# Cross-regulation of (p)ppGpp and c-di-GMP pathways controls a cell-cycle transition

Corentin Jaboulay, Chloé Dugelay, José P S Maio, Katell Beillevaire, Philippe Vogeleer, Daniel Vinella, Fabien Létisse, Régis Hallez, Boyuan Wang, Laurent Terradot, et al.

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Corentin Jaboulay, Chloé Dugelay, José P S Maio, Katell Beillevaire, Philippe Vogeleer, et al. Cross-regulation of (p)ppGpp and c-di-GMP pathways controls a cell-cycle transition. 2025. hal-05363575

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1 Cross-regulation of (p)ppGpp and c-di-GMP pathways controls a cell-

2 cycle transition

3 Corentin Jaboulay¹†, Chloé Dugelay¹, José P.S. Maio², Katell Beillevaire¹, Philippe Vogeleer³,

4 Daniel Vinella¹, Fabien Létisse³, Régis Hallez², Boyuan Wang⁴, Laurent Terradot¹, Mathilde

5 Guzzo¹#

6

7

8 ¹ Microbiologie Moléculaire et Biochimie Structurale (MMSB), Université Lyon 1, CNRS,

9 UMR5086, Lyon, France.

10 ²Bacterial Cell cycle & Development (BCcD), Biology of Microorganisms Research Unit (URBM),

11 Namur Research Institute for Life Science (NARILIS), University of Namur, Namur, Belgium.

12 ³Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS,

13 UPS, Toulouse, France

14 ⁴Department of Pharmacology, UT Southwestern Medical Center, Dallas, TX 75390, USA

15 †current address: Université de Lausanne, Département de Microbiologie Fondamentale

16

17

18

19

20

21 # corresponding author: mathilde.guzzo@cnrs.fr
## 22 Abstract

Bacteria must adapt their physiology to changing environments, a process often orchestrated by nucleotide messengers such as (p)ppGpp and cyclic di-GMP (c-di-GMP). While (p)ppGpp promotes survival under nutrient stress by suppressing growth, c-di-GMP supports sessile lifestyles and cell differentiation. In the α-proteobacterium *Caulobacter crescentus*, these messengers have opposite effects on the cell cycle, suggesting potential cross-regulations. Here, we uncover direct molecular cross-talk between (p)ppGpp and c-di-GMP signaling pathways. Using biochemical, structural and genetic approaches, we show that (p)ppGpp binds to the diguanylate cyclase PleD and inhibits c-di-GMP synthesis by competing with GTP at the active site. This interaction prevents c-di-GMP accumulation under nutrient-limiting conditions, linking metabolic stress to inhibition of developmental progression. Our findings reveal a new paradigm for second messenger interplay, in which one messenger directly suppresses synthesis of another. This work provides insight into how bacteria integrate metabolic and developmental cues through small-molecule cross-talk to guide adaptive behavior.

## 36 Introduction

All living organisms constantly sense and respond to their environment to adapt to changing conditions. In bacteria, these responses can lead to a temporary arrest of growth, followed by renewed proliferation when conditions improve. These adaptive responses are orchestrated by complex signaling networks that rely on a wide array of regulatory molecules. Among them, nucleotide-based second messengers have emerged as central coordinators of bacterial physiology, integrating environmental cues to regulate a broad spectrum of cellular processes.

Molecular cross-talk between small signaling molecules is increasingly recognized as a critical mechanism for integrating multiple environmental cues. Several mechanisms have already been identified, including competition for common molecular targets, direct allosteric regulation of
46 synthetic or degradative enzymes or metabolic competition (Fung et al., 2023). One example of
47 small-molecule cross-talk is the binding of c-di-AMP to DarB/CbpB, which prevents DarB/CbpB
48 from activating the *Bacillus subtilis* Rel synthetase/hydrolase (Ainelo et al., 2023; Krüger et al.,
49 2021; Peterson et al., 2020), thereby inhibiting (p)ppGpp synthesis. Conversely, (p)ppGpp inhibits
50 c-di-AMP degradation by targeting the phosphodiesterase GdpP (Corrigan et al., 2015; Rao et
51 al., 2010), although this mechanism has not yet been characterized *in vitro*. In *Pseudomonas
52 aeruginosa*, (p)ppGpp limits c-di-GMP synthesis by regulating purine salvage pathways (Kennelly
53 et al., 2024).

54 Two of the most extensively studied nucleotide messengers are guanosine tetra- and
55 pentaphosphate (collectively named (p)ppGpp) and cyclic di-GMP (c-di-GMP), each of which
56 governs distinct, though sometimes overlapping, aspects of bacterial adaptation. (p)ppGpp is
57 synthesized from GDP (ppGpp) or GTP (pppGpp) and ATP by members of the Rel/SpoT Homolog
58 (RSH) family (Atkinson et al., 2011; Ronneau & Hallez, 2019)(Fig. 1A) in response to
59 environmental stresses, particularly nutrient limitation (Battesti & Bouveret, 2006, 2009; Das et
60 al., 2009; Potrykus & Cashel, 2008; Ronneau et al., 2016, 2019). Acting as a global regulator,
61 (p)ppGpp represses growth-related processes and promotes survival, modulating transcription,
62 translation, DNA replication, and metabolism (Steinchen et al., 2020). This conserved alarmone
63 plays key roles in most bacterial species, as well as in plant chloroplasts and mitochondria
64 (Atkinson et al., 2011; Ito et al., 2020). Conversely, c-di-GMP governs transitions between motile
65 and sessile lifestyles. It is synthesized from GTP by diguanylate cyclases (DGCs) containing
66 GGDEF domains (Fig. 1A) and degraded by phosphodiesterases (PDEs) with EAL or HD-GYP
67 domains (Ryjenkov et al., 2005; Tal et al., 1998). High levels of c-di-GMP promote surface
68 attachment, biofilm formation, and cellular differentiation, while low levels support motility and
69 planktonic growth (Jenal et al., 2017).
The α-proteobacterium *Caulobacter crescentus* provides a powerful model to investigate how such signalling pathways integrate environmental information to control developmental transitions. *C. crescentus* undergoes a well-characterized asymmetric cell division, producing a motile, non-replicative swarmer cell in G1 phase and a sessile, replicative stalked cell in S phase (Barrows & Goley, 2023; Collier, 2019)(Fig. 1B). Cell-cycle progression requires the swarmer cell to differentiate into a stalked cell. This transition is tightly controlled by (p)ppGpp and c-di-GMP: (p)ppGpp accumulates under carbon starvation, carbon exhaustion, nitrogen starvation or glutamine deprivation (Boutte et al., 2012; Boutte & Crosson, 2011; Britos et al., 2011; Hallgren et al., 2023; Lesley & Shapiro, 2008; Ronneau et al., 2016) and prolongs the G1 phase (Ronneau et al., 2016), blocking differentiation. Conversely, under favorable conditions, rising levels of c-di-GMP promote the swarmer-to-stalked transition and S-phase entry (Abel et al., 2013; Duerig et al., 2009; Lori et al., 2015) mainly through the activation of the PleD diguanylate cyclase (Christen et al., 2010; Kaczmarczyk et al., 2020, 2024; Paul et al., 2007, 2008), reinforcing their opposing roles in coupling environmental sensing to proliferation (Fig. 1B). Both second messengers are derived from GTP (or GDP for ppGpp) (Fig. 1A), suggesting a need for tight metabolic prioritization and temporal separation of their synthesis during the cell cycle.

Due to its genetic tractability and well-characterized cell cycle, *C. crescentus* offers a powerful model for dissecting nucleotide signaling cross-talk. In this study, we uncover a direct molecular antagonism between (p)ppGpp and c-di-GMP. Using biochemical, structural, and genetic approaches, we show that (p)ppGpp binds directly to the active site of the diguanylate cyclase PleD and competes with its GTP substrate, thereby inhibiting c-di-GMP synthesis. This mechanism reveals a direct link between nutrient stress signaling and developmental regulation in *C. crescentus* and provides new insight into how bacteria integrate multiple inputs to fine-tune growth and differentiation.

94
## 95 Results

### 96 (p)ppGpp directly binds to a c-di-GMP-producing enzyme

To investigate potential molecular connections between (p)ppGpp and c-di-GMP signaling, we employed a ppGpp capture-compound strategy (Wang et al., 2019) to search for ppGpp-binding proteins in *C. crescentus* (Fig. 2AB, Fig. S1). This approach uses a trifunctional molecule where ppGpp is derivatized to include a cross-linkable moiety and a biotin handle (Wang et al., 2019).

The cross-linkable ppGpp variants were incubated with two *C. crescentus* cell lysates: one grown in minimal medium with ¹⁵N-labeled ammonium chloride and another in standard minimal medium (see Methods, Fig. 2A). Unlabeled lysates were outcompeted with excess of unmodified ppGpp.

Labeling with ¹⁵N allows the calculation of heavy-to-light peptide ratios, thereby identifying proteins that remain bound to ppGpp even in the presence of excess unmodified competitor.

Following cross-linking, proteins were isolated via streptavidin pull-down, digested with trypsin, and analyzed by mass spectrometry (see Methods, Fig 2AB). This screen identified homologs of known ppGpp targets in other bacteria, including hypoxanthine-guanine phosphoribosyltransferase (HPRT), a MutT-like protein, and the HflX GTPase (Bennison et al., 2021; Corrigan et al., 2016; Wang et al., 2019; Yang et al., n.d.; Zhang et al., 2018)(Fig. 2C).

Additionally, we identified PleD, a diguanylate cyclase (DGC), as a potential ppGpp-binding protein with an average heavy-to-light ratio of 11 (log₂ H/L = 3.5, Fig. 2C, see Methods).

PleD is a response regulator that consists of a phosphorylatable N-terminal receiver domain (D1), a receiver-like adaptor domain (D2) and a DGC domain. PleD catalyzes the synthesis of c-di-GMP from two GTP molecules in its active site within the DGC domain. Phosphorylation of PleD at aspartate D53 of the D1 domain enhances PleD activity, likely by promoting dimerization (Wassmann et al., 2007), and is mediated by the PleC and DivJ histidine kinases during *C. crescentus* differentiation (Chong et al., 2024; Kaczmarczyk et al., 2020; Paul et al., 2004). PleD activation during cell differentiation triggers the accumulation of intracellular c-di-
120 GMP which drives the morphological changes from swarmer to stalked cell and the transition to
121 a replicative cell. Since accumulation of (p)ppGpp has the opposite effect of blocking cell
122 differentiation in C. crescentus, a potential PleD-(p)ppGpp interaction suggests an antagonistic
123 cross-talk between c-di-GMP and (p)ppGpp signaling networks, that we explore below.

124 (p)ppGpp directly binds PleD and inhibits its diguanylate cyclase activity

125 To investigate if PleD is a (p)ppGpp target, we purified recombinant C. crescentus PleD from E.
126 coli (Chan et al., 2004) and assessed binding to ppGpp and pppGpp using nano differential
127 scanning fluorimetry (nanoDSF) and isothermal titration calorimetry (ITC) (Table S1-S2). Of note,
128 during purification, a fraction of DGCs was recovered with c-di-GMP bound, most likely at their
129 inhibitory sites (I-site) (Fig. S2A). Similar observations have been reported for purified GGDEF
130 enzymes (Chan et al., 2004; De et al., 2009; Wassmann et al., 2007), although the extent varies.

131 NanoDSF assays revealed significant increases in the melting temperature of PleD in the
132 presence of both ppGpp and pppGpp (Fig. S2BC). Notably, pppGpp induced a larger shift (ΔTm
133 = 11.52°C ± 0.23) than ppGpp (ΔTm = 6.332°C ± 2.323) (Fig. S2BC, Table S2). No significant
134 shift was observed with another purine nucleotide, ATP, highlighting nucleotide specificity (Fig.
135 S2BC). ITC revealed a 1:1 binding stoichiometry for both ppGpp (K_D = 45.4 µM ± 22.3) and
136 pppGpp (K_D = 9.31 µM ± 1.16) (Fig. 3A). Interestingly, pppGpp bound PleD with an affinity
137 comparable to GMPCPP, a non-hydrolyzable GTP analog and substrate mimic (Fig. 3A, Fig.
138 S3A). These data reveal a strong and specific interaction between PleD and (p)ppGpp, with tighter
139 binding by pppGpp than ppGpp.

140 To explore a potential role of (p)ppGpp on PleD's enzymatic activity, we assessed PleD's *in vitro*
141 c-di-GMP production using reverse phase (or C18)-HPLC (Paul et al., 2004). In the presence of
142 GTP at 100 µM, PleD produced c-di-GMP (Paul et al., 2004) (Fig. S3B). Addition of either ppGpp
143 or pppGpp significantly impaired the enzymatic activity of PleD *in vitro* (Fig. 3BC). At a 1:5 GTP-
144 to-ppGpp ratio, the c-di-GMP synthesis activity of PleD was reduced by more than 50% (Fig.
145 3BC). In the same conditions, pppGpp exhibited an even stronger inhibitory effect, nearly 146 abolishing c-di-GMP production with over 90% inhibition (Fig. 3BC). Under varying alarmone 147 concentrations in the reaction mixture, pppGpp demonstrated potent inhibition at a 1:1 ratio with 148 GTP (Fig. 3C), and showed a greater efficacy in suppressing PleD activity compared to ppGpp at 149 a similar ratio. These results indicate that pppGpp is a more effective inhibitor of PleD than ppGpp.

150 In contrast to (p)ppGpp, ATP had no detectable effect on PleD activity (Fig. S3A), underscoring 151 the enzyme's specificity for guanosine-derived nucleotides. Phosphorylation of PleD at residue 152 D53, mediated by the truncated DivJ kinase *in vitro* (Paul et al., 2004), led to an approximately 153 2.5-fold increase in enzymatic activity (Fig. S3A), aligning with previous findings (Paul et al., 2004).

154 Despite this activation, (p)ppGpp remained potent inhibitors (at a 1:5 GTP:(p)ppGpp ratio), 155 significantly reducing the activity of PleD in its phosphorylated state (Fig. 3D, Fig. S3C). These 156 findings emphasize that the inhibitory effect of (p)ppGpp operates independently of PleD's 157 phosphorylation status, indicating a robust and direct mechanism of action.

158 **Structural basis of PleD inhibition by (p)ppGpp**

159 To gain insights into the mechanism of PleD's inhibition by these alarmones, we solved PleD's 160 crystal structure in complex with ppGpp (PleD<sup>ppGpp</sup>) or pppGpp (PleD<sup>pppGpp</sup>) at resolutions of 2.9 Å 161 and 3 Å, respectively (Table S3). The complexes crystalized in the space group P6<sub>1</sub>22, with four 162 molecules per asymmetric unit, arranged as dimers of dimers (Fig. S4AB). In both structures, the 163 overall conformation of the PleD dimer resembles the inactive conformation described in the 164 PleD:c-di-GMP binary complex structure previously published (Chan et al., 2004) (Fig. 4A, 165 Fig.S4C), albeit domain swapping between the D1-D2 and the DGC domain was observed in our 166 structures. Compared to the inactive conformation, the stem α5 helices undergo a small 167 conformational change with helices extended by 4-5 residues, reminiscent of what was observed 168 upon activation by phosphorylation (Wassmann et al., 2007) (Fig. S4C). Of note, both the 169 PleD<sup>ppGpp</sup> and the PleD<sup>pppGpp</sup> structures were solved in the presence of c-di-GMP, thus each
170 monomer of the (p)ppGpp-PleD structures is bound by two cyclic di-GMP molecules at its I-site,
171 i.e. between the DGC and the D2 domains (Chan et al., 2004) (Fig. 4AB). Each PleD DGC domain
172 is bound by a (p)ppGpp and a Mg²⁺ ion in the active site (Fig. 4AB).

173 The binding of the alarmones is at the interface of the dimer of dimers, bringing together two DGC
174 domains, with each of them interacting with two (p)ppGpp (Fig. 4C, Fig. S4A-C). Consequently,
175 these two DGC domains are in a "closed" conformation, contrasting with the open conformation
176 identified in the inactive, product-bound PleD (Fig. 4C). This is illustrated by the difference of
177 position of helices α4 (following the nomenclature in (Chan et al., 2004)) from the two DGC
178 domains. While these are 20 Å away from each other in the product-bound form, they are less
179 than 5 Å in the (p)ppGpp-bound structures (Fig. 4C). The binding mode of GDP and GTP moieties
180 of ppGpp and pppGpp into the individual DGC domain is similar to the one described for the non-
181 hydrolyzable GTP-αS (Wassmann et al., 2007) (Fig. 4D). The guanine moiety sits in a pocket
182 formed by DGC α1 and α2 and is hydrogen-bonded by D344 and N335. F331 main chain and
183 K332 side chain interact with the β-pyrophosphate (Fig. 4D) while I328 main chain, D327 and
184 E370 side chains bind the magnesium ion (Fig. 4D). The 3' pyrophosphates of both alarmones
185 point away from the active site although density for the second phosphate was rather poor (Fig.
186 4AB). The α-phosphate interacts with N335 but also with R300 from the other DGC domain (Fig.
187 4D). As a consequence, the 3' pyrophosphate might interact with H340 side chain but this
188 interaction is not absolutely conserved in the four subunits of the asymmetric unit. This might be
189 explained by the charge of the surface at this site being predominantly negative and thus
190 unfavourable to binding of negatively charged pyrophosphate. Residue N335 has been shown to
191 be important for PleD DGC activity (Chan et al., 2004; Christen et al., 2006). We also observed
192 that residue R300, which is highly conserved in DGC domains (Fig. S5), plays an important role
193 in PleD's activity. Indeed, substituting R300 with either alanine or lysine significantly reduced
194 enzymatic activity (Fig. S4D). Thus, (p)ppGpp binding reveals key residues involved in PleD's
195 catalytic function, potentially stabilizing intermediate reaction conformations that were not
196 captured in previous PleD structures. The main difference between the binding of ppGpp and
197 pppGpp occurs with pppGpp 5' γ-phosphate being bound by R446 and also by Y439, two highly
198 conserved residues in DGC domains and the latter from the second DGC α4 helix (Fig. 4CD, Fig.
199 S5). This binding adds two hydrogen bonds, explaining the increased affinity of pppGpp for PleD
200 and its higher inhibitory effect compared to ppGpp.

201 Conservation of (p)ppGpp-mediated inhibition across PleD orthologs

202 Diguanylate cyclases are widely conserved across bacteria and in some eukaryota (Kawabe et
203 al., 2023; Römling et al., 2013). The active site of the DGC domain is particularly conserved
204 among DGC enzymes of different families (Fig. S5), with numerous conserved residues that have
205 been shown to be involved in the enzymatic reaction and/or dimerization. As we showed that
206 (p)ppGpp ligands can bind in the active site of the PleD<sub>CC</sub> DGC, interact with conserved residues
207 and affect its activity, we investigated the conservation of (p)ppGpp sensitivity in this enzyme
208 family and beyond. We purified PleD orthologs from *Agrobacterium tumefaciens* and
209 *Sinorhizobium meliloti*, both part of the α-proteobacteria class, and assessed their activities in
210 *vitro*. In both cases, ppGpp and pppGpp strongly inhibited c-di-GMP production, with pppGpp
211 showing greater potency (Fig. 3EF, Fig. S3DE). NanoDSF assays further confirmed PleD<sub>AT</sub>
212 binding to (p)ppGpp, indicating conserved interaction mechanisms (Fig. S2DE; Table S2).

213 We expanded these studies to WspR, a PleD ortholog from *Pseudomonas aeruginosa*,
214 representing γ-proteobacteria. We observed that ppGpp had minimal impact on WspR activity
215 while pppGpp inhibited more than 50% of its enzymatic function (Fig. 3G, Fig. S3F). In contrast,
216 DgcZ, a diguanylate cyclase with a different domain organization showed no reduction in activity
217 (Fig. 3H, Fig. S3G). These results suggest that (p)ppGpp-mediated inhibition is a conserved
218 feature of PleD orthologs, with pppGpp exhibiting greater inhibitory efficacy and potential
219 conserved activity beyond α-proteobacteria.
220 **(p)ppGpp inhibition of PleD activity prevents cell differentiation under glucose**
221 **exhaustion in C. crescentus**

222 Having established biochemically that (p)ppGpp directly inhibits PleD activity, we next examined
223 the physiological relevance of this interaction *in vivo*. Under glucose exhaustion conditions
224 (M2G₁/₁₀*), C. crescentus cells remain arrested at the swarmer, non-replicative stage of the cell
225 cycle, preventing differentiation into stalked cells and the initiation of DNA replication until
226 conditions become favorable again (Boutte et al., 2012)(Fig. S6A). Previous studies have shown
227 that glucose exhaustion triggers the accumulation of ppGpp and pppGpp in C. crescentus cells
228 (Boutte et al., 2012; Hallgren et al., 2023; Leslie et al., 2015). Based on our *in vitro* data, we
229 propose that the starvation-induced accumulation of (p)ppGpp inhibits PleD-mediated c-di-GMP
230 synthesis, thereby involved in blocking the swarmer-to-stalked cell transition.

231 To investigate this model *in vivo*, we quantified for the first time in C. crescentus the intracellular
232 concentrations of ppGpp, pppGpp, c-di-GMP, GTP, and GDP in mixed populations of cells grown
233 under nutrient-rich conditions (M2G), nitrogen starvation (P2G), carbon exhaustion (M2G₁/₁₀*),
234 and carbon starvation (M2) using LC-MS (Fig. 5A–C, Fig. S6BC; Table S4; see Methods). Under
235 nitrogen starvation, ppGpp levels increased to ~85 µM, and to ~55 µM under carbon starvation,
236 compared to ~30 µM in nutrient-replete conditions (Fig. 5A, Table S4). Interestingly, ppGpp levels
237 remained largely unchanged under carbon exhaustion. By contrast, pppGpp concentrations rose
238 markedly to ~10 µM under carbon exhaustion, whereas they remained around ~4 µM in all other
239 tested conditions (Fig. 5B, Table S4). Notably, the intracellular concentrations of ppGpp and
240 pppGpp are comparable to the K_D values determined by ITC (Fig. 3A), suggesting that PleD
241 binding by (p)ppGpp may be finely tuned under physiological conditions.

242 GTP concentrations decreased from ~440 µM in nutrient-rich conditions to ~271 µM under carbon
243 starvation and ~119 µM under carbon exhaustion (Fig. 5C, Table S4), consistent with a global
244 metabolic downshift under nutrient depletion. GDP levels also decreased similarly to the GTP
245 concentrations (Fig. S6B, Table S4). In contrast, c-di-GMP levels remained largely unchanged
246 across all tested conditions (Fig. S6C, Table S4), despite marked alterations in GTP and
247 (p)ppGpp pools.

248 To test our *in vitro* model of (p)ppGpp-c-di-GMP cross-regulation, we turned to genetic
249 approaches and *in vivo* analysis of c-di-GMP-dependent phenotypes. The degradation of CtrA,
250 a master regulator of cell-cycle progression in *C. crescentus*, is tightly regulated by c-di-GMP
251 levels. Briefly, c-di-GMP binding to the PopA adaptor drives CtrA degradation by the ClpXP
252 protease at the swarmer-to-stalked transition (Duerig et al., 2009; Ozaki et al., 2014). Under
253 favorable conditions, while PleD is not essential, its activation and the resulting c-di-GMP
254 synthesis has been shown to promote both cell differentiation and CtrA degradation to drive cell-
255 cycle progression (Fig. 5D) (Abel et al., 2013; Kaczmarczyk et al., 2020; Lori et al., 2015; Paul et
256 al., 2007). Conversely, under glucose exhaustion conditions, wild-type cells maintain CtrA protein
257 levels, which effectively blocks differentiation (Boutte et al., 2012)(Fig. 5D, Fig. S6A). This
258 observation correlates with the hypothesis that (p)ppGpp accumulation in this condition inhibits
259 PleD's activity, preventing the rise in c-di-GMP levels required for CtrA degradation.

260 To test this model, we examined CtrA levels in a *rel* mutant, which does not produce (p)ppGpp.
261 In a *rel* mutant, *i.e.* in the absence of (p)ppGpp, CtrA follows a typical cell-cycle dependent profile
262 under favorable conditions, while under glucose exhaustion, CtrA is degraded (Fig. 5D). This
263 suggests that, without (p)ppGpp, the burst in c-di-GMP synthesis occurs which triggers CtrA
264 degradation. In support of that, inactivation of *pleD* in *rel* mutant cells suppressed the CtrA
265 degradation under glucose exhaustion, with the *rel* *pleD* double mutant behaving like the wild-
266 type and the *pleD* mutant (Fig. 5D, Fig. S6D). In contrast, CtrA oscillations were maintained under
267 favorable conditions in both the *pleD* single mutant and the *rel* *pleD* double mutant (Fig. 5D; Fig.
268 S6D). Notably, despite CtrA degradation under glucose exhaustion, *rel* mutant cells do not start
269 DNA replication (Fig. S6A), likely due to repression of *dnaA* expression and rapid degradation of
270 the protein under starvation conditions, as previously reported (Hallgren et al., 2023; Leslie et al.,
271 2015).

272 Additionally, we compared the growth of wild-type, *rel* mutant, and *rel pleD* mutant strains under
273 glucose exhaustion conditions. We found that in both asynchronous populations and
274 synchronized cells, growth halted only in the wild-type and *rel pleD* mutant strains, while the *rel*
275 mutant continued to grow slightly (Fig. S6EF). This observation aligns with our hypothesis that
276 the accumulation of (p)ppGpp in wild-type cells under starvation conditions prevents cell-cycle
277 transition to growth and differentiation by inhibiting PleD. Moreover, the fact that unsynchronized
278 *rel* mutant cells grow more than one doubling time under glucose exhaustion conditions, whereas
279 wild-type cells (and *rel pleD* mutant cells) stop after one doubling time (Fig. S6E), indicates that
280 (p)ppGpp production is responsible for halting growth before glucose or other carbon sources are
281 fully depleted, and suggests a PleD-dependent mechanism. Of note, this also explains our choice
282 to resuspend synchronized cells in spent medium rather than M2 medium for experiments: spent
283 medium retains residual glucose and carbon sources, allowing cells to continue progressing
284 through the cell cycle without triggering abrupt starvation, as M2 medium would.

285 To further test that elevated c-di-GMP levels are responsible for the CtrA degradation observed
286 in glucose exhausted *rel* mutant cells, we expressed the phosphodiesterase *proE* from *P. aeruginosa* in the *rel* mutant under glucose exhaustion conditions. Ectopic expression of *proE* has
287 been shown to reduce c-di-GMP levels in C. crescentus (Duerig et al., 2009) and we observed
288 that it restored CtrA stabilization under glucose exhaustion conditions whereas a catalytic mutant
289 (*proE*\*) could not (Fig. 5E; Fig. S6A), further supporting the idea that PleD-dependent c-di-GMP
290 synthesis drives CtrA degradation in *rel* mutant cells in carbon limiting conditions.
291

292
293 **Discussion**

294 Our results demonstrate that (p)ppGpp directly inhibits the diguanylate cyclase activity of PleD by
295 competing with its GTP substrate at the enzyme's active site. *In vitro*, both ppGpp and pppGpp
296 bind PleD (Fig. 3A, Fig. S2) and significantly reduce c-di-GMP synthesis in a concentration-
297 dependent manner (Fig. 3BC, Fig. S3). This inhibition occurs independently of PleD's
298 phosphorylation state (Fig. 3D, Fig. S3C), indicating that (p)ppGpp modulates PleD activity
299 outside of its canonical activation pathway. Structural data further support a mechanism where
300 (p)ppGpp binds within the GGDEF catalytic site and stabilizes an inactive dimeric conformation
301 of PleD (Fig. 4, Fig. S4).

302 *In vivo*, pppGpp accumulates under carbon exhaustion while GTP levels decline (Fig. 5BC),
303 consistent with a model in which pppGpp links nutrient limitation to cell-cycle control by
304 suppressing PleD-dependent c-di-GMP synthesis. Under carbon exhaustion, pppGpp
305 concentrations rise to ~10 µM, which remains lower than ppGpp concentrations in the same
306 conditions (Fig. 5AB) but remains in the same range as the K<sub>D</sub> measured in the ITC assays we
307 performed (Fig. 3A). We observed that (p)ppGpp concentrations in *C. crescentus* remain well
308 below those typically reported in *E. coli* using quantitative LC-MS methods, where measured
309 basal levels were ~140 µM for ppGpp and ~4 µM for pppGpp, and could rise to several hundred
310 micromolar under stringent response (Patacq et al., 2018, 2020; Fig. 5B). This may reflect the
311 oligotrophic lifestyle of *C. crescentus*, which may rely on a dampened alarmone response to cope
312 with frequent nutrient variation. Supporting this model, and as previously published (Boutte et al.,
313 2012; Hallgren et al., 2023), we observed a Rel-dependent stabilization of the master cell-cycle
314 regulator CtrA under carbon exhaustion in synchronized cultures (Fig. 5D). Conversely, in *rel*
315 mutant cells, which lack (p)ppGpp accumulation, CtrA was degraded (Fig. 5D). This phenotype
316 was PleD- and c-di-GMP-dependent, as CtrA was stabilized in *rel pleD* double mutants and in *rel*
317 mutant cells ectopically producing the c-di-GMP-degrading enzyme ProE (Fig. 5DE).
318 Our structural and biochemical analyses indicate that direct inhibition of DGCs by (p)ppGpp may
319 be conserved across enzymes with shared architecture. In *C. crescentus*, (p)ppGpp binds within
320 the active site of PleD, competing with GTP and stabilizing an inactive dimeric conformation (Fig.
321 4C). This mechanism involves a conformational shift where the α4 helices of the GGDEF domains
322 are <5 Å apart in the (p)ppGpp-bound form, compared to ~20 Å in the product-bound structure
323 (Fig. 4C). This "closed" arrangement strongly supports an active and specific mechanism of
324 inhibition, rather than simple competitive binding. Notably, residues mediating (p)ppGpp binding,
325 including R300, are conserved across PleD-family DGCs (Fig. S5), suggesting that this regulatory
326 interaction may be broadly conserved. In addition to R300, our structural analysis identifies Y439
327 and R446 as residues that specifically engage the γ-phosphate of pppGpp. All three residues are
328 highly conserved features of GGDEF domains across DGCs (Fig. S5), and R300 and R446 have
329 been shown to be important for catalytic function (Wassmann et al., 2007; Fig. S4D), contributing
330 to both GTP binding and (p)ppGpp-mediated inhibition. This dual involvement suggests that
331 mutations disrupting (p)ppGpp binding are likely to impair PleD activity itself, making it difficult to
332 uncouple the inhibitory and catalytic functions.

333 Orthologs of PleD from *A. tumefaciens* and *S. meliloti* are similarly inhibited by (p)ppGpp *in vitro*,
334 with pppGpp showing stronger effects, consistent with our findings in *C. crescentus* (Fig. 3BEF).
335 These enzymes share a conserved domain organization, with two N-terminal receiver domains
336 that promote dimerization upon phosphorylation and a C-terminal GGDEF domain responsible for
337 catalysis. This conservation suggests that coupling DGC activity to metabolic stress via (p)ppGpp
338 may be a widespread strategy among α-proteobacteria. WspR from *Pseudomonas aeruginosa* is
339 a PleD ortholog that also adopts this two-domain architecture and can be activated by
340 phosphorylation. However, unlike PleD, WspR can dimerize and become active independently of
341 phosphorylation (De et al., 2008). We found that WspR is partially inhibited by pppGpp *in vitro*,
342 suggesting that alarmone-mediated regulation may extend beyond α-proteobacteria, albeit with
343 different sensitivities. By contrast, DgcZ from E. coli, which features a Zn-sensing regulatory
344 domain and forms constitutive dimers (Zähringer et al., 2013), shows no significant activity change
345 in the presence of (p)ppGpp *in vitro* (Fig. 3H). This highlights that GGDEF domain conservation
346 alone is not sufficient to predict functional impact, which likely depends on additional factors such
347 as enzymatic turnover rate, oligomerization dynamics or other mechanisms. These differences
348 underscore the structural and kinetic complexity shaping how DGCs respond to intracellular
349 alarmone signals. In the case of PleD, (p)ppGpp binding not only blocks substrate access but
350 also locks the enzyme in an inactive conformation, providing a clear example of active inhibition.
351 These findings argue against a purely competitive model and underscore the need to consider
352 the structural and mechanistic diversity of (p)ppGpp regulation. Further studies will be needed to
353 determine how widespread this mechanism is across bacterial DGC families and how it integrates
354 with broader stress and signaling networks. Notably, when purifying PleD *in vitro*, we recovered a
355 fraction of PleD with c-di-GMP bound, likely at its I-site, consistent with observations for other
356 GGDEF enzymes (Chan et al., 2004; De et al., 2009; Wassmann et al., 2007). However, PleD<sub>SM</sub>
357 and DgcZ preparations contained little or no bound c-di-GMP (Fig. S2A). Of note, these DGCs
358 show higher c-di-GMP synthesis activity, which is consistent with the inhibitory effect of c-di-GMP
359 upon binding to the I-site (Fig. S3HI). However, the absence of pre-bound ligand in these enzymes
360 did not increase their sensitivity to (p)ppGpp inhibition, indicating that the inhibitory effect is not
361 simply due to displacement of I-site-bound c-di-GMP, but rather reflects a direct interference with
362 catalytic activity.

363 Interestingly, despite the strong inhibitory effect of (p)ppGpp on PleD activity *in vitro*, we did not
364 detect significant differences in c-di-GMP concentrations between nutrient-rich and starvation
365 conditions at the population level (Fig. S6C). Similar measurements in synchronized populations
366 have previously revealed clear cell-cycle oscillations of c-di-GMP, with levels rising during the
367 swarmer-to-stalked transition (Abel et al., 2013). Thus, the absence of changes in our bulk assays
368 may reflect averaging over heterogeneous subpopulations, masking temporal variations in c-di-
369 GMP accumulation. The absence of major changes in c-di-GMP levels could also reflect
370 compensatory activities or the activity of other diguanylate cyclases that remain functional under
371 nutrient limitation, or alternatively, a spatial compartmentalization of c-di-GMP synthesis and
372 degradation. Such subcellular organization of c-di-GMP pools has already been described in C.
373 *crescentus* (Christen et al., 2010; Kaczmarczyk et al., 2020, 2024). In *C. crescentus*, (p)ppGpp
374 levels may fluctuate across the cell cycle and may be higher in swarmer cells than in stalked cells
375 (Ronneau et al., 2016; Shyp et al., 2020). At the same time, PleD is transiently recruited to the
376 cell pole during the swarmer-to-stalked transition, where it triggers a burst of c-di-GMP synthesis
377 (Abel et al., 2011; Christen et al., 2010; Paul et al., 2008). This spatiotemporal coordination raises
378 the possibility that PleD activity is controlled by local or transient changes in the GTP:(p)ppGpp
379 ratio, which may not be reflected in bulk measurements. While this hypothesis remains to be
380 tested, it highlights the importance of considering spatial and temporal dynamics when
381 interpreting second messenger signaling. Similar principles have been observed in other bacteria,
382 where local control of nucleotide signaling enables precise regulation of specific outputs,
383 independently of global nucleotide levels (Nicastro et al., 2020; Richter et al., 2020).

384 Beyond its direct inhibition of PleD, (p)ppGpp may also influence c-di-GMP signaling indirectly by
385 reshaping nucleotide metabolism. As a central regulator of purine biosynthesis, (p)ppGpp lowers
386 intracellular GTP levels during nutrient stress, reducing substrate availability for DGCs (Fig. 5C).
387 Such interplay between purine metabolism and c-di-GMP signaling has been described in P.
388 *aeruginosa* (Kennelly et al., 2024). Although we did not observe major changes in bulk c-di-GMP
389 levels in *C. crescentus*, this dual-layer regulation, combining enzyme inhibition and precursor
390 limitation, may contribute to the fine-tuning of local signaling.

391 While our proposed model is that (p)ppGpp directly inhibits PleD to modulate cell differentiation,
392 this is likely only one facet of its broader regulatory role in *C. crescentus*. As in other bacteria,
393 (p)ppGpp likely influences multiple core processes, including transcription, translation, and
394 metabolism in *C. crescentus*. Thus, while the PleD pathway provides a clear example of how
395 metabolic signals are coupled to developmental outcomes, it likely operates in parallel with
396 additional layers of (p)ppGpp-dependent regulation that collectively shape the growth and cell
397 cycle dynamics of *C. crescentus*.

398 The interaction between (p)ppGpp and DGCs such as PleD adds to a growing body of evidence
399 that alarmone signaling intersects with c-di-GMP pathways at multiple levels. In addition to
400 modulating DGC activity, (p)ppGpp also regulates effector proteins like SMB, which has been
401 shown to bind both (p)ppGpp and c-di-GMP at the same site to control growth in *C. crescentus*
402 through a metabolic switch(Shyp et al., 2020). This work underscores the potency of second
403 messengers to function as molecular switches at the interface between metabolism and
404 development, and highlights how direct cross-talk between signaling pathways can coordinate
405 complex cellular decisions in bacteria.

406 Acknowledgments

407 We acknowledge the contribution of SFR Biosciences (Université Claude Bernard Lyon 1, CNRS
408 UAR3444, Inserm US8, ENS de Lyon) at the Protein Science Facility, especially Eric Diesis and
409 Virginie Gueguen-Chaignon. We acknowledge the contributions of the CELPHEDIA Infrastructure
410 (http://www.celphedia.eu/), especially Sébastien Dussurgey at the center AniRA in Lyon. We
411 thank Proxima 2 beamline staff scientists from SOLEIL and Dr. Max Nanao from beamline
412 ID23EH2 of the ESRF for assistance during data collection. We are grateful to Michael Laub's lab
413 and the Koch Institute Biopolymer & Proteomics Core at MIT for helping us with the chemical
414 proteomics work. We thank the Jault and Lesterlin labs at MMSB for technical support and access
415 to equipment, especially Kevin Lang, Elise Kaplan, Cécile Gonzalez and Annick Dedieu, as well
416 as Marie-Laure Fogeron for assistance with ITC. This research was supported by an ATIP Avenir
417 grant funded by the Centre National de la Recherche Scientifique (CNRS) and the Fondation
418 Bettencourt-Schueller awarded to MG, by an Amorçage Europe grant of the Région Auvergne-

419 Rhône-Alpes awarded to MG and by a FEBS Excellence Award from the Federation of European

420 Biochemical Societies awarded to MG. We thank Michael Laub and Justine Collier for critical

421 reading of the manuscript.

422 **Contributions**

423 Conceptualization: C.J and M.G - Methodology: C.J, C.D, J.P-S.M, P.V, F.L, R.H, B.W, L.T, M.G -

424 Formal analysis: C.J, C.D, J.P-S.M, P.V, F.L, R.H, B.W, L.T, M.G - Investigation: C.J, C.D, J.P-

425 S.M, P.V, B.W, L.T, M.G - Writing: original draft from C.J and M.G with contributions of all authors-

426 Resources: L.T., R.H., F. L. and M.G. - Funding acquisition: M.G

427 **Declaration of interests**

428 The authors declare no competing interests.

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664 17/SUPPL_FILE/MBO001183765ST3.DOCX

665

## 666 Methods

### 667 Growth conditions

668 *Caulobacter crescentus* strains were grown in PYE (2 g/L bactopeptone, 1g/L yeast extract, 0.3

669 g/L MgSO₄, 0.5 mM CaCl₂) or in fresh M2 medium (0.87 g/L Na₂HPO₄, 0.53 g/L KH2PO₄, 0.5 g/L

670 NH₄Cl, 0.5 mM MgSO₄, 10 mM FeSO₄, 0.1 mM CaCl₂) supplemented with 0.2% or 0.02% glucose

671 at 30°C with constant shaking (210 rpm). Expression from the P<sub>van</sub> promoter was induced with

672 vanillate (100 µM). To maintain plasmids, antibiotics were added at the following concentrations

673 (liquid/plate): kanamycin (5 µg mL⁻¹ / 25 µg mL⁻¹), oxytetracycline (1 µg mL⁻¹ / 2 µg mL⁻¹),

674 gentamycin (2.5 µg mL⁻¹ / 5 µg mL⁻¹), chloramphenicol (2 µg mL⁻¹ / 1 µg mL⁻¹).
Escherichia coli strains were grown in LB (10 g/L NaCl, 10 g/L tryptone, 5 g/L yeast extract) and
supplemented with antibiotics at the following concentrations unless noted (liquid/plate):
kanamycin (30 µg mL⁻¹ / 50 µg mL⁻¹), oxytetracycline (12 µg mL⁻¹ / 12 µg mL⁻¹), gentamycin (15
µg mL⁻¹ / 20 µg mL⁻¹), chloramphenicol (20 µg mL⁻¹ / 30 µg mL⁻¹), ampicillin (100 µg mL⁻¹ / 100 µg
mL⁻¹).

680 Strain construction

681 *Escherichia coli* strains were derivative of the BL21(DE3) strain for the production of recombinant
682 proteins. All BL21 derivative strains were constructed by transforming the replicative plasmid by
683 heat-shock. All *C. crescentus* strains were derivative of the wild-type isolate MGC001 from the
684 CB15N/NA1000 strain. All strains, plasmids and oligonucleotides used in this study are listed in
685 Tables S5-S7.

686 To construct strains MGC015 and MGC016 and strains MGC564 and MGC565, MGC001 and
687 MGC533 were electroporated with pBVMCS-4-*proE* or pBVMCS-4-*proE*\* respectively, selecting
688 on gentamycin plates.

689 Strain MGC636 (NA1000 *Δrel ΔpleD*) was constructed using two-step recombination [Skerker JM,
690 Prasol MS, Perchuk BS, Biondi EG, Laub MT (2005)]. First, pNPTS138-*pleD::tet* was
691 electroporated into MGC533 (NA1000 *Δrel*) followed by selection on kanamycin. Single colonies
692 obtained were grown overnight in liquid PYE + tetracycline. After 10-15 hour-growth, 5 µL in 45
693 µL PYE was plated for counter-selection on PYE containing tetracycline and 0.3% sucrose.
694 Sucrose resistant clones were isolated on PYE + tetracycline and PYE + tetracycline + kanamycin
695 plates and isolated clones that lost kanamycin resistance were selected. *pleD* deletion was
696 confirmed by PCR.

697 Plasmid construction
698 pNTPS138-*pleD*: The *pleD* deletion cassette was amplified from MGE477 chromosomal DNA
699 using primers MGP841 and MGP842. Fragment was cloned into EcoRV site of the pNTS138
700 plasmid using DNA ligation. The construction was verified by sequencing.

701 pET28a-*divJ*₁₋₂₄₆: The *divJ*₁₋₂₄₆ fragment encoding DivJ deleted of the first 246 amino acids was
702 amplified from *Caulobacter crescentus* NA1000 chromosomal DNA using primers MGP817 and
703 MGP821. Fragment was cloned into BamHI restriction site of the pET28a plasmid using
704 Sequence and Ligation Independent Cloning (SLIC). The construction was verified by
705 sequencing.

706 pET28a-*pleD*AT: The *pleD*AT fragment was amplified from *Agrobacterium tumefaciens* C58
707 chromosomal DNA using MGP856 and MGP857. The pET28a plasmid was amplified using
708 MGP730 and MGP855. Fragment was cloned into amplified pET28a using SLIC. The construction
709 was verified by sequencing.

710 pET28a-*pleD*SM: The *pleD*SM fragment was amplified from *Sinorhizobium meliloti* 1021
711 chromosomal DNA using MGP899 and MGP900. The pET28a plasmid was amplified using
712 MGP730 and MGP855. Fragment was cloned into amplified pET28a using SLIC. The construction
713 was verified by sequencing.

714 pET28a-*dgcZ*: The *dgcZ* fragment was amplified from *Escherichia coli* MG1655 chromosomal
715 DNA using MGP 858 and MGP 859. The pET28a plasmid was amplified using MGP730 and
716 MGP855. Fragment was cloned into amplified pET28a using SLIC. The construction was verified
717 by sequencing.

718 pET28a-*wspR*: The *wspR* fragment was amplified from *Pseudomonas aeruginosa* strain PA01
719 chromosomal DNA using MGP 860 and MGP 861. The pET28a plasmid was amplified using
720 MGP730 and MGP855. Fragment was cloned into amplified pET28a using SLIC. The construction
721 was verified by sequencing.
722 pET11b-*pleD*<sub>R300A</sub>: The pET11b-*pleD* plasmid was amplified using MGP1013 and MGP1014. The synthetic gBlock *pleD*<sub>R300A</sub> (Table S7) was cloned into amplified pET11b-*pleD* using SLIC. The construction was verified by sequencing.

725 pET11b-*pleD*<sub>R300K</sub>: The pET11b-*pleD* plasmid was amplified using MGP1013 and MGP1014. The synthetic gBlock *pleD*<sub>R300K</sub> (Table S7) was cloned into amplified pET11b-*pleD* using SLIC. The construction was verified by sequencing.

728 Solid-phase peptide synthesis

729 We synthesized four peptides with the following sequence: biotinyl-Gly-Lys(bromoacetyl-X-pMet-)-Gly.NH<sub>2</sub>(Wang et al., 2019). X is a linker amino acid varying in each peptide.

<table><thead><tr><td>Peptide #</td><td>X</td><td>Free amino-acid structure for X</td></tr></thead><tbody><tr><td>1</td><td>Gly</td><td>H<sub>2</sub>N-CH<sub>2</sub>COOH</td></tr><tr><td>2</td><td>PEG2</td><td>H<sub>2</sub>N-(CH<sub>2</sub>CH<sub>2</sub>O)<sub>2</sub>-CH<sub>2</sub>COOH</td></tr><tr><td>3</td><td>PEG4</td><td>H<sub>2</sub>N-(CH<sub>2</sub>CH<sub>2</sub>O)<sub>4</sub>-CH<sub>2</sub>COOH</td></tr><tr><td>4</td><td>PEG6</td><td>H<sub>2</sub>N-(CH<sub>2</sub>CH<sub>2</sub>O)<sub>6</sub>-CH<sub>2</sub>CH<sub>2</sub>COOH</td></tr></tbody></table>

731
732 The general procedure for a coupling-deprotection cycle: 5 eq. Fmoc protected amino acid and 4.8 eq. of 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b] pyridinium 3-oxide 733 hexafluorophosphate (HATU) dissolved in N,N-dimethylformamide (DMF) to achieve a final 734 concentration of 0.4 M amino acid and 0.38M HATU. 5 eq. of diisopropylethylamine (DIEA) was 735 then added and the mixture was applied to the solid support and left to react at room temperature 736 (RT) for 20 minutes, followed by 3 washes with DMF, 2 x 8 min deprotection with 20% (v/v) 737 piperidine in DMF and 5 washes with DMF. Biotin was coupled following the same procedure but 738 without deprotection. For coupling of Fmoc-pMet-OH, usage of the amino acid, HATU and DIEA 739 were reduced to 2.0, 1.9, and 2 eq., respectively, and coupling time extended to 1 hour at RT. 740
741 The Biotinyl-GKG structure first synthesized at 0.1-mmol scale on Rink-amide resin (ChemMatrix). The resin was swelled in DMF prior to the first coupling. Coupling-deprotection 742 cycles were performed with Fmoc-Gly-OH, Fmoc-Lys(Alloc)-OH, and Fmoc-Gly-OH (in this 743 order), and the N-terminal amine was biotinylated. Thereafter, The Alloc protecting group was 744 quantitatively removed by a 20-minute, RT treatment with 598 µL of phenylsilane (4.85 mmol) and 745 45.63 mg (39.5 µmol) of tetrakis(triphenylphosphine)palladium(0) in 4 mL of dichloromethane 746 (DCM). The resin was then thoroughly washed with dichloromethane (DCM), air-dried, and stored 747 in a desiccator for further use. 748

749 Syntheses was continued on a 0.01-mmol scale. Resin bearing the Biotinyl-GKG fragment was 750 weighed into individual vessels and re-swelled with DMF. Fmoc-pMet-OH (Prepared in-house 751 from L-Photo-Methionine, Thermo Fisher) was coupled to the lysine side chain followed by the 752 linker amino acid X: Fmoc-Gly-OH (Sigma-Aldrich), Fmoc-NH-PEG2-CH₂COOH, Fmoc-NH- 753 PEG4-CH₂COOH or Fmoc-NH-PEG6-CH₂CH₂COOH (ChemPep). The deprotected N-terminus 754 of the linker residue was then bromoacetylated by 500 µL DMF solution containing 25 mg 755 bromoacetic anhydride (10 eq) and 3.5 µL DIEA (2 eq) for 10-minute incubation at RT. Finally, the 756 resin bearing each peptide was washed thoroughly with DCM, air dried, and the product peptide 757 was cleaved from the resin with 1 mL neat trifluoroacetic acid (TFA) containing 2.5% (v/v) water 758 and 2.5% (v/v) triisopropylsilane at 60 °C for 8 min. TFA in the resulting solution was evaporated 759 and the residual was triturated with 1+1 (v/v) mixture of diethyl ether and hexane to precipitate 760 the peptide. The crude peptide was washed once with the ether-hexane mixture, air-dried and 761 lyophilized. The crude peptide was refined over a semi-preparative reverse phase (RP)-HPLC 762 (Discovery BIO wide pore C18 column: 10 x 250 mm, 5 µm) using a linear gradient of solvent A 763 (0.1% TFA in 90% acetonitrile) and B (0.1% TFA) with the percentage of B increasing from 5% to 764 25% over 10 minutes. Flow rate was 6 mL/min. Pure fractions of the product peptide were 765 combined and lyophilized. 766
766 **Synthesis of ppGpp photo-affinity probes**

767 Purified *B. subtilis* SasB was used to synthesize [5'-β-thio]-ppGpp (ppGpp-5βS) and [3'-β-thio]-
768 ppGpp (ppGpp-3βS). The reactions were conducted in 2mL buffer containing 20mM Tris-HCl 8.0,
769 150 mM NaCl, 25mM MgCl2 and 1mM TCEP. Final concentrations were 12 mM ATP and 10 mM
770 [5'-β-thio]-GDP (Sigma-Aldrich) for ppGpp-5βS synthesis and 10 mM [5'-β-thio]-ATP (Sigma-
771 Aldrich) and 10 mM GDP for ppGpp-3βS synthesis. 250 µM pppGpp was included to activate
772 SasB(Steinchen et al., 2015). This mixture was incubated with 5 µM SasB at 37°C for 3 hours
773 and completion of reaction confirmed by anion-exchange (ANIEX) chromatography on a MonoQ
774 5/50 column using buffers A: 5 mM Tris-HCl 8.0 and B: 5mM Tris-HCl 8.0 and 1mM NaCl. A linear
775 gradient of 10-40%B in 15 mL was used. All ANIEX runs were completed with Upon completion,
776 the reaction was vortexed with 300uL chloroform for 1 minute, and spun at 21,000 g for 5 min.
777 The aqueous layer was diluted 20-fold in water and resolved on MonoQ 10/100 with 10-40%B in
778 80mL. Fractions containing the product were combined, lyophilized and re-dissolved in 5 mL
779 water. LiCl powder was then added to 1 M concentration, and the products were precipitated by
780 addition of 20 mL ice-cold 100% ethanol. After staying in ice-water bath for 30 min, precipitate
781 was spun down, mother liquor aspirated, and pellet was washed with 5 mL 95% EtOH. The final
782 product was collected through centrifugation, redissolved in water, quantified based on absorption
783 at 252 nm (ε = 13,600 M⁻¹ cm⁻¹) and then lyophilized. Dried, colorless powder was dissolved with
784 Milli-Q water for 100mM stocks and stored frozen -80°C. ppGpp photo-affinity probes were
785 synthesized by conjugating 2.2 µmol ppGpp-5βS or ppGpp-3βS to 2 µmol peptide at pH = 6.5 at
786 42 °C for 1 hr. Conjugation to peptide **1-4** were carried out in separate reactions. Products were
787 purified on a MonoQ (5/50) column at 4 °C using buffer A (5 mM HEPES-Na 7.0, 10% acetonitrile)
788 and buffer B (5 mM HEPES-Na pH 7.0, 1M NaCl, 10% acetonitrile). All Four ppGpp-5βS
789 conjugates were then combined into an equimolar mixture and diluted to 0.5 mM total
790 concentration. A similar stock was prepared for all four ppGpp-3βS conjugates.
791 Effector capture and enrichment from *C. crescentus* lysate

792 M2G medium containing 0.4% glucose and 0.50 g/L regular NH₄Cl (light medium) or 0.51 g/L

793 NH₄Cl (¹⁵N, 99%, Cambridge Isotope Laboratories, heavy medium) were used. *C. crescentus*

794 NA1000 was cultured at 30°C in 60-mL batches in 500-mL baffled flask with vigorous shaking to

795 OD₆₀₀ = 0.38 and then chilled on ice. Cells were collected, washed with 1 mL lysis buffer (20 mM

796 HEPES-Na 7.0, 300 mM NaCl), and resuspended in lysis buffer to 600 µL total volume. This

797 sample was sonicated and cell debris removed through centrifugation at 20,000G for 10 minutes

798 to yield a cleared lysate containing approximately 6 mg/mL total protein. Two reactions were

799 assembled in adjacent wells on a 96-well plate chilled on ice for each photo-affinity probe mixture.

800 The effector-capture reaction consisted of 400 µg protein from the light lysate, 100 µM photo-

801 affinity probe, 1.8 mM MgCl₂, and 0.2 mM MnCl₂. The control reaction consisted of 800 µg protein

802 from the heavy lysate, 100 µM capture compound, 5 mM ppGpp, 6.3 mM MgCl₂ and 0.2 mM

803 MnCl₂. After exposure to 365-nm UV lamp for 2 minutes, reactions were sampled for western

804 blotting and all four reactions (one pair each for ppGpp-5βS and ppGpp-3βS probes) were

805 combined and extensively exchanged in a 10kDa-MWCO concentrator into a buffer containing 20

806 mM HEPES-Na pH 7.5 and 200 mM NaCl. The final concentrate (approximately 100 µL) was

807 diluted with 400 µL RIPA buffer (50 mM Tris 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium

808 deoxycholate, 1% Triton X-100) and incubated overnight with 200 µL MyOne Streptavidin C1

809 dynabeads (Thermo Fisher Scientific) at 4 °C. On the second day, the protein in supernatant

810 (flowthrough) was sampled for Western blotting and then removed, and the dynabeads were

811 washed at 4 °C twice with RIPA buffer, once with 0.1 M Na₂CO₃, once with 100 mM Tris 8.0, 4 M

812 guanidinium chloride (GuHCl) and twice more with RIPA buffer. Bound protein was eluted through

813 boiling the dynabeads in 40 µL SDS-PAGE loading dye containing 2 mM biotin. The eluate was

814 lyophilized and re-diluted using water to 15 µL, resolved on 12% TGX-PAGE.

815 LC-MS2 based proteomics
816 SDS-PAGE of eluates from streptavidin dynabeads was stained with Coomassie Brilliant Blue
817 (CBB), revealing three major protein bands, the streptavidin at 13 kDa, EF-Tu at 40 kDa and EF-
818 G at 80 kDa. The gel lane for each eluate was excised and regions below the 15-kDa or above
819 the 200-kDa marker were trimmed off. The lane was further divided into three samples and
820 processed separately: the EF-Tu and EF-G bands (sample A), the fragment below EF-Tu (sample
821 B), and the rest (sample C). The gel fragments were diced into 1-mm pieces and then treated with
822 10 mM DTT at 56 °C for 1 hr followed by 55 mM iodoacetamide at RT for 1 hr in the dark. After a
823 brief wash with 0.1 M NH₄HCO₃, gel pieces were dehydrated with acetonitrile, dried in a speedvac,
824 and re-hydrated with a minimal volume of 6 ng/µL trypsin in 0.1 M NH₄HCO₃ for digestion at RT
825 overnight. Thereafter, tryptic fragments were extracted by four shrinking-swelling iterations. In
826 each iteration, gel pieces were first dehydrated with 5% formic acid in 50% (v/v) acetonitrile (first
827 two iterations) or neat acetonitrile (second two). Supernatant containing tryptic fragments was
828 then removed for collection and gel pieces were re-swelled with 0.1 M NH₄HCO₃ in water. The
829 combined supernatant was dried in a speedvac and reconstituted in 50 µL 0.1% formic acid. 5 µL
830 was used for LC-MS2 analysis.

831 Tryptic fragments were separated by RP-HPLC (Thermo Easy nLC1000) using a precolumn
832 (made in house, 6 cm of 10 µm C18) and a self-pack 5 µm tip analytical column (12 cm of 5 µm
833 C18, New Objective) over a 140-minute gradient before nanoelectrospray using a QExactive
834 mass spectrometer (Thermo). Raw mass spectral data files (.raw) were searched using Proteome
835 Discoverer 2.1 (Thermo) and Mascot version 2.4.1 (Matrix Science). Mascot search parameters
836 were: 10 ppm mass tolerance for precursor ions; 15 mmu for fragment ion mass tolerance; 2
837 missed cleavages of trypsin; fixed modification was carbamidomethylation of cysteine. A separate
838 search was carried out for heavy peptides whereby replacement of all ¹⁴N by ¹⁵N in all amino acids
839 were included as fixed modification. Signals of heavy and light fragments of the same sequence
840 are related by expected mass difference and retention time. Quantification was then obtained
841 using the area of the precursor-ion peaks. SILAC ratio of a unique peptide sequence was
842 calculated as the mean of all precursor ions that match the sequence regardless of modifications.

843 SILAC ratio of a protein was calculated as the mean of all unique peptides mapped to the protein.

844 SILAC ratios were converted to log₂ units. Only proteins detected in both replicates each by at
845 least two unique tryptic fragments were reported.

846 **Purification of His-tagged proteins**

847 All Histidine-tagged proteins were purified with the standard protocol described below. *E. coli*
848 BL21(DE3) were used to express proteins from pET11b and pET28a expression plasmids (Table
849 S5, S6). Cells were grown in 1-4 L of LB medium supplemented with ampicillin for cells carrying
850 pET11b-derived plasmid or kanamycin (25 µg/mL) for cells carrying pET28a-derived plasmid.

851 Expression was induced by adding isopropyl 1-thio-D-galactopyranoside (IPTG) to 0.5 mM final
852 concentration and the cultures were incubated overnight at 20°C. After harvesting by
853 centrifugation, the cell pellets were resuspended in 40 mL lysis buffer containing 50 mM Tris-HCl,
854 pH 8.0, 500 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol, 50 µg mL⁻¹ lysozyme (Sigma),
855 5 µg/mL DNase I (Roche) and Complete protease inhibitor (Roche). Cells were disrupted through
856 sonication, and the lysates were cleared at 100,000 x g for 1 hr. at 4°C. Cleared lysate was applied
857 to 5 mL Ni-NTA resin equilibrated with wash buffer 1 (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10
858 mM imidazole, 5 mM β-mercaptoethanol) on an ÄKTA pure™ system (GE Healthcare). The Ni-
859 NTA resin was washed with ten column volumes of wash buffer 1. The bound protein was eluted
860 with a linear gradient of elution buffer (50 mM Tris-HCl pH 8, 500 mM NaCl, 5 mM β-
861 mercaptoethanol and 400 mM imidazole). Elution fractions were pooled and dialyzed overnight in
862 SEC buffer (25 mM Tris-HCl pH 7.8, 100 mM NaCl, 5 mM MgCl₂, glycerol 5% and 1 mM β-
863 mercaptoethanol). Dialyzed proteins were concentrated using a 30kDa Amicon® Ultra Centrifugal
864 concentrator (Merck Millipore). Then, the concentrated proteins were centrifuged at 16,000 x g
865 for 15 min. at 4 °C and loaded onto a Superdex™ Increase 200 (10/300) gel filtration column (GE
866 Healthcare) equilibrated in SEC buffer. The fractions were pooled and concentrated to the desired
867 concentration (around 10 mg ml⁻¹) for further experiments and frozen at -80°C.

868 Isothermal Titration Calorimetry (ITC)

869 Experiments were carried out at 25 °C, a syringe stirring speed of 700 r.p.m., a pre-injection delay
870 of 60 sec. and a recording interval of 150 sec. in an ITC200 MicroCalorimeter (Malvern
871 Panalytical). SEC purified PleD at 100 µM was loaded in the sample and the titration of the protein
872 performed in SEC buffer (25 mM Tris-HCl pH 7.8, 100 mM NaCl, 5 mM MgCl₂, glycerol 5% and
873 1 mM β-mercaptoethanol). ppGpp, pppGpp or GpCpp (Jena Biosciences) in SEC buffer were
874 injected in the sample cell. Blank titrations were performed with protein-free SEC buffer in the
875 sample cell. Baseline correction and integration of the raw differential power data and fitting of
876 the resulting binding isotherms to obtain the K_D values were performed using the Microcal ORIGIN
877 software. The parameters and the concentration of protein and ligands used are described in
878 Table S1.

879 nano Differential Scanning Fluorimetry (nanoDSF)

880 For nanoDSF experiments, 100 µM purified protein in 25mM Tris pH 7.8, 100mM NaCl, 5% (v/v)
881 glycerol, 5mM MgCl₂ and 1 mM β-mercaptoethanol was incubated without or with 1 mM ATP ,
882 GDP (Sigma), ppGpp, pppGpp, or GTP (Jena Biosciences), respectively, at room temperature for
883 5 min. Reactions were subsequently analyzed in a Prometheus NT.48 (NanoTemper
884 Technologies) device with a 1°C/min. thermal ramp (from 20°C to 95°C) and the internal
885 fluorescences at 330 nm and 350 nm were recorded. Data analysis and calculation of derivatives
886 were done using the PR.ThermControl software (NanoTemper Technologies).

887 Enzymatic assays

888 Diguanylate cyclase assays were adapted from procedures described previously (Paul et al,
889 2004). The reaction mixtures contained purified PleD<sub>CC</sub>, PleD<sub>AT</sub>, PleD<sub>SM</sub>, WpsR or DgcZ (40 µM)
890 in 25 mM Tris-HCl pH 7.8, 100 mM NaCl, 10 mM MgCl₂ in a 50 µL final volume. The reactions
891 were started by the addition of 100 µM GTP to the mixture. For enzymatic assays of
892 phosphorylated PleD, the protein was preincubated for 25 min. with purified DivJ₁-₂₄₆ (10 µM) and
893 200 µM ATP in the same buffer previously described. When added, the (p)ppGpp, ATP, GDP or
894 GpCpp molecules (Jena Biosciences) were supplemented at the indicated concentration at the
895 same time as the GTP substrate and incubated at 25°C for 2 hours. The reactions were stopped
896 by addition of EDTA at a final concentration of 10 mM and transferred into the HPLC system
897 (UltiMate3000, Thermo Fischer) for separation on a Nucleosil C18 column (Macherey-Nagel) with
898 a 10 min. gradient of 70% acetonitrile – 0.09% trifluoroacetic acid. The c-di-GMP peak was
899 detected using the 253 nm UV absorption.

900 **Crystallization, data collection, and structure determination**

901 The screening was conducted using a Mosquito workstation (TTP Labtech) on commercial
902 crystallization solutions with the sitting-drop vapor diffusion method. Drop were set up at 19°C
903 and consisted of 150 nL of reservoir solution and 150 nL of protein solution (5 mg/mL)
904 supplemented with 0.5 µM c-di-GMP and 5 mM ppGpp or pppGpp. All crystallization trials were
905 visualized on RockImager 182 (Formulatrix) and crystals appeared after one month. Crystal of
906 PleD in complex with ppGpp and c-di-GMP grew in 0.1 M NaCl, 50 mM sodium cacodylate pH
907 6.5, 10% w/v PEG 4000 and 500 µM spermine (Natrix, Hampton research, E7 condition). Crystal
908 of PleD in complex with pppGpp and c-di-GMP were obtained in 0.2 M Lithium citrate tribasic
909 tetrahydrate, 20% w/v Polyethylene glycol 3.350 (PEG ION HT (Hampton research), D9
910 condition). PleD-ppGpp crystals were frozen in a reservoir solution supplemented by 15%
911 Glycerol, and PleD-pppGpp crystals were frozen in a reservoir solution supplemented by 15%
912 Glycerol and 5mM pppGpp. X-ray Data for PleD-(p)ppGpp crystals were collected at the ID23EH2
913 beamline of the European synchrotron Radiation Facility and at Proxima 2 beamline of
914 synchrotron SOLEIL. PleD-ppGpp crystals diffracted at a resolution 2.9 Å and PleD-pppGpp
915 diffracted at 3 Å and both belonged to the space group P 6₁₂₂ with 4 molecules per asymmetric unit. Diffraction data were processed using XDS ((Kabsch, 2010) and with AIMLESS (Evans & Murshudov, 2013) from the CCP4 program suite (Hough & Wilson, 2018; "The CCP4 Suite: Programs for Protein Crystallography," 1994). Data collection statistics are indicated in supplementary data Table S3.

920 Both the PleD-ppGpp and pppGpp structures were solved by molecular replacement using the coordinates of PleD monomer in complex with c-di-GMP (PDB ID: 1W25, (Chan et al., 2004)) as a probe in PHASER (Hough & Wilson, 2018). Models were re-built manually in COOT (Casañal et al., 2020) and refined using REFMAC (Murshudov et al., 2011) and PHENIX (van Zundert et al., 2021). The models were refined with final R<sub>work</sub>/R<sub>free</sub> of 0.20/0.24 and 0.19/0.23 for PleD-ppGpp and PleD-pppGpp, respectively. The coordinates and structure factors were deposited in the Protein Data Bank with accession code 9G85 (PleD-ppGpp) and 9G86 (PleD-pppGpp). Figures were generated with Pymol, molecular Graphic System, Version 3 Schrödinger, LLC.

928 **Time courses and synchronization**

929 Cells were grown overnight in PYE with appropriate antibiotics. Cells were diluted in M2 supplemented with 0.2% glucose and appropriate antibiotics when necessary and grown to OD<sub>600</sub> 0.1-0.4. Cells were either diluted in M2 supplemented with 0.2% glucose to an OD<sub>600</sub> of 0.06 or switched to M2 supplemented with 0.02% glucose (M2G<sub>1/10</sub>) (for glucose exhaustion) to an OD<sub>600</sub> of 0.13 and grown for 5 hours to both reach an OD<sub>600</sub> of ~0.35.

934 For synchronization, cells were first centrifuged at 10,000 × g for 10 minutes at 4°C. Swarmer cells were then isolated using a Percoll density gradient centrifugation. The cell pellets were resuspended in equal volumes of cold M2 buffer (0.87 g/L Na₂HPO₄, 0.53 g/L KH₂PO₄, 0.5 g/L NH₄Cl) and Percoll (GE Healthcare), followed by centrifugation at 10,000 × g for 20 minutes. The upper ring was removed, while the lower ring, containing swarmer cells, was transferred to a new 939 15-mL Falcon tube. The swarmer cells were washed with 13 mL of cold M2 buffer and centrifuged
940 at 10,000 × g for 5 minutes at 4°C. Finally, the pellet was resuspended in 2 mL of cold M2 buffer
941 and subjected to a final centrifugation at 21,000 × g for 1 minute.

942 Synchronized swarmer cells were released into either M2 medium supplemented with appropriate
943 antibiotics and 0.2% glucose (M2G condition) or into filtered M2G 0.02% glucose spent medium
944 used during the 5-hour exhaustion step before synchronization (M2G₁/₁₀*). OD₆₀₀ was adjusted to
945 0.1-0.2 for each condition. When appropriate, cultures were supplemented with inducers. At the
946 indicated time points, samples were harvested from each flask for flow cytometry (0.15 ml) or
947 immunoblotting (2 ml). For flow cytometry, samples were stored in 30% ethanol at 4°C. For
948 immunoblotting, cells were centrifuged for 1 min. at 14,000g, supernatant was aspirated, and
949 frozen in liquid nitrogen.

950 **Immunoblotting**

951 Frozen pellets were resuspended on ice in 40 µl Laemmli sample buffer (2% [w/v] SDS, 10%
952 glycerol, 60 mM Tris-Cl [pH 6.8], 0.01% [w/v] bromophenol blue, 1% β-mercaptoethanol) per
953 OD₆₀₀ unit and denatured at 95°C for 10 min. Samples were separated by 12% SDS-PAGE for
954 60 min. at 130 V at room temperature. Blocking was done in TBST buffer (TBS containing 0.1%
955 Tween-20) with 5% (w/v) non-fat milk powder and antibodies were used at the following
956 concentrations: anti-RpoA (1:7,500) (Bio-Legend), anti-CtrA (1:5,000), anti-FLAG (1:7500)
957 (Merck) diluted in TBST 5% non-fat milk. Horseradish peroxidase (HRP)-conjugated secondary
958 antibodies (ThermoFisher) were used at the following concentrations: anti-mouse (1:10,000) for
959 anti-RpoA and anti-Flag immunoblotting and anti-rabbit (1:10,000) for anti-CtrA immunoblotting.
960 The membranes were developed with SuperSignal™ West Pico PLUS Chemiluminescent
961 Substrate (ThermoFisher) and visualized with a Fusion FX (Vilber). Band intensities were
962 quantified using ImageJ software.

963 **Flow cytometry**
964 A fraction of fixed cells from the time course sampling (corresponding to an OD₆₀₀ of ~0.005) were 965 centrifuged at 6,000 rpm for 4 min. Pelleted cells were resuspended in 1 mL of Na₂CO₃ buffer 966 containing 3 mg ml⁻¹ RNase A (Qiagen) and incubated at 50°C for at least 4 hours or overnight to 967 digest RNA. 968

968 Diluted samples were stained with 0.25 µM SYTOX Green (Invitrogen) for flow cytometric 969 quantification of cellular DNA content. Cells were run on a Attune Nxt (Invitrogen) (FSC = 360, 970 SSC = 460, FITC = 320; Threshold: FITC > 1000) / MASCQuant (Miltenyi) (param: FSC = 290, 971 SSC = 574, FITC = 458; Threshold: FITC > 1000) and 50,000 total SYTOX positive events were 972 collected for each experiment. 973

## Sampling and Intracellular Metabolite Extraction

974 Cells were cultured until the desired growth phase (OD₆₀₀ = 0.4), then harvested by withdrawing 975 an appropriate volume corresponding to 8 x 10⁷ cells. The collected samples were immediately 976 quenched by mixing with 5 mL of a precooled methanol:acetonitrile:water solution (4:4:2, v/v/v) at 977 -20°C to rapidly stop metabolic activity (Millard et al., 2014). An internal standard consisting of 50 978 µL of a uniformly ¹³C-labeled *E. coli* metabolite extract (prepared as described in Patacq et al., 979 2018) was added to each sample (Millard et al., 2014). Samples were vortexed vigorously and 980 incubated at -20°C for 2 hours to ensure metabolite extraction. Cell debris was removed by 981 centrifugation at 16,000 × g for 5 minutes at 4°C, and the supernatants were dried overnight using 982 a SpeedVac concentrator (Eppendorf Concentrator plus, Eppendorf AG, Hamburg, Germany). 983 Dried extracts were stored at -80°C until analysis. Prior to analysis, samples were reconstituted 984 in 250 µL of ultrapure water and analyzed using ion chromatography (IC; Thermo Scientific Dionex 985 ICS-5000+, Dionex, Sunnyvale, CA, USA) coupled to a Q Exactive Orbitrap mass spectrometer 986 (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a heated electrospray ionization 987 (HESI) source. Separation was achieved with a KOH gradient as follows: 7 mM (0-1 min), linear 988 increase to 15 mM (1-9.5 min), held at 15 mM (9.5-20 min), ramped to 45 mM (20-30 min), then 989
989 to 70 mM (30–33 min), followed by 100 mM (33.1–42 min), and re-equilibrated at 7 mM (42.5–50
990 min). The total run time was 50 minutes, and 10 µL were injected. Mass spectra were acquired in
991 negative ion mode using full-scan acquisition at a resolution of 70,000 (m/z 200), with the following
992 source parameters: capillary temperature 300°C, heater temperature 450°C, sheath gas flow 40
993 AU, auxiliary gas flow 20 AU, S-lens RF level 60%, and spray voltage -3.5 kV. Metabolites were
994 identified based on exact m/z values with a 5 ppm mass tolerance, using Xcalibur software
995 (Thermo Fisher Scientific) and processed with El-Maven (version 0.12.0, (Agrawal et al., 2019)).
996 Four independent biological replicates were analyzed.

997 **Intracellular Metabolite Concentrations**

998 First, metabolite concentrations in extracts were determined using calibration curves and the ratio
999 of ¹²C- to ¹³C-labeled peak areas for GDP, GTP and (p)ppGpp; c-di-GMP was quantified using the
1000 ¹²C-labeled form only due to absence of ¹³C-labeled isotopologue in the ¹³C-labeled *E. coli* extract.
1001 Then, total cell volume was estimated from the cell counts on OD₆₆₀ (1 OD = 2 × 10⁹ CFU/mL)
1002 and an average *C. crescentus* cell volume of 6.46 × 10⁻¹⁶ L (Snyder et al., 2020). Finally,
1003 intracellular concentrations were calculated by dividing metabolite concentration in extracts by the
1004 total cell volume.
<figure><img src="image_2.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_3.png" /></figure>
**Figure 1: (p)ppGpp/c-di-GMP antagonism**

(A) *Top*: Schematic of *Caulobacter crescentus* cell-cycle. Cells divide asymmetrically generating a swarmer daughter cell (with indicated flagellum and pili) and a stalked daughter cell (with indicated stalk). (p)ppGpp (green) and c-di-GMP (purple) accumulate at different steps of the cell-cycle and have antagonistic activities on cell differentiation respectively blocking and triggering transition from swarmer-to-stalked cell. *Bottom*: (p)ppGpp and c-di-GMP predicted oscillations during the cell-cycle.

(B) Diagrams of enzymatic reactions leading to the synthesis of (p)ppGpp and c-di-GMP catalyzed by Rel enzyme or diguanylate cyclases (DGC) respectively. Both are made from guanosine-based nucleotides (GTP or GDP).
A. photo-affinity probes

<figure><img src="image_4.png" /></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2025-08-26-671821; this version posted August 23, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license https://doi.org/10.1101/2025-08-26-671821

B. Probes

<figure><img src="image_5.png" /></figure>

WB:
Strept-
avidin

MW
standard

CBB

C

<table><thead><tr><th>Accession</th><th>Protein</th><th>CCNA number</th><th>Function/annotation</th><th>log<sub>2</sub>(heavy/light)</th></tr></thead><tbody><tr><td>A0A0H3CA25</td><td></td><td>CCNA_02297</td><td>Hypoxanthine-guanine phosphoribosyltransferase</td><td>5.94</td></tr><tr><td>A0A0H3C5J2</td><td></td><td>CCNA_00267</td><td>MutT-like protein</td><td>4.01</td></tr><tr><td>A0A0H3C7H6</td><td>HflX</td><td>CCNA_01820?</td><td>GTPase</td><td>3.63</td></tr><tr><td>B8GZM2</td><td>PleD</td><td>CCNA_02546</td><td>GGDEF/response regulator protein</td><td>3.45</td></tr></tbody></table>
Figure 2: ppGpp-target identification in *Caulobacter crescentus*

(A) Chemical proteomic approach for ppGpp-target identification based on metabolic universal ¹⁵N-labeling of *C. crescentus* in M2G medium.

(B) *C. crescentus* proteome tagged by indicated probes visualized using streptavidin western blotting. See Figure S1 for probe structure and synthesis. Lysate containing 100 µM probe (ppGpp) or 100 µM probe and 5 mM native ppGpp (+ ppGpp) was illuminated with 360-nM UV. Aliquots containing 10 µg protein were resolved on 12% SDS-PAGE for immunoblotting

(C) Putative ppGpp-binding proteins identified. Shown are homologs of known ppGpp targets and the diguanylate cyclase PleD investigated in this study.
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /><figcaption>**ppGpp-3βS probes**
diverse linker length
used as a cocktail</figcaption></figure>

<caption>**ppGpp-5βS probes**
diverse linker length
used as a cocktail</caption>
**Figure S1 related to Figure 2:**

Synthesis of photo-affinity ppGpp probes¹. Chemical structures of (A) bromoacetylated peptides containing a diazirine photocrosslinker and a biotin handle and (B) thiophosphate analogs of ppGpp are shown. (C) Conjugation of building blocks in (A) and (B) yields the tripartite photoaffinity probes used in Figure 2A-B.
<figure><img src="image_11.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_16.png" /></figure>
Figure 3: Inhibition of PleD's activity by ppGpp and pppGpp through direct binding

(A) ITC traces (top) and fitted isotherms (bottom) for the titration of 100 µM PleD with 1 mM ppGpp, pppGpp, or GpCpp (left to right). K_D: dissociation constant; N: stoichiometry of the PleD-ligand complex.

(B) *In vitro* enzymatic assay measuring c-di-GMP synthesis by PleD from *Caulobacter crescentus*. Reactions contain 40 µM PleD and 100 µM GTP, either alone (black) or with 500 µM ppGpp (orange) or pppGpp (blue). The relative c-di-GMP production shows the amount of c-di-GMP generated under each condition, normalized to the production observed in the absence of competitor. Data are expressed as mean ± SD.

(C) Dose-response curves for c-di-GMP production, normalized to the control, with 40 µM PleD and 100 µM GTP in the presence of increasing concentrations of ppGpp (orange) or pppGpp (blue): 50 µM, 75 µM, 100 µM, 250 µM, or 500 µM. The relative c-di-GMP production was calculated by comparing each condition to the control where no competitor was present. Data represent mean ± SD from 3 independent experiments (for 50 µM, 75 µM, 100 µM and 250 µM conditions) and 12 independent experiments (for 500 µM condition). Asterisks indicate statistical significance (*= p <0.05; **= p <0.01; ***= p <0.001; ****= p <0.0001) based on one-sample Student's t-test.

(D) Same as (B), but for phospho-activated PleD pre-incubated with 10 µM DivJ1-246 and 200 µM ATP for 25 minutes prior to the addition of 500 µM ppGpp (orange) or pppGpp (blue).

(E) Same as (B) but for PleD ortholog from *Agrobacterium tumefaciens* (PleDAT).

(F) Same as (B) but for PleD ortholog from *Sinorhizobium meliloti* (PleDSM).

(G) Same as (B) but for WspR, a PleD ortholog from *Pseudomonas aeruginosa*.

(H) Same as (B) but for DgcZ from *Escherichia coli*.
A

<figure><img src="image_17.png" /></figure>

B

<figure><img src="image_18.png" /></figure>

C

<figure><img src="image_19.png" /></figure>

D

<figure><img src="image_20.png" /></figure>

E

<figure><img src="image_21.png" /></figure>
**Figure S2 related to Figure 3:**

(A) HPLC profiles of different DGCs following purification from *E. coli* containing 40 µM PleD<sub>cc</sub> (black), 40 µM PleD<sub>AT</sub> (pink), 40 µM PleD<sub>SM</sub> (blue), 40 µM WspR (green) or 40 µM DgcZ (red).

(B) First-derivative curves at 350 nm from nanoDSF assays of 100 µM PleD from *Caulobacter crescentus* (PleD<sub>cc</sub>) without any ligand (black) or with 1 mM ATP (pink), 1 mM GDP (red), 1 mM ppGpp (orange), 1 mM GTP (green), or 1 mM pppGpp (blue).

(C) Melting temperature (Tm) determination from nanoDSF assays of 100 µM PleD<sub>cc</sub> (left). ΔTm values indicate the difference in Tm between PleD<sub>cc</sub> without any ligand (black) and with GDP (red), ppGpp (orange), GTP (green), or pppGpp (blue). Data are presented as mean ± SD.

(D) Same as (B) for 100 µM PleD from *Agrobacterium tumefaciens* (PleD<sub>AT</sub>).

(E) Same as (C) for PleD<sub>AT</sub>.
bioRxiv preprint doi: https://doi.org/10.1101/2025.08.22.671821; this version posted August 23, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under bioRxiv's terms of use, available at http://bioRxiv.org/RRULES. This license does not transfer any rights to other parties.

A

<figure><img src="image_22.png" /></figure>

B

<figure><img src="image_23.png" /></figure>

C

<figure><img src="image_24.png" /></figure>

PleD<sub>CC</sub> + DivJ<sub>1-246</sub>

D

PleD<sub>AT</sub>

<figure><img src="image_25.png" /></figure>

E

PleD<sub>SM</sub>

<figure><img src="image_26.png" /></figure>

F

WspR

<figure><img src="image_27.png" /></figure>

G

DgcZ

<figure><img src="image_28.png" /></figure>

H

c-di-GMP production ratio
(mAu*min)

<figure><img src="image_29.png" /></figure>

I

c-di-GMP production
(mAu*min)

<figure><img src="image_30.png" /></figure>

J

<figure><img src="image_31.png" /></figure>
**Figure S3 related to Figure 3:**

(A) *In vitro* enzymatic assay measuring c-di-GMP production by PleD or the catalytically-inactive PleDE370Q mutant from *Caulobacter crescentus*. Reactions contain 40 µM PleD, 40 µM PleD pre-incubated with 10 µM DivJ1-246 and 200 µM ATP for 25 minutes or PleDE370Q. Bars represent 100 µM GTP alone (black), or with the addition of 500 µM ATP (pink), GpCpp (green), 100 µM GTP with PleDE370Q (blue), or 100 µM GTP with phosphoactivated PleD (red). The relative c-di-GMP production represents the amount of c-di-GMP produced in each condition compared to the baseline production by PleD without competitor. Data are shown as mean ± SD.

(B) High-performance liquid chromatography (HPLC) profiles of reaction mixtures containing 40 µM PleD alone (black), 40 µM PleD with 100 µM GTP (red), or 100 µM GTP with 500 µM ATP (pink), GpCpp (green), ppGpp (orange), or pppGpp (blue). A zoomed-in view of the specific c-di-GMP peaks (retention time ~3.35 min) for each condition is highlighted in the black square.

(C) Same as (B) for phospho-activated PleD pre-incubated with 10 µM DivJ1-246 and 200 µM ATP for 25 minutes. Reaction mixtures contain 40 µM PleD alone (black), 40 µM PleD with 100 µM GTP (pink), 40 µM phospho-activated PleD with 100 µM GTP (red), or 100 µM GTP with 500 µM ppGpp (orange), or pppGpp (blue).

(D) Same as (B) for PleD ortholog from *Agrobacterium tumefaciens* (PleDAT).

(E) Same as (B) for PleD ortholog from *Sinorhizobium meliloti* (PleDSM).

(F) Same as (B) for WspR from *Pseudomonas aeruginosa*.

(G) Same as (B) for DgcZ from *Escherichia coli*.

(H) *In vitro* enzymatic assay measuring c-di-GMP production by different diguanylate cyclases (DGCs), presented as the ratio of c-di-GMP production with 100 µM GTP to the condition with protein only (not normalized). Data are presented as mean ± SD.

(I) *In vitro* enzymatic assay measuring c-di-GMP production by DgcZ with 100 µM GTP. Data are presented as mean ± SD.
<figure><img src="image_32.png" /><figcaption>A</figcaption></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2025.08.22.671821; this version posted August 23, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

<figure><img src="image_33.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_34.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_35.png" /></figure>
Figure 4: Structural insights into PleD inhibition by (p)ppGpp.

(A) Ribbon representation of the crystal structure of PleD in complex with pppGpp (left) and ppGpp (right). The receiver domains D1 and D2 are colored in magenta an orange, respectively. The DGC domains are colored in cyan for PleD:pppGpp and slate for for PleD:ppGpp. The alarmones and c-di-GMP are represented as sticks. Carbones of alarmones are colored according to their DGC domain, c-di-GMP carbons are colored in green, oxygens are colored in red, phosphorus in orange and magnesium in magenta. The active and inhibitory sites (I-site) are indicated.

(B) Representative close-up view of the alarmone and cyclic-di-GMP molecules colored as in A) with corresponding 2Fo-Fc electron density maps contoured at 1.5 sigma.

(C) Comparison of the DGC domain's position of cyclic-di GMP-bound PleD (PDB code 1W25, left) with the DGC domain's position of pppGpp-bound PleD.

(D) Comparison of the active site from PleD:GTPαS structural data (PDB code 2V0N) and the PleD:(p)ppGpp active sites. Ligands are shown in stick representation with carbons colored in purple (ppGpp), magenta (pppGpp) or green (GTPαS). The DGC domains contributing to the binding are colored in cyan and slate (alarmones) or pink (GTPαS). Magnesium atom is represented as a green sphere. Side chains involved in the binding are shown as sticks and hydrogen bonds are indicated by grey dashes.
<caption>A</caption>

bioRxiv preprint doi: https://doi.org/10.1101/2025.08.22.671921; this version posted August 23, 2025. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

<figure><img src="image_36.png" /></figure>

PleD:ppGpp

<figure><img src="image_37.png" /></figure>

PleD:pppGpp

<figure><img src="image_38.png" /></figure>

PleD:c-di-GMP

DGC

<figure><img src="image_39.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_42.png" /></figure>

PleD:c-di-GMP

<figure><img src="image_43.png" /></figure>

PleD:pppGpp

<figure><img src="image_44.png" /></figure>

PleD:ppGpp

<figure><img src="image_45.png" /></figure>

PleD:GTPαS

Inactive

Active

<figure><img src="image_46.png" /><figcaption>D</figcaption></figure>
Figure S4 related to Figure 4:

(A) Comparison of the PleD:c-di-GMP tetramer (PDB code 1W25, left) with the crystal structure of PleD in complex with ppGpp (middle) or pppGpp (right). Receiver domains D1 and D2 are colored in magenta and orange, DGC domains are colored according the crystal structure.

(B) View of the dimer-dimer interface showing the two DGC domains facing each other.

(C) Comparison of the dimer in « inactive » conformation and « activated » conformation in the different crystal structures colored as in (A).

(D) *In vitro* enzymatic assay measuring c-di-GMP production by unphosphorylated PleD, PleD<sub>R300K</sub>, and PleD<sub>R300A</sub>. The reactions contained either 40 µM PleD (black), 40 µM PleD<sub>R300K</sub> (dotted lines) or 40 µM PleD<sub>R300A</sub> (dashed lines) with 100 µM GTP alone. Normalized c-di-GMP production is represented relative to the production by wild-type PleD.
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

<figure><img src="image_47.png" /></figure>
**Figure S5 related to Figure 4:**

ClustalW sequence alignment of PleD<sub>CC</sub>, PleD<sub>AT</sub>, PleD<sub>SM</sub>, WspR and DgcZ. UNIPROT accession codes are B8GZM2, A9CJ98, Q92QM5, Q9HXT9 and P31129 respectively. Residue colors correspond to the Clustal color scheme: hydrophobic (blue), positively charged (red), negatively charged (magenta), polar (green), glycine (orange), proline (yellow), aromatic (cyan) and non-conserved (white) residues. Red arrows indicate highly conserved R300, Y439 and R446 residues.
A

Intracellular concentration [µM]

<figure><img src="image_48.png" /></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2025.08.22.671821; this version posted August 23, 2025. The copyright holder for this preprint (which is not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

B

C

D

<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /></figure>

E

<figure><img src="image_51.png" /></figure>
Figure 5: (p)ppGpp inhibition of PleD activity prevents cell differentiation under glucose starvation in *Caulobacter*

(A) Targeted metabolomics measuring the intracellular concentration of ppGpp in a asynchronous population of wild-type cells grown in M2G medium (black), P2G medium (pink), M2 with 0.02% glucose (M2G₁/₁₀*) (green) or M2 without glucose (purple). Data represent the mean ± SD of three independent biological replicates. Asterisks indicate statistical significance (** = p < 0.01; *** = p < 0.001) based on a Student's t-test.

(B) Same as (A) but representing pppGpp intracellular concentration.

(C) Same as (A) but representing GTP intracellular concentration.

(D) *Top*: Immunoblots showing CtrA protein levels at the indicated times post-synchronization in wild-type (WT), *rel* mutant, and *rel* *pleD* mutant strains grown in M2G medium or grown in M2 with 0.02% glucose spent medium (M2G₁/₁₀*). *Bottom*: Quantification of CtrA band intensity normalized to the RpoA loading control. Data represent the mean ± SD of three independent biological replicates.

(E) Immunoblots showing CtrA protein levels at the indicated times post-synchronization in WT and *rel* mutant strains grown in M2 with 0.02% glucose spent medium (M2G₁/₁₀*), with ectopic expression of either the phosphodiesterase *proE* or its catalytically inactive mutant *proEE328A* (*proE*\*) (induced with 100 µM vanillate post-synchronization) (left). Quantification of CtrA band intensity normalized to the RpoA loading control (right). Data represent the mean ± SD of three independent biological replicates. Asterisks indicate statistical significance (** = p < 0.01; *** = p < 0.001) based on a Student's t-test.
<figure><img src="image_52.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_53.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_54.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_55.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_56.png" /></figure>

<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_59.png" /></figure>
Figure S6 related to Figure 5:

(A) Flow cytometry profiles after SYTOX staining showing DNA content of wild-type, *rel* mutant, *rel pleD* mutant and *pleD* mutant cells grown post-synchronization in M2 supplemented with 0.2% glucose (M2G) or 0.02% glucose spent medium (M2G₁/₁₀*) and of wild-type and *rel* mutant cells grown post-synchronization in M2 supplemented with 0.02% glucose spent medium with ectopic expression of *proE* or its inactive mutant *proE*<sub>*E328A*</sub> (induced with 100 µM vanillate post-synchronization).

(B) Targeted metabolomics measuring the intracellular concentration of GDP in a asynchronous population of wild-type cells grown in M2G medium (black), P2G medium (pink), M2 with 0.02% glucose (M2G₁/₁₀*) (green) or M2 without glucose (purple). Data represent the mean ± SD of three independent biological replicates. Asterisks indicate statistical significance (**) = p < 0.01; *** = p < 0.001) based on a Student's t-test.

(C) Same as (B) but representing c-di-GMP intracellular concentration.

(D) CtrA immunoblots showing protein levels at the indicated times post-synchronization in wild-type and *pleD* mutant cells grown in supplemented with 0.2% glucose (M2G) or 0.02% glucose spent medium (M2G₁/₁₀*) over 90 minutes (left). Quantification of CtrA band intensity normalized to the RpoA loading control (right). Data represent mean ± SD of three independent biological replicates. Asterisks indicate statistical significance (* = p < 0.05; ** = p < 0.01) based on a Student's t-test.

(E) Growth curves of asynchronous wild-type, *rel* mutant, and *rel pleD* mutant cells cultivated in M2 supplemented with 0.2% glucose (M2G) or 0.02% glucose (M2G₁/₁₀*) over 8 hours. Data represent mean ± SD of three independent biological replicates.

(F) Growth curves of wild-type, *rel* mutant, and *rel pleD* mutant cells cultivated post-synchronization in M2 supplemented with 0.2% glucose (M2G) or 0.02% glucose spent medium (M2G₁/₁₀*) over 90 minutes. Data represent mean ± SD of three independent biological replicates.
<table><caption>Table S1: Fitting results of ITC experiments</caption><thead><tr><th>ligand</th><th>[ligand] (µM)</th><th>[PleD] (µM)</th><th>ratio</th><th>Chi²</th><th>N (sites)</th><th>K (M⁻¹)</th><th>Kd (M)</th><th>ΔH (cal/mol)</th><th>ΔS (cal/mol/deg)</th></tr></thead><tbody><tr><td>GpCpp</td><td>1000</td><td>95</td><td>10,5-1</td><td>1.06E+03</td><td>0.8711</td><td>2.22E+05</td><td>4.50E-06</td><td>1442</td><td>29.28</td></tr><tr><td>GpCpp</td><td>1000</td><td>80</td><td>12,5-1</td><td>725.44</td><td>0.9047</td><td>1.33E+05</td><td>7.53E-06</td><td>1273</td><td>27.71</td></tr><tr><td>ppGpp</td><td>800</td><td>100</td><td>8-1</td><td>2145</td><td>0.95</td><td>6.28E+04</td><td>1.59E-05</td><td>2993</td><td>31.99</td></tr><tr><td>ppGpp</td><td>1000</td><td>100</td><td>10-1</td><td>619</td><td>0.9</td><td>1.43E+04</td><td>6.99E-05</td><td>3586</td><td>31.04</td></tr><tr><td>ppGpp</td><td>1000</td><td>80</td><td>10-1</td><td>414</td><td>0.875</td><td>1.98E+04</td><td>5.05E-05</td><td>3008</td><td>29.75</td></tr><tr><td>pppGpp</td><td>800</td><td>80</td><td>10-1</td><td>2.52E+04</td><td>0.8769</td><td>9.76E+04</td><td>1.02E-05</td><td>3183</td><td>33.5</td></tr><tr><td>pppGpp</td><td>1000</td><td>100</td><td>10-1</td><td>1.37E+04</td><td>1.18</td><td>1.00E+05</td><td>1.00E-05</td><td>4383</td><td>37.6</td></tr><tr><td>pppGpp</td><td>750</td><td>75</td><td>10-1</td><td>3.12E+03</td><td>0.95</td><td>1.30E+05</td><td>7.67E-06</td><td>2506</td><td>31.81</td></tr></tbody></table>
<table><caption>Table S2: NanoDSF measurements</caption><thead><tr><th>Tm (°C)</th><th>control</th><th>ppGpp</th><th>pppGpp</th><th>ATP</th></tr></thead><tbody><tr><td>PleD C. crescentus</td><td>46,39 ± 2,615</td><td>52,85 ± 2,146</td><td>59,21 ± 0,5707</td><td>47,5 ± 1,768</td></tr><tr><td>PleD A. tumefaciens</td><td>48,58 ± 0,2121</td><td>52,72 ± 0,601</td><td>55,72 ± 0,05657</td><td></td></tr></tbody></table>

<table><thead><tr><th>Δ Tm (°C)</th><th>ppGpp</th><th>pppGpp</th></tr></thead><tbody><tr><td>PleD C. crescentus</td><td>6,332 ± 2,323</td><td>11,52 ± 0,2318</td></tr><tr><td>PleD A. tumefaciens</td><td>4,135 ± 0,3815</td><td>7,092 ± 0,2275</td></tr></tbody></table>
<table><caption>Table S3: Data collection and refinement statistics.</caption><thead><tr><th></th><th>PleD:ppGpp</th><th>PleD:pppGpp</th></tr></thead><tbody><tr><td>Wavelength</td><td></td><td></td></tr><tr><td>Resolution range</td><td>47.7 - 2.9 (2.95 - 2.9)</td><td>48.9 - 3.0 (3.09 - 3.0)</td></tr><tr><td>Space group</td><td>P 61 2 2</td><td>P 61 2 2</td></tr><tr><td>Unit cell</td><td>133.0 133.0 510.7 90 90 120</td><td>132.5 132.5 508.4 90 90 120</td></tr><tr><td>Total reflections</td><td>2586899 (118101)</td><td>1825849 (141102)</td></tr><tr><td>Unique reflections</td><td>66317 (2917)</td><td>54311 (4359)</td></tr><tr><td>Multiplicity</td><td>39.0 (40.5)</td><td>33 (32)</td></tr><tr><td>Completeness (%)</td><td>99.9 (99.9)</td><td>100 (100)</td></tr><tr><td>Mean I/sigma(I)</td><td>10.8 (1.2)</td><td>12.6 (1.4)</td></tr><tr><td>Wilson B-factor</td><td>76.0</td><td>71.7</td></tr><tr><td>R-merge</td><td>0.33 (5.48)</td><td>0.35 (3.30)</td></tr><tr><td>R-meas</td><td>0.34 (5.55)</td><td>0.36 (3.35)</td></tr><tr><td>R-pim</td><td>0.05 (0.86)</td><td>0.08 (0.80)</td></tr><tr><td>CC1/2</td><td>0.99 (0.67)</td><td>1.00 (0.47)</td></tr><tr><td>CC*</td><td>1.00 (0.71)</td><td>1.00 (1)</td></tr><tr><td>Reflections used in refinement</td><td>60430 (2686)</td><td>54162 (2628)</td></tr><tr><td>Reflections used for R-free</td><td>3013 (143)</td><td>2789 (140)</td></tr><tr><td>R-work</td><td>0.20 (0.39)</td><td>0.19 (0.33)</td></tr><tr><td>R-free</td><td>0.24 (0.45)</td><td>0.23 (0.35)</td></tr><tr><td>Number of non-hydrogen atoms</td><td>14054</td><td>14126</td></tr><tr><td>macromolecules</td><td>13500</td><td>13572</td></tr><tr><td>ligands</td><td>520</td><td>532</td></tr><tr><td>solvent</td><td>34</td><td>22</td></tr><tr><td>Protein residues</td><td>1774</td><td>1789</td></tr><tr><td>RMS(bonds)</td><td>0.003</td><td>0.004</td></tr><tr><td>RMS(angles)</td><td>0.56</td><td>0.72</td></tr><tr><td>Ramachandran favored (%)</td><td>98.4</td><td>98.6</td></tr><tr><td>Ramachandran allowed (%)</td><td>1.6</td><td>1.4</td></tr><tr><td>Ramachandran outliers (%)</td><td>0.00</td><td>0.00</td></tr><tr><td>Rotamer outliers (%)</td><td>1.33</td><td>0.00</td></tr><tr><td>Clashscore</td><td>2.82</td><td>5.85</td></tr><tr><td>Average B-factor</td><td>79.13</td><td>71.79</td></tr><tr><td>macromolecules</td><td>79.24</td><td>72.06</td></tr><tr><td>ligands</td><td>70.41</td><td>65.87</td></tr><tr><td>solvent</td><td>60.97</td><td>50.27</td></tr></tbody></table>

Statistics for the highest-resolution shell are shown in parentheses.
<table><caption>Table S4: Calculated concentrations of GTP, GDP, ppGpp, pppGpp, c-di-GMP in *C. crescentus* grown in different media</caption><thead><tr><th>Medium</th><th>GTP [µM]</th><th>GDP [µM]</th><th>ppGpp [µM]</th><th>pppGpp [µM]</th><th>c-di-GMP [µM]</th></tr></thead><tbody><tr><td>M2G</td><td>440,24 ± 50,447</td><td>483,76 ± 48,091</td><td>29,51 ± 8,815</td><td>4,60 ± 1,551</td><td>5,35 ± 1,610</td></tr><tr><td>P2G</td><td>378,46 ± 42,388</td><td>368,62 ± 34,331</td><td>85,51 ± 21,577</td><td>4,04 ± 0,937</td><td>4,72 ± 1,729</td></tr><tr><td>M2G<sub>1/10</sub>*</td><td>271,67 ± 30,445</td><td>273,62 ± 52,032</td><td>37,92 ± 8,035</td><td>10,37 ± 2,948</td><td>5,27 ± 3,449</td></tr><tr><td>M2</td><td>119,12 ± 43,393</td><td>161,14 ± 36,616</td><td>55,43 ± 20,673</td><td>4,37 ± 1,799</td><td>4,02 ± 1,017</td></tr></tbody></table>

Data represent mean ± SD of four independent biological replicates.
<table><caption>Table S5: Bacterial strains used in this study</caption><thead><tr><th colspan="3">Escherichia coli strains</th></tr></thead><tbody><tr><td>MGE407</td><td>BL21(DE3) fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS λ DE3 = λ sBamH1o ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5</td><td>New England Biolabs</td></tr><tr><td>MGE414</td><td>BL21 pET11b-pleD</td><td>This study</td></tr><tr><td>MGE417</td><td>BL21 pET11b-pleD<sub>E370Q</sub></td><td>This study</td></tr><tr><td>MGE463</td><td>BL21 pET28a-divJ<sub>1-246</sub></td><td>This study</td></tr><tr><td>MGE472</td><td>BL21 pET28a-pleD<sub>AT</sub></td><td>This study</td></tr><tr><td>MGE560</td><td>BL21 pET28a-pleD<sub>SM</sub></td><td>This study</td></tr><tr><td>MGE475</td><td>BL21 pET28a-dgcZ</td><td>This study</td></tr><tr><td>MGE476</td><td>BL21 pET28a-wspR</td><td>This study</td></tr><tr><td>MGE484</td><td>BL21 pET11b-pleD<sub>R300A</sub></td><td>This study</td></tr><tr><td>MGE485</td><td>BL21 pET11b-pleD<sub>R300K</sub></td><td>This study</td></tr><tr><th colspan="3">Caulobacter crescentus strains</th></tr><tr><td>MGC001</td><td>NA1000</td><td>Evinger, M. & Agabian, 1977</td></tr><tr><td>MGC477</td><td>NA1000 ΔpleD::tet</td><td>Skerker et al. 2005</td></tr><tr><td>MGC533</td><td>NA1000 Δrel</td><td>Ronneau et al, 2016</td></tr><tr><td>MGC015</td><td>NA1000 pBVMCS-4-proE</td><td>This study</td></tr><tr><td>MGC016</td><td>NA1000 pBVMCS-4-proE<sub>E328A</sub></td><td>This study</td></tr><tr><td>MGC564</td><td>NA1000 Δrel pBVMCS-4-proE</td><td>This study</td></tr><tr><td>MGC565</td><td>NA1000 Δrel pBVMCS-4-proE<sub>E328A</sub></td><td>This study</td></tr><tr><td>MGC636</td><td>NA1000 Δrel ΔpleD</td><td>This study</td></tr></tbody></table>
<table><caption>Table S6: Plasmids used in this study</caption><tr><td>MGE477</td><td>pNTPS138-ΔspoT</td><td>Ronneau et al, 2016</td></tr><tr><td>MGE351</td><td>pBVMCS-6</td><td>Thanbichler et al, 2007</td></tr><tr><td>MGE003</td><td>pBVMCS-4-proE</td><td>Duerig et al, 2009</td></tr><tr><td>MGE004</td><td>pBVMCS-4-proE<sub>E328A</sub></td><td>Duerig et al, 2009</td></tr><tr><td>MGE409</td><td>pET11b-pleD</td><td>Paul et al, 2004</td></tr><tr><td>MGE412</td><td>pET11b-pleD<sub>E370Q</sub></td><td>Paul et al, 2007</td></tr><tr><td>MGE406</td><td>pET28a</td><td>Novagen</td></tr><tr><td>MGE506</td><td>pNTPS138-pleD</td><td>This study</td></tr><tr><td>MGE462</td><td>pET28a-divJ<sub>1-246</sub></td><td>This study</td></tr><tr><td>MGE470</td><td>pET28a-pleD<sub>AT</sub></td><td>This study</td></tr><tr><td>MGE473</td><td>pET28a-dgcZ</td><td>This study</td></tr><tr><td>MGE474</td><td>pET28a-wspR</td><td>This study</td></tr><tr><td>MGE481</td><td>pET11b-pleD<sub>R300A</sub></td><td>This study</td></tr><tr><td>MGE482</td><td>pET11b-pleD<sub>R300K</sub></td><td>This study</td></tr><tr><td>MGE643</td><td>pBVMCS-6-dgcZ</td><td>This study</td></tr></table>
<table><caption>**Table S7: Oligonucleotides and synthetic DNA sequences used in this study**</caption><tr><td>MGP 730</td><td>gcccatggtatatctcct</td></tr><tr><td>MGP 817</td><td>aatgggtcgcgatcccgcacgcacggagg</td></tr><tr><td>MGP 821</td><td>aatgggtcgcgatcccgcctgaccgccgctatc</td></tr><tr><td>MGP 841</td><td>ggatagtccaagccgtggg</td></tr><tr><td>MGP 842</td><td>gatcggtatacgggctggc</td></tr><tr><td>MGP 855</td><td>tgagatccggctgctaacaag</td></tr><tr><td>MGP 856</td><td>agatataccatgggcatgacggcgagagttctggt</td></tr><tr><td>MGP 857</td><td>tagcagccggatctcagtggtgggtggtggtggtgcgcggcggtacgac</td></tr><tr><td>MGP 858</td><td>agatataccatgggcatgatcaagaagacaacggaaattgatgc</td></tr><tr><td>MGP 859</td><td>tagcagccggatctcagtggtgggtggtggtggtgtaactcggttaatcacattttgttcgtc</td></tr><tr><td>MGP 860</td><td>agatataccatgggcatgcacaaccctcatgagagcaagaccgacctggg</td></tr><tr><td>MGP 861</td><td>tagcagccggatctcagtggtgggtggtggtggccgccggggccggcgga</td></tr><tr><td>MGP 867</td><td>catatgcgttcctcgcatc</td></tr><tr><td>MGP 871</td><td>cggcgactacaaggaccacgacatcgactacaaggacgacgacaagtagctcgaggtcaggtatcg</td></tr><tr><td>MGP 872</td><td>tgtcgtggtccttgtagtcgccgtcggtgcctttagtcaactcggttaatcacattttgttcgtc</td></tr><tr><td>MGP 873</td><td>gaggaaacgcatatgatcaagaagacaacggaaattgatgc</td></tr><tr><td>MGP 899</td><td>agatataccatgggcatgactgcgcgcacatcctgcgtc</td></tr><tr><td>MGP 900</td><td>tagcagccggatctcagtggtgggtggtggtggtgggcagcggcagcgacgac</td></tr><tr><td>MGP 1013</td><td>gtcggcaaggccgcccatac</td></tr><tr><td>MGP 1014</td><td>ggtcgggtacggccagctc</td></tr></table>

### pleD<sub>R300A</sub> gBlock (IDT)

ggcggccttgccgaccaccgcgttccgacccgaggccttggcctgataaacgccttcgtcggcgcgcttgagcagggctt cgggctgtcgcgcctcgcccgccgtggccgagacgccgatcgagatggtgacgttcagcatttcgcggccatgggcac cgtaagggcgagccggagacatgcacccgatccgctcggcgatgcgcagggcgtcagccagggcgtgtcgggc atgatcaccacgaactcttccccgccatagcggaagcagatcaatggcggacgttcgaggccagacgcaaggc gaactgcgcagcacctcgtcgcgcgatatcgtgaccgaaggtgtcgttgattttcttgaagaaatcgatgtcgatcagcagg gccgaaaccggatgcgccccagtgtcgcgcgcttgaccagcgagtcgagctgaccggtcatgtagcgggcattgtgca ggccggtcagctggtcggtgacggcca

### pleD<sub>R300K</sub> gBlock (IDT)

ggcggccttgccgaccaccgcgttccgacccgaggccttggcctgataaacgccttcgtcggcgcgcttgagcagggctt cgggctgtcgcgcctcgccgcgcgtggccgagacgccgatcgagatggtgacgttcagcatttcgcggccatgggcac cgtaagggcgagccggagacatgcacccgatccgctcggcgatgcgcagggcgtcagccagggcgtgtcgggc atgatcaccacgaactcttccccgccatagcggaagcagatcaatggcggacgttcgaggccagacgcaaggc gaactgcgcagcacctcgtcgcgcgatatcgtgaccgaaggtgtcgttgattttcttgaagaaatcgatgtcgatcagcagg gccgaaaccggatgcgccccagtgtcgcgcgcttgaccagcgagtcgagctgaccggtcatgtagcgccttattgtcag gccggtcagctggtcggtgacggcca