New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus

Abstract

Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function, and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane-bound Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5′-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared with the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism.

Cyclic nucleotides in archaea: Cyclic di‐AMP in the archaeonHaloferax volcaniiand its putative role

“…Various studies on intracellular c‐di‐AMP levels of bacteria have been conducted so far and a comparison with results presented here suggests that the intracellular concentration of c‐di‐AMP in H. volcanii (10.09 ng/mg protein on average under the tested conditions) is comparable to that reported for bacterial species. It was similar to levels in wild type Bacillus subtilis (5.6 ± 2.82 ng/mg protein) (Mehne et al, ), however, considerably lower than the measurements in Staphylococcus aureus (~140 ng/mg protein) (Bowman, Zeden, Schuster, Kaever, & Gründling, ).…”

The lost language of the RNA World

“…Cyclic dinucleotides with mixed sequence (c-AMP-GMP) and mixed phosphodiester linkages (cGAMP) serve as modern examples of the greater diversity of RNA words harnessed by present cells that could also have been exploited billions of years ago in the RNA World. Finally, evidence is currently emerging for the biological roles of the linear dinucleotides pGpG and pApA (67–69). These compounds can result from the degradation of c-di-GMP and c-di-AMP, suggesting that their linear degradation products might also be sensed as an extension of the c-di-GMP or c-di-AMP signaling networks, respectively.…”

“…Annotations i , a , b , c , and i a/b/c represent all possible combinations of isomers with variation in the phosphodiester linkage connectivity and variation in the location and connectivity of the terminal phosphate. Red highlights the compounds pGpG (67) and pApA (68, 69), for which there is evidence of biological roles in modern cells. ( B ) Matrix depicting all 10 possible cyclic dinucleotide RNAs formed by the four common ribonucleotides joined by 3′,5′-phosphodiester linkages.…”

Identification of the main glutamine and glutamate transporters inStaphylococcus aureusand their impact on c-di-AMP production

“…Intracellular c-di-AMP levels in WT LAC* and the indicated S. aureus mutant strains were determined using a previously described competitive ELISA method (Underwood et al ., 2014) and a slightly modified method for the preparation of S. aureus samples (Bowman et al ., 2016). Briefly, a single colony of the WT and different S. aureus mutant strains were picked from TSA plates and used to inoculate 5 ml of either CDM+Gln or CDM+Glu and the cultures were incubated for 18 h at 37°C with shaking.…”