# A toxin/antitoxin system targeting the replication sliding-clamp induces competence in Streptococcus pneumoniae.

Maziero Mickaël, Dimitri Juillot, Isabelle Mortier-Barrière, Rut Carballido-Lopez, Nathalie Campo, Pierre Genevaux, Patricia Bordes, Patrice Polard, Mathieu Berge

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Maziero Mickaël, Dimitri Juillot, Isabelle Mortier-Barrière, Rut Carballido-Lopez, Nathalie Campo, et al.. A toxin/antitoxin system targeting the replication sliding-clamp induces competence in Streptococcus pneumoniae.. 2025. hal-05238647

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1 **A toxin/antitoxin system targeting the replication sliding-clamp**
2 **induces competence in Streptococcus pneumoniae.**

3 Maziero Mickaël¹, ², Juillot Dimitri³, Mortier-Barrière Isabelle¹, ², Carballido-Lopez Rut³,
4 Campo Nathalie¹, ², Genevaux Pierre¹, ², Bordes Patricia¹, ², Patrice Polard¹, ²* and Mathieu
5 Bergé¹, ²*.

6 ¹ Laboratoire de Microbiologie et Génétique Moléculaires (LMGM), Centre de Biologie
7 Intégrative (CBI), CNRS, Toulouse, France.

8 ² Université de Toulouse, Toulouse, France.

9 ³ Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350, Jouy-en-Josas, France
10 *Corresponding authors

11 **Abstract:**

12 *Streptococcus pneumoniae* is a pathogenic bacterium capable of entering a cellular
13 differentiation state, called competence, which enables it to acquire new genetic functions by
14 natural transformation, as well as physiological functions such as tolerance to a number of
15 antibiotics. The transition to this state is regulated by various environmental or intracellular
16 signals that converge on the *comCDE* operon, which groups together the competence
17 initiation genes. A fraction of activated cells is sufficient to propagate competence to the
18 whole population via the product of the *comC* gene, the competence stimulating peptide
19 (CSP). Remarkably, depletion of the essential ClpX/ ClpP AAA+ protease has been shown to
20 induce the *comCDE* operon.
Here we demonstrate that the ClpX-dependent induction of competence relies on the Spr1630 toxin (RipA), part of a Rosmer toxin-antitoxin system. We show that this toxin generates replicative stress by acting on the sliding clamp of replication, inducing transcription of the *comCDE* operon. Bacteria that produce RipA appear to lose their viability but remain metabolically active and able to produce CSP, thereby transferring competence to viable neighbouring cells.

## Authors' summary:

The environment in which bacteria live puts them under a great deal of stress, forcing them to adapt constantly, either temporarily or permanently. *Streptococcus pneumoniae*, a pathogenic bacterium implicated in various pathologies such as otitis, meningitis and pneumonia, is also subject to stress, whether from its host, antibiotic treatments or the microbiota in which it lives. In response to this, *S. pneumoniae* is able to switch to a differentiated state called competence. This allows it to acquire new genetic characteristics through natural transformation, but also to better tolerate stresses such as antibiotics pressure.

The underlying signals and signaling pathways of this phenotypic switch remain poorly characterized. In this study, we identified a novel toxin-antitoxin system that, when activated, causes a subset of the population to self-sacrifice by disrupting its own DNA replication. This self-induced arrest serves as a signal that promotes the transition to competence in neighboring cells, thereby improving the capacity for adaptation at the populational level.

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## Introduction:

Streptococcus pneumoniae is a commensal bacterium of the human nasopharynx, but can become an invasive or non-invasive pathogen leading to a wide range of diseases such as otitis, pneumonia, meningitis [1,2]. The emergence of antibiotic resistance in S. pneumoniae is making treatment more complex, and led to the deaths of around 600,000 people in 2019 [3]. This is one of the reasons why S. pneumoniae was ranked among the 12 priority pathogens by the World Health Organization in 2017. Several studies have strongly suggested that the emergence and rapid spread of antibiotic resistance is due to the ability of S. pneumoniae to undergo natural transformation [4].

Natural transformation is a horizontal gene transfer mechanism that enables bacteria to capture exogenous DNA and integrate it into their chromosomes through homologous recombination. First identified in S. pneumoniae [5,6], then in a wide range of bacteria, this mechanism has been relatively well characterized at the molecular level in a variety of bacteria [7–9]. In S. pneumoniae, most of the proteins required for natural transformation are only expressed during a period when the bacterium enters a particular physiological state called competence. The induction of competence is regulated by the level of transcription of two operons, comCDE and comAB. The comC gene encodes for a peptide [10] which is exported and matured by the ABC-transporter ComAB; the externalized product is called *Competence Stimulating Peptide (CSP)*. CSP is able to activate the ComDE two component system [11]. In turn, phosphorylated ComE activates transcription of the comAB and comCDE operons as part of the early competence regulon, inducing an autocatalytic loop [11,12]. Two waves of genes are then successively induced: late and delayed competence genes [13,14], resulting in ~14% of the transcriptome being modified during competence.
The transition to competence is initially an individual behavior and when a sufficient fraction of the population has reached competence, the entire population is converted to competence following transmission of CSP via cell contact [15] by the self-inducing fraction [16]. The time required for this inductive fraction to appear is highly dependent on the stimuli received individually by the cells, which will affect the transcription of the early *comCDE* operon. Several stresses are known to promote competence, such as drugs that alter replication or genome integrity [17–19], temperature [20,21] and ribosome decoding [22]. At least two transduction pathways from these signals to *comCDE* have been described [20], but it is clear that not all pathways have been identified, and those that have remain poorly characterized. Early on in the molecular characterization of competence, it was noted that ClpP appeared to repress its development [23]. The *clpP* gene encodes the proteolytic subunit that forms the Clp ATP-dependent protease complex, together with an ATPase subunit ClpC, ClpE, ClpL, or ClpX. Systematic CRISPRi (clustered regularly interspaced short palindromic repeats interference) of *clpP* or any of the genes encoding the ATPase subunits demonstrated that only depletion of ClpP or ClpX induced competence development [24]. It was logically proposed that ClpX is the main ATPase implicated with ClpP to repress competence. Interestingly, ClpX is the only ATPase subunit of the ClpP complex that is essential for S. pneumoniae viability [25]. The essentiality of *clpX* was shown to be due to the presence of the gene *spr1630* which is proposed to encode the toxin of a so far uncharacterized toxin-antitoxin (TA) system [26].

The aim of the present work was to characterize the molecular pathway inducing competence under the control of ClpXP. Using genetic approaches, we demonstrated that competence induction by depletion of ClpX but not ClpP is dependent on *spr1630*. We demonstrated that Spr1630 is able to induce competence alone. We experimentally demonstrated that Spr1629
antagonizes the effects of Spr1630 leading us to propose that Spr1630 and Spr1629 form a
toxin-antitoxin system (TA) that belongs to the large Rosmer TA family. Furthermore, our data
strongly suggest that Spr1630 targets DnaN, the sliding clamp in DNA replication, and as such
we propose to rename this TA system RipAB (Replication Interfering Protein). Finally, we place
the impact of this TA system in the population context of the onset of competence and show
that production of the RipA toxin gives rise to an inducing fraction that acts as a group of
sentinels that warn the rest of the population of a stress by propagating competence
development.

## Results:

### Competence induction by *clpX* depletion is RipA-dependent.

We first sought to reproduce the induction of competence by transcriptional depletion of *clpX*
or *clpP* as described previously [23,24]. To monitor the development of competence, we used
a transcriptional fusion of the *comCDE* early operon promoter with the luciferase gene as a
reporter [17,27]. We then constructed strains carrying constructions that allow Clustered
Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) in *S. pneumoniae* [24].
Briefly, the defective *Streptococcus pyogenes* Cas9 (dCas9) controlled by an IPTG-dependent
promoter was integrated into the *S. pneumoniae* chromosome together with the *lacI* gene and
a single-guide RNA (sgRNA), targeting *clpX* or *clpP*, under the control of constitutive promoters
[24]. When these cells were cultured in an unfavourable environment for the spontaneous
development of competence (pH 7.0), no changes in the transcription of the *comCDE* operon
were observed (Fig 1A, black lines). The addition of IPTG induced a strong increase in luciferase
production reflecting the induction of transcription of the *comCDE* operon in either ClpX and
112 ClpP depletion (Fig 1A, red lines), suggesting that the depletion of ClpX or ClpP is a competence trigger, as previously published [24]. Notably, the increase of *comCDE* transcription was concomitant with a reduction in growth, which reports on the depletion of the Clp proteins.
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115 Since the biological essentiality of ClpX has previously been linked to the *ripA* gene [26], we explored the possibility that RipA is also involved in triggering competence. As expected, deletion of *ripA* significantly improved growth of ClpX depleted cultures (Fig 1B). Importantly, competence induction was completely abolished in the ClpX depleted culture lacking *ripA* (Fig 1B, *top left panel*), indicating that competence depends solely on RipA when ClpX is depleted.
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120 In an interesting way, and in contrast to the ClpX depletion background, ClpP depletion was still able to trigger competence in the *ripA* null mutant strain (Fig 1B, *top right panel*), suggesting that ClpP dependent induction of competence is not functionally related to RipA.
121
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123 However, as the absence of *ripA* consistently enhanced growth of the ClpP-depleted strain (Fig 1B, *bottom right panel*), we cannot rule out the involvement of ClpP in RipA toxicity management. Altogether, these results confirmed the link between ClpX and RipA, and revealed a ClpX-dependent role of RipA in competence induction.
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125
126

127 The RipA toxin induces competence and is antagonized by the RipB antitoxin.

128 To test the ability of RipA to induce competence, we produced the protein under the control of an IPTG inducible promoter [28]. To ensure tight repression of the IPTG-sensitive promoter, a second copy of the *lacI* gene driven by the constitutive PF6 promoter was integrated into the *S. pneumoniae* chromosome [24]. As *ripA* is part of an operon together with the essential *ripB* gene (*spr1629*) (Fig 2A), we also constructed strains containing the whole operon or *ripB* alone under IPTG induction. Fig 2B shows that expression of *ripA* induces competence development in an IPTG-dependent manner whereas *ripB* expression does not. Interestingly,
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134
expression of the *ripAB* operon does not lead to competence development either, suggesting that *ripB* expression inhibits the action of RipA. We also note that *ripA* expression alone causes growth retardation in correlation with IPTG concentration and again co-expression of *ripB* impairs this growth defect (Fig 2B). We confirmed this by assessing the growth capacity of these strains on agarose medium in presence of increasing IPTG concentrations (Fig 3AB). From 10 µM IPTG, the strain expressing only *ripA* began to lose viability. Growth ability was almost completely lost from 20 µM IPTG. Production of *ripB* in operon with *ripA* completely cancelled out this toxicity. To test whether RipB could exert its protective effect in trans, we expressed *ripA* and *ripB* under IPTG inducible promoters, but at two different chromosomal loci. In this condition, RipB again antagonised the effect of RipA (Fig 3C). These results are consistent with the idea that RipA and RipB form a TA system as previously proposed [26]. We next tested whether RipA could be responsible for the previously observed essential nature of *ripB* [29] by attempting to delete *ripB* in different genetic backgrounds. Transformation results shown in Fig 3D demonstrate that *ripB* deletion could be obtained only when *ripA* was absent, further supporting that RipAB is a bona fide TA system in *S. pneumoniae*. Since RipA is responsible for the essentiality of both *clpX* [26] and *ripB* in *S. pneumoniae*, these data suggest that both proteins are controlling RipA activity.

## Suppressor mutations in *dnaN* antagonize RipA toxicity

In search for RipA potential target(s), we took advantage of a *S. pneumoniae* interactome study based on yeast two-hybrid (Y2H) approach, which suggests that RipA interacts with DnaN [30]. As DnaN encodes the sliding clamp, an essential element in DNA replication, we considered that DnaN could be the target of RipA. To test this hypothesis, we searched for genetic suppressors of RipA toxicity in the *dnaN* locus. To this end, the strain producing RipA
under the control of an IPTG-inducible promoter was transformed by error-prone ~ 4kb PCR
fragments centred on the *dnaN* locus and plated on agar medium supplemented with 20 µM
IPTG. Interestingly, transformation with error-prone *dnaN* PCR fragments resulted in a higher
number of colonies (about tenfold) compared to non-transformed control cells (Fig 4A).
Sequencing the 4kb *dnaN* region of these transformants revealed that all mutations were
located in the *dnaN* gene (Fig 4B). Seven strains with single mutations were obtained, leading
to seven different amino acid substitutions affecting 5 different residues of DnaN (H183, L185,
Y249, Y333, L371). Remarkably, these five residues are clustered in the same region of the
DnaN structure (Fig 4B), a hydrophobic pocket implicated in DnaN protein-protein interactions
[31]. We then characterized the ability of these mutations to suppress the toxicity linked to
increased production of RipA (Fig 4C). We found that, the majority of the mutations allow
significant growth at IPTG concentrations similar to the chosen selection threshold (20 µM
IPTG). Beyond this concentration, their effect diminishes drastically as *ripA* transcription
increases. However, two mutations (Y249N) and (Y333H) enable growth at the highest IPTG
concentration tested (200µM IPTG). This suggests that the various mutations do not exhibit
the same ability to prevent toxicity of RipA. We have therefore categorised them as strong
(Y249N, Y333H), intermediate (L185R) or weak (H183Q, H183L, H183P, L371F) suppressors.

DnaN substitutions impair DnaN-RipA interaction in yeast two-hybrid assay.

Since RipA interacts with DnaN in yeast two-hybrid assays (Y2H) [30], we investigated whether
the single nucleotide polymorphism suppressive mutations in *dnaN* alter this interaction. We
decided to test this interaction with one of the strongest suppressive mutations (Y249N), as
well as a representative of the weaker mutations (H183P), and a mutation that displays an
intermediate suppressive phenotype (L185R). First, we detected an interaction between DnaN
181 with itself as expected, but also for RipA with itself, suggesting a potential dimerization of the
182 protein RipA (Fig 5A). We also reproduced the Y2H interaction between RipA and DnaN in both
183 directions of interactions. However the RipA:DnaN interaction was completely abolished
184 whatever the suppressive mutation analysed, (Fig 5B) suggesting a loss or reduction of
185 physical interaction between RipA and DnaN proteins that express suppressive mutations.
186 Together, these data suggest that RipA targets DnaN through a physical interaction.

187 DnaN substitutions impair competence development mediated by RipA.

188 We next investigated whether the interaction between RipA and DnaN was responsible for
189 the induction of competence in cells overexpressing RipA. To answer this, we tested the
190 induction of competence in strains carrying the three categories of suppressive mutations
191 described above. All strains carrying a suppressive mutation showed better growth in liquid
192 medium compared to the reference strain in IPTG+ conditions (Fig 6A). Mutant strains
193 displayed a gradation in the suppressive effect of the toxicity, as observed in spot test assays
194 (Fig 4C). Thus, the strain carrying the most effective suppressive mutation (DnaN<sup>Y249R</sup>) did not
195 show any growth problems whatever the IPTG concentration. Conversely, the weakest
196 suppressor mutant (DnaN<sup>H183P</sup>) remained quite strongly impacted at the highest RipA
197 production level. The DnaN<sup>L185R</sup> mutant exhibited an intermediate growth behaviour (Fig 6A).
198 Overall, competence induction of the mutant strains was strikingly impaired compared to the
199 WT reference strain (Fig 6A). However, enlarging the *comCDE* transcription observation scale
200 by a logarithm (Fig 6B) revealed a gradation in behaviour during the development of
201 competence in the three representative mutant strains. Indeed, DnaN<sup>Y249R</sup> never induced
202 competence whatever the concentration of IPTG whereas, DnaN<sup>H183P</sup> and DnaN<sup>L185R</sup> strains
203 only displayed a residual competence burst at high ITPG concentration (Fig 6B). It is interesting
to note that the ability to trigger competence appears inversely proportional to the categorisation proposed to describe the ability to withstand the toxicity of RipA.

206 "Doomed cells" are able to promote competence of naive cells.

207 All the above elements support a link between the toxicity of RipA and its ability to induce competence. To test this, we compared the toxicity and competence induction kinetics. When 208 RipA-producing cells were observed by microscopy, it was clear that bacterial multiplication 209 was affected after 90 minutes of IPTG exposition, but that cell integrity was maintained for 30 210 additional minutes (Fig 7A). To complete this observation, we evaluated the ability of the same 211 culture to generate colonies on IPTG-free agarose medium after different RipA exposure 212 duration. Fig 7B shows that most cells were unable to generate colonies after 45 minutes of 213 exposure. However, as competence development occurred after about 120 minutes of RipA 214 toxicity exposure (Fig 2B), we wondered about the physical state of these cells. To test 215 whether these cells are physiologically active, at least regarding competence regulation, we 216 incubated RipA-producing cells under the control of IPTG for more than 60 minutes, with 217 synthetic CSP to trigger rapid competence induction in the culture. These cells carrying the 218 comC::luc reporter gene were still able to respond to CSP at a higher level than unstressed 219 cells that did not produce RipA (Fig 7C). We then tested if RipA-exposed cells were also able 220 to produce enough CSP to propagate competence to naive cells. For this, we grew a strain 221 expressing ripA under IPTG promoter but lacking the comC::luc reporter for 120 minutes in 222 medium containing IPTG to allow induction of RipA-mediated competence. As a negative 223 control, we also used a strain with the same background, but unable to produce CSP (comCO 224 strain). After the allotted time, these cells were mixed (1:1 ratio) with naive wild-type cells 225 carrying the comC::luc reporter (strain R825). Almost instantaneously, wild-type cells switched 226
227 to competence when mixed with RipA stressed cells capable of producing CSP, whereas no
228 change was observed for wild-type cells alone or mixed with *comCO* cells (Fig 7D). These
229 observations were complemented by measuring the ability of strain R825 to transform. Under
230 these conditions, transformation can only occur in the presence of cells that produce *ripA* and
231 are capable of producing CSP (Fig 7D, bottom panel). Altogether these results show that the
232 RipA-producing cells, although rapidly losing their viability, remain metabolically active and
233 produce sufficient CSP to propagate the competence to naive cells and promote their ability
234 to transform.

235 Discussion:

236 ClpX and ClpP use independent pathways to repress competence.

237 We found that the RipA toxin is responsible for the induction of competence generated by
238 ClpX depletion but not ClpP depletion. Since deletion of *ripA* slightly improves the growth of a
239 *clpP* strain, it is not possible to exclude the possibility of a partial link between RipA and ClpP,
240 but ClpP-mediated induction of competence appears to be almost independent of RipA. It is
241 very likely that ClpP, in association with other accessory proteins like ClpE and ClpC, ensures
242 the homeostasis of several competence-regulating proteins, such as ComX or ComW
243 respectively as previously demonstrated [32] or hypothesized, such as ComE [23]. Liu *et al.*,
244 demonstrated that among the ClpP accessory proteins, only depletion of ClpX by CRISPRi was
245 capable of inducing competence [24]. However, it should be kept in mind that the intensity of
246 protein depletion generated by CRISPRi can be extremely variable from one gene to another
247 or from one guide to another [33]. In addition, the quantity of accessory proteins required to
248

ensure repression of competence can also be extremely variable depending on the protein.

249

Understanding the role of ClpP in competence repression will require further investigation.

250

## How are RipA, RipB and ClpX related?

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As *ripA* is responsible for *clpX* essentiality, it has already been hypothesised that RipA is a toxin that is impaired in its activity by ClpX [26]. We have furthermore shown here that the essential nature of RipB is also linked to the presence of RipA. The C-terminus of RipB protein shares homology with ImmA, a Zn metalloprotease involved in regulation of excision and transfer of the mobile genetic element ICEBs1 [34,35]. The N-terminus contains a transcriptional regulator domain of the XRE-family, sharing homology with the HipB antitoxin. As ectopic expression of *ripA* was impaired by trans-expression of *ripB*, it is reasonable to suggest that RipB acts as an antitoxin and that RipAB belongs to the large Rosmer TA family [36]. However, the RipAB system is not autonomous, since the action of RipB is strictly dependant on *clpX* integrity. This could be explained by different mechanisms. Firstly, we could assume that ClpX somehow modulates the toxin/antitoxin activation cycle. The involvement of ClpX in managing the function of a toxin-antitoxin system has already been reported, but until now its role has been described as activating the degradation of the antitoxin and therefore releasing the toxin [37–39]. In this work, ClpX plays a negative role in the action of the toxin. Other more indirect relationships might also be involved, for example, in a ClpX-deficient genetic context, DnaN appeared to be more sensitive to the action of RipA.

267

## The DnaN sliding clamp is a target of RipA

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Given that RipA toxicity can be counteracted by mutations in the DnaN gene and that the same mutations abolish the Y2H interaction between RipA and DnaN, it is reasonable to propose
that the sliding clamp is the target of RipA. The most obvious molecular action would be that RipA affects the main action of the sliding clamp, by disrupting DNA replication. Replicative stresses are clearly known to be factors that induce competence [16,17,19,40], which is in line with our results showing that RipA production induces competence and that suppressive mutations in *dnaN* strongly antagonise it. However, we cannot rule out the hypothesis that DnaN is an intermediate target of RipA and that its final target would be a partner of the sliding clamp. DnaN is known to act as a hub for interactions between different proteins such as MutL and MutS, DNA polymerases or ligases [41]. The majority of these interactions occur via a hydrophobic pocket localized on the anti-Pol face of the sliding clamp [31]. All the suppressive mutations uncovered in this work are localized in the hydrophobic pocket close to the interaction zone. It is therefore conceivable that RipA disrupts the dynamics of the sliding clamp interactome, thereby inducing perturbations in the replicative machinery.

## Biological role of RipA in S. pneumoniae

Several TA systems affecting replication have been discovered mostly affecting topoisomerases [42,43], but very few directly affect the sliding-clamp [44]. For example, SocB, the toxin of the SocAB type VI TA, was the first toxin described to target the sliding-clamp in *Caulobacter crescentus* [45]. The discovery of its target was also achieved using genetic approaches. The mutations suppressing the action of SocB were located in the same hydrophobic pocket of the sliding clamp as the one suppressing RipA toxicity. In addition, it is interesting to note that SocB induces the SOS response system by altering replication in *C. crescentus*; while in *S. pneumoniae*, a bacterium lacking an SOS response system [45], *ripA* expression induces the development of competence, a physiological state frequently proposed as a replacement for the SOS system [19,40,46,47]. However, consultation of
293 alignment tools did not reveal any sequence or structural homology between SocB and RipA,
294 nor between their respective antitoxins (https://www.rcsb.org/alignment). Taken together,
295 these elements suggest a functional convergence of these TA systems towards a central target
296 of the cell cycle.

297 However, one notable difference needs to be highlighted: while the induction of the SOS
298 system remains an individual behaviour, the induction of competence leads to a population
299 behaviour. In fact, it is well established that certain replicative stresses induce competence in
300 S. pneumoniae and in this way, a fraction of the population subjected to RipA stress could
301 transmit this signal through the production of CSP to the whole of the unstressed population,
302 through a potential sacrificial behaviour (Fig 8). In the end, the switch to competence allows
303 the generation of a heterogeneous population, both physiologically and genetically, allowing
304 the emergence of potentially better adapted individuals [16]. The fate of the inducer cells is
305 of particular interest, as it will provide clues as to the biological signals that activate RipA.
306 Indeed, the transition of these cells to a stasis state could be consistent with the ability of
307 antibiotics to induce competence [17]. In such a model, stressed cells that activate RipA trigger
308 two mechanisms that allow them to transition to a potentially persistent state while enabling
309 other cells to become competent. This could be relevant, given that competence enables the
310 population to tolerate certain antibiotics more effectively [16]. Alternatively, sacrificial
311 behaviour could limit the spread of danger while warning neighbouring cells of potential
312 stress. This behaviour is reminiscent of phage resistance mechanisms, in which TA systems are
313 involved [44,48]. This suggests that we should explore the possibility of a previously unknown
314 link between competence and phages in S. pneumoniae.

315

**Material and methods:**
316 **Strains and growth media.**

317 All *S. pneumoniae* strains used were derived from R800 strain [49] and are listed in the
318 supporting tables file.

319 Standard procedures for transformation and growth media were used [50]. Briefly, pre-
320 competent cells were treated at 37°C for 10 min with synthetic CSP1 (100ng mL⁻¹) to induce
321 competence, then exposed to transforming DNA for 20 min at 30°C. Transformants were then
322 plated on CAT agar supplemented with 4% horse blood and incubated for 120min at 37°C.
323 Transformants were then selected by addition of a second layer of agar medium containing
324 the appropriate antibiotic and incubated overnight at 37°C. Antibiotic concentrations (µg mL⁻¹) used for the selection were: chloramphenicol (Cm), 4.5; kanamycin (Kan), 250; spectinomycin (Spc), 100; streptomycin (Sm), 200, gentamycin (G), 40 and erythromycin (E),
325 326 327 0.1. Unless otherwise described, pre-competent cultures were prepared by growing cells to
328 an OD₅₀nm of 0.1 in C+Y medium (pH 6.8). Then cells were 10-fold concentrated in C+Y
329 medium supplemented with 15% glycerol and storage at -80°C.

330 **Deletion and invalidation mutagenesis.**

331 Deletion or invalidation mutagenesis was based on strand overlap extension (SOE) [51].
332 Briefly, primers MP170 and MP173 were used to generate PCR fragments carrying
333 spectinomycin or kanamycin resistance gene from plasmids pr412 (SpcR) or pr413 (KanR)
334 previously described [52]. The two PCR fragments that flank the integration site of the
335 resistance gene were amplified with the specific primer pairs described in the supporting
336 tables file. These pairs are composed of a primer defining the integration site and carrying the
337 sequence complementary to MP170 or MP173 in its 5' region and a distal primer at around 1
338 to 2 kb. The three PCR amplified fragments were purified and used as template to produce a
339 unique PCR fragment using the two distal primers. The resulting fragment was used to
340 transform a recipient strain as described above. The transformed strains were selected by
341 appropriate antibiotic selection and sequenced (Eurofins genomics).

342 **IPTG-dependent expression platform construction.**

343 SOE was also used to generate expression platforms CEPlac-*spr1630*, CEPlac *spr1629* or
344 CEPlac-*spr1630-1629*. Primers pairs MB293-MB294 and MB295-MB291 were used to amplify
345 an expression platform from strain R3833 previously described [28]. Internal PCR fragments
346 carrying genes of interest were amplified from R800 strain using the following primers pairs
347 MB296-MB297 (*spr1629-1630*), MB296-MB298 (*spr1630*), and MB299-MB297 (*spr1629*). Each
348 of these fragments was used with MB293-MB294 and MB295-MB291 primer pairs as
349 templates to generate a unique PCR fragment with primers MB293 and MB291. This final
350 fragment was used to transform strains of interest as described above (kanamycin selection).

351 The PcepII-lac platform was generated by transferring the Pcep-lac platform to the cpsN locus.
352 Briefly, MM56 and MM57 were used to amplify the platform from strain R5139. Flanking cpsN
353 *loci* were amplified using YA09-MM58 et YA14-MM59 primer pairs with R4631 strain as a
354 template [53]. These three PCR fragments were used as templates to amplify a single PCR
355 fragment using primers YA09-YA14. This fragment was used to transform strain R5198.

356 **CRISPRi depletion**

357 Strains carrying the CRISPRi system were constructed as described previously [24]. Plasmids
358 carrying *lacI* repressor (pPEPY-PF6-*lacI*), Cas9 enzyme (pJWV102-PL-*dCas9*) and expressing
359 interfering sgRNA (pPEPX-P3-*sgRNA luc*) were purchased from addgene. Plasmids pPEPX-P3-
360 sgRNA *clpX* and pPEPX-P3-*clpP* were constructed using the In-Fusion HD cloning kit (Takara)
361 as previously described [24]. Primers OIM134 and OIM135 were used to amplify pPEPX-P3-
362 sgRNA backbone. Primers pairs used to generate the RNA guide, MB551-MB552 (*clpX*), MM54-
363 MM55 (*clpP*), were published previously [24]. Generated plasmids were sequentially
364 integrated into the genome of strain R800 by transformation and homologous recombinaison.

365 Monitoring of growth and luciferase expression.

366 For monitoring operon *comCDE* expression, a transcriptional fusion was used, inserting a
367 *S. pneumoniae* *comCDE* promoter fragment (*Hind*III - *Bam*HI) upstream of the *luc* gene and
368 carried by pR414 plasmid. Homology-dependent integration of the non-replicative
369 recombinant plasmid into the pneumococcal chromosome was selected using erythromycin
370 resistance [52].

371 For the monitoring of growth and luciferase expression, precultures were gently thawed and
372 aliquots were inoculated (unless otherwise described) at OD<sub>550nm</sub> of 0.005 in luciferin-
373 containing C+Y medium (pH 7) as previously described [50] and distributed into a 96-wells
374 (300 µl per well) white microplate with clear bottom (Corning). Relative luminescence units
375 (RLU) and OD<sub>492nm</sub> values were recorded at defined time points throughout incubation at 37°C
376 in a Varioskan luminometer (ThermoFisher).

377 DnaN error prone PCR and screening.

378 DnaN *locus* was amplified from R800 genomic DNA using primers MB313 and MB314 and
379 DreamTaq DNA Polymerase (thermoFisher scientific) as recommended by the supplier with or
380 without adding 25µM of MnCl₂ to reduce the fidelity of the enzyme. The resulting fragments
381 were used to transform strain R5086 as described above. After an incubation of 120min in CAT
medium at 37°C, transformant cells were plated and grown 24 hours on CAT agar
supplemented with 4% horse blood and 20µM IPTG. DnaN locus was amplified on potential
suppressor clones using primers MB313 and MB314 and a high-fidelity DNA polymerase,
Primestar max DNA polymerase (Takara). PCR fragments were used to transform R5086 as
previously described to confirm that suppressive mutations were linked to DnaN locus.
Relevant PCR fragments were sequenced (Eurofins genomics).

3D mapping of suppressive mutations on DnaN.

Suppressive mutations were positioned on 3D DnaN structure from Protein Data Bank (PDB),
www.pdb.org (PDB ID code 2AWA) using Pymol software V0.99.

Yeast Two hybrid.

Gene-coding sequences of S. pneumoniae Spr1630 and DnaN proteins were PCR amplified
using R1501 DNA as template, and inserted in Gal4-based plasmids, PGAD-C1 and PGBD-C1
[54], using the In-Fusion HD cloning kit (Takara). Primers used were MM30 and MM31
(Spr1630), MM28 and MM29 (DnaN) and OCN424 and OCN425 (PGAD-C1 and PGBD-C1).

Resulting plasmids were then transformed independently in the yeast strains PJ69-4a and
PJ69-4α. Saccharomyces cerevisiae cells expressing S. pneumoniae proteins as GAL4 Binding
Domain (BD) fusions were mated with cells expressing some of these proteins as GAL4
Activating Domain (AD) fusions. Binary interactions were identified by growth of diploid cells
after 8 or 20 days at 30°C on synthetic complete medium [55] lacking leucine, uracil and
histidine (to select expression of the HIS3 interaction reporter). Controls with empty vector
plasmids (i.e., carrying only the BD or AD domain) were systematically included.
403 **Mixed culture Assay**

404 Strains R5204 and R5086 were inoculated at 0.04 OD₅₀₅nm in C+Y medium and grown in the
405 presence of 80 µM of IPTG for 120 minutes. In parallel R825 strain was inoculated at 0.04
406 OD₅₀₅nm in C+Y medium (pH 7) and grown for 60 minutes. Cells R5204 or R5086 were mixed
407 with R825 (volume to volume) to an expected ratio of 1 to 1. R825 luciferase activity was then
408 monitored. PCR amplified DNA carrying *rpsL41* allele (Streptomycin resistance) was added at
409 the time of mixing. 100 minutes after mixing a culture sample was collected and plated on
410 agar medium supplemented with erythromycin and streptomycin to determine the
411 percentage of transformation of strain R825.

412 Statistical tests:

413 Pairwise comparisons were done with a nonparametric MannWhitney test. P values were
displayed as follows: ***, 0.0001 < P < 0.001; **, 0.001 < P < 0.01; *, 0.01 < P < 0.05; ns,
P > 0.05.

416 Acknowledgements.

417 We would like to extend our special thanks to Calum Johnston and Hélène Cordier for their
critical review of the manuscript.

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**Figure Legends.**
Fig. 1: Depletion of *clpX* or *clpP* induces competence.

579 A. *comCDE* expression was monitored in strains carrying CRISPRi guide RNA targeting *clpX*
580 R4993 (left) or *clpP* R5203 (right). Cells were grown in C+Y medium at 37°C with or without
581 IPTG, as shown in the colour key. Culture was initiated 100 minutes prior to the first
582 measurement at time 0. *comCDE* expression values are expressed in relative light units per
583 OD (RLU/OD) in the top panel and the corresponding growth curves are reported as OD<sub>492nm</sub>
584 in the bottom panel.

585 B. Identical to A in Δ*ripA* strains carrying CRISPRi guide RNA targeting respectively 4995 (*clpX*
586 depletion, left) and R5250 (*clpP*, right).

587 For the sake of clarity, only a single data set, representative of at least three independent
588 experiments carried out on different days, is presented.

589

Fig 2: *ripA* expression induces competence development

591 A. Schematic representation of *ripA-ripB* locus and its chromosomal surroundings.

592 B. *comCDE* expression was monitored in strains expressing *ripA* (R5139), *ripB* (R5138) or *ripA-ripB* (R5140) under the control of an IPTG inducible promotor. Cells were grown in C+Y medium at 37°C with increasing concentration of IPTG from the first measurement at time 0.
593
594 Top panels: luciferase activity expressed in relative light units per OD (RLU/OD). Bottom
595 panels: corresponding growth curves.

597

Fig 3: Trans-dependant suppression of RipA toxicity by RipB.
599 A. Strains expressing *ripA* (R5139), *ripB* (R5138) or *ripA-ripB* (R5140) under the control of an
600 IPTG inducible promotor were serially diluted and spotted on CAT agar supplemented with 4%
601 horse blood containing different concentrations of IPTG. Plates were incubated at 37 °C
602 overnight.

603 For clarity, only a single spot test is presented and also represented as a histogram form in B
604 to reflect the efficiency of plating. Three independent spot tests were carried out on different
605 days displaying same results.

606 C. Same as B. for strains expressing *ripA* under the control of *CepII*<sub>Iac</sub> IPTG inducible promotor
607 at the *cps* *locus* (see material and methods), (R5239 and R5259). In strain R5259, *ripB* is
608 expressed under the control of *CEP*<sub>Iac</sub> IPTG inducible promotor at the "*ami locus*".

609 D. Percentage of transformation of strain wild type (wt) (R800) or invalidated for *ripA* (R4796)
610 with DNA carrying *ripB* invalidation (spectinomycin insertion). In the control, these two strains
611 were transformed with DNA carrying a point mutation conferring resistance to
612 streptomycin(*rpsL41*).

613 **Fig 4: Mutations in DnaN confer different levels of resistance to RipA toxicity.**

614 A. Strain R5086, expressing *ripA* under the control of an IPTG inducible promotor was transformed
615 with different error prone PCR fragments amplified from *dnaN locus* (see material and methods).

616 Transformant cells were plated on CAT agar supplemented with 4% horse blood containing 20µM
617 IPTG. CFU were numerated after overnight incubation at 37 °C.

618 B. Top panel. Schematic representation of the *dnaN locus* and its environment amplified by
619 primers MB313 and MB314. Red vertical lines indicate the position of mutated residues that
suppress RipA toxicity. Positioning of the different suppressive mutations on the primary structure (middle panel) and on the tertiary 3D structure of DnaN (bottom panel).

C. Efficiency of plating of strains expressing *ripA* under the control of an IPTG inducible promotor in a *dnaN* wild type genetic background (R5139) or in a DnaN suppressor mutations genetic background (R5165 to R5171). The strain expressing *ripA-ripB* under the control of IPTG was used as a growth control (R5140). Cells were plated on CAT agar supplemented with 4% horse blood containing different concentrations of IPTG. For clarity, only a single spot test is presented as a histogram. Three independent determinations were made on different days displaying same results.

**Fig 5: Suppressor mutations in DnaN reduce interactions with RipA in Yeast-two-hybrid system.**

A. Yeast-two-hybrid matrices produced to test interactions between RipA and DnaN.

B. Yeast-two-hybrid matrices performed to test interactions between RipA and DnaN suppressive alleles.

**Fig 6: Suppressor mutations in DnaN impair RipA dependant competence development.**

A. *comCDE* expression was monitored in strains expressing *ripA* under the control of an IPTG inducible promotor in a wild type *dnaN* genetic background (R5138) or in strains carrying suppressive alleles of dnaN, H183P (R5167), L185R (R5168), Y249N (R5169). Cells were grown in C+Y medium at 37°C with
641 increasing amounts of IPTG from the first measurement at time 0. Top panels: luciferase activity
642 expressed in relative light units per OD (RLU/OD). Bottom panels: corresponding growth curves.

643 B. zoom in on transcription induction curves (A) in strains carrying suppressor mutations.

644 **Fig 7: RipA toxicity allows propagation of competence to naive cells.**

645 A. Phase contrast time-lapses of the strain expressing *ripA* under the control of an IPTG
646 inducible promotor (R5139) without or with 80µM of IPTG (respectively top and bottom
647 panels).

648 B. Ability of R5139 strain to generate colony after IPTG exposure. R5204 strain was exposed
649 or not to 80µM of IPTG for different times (X-axis), washed with fresh medium and plated on
650 Agar medium without IPTG.

651 C. Responsiveness of R5139 strain to CSP after one hour exposition or not to IPTG.

652 D. Mixed culture of strains expressing *ripA* under the control of an IPTG inducible promotor,
653 R5204 (*comC*⁺) or R5086 (*comC*₀), with R825 strain used as competence reporter cells
654 through its *PcomCDE::luc* construct. Top panels: luciferase activity of R825 strain alone (grey)
655 or mixed with R5204 (red) or R5086 (blue). Middle panels: corresponding growth curves. For
656 clarity, only a single data set, representative of at least three independent determinations
657 made on different days, is presented.

658 Bottom panel. R825 transformation frequency that occurs during mixed cultures.

659 **Fig 8: Working model of RipA action on competence development at single cell**
660 **and population level in S. pneumoniae.**
A

<figure><img src="image_2.png" /></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2025.09.02.673660; this version posted September 2, 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.

B

<figure><img src="image_3.png" /><figcaption>Figure1</figcaption></figure>
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>Figure2</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 3</figcaption></figure>
A

B

C

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

error prone *dnaN* PCR product
amplified with XµM MnCl2

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

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

<figure><img src="image_10.png" /><figcaption>Figure4</figcaption></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2025.09.02.673660; this version posted September 2, 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.
A

<figure><img src="image_11.png" /></figure>

B

<figure><img src="image_12.png" /><figcaption>Figure5</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>Figure 6</figcaption></figure>
<figure><img src="image_14.png" /></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2025.09.02.673660; this version posted September 2, 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_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

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

<figure><img src="image_18.png" /></figure>
<figure><img src="image_19.png" /><figcaption>Figure 8</figcaption></figure>