. In this manner, we identified the ribonucleotide reductase as a novel factor altering cellular c-di-AMP levels and showed that reducing its expression leads to increased cellular c-di-AMP levels. As methicillin-resistant <i>S. aureus</i> strains are considered as a global health threat, it is important to study processes that dictate cellular c-di-AMP levels, which are associated with antibiotic resistance.