Are there additional putative targets?

It has been argued previously that the NAGK ArgB would be a suitable interaction target for DarA since the context as well as the structure fit nicely (Richts, 2018). The NAGK catalyzes the rate-limiting step of arginine biosynthesis and is already known to be regulated by a PII

protein in unicellular cyanobacteria. It has been shown that binding of PII to the NAGK increases the activity of the enzyme and releaves it from feedback inhibition by arginine in S. elongatus (Maheswaran et al., 2004). NAGK–PII interactions have also been reported for PIIhomologues in eukaryotes like the microalgae Myrmecia incisa, the red algaePorphyra purpurea and different plants (Burillo et al., 2004; Chenet al., 2006; Lapina et al., 2018; Li et al., 2017; Sugiyama et al., 2004). Our results exclude ArgB as a potential target candidate for an interaction since DarA has to act upstream of the arginine biosynthesis pathway. This is in agreement with BACTH assays with and without c-di-AMP present and SPINEs where no interaction of DarA with ArgB was detectable. Several in vivo andin vitro experiments conducted in parallel suggest the same (Richts, 2018).

At a glance, suitable possibilities for DarA to promote arginine synthesis by modulating glutamate production appear to be sparse (see also Figure 4.1 for an overview about the metabolic context). For example, DarA neither interacts with the transcriptional activator GltC nor influences the expression of gltAB in SPINEs andβ-galactosidase activity assays, respectively (Krammer, 2017). However, the experiments by Krammer (2017) were not done under genetic/experimental conditions where an interaction with DarA is crucial which should be repeated immediately. Similar to the interaction of GlnK with TnrA, DarA could potentially stabilize GltC and thus enhance the transcription of gltAB (Schumacher et al., 2015).

The glutamine synthetase GlnA works in concert with GltAB (GS/GOGAT system) and utilizes glutamate and NH4⁺ as substrates. In B. subtilis NH4⁺ assimilation is solely achieved by the GS/GOGAT system and not by a glutamate dehydrogenase (Gunka and Commichau, 2012). Recently, GlnA was linked to c-di-AMP and K⁺ homeostasis via the c-di-AMP regulated, high-affinity K⁺ importer KimA. Expression of kimA was increased by 4-fold in a ∆glnA strain when cells were grown with low amounts of potassium in the presence of ammonium and glutamine (Gundlach, 2017). Initially, expression of kimA has been described to be glutamate dependent (Gundlachet al., 2017b). Although the interplay needs more clarification, this adds yet another facet to the already highly complex regulation of glutamate and c-di-AMP metabolism and even DarA might contribute to this. However, GlnA belongs to the 100 most abundant proteins in B. subtilis and DarA protein amounts are lower by roughly 10 to 100-fold which makes a regulation by physical interaction impossible (Eymann et al., 2004; Maass et al., 2011; Maaß et al., 2014). As mentioned before, activity of the GS in enteric bacteria like E. coli is governed by its adenylation state and the enzyme is inactive when fully adenylated (van Heeswijk et al., 2013). This process is regulated by the PII protein GlnB which interacts with the ATase GlnE and either stimulates (unmodified) or inhibits (uridylated) the adenylation of the GS (Leigh and Dodsworth, 2007). However, in

B. subtilis the GS is not regulated by adenylation but instead by feedback inhibition (Fisher, 1999). When glutamine is present, GS activity is not needed and feedback inhibited by binding of glutamine to the GS (FBI-GS). Furthermore, the FBI-GS binds the transcriptional repressor GlnR and in complex represses the expression of theglnARoperon by binding to the promoter region, thus regulating its own expression (Fisher, 1999; Fisher and Wray, 2008).

In principle, inhibition of the glutamate degrading GDH by DarA could increase the available glutamate and indeed the hexamaric RocG also fits structurally (Gunkaet al., 2010).

However, rocG is not expressed under the conditions where DarA is needed since the cells are growing with glucose and ammonium as the carbon and nitrogen source, respectively. In the presence of glucoserocG is subject to CCR to prevent futile, ATP-consuming cycling of glutamate synthesis and degradation. This is achieved by the pleiotropic transcription factor CcpA. Under this condition CcpA forms a complex with the histidine-carrier protein HPr from the phosphotransferase system and inhibits the transcription ofrocG and many other catabolic genes (Belitskyet al., 2004; Commichauet al., 2008; Görke and Stülke, 2008). Furthermore, CcpA inhibits transcription of the alternative sigma factor σ^L which is needed for rocG expression (Choi and Saier, 2005; Débarbouilléet al., 1991; Gunka and Commichau, 2012).

In addition, the transcription factors AhrC and RocR are needed to activate transcription ofrocG when arginine is present (Belitsky and Sonenshein, 1999; Belitsky and Sonenshein, 1998; Commichauet al., 2007). However, arginine is not present in the medium and ahrC is deleted in the strain. In summary, RocG cannot be a target of DarA. AlthoughB. subtilis additionally encodes GudB, this second GDH can also be excluded. In the laboratory strain 168, a perfect direct repeat is inserted in the gudB gene and the enzyme is not functional (Belitsky and Sonenshein, 1998).

In one suppressor of thektrAB kimA ahrC darAdeletion mutant we also found a mutation inodhB which codes for the E2 subunit of the 2-oxoglutarate dehydrogenase complex (ODH).

TheB. subtilis ODH is composed of several monomers from the subunits OdhA (E1), OdhB (E2) and PdhD (E3) and catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl coenzyme A in the TCA cycle (Carlsson and Hederstedt, 1989). The mutation inodhB leads to an amino acid exchange within the lipoyl-binding domain and might impair the function of OdhB/ODH (Saieret al., 2016). This would arguably increase the available 2-oxoglutarate pool for glutamate synthesis by GltAB. However, inhibition of the huge ODH by DarA to promote glutamate synthesis seems highly unlikely from a structural point of view. InE. coli the structure has been resolved and the stoichiometry has been determined to 12:24:12 of the subunits E1:E2:E3, respectively (Pettit et al., 1973). In Gram-positive bacteria 2-oxo acid dehydrogenase complexes even show a 30:60:6 subunit arrangement, although no structure forBacillus ODH is available (Mattevi et al., 1992; Neveling et al., 1998). In addition, the genes for all three subunits are highly expressed and the proteins are way more abundant than DarA even when the genes are subject to CCR (Blenckeet al., 2003; Maasset al., 2011;

Maaßet al., 2014).

Recently, the pyruvate dehydrogenase complex (PDH) has been suggested as a potential target of PstA (DarA) in L. monocytogenes since subunits interacted with PstA in yeast two-hybrid and pull-down assays (Whiteley et al., 2017). The PDH catalyzes the oxidative decarboxylation of pyruvate to form acetyl-CoA which is fed into the TCA cycle, thus linking it with glycolysis (Gaoet al., 2002). As mentioned before, the authors found mutations blocking the allosteric activation of the pyruvate carboxylase PycA by acetyl-CoA. These mutations phenocopied the deletion of pstAin the ∆dacAstrain which rescued growth on rich medium.

Since no interaction of PstA with PycA was detectable, it was argued that a stimulating effect of apo-PstA on acetyl-CoA production might be reasonable (Whiteley et al., 2017). However, a physical interaction of DarA with the PDH is highly unlikely. Similar to the ODH, PDHs are enormous multienzyme complexes of 5 to 10 MDa. In Gram-positive bacteria like the close relativeBacillus stearothermophilus the PDH consists of a dodecahedron-like core composed of 60 E2 subunits that bind 30 E1 and 6 E3 subunits (Mattevi et al., 1992; Neveling et al., 1998). In addition, all subunits are among the 100 most abundant proteins inB. subtilis which makes regulation by physical binding of DarA impossible (Eymann et al., 2004).

In the aforementioned report the authors suggested that misregulation of the TCA cycle in the absence of c-di-AMP leads to a detrimental alteration of osmotic pressure. In L. monocytogenesPycA is hyperactive without c-di-AMP and the cells accumulate oxaloacetate, citrate and glutamate/glutamine which can be prevented by deletion of citZ (coding for the citrate synthase) (Sureka et al., 2014; Whiteley et al., 2017). Interestingly, Whiteley et al. (2017) showed that a lack of the citrate synthase, but not a lack of aconitase or isocitrate dehydrogenase which catalyze the next steps in the TCA cycle, also phenocopies the ∆dacA∆pstAstrain. Consequently, the authors stated that the accumulation of citrate and not downstream metabolites like glutamate is toxic for the ∆dacA strain. However, L. monocytogenes only contains a truncated TCA cycle and oxaloacetate is mainly produced by PycA, which is not the case for B. subtilis so the phenotypes are likely to be different (Schär et al., 2010). Unfortunately, intracellular amounts of citrate or glutamate of the ∆dacA

∆pstA strain were not provided by the authors so it is not clear which metabolite is causative for the ∆pstAphenotype.

Several reports of PII protein interactions suggest that there is a unified mechanism by which these small proteins interact with their targets. In almost all cases, the loop regions play a pivotal role and govern the interaction. The B-loops of DarA are unusually long and even longer than the usual T-loops in GlnK. Furthermore, they are reoriented upon c-di-AMP binding which suggests these are most crucial for an interaction (Gundlachet al., 2015a; Müller et al., 2015). As mentioned, prime targets of PII proteins are often trimeric or hexameric and interactions with AmtB or the NAGK have been analyzed extensively (Forchhammer and Lüddecke, 2016). Interestingly, even interactions with monomeric targets are mechanistically similar, like the interaction of PII with PipX, the cyanobacterial coactivator of NtcA. One PIItrimer binds three PipX monomers between the protruding T-loops (Llácer et al., 2010;

Zhao et al., 2010). In cyanobacteria NtcA, together with the coactivator PipX, acts as global

transcriptional regulator for genes involved in nitrogen assimilation and PII competes with NtcA for the binding of PipX (Espinosaet al., 2014). Only few exceptional mechanisms have been resolved. For example the previously mentioned interaction of PIIwith three monomers of the ADP-ribosylglycohydrolase DraG inAzospirillum brasilense. In this unique case the loop regions are not involved in target-binding but the lateral body of PII (Rajendranet al., 2011).

Another example is the aforementioned interaction ofB. subtilis GlnK with TnrA where the T-loop regions are again not involved and the lateral α-helices mediate the interaction which stabilizes the global nitrogen regulator. This interaction is also exceptional since TnrA is bound as an active dimer (Schumacheret al., 2015). To conclude, the primary target of most known PII proteins appears to be trimeric/hexameric which of course fits to their structural assembly.

However, some examples of interactions with monomers and even dimers have been reported in the last years. The results of the unbiasedin silico screening for putative DarA interaction partners, together with future experiments, might yield more promising candidates in addition to GltAB and one should be open for potential monomeric/dimeric targets. However, there data on the structure and/or localization for many proteins are missing. After accounting for our rational requirements still 160 proteins were in our list of putative target candidates simply because we could not exclude more of them for certain.