Targets of cyclic di-AMP

The essentiality of the second messenger c-di-AMP in many bacteria is an unique feature and has attracted much attention during the last decade (Commichau et al., 2015a; Corrigan and Gründling, 2013). Consequently, many research groups tried to elucidate the essential role of the nucleotide in various bacteria. Several new methods were developed in the process to aid the elucidation, among them pull-down assays, high-throughput screenings and conditional generation and analyses of suppressor mutants (Corrigan et al., 2013; Kampf et al., 2017; Orr and Lee, 2017; Rubin et al., 2018). Indeed, a plethora of c-di-AMP targets (see Figure 1.3) has been identified in a diverse set of bacteria, among them B. subtilis, the pathogenic firmicutes L. monocytogenes,S. aureus and S. pneumoniae, the genome-reduced Mollicute M. pneumoniae as well as lactic acid bacteria and actinobacteria (Blötz et al., 2017; Corrigan et al., 2013; Gundlach, 2017; Gundlach et al., 2015a; Pham et al., 2018; Surekaet al., 2014;

Zarrellaet al., 2018; Zhanget al., 2013).

The first identified c-di-AMP receptor was the TetR-like transcription factor DarR in Mycobacterium smegmatis. DarR represses the expression of its own gene, ofcspA, encoding a cold shock family protein, and of two genes associated with fatty acid metabolism (Zhang et al., 2013). However, DarR homologues are only present in few actinobacteria suggesting a

minor, specialized role of DarR in c-di-AMP signal transduction (Corrigan and Gründling, 2013; Zhang et al., 2013).

Interestingly, several c-di-AMP targets from different bacteria are involved in various ways in the regulation of the cellular potassium homeostasis (Commichauet al., 2018b). One major target is the cytoplasmic gating component KtrA of the high-affinity K⁺ importer KtrAB, which was first identified inS. aureus(Corriganet al., 2013). KtrA can also be found in L. monocytogenes, B. subtilis as well as in close relatives, and binding of c-di-AMP to B. subtilisKtrA was shown recently (Commichauet al., 2015a; Gundlach, 2017). KtrA contains two highly conserved regulator of conductance K⁺ (RCK) domains, RCK^_C and RCK^_N (Albrightet al., 2006). While the RCK^_N domain binds ADP/ATP and NAD⁺/NADH, the RCK^_C domain binds c-di-AMP (Albrightet al., 2006; Corrigan et al., 2013). Another K⁺ uptake system, highly similar to KtrAB, is the low-affinity K⁺ importer KtrCD inB. subtilis (Holtmannet al., 2003). KtrC also exhibits both RCK domains and binds c-di-AMP (Corrigan et al., 2013; Gundlach, 2017). Yet another pair of orthologues are CabP (for KtrA/C) and TrkH (for KtrB/D) fromS. pneumoniae. Both proteins are required for growth of the bacteria at low K⁺ concentrations. It is assumed that direct binding of c-di-AMP to CabP, KtrA or KtrC leads to inhibition of K⁺ uptake by CabP/TrkH, KtrAB or KtrCD, respectively (Bai et al., 2014; Zarrellaet al., 2018). Interestingly, c-di-AMP levels in a S. pneumoniae cabP deletion mutant were significantly reduced, which was not the case for atrkH deletion mutant (Zarrellaet al., 2018).

Furthermore, the novel high-affinity K⁺ importer kimAwas just identified in B. subtilis and shown to be negatively regulated by c-di-AMP binding to the protein (Gundlach, 2017).

Interestingly, c-di-AMP also binds to thekimA (ydaO) riboswitch that is located upstream ofkimA and inhibits expression upon ligand-binding (genetic OFF-switch) (Gundlachet al., 2017b; Nelsonet al., 2013). ThekimA riboswitch is also found in front of the ktrAB operon, encoding a high-affinity K⁺ transporter as described before. This again makes c-di-AMP a unique second messenger. The dinucleotide governs one physiological process (K⁺ uptake) by regulating both the protein activity and gene expression through binding to the protein and the corresponding mRNA riboswitch (Gundlachet al., 2017b; Nelsonet al., 2013).

Another target protein involved in K⁺ homeostasis is the sensor histidine kinase KdpD inS. aureus (Commichauet al., 2018b; Corriganet al., 2013). The membrane-bound KdpD together with the cytosolic response regulator KdpE constitute a classical two-component system. Under severe K⁺ limitation KdpD is autophosphorylated and the phosphate group is transferred to KdpE which triggers expression of the high-affinity K⁺uptake systemkdpFABC (Ballalet al., 2007; Greie, 2011). The bacterial KdpDE/FABC system is widely distributed and also found in other c-di-AMP-producing bacteria such asM. smegmatis and the well studied L. monocytogenes, but not inB. subtilis(Aliet al., 2017; Ballalet al., 2007; Commichauet al., 2015a; Commichau et al., 2018a; Corrigan et al., 2013). Binding of c-di-AMP to S. aureus KdpD occurs at the universal stress protein domain, which impairs the expression of KdpFABC and consequently inhibits K⁺ import (Moscoso et al., 2016).

Another protein containing both RCK domains and that binds c-di-AMP is the cation/H⁺ antiporter CpaA from S. aureus (Corrigan et al., 2013). Recently, c-di-AMP binding was also shown for the orthologue YjbQ from B. subtilis (Gundlach, 2017). Binding of c-di-AMP stimulates the activity of CpaA. However, CpaA has no specificity for K⁺ over Na⁺ ions (Chin et al., 2015; Corrigan et al., 2013). Consequently, it is not entirely conclusive whether

the K⁺, Na⁺ or H⁺ gradient, or a combination, is the relevant function.

Osmolyte uptake

Figure 1.3: Targets known to bind c-di-AMP.(adapted from Commichauet al., 2018b). Major targets of c-di-AMP are proteins and RNA molecules that are involved in K⁺ or osmolyte homeostasis. Another target is the pyruvate carboxylase PycA in some bacteria. Several c-di-AMP-binding proteins are still of unknown function like CbpA, CbpB (YkuL) and DarA. More target proteins await characterization.

Other targets of c-di-AMP are involved in osmolyte uptake. c-di-AMP inhibits the OpuC osmolyte uptake system in S. aureus and L. monocytogenes (Huynhet al., 2016; Schuster et al., 2016). OpuC is an ATP binding cassette (ABC) osmoprotectant import system of the Opu family. OpuC consists of four different subunits, OpuCA, OpuCB, OpuCC and OpuCD (Hoffmann and Bremer, 2017). c-di-AMP binds to the cystathionine-β-synthase (CBS) domain of the adenosine triphosphatase (ATPase) subunit OpuCA. Subsequently, import of the compatible solute carnitine is inhibited as shown for S. aureus and L. monocytogenes (Huynh et al., 2016; Schuster et al., 2016). Binding of c-di-AMP to OpuCA inB. subtilis was

also shown recently (Gundlach, 2017).

Inhibition of osmolyte uptake by c-di-AMP is also found in lactic acid bacteria, however the mode of action is different (Devaux et al., 2018; Phamet al., 2018). Expression ofbusAA–

AB, encoding a glycine betaine osmoprotectant transporter, is inhibited by the transcriptional repressor BusR (Romeo et al., 2003). Binding of c-di-AMP to the TrkA^_C domain of BusR enhances the repression of busAA–AB, leading to the inhibition of glycine betaine uptake (Devaux et al., 2018; Phamet al., 2018).

In addition to targets involved in potassium and osmolyte homeostasis, c-di-AMP binding has also been reported for a variety of other proteins. c-di-AMP binds to the pyruvate carboxylase PycA in Enterococcus faecalis,S. aureus, L. monocytogenes andL. lactis and inhibits its activity (Choiet al., 2017; Sureka et al., 2014; Whiteleyet al., 2017). PycA acts in the tricarboxylic acid (TCA) cycle and catalyzes the ATP-dependent conversion of pyruvate to oxaloacetate (Jitrapakdee et al., 2008). Interestingly, PycA is of special importance in L. monocytogenesandL. lactis since both bacteria only contain a truncated TCA cycle (Schär et al., 2010; Wanget al., 2000). c-di-AMP binding toB. subtilis PycA has not been addressed so far. Recently, the mycobacterial recombinase RecA was shown to bind both c-di-AMP and c-di-GMP. It was further demonstrated that c-di-AMP repressed the translation ofrecA mRNA and influenced homologous recombination and DNA damage repair (Manikandan et al., 2018). In addition to the aforementioned OpuCA, the poorly characterized proteins CbpA and CbpB (YkuL inB. subtilis) from L. monocytogenes also contain CBS domains which bind c-di-AMP (Gundlach, 2017; Surekaet al., 2014). However, their function remains to be elucidated (Commichauet al., 2018b). Another c-di-AMP-binding protein of unknown function is the PII-like protein DarA (PstA in some organisms), which is highly conserved among Gram-positive, c-di-AMP-producing firmicutes (Gundlachet al., 2015a). DarA itself is subject of this thesis and will be discussed later in detail. Even more c-di-AMP-binding proteins have been identified recently inB. subtilis, among them KhtT and MgtE (Gundlach, 2017). KhtT is part of the K⁺/H⁺ antiporter KhtSTU and contains an RCK^_C domain, which is known to bind c-di-AMP. KhtT has no RCK^_N domain, in contrast to KtrA and KtrC (Albrightet al., 2006; Corrigan et al., 2013; Fujisawa et al., 2004). MgtE is the main Mg²⁺

importer inBacillus and contains a CBS domain, which is already known to bind c-di-AMP (Surekaet al., 2014; Wakeman et al., 2014). Interplay of the two proteins in the c-di-AMP

signaling network still remains to be unraveled.