Manon Lang

Evelyne Krin

Chloé Korlowski

Odile Sismeiro

Hugo Varet

Jean-Yves Coppée

Didier Mazel
email: mazel@pasteur.fr

Zeynep Baharoglu
email: zeynep.baharoglu@pasteur.fr

Sleeping ribosomes: bacterial signaling triggers RaiA mediated persistence to aminoglycosides

Indole is a molecule proposed to be involved in bacterial signaling. We find that indole secretion is induced by sublethal tobramycin concentrations and increases persistence to aminoglycosides in V. cholerae. Indole transcriptomics showed increased expression of raiA, a ribosome associated factor. Deletion of raiA abolishes the appearance of indole dependent persisters to aminoglycosides, while its overexpression leads to 100-fold increase of persisters, and a reduction in lag phase, evocative of increased active 70S ribosome content, confirmed by sucrose gradient analysis. We propose that, under stress conditions, RaiA-bound inactive 70S ribosomes are stored as so-called "sleeping ribosomes", and are rapidly reactivated upon stress relief. Our results point to an active process of persister formation, through ribosome protection during translational stress (e.g. aminoglycoside treatment), and reactivation upon antibiotic removal. Translation is a universal process, and these results could help elucidate a mechanism of persistence formation in a controlled, thus inducible way.

Introduction

Antibiotic resistance is a major public health concern leading to increased health care costs and mortality [START_REF] Opatowski | Hospitalisations with infections related to antimicrobial-resistant bacteria from the French nationwide hospital discharge database[END_REF][START_REF] Touat | A Payer Perspective of the Hospital Inpatient Additional Care Costs of Antimicrobial Resistance in France: A Matched Case-Control Study[END_REF]. Although the majority of studies address the response of bacteria to lethal doses of antibiotics, the effect of low doses of antibiotics on bacteria has also recently started to draw attention. Antibiotic concentrations lower than the minimal inhibitory concentration (sub-MICs) have historically been proposed to serve as signaling molecules [START_REF] Davies | The world of subinhibitory antibiotic concentrations[END_REF], provoking considerable changes in transcription and triggering a wide variety of cellular responses in different bacterial species [START_REF] Andersson | Microbiological effects of sublethal levels of antibiotics[END_REF] and mutagenesis [START_REF] Gutierrez | beta-lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity[END_REF]. In V. cholerae, sub-MIC aminoglycosides (AGs) are known to activate various stress response pathways, such as the SOS [START_REF] Baharoglu | Identification of genes involved in low aminoglycoside-induced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase[END_REF][START_REF] Baharoglu | Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance[END_REF] and RpoS stress responses [START_REF] Baharoglu | RpoS Plays a Central Role in the SOS Induction by Sub-Lethal Aminoglycoside Concentrations in Vibrio cholerae[END_REF], allowing cells to cope with increased reactive oxygen species (ROS) levels and DNA breaks [START_REF] Negro | Formation and Interrelationships of Tryptophanase and Tryptophan Synthetases in Escherichia Coli[END_REF]. AGs are bactericidal antibiotics that are known to enter the bacterial cell through the proton motive force [START_REF] Fraimow | Tobramycin uptake in Escherichia coli is driven by either electrical potential or ATP[END_REF][START_REF] Herisse | Silver potentiates aminoglycoside toxicity by enhancing their uptake[END_REF][START_REF] Taber | Bacterial uptake of aminoglycoside antibiotics[END_REF]. AGs target the ribosome, leading to mistranslation and eventually cell death [START_REF] Davis | Mechanism of bactericidal action of aminoglycosides[END_REF]. Interestingly, sub-MIC antibiotics, among which AGs, have been shown to stimulate the production of a small molecule, indole [START_REF] Han | Environmental factors affecting indole production in Escherichia coli[END_REF]. Indole is a byproduct of tryptophan degradation by tryptophanase TnaA [START_REF] Evans | The tryptophanase-indole reaction: Some observations on the production of tryptophanase by Esch. coli; in particular the effect of the presence of glucose and amino acids on the formation of tryptophanase[END_REF] in both Gram+ and Gram-bacterial species (Lee and Lee, 2010), together with pyruvate and ammonia. While pyruvate and ammonia are respectively sources of carbon and nitrogen, the role of indole is not well understood. Indole is also found in plants and animals, and was linked with signaling and human diseases (for a review, [START_REF] Lee | Roles of indole as an interspecies and interkingdom signaling molecule[END_REF]). Common indole concentrations in the human gut are in the order of 250-1100 µM, and up to 200 µM in blood and other tissues. Regulation of indole production has been described, namely through carbon source utilization [START_REF] Botsford | Catabolite repression of tryptophanase in Escherichia coli[END_REF]) and catabolic repression [START_REF] Yanofsky | Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli[END_REF], amino acid availability (Newton and Snell, 1965), cold temperature [START_REF] Lee | Indole cell signaling occurs primarily at low temperatures in Escherichia coli[END_REF], heat shock [START_REF] Li | The effect of a single, temperature-sensitive mutation on global gene expression in Escherichia coli[END_REF] and growth phase [START_REF] Kobayashi | Growth phasedependent expression of drug exporters in Escherichia coli and its contribution to drug tolerance[END_REF]. Particularly, indole is produced during transition from exponential to stationary phase [START_REF] Lelong | The Crl-RpoS regulon of Escherichia coli[END_REF]. In E. coli, indole is nontoxic at physiologic concentrations (below 1 mM) [START_REF] Lee | Indole is an inter-species biofilm signal mediated by SdiA[END_REF], and does not change the growth rate [START_REF] Lee | Indole cell signaling occurs primarily at low temperatures in Escherichia coli[END_REF]. At high concentrations however (above 1-3 mM), indole inhibits cell division [START_REF] Chant | Indole signalling contributes to the stable maintenance of Escherichia coli multicopy plasmids[END_REF][START_REF] Chimerel | Indole prevents Escherichia coli cell division by modulating membrane potential[END_REF]. In V. cholerae, indole secretion reaches its maximum at 600 µM during transition from midlog to stationary phase [START_REF] Howard | Indole inhibits ToxR regulon expression in Vibrio cholerae[END_REF][START_REF] Mueller | Indole acts as an extracellular cue regulating gene expression in Vibrio cholerae[END_REF] and was not observed to have any effect on the polarity of the V. cholerae cell membrane at this concentration [START_REF] Mueller | Indole acts as an extracellular cue regulating gene expression in Vibrio cholerae[END_REF]. Indole can pass across the cell membrane without the need for a transporter [START_REF] Pinero-Fernandez | Indole transport across Escherichia coli membranes[END_REF], and was proposed to act as an interkingdom signaling molecule [START_REF] Martino | Indole can act as an extracellular signal to regulate biofilm formation of Escherichia coli and other indole-producing bacteria[END_REF][START_REF] Wang | Indole can act as an extracellular signal in Escherichia coli[END_REF]). An effect of indole in persistence to antibiotics has also been observed. At toxic concentrations (1-2 mM), where indole behaves as a membrane ionophore, it was observed to reduce persistence of stationary phase cultures to tested antibiotics (ciprofloxacin, ampicillin) [START_REF] Hu | Toxin YafQ increases persister cell formation by reducing indole signalling[END_REF]. However, studies in E. coli show that lower concentrations of indole increase survival/persistence to lethal concentrations of ofloxacin, ampicillin and kanamycin [START_REF] Vega | Signaling-mediated bacterial persister formation[END_REF][START_REF] Vega | Salmonella typhimurium intercepts Escherichia coli signaling to enhance antibiotic tolerance[END_REF], suggesting that the protective effect of indole is not specific to one family of antibiotic. Studies also pointed to the involvement of indole secretion in the cooperation between antibiotic resistant and sensitive populations during antibiotic stress (Lee et al., 2010). Notably, a recent study identified indole production as a potential target for the increased activity of quinolones against persisters in E. coli [START_REF] Zarkan | Inhibition of indole production increases the activity of quinolone antibiotics against E. coli persisters[END_REF]. Since indole appears to be beneficial for bacteria in the presence of antibiotics, we addressed whether indole production is increased upon sub-MIC AG treatment in V. cholerae and whether this can lead to improved response to lethal antibiotic concentrations. Importantly, we find that indole strongly increases persistence to AGs through the action of RaiA. We find that transcription from the raiA gene promoter is highly upregulated in the presence of indole in exponential phase V. cholerae cells. RaiA was shown to be a ribosome associated protein, in the same conditions as Rmf (ribosome modulation factor) and Hpf (hibernation promoting factor) [START_REF] Maki | Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli[END_REF]. The two latter factors cause dimerization of vacant 70S into inactive 100S ribosome dimers, in a process called ribosome hibernation during stationary phase [START_REF] Gohara | Survival of the drowsiest: the hibernating 100S ribosome in bacterial stress management[END_REF], whereas RaiA mostly binds to free 70S monosomes [START_REF] Maki | Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli[END_REF][START_REF] Sabharwal | The VrrA sRNA controls a stationary phase survival factor Vrp of Vibrio cholerae[END_REF]. E. coli mutants lacking these ribosome-associated factors do not show any growth defect during exponential growth, which is consistent with the fact that their expression is specific to stationary phase and stress [START_REF] Prossliner | Ribosome Hibernation[END_REF]. RaiA was observed to protect the 70S ribosome from degradation [START_REF] Agafonov | A protein residing at the subunit interface of the bacterial ribosome[END_REF][START_REF] Di Pietro | Role of the ribosome-associated protein PY in the cold-shock response of Escherichia coli[END_REF], and was also observed to block the binding of tRNA to the ribosomal A site in a cell free translation system [START_REF] Agafonov | Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage[END_REF], and during cold shock (Vila-Sanjurjo et al., 2004). In the present study, characterization of the raiA deletion mutant shows that RaiA is instrumental in the appearance of persister cells to AGs. We propose here a new mechanism of induced persistence to AGs by which RaiA positively affects the intact ribosome content of the cell, and facilitates regrowth after removal of the antibiotic.

Results

Indole is produced in response to sub-MIC tobramycin and increases persistence to AGs

Since the antibiotics ampicillin and kanamycin increase indole levels in E. coli [START_REF] Han | Environmental factors affecting indole production in Escherichia coli[END_REF], we measured indole secretion [START_REF] Saint-Ruf | Massive diversification in aging colonies of Escherichia coli[END_REF] in V. cholerae to determine whether the aminoglycoside tobramycin also impacts indole levels in this case. We found increased extracellular indole concentrations in the presence of sub-MIC tobramycin (TOB 0.15 µg/ml, 20% of the MIC, Figure 1). We next addressed whether indole has an impact on the growth of V. cholerae, using an indole concentration of 350 µM. This concentration was previously shown to be physiologically relevant in V. cholerae and was observed to have no inhibitory effect on growth, and to complement the biofilm formation defect of a ∆tnaA mutant deficient for indole production [START_REF] Mueller | Indole acts as an extracellular cue regulating gene expression in Vibrio cholerae[END_REF]. We found that 350 µM indole does not affect growth in the absence of antibiotics, but improves growth in sub-MIC antibiotics tobramycin (Figure S1A).

We next addressed the effect of indole in the response to lethal concentrations of antibiotics, by measuring persister cells formation in V. cholerae. In order to do so, we adapted to V. cholerae, a protocol developed for E. coli (Ivan Matic and Wei-Lin Su, personal communication). Early exponential phase cultures were treated with lethal doses of antibiotics (5 to 10 times the MIC) for 20 hours. We first confirmed that cells surviving after 20 hours of antibiotic treatment and which grow upon antibiotic removal, were indeed persister cells, by performing survival curves (Figure S2A). The biphasic profiles of the killing curves we obtained was consistent with the formation of persister cells after 5 hours [START_REF] Brauner | Distinguishing between resistance, tolerance and persistence to antibiotic treatment[END_REF]. Furthermore, these cells were not resistant to the antibiotic, as no growth was observed when streaked on antibiotic containing plates (not shown). We thus carried on with the quantification of persisters at 20 hours of antibiotic treatment. We found that V. cholerae cultures grown in the presence of indole yielded higher numbers of persister cells to the AGs TOB and gentamicin (GEN) (Figure 2AB), as previously observed for E. coli treated with kanamycin and ofloxacin [START_REF] Vega | Signaling-mediated bacterial persister formation[END_REF]), but we observed no effect for persistence to carbenicillin (CRB), or trimethoprim (TMP) (Figure 2CD). Furthermore, a strain deleted for tnaA yielded less persisters to tobramycin than the WT strain (Figure S2B), and the effect was reversed by indole complementation, consistent with a link between indole and persistence.

Indole, at concentrations allowing growth, does not affect AG entry

We next addressed whether the beneficial effect of indole treatment in the presence of AGs is due to modifications in membrane potential, which could lead to decreased AG entry into the cell. AG uptake by the bacterial cell is known to be linked to the proton motive force (PMF). At high concentrations (5 mM) indole is a proton ionophore which blocks cell division by dissipating the PMF [START_REF] Kralj | Electrical spiking in Escherichia coli probed with a fluorescent voltage-indicating protein[END_REF], and was observed to interact with the cell membrane and change its physical structure [START_REF] Mitchell | Indole adsorption to a lipid monolayer studied by optical second harmonic generation[END_REF]. In order to measure AG entry in the bacterial cell, we used the aminoglycoside neomycin coupled to the fluorophore Cy-5, which was previously synthesized for aminoglycoside uptake studies in bacteria and demonstrated to bear the properties of aminoglycosides for uptake, mode of action and activity against Gram negative bacteria (Sabeti Azad et al., 2020), (Pierlé et al, unpublished). We found that indole at the physiological concentration of 350 µM does not affect AG entry into the bacterial cell (Figure S3A), ruling out the possibility of decreased AG entry due to modifications of PMF in the presence of indole. Moreover, the presence of 350 µM indole did not change the MIC of two aminoglycosides: tobramycin and gentamicin (Figure S3B). The beneficial effect of indole in the presence of AGs is thus not through reduced antibiotic entry.

Indole induces RaiA (VC0706) expression.

In order to shed light into mechanisms allowing for more efficient response to antibiotic stress upon indole treatment, we decided to study the transcriptomic changes of V. cholerae in response to 350 µM indole. mRNA sequencing of exponential phase cultures shows differential regulation of 260 genes shown in Table S1 and represented in Figure 3A (>2-fold change, adjusted p value <0.01 as in [START_REF] Krin | Expansion of the SOS regulon of Vibrio cholerae through extensive transcriptome analysis and experimental validation[END_REF]). The most affected categories were respiration (31 genes upregulated), electron transfer and iron uptake (55 genes downregulated). Indole mediated protection against antibiotic killing was previously proposed to be through upregulation of efflux pumps [START_REF] Blair | Alternative explanation for indoleinduced antibiotic tolerance in Salmonella[END_REF][START_REF] Hirakawa | Indole induces the expression of multidrug exporter genes in Escherichia coli[END_REF][START_REF] Kobayashi | Growth phasedependent expression of drug exporters in Escherichia coli and its contribution to drug tolerance[END_REF]Lee and Lee, 2010;[START_REF] Nikaido | Effects of indole on drug resistance and virulence of Salmonella enterica serovar Typhimurium revealed by genome-wide analyses[END_REF], or through an increase in OxyR associated oxidative stress response [START_REF] Vega | Salmonella typhimurium intercepts Escherichia coli signaling to enhance antibiotic tolerance[END_REF]. However, our RNA-seq data show no induction of oxidative stress response related genes in V. cholerae (oxyR, soxRS, katG) by indole, and rather suggest decreased expression of proteins linked to iron uptake, suggesting decreased iron and ROS levels upon indole treatment [START_REF] Baharoglu | RpoS Plays a Central Role in the SOS Induction by Sub-Lethal Aminoglycoside Concentrations in Vibrio cholerae[END_REF][START_REF] Mehi | Perturbation of iron homeostasis promotes the evolution of antibiotic resistance[END_REF]. The second most affected category belongs to translation related genes (31 genes, approximately 10% of total differentially regulated genes, with p<3.00E-4). Notably, one translation related gene was markedly upregulated: raiA (VC0706, 20-fold up), together with rmf (VC1484, 3-fold up), which are both described as factors associated with inactive ribosomes in stationary phase [START_REF] Agafonov | A protein residing at the subunit interface of the bacterial ribosome[END_REF][START_REF] Di Pietro | Role of the ribosome-associated protein PY in the cold-shock response of Escherichia coli[END_REF]. raiA expression is known to be triggered by transition to stationary phase [START_REF] Maki | Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli[END_REF], and RaiA is usually weakly expressed during exponential phase. RT-qPCR on raiA (Figure 3B) and fluorescence associated flow cytometry on cells carrying GFP fused to the raiA promoter (Figure 4A andS4AB) confirmed upregulation of raiA by indole during exponential phase, and increased expression of raiA in stationary phase. Since transcriptomic data pointed RaiA as one of the most differentially regulated genes by indole, we next decided to address the contribution of RaiA in the indole associated phenotypes in V. cholerae.

Absence of raiA reduces persistence to AGs and abolishes induction by indole.

In order to address the involvement of RaiA in indole induced persistence, we constructed a V. cholerae ΔraiA mutant, and measured the frequency of persistence to tobramycin (TOB), gentamicin (GEN), carbenicillin (CRB) and trimethoprim (TMP) in exponential phase cultures, grown in the absence and presence of indole (Figure 2ABCD). We found that V. cholerae ΔraiA generally formed slightly less persister cells to TOB, although the difference was not statistically significant when we compare persistence frequency, probably because at exponential phase raiA expression is not strong enough in the untreated WT strain, and due to variability between experiments. However, the ratio of persisters calculated separately for each experiment shows significant 10-fold decrease in ΔraiA compared to WT (Figure S5A). Strikingly, the deletion of raiA completely abolished induction of persistence by indole to both tested AGs (TOB, GEN, Figure 2AB), pointing to a role of RaiA in persister cell formation. No effect of neither indole nor raiA was observed in the formation of persisters to CRB or TMP (Figure 2CD), suggesting that the effect of RaiA in persister formation is specific to AGs. The involvement of RaiA in persistence to AGs also appears to be conserved in E. coli as the raiA deficient mutant yielded less persisters to TOB (Figure S5B). Finally, as performed above in the WT V. cholerae strain, we confirmed that the presence of indole does not affect the AGs MIC of the ΔraiA strain, and that deletion of raiA does not affect the MIC and AG uptake (Figure S3AB), meaning that the phenotypes we observe are not due to reduced entry of AGs or increased resistance. On the other hand, RaiA is dispensable for growth improvement by indole (Figure S1B), because indole still improves growth in TOB when raiA is deleted, showing that the mechanism of AG persister induction by indole (antibiotic concentration >MIC) is different than the mechanism of growth improvement in sub-MIC AGs. It is worth mentioning here that the growth of ∆raiA strain appeared to be slightly slower than the WT strain, suggesting that RaiA may also have a role during exponential growth, despite its low level of expression.

RaiA overexpression increases persistence to AGs and promotes earlier exit from stationary phase.

In order to mimic conditions of RaiA induction, we cloned it under a controlled Para promoter, which is repressed by glucose and induced by arabinose. Since the presence of different carbon sources may differentially affect growth and the response to aminoglycosides (Pierlé et al., unpublished), and also because arabinose was shown to have an impact on growth in V. cholerae, we compared the persistence levels of cells carrying the empty vector (p0 in Figure 5ABCDEF) or the pBAD-RaiA plasmid, in the presence of arabinose (RaiA overexpression conditions). Overexpression of RaiA strongly increased persisters formation in three aminoglycosides: TOB, GEN and NEO (Figure 5ABC), but not in three non-aminoglycoside antibiotics from different families: CRB, TMP and ciprofloxacin (CIP) (Figure 5DEF), indicating that RaiA is directly and specifically involved in the persistence mechanism to aminoglycosides. Additionally, the MIC showed no difference between the raiA overexpression strain compared to the strain with empty plasmid p0 and pRaiA+ (MIC = 0.75 µg/ml for both on MH plates containing arabinose), ruling out a potential effect on resistance. We next asked whether such increased persistence could be due to slower growth when RaiA is overexpressed. We monitored growth in conditions where 1) RaiA is not overexpressed previous to inoculation and only overexpressed during the growth curve, and 2) RaiA is previously overexpressed in cells used for inoculation (Figure S6A). First, our results show no difference in growth rate (slope) in presence or absence of RaiA overexpression (Figure 6AD and S6B), indicating that RaiA overexpression does not cause a slow growth phenotype, which discards the hypothesis linking RaiA-mediated persistence to slow growth/dormancy. Interestingly, we observe in cells where RaiA overexpression was pre-induced, that these cells start growing faster due to a reduction in lag phase (Figure 6BC and Figure S6B). Such a reduction in lag phase is reminiscent of increased active ribosome content which allows faster resumption of growth at the exit of stationary phase [START_REF] Condon | rRNA operon multiplicity in Escherichia coli and the physiological implications of rrn inactivation[END_REF]. As a corollary, when raiA was deleted, the lag phase was increased (Figure 7A and Figure S6C), suggesting that RaiA levels affect inactive but "ready to use" ribosome content, which we called sleeping ribosomes, in stationary phase.

The effect of RaiA on lag phase and persistence is independent of ribosome hibernation factors Rmf/Hpf

RaiA was previously found to be associated with the inactive ribosomes at stationary phase, in the same conditions as Rmf and Hpf factors. Rmf and Hpf are known to dimerize ribosomes into so called hibernating 100S ribosomes, whereas RaiA associates with monomeric 70S ribosomes [START_REF] Gohara | Survival of the drowsiest: the hibernating 100S ribosome in bacterial stress management[END_REF][START_REF] Maki | Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli[END_REF][START_REF] Prossliner | Ribosome Hibernation[END_REF]. In order to address whether Hpf/Rmf dependent ribosome hibernation also favors rapid exit from stationary phase, we performed deletion and overexpression experiments similar to what we did for RaiA. Deletion of rmf or hpf has no effect on lag phase nor growth (Figure 7A andS6C). Our results showed that in contrast to overexpression of RaiA which decreases the lag phase, overexpression of Rmf or Hpf rather increase lag phase (Figure 7B andS6C). These findings are consistent with a model where increased ribosome dimerization by Hpf/Rmf would require action of dissociation factors in order to resume growth whereas spontaneous dissociation of RaiA from inactive 70S ribosomes [START_REF] Agafonov | Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage[END_REF][START_REF] Maki | Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli[END_REF], is sufficient for growth restart. Furthermore, when we overexpressed RaiA in ∆hpf or ∆rmf mutants, we observed a reduction of lag phase similar to what is observed upon overexpression of RaiA in the WT strain (Figure 7DC), meaning that RaiA action is not dependent of Hpf/Rmf mediated ribosome hibernation.

In addition, we addressed the effect of hibernation factors on persistence in exponential phase. Unlike for RaiA overexpression, no increase in persistence to TOB was detected upon overexpression of Hpf or Rmf, excluding an effect of 100S ribosome dimer formation on persistence to AGs in exponential phase (Figure 5A). Unexpectedly, persistence levels even decreased upon overexpression of Hpf, suggesting that 70S-RaiA (sleeping ribosome) and 100S-Rmf/Hpf (hibernating ribosome) complexes may have opposite effects on persistence to AGs in exponentially growing bacteria. We also tested the persistence levels of rmf and hpf deletion mutants. Surprisingly again, and consistent with overexpression results, we found that deletion of rmf or hpf hibernation factors increases persistence to AGs (Figure S7). The increase of persistence due to deletion of rmf is dependent on the presence of raiA, as the double mutant raiA rmf shows reduced persistence compared to WT, and similar to the raiA mutant. This can be due to the observed increased expression of raiA in the absence of rmf [START_REF] Sabharwal | The VrrA sRNA controls a stationary phase survival factor Vrp of Vibrio cholerae[END_REF] or amplified ribosome-RaiA complex formation in the absence of rmf, due to decreased 100S formation [START_REF] Ueta | Ribosome binding proteins YhbH and YfiA have opposite functions during 100S formation in the stationary phase of Escherichia coli[END_REF]. Finally, it is important to note that the hpf raiA double mutant could not be constructed despite the use of two different strategies, implying synthetic lethality. Overall these results suggest an equilibrium between Rmf/Hpf and RaiA actions, consistent with previous literature that showed a combined role for these proteins in ribosome hibernation and antagonizing regulation of rmf/hpf and RaiA in V. cholerae [START_REF] Sabharwal | The VrrA sRNA controls a stationary phase survival factor Vrp of Vibrio cholerae[END_REF].

RaiA overexpression increases 70S ribosome proportion over 50S and 30S subunits in stationary phase.

In order to address whether intact 70S ribosomes are protected/stored upon RaiA overexpression, we measured the ribosome contents of WT and RaiA overexpressing cells, as well as the strain deleted for raiA, by performing 10-50% sucrose gradients on cellular extracts from 24h stationary phase cultures. The profiles obtained were consistent with well described peaks for 30S and 50S subunits followed by a third peak corresponding to the 70S ribosome [START_REF] Ueta | Conservation of two distinct types of 100S ribosome in bacteria[END_REF]. No clear 100S peak was observed in any of the experiments. Deletion of raiA leads to a slight increase in the proportion of dissociated subunits compared to 70S ribosome. We found that the proportion of 70S ribosomes is increased compared to 30S+50S dissociated subunits upon RaiA overexpression (Figure 8AB). This agrees with the hypothesis that RaiA would stabilize the intact 70S ribosome and increase functional ribosome pools, reducing the lag phase of the population upon growth restart.

RaiA expression is under environmental control and linked to the Fur iron sensing regulon in V. cholerae

RaiA is known to be expressed in stationary phase and upon temperature stress [START_REF] Agafonov | A protein residing at the subunit interface of the bacterial ribosome[END_REF][START_REF] Agafonov | Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage[END_REF][START_REF] Maki | Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli[END_REF][START_REF] Sabharwal | The VrrA sRNA controls a stationary phase survival factor Vrp of Vibrio cholerae[END_REF][START_REF] Slamti | Global gene expression and phenotypic analysis of a Vibrio cholerae rpoH deletion mutant[END_REF]. Regulation of raiA was also described to occur through carbon catabolite control (CRP-cAMP) [START_REF] Manneh-Roussel | cAMP Receptor Protein Controls Vibrio cholerae Gene Expression in Response to Host Colonization[END_REF][START_REF] Shimada | Involvement of cyclic AMP receptor protein in regulation of the rmf gene encoding the ribosome modulation factor in Escherichia coli[END_REF]. Our transcriptomic data suggests that indole affects genes from the Fur regulon (Table S1). Fur responds to iron levels and is generally known to be a repressor of iron/heme transport genes (such as hutA, hutXW, tonB, fbpA, viuB, among others) and also activates a small number of genes (namely the napABC operon and menB in V. cholerae) [START_REF] Mey | Iron and fur regulation in Vibrio cholerae and the role of fur in virulence[END_REF]. The V. cholerae raiA gene promoter, appears to carry sequences similar to Fur boxes described in V. cholerae [START_REF] Davies | Mapping the regulon of Vibrio cholerae ferric uptake regulator expands its known network of gene regulation[END_REF]. In order to shed light in the means by which indole induces expression from the raiA promoter in exponential phase, we constructed deletion mutants for fur, crp and for the stationary phase sigma factor rpoS. We used our PraiA-gfp transcriptional fusion to measure expression in the presence and absence of indole in the mutants compared to wild type strain.

As expected, in WT V. cholerae, fluorescence was triggered by indole at 3 hours of culture which corresponds to early exponential phase (OD 0.2 to 0.3), and accumulated in stationary phase (Figure 4A andS4AB). Deletion of crp strongly decreases raiA expression (25x in exponential phase, Figure 4B and S4AB), confirming that CRP is a prevailing activator of V. cholerae raiA. On the other hand, deletion of rpoS had no major effect on V. cholerae raiA expression (1.25x decrease). In the ∆fur strain, raiA expression was increased 2-fold (Figure 3B and S4AB), suggesting Fur dependent repression of the raiA promoter. No major effect on raiA promoter was observed upon treatment with iron, possibly because iron is already in excess levels for the cells during growth. Strikingly, treatment with dipyridyl (DP), an iron chelator which mimics conditions of iron starvation strongly induced fluorescence (Figure 4A). To confirm that these effects were specific to the raiA promoter, we introduced constitutively expressed gfp using the same plasmid vector in the strains WT, ∆crp and ∆fur and observed no effect on fluorescence (Figure S8). This was also the case upon indole treatment of the WT strain. DP appears to decrease fluorescence for constitutive promoter, while increasing it 4x from raiA promoter (Figure 3B). Overall, these data confirm that decreased fluorescence in ∆crp and increased fluorescence in ∆fur or upon indole and DP treatments are specific to an effect on transcription from the raiA promoter.

These results show a link between extracellular iron levels and RaiA expression. Together with the CRP-cAMP control, RaiA expression appears to be under environmental control, highlighting a link between bacterial persistence and environmental stress.

The effect of RaiA overexpression is conserved in the Gram-negative pathogen Pseudomonas aeruginosa.

We next asked whether RaiA could have a similar function in other Gram-negative pathogens such as Pseudomonas aeruginosa, an organism associated with antibiotic resistance and persistence [START_REF] Koeva | An Antipersister Strategy for Treatment of Chronic Pseudomonas aeruginosa Infections[END_REF][START_REF] Spoering | Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials[END_REF] and of high concern regarding resistant infections. P. aeruginosa RaiA exhibits 37% protein identity with RaiA from V. cholerae. We overexpressed V. cholerae RaiA in P. aeruginosa and assessed lag phase and persistence, as we performed for V. cholerae. We found that upon RaiA overexpression, lag phase is also decreased in P. aeruginosa (Figure 9A and S6D), and strikingly, persistence to tobramycin is increased (Figure 9B). These results show that RaiA-protected sleeping ribosomes can be involved in persistent infections by various pathogenic bacteria.

Discussion

We show here that indole is produced upon sub-MIC aminoglycoside treatment in V. cholerae and at physiological concentrations, and increases appearance of persister cells in lethal concentrations of AGs. We find that such increase in persistence occurs through an inducible mechanism involving RaiA (previously called pY or yfiA in E. coli, and vrp in V. cholerae). While indole improved growth in the presence of sub-MIC antibiotics seems to be nonspecific to an antibiotic class and independent of RaiA, increased persistence involving RaiA is specific to AGs in exponentially growing bacteria. Since RaiA is regulated by several environmental cues and signaling molecules, our findings highlight a new, inducible mechanism of persistence, based on increased protection of ribosomes during stress, rather than slowdown of the metabolism. In vitro characterization of RaiA in E. coli, has previously shown association with ribosomes during cold shock and stationary phase, but not during growth at 37°C, suggesting that binding of RaiA is prompted by stress [START_REF] Agafonov | Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage[END_REF]. Based on crystal structures, RaiA was suggested to arrest translation (Vila-Sanjurjo et al., 2004). However, our results in V. cholerae do not support such a role, since there is no impact of RaiA overexpression on growth, and rather point to a protective effect of RaiA under ribosomal stress caused by AG treatment. Alternatively, since RaiA is able to stabilize the 70S ribosome monomers against dissociation in vitro [START_REF] Agafonov | A protein residing at the subunit interface of the bacterial ribosome[END_REF][START_REF] Di Pietro | Role of the ribosome-associated protein PY in the cold-shock response of Escherichia coli[END_REF], it was proposed to constitute a pool of inactive 70S ribosomes preserved from degradation in bacteria [START_REF] Giuliodori | Cold-shock response in Escherichia coli: A model system to study posttranscriptional regulation[END_REF][START_REF] Giuliodori | Cold-stress-induced de novo expression of infC and role of IF3 in cold-shock translational bias[END_REF][START_REF] Gualerzi | Translation initiation at the root of the cold-shock translational bias[END_REF].

In vivo effects of RaiA on bacterial phenotypes are less well described. A protective effect of RaiA during stress, such as starvation, was previously observed in the Gram-positive species Mycobacterium tuberculosis [START_REF] Li | Zinc depletion induces ribosome hibernation in mycobacteria[END_REF] and Lactococcus lactis [START_REF] Puri | Lactococcus lactis YfiA is necessary and sufficient for ribosome dimerization[END_REF]). Yet, the mechanisms remained enigmatic. Our results show a role of RaiA on survival to antibiotic stress in Gram-negative pathogens. One known mechanism of protection of non-translating ribosomes is ribosome hibernation. The ribosome hibernation factors, Rmf (ribosome modulation factor) and Hpf (hibernation promoting factor) [START_REF] Maki | Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli[END_REF], dimerize 70S ribosomes (monosomes) into 100S hibernating ribosome dimers. The importance of ribosome hibernation in stress survival is well established in various bacteria [START_REF] Mckay | Ribosome hibernation facilitates tolerance of stationary-phase bacteria to aminoglycosides[END_REF][START_REF] Tkachenko | Stationary-phase genes upregulated by polyamines are responsible for the formation of Escherichia coli persister cells tolerant to netilmicin[END_REF], as 100S dimers are less susceptible to degradation by RNases [START_REF] Feaga | Ribosome Dimerization Protects the Small Subunit[END_REF][START_REF] Prossliner | Ribosome Hibernation[END_REF][START_REF] Prossliner | Hibernation factors directly block ribonucleases from entering the ribosome in response to starvation[END_REF][START_REF] Wada | Growth phase-coupled changes of the ribosome profile in natural isolates and laboratory strains of Escherichia coli[END_REF][START_REF] Yamagishi | Regulation of the Escherichia coli rmf gene encoding the ribosome modulation factor: growth phaseand growth rate-dependent control[END_REF]. Ribosome hibernation factors were even proposed as potential new targets for antibiotics [START_REF] Matzov | Stress response as implemented by hibernating ribosomes: a structural overview[END_REF]. However, in some cases, 70S particles appear to be more robust during heating than 100S dimers which dissociate into 30S and 50S subunits more rapidly [START_REF] Niven | Ribosome modulation factor protects Escherichia coli during heat stress, but this may not be dependent on ribosome dimerisation[END_REF]. RaiA was previously identified as bound to the ribosome together with Rmf and Hpf, but its role in relation with ribosome hibernation is unclear. In V. cholerae, RaiA shows a synergistic effect with Hpf for survival to starvation [START_REF] Sabharwal | The VrrA sRNA controls a stationary phase survival factor Vrp of Vibrio cholerae[END_REF], and we observe a synthetic lethal phenotype for the deletion of raiA and hpf. Despite such apparent synergy with hibernation factors, RaiA was shown to inactivate 70S ribosomes without forming 100S dimers [START_REF] Polikanov | How hibernation factors RMF, HPF, and YfiA turn off protein synthesis[END_REF][START_REF] Ueta | Ribosome binding proteins YhbH and YfiA have opposite functions during 100S formation in the stationary phase of Escherichia coli[END_REF]. RaiA thus appears to act in a process different than ribosome hibernation, maybe by blocking breaking down of ribosomes into 30S and 50S subunits by ribosome recycling factors (Rrf/EF-G) [START_REF] Agafonov | A protein residing at the subunit interface of the bacterial ribosome[END_REF][START_REF] Janosi | Ribosome recycling by ribosome recycling factor (RRF)--an important but overlooked step of protein biosynthesis[END_REF]. In E. coli, RaiA can even prevent 100S dimer formation by Hpf and Rmf [START_REF] Maki | Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli[END_REF][START_REF] Ueta | Ribosome binding proteins YhbH and YfiA have opposite functions during 100S formation in the stationary phase of Escherichia coli[END_REF]. According to cryo-EM data, RaiA can compete with Rmf for ribosome binding, hence shifting the ribosome content from a Rmf-mediated dimeric inactive form to a RaiA-bound monomeric inactive form [START_REF] Franken | A general mechanism of ribosome dimerization revealed by single-particle cryoelectron microscopy[END_REF]. Recent work has identified RaiA as a player in persister formation together with hibernation factors, through a dormancy related mechanism linked to stringent response/ppGpp [START_REF] Song | ppGpp ribosome dimerization model for bacterial persister formation and resuscitation[END_REF][START_REF] Wood | Forming and waking dormant cells: The ppGpp ribosome dimerization persister model[END_REF]. In these studies, persister formation was assessed on near stationary cell cultures (OD 0.8), where these factors are highly expressed, and persistence is largely due to dimerization of inactive ribosomes, leading to a general dormancy state which also favors persister formation to other antibiotics like ampicillin and ciprofloxacin. In contrast, the present study shows in exponentially growing cells and without nutrient limitation, a specific mechanism involving the induction of RaiA, triggering persistence specifically to aminoglycosides (and not to other antibiotics), without affecting growth (Figure 10A). The mechanism we describe is also different than the ppGpp-independent persistence caused in slow growing bacteria [START_REF] Pontes | Slow growth determines nonheritable antibiotic resistance in Salmonella enterica[END_REF], since increased RaiA does not affect growth rate in our study. In this regard, the RaiA-dependent persistence mechanism observed in the present study is not a dormancy-like or slow growth mechanism, unlike the increased persistence triggered by ppGpp to antibiotics from various families, in stationary phase. Moreover, no effect of hibernation factors Rmf/Hpf is observed, suggesting different persistence mechanisms than ribosome dormancy formation in growing cells. Recent studies also show the existence of mechanisms triggering persistence independently of ppGpp or hibernation factors in bacteria [START_REF] Hossain | Antibiotic tolerance, persistence, and resistance of the evolved minimal cell, Mycoplasma mycoides JCVI-Syn3[END_REF][START_REF] Wood | Deacylated tRNA Accumulation Is a Trigger for Bacterial Antibiotic Persistence Independent of the Stringent Response[END_REF]. We show here that RaiA is one player enhancing persister formation in the Gram-negative bacterium V. cholerae.

While RaiA, Hpf and Rmf are rapidly released from ribosomes when normal growth conditions are restored [START_REF] Agafonov | Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage[END_REF][START_REF] Maki | Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli[END_REF], ribosome reactivation necessitates dissociation of hibernating 100S ribosome dimers into monomers by HflX and other factors [START_REF] Basu | Disassembly of the Staphylococcus aureus hibernating 100S ribosome by an evolutionarily conserved GTPase[END_REF], whereas no dissociation factor is needed for the reactivation of the RaiA inactivated 70S ribosome. There may thus be an interplay and equilibrium between hibernating 100S-Hpf/Rmf and "sleeping" 70S-RaiA forms. Such synergy or antagonism between inactive 70S and 100S ribosome pools can however depend on the nature of the stress and the bacterial species. We show here that persistence to aminoglycosides is better achieved in the presence of RaiA (70S), rather than hibernation factors (100S) in V. cholerae.

Here, we propose that increased production of RaiA (e.g. upon stress) leads to preservation of nontranslating ribosomes in a pool of inactive and intact 70S "sleeping" ribosomes. In that scenario, bacteria can keep growing in the absence of the antibiotic, since RaiA will not bind to actively translating ribosomes. Upon stress affecting translation (e.g. aminoglycosides), ribosomes may stall or stop, which would lead to the dissociation from the mRNA. If RaiA is induced, we propose that such non-translating 70S ribosomes will be bound and protected by RaiA, and avoid death upon antibiotic treatment, not by entering a dormant state, but by inducing protection of ribosomes. We propose that, in contrast to hibernating ribosomes which need recycling factors to dissociate into 70S monosomes, such sleeping ribosomes can be rapidly reactivated upon stress relief through spontaneous dissociation of RaiA from the intact 70S monosome [START_REF] Agafonov | Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage[END_REF][START_REF] Maki | Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli[END_REF], conferring an advantage for stress survival (Figure 10AB). In line with this, a recent study showed that the greater the ribosome content of the cell, the faster persister cells resuscitate [START_REF] Kim | Single cell observations show persister cells wake based on ribosome content[END_REF]. In parallel, protein synthesis was shown to be necessary for persister cell formation in V. cholerae [START_REF] Paranjape | Inhibition of protein synthesis eradicates persister cells of V. cholerae[END_REF], consistent with the fact that we see no effect of RaiA overexpression on growth rates, thus on protein synthesis during growth. Our results do not exclude however another role for RaiA on translating ribosomes. Increased RaiA levels thus allow higher persistence, but what is controlling RaiA expression? RaiA is expressed in stationary phase and during cold shock [START_REF] Agafonov | A protein residing at the subunit interface of the bacterial ribosome[END_REF][START_REF] Agafonov | Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage[END_REF][START_REF] Maki | Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli[END_REF][START_REF] Sabharwal | The VrrA sRNA controls a stationary phase survival factor Vrp of Vibrio cholerae[END_REF]. Other known factors are the heat shock in V. cholerae [START_REF] Slamti | Global gene expression and phenotypic analysis of a Vibrio cholerae rpoH deletion mutant[END_REF], stringent response [START_REF] Prossliner | Ribosome Hibernation[END_REF], envelope stress and the carbon catabolite response [START_REF] Manneh-Roussel | cAMP Receptor Protein Controls Vibrio cholerae Gene Expression in Response to Host Colonization[END_REF][START_REF] Shimada | Involvement of cyclic AMP receptor protein in regulation of the rmf gene encoding the ribosome modulation factor in Escherichia coli[END_REF]. We additionally show that raiA expression is linked to iron levels and responds to Fur regulation. Interestingly, iron associates with ribosomes [START_REF] Bray | Multiple prebiotic metals mediate translation[END_REF] and a link between iron and modulation of ribosome function during stress has been described [START_REF] Zinskie | Irondependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae[END_REF]. Iron control of RaiA may thus allow protection of ribosomes from iron related damage. Altogether, RaiA appears to be part of the bacterial response to environmental stress. Results of the present study constitute a link between bacterial signaling, ribosome protection and persistence. Moreover, since RaiA action appears to be conserved in various pathogens (Gramnegatives and positives, such as S. aureus and M. tuberculosis), it may be one factor involved in the failure of treatment in persistent infections. It would be interesting to ask now whether RaiA can be used as an early indicator of persistence, which would allow isolation of persisters within a heterogeneous population and further studies using single cell approaches.

Limitation of Study

This study shows that indole is secreted under sub-MIC aminoglycosides treatment and increases raiA expression. It also shows that indole is implicated in raiA-dependent persister formation under lethal aminoglycosides treatment. However, we do not address whether indole is also produced under lethal treatment because it is challenging to quantify indole production or tnaA expression on dying cells. Development of this method in our lab could be the purpose of a future work. In addition, raiA-dependent persister formation is shown here to be specific to aminoglycosides (bactericidal), which target translation. Bacteriostatic antibiotics such as chloramphenicol or tetracycline also target the ribosome, but persistence assays are usually not performed using these antibiotics. Our study does not exclude a role for raiA protection on sleeping in the response to such translation targeting antibiotics.

Author Contributions

Experiments were designed by Z.B. Experiments were conducted by M.L., E. K., C.K. and Z. B., and results were interpreted by Z.B. and M.L. RNA-seq library preparation, sequencing and statistical analysis was performed by O. S., H.V. and J-Y. C. The manuscript was written and reviewed by Z.B., M.L. and D.M. All authors read and approved the final manuscript.

Main Figure Titles and Legends

Figure 1. Indole is produced during growth in sub-MIC tobramycin and induces raiA. Measure of extracellular indole concentrations of bacterial cultures grown overnight in rich medium MOPS (Teknova EZ rich defined medium) with and without tobramycin at 0.10 µg/ml (TOB 0.10) or 0.15 µg/ml (TOB 0.15) using the Kovacs reagent [START_REF] Saint-Ruf | Massive diversification in aging colonies of Escherichia coli[END_REF]. ∆tnaA strain was used as negative control without tobramycin. Experiments were performed in triplicates, and statistical analysis was performed (***: p<0.001). Error bars represent standard deviation. Early exponential phase of wild-type (WT) V. cholerae carrying either the empty pBAD vector (p0) or with specified gene (pGene) cultures were treated with lethal doses of the specified antibiotics for 20 hours. The y axis represents survival, as the number of CFU growing after antibiotic treatment and removal divided by the total number of CFU at time zero (before antibiotic treatment). Tobramycin (TOB): 10 µg/ml, gentamicin (GEN): 5 µg/ml, carbenicillin (CRB): 100 µg/ml, ciprofloxacin (CIP): 0.025 µg/ml, trimethoprim (TMP): 50 µg/ml, neomycin (NEO): 30 µg/ml, indole (IND): 350 µM. Experiments were performed 3 to 6 times, and statistical analysis was performed (*: p <0.05; **: p<0.01). Figure 6. RaiA influences lag phase upon growth restart after the stationary phase. Growth is measured on a TECAN plate reader. The curves of wild type V. cholerae with the empty plasmid are compared with plasmid carrying raiA under inducible promoter in MH media containing either glucose or arabinose. The promoter is repressed using glucose (GLC) and induced using arabinose (ARA). Growth was performed as specified, with RaiA expression repressed or induced in the overnight culture used for inoculum (overnight culture supplemented or not with ARA; indicated as "during precultures"), or/and with RaiA expression induced or not during growth in the microplate reader (growth media supplemented or not with ARA in the microplate; indicated as "during sub-cultures") : Figure 8. RaiA levels influence stationary phase 70S ribosome content relative to 50S+30S subunits in V. cholerae. A. Cellular extracts of 24 hours cultures (ΔraiA and WT V. cholerae carrying the p0/pBAD-RaiA vector, in MH media containing spectinomycin and arabinose) were separated on 10-50% sucrose density gradient. Ribosomal RNA content was measured at OD 260 nm using a spectrometer coupled to a pump and time on X axis represents samples from less dense (upper fragments, smaller complexes) to denser (bottom of the tube, heavier complexes). Lysis was performed in the presence of 10 mM MgCl2. Cell debris eluting before 550 seconds are not shown. During exponential growth, treatment with lethal doses of aminoglycosides leads to disruption of translation and stalling of translating 70S ribosomes, and eventually to their dissociation into 50S+30S subunits and degradation of these ribosomes, resulting in cell death. Upon induction of RaiA (e.g. by indole signaling), we propose that RaiA does not affect translating ribosomes, but binds non-translating 70S ribosomes to form sleeping ribosomes, and protects them from dissociation into subunits and degradation. This results in increased persistence. B. Length of lag phase. In a stationary phase culture (e.g. overnight culture), translation is highly decreased, leading to dissociation from the mRNA ribosome degradation. Upon growth restart, a lag phase thus occurs where ribosomes are resynthesized. We propose that when RaiA production is increased before entering the stationary phase, an increased proportion of non-translating ribosomes are bound and are preserved as 70S by RaiA, rather than being degraded. The presence of such an increased pool of intact 70S ready-to-use sleeping ribosomes gives an advantage on translation reactivation, while degraded ribosome have to be recycled or de novo synthetized, decreasing the lag phase necessary for the synthesis of a sufficient number of ribosomes.

STAR METHODS

RESOURCE AVAILABILITY

Lead contacts

Further informations and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contacts, Didier Mazel (didier.mazel@pasteur.fr) or Zeynep Baharoglu (baharogl@pasteur.fr).

Materials availability

This study did not generate new unique reagents.

Data and code availability

RNA-seq data are deposited at GEO: GSE182561

EXPERIMENTAL MODEL AND SUBJECT DETAILS Bacterial Strains and Plasmids

All V. cholerae strains used in this study are derivative of V. cholerae N16961 hapR+, and were constructed by allelic exchange. All E. coli strains used in this work are derivatives of E. coli MG1655, and were constructed by transduction using E. coli Keio knockouts strains. Strains and plasmids are listed in Key Resources Table , andTable S2 for more details.

Media and Growth Conditions

Vibrio cholerae: Colonies on plates grew at 37°C, in MH media. Plates are conserved at room temperature and should not be placed at 4°C. Liquid cultures grew at 37°C in appropriate media (see Methods), in aerobic conditions, with 180 rotations per minute. E. coli and P. aeruginosa: Colonies on plates grew at 37°C, in MH media, and are conserved at 4°C. Liquid cultures grew 37°C in aerobic condition in appropriate media (see Methods), with 180 rotations per minute.

METHOD DETAILS

Persistence tests. Persistence tests were performed on early exponential phase cultures. In order to clear the culture from previously non-growing cells that could potentially be present from the stationary phase inoculum, we performed a two-step dilution protocol, before antibiotic treatment. For overexpression experiments, glucose 1% was added to the overnight cultures to repress the pBAD promoter. Overnight V. cholerae cultures were first diluted 1000x in 4 ml fresh Mueller-Hinton (MH) medium, without indole (with the antibiotic allowing to maintain the plasmid and incubated at 37°C with shaking. For overexpression experiments, arabinose 0.2% was added at this first dilution step to the fresh MH media to induce the pBAD promoter. When the OD 620 nm reached ~0.2, cultures were diluted 1000x a second time, in order to clear them from non-growing cells, in Erlenmeyers containing 25 ml fresh MH medium, without or with indole at 350 µM (with the antibiotic allowing to maintain the plasmid, when the strain carried a plasmid), and were allowed to grow at 37°C. For overexpression experiments, arabinose 0.2% was added again at this second dilution step to the fresh MH media to induce the pBAD promoter. When cultures reached an OD 620 nm between 0.25 and 0.3 (early exponential phase), appropriate dilutions were plated on MH plates to determine the total number of CFUs in time zero untreated cultures. Note that for V. cholerae, it was important to treat cultures at the precise OD 620 nm 0.25-0.3, as persistence levels seem to be particularly sensitive to growth phase in this species, where they decline in stationary phase, and because we wanted to avoid any stationary phase protein expression such as raiA or rpoS at later growth. 5 ml of cultures were collected into 50 ml Falcon tubes and treated with lethal doses of desired antibiotics (5-10 times the MIC: tobramycin 10 µg/ml, gentamicin 5 µg/ml, neomycin 30 µg/ml, carbenicillin 100 µg/ml, ciprofloxacin 0.025 µg/ml, trimethoprim 50 µg/ml) for 20 hours at 37°C with shaking in order to guarantee oxygenation. The same protocol was used for persistence assays on P. aeruginosa. Appropriate dilutions were then plated on MH agar without antibiotics and proportion of growing CFUs were calculated by doing a ratio with total CFUs at time zero. Experiments were performed 3 to 6 times.

Quantification of extracellular indole concentrations.

Extracellular indole concentration was measured on bacterial cultures grown overnight with and without antibiotics using the Kovacs reagent [START_REF] Saint-Ruf | Massive diversification in aging colonies of Escherichia coli[END_REF]. First, we established an indole concentration standard curve using 1 ml culture medium (without bacteria) supplemented with indole 0 to 1000 µM (100 µM steps). After adding 500 µl KOVACS reagent, 100 µl of the top layer of the reaction was mixed with 800 µl isoamyl-HCl and OD was read at 570 nm. For indole measurement on bacterial samples, 1 ml of culture was subjected at the same protocol. Measured indole concentration was normalized to the bacterial dry mass based on the assumption that for an OD 600 nm = 1 bacterial dry mass is 0.3 mg/ml [START_REF] Soini | High cell density media for Escherichia coli are generally designed for aerobic cultivations -consequences for large-scale bioprocesses and shake flask cultures[END_REF]. We detected no indole production in MH medium in any condition. In order to quantify secreted indole in the presence and absence of sub-MIC antibiotics, we thus used the defined rich MOPS transparent medium (Teknova EZ rich defined medium), where MIC TOB is 0.75 µg/ml. All the following experiments (persistence, growth) were conducted in MH, as it allows to study the impact of defined indole concentrations added to the media.

Quantification of fluorescent neomycin uptake was performed as described (Pierlé et al, manuscript in revision, [START_REF] Okuda | Mechanism of action of a class of antibiotics from their entry to their target in bacteria : a real time visualization[END_REF][START_REF] Sabeti Azad | Fluorescent aminoglycoside antibiotics and methods for accurately monitoring uptake by bacteria[END_REF]). Neo-cy5 is an aminoglycoside coupled to the fluorophore Cy5, and has been shown to be active against Gram-bacteria (Okuda, 2015;[START_REF] Sabeti Azad | Fluorescent aminoglycoside antibiotics and methods for accurately monitoring uptake by bacteria[END_REF]. Briefly, overnight cultures were diluted 100-fold in rich MOPS (Teknova EZ rich defined medium). When the bacterial strains reached an OD 620 nm of 0.25, they were incubated with 0.4 µM of Cy5 labeled Neomycin for 15 minutes at 37°C. 10 µl of the incubated culture were then used for flow cytometry, diluting them in 250 μl of PBS before reading fluorescence. WT V. cholerae, was incubated simultaneously without Neo-Cy5 as a negative control. Flow cytometry experiments were performed as described [START_REF] Baharoglu | Conjugative DNA transfer induces the bacterial SOS response and promotes antibiotic resistance development through integron activation[END_REF]) and repeated at least 3 times. For each experiment, 100,000 events were counted on the Miltenyi MACSquant device.

MIC determination using etests. Stationary phase cultures were diluted 20 times in PBS, and 300 µL were plated on MH plates and dried for 10 minutes. etests (Biomérieux) were placed on the plates and incubated overnight at 37°C.

RNA-seq.

Overnight cultures of the O1 biovar El Tor N16961 hapR+ V. cholerae strain were diluted 100x and grown in triplicate in MH medium until an OD 620 nm = ~0.4 with or without 350 µM indole. Sample collection, total RNA extraction, library preparation, sequencing and analysis were performed as previously described [START_REF] Krin | Expansion of the SOS regulon of Vibrio cholerae through extensive transcriptome analysis and experimental validation[END_REF]. raiA qRT-PCR. Total RNA was extracted and purified from exponential phase cultures in MH in presence or absence of indole, as previously described [START_REF] Krin | Expansion of the SOS regulon of Vibrio cholerae through extensive transcriptome analysis and experimental validation[END_REF]. Reverse transcription (RT) was performed on 100 ng total RNA using SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen). Quantitative PCR was performed on 2 µl RT sample diluted 10-fold using SYBR Green PCR Master Mix (APPLIED) and QuantStudio 6. Quantification was performed using standard range. Expression values were normalized against gyrA as previously described in V. cholerae [START_REF] Liu | Identification and characterization of a phosphodiesterase that inversely regulates motility and biofilm formation in Vibrio cholerae[END_REF][START_REF] Lo Scrudato | The regulatory network of natural competence and transformation of Vibrio cholerae[END_REF].

Quantification of raiA expression by fluorescent flow cytometry using a gfp fusion. gfp was amplified by PCR using primers carrying the raiA promoter region and cloned into pTOPO-TA cloning vector. The PraiA-gfp fragment was then extracted using EcoRI and cloned into the low copy plasmid pSC101 (1 to 5 copies per cell). The plasmid was introduced into desired strains, and fluorescence was measured on Figure S2. Persistence and the effect of indole. A. Kinetics of survival of WT V. cholerae to tobramycin 10 µg/ml in MH media. Time zero corresponds to the total number of CFU before addition of antibiotics, to an early exponential phase culture (OD 620 nm 0.25-0.3), as described in the methods section. The proportion of surviving cells is calculated after plating and counting growing colonies, and is represented for each time point. Curves represent geometric means of at least 3 replicates for each time point and error bars represent geometric standard deviation. B. Persistence of V. cholerae WT and ∆tnaA mutants in exponential phase (in LB instead of MH, to allow indole production, see methods) after 20 hours treatment with specified antibiotics. Tobramycin (TOB): 10 µg/ml, carbenicillin (CRB): 100 µg/ml. Related to Figure 2. Representative acquisitions are shown. Each plot represents one experiment. Each experiment was performed at least 3 times and data and statistical significance are shown in the histograms in the main manuscript. A: histogram curves: GFP fluorescence is represented in the x-axis (FITC channel), the yaxis represents the number of events corresponding to the number of cells, normalized to height (same number of total cells for both conditions). B: dot plots: Bacterial cells' size and shape (rugosity). FSC: forward scatter. SSC: size scatter. The SSC (size scatter) vs FSC (forward scatter) graphs show the distribution of cells by size and shape and no major difference in cell size/shape is observed between the WT and mutants with or without indole. Related to Figure 4. Figure S5. RaiA is involved in persistence in V. cholerae and E. coli. A. Increase of surviving colonies (persisters) after 20 hours of treatment with tobramycin (TOB) 10 µg/ml (ratio over WT surviving colonies) in V. cholerae. Error bars represent geometric standard deviation B. Persistence of E. coli WT and raiA mutant, at late exponential phase (OD 620nm 0.5), after 20 hours treatment with 10 µg/ml tobramycin (TOB) in MH media. Serial dilutions are spotted on plates for estimation of survival. Spot assays were performed at least 3 times. Related to Figure 2. Figure S6. Influence of RaiA on the length of lag phase upon growth restart after the stationary phase. A. Experimental set-up. Repression was achieved with 1% glucose (GLC), induction was achieved with 0.2% arabinose (ARA). B. Lag time (minutes) defined as time to reach OD 620 nm 0.15 in WT V. cholerae carrying empty vector (p0) or pBAD-RaiA vector (see ABCD in Figure 6). C. Lag time (in minutes) defined as time to reach OD 620 nm 0.15 in V. cholerae ΔraiA, Δrmf and Δhpf and WT V. cholerae carrying empty vector (p0) or hibernation factors (pRaiA, pRmf, pHpf) (Figure 7AB) D. Lag time (in minutes) defined as time to reach OD 620 nm 0.15 in WT P. aeruginosa (Figure 9A) carrying empty vector (p0) or pBAD-RaiA vector. Experiments were performed in triplicates and statistical analysis was performed (**: p<0.01; ***: p<0.001; ****: p<0.0001; ns: not significant). Related to Figure 6, 7AB and 9A. Figure S7. Influence of RaiA, Rmf and Hpf on persistence. Persistence of V. cholerae WT and mutants in early exponential phase, after 20 hours treatment with 10 µg/ml tobramycin (TOB) in MH media.

Serial dilutions are spotted on plates for estimation of survival. Spot assays were performed at least 3 times. Related to PCR amplification of 500 bp up and down regions of VC0706 using primers ZIP381/384 and ZIP382/383. PCR amplification of aadA7 conferring spectinomycin resistance on pAM34 using ZB47/48. PCR assembly of the VCA0706::aad7 fragment using ZIP383/384 and allelic exchange by natural transformation, as described previously (Val et al., 2012[START_REF] Negro | Formation and Interrelationships of Tryptophanase and Tryptophan Synthetases in Escherichia Coli[END_REF] ∆rmf (VC1484) L557 allelic exchange by integration and excision of conjugative suicide plasmid pMP7 L045, replacing the gene with frt::aph::frt as described previously (Val et al., 2012[START_REF] Negro | Formation and Interrelationships of Tryptophanase and Tryptophan Synthetases in Escherichia Coli[END_REF] ∆hpf (VC2530) M566 allelic exchange by integration and excision of conjugative suicide plasmid pMP7 M472, replacing the gene with frt::aph::frt as described previously (Val et al., 2012[START_REF] Negro | Formation and Interrelationships of Tryptophanase and Tryptophan Synthetases in Escherichia Coli[END_REF] ∆raiA ∆rmf L790 allelic exchange by integration and excision of conjugative suicide plasmid pMP7 L045 in J251, replacing the gene with frt::aph::frt as described previously (Val et al., 2012[START_REF] Negro | Formation and Interrelationships of Tryptophanase and Tryptophan Synthetases in Escherichia Coli[END_REF] ∆crp 9950 allelic exchange by integration and excision of conjugative suicide plasmid pMP7 8348 , replacing the gene with aad7 (Val et al., 2012[START_REF] Negro | Formation and Interrelationships of Tryptophanase and Tryptophan Synthetases in Escherichia Coli[END_REF] ∆fur N541 allelic exchange by integration and excision of conjugative suicide plasmid pMP7 N450, replacing the gene with frt::aph::frt as described previously (Val et al., 2012, Negro et al., , 1995) pBAD43-RaiA+ L907 raiA (VC0706) amplified using primers raiAeco/raiAxba and cloned under Pbad using 5' EcoRI and 3' XbaI restriction sites pBAD43-Hpf+ M834 hpf (VC2530) amplified using primers hpfeco/hpfxba and cloned under Pbad promoter using 5' EcoRI and 3' XbaI restriction sites pBAD43-Rmf+ M831 rmf (VC1484) amplified using primers rmfeco/rmfxba and cloned under Pbad using 5' EcoRI and 3' XbaI restriction sites pSC101-PraiAgfp L707

raiA promoter region (500 bp) was amplified using primers ZIP383/446 and gfp was amplified using primers zip443/444. 2 fragments were assembled by PCR assembly using primers ZIP383/ZIP443, so that gfp is fused to the raiA promoter at the ATG start codon of raiA. The assembled fragment was cloned into pTOPO and extracted using EcoRI and cloned into pSC101 low copy plasmid (carbenicillin resistant).

pSC101-Pc-gfp N110 gfp was amplified using primers carrying the promoter zip513/200. The fragment was cloned into pTOPO and extracted using EcoRI and cloned into pSC101 low copy plasmid (carbenicillin resistant).

pMP7-∆hpf::kan M472 gibson assembly using primers MV450/451 for the amplification of pMP7 vector, primers VC2530hpf5bis/7 and VC2530hpf6bis/8bis for up and down regions of the gene, and primers MV268/269 on pKD4 plasmid for the resistance gene (frt::kan::frt

Figure 2 .

 2 Figure 2. Modulation of persistence of exponential phase WT and ∆raiA V. cholerae by indole. A to D: Early exponential phase of wild-type (WT) and ∆raiA V. cholerae cultures were treated with lethal doses of the specified antibiotics for 20 hours. The y axis represents survival, as the number of CFU

Figure 3 .

 3 Figure 3. Indole production during growth in sub-MIC tobramycin induces raiA. A. Volcano plot showing differentially expressed genes upon indole treatment. X axis represents log 2-fold change, Y axis represents the negative log 10 of the p value. raiA, rmf and hpf are indicated. The dashed line represents a p value of 0.01, all of the dots above thus show a p value<0.01. Red dots indicate genes linked with respiration, green with sugar metabolism, dark blue with iron, purple with amino-acid metabolism, and light blue with translation. RNA-seq was performed in triplicates for each condition. See also Table S1. B. raiA mRNA levels measured by RT-qPCR on exponential phase V. cholerae cultures in presence or absence of indole. Statistical analysis was performed (**: p<0.01). Error bars represent standard deviation.

Figure 4 .

 4 Figure 4. Environmental stress induce raiA expression in exponential phase V. cholerae. Fluorescence quantification of GFP expression from the raiA promoter by flow cytometry in MH media in exponential phase (except for "Stat"). A. in WT V. cholerae. NT: Non-treated, IND: indole (350 µM), Fe: iron (18 µM), DP: 2,2′-Dipyridyl (500 µM), Glc: Glucose (1%), Mal: Maltose (1%), Stat: stationary phase. The y axis represents the fluorescence ratio of the treated over non-treated (NT) strain. B. in indicated V. cholerae deletion mutants. The y axis represents the fluorescence ratio of the mutant over wild type (WT) strain. Mean fold change values are indicated within histogram bars. Experiments were performed at least 3 times, and statistical analysis was performed (**: p <0.01; ****: p<0.0001). Error bars represent standard deviation. See also Figure S4.

Figure 5 .

 5 Figure5. Modulation of persistence of exponential phase V. cholerae by RaiA. Early exponential phase of wild-type (WT) V. cholerae carrying either the empty pBAD vector (p0) or with specified gene (pGene) cultures were treated with lethal doses of the specified antibiotics for 20 hours. The y axis represents survival, as the number of CFU growing after antibiotic treatment and removal divided by the total number of CFU at time zero (before antibiotic treatment). Tobramycin (TOB): 10 µg/ml, gentamicin (GEN): 5 µg/ml, carbenicillin (CRB): 100 µg/ml, ciprofloxacin (CIP): 0.025 µg/ml, trimethoprim (TMP): 50 µg/ml, neomycin (NEO): 30 µg/ml, indole (IND): 350 µM. Experiments were performed 3 to 6 times, and statistical analysis was performed (*: p <0.05; **: p<0.01).

  Figure6. RaiA influences lag phase upon growth restart after the stationary phase. Growth is measured on a TECAN plate reader. The curves of wild type V. cholerae with the empty plasmid are compared with plasmid carrying raiA under inducible promoter in MH media containing either glucose or arabinose. The promoter is repressed using glucose (GLC) and induced using arabinose (ARA). Growth was performed as specified, with RaiA expression repressed or induced in the overnight culture used for inoculum (overnight culture supplemented or not with ARA; indicated as "during precultures"), or/and with RaiA expression induced or not during growth in the microplate reader (growth media supplemented or not with ARA in the microplate; indicated as "during sub-cultures") : A. GLC/ARA, B. ARA/GLC, C. ARA/ARA, D. GLC/GLC. A scheme of the experimental setup is found on Figure S5A. Experiments were performed in triplicates and geometric means are represented. Error bars represent the geometric standard deviation. See also Figure S6B. Figure 7. The effect of RaiA on lag phase is independent of ribosome hibernation factors Rmf/Hpf. A. Growth in MH media of WT and mutant strains. B. Growth of V. cholerae overexpressing hibernation

  Figure8. RaiA levels influence stationary phase 70S ribosome content relative to 50S+30S subunits in V. cholerae. A. Cellular extracts of 24 hours cultures (ΔraiA and WT V. cholerae carrying the p0/pBAD-RaiA vector, in MH media containing spectinomycin and arabinose) were separated on 10-50% sucrose density gradient. Ribosomal RNA content was measured at OD 260 nm using a spectrometer coupled to a pump and time on X axis represents samples from less dense (upper fragments, smaller complexes) to denser (bottom of the tube, heavier complexes). Lysis was performed in the presence of 10 mM MgCl2. Cell debris eluting before 550 seconds are not shown. Graphs are normalized to total OD 260 nm =1 for each sample. Mean values are indicated within histogram bars. Error bars represent standard deviation. B. Percentage of 70S ribosomes over total ribosome subunits (70S)/(70S+50S+30S). Error bars represent standard deviation (**: p<0.01).

Figure 9 .

 9 Figure 9. RaiA overexpression in Pseudomonas aeruginosa increases tolerance to tobramycin in exponential phase and promotes earlier exit from stationary phase. A. Growth is measured on a TECAN plate reader. The curves of WT P. aeruginosa with the empty plasmid (p0) are compared with the plasmid carrying raiA of V. cholerae under inducible promoter in MH media containing arabinose. See also Figure S6D B. Early exponential phase of WT P. aeruginosa cultures were treated with lethal doses of tobramycin (TOB: 10 µg/ml) for 20 hours. The y axis represents survival, as the number of CFU growing after antibiotic treatment and removal divided by the total number of CFU at time zero (before antibiotic treatment) (*: p <0.05). Experiments were performed in triplicates.

Figure 10 .

 10 Figure10. Summary model: RaiA mediated ribosome protections increases persistence to aminoglycosides and decreases lag phase during growth restart. Diagrams depict RaiA induced conditions. A. Persistence. During exponential growth, treatment with lethal doses of aminoglycosides leads to disruption of translation and stalling of translating 70S ribosomes, and eventually to their dissociation into 50S+30S subunits and degradation of these ribosomes, resulting in cell death. Upon induction of RaiA (e.g. by indole signaling), we propose that RaiA does not affect translating ribosomes, but binds non-translating 70S ribosomes to form sleeping ribosomes, and protects them from dissociation into subunits and degradation. This results in increased persistence. B. Length of lag phase. In a stationary phase culture (e.g. overnight culture), translation is highly decreased, leading to dissociation from the mRNA ribosome degradation. Upon growth restart, a lag phase thus occurs where ribosomes are resynthesized. We propose that when RaiA production is increased before entering the stationary phase, an increased proportion of non-translating ribosomes are bound and are preserved as 70S by RaiA, rather than being degraded. The presence of such an increased pool of intact 70S ready-to-use sleeping ribosomes gives an advantage on translation reactivation, while degraded ribosome have to be recycled or de novo synthetized, decreasing the lag phase necessary for the synthesis of a sufficient number of ribosomes.

Figure S3 .

 S3 Figure S3. Indole does not apparently influence aminoglycoside entry and resistance in V. cholerae WT and ∆raiA. A. Intracellular level of neomycin coupled to the fluorophore Cy5 measured by fluorescence associated flow cytometry. Error bars represent standard deviation. B. Minimal inhibitory concentrations of tobramycin (TOB) and gentamicin (GEN) measured using etests in V. cholerae, in the absence (MH) and presence of indole (IND), and indicated in µg/ml by a numeral on each image. Related to Figure 2.

Figure S4 .

 S4 Figure S4. Fluorescence detection from the raiA promoter-gfp fusion by flow cytometry.Representative acquisitions are shown. Each plot represents one experiment. Each experiment was performed at least 3 times and data and statistical significance are shown in the histograms in the main manuscript. A: histogram curves: GFP fluorescence is represented in the x-axis (FITC channel), the yaxis represents the number of events corresponding to the number of cells, normalized to height (same number of total cells for both conditions). B: dot plots: Bacterial cells' size and shape (rugosity). FSC: forward scatter. SSC: size scatter. The SSC (size scatter) vs FSC (forward scatter) graphs show the distribution of cells by size and shape and no major difference in cell size/shape is observed between the WT and mutants with or without indole. Related to Figure4.

Figure 7 Figure S8 .

 7S8 Figure S8. Expression of gfp from constitutive promoter (Pc) in conditions where expression from the raiA promoter is up or down-regulated. Fluorescence quantification of GFP expression from constitutive promoter by flow cytometry in MH media in exponential phase, in WT and indicated V. cholerae deletion mutants. IND: indole (350 µM), DP: 2,2′-Dipyridyl (500 µM). The Y axis represents fluorescence ratio of the mutant over wild type (WT) strain. Error bars represent standard deviation. Related to Figure 4.

Figure S1 .

 S1 Figure S1. Indole improves growth of V. cholerae in the presence of tobramycin. A. WT. B. ∆raiA. Growth is measured on a TECAN plate reader. IND: indole 350 µM. TOB: tobramycin sub-MIC (0.6 µg/ml). Experiments were performed in triplicates and geometric means are represented. Error bars represent the geometric standard deviation. Related to Figure 1.

Figure S2 .

 S2 Figure S2. Persistence and the effect of indole. A. Kinetics of survival of WT V. cholerae to tobramycin 10 µg/ml in MH media. Time zero corresponds to the total number of CFU before addition of antibiotics, to an early exponential phase culture (OD 620 nm 0.25-0.3), as described in the methods section. The proportion of surviving cells is calculated after plating and counting growing colonies, and is represented for each time point. Curves represent geometric means of at least 3 replicates for each time point and error bars represent geometric standard deviation. B. Persistence of V. cholerae WT and ∆tnaA mutants in exponential phase (in LB instead of MH, to allow indole production, see methods) after 20 hours treatment with specified antibiotics. Tobramycin (TOB): 10 µg/ml, carbenicillin (CRB): 100 µg/ml. Related to Figure 2.

Figure S3 .

 S3 Figure S3. Indole does not apparently influence aminoglycoside entry and resistance in V. cholerae WT and ∆raiA. A. Intracellular level of neomycin coupled to the fluorophore Cy5 measured by fluorescence associated flow cytometry. Error bars represent standard deviation. B. Minimal inhibitory concentrations of tobramycin (TOB) and gentamicin (GEN) measured using etests in V. cholerae, in the absence (MH) and presence of indole (IND), and indicated in µg/ml by a numeral on each image. Related to Figure 2.

Figure S4 .

 S4 Figure S4. Fluorescence detection from the raiA promoter-gfp fusion by flow cytometry.Representative acquisitions are shown. Each plot represents one experiment. Each experiment was performed at least 3 times and data and statistical significance are shown in the histograms in the main manuscript. A: histogram curves: GFP fluorescence is represented in the x-axis (FITC channel), the yaxis represents the number of events corresponding to the number of cells, normalized to height (same number of total cells for both conditions). B: dot plots: Bacterial cells' size and shape (rugosity). FSC: forward scatter. SSC: size scatter. The SSC (size scatter) vs FSC (forward scatter) graphs show the distribution of cells by size and shape and no major difference in cell size/shape is observed between the WT and mutants with or without indole. Related to Figure 4.

Figure S5 .

 S5 Figure S5. RaiA is involved in persistence in V. cholerae and E. coli. A. Increase of surviving colonies (persisters) after 20 hours of treatment with tobramycin (TOB) 10 µg/ml (ratio over WT surviving colonies) in V. cholerae. Error bars represent geometric standard deviation B. Persistence of E. coli WT and raiA mutant, at late exponential phase (OD 620nm 0.5), after 20 hours treatment with 10 µg/ml tobramycin (TOB) in MH media. Serial dilutions are spotted on plates for estimation of survival. Spot assays were performed at least 3 times. Related to Figure 2.

Figure S7 .

 S7 Figure S7. Influence of RaiA, Rmf and Hpf on persistence. Persistence of V. cholerae WT and mutants in early exponential phase, after 20 hours treatment with 10 µg/ml tobramycin (TOB) in MH media. Serial dilutions are spotted on plates for estimation of survival. Spot assays were performed at least 3 times. Related to Figure 7.

Figure S8 .

 S8 Figure S8. Expression of gfp from constitutive promoter (Pc) in conditions where expression from the raiA promoter is up or down-regulated. Fluorescence quantification of GFP expression from constitutive promoter by flow cytometry in MH media in exponential phase, in WT and indicated V. cholerae deletion mutants. IND: indole (350 µM), DP: 2,2′-Dipyridyl (500 µM). The Y axis represents fluorescence ratio of the mutant over wild type (WT) strain. Error bars represent standard deviation. Related to Figure 4.

Table S1 : Genes differentially regulated by indole. Related to Figure 3.

 S1

	Id VCA0230 VCA0229 VC0731 VC2559 VC0034 VC0573 VC2414 VC1440	norm MH-A 883 1120 norm MH-B 1866 545 636 9328 7 16354 norm MH-C 957 685 1531 1298 1238 1465 2168 3551 2065 4031 5106 3159 2573 7 6 21516 803 1073 578	norm IND-A 128 68 5035 326 936 2130 6744 467	norm IND-B 53 15 7322 158 1054 1225 3908 268	norm IND-C 173 72 4748 308 1174 2439 6762 424	Fold chang e 0,1 0,1 0,4 0,2 0,5 0,5 0,3 0,5	p value 5,5E-19 3,1E-17 7,7E-05 5,8E-04 6,8E-19 3,7E-17 1,2E-04 8,2E-04 1,3E-16 6,1E-15 1,4E-04 9,8E-04 adj p value 2,5E-16 1,2E-14 1,7E-04 1,1E-03
	UP VC1434 VCA0907	5799 4818 1430 2408	6784 1943	1544 1139	2021 688	1387 868	0,3 0,5	1,6E-15 6,9E-14 2,0E-04 1,3E-03
	VC1544 VC2089	860 762 1693 841	702 896	211 567	92 307	14508 200 554	0,2 0,5	5,8E-15 2,3E-13 2,2E-04 1,4E-03
	VC0706 VC1266 VC1441	3903 4103 992 730 3956 4982	3710 80171 68404 899 180 81 2505 2010 1104	6 20,1 218 0,2 2188 0,5	8,0E-03 8,9E-03 2,8E-14 1,1E-12 3,7E-04 2,2E-03
	VC1484 VC1548 VCA0496	120 828 1389	111 977 942	142 762 2071	570 200 426	232 71 1108	501 185 411	3,1 0,2 0,5	2,6E-05 1,3E-01 3,5E-14 1,3E-12 4,2E-03 1,6E-02
	Motility-Chemotaxis VC1168 Sugar metabolism	2427 1657	3276	600	422	657	0,2	4,4E-14 1,6E-12
	VC0410 VC2560 VCA0896	1779 1337 938 837 3304 3007	1477 656 3497	3761 178 889	3910 57 693	3890 151 872	2,5 0,2 0,3	1,7E-14 8,0E-03 1,6E-13 5,6E-12 1,6E-32 1,8E-30
	VC1049 VC2370 VC0414 VC0413 VC0411 VCA0536 VCA0954 VC0409 VC0827 VC0826 VC1763 VC0820 Respiration -energy VC1115 VC0966 VC2371 VC0575 VC0540 VC1126 VC1890 VCA0657 VC1267 VC1004 VC1625 VC2024 VC1547 VCA0897 VC1623 VCA0898 VC0608 VC0910 VC1543 VC0911 VC1264 VC2480 VC0541 VCA1060 VC0627 VC2183 VC2413 Aminoacid metabolism VC0574 VC1658 VCA0779 VC0008 VC0009	413 316 3748 3638 473 270 658 597 1939 1418 508 934 1750 2718 9371 6775 1152 1207 1967 1966 428 1057 369 623 663 546 7486 7229 163 157 3481 4499 1025 1091 965 7293 0 4135 4112 685 529 1006 697 6034 4770 6073 5424 3794 3293 1186 1193 3467 2804 2356 1972 5767 4943 1724 3895 3175 3395 1647 1544 4479 3736 3449 2931 1448 1607 2020 1993 2171 771 1608 1342 5155 4222 1118 4 1287 6 15480 450 299 2441 376 1067 598 1721 5433 26356 19176 30086 2398 2145 2322 1469 1694 1286 7061 7532 6276 1239 1302 1053 3411 3912 2746 1748 2438 2062 3886 5756 4105 1001 3000 7845 2896 1336 5009 13957 5414 267 1554 1847 1495 278 1133 751 2000 508 6006 6126 5482 6500 15713 18048 21828 107 1820 1907 1910 12,1 4,9 4,8 2,1 2,1 2,2 2,9 2,1 3,3 3,6 4,0 2,5 2,6 9,8 2,6 4787 1741 1310 1531 0,4 938 257 204 349 0,3 7992 2766 2625 2583 0,3 4697 2193 1882 2160 0,5 732 221 201 201 0,3 951 208 95 240 0,2 6826 1934 1231 1837 0,3 5715 1808 1036 2011 0,3 4602 1688 1965 1679 0,5 907 270 101 276 0,2 1622 845 579 943 0,3 1754 913 895 853 0,4 6369 2709 1746 2587 0,4 2700 607 375 613 0,2 3392 1601 1326 1539 0,5 1204 468 228 484 0,3 4242 1881 2225 1776 0,5 2531 663 257 830 0,2 2584 982 741 885 0,5 2192 759 645 969 0,4 1674 633 563 664 0,4 1589 479 458 718 0,4 6751 2904 1660 3070 0,5 4976 2760 5137 0,3 4963 6302 6116 2629 1626 2374 0,4 2116 1124 2478 185 194 252 0,1 1014 1043 1299 573 473 544 0,5 724 505 822 117 118 140 0,2 1872 1856 679 385 742 88 83 99 0,2	1,1E-38 9,0E-03 2,6E-35 1,0E-02 1,4E-06 1,9E-02 1,2E-05 3,0E-02 1,6E-05 3,9E-02 6,5E-09 4,6E-02 2,7E-04 5,9E-02 3,7E-09 6,3E-02 4,4E-07 1,4E-01 4,7E-08 1,5E-01 3,3E-03 2,0E-01 1,5E-03 2,2E-01 6,1E-84 7,9E-03 2,3E-14 1,1E-02 6,9E-67 1,3E-02 1,7E-13 5,9E-12 2,4E-18 1,2E-16 1,9E-13 6,4E-12 7,2E-14 2,5E-12 2,8E-13 9,2E-12 4,2E-13 1,4E-11 1,2E-12 3,6E-11 4,2E-09 8,0E-08 1,3E-12 3,8E-11 4,9E-09 8,9E-08 2,0E-12 5,9E-11 7,2E-09 1,3E-07 2,2E-12 6,6E-11 1,1E-05 1,0E-04 2,0E-11 5,5E-10 2,5E-05 2,2E-04 2,7E-11 7,1E-10 5,1E-05 4,0E-04 3,0E-11 7,8E-10 1,4E-04 9,5E-04 6,9E-11 1,7E-09 2,8E-04 1,7E-03 3,9E-10 8,7E-09 4,8E-10 1,0E-08 8,2E-26 7,4E-24 5,1E-10 1,1E-08 1,2E-20 7,9E-19 6,7E-10 1,4E-08 4,2E-20 2,6E-18
	VC0477 VC1425 VC0472	4301 4841 8 8 11818 4095 27291 30017 20290 6031 6511 6308 6447 7377 9080 2458 1906 2154	5,6 0,4 0,3	4,2E-39 1,5E-02 2,6E-09 4,9E-08 3,0E-19 1,7E-17
	VCA0511 VC1190 VC1312	174 3535 3491 249 2358 2539	197 3519 3364	1325 1105 624	1518 534 800	1124 1290 669	6,0 0,3 0,3	4,6E-35 1,8E-02 4,7E-09 8,7E-08 1,9E-18 9,8E-17
	VCA0665 VC0539 VC0010 VC2031 VC0880	329 816 3040 2440 253 759 1195 956 758 657	320 978 3330 1683 1076	9542 299 550 516 241	8962 125 251 398 122	8507 27,0 291 0,3 670 0,2 518 0,4 270 0,3	5,2E-5,5E-09 1,0E-07 2,0E-14 7,7E-13 122 1,8E-02 5,2E-6,3E-09 1,1E-07 2,2E-10 5,2E-09
	VCA0665 VC0538 VCA1073	329 2463 3016 253 509 701	320 3026 623	9542 969 250	8962 418 182	8507 27,0 946 0,3 291 0,4	122 1,8E-02 6,6E-09 1,2E-07 3,3E-09 6,3E-08
	VC1951 VC2044 VC0743	347 4468 3773 334 6022 4953	242 4970 6483	5920 2081 2669	6998 1443 1673	5337 17,9 2069 0,4 2698 0,4	1,2E-87 2,0E-02 6,9E-09 1,2E-07 7,6E-08 1,1E-06
	VC1114 VC1624 VC1995	430 2450 2405 337 1199 1731	365 2567 2309	1584 1013 639	1201 552 415	1615 1133 724	3,7 0,4 0,4	1,7E-17 2,3E-02 3,1E-08 5,0E-07 8,9E-08 1,3E-06
	VC0356 VC0384 VCA0815	583 2708 2991 766 3885 3098	592 1808 3980	1725 989 1836	2375 444 1216	1675 818 1630	2,9 0,3 0,4	7,9E-12 2,4E-02 5,1E-08 8,1E-07 1,7E-07 2,4E-06
	VC2145 VC1350 VC0907	931 1809 1972 952 596 504	989 2095 737	7482 700 298	9974 899 182	5936 926 300	7,6 0,4 0,4	1,1E-42 2,4E-02 6,0E-08 9,2E-07 2,0E-06 2,3E-05
	VCA0563 VC2368 VC0278 VC0485 VCA0564 VC2656 VC2646 VC1687 VC2099 VC1299 VC0941 VC2389 VC2682 VC0168 VC0947 VC1439 VCA0772 VC0385 VC0275 VC0386 VC2485 VCA0554 VCA0088 VC0662	3532 3857 8558 1112 4 512 493 8659 1108 5 11206 35439 45566 26147 3297 10041 12683 8503 8314 25789 18044 26995 641 1311 1930 1207 4137 4464 3746 10730 12961 8158 941 1355 964 33710 22990 28100 22,5 2,8 2,4 2,6 3,3 2,5 2110 1858 1643 4701 6486 4176 2,6 61 69 112 1751 1301 1331 15,5 4134 4299 6134 2367 2308 2392 0,5 1007 2194 1529 1452 766 807 883 0,5 6 7756 8318 4801 2914 4531 0,5 1233 5 2219 1588 1853 772 900 1076 0,5 1044 6 14700 5767 2538 5581 0,4 1067 1309 4710 4663 3918 2115 1538 2593 0,5 1 6 9872 4646 6635 4962 0,5 3658 4680 4161 2062 1186 2295 0,5 607 666 968 364 202 431 0,5 2632 2744 1443 910 465 974 0,4 3965 3080 4200 1403 423 1669 0,4 1716 1717 944 651 435 748 0,4 1370 3351 2132 1019 971 914 0,5 3640 1975 4155 1213 845 1601 0,4 2895 3412 2191 1471 647 1654 0,5 1746 1104 2625 973 890 798 0,5	3,1E-11 2,7E-02 4,1E-08 2,9E-02 1,2E-08 3,1E-02 4,0E-13 3,2E-02 8,6E-08 3,4E-02 2,0E-76 4,1E-02 1,7E-07 4,6E-02 6,7E-48 5,0E-02 1,8E-07 2,5E-06 2,3E-06 2,6E-05 3,3E-06 3,5E-05 5,2E-06 5,2E-05 3,7E-06 3,8E-05 1,5E-05 1,4E-04 5,0E-06 5,0E-05 8,6E-05 6,4E-04 5,5E-06 5,5E-05 1,0E-04 7,2E-04 6,3E-06 6,3E-05 1,1E-04 8,0E-04 2,9E-05 2,5E-04 3,7E-04 2,2E-03 3,1E-05 2,6E-04 3,8E-04 2,2E-03

Table S2 :

 S2 Strains and plasmids. Related to STAR Methods.

		Strain
		numbe
	Strain	r	Construction
	Vibrio
	cholerae
	N16961 wt
	strain
	N16961 hapR+
	wt strain	F606	Gift from Melanie Blokesch
	∆raiA (VC0706) J251

  primers MV450/451 for the amplification of pMP7 vector, primers VC2106fur5/7 and VC2106fur9/8 for up and down regions of the gene, and primers MV268/269 on pKD4 plasmid for the resistance gene (frt::kan::frt). ACA ATT AAT CAT CCG GCT CGT ATA ATG TGT GGA ATT GTG AGC GGA TAA CAA TTT CAC ACA GGA AAC ACA TAT GCG TAA AGG AGA AGA AC

	VC2530hpf7		CTACACAATCGCTCAAGACGTGagactttccttctctagtttagg
	VC2530hpf6bis		TACGTAGAATGTATCAGACTcttcacactgcaatagcacg
	VC2530hpf8bis		CTAATTCCCATGTCAGCCGTGCGCCAGATCGCCGAAGCTA
	VC1484rmf5		CTATTATTTAAACTCTTTCCggcttggttgttagatgatc
	VC1484rmf7		CTACACAATCGCTCAAGACGTGagttctgtcctcatacggta
	VC1484rmf6		TACGTAGAATGTATCAGACTgttgatgatggatcgcattacc
	VC1484rmf8		CTAATTCCCATGTCAGCCGTttcccatacaacaaagcttga
	VC2106fur5		CTATTATTTAAACTCTTTCCAAGCGGATGCGAACTTCGC
	VC2106fur7		CTACACAATCGCTCAAGACGTGATACTTTCCTGTTGATGTTCTGC
	VC2106fur8 VC2106fur9		CTAATTCCCATGTCAGCCGTGCTCACAAGCCGAAGAAATAA ). gibson assembly using primers MV450/451 for the amplification of pMP7 TACGTAGAATGTATCAGACTccacaaatcgatcagtttatgg
	ZIP200		vector, primers VC1484rmf5/7 and VC1484rmf6/8 for up and down TATCAAGCTTATTTGTATAGTTCATCCATGCC
	pMP7-		regions of the gene, and primers MV268/269 on pKD4 plasmid for the GAG CTG TTG
	∆rmf::kan	L045	resistance gene (frt::kan::frt).
	pMP7-ZIP513
	∆crp ::aad7	8348	Baharoglu et al., 2012
	pMP7-∆fur::kan N450 gibson assembly using Primers
	raiAecoRI		ggaattcaccATGAAAATCAACATCACTGGTAA
	raiAxba		gctctagaTTATTCCACTTCTTCGCTCAG
	hpfeco		ggaattcaccATGCAAATCAACATTCAAGGCC
	hpfxba		gctctagaTTAATGACTACTTAGCTTTTCTTT
	rmfeco		ggaattcaccATGAAGAGACAAAAGCGTGAT
	rmfxba		gctctagaTTATTTGCAGAGACCAGAAAGTTT
	ZB47		CCCGTTCCATACAGAAGCTGGGCGAACAAACGATGCTCGC
	ZB48		GACATTATTTGCCGACTACCTTGGTGATCTCGCCTTTCACG
			GCGAGCATCGTTTGTTCGCCCAGCTTCTGTATGGAACGGGTAAATA
	ZIP381		GAATGATGGGAGATAGCGC
			CGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCAATGC
	ZIP382		TTTTTCCTCTGTGTCATCCCTTATGG
	ZIP383		AACCTATCCAGATACCGAAGCGGC
	ZIP384		GTTTTGCAGATAGGATTGCTGAAGC
	ZIP93		GTCGAATGCCAAACGAAAGCGGAAAATACC
			GCGAGCATCGTTTGTTCGCCCAGCTTCTGTATGGAACGGGTACGGT
	ZIP94		ATTGAAAAAGTGCAGCCTGC
			CGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCGCTGG
	ZIP95		GTATTCCTAAAAATTAAAATATAAAGTCATGC
	ZIP96		CACAACAACTCTAGTATCTGGTTTACCCTCG
			GGCAGCCTATGCAGGCTGCACTTTTTCAATACCGTCCCGTTCCATAC
	ZIP97		AGAAGCTGGGCGAACAAACGATGCTCGC
			ATGACTTTATATTTTAATTTTTAGGAATACCCAGCGACATTATTTGCC
	ZIP98		GACTACCTTGGTGATCTCGCCTTTCACG
	ZIP443		TTATTTGTATAGTTCATCCATGCCATGTGTAATCCCAGC
	ZIP444		ATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCC
			GGACAACTCCAGTGAAAAGTTCTTCTCCTTTACGCATAATGCTTTTTC
	ZIP446		CTCTGTGTCATCCCTTATGG
	VC2530hpf5bis		CTATTATTTAAACTCTTTCCgccgatggtgctcaacgatg

Acknowledgements

We are thankful to Micheline Fromont-Racine for her valuable help with the experiments for ribosome content analysis by sucrose density gradients. We thank Ivan Matic, Wei-Lin Su and Sébastien Fleurier for helpful discussions and Ivan Matic for critical reading of the manuscript. We also thank Sebastian Aguilar Pierlé for advice for Neo-Cy5 uptake experiments. We thank Louna Fruchard for help with persistence assays. This work was supported by the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS-UMR 3525), the Fondation pour la Recherche Médicale (FRM Grant No. DBF20160635736), ANR Unibac (ANR-17-CE13-0010-01) and Institut Pasteur grant PTR 245-19.

Declaration of Interests

The authors declare no competing interests. indicated conditions, by counting 100,000 cells on the Miltenyi MACSquant device. Likewise, the control plasmid Pc-gfp (constitutive) was constructed using primers ZIP513/ZIP200 and similarly cloned in pSC101.

Growth curves. Overnight cultures were diluted 100x in fresh medium, on 96 well plates. Each well contained 200 µl. For overexpression lag experiments, glucose 1% was added to the media to repress the pBAD promoter while arabinose 0.2% was added to the media to induce the pBAD promoter, and same growth conditions (glucose or arabinose) were compared in order to avoid noise due to the effects of arabinose on growth and cells shape in V. cholerae [START_REF] Espinosa | Larabinose induces the formation of viable non-proliferating spheroplasts in Vibrio cholerae[END_REF]. Spectinomycin was also added during each experiment to maintain the plasmid. Plates were incubated with shaking on TECAN plate reader device at 37°C, OD 620 nm was measured every 15 minutes.

Preparation of cell lysate for the analysis of ribosome content. The protocol was adapted from [START_REF] Qin | Analysis of polysomes from bacteria[END_REF]. Since we used stationary phase cultures instead of exponential phase, presence of polysomes is not expected. 10 ml of 20 hours cultures were centrifuged in ice cold 50 ml Falcon tubes for 15 minutes at 5000 rpm at 4°C. Pellets were resuspended in 500 µl lysis buffer (10 mM Tris-HCl, pH 8, 10 mM MgCl2, Lysozyme 1 mg/ml, protease inhibitor), transferred in ice cold 1.5 ml tubes and incubated with 12 µl Ribolock RNase inhibitor (Thermo scientific) and DNaseI (5 U/ml) at 4°C for 15 minutes. Cell lysis was performed through 3 cycles of flash-freezing in dry ice and thawing in a water bath at 4°C. 15 µl of 10% sodium deoxycholate were added and cell lysate was obtained after centrifugation at 10,000 rpm for 10 minutes at 4°C. The pellet containing cell debris was discarded. Lysate was kept at -80°C until sucrose gradient ultracentrifugation.

Sucrose gradient. 10-50% sucrose gradient tubes (Beckman ULTRA CLEAR) were prepared. 2U of OD 260 nm of each cell extracts were deposited on sucrose gradient tubes. Ultracentrifugation was performed at 39,000 rpm at 4°C for 2 hours 45 minutes. Fractions were collected using a pump coupled to a spectrometer at OD 260 nm, and plotted as a function of time (seconds).

Quantification and statistical analysis. First an F-test was performed in order to determine whether variances are equal or different between comparisons. For comparisons with equal variance, Student's t-test was used. For comparisons with significantly different variances, we used Welch's t-test. For multiple comparisons, we used one-way ANOVA. We used GraphPad Prism to determine the statistical differences between groups. **** means p<0.0001, *** means p<0.001, ** means p<0.01, * means p<0.05. Number of replicates for each experiment was 3<n<6. Means and geometric means for logarithmic values were also calculated using GraphPad Prism. For persistence tests, data were first log transformed in order to achieve normal distribution, and statistical tests were performed on these log-transformed data.

Supplemental Data