The first identified bacterial c-di-AMP-regulated protein was the TetR-like transcriptional factor DarR in Mycobacterium smegmatis (Zhang et al., 2013). Binding of c-di-AMP has been shown to stimulate DNA-binding of DarR that in consequence negatively regulates expression of an operon coding for three genes, including a major facilitator family transporter, a medium chain fatty acyl-CoA ligase and a homolog of the cold shock protein CspA. Since this study, c-di-AMP was found to regulate a variety of cellular targets in bacteria, of, which many are highlighted in Fig. 1.2.
c-di-AMP was identified to bind and regulate the potassium import system KtrAB by binding to the RCK_C domain of the cytosolic KtrA and cation/proton antiporter CpaA in S. aureus that putatively functions as a potassium export system, which were also subjects of further studies illuminating the molecular mechanisms (Chin et al., 2015; Corrigan et al., 2013; Kim et al., 2015). Interestingly, c-di-AMP would thereby control potassium homeostasis in S. aureus by inhibiting import and stim-ulating export. In the same study, the PII-like protein PstA of unknown function and the histidine kinase KdpD of the KdpDE two component system were identified as c-di-AMP binding proteins (Corrigan et al., 2013). While the function of PstA is unknown, KdpDE regulated expression of the Kdp(F)ABC potassium import system, indicating again an important role for c-di-AMP in potassium homeostasis. Intriguingly, translation of KtrAB and YdaO (which was later identified as a high-af-finity potassium importer in B. subtilis and renamed KimA) are regulated by a c-di-AMP responsive riboswitch. Binding of c-di-AMP to the ydaO (kimA) riboswitch prevents translation of the potas-sium importers. This makes c-di-AMP a special regulator and the first of its kind by regulating a biological process by controlling synthesis and activity (Gao & Serganov, 2014; Gundlach et al., 2017; Moscoso et al., 2015; Nelson et al., 2013).
To emphasize the importance of c-di-AMP on potassium homeostasis, it should be noted that it was identified to regulate potassium import in a whole variety of different bacteria. It was found to inhibit uptake in S. pneumoniae via binding to the CabP protein that interacts with the potas-sium importer TrkH and thereby inhibits import (Bai et al., 2014). Interestingly, a later study showed that CabP deletion leads to reduced c-di-AMP concentrations in S. pneumoniae, indicating that either CabP or the resulting altered potassium homeostasis is able to be senses and thereby to modulate c-di-AMP homeostasis (Zarrella et al., 2018). Moreover, c-di-AMP concentrations were recently identified to be light-dependent in the cyanobacterium Synechococcus elongatus and a cdaA deletion mutant was more susceptible to oxidative stress during the nighttime part of a day-night cycle. Interestingly, S. elongatus encodes several putative c-di-AMP regulated potas-sium importers and an impairment in potaspotas-sium transport leads to a similar susceptibility to oxi-dative stress, as it was shown for the cdaA mutant (Rubin et al., 2018). In L. lactis, c-di-AMP has been shown to inhibit potassium uptake via KupA and KupB of the Kup family potassium importer and even in the minimal organism M. pneumoniae, c-di-AMP was identified as a regulator of po-tassium uptake (Blötz et al., 2017; Quintana et al., 2019). Eventually, c-di-AMP was shown to be essential due to its central role in regulating potassium homeostasis in B. subtilis (Gundlach et al.,2017). Taken together this demonstrates the critical role of c-di-AMP in potassium homeostasis in a broad spectrum of different bacteria.
c-di-AMP was also found to regulate other cellular processes or bind to proteins whose function is not yet elucidated. It was shown to allosterically inhibit the pyruvate carboxylase PycA in
Fig. 1.2 Synthesis and degradation of the essential second messenger c-di-AMP and its cellular targets. c-di-AMP is synthesized by DACs. Shown are members of the main three types of DACs: CdaA, DisA and CdaS. CdaA activity is mod-ulated by the YbbR-domain containing protein CdaR by the cell wall precursor synthesizing glucosamine mutase GlmM.
c-di-AMP is degraded by PDEs to pApA. Shown are members of the main types of PDEs, GdpP and PgpH and also of the DhhP- (that can also degrade pApA further to AMP) and the CdnP-type, (that degrades c-di-AMP to AMP) as well as the pApA to AMP degrading nano-RNase NrnA. Shown is also MdrT, a member of c-di-AMP secreting multidrug resistance transporters. Furthermore, targets that have been shown to bind c-di-AMP are depicted: the pyruvate carboxylase PycA, which is allosterically inhibited by c-di-AMP and the proteins of unknown function: DarA, CbpA and CbpB of L. mono-cytogenes. Moreover, the known c-di-AMP-controlled osmolyte transporters are shown: The high-affinity potassium uptake systems KtrAB and KimA that are controlled on protein level and via a c-di-AMP-binding riboswitch in B. subtilis, the Kdp(F)ABC high-affinity potassium transporter, whose transcription is regulated via the c-di-AMP inhibited two com-ponent system KdpDE in S. aureus and the low-affinity potassium transporter KtrCD. Two novel high-affinity potassium transporters, KupA and KupB, which were recently identified as c-di-AMP regulated in L. lactis are depicted, as well.
c-di-AMP, moreover, regulates the putative potassium exporter CpaA in S. aureus and binds the BusR transcription fac-tor that inhibits expression of the glycine betaine importer BusAB in L. lactis and S. agalactiae. Finally, the OpuC glycine betaine transporter from B. subtilis is depicted, too (modified from Commichau et al., 2018).
c-di-AMP Activation
L. monocytogenes and L. lactis or bind to the S. aureus PstA homologs in B. subtilis (DarA) or L. monocytogenes (PstA). The same is also true for two other proteins of unknown function, CbpA and CbpB in L. monocytogenes and NrdR, the negative regulator of the ribonucleotide reductase operon nrd (Campeotto et al., 2015; Choi et al., 2015; Choi et al., 2017; Gundlach et al., 2015a;
Müller et al., 2015a; Sureka et al., 2014). While the function of PstA/DarA or the c-di-AMP binding proteins CbpA or CbpB is not yet understood, recent studies identifying novel binding proteins or genetic linkages via c-di-AMP dependent suppressor mutations demonstrate that c-di-AMP is not only regulating potassium homeostasis, but homeostasis of osmolytes in general. c-di-AMP can therefore be described as a major regulator for osmoregulation in Firmicutes. Whiteley et al. iden-tified the growth of L. monocytogenes in a defined minimal medium as the first reported condition where c-di-AMP was non-essential (Whiteley et al., 2015). They could show that the limitation of osmolytes allows deletion of cdaA and that accumulation of suppressor mutations in genes en-coding oligopeptide uptake systems (OppABCDF), the PII-like protein PstA, the alarmone syn-thase/hydrolase RelA, a glycine betaine importer (GbuABC), the pyruvate carboxylase, the potas-sium importer homolog Lmo0993 (KtrD) or the CbpB protein contributed, among others, to sup-pression of c-di-AMP essentiality on complex media. In their second study they could, further-more, show that the mutations in the opp and gbu genes are sufficient to allow cdaA deletion mutants to grow on complex medium, resulting in the hypothesis that uncontrolled accumulations of oligopeptides and glycine betaine under these conditions is detrimental for growth of L. mono-cytogenes. Interestingly, they could further show that c-di-AMP impacts regulation of the flux in the citric acid cycle, an import cross-section between carbon and nitrogen metabolism and there-fore also biosynthesis of amino acids that also act as osmolytes (Whiteley et al., 2017). c-di-AMP, furthermore, has been identified to regulate uptake of the osmolyte carnitine in L. monocytogenes by binding to the CBS domain of OpuCA of the OpuC transporter but not to the CBS domain in GbuA of the GbuABC glycine betaine importer or the CBS domain of BilEA, of the bile salt specific exporter BilE (Huynh, et al., 2016). Binding of c-di-AMP to OpuCA was also confirmed for S. aureus but again other CBS domain containing proteins, including the magnesium transporter MgtE and the transcription factor CcpN, where not identified as c-di-AMP binding proteins (Schuster et al., 2016). Interestingly, although c-di-AMP has been established as a major determinant for osmotic regulation in S. aureus, due to its effect on osmolyte (glycine betaine via OpuD) and amino acid uptake (AlsT), it also seems to have a great impact on cell respiration and is dispensable under anaerobic conditions in this bacterium, highlighting that the complex regulatory effects of c-di-AMP are even with the increased knowledge not yet fully understood (Zeden et al., 2018).
c-di-AMP has furthermore been identified to regulate osmotic homeostasis in Streptococcus aga-lactiae and L. lactis, by binding to BusR, that upon binding negatively regulates expression of the genes encoding BusAB, a glycine betaine importer (Devaux et al., 2018; Pham et al., 2018). Inter-estingly, in the study of Pham and colleagues, impact of osmotic up- and downshifts on c-di-AMP concentrations in different bacteria and mutants was investigated, too. They could show, that upon osmotic upshift (hyperosmotic shock) c-di-AMP levels rapidly decrease and vice versa in-crease upon hypoosmotic shock. Intriguingly, energizing of the cells that is required for ATP syn-thesis and presence of the DAC CdaA and the PDE GdpP/PdeA seemed to have the greatest influ-ence on the bacteria to adapt their c-di-AMP concentration accordingly (Pham et al., 2018). Taken together, c-di-AMP has been shown to influence many cellular processes, predominantly osmotic homeostasis, in many bacteria that produce this nucleotide second messenger, with L. monocyto-genes being a prominent bacterium of c-di-AMP research (Commichau et al., 2015; Commichau et al., 2018).