Evelyne Krin

André Carvalho

Manon Lang

Anamaria Babosan

Didier Mazel
email: didier.mazel@pasteur.fr

Zeynep Baharoglu
email: zeynep.baharoglu@pasteur.fr

Vc2691cpxp7 Ctacacaatcgctcaagacgtgcgttcgttcctctacatttc

Vc0747iscr5 Ctattatttaaactctttccaacgtactggcttgaccaat

Vca0752trxc5 Ctattatttaaactctttccgcgttgagatcaaaggcggc

RavA-ViaA antibiotic response is linked to Cpx and Zra2 envelope stress systems in Vibrio cholerae

Keywords:

RavA-ViaA were reported to play a role in aminoglycoside sensitivity but the mechanisms remain elusive. Here, we performed competition and survival experiments to confirm that deletion of ravA-viaA increases tolerance of the Gram-negative pathogen Vibrio cholerae to low and high aminoglycoside concentrations, during aerobic growth. Using high throughput strategies in this species, we identify Cpx and Zra2 two-component systems as new partners of RavA-ViaA. We show that the aminoglycoside tolerance of ∆ravvia requires the presence of these membrane stress sensing two-component systems. We propose that deletion of the RavA-ViaA function facilitates the response aminoglycosides because of a pre-activated state of Cpx and Zra2 membrane stress response systems. We also find an impact of these genes on vancomycin resistance, and we show that simultaneous inactivation of ravvia function together with envelope stress response systems leads to outer membrane permeabilization. Vancomycin is mostly used for Gram-positive because of its low efficiency for crossing the Gram-negative outer membrane. Targeting of the ravA-viaA operon for inactivation could be a future strategy to allow uptake of vancomycin into multidrug resistant Gramnegative bacteria.

Importance

The RavA-ViaA complex was previously found to sensitize Escherichia coli to aminoglycosides in anaerobic conditions, but the mechanism is unknown. Aminoglycosides are antibiotics known for their high efficiency against Gram-negative bacteria. In order to elucidate how the expression of the RavA-ViaA genes increases bacterial susceptibility to aminoglycosides, we aimed at identifying partner functions necessary for increased tolerance in the absence of RavA-ViaA, in V. cholerae. We show that membrane stress response systems Cpx and Zra2 are required in the absence of ravA-viaA, for the tolerance to aminoglycosides and for outer membrane integrity. In the absence of these systems, the ∆ravvia strain's membrane become permeable to external agents such as the antibiotic vancomycin.

Introduction

Antibiotic resistance is a growing public health problem. Common resistance mechanisms developed by bacteria consist of limiting antibiotic entry, increasing efflux, degrading the antibiotic or mutating the target. Decreasing intracellular concentrations of antibiotics is one of the most frequent resistance strategies. The aminoglycoside (AG) class of antibiotics targets the ribosome, leading to mistranslation, protein misfolding and eventually cell death. AGs are highly efficient against Gramnegative bacteria [START_REF] Lortholary | Aminoglycosides[END_REF]. They comprise kanamycin, tobramycin, gentamicin, neomycin, amikacin and streptomycin and are commonly used worldwide. The currently accepted model for AG entry [START_REF] Davis | Mechanism of bactericidal action of aminoglycosides[END_REF][START_REF] Davis | Mechanism of bactericidal action of aminoglycosides[END_REF] starts with the PMF-dependent [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] and respiration-dependent [START_REF] Bryan | Effects of membrane-energy mutations and cations on streptomycin and gentamicin accumulation by bacteria: a model for entry of streptomycin and gentamicin in susceptible and resistant bacteria[END_REF][START_REF] Nichols | Respiration-dependent uptake of dihydrostreptomycin by Escherichia coli. Its irreversible nature and lack of evidence for a uniport process[END_REF][START_REF] Alper | Transport of antibiotics and metabolite analogs by systems under cyclic AMP control: positive selection of Salmonella typhimurium cya and crp mutants[END_REF] entry of a small AG quantity, leading to mistranslation by the ribosome. Incorporation of mistranslated proteins would then lead to membrane damage and a subsequent second step AG uptake in large amounts [START_REF] Taber | Bacterial uptake of aminoglycoside antibiotics[END_REF][START_REF] Davis | Misread protein creates membrane channels: an essential step in the bactericidal action of aminoglycosides[END_REF].

The ravA-viaA operon (formerly yieMN) was found to sensitize Gram-negative bacteria to the AGs [START_REF] Wong | The MoxR ATPase RavA and its cofactor ViaA interact with the NADH:ubiquinone oxidoreductase I in Escherichia coli[END_REF], but the mechanism remains elusive. The presence of RavA-ViaA has been reported mostly in ɣ-proteobacteria. Notably, ravA-viaA were found in 37 out of 50 randomly chosen enterobacteria [START_REF] Kandiah | Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA[END_REF]. RavA (regulatory ATPase variant A) belongs to the classe of P-loop AAA+ proteins, which hydrolyze ATP and are involved in diverse molecular processes such as protein degradation, assembly of membrane complexes, DNA repair, and others. ViaA (VWA interacting with AAA+ ATPase) carries a von Willebrand factor type A (VWA) domain and interacts with RavA and stimulates its ATPase activity [START_REF] Snider | Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase[END_REF]. Structural work revealed that RavA and ViaA interact with each other [START_REF] Snider | Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase[END_REF], and form a complex with a third partner, LdcI (or CadA) [START_REF] Kandiah | Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA[END_REF][START_REF] Snider | Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase[END_REF], leading to a cage-like structure up to 1 MDa in size [START_REF] Jessop | Structural insights into ATP hydrolysis by the MoxR ATPase RavA and the LdcI-RavA cage-like complex[END_REF]. It was also shown that the RavA-ViaA complex interacts with phospholipids at the inner membrane [START_REF] Felix | The AAA+ ATPase RavA and its binding partner ViaA modulate E. coli aminoglycoside sensitivity through interaction with the inner membrane[END_REF].

The involvement of ravA-viaA genes in AG susceptibility was originally identified by independent approaches in different proteobacteria, Escherichia coli [START_REF] Wong | The MoxR ATPase RavA and its cofactor ViaA interact with the NADH:ubiquinone oxidoreductase I in Escherichia coli[END_REF] and Vibrio cholerae [START_REF] Baharoglu | Identification of genes involved in low aminoglycosideinduced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase[END_REF]. In E. coli, overexpression of ravA-viaA sensitizes to gentamicin, and its deletion was shown to increase AG resistance [START_REF] Wong | The MoxR ATPase RavA and its cofactor ViaA interact with the NADH:ubiquinone oxidoreductase I in Escherichia coli[END_REF][START_REF] Girgis | Genetic architecture of intrinsic antibiotic susceptibility[END_REF] but only in anaerobic conditions and low energy state [START_REF] Felix | The AAA+ ATPase RavA and its binding partner ViaA modulate E. coli aminoglycoside sensitivity through interaction with the inner membrane[END_REF][START_REF] Khoury | Bioenergetic State of Escherichia coli Controls Aminoglycoside Susceptibility[END_REF]. In V. cholerae, the ravA-viaA operon (VC_A0762-VC_A0763) is also involved in the response to low doses (below MIC, or sub-MIC) of AGs, this time in aerobic conditions. While studying the response of V. cholerae to sub-MIC tobramycin we have conducted a high throughput transposon insertion sequencing (TN-seq) screen [START_REF] Babosan | Non-essential tRNA and rRNA modifications impact the bacterial response to sub-MIC antibiotic stress[END_REF][START_REF] Negro | RadD Contributes to R-Loop Avoidance in Sub-MIC Tobramycin[END_REF] where the most enriched insertions were detected in the VC_A0762 (viaA) and VC_A0763 (ravA) genes (respectively 60x and 30x enrichment), suggesting that inactivation of the operon confers a growth advantage in the presence of AGs in V. cholerae. This is consistent with our previous results showing that genotoxic stress induced by tobramycin is limited in the absence of this operon [START_REF] Baharoglu | Identification of genes involved in low aminoglycosideinduced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase[END_REF].

The PMF is involved in the first phase of AG uptake [START_REF] Eisenberg | Quantitative association between electrical potential across the cytoplasmic membrane and early gentamicin uptake and killing in Staphylococcus aureus[END_REF], while the second phase occurs in response to mistranslation (or through sugar transporters). PMF is produced by the activity of electron transfer chains in respiratory complexes, where Fe-S cluster are key actors. It was shown that Fe-S biogenesis and their fueling to respiratory complexes have a direct impact on AG uptake [START_REF] Ezraty | Fe-S cluster biosynthesis controls uptake of aminoglycosides in a ROS-less death pathway[END_REF]. Previous studies reported that RavA and ViaA interact with Fe-S cluster biogenesis machineries and also with components of the major Fe-S cluster containing Nuo respiratory complex [START_REF] Wong | The MoxR ATPase RavA and its cofactor ViaA interact with the NADH:ubiquinone oxidoreductase I in Escherichia coli[END_REF][START_REF] Babu | Quantitative genome-wide genetic interaction screens reveal global epistatic relationships of protein complexes in Escherichia coli[END_REF], suggesting that RavA-ViaA could contribute to folding of the respiratory complex I. Therefore, it was proposed that RavA and ViaA sensitize E. coli to AGs by facilitating Fe-S targeting to complex I and as a consequence an increased level of PMF. Note that E. coli and V. cholerae are very dissimilar in respect to the respiration complexes, Fe-S biogenesis and oxidative stress response pathways. For instance, V. cholerae lacks the above mentioned Cyo/Nuo complex I, and lacks also the SUF Fe-S biogenesis system used under oxidative stress. RavA-ViaA complex, was thus proposed to allows AGs to accumulate inside the cells, presumably by enhancing their uptake of AGs [START_REF] Khoury | Bioenergetic State of Escherichia coli Controls Aminoglycoside Susceptibility[END_REF].

In order to shed light into how these genes modulate bacterial susceptibility to AGs, it's important to understand in which conditions these genes are expressed, which bacterial functions are necessary for AG sensitization, and which bacterial processes are affected by these genes.

Here, we first extensively confirmed the RavA-ViaA dependent AG susceptibility and tolerance phenotypes in V. cholerae. Next, high throughput approaches identified the involvement of envelope stress responses in these phenotypes. TN-seq showed that inactivation of cpxP repressor, i.e. activation Cpx response, is beneficial in ∆ravvia. Cpx responds to conditions that cause misfolding of inner membrane (IM) and periplasmic proteins, and subsequent membrane defect (for review [START_REF] Grabowicz | Envelope Stress Responses: An Interconnected Safety Net[END_REF]). Cpx also down-regulates outer membrane proteins (OMPs). In parallel, transcriptomic data in ∆ravvia showed strong induction of VC_1314 and the VC_1315-VC_1316 operon which presents similarities with the Cpx and Zra two-component envelope stress response systems. In E. coli, ZraSR contributes to antibiotic resistance and is important for membrane integrity [START_REF] Rome | The Two-Component System ZraPSR Is a Novel ESR that Contributes to Intrinsic Antibiotic Tolerance in Escherichia coli[END_REF]. ∆zra has increased membrane disruption during treatment with membrane targeting antibiotics. Zra chaperone activity is enhanced in the presence of zinc ions. In this case, zinc was proposed to be a marker of envelope stress perturbation where ZraPSR is a sentinel sensing and responding to zinc entry into the periplasm [START_REF] Petit-Hartlein | Biophysical and physiological characterization of ZraP from Escherichia coli, the periplasmic accessory protein of the atypical ZraSR two-component system[END_REF].

We find that the AG tolerance conferred by ravA-viaA deletion in V. cholerae requires the presence of the Cpx system and Zra-like system, which we propose to name zraP2-zraS2-zraR2. We further show for the first time that ravA-viaA deletion together with inactivation of Cpx or Zra2 envelope stress responses leads to outer membrane permeabilization and may confer vancomycin sensitivity to Gram-negative bacteria.

Results

ravvia deletion decreases sub-MIC aminoglycoside susceptibility in V. cholerae

In order to evaluate AG related roles of RavA-ViaA, we assessed the effect of ravA-viaA deletion (referred to as ∆ravvia below) and overexpression (chromosomal extra-copy, referred to as WT::ravviaOE+, where OE stands for overexpression). We first performed competitions against V. cholerae WT, in the absence and presence of sub-MIC doses of AGs: tobramycin (TOB) and gentamicin (GEN), and also of antibiotics from families other than AGs: chloramphenicol (CM) that targets translation and ciprofloxacin (CIP) that targets DNA replication.

Competition results show that (Figure 1A) (i) V. cholerae ∆ravvia has a growth advantage compared to WT during growth with AGs TOB and GEN (40 and 50 % MIC); (ii) V. cholerae WT::ravviaOE+ has a growth disadvantage compared to WT with TOB and GEN; (iii) no significant effect is observed in the presence of sub-MICs of tested antibiotics other than AGs. This suggests the specificity of the mechanism of action of RavA-ViaA to AGs, in V. cholerae. Note that the beneficial effect of ∆ravvia is observed here during aerobic growth, while in E. coli, it can only be observed in anaerobic conditions.

ravvia deletion increases tolerance to high doses of aminoglycosides in V. cholerae

Next, we tested the effect of ravvia deletion on resistance by measuring the minimal inhibitory concentration (MIC), and on survival to lethal treatment with AGs. The MIC of ∆ravvia seemed unchanged, or slightly increased compared to WT (1.2 to 1.5 µg/ml instead of 1.2 µg/ml) (Table 1). Single ∆ravA or ∆viaA show the same MIC as the deletion of the whole operon, as expected, since the two proteins form a complex. The ravviaOE+ mutant shows lower resistance (MIC at 0.75 µg/ml) (Table 1).

For survival to lethal treatment, we tested the tolerance of the strains to 5x and 10x MIC TOB doses for 6 hours. Deletion of ravvia strongly increases survival to lethal TOB doses, when compared to the WT strain (Figure 1B). No effect is observed upon treatment with CIP, Trimethoprim and Carbenicillin (Figure S1). Treatment to TOB 5 µg/ml during 3 hours shows that ∆ravvia is not affected by TOB during this treatment window (Figure 1B), but these cells still die upon longer treatment periods (6 hours) (Figure 1B), excluding any bacteriostatic action of AGs in ∆ravvia mutant. Thus, ∆ravvia is killed upon lethal AG treatment, even though at a much slower rate than the WT, which is the definition of a tolerant population [START_REF] Balaban | Definitions and guidelines for research on antibiotic persistence[END_REF].

The effect of ravvia is only partly due to differential aminoglycoside uptake

We tested whether RavA-ViaA complex impacts AG entry, using the AG neomycin coupled to the fluorophore Cy5 (Neo-cy5), as previously done [START_REF] Carvalho | Deficiency in cytosine DNA methylation leads to high chaperonin expression and tolerance to aminoglycosides in Vibrio cholerae[END_REF][START_REF] Lang | Sleeping ribosomes: Bacterial signaling triggers RaiA mediated persistence to aminoglycosides[END_REF][START_REF] Pierlé | Identification of the active mechanism of aminoglycoside entry in V. cholerae through characterization of sRNA ctrR, regulating carbohydrate utilization and transport[END_REF]. Neo-cy5 is a fluorescent AG specifically designed for bacterial uptake studies retains the properties of AGs in terms of uptake, mode of action, and activity against Gram-negative bacteria [START_REF] Azad | Fluorescent aminoglycoside antibiotics and methods for accurately monitoring uptake by bacteria[END_REF] . In this assay, cell fluorescence is proportional to Neo-cy5 uptake. AG uptake increased in ravviaOE+ (Figure 2A), suggesting that RavA-viaA overexpression facilitates AG entry into the bacterial cell. Surprisingly, AG uptake is not decreased in V. cholerae ∆ravvia (Figure 2A). Since AG uptake depends on PMF, we tested the effect of ∆ravvia on PMF. We used Mitotracker assay [START_REF] Lang | Sleeping ribosomes: Bacterial signaling triggers RaiA mediated persistence to aminoglycosides[END_REF][START_REF] El Mortaji | A peptide of a type I toxin-antitoxin system induces Helicobacter pylori morphological transformation from spiral shape to coccoids[END_REF], based on a fluorescent dye which accumulates inside the cell in a PMF dependent way. We observed increased PMF in ravviaOE+ strain (Figure 2B), strengthening the notion that increased AG entry of ravviaOE+ is due to increased PMF. Conversely, no significant decrease of PMF was detected in the AG tolerant ∆ravvia strain (Figure 2B), suggesting that the effect of ravvia on AG susceptibility may require additional explanation as simply PMF modulation. Moreover, as expected, sdh (succinate dehydrogenase) deletion increased fitness in AGs, because of a decrease in PMF [START_REF] Herisse | Silver potentiates aminoglycoside toxicity by enhancing their uptake[END_REF]. Simultaneous deletion of sdh and ravvia shows an additive fitness advantage (Figure 2C), suggesting that the mechanisms of increased fitness of ∆ravvia in sub-MIC TOB is not through a common pathway with the ∆sdh-dependent PMF decrease.

High throughput approaches point to a role of membrane stress two-component systems in ∆ravvia

In order to further understand changes due to ravvia deletion and to search for potential partners of RavA-ViaA, we decided to adopt transcriptomic and TN-seq approaches (Figure 3). RNAseq was performed on exponentially growing WT and ∆ravvia. Major changes in ∆ravvia compared to WT include more than 10-fold upregulation of sugar transporters, anaerobic respiration, consistent with recent study published by the Barras and Py laboratories [START_REF] Khoury | Bioenergetic State of Escherichia coli Controls Aminoglycoside Susceptibility[END_REF] and [START_REF] Wong | The RavA-ViaA Chaperone-Like System Interacts with and Modulates the Activity of the Fumarate Reductase Respiratory Complex[END_REF], and the VC_1314-1315-1316 genes (Table S1).

The VC_1314 gene and the VC_1315-VC_1316 operon show 8-to 10-fold upregulation. VC_1315 presents 35 % sequence identity which E. coli zraS gene. The ZraP regulator and ZraSR twocomponent membrane stress response system is involved in antibiotic resistance [START_REF] Rome | The Two-Component System ZraPSR Is a Novel ESR that Contributes to Intrinsic Antibiotic Tolerance in Escherichia coli[END_REF]. VC_1315 also presents 29 % (and 21 %) sequence identity with the V. cholerae (and E. coli) cpxA gene. The periplasmic CpxP is the negative regulator of the envelope stress response CpxRA system [START_REF] Raivio | The Escherichia coli Cpx envelope stress response regulates genes of diverse function that impact antibiotic resistance and membrane integrity[END_REF]. VC_1316 presents 26 % sequence identity with the E. coli cpxR and 23 % with the V. cholerae cpxR. VC_1314 does not show any sequence similarity neither to zraP, nor to cpxP. In E. coli, The CpxP-CpxAR system and the ZraP-ZraRS systems, were proposed to be functional homologues [START_REF] Petit-Hartlein | Biophysical and physiological characterization of ZraP from Escherichia coli, the periplasmic accessory protein of the atypical ZraSR two-component system[END_REF]. We called VC_1314-1315-1316, the zra-like system below in this manuscript.

In parallel to the transcriptomic study, we applied our previously described comparative TNseq approach [START_REF] Baharoglu | Identification of genes involved in low aminoglycosideinduced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase[END_REF][START_REF] Babosan | Non-essential tRNA and rRNA modifications impact the bacterial response to sub-MIC antibiotic stress[END_REF], to V. cholerae ∆ravvia, to search for genes that are important for survival in the presence of sub-MIC TOB, again in aerobic conditions. We sequenced mutant libraries before and after 16 generations without and with TOB at 50% of the MIC. After sequencing, comparative analysis of the number of detected gene inactivations between the two conditions, indicates whether a given gene is important for growth in the antibiotic (decreased number of reads), or wether its inactivation is beneficial (increased number of raeds), or unchanged. Tables S2, S3 and S4 show the exhaustive lists of at least 2-fold differentially detected genes with transposon insertions in WT and ∆ravvia, with and without TOB. Deletion of ravvia leads to changes in factors involved in carbon metabolism, iron and respiration, and membrane stress. We constructed deletion mutants in WT and ∆ravvia contexts and performed competition experiments (Figure S2) for 21 of these genes to validate TN-seq results and to identify factors necessary for AG tolerance of ∆ravvia. Competition results were mostly consistent with TN-seq data. Among identified factors, one, cpxP, has particularly caught our attention because its inactivation is beneficial in ∆ravvia (Figure S2), and because inactivation of ravvia in ∆cpxP does not cause an additional increase in fitness, suggesting that they may act in the same pathway. Note that unlike CpxP, the E. coli ZraP represses the expression of the zraPSR operon only when bound to zinc [START_REF] Petit-Hartlein | Biophysical and physiological characterization of ZraP from Escherichia coli, the periplasmic accessory protein of the atypical ZraSR two-component system[END_REF]. In the conditions of our TN-seq experiments in V. cholerae, there is no zinc supplementation, so if VC_1314 is a ZraP-like protein, it is not necessarily a repressor under the tested conditions. Upon envelope stress, E. coli ∆zraP shows increased membrane disruption [START_REF] Rome | The Two-Component System ZraPSR Is a Novel ESR that Contributes to Intrinsic Antibiotic Tolerance in Escherichia coli[END_REF]. At T0, we counted 1.4-fold less insertions in zraP-like (VC_1314) in ∆ravvia, than the WT, suggesting that in the absence of ravvia, ZraP function is more important. This is statistically significant (padj= 7.3x10 -5 ) but below the 2-fold change threshold and that's why it does not appear in the TN-seq results tables. Because Cpx and Zralike systems were identified both in TN-seq, and in RNA-seq, we decided to focus on the link between the AG tolerant phenotypes of ∆ravvia and envelope stress response through the Cpx and Zra-like systems.

Cpx and Zra-like system mediated envelope stress response systems are necessary for the fitness advantage of ∆ravvia during growth with sub-MIC AGs and AG tolerance

In order to assess the importance of the Cpx and the putative Zra-like systems in the response to AGs of ∆ravvia, we tested fitness and tolerance in competition and survival experiments in the absence of one or both of these systems. Figure 4ABC shows competition experiments with cpx and zra-like operons inactivation (Figure S3 shows the same competitions with statistical significance compared to the WT strain). Inactivation of cpx alone does not affect fitness in TOB (Figure 4A) while inactivation of zra-like alone increases fitness in TOB (Figure 4B). The fitness advantage of ∆zra-like depends on the presence of cpx, since deletion of cpx in ∆zra-like suppresses its fitness advantage (Figure 4C). Strikingly, the deletion of cpx or zra-like in ∆ravvia leads to, respectively, loss or strong decrease of the fitness advantage of ∆ravvia in TOB (Figure 4A andB). The triple mutant ∆ravvia ∆cpx ∆zra shows a phenotype similar to the ∆ravvia ∆cpx double mutant (Figure 4C). We performed additional competitions to ascertain the effect of deletion of zra in ∆ravvia ∆cpx, this time by competing the triple ∆ravvia ∆cpx ∆zra with ∆ravvia ∆cpx. We observe that deletion of zra has in fact an additional negative impact on the fitness of ∆ravvia ∆cpx (Figure 4D). These results show that Cpx envelope stress response system is necessary for the enhanced tolerance of ∆ravvia to TOB, and that the Zra-like system also contributes significantly to this fitness advantage.

We next performed TOB tolerance tests using a concentration of 5x MIC for 3 hours (Figure 4E). Under these conditions, the survival of the single ∆zra-like system mutant is slightly lower than WT, the survival of ∆cpx is lower and both systems seem to be additive as the double mutant appears to show even lower tolerance than the single ∆cpx. Strikingly, the high level of tolerance of ∆ravvia is completely lost upon deletion of zra-like and goes even lower than WT upon deletion of cpx and in the triple mutant. For unknown reasons, the decrease of tolerance is stronger in ∆zra-like ∆cpx than in ∆zra-like ∆cpx ∆ravvia, as if deletion of zra-like in ∆ravvia ∆cpx was beneficial. In any case, the AG tolerance conferred by the deletion of ravvia necessitates the presence of both Cpx and Zra-like systems.

∆avvia related phenotypes are linked to extracellular zinc concentrations

Since Cpx and Zra systems have previously been associated with metals such as iron and zinc, we next performed competition experiments in the presence of these metals. The presence of supplemented iron did not affect the fitness of the ∆ravvia derivatives in any condition (Figure S4ABC). Zinc supplementation (Figure 5ABC) restores fitness in TOB for the ∆ravvia ∆cpx and ∆ravvia ∆cpx double mutants but not for the triple mutant, suggesting that the effect of zinc in ∆ravvia is somehow linked to the Cpx and Zra-like systems, which may act in a redundant way. Moreover, while zinc has no effect on the TOB tolerance phenotype of ∆ravvia or ∆ravvia ∆zralike, it restores high tolerance to the ∆ravvia ∆cpx mutant (Figure 5D), which is consistent with the zinc-dependent increase of fitness of the ∆ravvia ∆cpx mutant shown in Figure 5C. We wondered whether these genes could be regulated by zinc. We found that cpx, but not zra, mRNA levels are increased in the presence of zinc (Figure 5E). Ravvia expression from Pravvia promoter fused to gfp is also induced by zinc (Figure 5F). Overall, results indicate that the ∆ravvia mutant's AG tolerance is dependent on the Cpx and Zra-like systems, and that ravvia function is also somehow associated with the sensing of zinc levels.

∆ravvia has a low ROS phenotype which depends on Cpx/Zra

We have previously shown that sub-MIC TOB leads to reactive oxygen species (ROS) formation in V. cholerae, which induces the bacterial SOS stress response [START_REF] Baharoglu | RpoS Plays a Central Role in the SOS Induction by Sub-Lethal Aminoglycoside Concentrations in Vibrio cholerae[END_REF]. However ∆ravvia is not more resistant to H2O2 (not shown). Considering that ∆ravvia is more tolerant to TOB and that ∆ravvia fails to induce SOS response in presence of TOB [START_REF] Baharoglu | Identification of genes involved in low aminoglycosideinduced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase[END_REF], we hypothesized that these two observations could be explained by a diminished ROS formation in ∆ravvia. We used the CellROX dye that, upon increased levels of ROS (O2 -and •OH), emits green fluorescence. We observed that lack of ravvia in V. cholerae leads to decreased ROS generation, both in the absence and presence of sub-MIC TOB (Figure 6, MH and sub-MIC TOB). We similarly observed that ∆ravvia produces decreased levels of ROS upon treatment with lethal doses of TOB (Figure S5).

We next tested whether the function of Cpx/Zra-like systems in ∆ravvia could be involved in the "low ROS" phenotype observed for ∆ravvia. Figure 6 shows that the deletions of either cpx or zralike (or both) suppress this phenotype, meaning that both Zra-like and Cpx are involved in the low ROS phenotype of ∆ravvia. This is consistent with the fact that both systems are also necessary for AG tolerant phenotype of ∆ravvia. As a control, we also tested the double mutant ∆ravvia ∆bcp. Bcp is a thiol peroxidase responding to oxidative stress. We see no effect of bcp deletion on the low ROS phenotype of ∆ravvia. In conclusion, ∆ravvia shows a low ROS phenotype, which is reversed upon inactivation of Cpx and Zra-like stress responses.

Simulatenous deletion of Cpx or Zra-like systems with ∆ravvia leads to outer membrane permeability

Since Cpx and Zra systems, have been known for their involvement in envelope stress response, we decided to test the effect of ravvia deletion mutant and derivatives on outer membrane permeability.

Vancomycin is an antibiotic targeting the synthesis of the peptidoglycan, but which cannot be used to treat gram-negative bacteria, because its large molecular weight prevents it from crossing the outer membrane (OM) through porins, and penetrate into the cell [START_REF] Delcour | Outer membrane permeability and antibiotic resistance[END_REF]. When the OM is damaged however, vancomycin uptake by gram negative bacteria is possible [START_REF] Song | A broad-spectrum antibiotic adjuvant reverses multidrug-resistant Gram-negative pathogens[END_REF] [START_REF] Lei | Potentiation of Vancomycin: Creating Cooperative Membrane Lysis through a "Derivatization-for-Sensitization" Approach[END_REF]. We tested whether deletion of ravvia has an impact on vancomycin entry, by measuring the MICs of the different mutants. Our results show that single deletions of ∆ravvia, ∆cpx or ∆zra-like do not affect the MIC to vancomycin, while double deletions of ∆ravvia together with ∆cpx or ∆zra-like or both decreases the MIC from >256 µg/ml to about 64-100 µg/ml (Figure 7A), suggesting that simultaneous inactivation of ravvia function together with envelope stress response systems leads to OM damage or permeabilization.

Changes in OM permeability can be quantified using nitrocefin [START_REF] Mathur | The Vibrio cholerae ToxR-regulated porin OmpU confers resistance to antimicrobial peptides[END_REF], a chromogenic probe which develops color (at 490 nm) upon entry into the periplasm, in the presence of β-lactamase. We thus measured permeability of ∆ravvia and ∆cpx/∆zra-like derivatives transformed with the low-copy pSC101 plasmid carrying the bla gene. Again, results show that neither single deletions of ∆ravvia, ∆cpx or ∆zra-like, nor the double ∆cpx ∆zra-like deletion affect nitrocefin entry, while double deletions of ∆ravvia together with ∆cpx or ∆zra-like or both, strongly increases it, consistent with increased OM permeability (Figure 7B). Since AGs also are also expected to cross the cell envelope with higher efficacy with increased OM permeability, we checked whether AG uptake is increased in ∆ravvia ∆cpx or ∆ravvia ∆zra-like or the triple mutant (Figure 7C). Consistent with vancomycin and nitrocefin entry results, neo-cy5 uptake is also increased in ∆ravvia ∆cpx, ∆ravvia ∆zra-like, but not in single mutants or the ∆cpx ∆zra-like mutant. Altogether, these results point to increased outer membrane permeability when either Cpx or Zra-like system is inactivated in ∆ravvia.

Discussion

This study presents the first evidence of a link between RavA-ViaA function and the response to envelope stress. In fact, we identify the Cpx and a Zra-like two-component systems to be involved in the increased tolerance of V. cholerae ∆ravvia to AGs. Since Cpx and Zra systems are known to respond to protein misfolding at the periplasm, one can speculated that inactivationb of ravvia could generate an increase in misfolded periplasmic proteins.

In E. coli, ∆cpx mutants show alterations in conjugational plasmid transfer, transport, ability to grow on some carbon sources and resistance to AGs. More generally, the E. coli Cpx transcriptome impacts inner membrane associated processes such protein secretion, and other processes like iron homeostasis, translation , and interestingly, ribosome protection factors raiA and rmf [START_REF] Raivio | The Escherichia coli Cpx envelope stress response regulates genes of diverse function that impact antibiotic resistance and membrane integrity[END_REF]. Cpx also negatively regulates respiration, energy, and TCA cycle genes. In V. cholerae, Cpx senses and responds to low iron, and induces iron transporters and efflux pumps [START_REF] Acosta | The Vibrio cholerae Cpx envelope stress response senses and mediates adaptation to low iron[END_REF]. Disruption of the OM and accumulation of misfolded proteins in the periplasm were shown to induce the Cpx regulon [START_REF] Buelow | Cpx signal transduction is influenced by a conserved N-terminal domain in the novel inhibitor CpxP and the periplasmic protease DegP[END_REF][START_REF] Raivio | Tethering of CpxP to the inner membrane prevents spheroplast induction of the cpx envelope stress response[END_REF][START_REF] Raivio | The Cpx envelope stress response is controlled by amplification and feedback inhibition[END_REF]. The fact that our TNseq data also identified the tat operon as important for the growth advantage of ∆ravvia in TOB also supports the existence of a so far unknown link between RavA-ViaA and misfolded proteins in the periplasm. Cpx activity is regulated by successive phosphorylations (except for cpxP): envelope stress leads to phosphorylation and activation of CpxA, which phosphorylates and activates CpxR [START_REF] Digiuseppe | Signal detection and target gene induction by the CpxRA twocomponent system[END_REF]. Envelope stress in parallel inactivates the repressor CpxP, because CpxP binds misfolded membrane proteins and is thus titrated away from CpxA. Thus, the Cpx activity is not solely regulated by activation of the promoter.

Such protein misfolding at the periplasm can happen during AG treatment, and Cpx was in fact previously linked to AG resistance [START_REF] Bryan | Effects of membrane-energy mutations and cations on streptomycin and gentamicin accumulation by bacteria: a model for entry of streptomycin and gentamicin in susceptible and resistant bacteria[END_REF][START_REF] Thorbjarnardottir | Mutations determining generalized resistance to aminoglycoside antibiotics in Escherichia coli[END_REF]. Interestingly, Cpx was also shown to induce the heat-shock sigma factor RpoH in the presence of the AG gentamicin, indicating that the effect AG gentamicin at the membrane is sufficient to trigger the response to misfolded IM protein stress by Cpx [START_REF] Cudic | The role of the two-component systems Cpx and Arc in protein alterations upon gentamicin treatment in Escherichia coli[END_REF]. ∆cpxR is more susceptible to AGs in E. coli [START_REF] Guest | A Bacterial Stress Response Regulates Respiratory Protein Complexes To Control Envelope Stress Adaptation[END_REF], and in Salmonella independently of efflux pumps [START_REF] Jing | Role of cpxA Mutations in the Resistance to Aminoglycosides and beta-Lactams in Salmonella enterica serovar Typhimurium[END_REF], and also independently of oxygen consumption or PMF [START_REF] Guest | A Bacterial Stress Response Regulates Respiratory Protein Complexes To Control Envelope Stress Adaptation[END_REF], but involves altered protein composition at the membrane [START_REF] Raivio | Everything old is new again: an update on current research on the Cpx envelope stress response[END_REF]. The AG gentamicin was shown to activate the Cpx response [START_REF] Kashyap | Bactericidal peptidoglycan recognition protein induces oxidative stress in Escherichia coli through a block in respiratory chain and increase in central carbon catabolism[END_REF]. As a corollary, activation of Cpx leads to increased AG resistance. This was shown to be due to protection against AGs at the membrane [START_REF] Raivio | The Escherichia coli Cpx envelope stress response regulates genes of diverse function that impact antibiotic resistance and membrane integrity[END_REF][START_REF] Mahoney | The Cpx stress response confers resistance to some, but not all, bactericidal antibiotics[END_REF], partly through regulation of protein degradation at the inner membrane [START_REF] Raivio | Everything old is new again: an update on current research on the Cpx envelope stress response[END_REF] and partly to downregulation of electron transport chains and iron import [START_REF] Raivio | The Escherichia coli Cpx envelope stress response regulates genes of diverse function that impact antibiotic resistance and membrane integrity[END_REF]. Cpx was in fact described to repress nuo and cyo aerobic respiratory complexes in E. coli [START_REF] Guest | A Bacterial Stress Response Regulates Respiratory Protein Complexes To Control Envelope Stress Adaptation[END_REF], which could be toxic in conditions challenging membrane integrity, and such repression allows bacterial cells to adapt to conditions disrupting membrane integrity, among which AG induced protein misfolding. There are no described sequence homologues for nuo or cyo operons in V. cholerae. The functional equivalent may be the nqr system [START_REF] Agarwal | Impact of Na(+)-Translocating NADH:Quinone Oxidoreductase on Iron Uptake and nqrM Expression in Vibrio cholerae[END_REF](VC_2291-2-3-4), for which we see that inactivation is beneficial in WT TOB but not in ∆ravvia TOB, maybe because it is already down-regulated (approximately 2-fold but with p-value> 0,05) in ∆ravvia.

Since the Cpx response is involved in the biogenesis of large complexes present at the envelope (respiratory complexes, but also type 4 pili, and maybe others), one could envisage that the action of Cpx may be through reduced protein trafficking at the inner membrane, hence reduced membrane stress. These properties -decrease of respiration and iron import by Cpx -can explain how ROS formation increases in ∆ravvia upon cpx deletion, because ROS are mainly produced with oxygen and iron through the Fenton reaction. Cpx is also closely linked to energy status of the cell and regulates protein folding and degrading factors, which are involved in adaptation to stress caused by high level of respiration [START_REF] Tsviklist | The Cpx Stress Response Regulates Turnover of Respiratory Chain Proteins at the Inner Membrane of Escherichia coli[END_REF].

In E. coli, ZraPSR is involved in resistance to several drugs and is important for membrane integrity [START_REF] Rome | The Two-Component System ZraPSR Is a Novel ESR that Contributes to Intrinsic Antibiotic Tolerance in Escherichia coli[END_REF]. Zra chaperone activity is enhanced by zinc. There is often significant overlap between processes affected by Zinc and by ROS, with zinc having mostly antioxidant function [START_REF] Hubner | Interactions of zinc-and redox-signaling pathways[END_REF]. ∆zra has increased membrane disruption during treatment with membrane targeting antibiotics. ChIP-seq and transcriptomic studies found that Zra controls the expression of acr, raiA, rpoH, etc. In V. cholerae, we find that the operon VC_1314-VC_1315-VC_1316 is highly upregulated in ∆ravvia. Although VC_1314 (487 aa) does not present any sequence homology to neither zra nor cpx systems, VC_1315 (449 aa) and VC_1316 (158 aa) present homologies respectively with E. coli zraS (465 aa) and cpxR (232 aa). Since the V. cholerae VC_1314-VC_1315-VC_1316 zra-like system also responds to zinc, we propose to name it zraP2-zraS2-zraR2.

One question remains open: why does deletion of the RavA-ViaA function activate the Cpx/Zra2 systems? The answer could come from the fact that when both RavA-ViaA and Cpx or Zra2 are inactivated, membrane permeability increases as observed with vancomycin and nitrocefin entry. The mechanism could be a complex one, since we also observe that RNA modifications, which impact translation [START_REF] Babosan | Non-essential tRNA and rRNA modifications impact the bacterial response to sub-MIC antibiotic stress[END_REF], also impact ∆ravvia phenotypes. Note that V. cholerae harbors two groESL operons, with gro1 being essential and gro2 accessory. We have recently shown the involvement of gro2 in the response to AGs [START_REF] Carvalho | Deficiency in cytosine DNA methylation leads to high chaperonin expression and tolerance to aminoglycosides in Vibrio cholerae[END_REF]. Here, even in the absence of AGs, gro2 is already essential for ∆ravvia. A specific target of this chaperone may be important in the absence of ravvia. ∆ravvia increases resistance to AGs but the importance of gro2 and clpS may point to the existence of endogenous membrane protein stress in ∆ravvia. Thus, the presence of RavA-ViaA seems to be useful in order to maintain envelope integrity, and its function as a barrier against the entry of exogenous agents, such as the last resort antibiotic vancomycin.

Materials and methods

Table S5 shows strains used in this study and their construction. Table S6 shows primer sequences.

Media and Growth Conditions

Platings were done at 37°C, in Mueller-Hinton (MH) agar media. Liquid cultures were grown at 37°C in MH in aerobic conditions, with 180 rotations per minute. Competition experiments were performed as described [START_REF] Babosan | Non-essential tRNA and rRNA modifications impact the bacterial response to sub-MIC antibiotic stress[END_REF]: overnight cultures from single colonies of mutant lacZand WT lacZ+ strains were mixed 1:1 (500 μl + 500 μl). At this point 100 μl of the mix were serial diluted and plated on MH agar supplemented with X-gal (5-bromo-4-chloro-3-indolyl-β-Dgalactopyranoside) at 40 μg/ml to assess T0 initial 1:1 ratio. At the same time, 5 μl from the mix were added to 200 µl of MH or MH supplemented with sub-MIC antibiotics (concentrations, unless indicated otherwise: TOB: tobramycin 0.6 μg/ml; GEN: 0.5 µg/ml; CIP: ciprofloxacin 0.01 μg/ml, CRB: carbenicillin 2.5 μg/ml), PQ: paraquat 10 µM, or H2O2: 2 mM. Cultures were incubated in 96 well plates with agitation at 37°C for 24 hours, and then diluted and plated on MH agar plates supplemented with Xgal. Plates were incubated overnight at 37°C and the number of blue and white CFUs was assessed. Competitive index was calculated by dividing the number of white CFUs (∆lacZ-strain) by the number of blue CFUs (lacZ+ strain) and normalizing this ratio to the T0 initial ratio.

MIC determination

Stationary phase cultures grown in MH were diluted 20 times in PBS, and 300 μl were plated on MH plates and dried for 10 minutes. Etest straps (Biomérieux) were placed on the plates and incubated overnight at 37°C. Survival/tolerance tests were performed on early exponential phase cultures. The overnight stationary phase cultures were diluted 1000X and grown until OD 600 nm of 0.35 to 0.4, at 37°C with shaking, in Erlenmeyers containing 25 ml fresh MH medium. Appropriate dilutions were plated on MH plates to determine the total number of CFUs in time zero untreated cultures. 5 ml of cultures were collected into 50 ml Falcon tubes and treated with lethal doses of desired antibiotics (5 or 10 times the MIC: tobramycin 5 or 10 μg/ml, carbenicillin 50 μg/ml, ciprofloxacin 0.025 μg/ml) for 30 min, 1 hour, 2 hours and 4 hours if needed, at 37°C with shaking in order to guarantee oxygenation. 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 8 times.

Quantification of fluorescent neomycin uptake was performed as described [START_REF] Lang | Sleeping ribosomes: Bacterial signaling triggers RaiA mediated persistence to aminoglycosides[END_REF]. Neo-Cy5 is an aminoglycoside coupled to the fluorophore Cy5, and has been shown to be active against Grambacteria [START_REF] Azad | Fluorescent aminoglycoside antibiotics and methods for accurately monitoring uptake by bacteria[END_REF][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]. Briefly, overnight cultures were diluted 100-fold in rich MOPS (Teknova EZ rich defined medium). When the bacterial strains reached an OD 600 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, 100000 events were counted on the Miltenyi MACSquant device.

PMF measurements

Quantification of PMF was performed using the Mitotracker Red CMXRos dye (Invitrogen) as described [START_REF] El Mortaji | A peptide of a type I toxin-antitoxin system induces Helicobacter pylori morphological transformation from spiral shape to coccoids[END_REF], in parallel with the neo-Cy5 uptake assay, using the same bacterial cultures. 50 µl of each culture were mixed with 60 µl of PBS. Tetrachlorosalicylanilide TCS (Thermofischer), a protonophore, was used as a negative control with a 500 µM treatment applied for 10 minutes at room temperature. Then, 25 nM of Mitotracker Red were added to each sample and let at room temperature for 15 minutes under aluminium foil. 20 μL of the treated culture were then used for flow cytometry, diluted in 200 μL of PBS before reading fluorescence.

ROS measurements

Overnight cultures were diluted 1000X in MH medium and grown until an OD 600 nm of 0.3. Then, 100 µl of each culture was transferred to a 96-well plate, and treated with 1 µl of 250 µM CellRox Green (Thermofischer Scientific), for 30 minutes at 37 degrees, under aluminium foil. For flow cytometry, 10 µl were mixed into 200 µl of PBS. Fluorescence per cell was read on 100000 events, on the MACSquant device at 488 nm.

RNA purification and RNA-seq:

Cultures were diluted 1000X and grown in triplicate in MH to an OD 600 nm of 0.4. Frist, 1.5 ml of TriZol reagent was added to 500 µl of culture pellet followed by the addition of 300 µl of chloroforme. After centrifugation, the upper phase was mixed with a 1:1 volume of 70 % ethanol before column purification. RNA was purified with the RNAeasy mini kit (Qiagen) according to manufacturer instruction (from step 4 of the protocole Part 1). Quality of RNA was controlled using the Bioanalyzer. 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].

Transposon insertion sequencing

Libraries were prepared as previously described [START_REF] Baharoglu | Identification of genes involved in low aminoglycosideinduced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase[END_REF][START_REF] Chiang | Construction of a mariner-based transposon for epitope-tagging and genomic targeting[END_REF], to achieve a library size of 600000 clones, and subjected to passaging in MH and MH + TOB 0.5 µg/ml for 16 generations [START_REF] Negro | RadD Contributes to R-Loop Avoidance in Sub-MIC Tobramycin[END_REF]. A saturated mariner mutant library was generated by conjugation of plasmid pSC1819 from E .coli to V. cholerae WT. Briefly, pSC189 [START_REF] Baharoglu | Identification of genes involved in low aminoglycosideinduced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase[END_REF][START_REF] Chiang | Construction of a mariner-based transposon for epitope-tagging and genomic targeting[END_REF] was delivered from E. coli strain 7257 (β2163 pSC189::spec, laboratory collection) into the V. cholerae WT strain. Conjugation was performed for 2 h on 0.45 µM filters. The filter was resuspended in 2 ml of MH broth. Petri dishes containing 100 µg/ml spectinomycin were then spread. The colonies were scraped and resuspended in 2 ml of MH. When sufficient single mutants were obtained (>600000 for 6X coverage of non-essential regions), a portion of the library was used for gDNA extraction using Qiagen DNeasy Blood & Tissue Kit as per manufacturer's instructions. This was used for library validation through insert amplification by nested PCR using a degenerate primer (ARB6), which contains 20 defined nucleotides followed by a randomized sequence. This was combined with a primer anchored in the edge of the transposon sequence (MV288) [START_REF] Baharoglu | Identification of genes involved in low aminoglycosideinduced SOS response in Vibrio cholerae: a role for transcription stalling and Mfd helicase[END_REF][START_REF] Negro | RadD Contributes to R-Loop Avoidance in Sub-MIC Tobramycin[END_REF]. After this, primer ARB3, which contains the first 20 nucleotides of ARB6 was used for nested amplification in combination with MV288. After validation, the libraries were passaged in MH media for 16 generations with or without 50 % MIC of TOB, in triplicates. gDNA from time point 0 and both conditions after 16 generation passage in triplicate was extracted. Sequencing libraries were prepared using Agilent's SureSelect XT2 Kit with custom RNA baits designed to hybridize the edges of the Mariner transposon. The 100 ng protocol was followed as per manufacturer's instructions. A total of 12 cycles were used for library amplification. Agilent's 2100 Bioanalzyer was used to verify the size of the pooled libraries and their concentration. HiSeq Paired-end Illumina sequencing technology was used producing 2 x 125 bp long reads. Reads were then filtered through transposon mapping to ensure the presence of an informative transposon/genome junction using a previously described mapping algorithm [START_REF] Pierle | Genetic Diversity of Tick-Borne Rickettsial Pathogens; Insights Gained from Distant Strains[END_REF]. Informative reads were extracted and mapped. Reads were counted when the junction was reported as mapped inside the CDS of a gene plus an additional 50 bp upstream and downstream. Expansion or decrease of fitness of mutants was calculated in fold changes with normalized insertion numbers. Normalization calculations were applied according to van Opijnen et al [START_REF] Van Opijnen | Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms[END_REF]. Expansion or decrease of fitness of mutants was calculated in fold changes with normalized insertion numbers. Baggerly's test on proportions [START_REF] Baggerly | Differential expression in SAGE: accounting for normal between-library variation[END_REF] was used to determine statistical significance as well as a Bonferroni correction for multiple hypotheses testing.

mRNA quantifications by digital-RT-PCR

qRT-PCR reactions were prepared with 1 μl of diluted RNA samples using the qScript XLT 1-Step RT-qPCR ToughMix (Quanta Biosciences, Gaithersburg, MD, USA) within Sapphire chips. Digital PCR was conducted on a Naica Geode (programmed to perform the sample partitioning step into droplets, followed by the thermal cycling program suggested in the user's manual. Image acquisition was performed using the Naica Prism3 reader. Images were then analyzed using Crystal Reader software (total droplet enumeration and droplet quality control) and the Crystal Miner software (extracted fluorescence values for each droplet). Values were normalized against expression of the housekeeping gene gyrA as previously described [START_REF] Scrudato | The regulatory network of natural competence and transformation of Vibrio cholerae[END_REF].

Quantification of gfp fusion expression by fluorescent flow cytometry

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] on overnight cultures and repeated at least 3 times. For each experiment, 50000 to 100000 events were counted on the Miltenyi MACSquant device. Transcriptional fusion: ravvia promoter sequence fused to gfp by amplification of Pravvia-gfp from pZE1-gfp [START_REF] Elowitz | A synthetic oscillatory network of transcriptional regulators[END_REF] using primers ZIP537/ZIP200. The fragment was cloned into pTOPO-TA cloning vector. The Pravvia-gfp fragment was then extracted using EcoRI and cloned into the low copy plasmid pSC101. The plasmid was introduced into desired strains, and fluorescence was measured on indicated conditions, by counting 100000 cells on the Miltenyi MACSquant device Growth on microtiter plate reader Overnight cultures were diluted 1:500 in fresh MH medium, on 96 well plates. Each well contained 200 μl. Plates were incubated with shaking on TECAN plate reader device at 37°C, OD 600 nm was measured every 15 minutes. Tobramycin was used at sub-MIC: TOB 0.6 μg/ml. The concentrations of other antibiotics are specified on each figure.

Quantification of nitrocefin entry.

Nitrocefin, a chromogenic probe, 3-(2,4-dinitrostyryl)-(6 R,7 R)-7-(2-thienylacetamido)-ceph-3-em-4carboxyl acid (Calbiochem), was used to assess membrane permeability. Tested strains were transformed with the low copy pSC101 plasmid carrying the bla gene coding for a β-lactamase, a periplasmic protein which allows coloration of nitrocefin upon entry into the bacterial cell. Cells were grown to stationary phase, washed twice and resuspended at a concentration of 5×10 7 cells/ml in PBS. The reaction was performed with 175 μl of PBS buffer and 25 μl of the nitrocefin 0.5 mg/ml stock solution, in 96 well plates. 50 μl of the bacterial suspension was added and the OD at 490 nm was measured every 2 min for 45 min using a plate reader at 37°C, with shaking for 10 s every minute.

Data availability:

The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE196651 (GSM5897403, GSM5897404, GSM5897405, GSM5897412, GSM5897413, GSM5897414) for RNAseq data and GSE198341 for TNseq data (GSM5945317, GSM5945318, GSM5945319, GSM5945323, GSM5945324, GSM5945325, GSM5945329, GSM5945330, GSM5945331, GSM5945338, GSM5945339, GSM5945340, GSM5945341, GSM5945342, GSM5945343). S1. Pie-chart for TN-seq recapitulates gene categories for differentially detected transposon insertions in ∆ravvia compared to WT, in all conditions shown in Tables S2, S3 and S4.

Supplementary figures

Figure S2. Impact of selected gene deletions in WT and ∆ravvia on fitness during growth in sub-MIC antibiotics: in vitro competition experiments of V. cholerae WT and mutant strains in the absence or presence of TOB at sub-MICs (50% of the MIC).

We were looking for factors for which inactivation would lead to loss of the fitness advantage of ∆ravvia in AGs. We tested the effect of 21 gene deletions, selected because they are important in WT but not in ∆ravvia (panel with red square), important in ∆ravvia (green panel) or beneficial in WT only (purple panel). For 16 of them, none completely suppressed this phenotype. TN-seq screen identified functions that are necessary for AG resistance in ∆ravvia. (i) in the absence of TOB: several genes are no longer essential or as important in ∆ravvia as in WT. These genes include carbohydrate utilization and metabolism genes (crp), iron and respiration related factors (feoC, iscX, trxC), and envelope related genes (cpxP). This suggests that deletion of ravvia leads to changes in carbon metabolism, iron and respiration, and membrane stress. This is consistent with transcriptomic data. Several genes become essential or important in ∆ravvia: genes involved in respiration and iron utilization (e.g. rdx, bcp); carbohydrate metabolism (crr), translation (truC, rluE) and protein stress (groES2, clpS, not tested here in competition). (ii) after growth with TOB: several genes are no longer needed in ∆ravvia: electron transport/redox, carbohydrate metabolism (sgrR), stringent response and translation stress (e.g. raiA, hpf, not tested here), envelope and cell division, e.g. cspE, coding for a cold shock transcription antiterminator which interacts specifically with mRNAs that encode membrane proteins [START_REF] El Mortaji | A peptide of a type I toxin-antitoxin system induces Helicobacter pylori morphological transformation from spiral shape to coccoids[END_REF]. In summary, several functions appear to be less needed in ∆ravvia: proteins protecting ribosomes upon translation stress (e.g. hibernation factors), consistent with the that AGs cause less translation stress in ∆ravvia. For cytochromes, their inactivation probably decreases PMF and confers AG resistance in WT but since ∆ravvia is already more resistant, their effect on PMF may have little impact on AG tolerance of ∆ravvia. Notable phenotypes were conferred by deletion of the tat operon (export of folded proteins to the perisplasm) which leads to loss of ∆ravvia's growth advantage in TOB, as well as RNA modification factors dusB, rluB, rluE, for which deletion is known to be beneficial in TOB [START_REF] Khoury | Bioenergetic State of Escherichia coli Controls Aminoglycoside Susceptibility[END_REF], and cpxP which confers an advantage only to the WT strain. MH: no antibiotic treatment (black dots). TOB: tobramycin 0.6 μg/ml (blue dots). The Y-axis represents log2 of competitive index value calculated as described in the methods. A competitive index of 1 (i.

3.9

VC_0957 ybeL 3.6 VC_A0619 3.0 VC_1690 2.8 VC_2470 2.5 VC_1710 2.2 VC_1698 2.2 VC_A0971 2.1 VC_A0330 2.1 VC_1125 2

  Statistical significance. For multiople comparisons, we used one-way ANOVA. **** means p<0.0001, *** means p<0.001, ** means p<0.01, * means p<0.05. ns: non-significant. All statistical tests were corrected for multiple comparisons and false discovery using the Bonferroni correction.

Figure 2 :

 2 Figure 2: Effect of RavA-ViaA on AG uptake and membrane potential. A. Intracellular level of neomycin coupled to the fluorophore Cy5 measured by fluorescence associated flow cytometry. Error bars represent standard deviation. B. Quantification of changes in PMF using Mitotracker Red fluorescence measured by flow cytometry. Representative acquisitions are shown: fluorescence is represented in the x-axis (FITC channel), the y-axis represents the number of events corresponding to the number of cells, normalized to height (same number of total cells for both conditions). Each plot represents one experiment. C. Competition experiments of V. cholerae WT and indicated mutants. MH: no antibiotic treatment (black). TOB: tobramycin 0,6 µg/ml (blue). The Y-axis represents log 2 of competitive index value calculated as described in the methods. A competitive index of 1 (i.e. log2 value of 0) indicates equal growth of both strains. For statistical significance calculations, we used oneway ANOVA. **** means p<0.0001, *** means p<0.001, ** means p<0.01, * means p<0.05. ns: nonsignificant. Number of replicates for each experiment: n=3. Only significant p values are shown.

Figure 3 :

 3 Figure 3: High throughput methods identify factors that are differentially detected in ∆ravvia compared to WT. Pie-chart for RNA-seq recapitulates differentially (up-or down-) expressed gene categories in ∆ravvia, as shown in TableS1. Pie-chart for TN-seq recapitulates gene categories for differentially detected transposon insertions in ∆ravvia compared to WT, in all conditions shown in Tables S2, S3 and S4.

Figure 4 :

 4 Figure 4: Cpx and Zra-like two component envelope stress response systems are involved in fitness increase of ∆ravvia with TOB and TOB tolerance. ABC. Competitions. The effect of deletion of cpx (A),or zra-like (B), or both (C) on competitive index in MH without and with TOB, where MH is the untreated growth medium. In vitro competition experiments of V. cholerae WT and indicated mutants in specified media: in black: MH: no antibiotic treatment. In blue: TOB: tobramycin 0,6 µg/ml. The Yaxis represents log2 of competitive index value calculated as described in the methods. A competitive index of 1 (i.e. log2 value of 0) indicates equal growth of both strains. D. Competitive index in MH without and with TOB, of the triple mutant (indicated as ∆∆∆) ∆ravvia ∆cpx ∆zra against ∆ravvia ∆cpx. E. Tolerance. Cultures were grown to exponential phase in MH medium. Survival of WT and ∆ravvia to 3-hours treatment with lethal TOB at 5x MIC 5 µg/ml was measured. The Y-axis represents log2 value of survival ratios, calculated as survival of the mutant over survival of the WT. A relative survival ratio of 1 (i.e. log2 = 0) indicates equal survival as the WT strain. For statistical significance calculations, we used one-way ANOVA. **** means p<0.0001, *** means p<0.001, ** means p<0.01, * means p<0.05. ns: non-significant. Number of replicates for each experiment: 3<n<8.

Figure 5 :

 5 Figure5: Effect of zinc supplementation. ABC. Competitions. Cultures were grown to exponential phase in MH medium supplemented with zinc during growth. "Zn" stands for ZnCl2: 1.5mM. The effect of deletion of cpx (A), or zra-like (B), or both (C) on competitive index in MH without and with TOB, where MH is the untreated growth medium. In vitro competition experiments of V. cholerae WT and indicated mutants in specified media: in black: MH: no antibiotic treatment. In blue: TOB: tobramycin 0,6 µg/ml. The Y-axis represents log2 of competitive index value calculated as described in the methods. A competitive index of 1 (i.e. log2 value of 0) indicates equal growth of both strains. D. Survival of WT and ∆ravvia to 3-hours treatment with lethal TOB at 5x MIC 5 µg/ml, in presence of zinc. The Y-axis represents log2 value of survival rates ratios, calculated as survival of the mutant over survival of the WT. A relative survival ratio of 1 (i.e. log2 = 0) indicates equal survival as the WT strain. E. Expression of cpx and zra. mRNA levels were measured using digital RT-PCR as explained in materials and methods. The Y-axis represents the fold change of induction in presence of zinc divided by the expression in the absence of zinc. F. Expression from promoter of ravvia was measured using fluorescent transcriptional fusion of gfp expressed from ravvia promoter, and quantified using flow cytometry. The Y-axis represents the relative fluorescence in the indicated conditions. Zn: zinc 1.5 mM. NT: non-treated. ∆zur: zur deletion strain. WT: wild type strain. For statistical significance calculations, we used one-way ANOVA. **** means p<0.0001, *** means p<0.001, ** means p<0.01, * means p<0.05. ns: non-significant. Number of replicates for each experiment: 3<n<6.

Figure 6 :

 6 Figure 6: Low ROS phenotype of ∆ravvia is dependent on the presence of Cpx and Zra-like stress response systems. Quantification of variation of reactive oxygen species using CellRox. The y-axis represents log2 fold-change of detected ROS fluorescence in the indicated strain over the WT strain. Each experiment was performed at least 3 times and data and statistical significance are shown in the histograms. For statistical significance calculations, we used one-way ANOVA. **** means p<0.0001, ** means p<0.01. ns means non-significant.

Figure 7 :

 7 Figure 7: Response of ∆ravvia to the membrane targeting antibiotics. A. Minimum inhibitory concentration of vancomycin. MIC values are represented in the Y-axis for the indicated strains. B. Outer membrane permeability to nitrocefin measured on stationary phase cultures diluted to 5 × 10 7 cells/ml by measuring the OD at 490 nm for 40 min. C. AG uptake quantified through Neo-cy5 entry into exponential phase cultures. Intracellular level of neomycin coupled to the fluorophore Cy5 measured by fluorescence associated flow cytometry. Error bars represent standard deviation. For statistical significance calculations, we used one-way ANOVA. **** means p<0.0001. Only significant p values are shown.

Figure S1 :

 S1 Figure S1: Effect of RavA-ViaA on tolerance to antibiotics. A. Survival of WT and ∆ravvia to treatment with antibiotics at 10x the MIC. Cultures were grown without antibiotics up to early exponential phase, and treated with antibiotics at lethal concentrations. TOB: tobramycin 10 µg/ml. Non-aminoglycoside antibiotics: CIP: ciprofloxacin. TRM: trimethoprim. CRB: carbenicillin. For statistical significance calculations, we used one-way ANOVA. *** means p<0.001, ns means non-significant. Number of replicates for each experiment: n>=3.

Figure S2

  Figure S2

  Figure S2. Impact of selected gene deletions in WT and ∆ravvia on fitness during growth in sub-MIC antibiotics:in vitro competition experiments of V. cholerae WT and mutant strains in the absence or presence of TOB at sub-MICs (50% of the MIC). We were looking for factors for which inactivation would lead to loss of the fitness advantage of ∆ravvia in AGs. We tested the effect of 21 gene deletions, selected because they are important in WT but not in ∆ravvia (panel with red square), important in ∆ravvia (green panel) or beneficial in WT only (purple panel). For 16 of them, none completely suppressed this phenotype. TN-seq screen identified functions that are necessary for AG resistance in ∆ravvia. (i) in the absence of TOB: several genes are no longer essential or as important in ∆ravvia as in WT. These genes include carbohydrate utilization and metabolism genes (crp), iron and respiration related factors (feoC, iscX, trxC), and envelope related genes (cpxP). This suggests that deletion of ravvia leads to changes in carbon metabolism, iron and respiration, and membrane stress. This is consistent with transcriptomic data. Several genes become essential or important in ∆ravvia: genes involved in respiration and iron utilization (e.g. rdx, bcp); carbohydrate metabolism (crr), translation (truC, rluE) and protein stress (groES2, clpS, not tested here in competition). (ii) after growth with TOB: several genes are no longer needed in ∆ravvia: electron transport/redox, carbohydrate metabolism (sgrR), stringent response and translation stress (e.g. raiA, hpf, not tested here), envelope and cell division, e.g. cspE, coding for a cold shock transcription antiterminator which interacts specifically with mRNAs that encode membrane proteins[START_REF] El Mortaji | A peptide of a type I toxin-antitoxin system induces Helicobacter pylori morphological transformation from spiral shape to coccoids[END_REF]. In summary, several functions appear to be less needed in ∆ravvia: proteins protecting ribosomes upon translation stress (e.g. hibernation factors), consistent with the that AGs cause less translation stress in ∆ravvia. For cytochromes, their inactivation probably decreases PMF and confers AG resistance in WT but since ∆ravvia is already more resistant, their effect on PMF may have little impact on AG tolerance of ∆ravvia. Notable phenotypes were conferred by deletion of the tat operon (export of folded proteins to the perisplasm) which leads to loss of ∆ravvia's growth advantage in TOB, as well as RNA modification factors dusB, rluB, rluE, for which deletion is known to be beneficial in TOB[START_REF] Khoury | Bioenergetic State of Escherichia coli Controls Aminoglycoside Susceptibility[END_REF], and cpxP which confers an advantage only to the WT strain. MH: no antibiotic treatment (black dots). TOB: tobramycin 0.6 μg/ml (blue dots). The Y-axis represents log2 of competitive index value calculated as described in the methods. A competitive index of 1 (i.e. log2 value of 0) indicates equal growth of both strains. Statistical comparisons are between the competition [∆gene vs WT] and [∆gene ∆ravvia vs WT]. For statistical significance calculations, we used one-way ANOVA. **** means p<0.0001, *** means p<0.001, ** means p<0.01, * means p<0.05. ns: non-significant. Number of replicates for each experiment: 3<n<6.

Figure S3 Figure S3 .

 S3S3 Figure S3

Figure S4

  Figure S4

Figure S4 :

 S4 Figure S4: Effect of iron supplementation. ABC. Competitions. The effect of deletion of cpx (A), or zra-like (B), or both (C) on competitive index in MH with addition of iron, without and with TOB. Cultures were grown to exponential phase in MH medium supplemented with iron during growth. "Fe" stands for FeSO4 18µM. In vitro competition experiments of V. cholerae WT and indicated mutants in specified media: in black: MH: no antibiotic treatment. In blue: TOB: tobramycin 0,6 µg/ml. The Y-axis represents log2 of competitive index value calculated as described in the methods. A competitive index of 1 (i.e. log2 value of 0) indicates equal growth of both strains. For statistical significance calculations, we used one-way ANOVA. **** means p<0.0001, *** means p<0.001, ** means p<0.01, * means p<0.05. ns: non-significant. Number of replicates for each experiment: 3<n<8.

Figure S5 :

 S5 Figure S5

Table 1 MIC

 1

	µg/ml	MH
	∆ravvia	1.2-1.5
	WT::ravviaOE+	0.75
	∆ravA	1.2-1.5
	∆viaA	1.2-1.5
	WT	1.2

Table S1 . RNA-seq Table S2. Transposon insertion sequencing at time T0: genes that cannot be inactivated only in WT or only in ∆ravvia Table S3. Transposon insertion sequencing at time T16, with no treatment: genes that tolerate more insertions either in WT or in ∆ravvia Table S4. Transposon insertion sequencing at time T16, with sub-MIC TOB: genes that tolerate more insertions either in WT or in ∆ravvia Table S5: Strains Table S6: Plasmids Main figure references Figure 1: Effect of RavA-ViaA on fitness and tolerance to aminoglycosides. A.

 S1 Competition experiments mixing ravvia deletion (∆) and extra-copy (OE+) mutants and WT in indicated conditions. NT: non-treated. TOB: tobramycin. GEN: gentamicin. CM: chloramphenicol. CIP: ciprofloxacin. The Yaxis represents log2 of competitive index value calculated as described in the methods. Survival of indicated strain to lethal tobramycin treatment. V. cholerae WT and deletion mutant cultures were grown without antibiotics up to early exponential phase, and serial dilutions were plated on MH medium without antibiotics. Exponential phase cultures were then treated with antibiotics at lethal concentrations for the indicated times. At each time point, dilutions were spotted on MH. Y-axis shows survival calculated as number of colonies at time TN divided by the initial number of colonies before antibiotic treatment. TOB: tobramycin 5 or 10 µg/ml.

	A competitive
	index of 1 (i.e. log2 value of 0) indicates equal growth of both strains. For statistical significance
	calculations, we used one-way ANOVA. **** means p<0.0001, *** means p<0.001, ** means p<0.01,
	* means p<0.05. Only significant p values are shown. Number of replicates for each experiment: 3<n<8.
	Concentrations are indicated in µg/ml. B.

Table S1 . RNA-seq Locus tag gene name Fold change* increase in ∆ravvia

 S1

	VC_0392 VC_A0689		2.0 2.7
	Envelope VC_0728	ppk2	2.7
	Carbohydrate metabolism *VC_1315 VC_A0221	zraS-like	11.4 2.6
	VC_1820 *VC_1316 VC_A0274	putative PTS system, zraR/cpxR-like	104.7 10.9 2.5
	VC_1314 VC_A0688	mannose specific IIA phaC	8.8 2.4
	VC_1821 VC_A0867 VC_0551	subunit putative PTS system, ompW	11.5 5.9 2.3
	VC_0972 VC_0550	maltose/mannose-chiP oadA	4.6 2.3
	VC_1081 VC_2507	specific IIBC component ntrC ybeZ	3.1 2.1
	VC_1822 VC_1122 VC_2473	putative PTS system ytfK	3.8 2.2 3.8
	VC_1522 Quorum Sensing, Competence	mannose-specific IIA/IIB/IIC atoC	2.1
	VC_1825 Respiration/redox VC_A0865	components Putative ARAC-type hapA	8.8 2.4
	*VC_2656 VC_1153	regulatory mannose frdA tfoX	6.1 4.2
	VC_1826 *VC_2657 Hypotetical proteins	dependent manP frdB	27.4 10.7
	VC_1827 *VC_2658 VC_A1065	manA frdC	9.9 9.9 4.7
	VC_1325 *VC_2659	mglB frdD	16.9 9.6
	VC_1327 VC_1950	mglA torZ	11.1 9.9
	VC_A0516 VC_0651	fruA ubiV	10.4 6.7
	VC_A0517 VC_1951	pfkB torY	10.7 6.1
	VC_A0518 VC_A0983	fruB lldP	9.2 6.0
	VC_0687 VC_A0984	cstA lldD	10.6 4.1
	VC_A0137 VC_A0665	glpT dcuC	14.0 5.4
	VC_A0747 VC_A0205	glpA dcuB	3.7 5.1
	VC_A0749 VC_0338	glpC	3.4 5.1
	VC_1596 VC_A0784	gal	7.8 4.6
	VC_1595 VC_A0610	galK elbB	9.8 4.3
	VC_1594 VC_A0691	galM	9.3 4.2
	VC_1898 VC_A0512	trg nrdG	4.7 3.6
	VC_1298 VC_A0690	trg	4.0 3.5
	VC_A1069 VC_1514	tar tatB	4.6 2.9
	VC_A0903 VC_1512	ynfG	3.2 2.8
	VC_2337 VC_1515	galR torD	3.2 2.5
	VC_1645 VC_1511		3.1 2.4
	VC_A0860 VC_1516	malS	3.0 2.3
	VC_A1041 Nucleotide metabolism		2.5
	VC_A1045 VC_A0053		2.2 3.3
	Aminoacid/protein metabolism VC_A0592	3.1
	VC_1343 VC_A0798	pepT yieH	11.5 2.9
	VC_0282 VC_1034	tsr udp	4.4 2.6
	VC_0027 Others	ilvA	3.9
	VC_1872 VC_2361	yeaG grcA	2.7 6.6
	VC_A0574 VC_0076	patZ uspA	2.7 4.1
	VC_2374 VC_A0904	gltD gntP	2.5 3.5
	VC_A0985 VC_A1015	htpG	3.3 3.3
	VC_A0885 VC_0737	tdh acuB	2.4 2.9
	VC_1605a		2.9

Locus tag gene name Fold change* decrease in ∆ravvia

	Aminoacid transport and synthesis
	VC_0907	metN	2.1
	VC_1312	alr	2.1
	VC_1658	sdaC	3.3
	VC_A0063	ptrB	3.1
	Iron-sulfur and respiration
	VC_0538	cysP	2.5
	VC_0384	cysJ	2.4
	VC_A0064	tonB	2.3
	VC_0749	iscU	2
	VC_1623	nspC	2.1
	VC_1624		2.4
	VC_A0779	puuB	2.4
	Others
	VC_2561	cobA	2.8
	VC_1040	cobO	2.3
	VC_1112	bioB	2.6
	VC_A0035		2.1
			.1
	VC_A0381		2.1
	VC_1697		2.0
	VC_2221		12.8
	Controls
	VC_A0763	ravA	250
	VC_A0762	viaA	142.9
	Purine biosynthesis
	VC_2227	purN	3.6
	VC_2226	purM	3.4
	VC_1004	purF	2.5
	Translation, tRNA synthesis
	VC_2706	yhhQ	3.5
	VC_1942	folD	2.8
	VC_0327	rplL	2.2
	VC_0564	rplS	1.9
	VC_0325	rplA	1.9
	VC_1259	trhO	2.7
	VC_t017	tRNA-Thr	2.1
	VC_A1059	trmY	2
	VC_0916	etp	2.7

*:

only values with adjusted pvalue<0.01 are shown Table S2. Transposon insertion sequencing at time T0: genes that cannot be inactivated only in WT or only in ∆ravvia Non-treated -No insertion detected in WT (no reads) but inactivation possible in ∆ravvia

	Carbohydrate metabolism
	csrA	Carbon storage regulator, stringent response
	bax (VC_1430)	Sugar metabolism (glycoside hydrolase)
	crp*	Carbon catabolite control (enriched in ravvia but still very low)
	Iron
	yggX (VC_0451)	Role in Fe-S cluster stability, redox sensitive
	feoC*(VC_2076)	Ferrous iron uptake, redox sensitive FE-S cluster, Fur regulation
	iscX*	Regulator of Fe-S assembly, function unclear
	Redox/respiration
	trxC*	Thioredoxin, redox sensitive
	dsbD (VC_A0325)	Respiration, thiol:disulfide interchange protein
	ubiG (VC_1262)	Respiration, 3-demethylubiquinone-9 3-methyltransfera
	Envelope
	cpxP*(VC_2691)	Resistance to extracytoplasmic stress, envelope stress (with degP)
	rffM (VC_0927)	Enterobacterial common antigen
	tatB* (	Export of folded proteins
	Others
	ligA VC_1542)	DNA ligase
	gspS2 (	Lipoprotein
	higB-1 (VC_A0391)	Toxin
	Unknown
	yggU	VC_0458
	yhbS	VC_0655
	hyp	VC_1613
	hyp	VC_1637
	hyp	VC_A0381
	pseudogene	VC_A0390
	hyp	VC_A0467
	hyp	VC_A0471
	hyp	VC_A0547
	hyp	VC_A1030
	hyp	VC_0868
	Non-treated -

No insertion detected in ∆ravvia (no reads) but inactivation possible in WT Expected control genes

	grx4 VC_2044	Glutaredoxin
	bcp* VC_2160	Thioredoxin-Dependent Thiol Peroxidase (homol. to selU YbbB tRNA 2-selenouridine synthase)
	VC_0382	Putative ABC-type Fe3+-hydroxamate transport system
	Carbohydrate metabolism
	crr*	Phosphotransfer protein in sugar uptake, regulates sugar metabolism
	ace	Isocitrate lyase (deletion =>carbon catabolite repression)
	creA* VC_A0800	Catabolite regulation protein
	Translation/protein stress
	rrf_9 VC_r022	5S Ribosomal RNA, polar on tRNA glu/lys/val
	epmC VC_2113	efp hydroxylase
	truC	tRNA pseudouridine65 synthase (ile, asp)
	rluE*	23S rRNA pseudouridine2457 synthase, P center of the ribosome, associated to AG resistance
	clpS	protease
	groES2 VC_A0819	chaperone
	Stress
	crl	stat phase/stress regulator (rpoS regulon)
	VC1615	UTP pyrophosphatase, prevent unspecific incorporation of modified bases into RNAs
	ytfK VC_2473	Stringent response
	Others
	flgJ	peptidoglycan hydrolase , creates holes in the peptidoglycan layer for flagella assembly
	VC_0246	LPS transport system permease protein
	trpR	tryptophan (trp) transcriptional repressor
	Unknown:
	VC_0124	putative lipoprotein L
	VC_1176	Trp operon leader peptide TrpL
	VC_1537	putative Lipoprotein NlpC
	VC_2040	hypothetical
	VC_2147	hypothetical
	VC_A0233	hypothetical
	VC_A0348	Toxin RelE
	VC_A0435	hypothetical
	VC_A0497	Toxin DhiT
	VC_A0652	hypothetical
	VC_A0831	hypothetical
	VC_A0966	hypothetical
	VC_A1061	hypothetical
	lacZ	No reads in ∆ravvia because the strain is ∆lacZ
	viaA
	ravA
	Redox/respiration/iron
	napC VC_A0680	Cytochrome c
	doxX VC_A1019	Oxydoreductase
	rdx* VC_0982	Oxidoreductase (selenoprotein W-related protein/selT motif)

Table S3 . Transposon insertion sequencing at time T16, with no treatment: genes that tolerate more insertions either in WT or in ∆ravvia

 S3

	pspG hyp	VC_A0342	56 59	194 112	113 125	561 189	579 133	421 113	10.0 3.2	3.0 1.2	3.7 0.9
			38	6	25	148	22	27	3.9	3.4	1.1
	Non-treated 16 gen-yhdZ	VC_A1037	55	93	128	177	165	165	3.2	1.8	1.3
	More insertions in ravvia carbohydrate metabolism
	gene name pfkA		T0 ravvia 31	T16 ravvia 13	T16 TOB ravvia 28	T0 WT 124	T16 WT 54	T16 TOB WT 62	FC T0 ravvia/wt 4.0	FC T16 * ravvia/wt 4.0	FC T16 TOB 2.2
	galU		68	48	23	215	100	16	3.2	2.1	ravvia/wt 0.7
	carbohydrate metabolism regulators
	araD cspA	VC_A0244 VC_A0166	194 30	174 24	143 28	58 115	110 162	97 32	3.3 3.8	1.6 6.7	1.5 1.1
	malK marR	VC_A1005	510 37	242 92	352 65	140 181	96 190	142 110	3.6 4.8	2.5 2.1	2.5 1.7
		VC_0549 VC_A0999	482 65	232 65	334 69	152 239	222 144	173 123	3.2 3.7	1.0 2.2	1.9 1.8
	redox FE-S/redox
	dsbD	VC_A0506 VC_A0389	234 39	88 99	212 101	69 155	165 195	108 103	3.4 4.0	0.5 2.0	2.0 1.0
	nucleotide pool ibaG	VC_2515	8	19	9	114	91	6	14.3	4.8	0.7
	surE/umpG grxB		409 36	352 43	323 47	140 129	152 119	69 68	2.9 3.6	2.3 2.7	4.7 1.5
	pnpP	VC_A0970 VC_1254	331 61	212 102	204 190	91 216	241 193	132 387	3.6 3.5	0.9 1.9	1.5 2.0
	envelope/membrane hutC		55	72	88	187	164	127	3.4	2.3	1.4
	speB	VC_A0152	410 26	422 41	344 19	112 109	320 73	150 35	3.7 4.2	1.3 1.8	2.3 1.9
	mshF thiS	VC_0407	869 63	525 38	618 78	154 202	127 157	151 105	5.6 3.2	4.1 4.1	4.1 1.3
	vpsQ electron transfer	VC_0939	901	679	783	292	313	283	3.1	2.2	2.8
	others ccmF	VC_A0368	88	136	103	311	224	201	3.5	1.6	2.0
	mobA ccmE		212 61	141 36	151 81	63 190	67 108	71 846	3.4 3.1	2.1 3.0	2.1 10.4
	citX cobO		411 73	331 77	311 82	117 221	203 180	86 165	3.5 3.0	1.6 2.3	3.6 2.0
	trpD DNA repair		149	142	187	63	60	59	2.4	2.3	3.2
	phhB xthA	VC_A0827	546 37	391 38	384 37	154 140	178 158	148 141	3.5 3.8	2.2 4.1	2.6 3.8
	unknown two component system
	alpA dpiB citA*	VC_1809 VC_0791	286 48	373 67	383 65	90 179	112 161	183 119	3.2 3.7	3.3 2.4	2.1 1.8
	yebG cheY	VC_2326 VC_A1096	340 61	237 39	286 62	107 207	156 154	110 36	3.2 3.4	1.5 4.0	2.6 0.6
	ynjD others	VC_1666	206	225	222	32	98	101	6.3	2.3	2.2
	hyp hfq	VC_A0743	259 33	241 38	258 0	50 117	81 116	67 0	5.2 3.5	3.0 3.1	3.9 no reads
	hyp unknown	VC_A0440	353	355	421	78	139	69	4.5	2.6	6.1
	yaeP hyp	VC_0872 VC_2367	395 22	348 103	271 85	91 199	184 173	201 160	4.3 9.0	1.9 1.7	1.3 1.9
	hyp yacL	VC_A0086 VC_0605	297 59	271 125	267 131	71 437	254 371	191 224	4.2 7.5	1.1 3.0	1.4 1.7
	hyp hyp	VC_A0649 VC_A0494	360 24	331 35	318 14	93 113	188 141	195 139	3.9 4.8	1.8 4.0	1.6 10.2
	hyp hyp	VC_A0032 VC_A0919	516 60	417 107	542 68	137 284	158 174	114 260	3.8 4.7	2.6 1.6	4.8 3.8
	? ybhG	VC_A0429-30 VC_1659	195 33	262 78	375 73	52 143	113 154	149 104	3.7 4.3	2.3 2.0	2.5 1.4
	hyp hyp	VC_A1030 VC_A0458	173 92	137 127	115 126	55 369	40 275	58 183	3.1 4.0	3.5 2.2	2.0 1.5
	yecM fliT	VC_2073	191 75	205 390	181 89	61 296	134 869	120 23	3.1 4.0	1.5 2.2	1.5 0.3
	Non treated 16 gen-hyp	VC_2046	143	202	153	539	562	281	3.8	2.8	1.8
	more insertions in WT gene name hyp VC_A0387	T0 145	T16 172	T16 TOB 183	T0 WT 545	T16 WT 363	T16 TOB 288	FC T0 3.7	FC T16* 2.1	FC T16 1.6
	rstA2	VC_1463	ravvia 68	ravvia 17	ravvia 0	253	205	WT 138	wt/ravvia 3.7	wt/ravvia 11.7	TOB no reads wt/ravvia in ravvia
	Translation/protein wbeT rfbT	VC_0258	37	46	36	136	154	93	3.7	3.3	2.5
	yjgA unfA	VC_2536 VC_1191	51 48	96 81	77 125	163 176	172 128	142 78	3.2 3.6	1.8 1.6	1.8 0.6
	membrane transport hyp	VC_A1024	136	227	217	483	597	369	3.6	2.6	1.7
	hyp	VC_1605a VC_0491	16 39	31 81	38 68	198 138	146 123	99 90	12.5 3.5	4.7 1.5	2.6 1.3

*:

FC T16, only values with adjusted pvalue<0.01 are shown Table S4. Transposon insertion sequencing at time T16, with sub-MIC TOB: genes that tolerate more insertions either in WT or in ∆ravvia

	gene name	T0	T16	T16	T0	T16	T16	fold	fold	FC
		ravvi	ravvi	TOB	WT	WT	TOB	decr	decr	T16
		a	a	ravvi			WT	ease	ease	TOB
				a				ravvi	WT	ravvi
								a	T16T	a/wt
								T16T	OB/T	**
								OB/T	0
								0
	TOB 16gen:

insertions decrease in wt but unchanged (or increased) in ravvia => factors needed in TOB in WT but not needed in ∆ravvia

	cspE*		164	261	135	60	118	1	1.9	185.	211	cold shock transcription antiterminator
										2		interact specifically with mRNAs that
												encode membrane proteins
	nhaB		380	356	279	203 224	76	1.3	2.9	3.7	Na+:H+ antiporter
	others
	hapR		397	645	381	295 477	54	1.7	8.9	7.1	Quorum sensing master regulator
	menC		376	328	341	178 136	71	1	1.9	4.8	menaquinone synth. (vitK2)
	bioD		234	180	135	160 194	37	1.3	5.3	3.7	biotin synth
	fklB		324	379	117	244 165	27	1	4.2	3.2	peptidyl-prolyl cis-trans isomerase
	diaA		173	193	202	138 110	59	1	1.9	3.4	DnaA initiator-associating factor for
												replication initiation
	unknown
	electron yfcL VC_2112 transport/redox VC_A0186 hyp VC_1 VC_A0463 810 hyp VC_A	83 22 80 76 112	126 92 134 177 129	236 138 156 128 203	60 44 55 39 41	43 60 109 89 114	51 21 33 39 60	0,4 0.7 0.5 1.4 0.6	121. 2.9 3 213. 2.3 8 206.	4.7 6.7 4.7 3.3 3.4	NADH:ubiquinone oxidoreductase subunit 2 glyoxalase
	ahpC	0003	219	221	174	179 107	17	1.3	6.2 7	10.1	peroxiredoxin C
	ypjD hyp	0302 VC_A	289 183	300 241	246 281	243 203 190 155	71 50	1.2 0.7	2.9 9 237.	3.5 5.6	cytochrome c assembly ypjD
	ridA hyp	VC_1	217 160	145 138	154 244	267 191 62 190	21 65	0.9 0.7	9 290.	7.2 3.7	RidA family redox-regulated chaperone
	ubiG tldD	531	120 262	153 334	146 358	187 172 210 272	41 87	1 0.7	4.2 4 372.	3.5 4.1
	potD plsY		470 167	425 144	314 116	249 266 251 173	82 36	1.4 1.4	3.2 0 120.	3.8 3.2	spermidine preferential ABC transporter
										5
	dksA yhbS	655 VC_0	117 329	299 261	233 275	111 310 139 78	31 32	1.3 1.2	10 65.8	7.5 8.5
	thiD	VC_A	200 118	178 230	217 121	89 212 270 136	64 6	0.8 1.0	2.1 277.	3.4 21.2
	trpB phnX	0808 VC_A	164 204	137 238	131 262	94 106 108 84	18 86	1 0.8	4.5 6 138.	7.1 3.1
		0606	Tran							4
	hyp	VC_1	slati 280	357	352	121 111	75	0.8	140.	4.7
		613	on							0
	raiA YeeX	VC_A	103 168	101 171	156 177	124 52 84 138	17 2	0.6 0.9	3.1 145.	9.2 74.7	ribosome protection
	yeiP hyp	0741 VC_1	214 143	208 298	211 171	277 271 77 63	50 23	1 0.8	5.4 7 75.5	4.2 7.4	EFP like protein
	hpf	262	261	170	114	155 81	22	1.5	3.6	5.1	ribosome hibernation
	hyp	VC_1	313	323	303	171 229	90	1.0	221.	3.4
		479								3
	sgrR hyp	VC_A	212 121	224 167	225 168	119 147 74 86	56 43	1 0.7	2.6 119.	4 3.9	Sugar transport-related Regulator
	0631 envelope/membrane/cell hyp VC_2 347 division ompR 278 434 hyp VC_1 213	294 211 219	297 167 286	216 176 198 174 156 151	97 40 93	1.2 1.3 0.7	6 150. 4.4 5 202.	3.1 4.2 3.1
	envC hyp	574 VC_1	357 192	349 149	294 185	271 316 144 151	92 49	1.2 1.0	3.4 4 146.	3.2 3.8	murein hydrolase activator (septum)
	envZ	310	393	340	298	310 258	71	1.1	3.6 2	4.2
	envZ hyp	0382 VC_A	275 262	289 232	161 251	175 235 220 146	47 75	1.8 1.0	5 7 140.	3.4 3.4	sensory histidine kinase
	zapD	VC_1	294 129	286 172	282 192	195 197 131 122	75 52	1 0.7	2.6 182.	3.8 3.7
	matP	536 VC_A	110 103	215 150	183 119	131 139 112 126	14 22	1.2 0.9	10 4 146.	13.2 5.5
	ftsE	0331-	464	476	331	388 421	63	1.4	6.7 1	5.3
	ftsX TOB 16gen:	2	493	477	447	427 470	77	1.1	6.1	5.8
	DedD		146	298	178	76	173	15	1.7	11.7	12	cell division protein
	RlpA		313	335	216	313 306	47	1.6	6.5	4.6	septal ring lytic transglycosylase
	YfbV		302	240	210	268 303	30	1.1	9.9	6.9	membrane protein
	Blc		372	424	384	195 286	94	1.1	3	4.1	OM lipoprotein
	glpG		243	227	176	333 323	33	1.3	9.8	5.3	serine proteases which cleave membrane
												proteins

insertions highly decrease in wt but slightly decreased in ravvia => factors needed in TOB in WT but less needed in ∆ravvia translation

	flhA	186	798	358	157 773	90	2.2	8.6	4
	fliF	181	729	340	266 994	88	2.1	11.3	3.9
	fliG	119	501	202	202 758	58	2.5	13	3.5
	FlgN	198	639	165	222 571	40	3.9	14.3	4.1	flagella synthesis protein
	flgF	178	594	266	243 885	77	2.2	11.5	3.4
	carbohydrate metabolism
	cpsB	640	1545 498	625 150	36	3.1	41.8	13.8	mannose-1-phosphate
						3					guanylyltransferase
	manB	382	1005 340	437 100	20	3	50.6	17.1	phosphomannomutase
						6
	wcaJ	163	378	132	269 640	22	2.9	29.3	6	UDP-glucose:undecaprenyl-phosphate
											glucose-1-phosphate transferase
	manA_1	608	630	299	665 587	30	2.1	19.4	9.9
	gmd	718	1230 131	402 989	38	9.4	25.7	3.4	GDP-mannose 4,6-dehydratase
	envelope/membrane/cell
	division
	phoB	229	307	145	151 192	23	2.1	8.2	6.2
	mepM	342	373	176	423 315	56	2.1	5.7	3.2	Peptidoglycan-specific endopeptidase
	others
	VC_2654	324	379	117	244 165	27	3.2	6.1	4.3	rhodanese-like domain-containing protein
											thiosulfate disproportionation
	aspA	251	488	197	358 559	44	2.5	12.7	4.5
	gene name	T0	T16	T16	T0	T16	T16	fold	fold	FC
		ravvi	ravvi	TOB	WT	WT	TOB	incre	incre	T16
		a	a	ravvi			WT	ase	ase	TOB
				a				ravvi	WT	wt/r
								a	T16T	avvia
								T16T	OB/T	**
								OB/T	16M
								16M	H
								H
	TOB 16gen:
	miaB*	251	234	119	253 233	39	2	6	3.1	tRNA modification
	rsmI	378	299	122	441 297	34	2.4	8.6	3.6	rRNA modification
	flagella
	fliR	153	663	314	118 684	73	2.1	9.4	4.3

insertions increase in wt but unchanged in ravvia => important for ∆ravvia growth in TOB or inactivation has no additional benefit in ∆ravvia => partners/linked processes? translation/protein stress

		ccmI	59	55	105	109 119	353	1.9	3	3.4	cytochrome c biogenesis
		arcA	147	47	57	113 23	289	1.2	12.6	5.1	response regulator, regulates cadA
		_2
		arcB	195	64	97	193 41	480	1.5	11.6	4.9	response regulator, regulates cadA
		dsbD	118	77	146	136 89	686	1.9	7.7	4.7	thiol:disulfide interchange
		dipZ
		ychF	72	21	57	99	58	737	2.7	12.7	12.9	redox-responsive ATPase
		cadA	95	39	115	98	109	1491	2.9	13.7	13.0
		=
		ldcI*
		iscR*	21	27	61	47	46	845	2.2	18.4	13.9
	carbohydrate metabolism
		crr*	32	16	14	156 23	489	0.9	21.3	33.9	glucose uptake
		ptsI	74	19	56	99	27	867	3	32.7	15.5
		ptsH	50	41	3	79	78	230	0.1	2.9	86.6	phosphocarrier protein HPr
		pykF	42	16	39	82	32	218	2.4	6.8	5.6	pyruvate kinase
		rpiR	115	64	103	84	32	497	1.6	15.4	4.8	carbohydrate utilization regulator
		rpe	174	32	62	175 39	536	1.9	13.9	8.6	ribulose-phosphate 3-epimerase
	envelope/membrane/motilit
	y
		bamC 255	100	174	180 88	555	1.7	6.3	3.2	outer membrane protein assembly
		mioC	175	50	161	113 69	827	3.3	12.1	5.1	flavoprotein, cell division
		VC_1	166	148	98	160 143	662	0.7	4.6	6.8	sodium:alanine symporter
		422
		hdfR	73	66	137	137 139	779	2.1	5.6	5.7	negative regulator of flagella
	others
		luxO	126	102	168	146 108	711	1.7	6.6	4.2	QS
		citG	193	191	191	296 283	633	1	2.2	3.3
	unknown
		VC_0	98	36	47	180 178	1016	1.3	5.7	21.7	putative lipoprotein L
		124
	VC_0803	trmH VC_2	130 61	87 34	148 112	221 147 183 126	801 1508	1.7 3.3	5.5 12	5.4 13.4	tRNA modification
		* 498
		rluB* VC_A	110 72	60 33	96 135	152 111 130 155	1049 1833	1.6 4.1	9.5 11.8	11.0 13.6	rRNA modification putative cobaltochelatase subunit CobN
	YfcZ ybfF	dusB 0039 VC_A * slyD 0919 VC_2	183 60 44 100	118 107 48 122	184 68 75 160	116 91 284 174 97 108 201 225	1118 260 348 518	1.6 0.6 1.6 1.3	12.3 1.5 3.2 2.3	6.1 3.8 4.7 3.2	tRNA modification Chaperone
	iron	097
		hemF 108 1061 VC_A 106	95 64	136 54	87 129 261 104	422 163	1.4 0.8	4 0.6	3.1 3.0
		fur* 476 VC_2	5 46	7 67	7 53	43 126 189 6	157 228	0.9 0.8	26.8 1.2	23.1 4.3
	electron transport/ Fe-
	S/redox
		ccoO	115	75	141	205 47	1405	1.9	29.9	9.9	cytochrome C oxidase
		ccoG	163	81	179	181 124	1938	2.2	15.6	10.8	cytochrome c
		VC_0	88	51	136	165 112	1613	2.7	14.4	11.9	cytochrome b
		574
		VC_0	136	37	146	114 53	1432	3.9	27	9.8	cytochrome c
		575
		VC_0	118	63	165	82	58	1859	2.6	32.1	11.3	cytochrome b
		168
		ccmG 94	61	92	92	28	441	1.5	15.8	4.8	cytochrome c biogenesis protein
		ccmE	61	36	81	190 108	846	2.2	7.8	10.4	cytochrome c
		ccmA	77	47	105	117 60	483	2.2	8.1	4.6	cytochrome c

**:

FC T16TOB, only values with adjusted pvalue<0.01 are shown Table S5.

	Strain	genotype	resistance	paren	construction	primers for gibson assembly
	#			tal		or details for cloning
				strain
	J085	WT V. cholerae	strep		lab collection
		N16961 hapR+
	K329	WT V. cholerae	strep	J085	laboratory	deletion of lacZ by
		N16961 hapR+ ∆lacZ			collection	conjugation J085*4850 and
						sucrose 15% excision
	L555	ΔravA-viaA::spec	spec	K329 pMP7 L571	p9344 digested with EcoRI
		ΔlacZ				and cloned into pMP7 EcoRI
						site
	M093	ΔravA-viaA::kan	frt::kan::frt	K329 pMP7 L911	yeiMN5/7 for up region and
		ΔlacZ				yeiMN6/8 for down region
	O390	ΔravA-viaA ΔlacZ		M093 excision of
					frt::kan::frt
	O506	ΔravA-viaA pGBts-	spec 30°C		transfomation	operon ravA-viaA with its own
		ravA-viaA+			of	promoter amplified with
					thermosensitive	primers Pravaviamonteco and
					low copy pGBts	Opravaviavaleco and clones
					plasmid	inside EcoRI of pGBts
					expressing
					ravvia into
					ravvia deleted
					strain.
	M145	WT::Pbla-ravA-viaA-	Cm5	J085	tn7	Pbla-ravA-viaA cloned in XmaI
		extracopy OE+			transposition	into pMVM4 (=L950)
					pM060
	N542	ΔravA::kan ΔlacZ	frt::kan::frt	K329 pMP7 N624	yeiMN5/7 for up region and
						yeiN6/8 for down region
	N601	ΔravA ΔlacZ	-	N542 excision of
					frt::kan::frt
	N544	ΔviaA::kan ΔlacZ	frt::kan::frt	K329 pMP7 N625	yeiM5/7 for up region and
						yeiMN6/8 for down region
	N620	ΔviaA ΔlacZ	-	N544 excision of
					frt::kan::frt
	S490	ΔcpxPAR::kan	frt::kan::frt	K329 pMP7 S448	cpxPAR5/7 for up region and
						cpxAR6bis/8 for down region
	Q080	∆zra2 (∆VC1315-	frt::kan::frt	K329 pMP7 P679	VC1315-165/7 for up region
		1316) ΔlacZ				and VC1315-166/8 for down
						region

Table S6 : Primers

 S6

	VC0281ldcIcada6	TACGTAGAATGTATCAGACTCCCTCGCCACTCGCACCCAA
	primers VC1315-165	CTATTATTTAAACTCTTTCCTTTATGTCACACACCGCGAC
	yeiMN5 VC1315-167	CTATTATTTAAACTCTTTCCACGACAATCTCGCCTTGGT CTACACAATCGCTCAAGACGTGAACGTTTCCTTAATGGCCGA
	yeiMN7 VC1315-168	CTACACAATCGCTCAAGACGTGCTCTTGATTCCTCAGACAAAG CTAATTCCCATGTCAGCCGTGCGATGTTAGGTGAGCTTAG
	VC1315-166	TACGTAGAATGTATCAGACTACTAATGAGGGGCATGTTTAT
	yeiMN8	CTAATTCCCATGTCAGCCGTCTTGAATGCTTCATACCCAAC
	cpxPAR5	CTATTATTTAAACTCTTTCCGTTGAGGATCAAAAAGCACG
	yeiMN6	TACGTAGAATGTATCAGACTATAAAAAAACGTCATAAGAATAGC
	cpxPAR7	CTACACAATCGCTCAAGACGTGCAAAAAACACGCTAGTCAATAA
	VC2106fur5	CTATTATTTAAACTCTTTCCAAGCGGATGCGAACTTCGC
	cpxAR8	CTAATTCCCATGTCAGCCGTAGCGCTTAACGCAGTAACTC
	VC2106fur7 cpxAR6bis	CTACACAATCGCTCAAGACGTGATACTTTCCTGTTGATGTTCTGC TACGTAGAATGTATCAGACTCGCTCAGTGGCCTATCTT
	VC2106fur8 VCA0184capB5	CTAATTCCCATGTCAGCCGTGCTCACAAGCCGAAGAAATAA CTATTATTTAAACTCTTTCCTTTTCGCCTTTTCCACTCGG
	VC2106fur9 VCA0184capB7	TACGTAGAATGTATCAGACTCCACAAATCGATCAGTTTATGG CTACACAATCGCTCAAGACGTGAATAAATATCCTAAAAACATTTTTTAAC
	yeiN8 VCA0184capB8	CTAATTCCCATGTCAGCCGTGCATTCGTAACCTCAACCAA CTAATTCCCATGTCAGCCGTCTGAAAGCATCAAAGTTCTGTAA
	yeiN6 VCA0184capB6	TACGTAGAATGTATCAGACTACGCTCTTTGTTGGCTTTAAG TACGTAGAATGTATCAGACTGCGACGCCCATGCAGGCTAT
	yeiM5	CTATTATTTAAACTCTTTCCCGTTATTGCAGAGCAATATGTC
	yeiM7	CTACACAATCGCTCAAGACGTGAATGACACCTAAGCAAAAAATTG
	Pravaviamonteco	GGAATTCTATTGAAACTATTGTTTATAGAGCG
	Opravaviavaleco	GGAATTCTTACCACTTCTTCATTAGCCG
	VCA0578sgrR5bis	CTATTATTTAAACTCTTTCCGACACGACAATCGCGTTACC
	VCA0578sgrR7	CTACACAATCGCTCAAGACGTGAAAGAGGAAATCTCATCTAACTT
	VCA0578sgrR8	CTAATTCCCATGTCAGCCGTCTTTTACTCACTCGTGGGAT
	VCA0578sgrR6bis	TACGTAGAATGTATCAGACTTTGTTCTCTGCCATCTCTTTTTTC
	VC0982rdx5	CTATTATTTAAACTCTTTCCATATTGGGGGAGTGACTTCA
	VC0982rdx7	CTACACAATCGCTCAAGACGTGCGTGACGTCCTTGTTATGTC
	VC0982rdx8	CTAATTCCCATGTCAGCCGTGCGATCCCCAAACGACTCAG
	VC0982rdx6	TACGTAGAATGTATCAGACTAATCCTATCCCGCAAGGGTA
	VC2160bcp5	CTATTATTTAAACTCTTTCCGAAGTGGTTCGATTAGTGAC
	VC2160bcp7	CTACACAATCGCTCAAGACGTGAATTATCCCTTTGATTACTGACT
	VC2160bcp8	CTAATTCCCATGTCAGCCGTATCGGTAGAAATGCCGATTTT
	VC2160bcp6	TACGTAGAATGTATCAGACTGCCGATGAAGTTCGGACGTT
	VC0964crr5	CTATTATTTAAACTCTTTCCAAGAAGTCTCTTTCTTCTCTATC
	VC0964crr7	CTACACAATCGCTCAAGACGTGTGTCATGCTCCTAACGTT
	VC0964crr8	CTAATTCCCATGTCAGCCGTGACCAAGTAATCGCTTGG

Acknowledgements.

We thank Louna Fruchard for her help with flow cytometry experiments. We thank Frédéric Barras for critical reading of the manuscript, Jessica El Khoury and Eduardo Rocha and Béatrice Py for valuable discussions, and Dominique Fourmy for the gift of Neo-Cy5. We also thank, for RNA-seq experiments, E. Turc, L. Lemée, T. Cokelaer, Biomics Platform and C2RT (Institut Pasteur, Paris, France, supported by France Génomique (ANR-10-INBS-09) and IBISA. We thank Sebastian Aguilar Pierlé for help with TNseq analysis. This research was funded by the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS-UMR 3525), the Institut Pasteur grant PTR 245-19, ANR ModRNAntibio (ANR-21-CE35-0012), ANR-LabEx [ANR-10-LABX-62-IBEID], the Fondation pour la Recherche Médicale (FRM EQU202103012569). AB was funded by Institut Pasteur Roux-Cantarini fellowship.