# Sleeping ribosomes: bacterial signaling triggers RaiA mediated persistence to aminoglycosides

Manon Lang, Evelyne Krin, Chloé Korlowski, Odile Sismeiro, Hugo Varet,

Jean-Yves J.-Y. Coppée, Didier Mazel, Zeynep Baharoglu

► To cite this version:

Manon Lang, Evelyne Krin, Chloé Korlowski, Odile Sismeiro, Hugo Varet, et al., Sleeping ribosomes: bacterial signaling triggers RaiA mediated persistence to aminoglycosides. *iScience*, 2021, pp.103128. 10.1016/j.isci.2021.103128. pasteur-03349141

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1 Sleeping ribosomes: bacterial signaling triggers RaiA mediated persistence to aminoglycosides.

2 Manon Lang¹,², Evelyne Krin¹, Chloé Korlowski¹, Odile Sismeiro³, Hugo Varet³,⁴, Jean-Yves Coppée³,

3 Didier Mazel¹*, Zeynep Baharoglu¹*

4

5 ¹Département Génomes et Génétique, Institut Pasteur, UMR3525, CNRS, Unité Plasticité du Génome

6 Bactérien, Paris, France.

7 ²Sorbonne Université, Collège Doctoral, F-75005 Paris, France.

8 ³Biomics technological plateform, Center for Technological Resources and Research, Institut Pasteur,

9 Paris, France.

10 ⁴Bioinformatics and Biostatistics Hub, Department of Computational Biology, USR 3756 CNRS, Institut

11 Pasteur, Paris, France.

12

13 * corresponding authors, mazel@pasteur.fr, zeynep.baharoglu@pasteur.fr

14

15 Abstract

16 Indole is a molecule proposed to be involved in bacterial signaling. We find that indole secretion is

17 induced by sublethal tobramycin concentrations and increases persistence to aminoglycosides in V.

18 *cholerae*. Indole transcriptomics showed increased expression of *raiA*, a ribosome associated factor.

19 Deletion of *raiA* abolishes the appearance of indole dependent persisters to aminoglycosides, while its

20 overexpression leads to 100-fold increase of persisters, and a reduction in lag phase, evocative of

21 increased active 70S ribosome content, confirmed by sucrose gradient analysis. We propose that,

22 under stress conditions, RaiA-bound inactive 70S ribosomes are stored as so-called “sleeping

23 ribosomes”, and are rapidly reactivated upon stress relief. Our results point to an active process of

24 persister formation, through ribosome protection during translational stress (e.g. aminoglycoside

25 treatment), and reactivation upon antibiotic removal. Translation is a universal process, and these

26 results could help elucidate a mechanism of persistence formation in a controlled, thus inducible way.

27 Introduction

28 Antibiotic resistance is a major public health concern leading to increased health care costs and

29 mortality (Opatowski et al., 2019; Touat et al., 2019). Although the majority of studies address the

30 response of bacteria to lethal doses of antibiotics, the effect of low doses of antibiotics on bacteria has

31 also recently started to draw attention. Antibiotic concentrations lower than the minimal inhibitory

32 concentration (sub-MICs) have historically been proposed to serve as signaling molecules (Davies et

33 al., 2006), provoking considerable changes in transcription and triggering a wide variety of cellular

34 responses in different bacterial species (Andersson and Hughes, 2014) and mutagenesis (Gutierrez et

35 al., 2013). In *V. cholerae*, sub-MIC aminoglycosides (AGs) are known to activate various stress response

36 pathways, such as the SOS (Baharoglu et al., 2014; Baharoglu and Mazel, 2011) and RpoS stress

37 responses (Baharoglu et al., 2013), allowing cells to cope with increased reactive oxygen species (ROS)

38 levels and DNA breaks (Negro et al., 2019). AGs are bactericidal antibiotics that are known to enter the

39 bacterial cell through the proton motive force (Fraimow et al., 1991; Herisse et al., 2017; Taber et al.,

40 1987). AGs target the ribosome, leading to mistranslation and eventually cell death (Davis, 1987).

41 Interestingly, sub-MIC antibiotics, among which AGs, have been shown to stimulate the production of

42 a small molecule, indole (Han et al., 2011). Indole is a byproduct of tryptophan degradation by

43 tryptophanase TnaA (Evans et al., 1941) in both Gram+ and Gram- bacterial species (Lee and Lee,

44 2010), together with pyruvate and ammonia. While pyruvate and ammonia are respectively sources of

45 carbon and nitrogen, the role of indole is not well understood. Indole is also found in plants and

46 animals, and was linked with signaling and human diseases (for a review, (Lee et al., 2015)). Common

47 indole concentrations in the human gut are in the order of 250-1100 µM, and up to 200 µM in blood

48 and other tissues. Regulation of indole production has been described, namely through carbon source

49 utilization (Botsford and DeMoss, 1971) and catabolic repression (Yanofsky et al., 1991), amino acid
50 availability (Newton and Snell, 1965), cold temperature (Lee et al., 2008), heat shock (Li et al., 2003)
51 and growth phase (Kobayashi et al., 2006). Particularly, indole is produced during transition from
52 exponential to stationary phase (Lelong et al., 2007).

53 In *E. coli*, indole is nontoxic at physiologic concentrations (below 1 mM) (Lee et al., 2007), and does
54 not change the growth rate (Lee et al., 2008). At high concentrations however (above 1-3 mM), indole
55 inhibits cell division (Chant and Summers, 2007; Chimerel et al., 2012). In *V. cholerae*, indole secretion
56 reaches its maximum at 600 µM during transition from midlog to stationary phase (Howard et al., 2019;
57 Mueller et al., 2009) and was not observed to have any effect on the polarity of the *V. cholerae* cell
58 membrane at this concentration (Mueller et al., 2009).

59 Indole can pass across the cell membrane without the need for a transporter (Pinero-Fernandez et al.,
60 2011), and was proposed to act as an interkingdom signaling molecule (Martino et al., 2003; Wang et
61 al., 2001). An effect of indole in persistence to antibiotics has also been observed. At toxic
62 concentrations (1-2 mM), where indole behaves as a membrane ionophore, it was observed to reduce
63 persistence of stationary phase cultures to tested antibiotics (ciprofloxacin, ampicillin) (Hu et al., 2015).

64 However, studies in *E. coli* show that lower concentrations of indole increase survival/persistence to
65 lethal concentrations of ofloxacin, ampicillin and kanamycin (Vega et al., 2012; Vega et al., 2013),
66 suggesting that the protective effect of indole is not specific to one family of antibiotic. Studies also
67 pointed to the involvement of indole secretion in the cooperation between antibiotic resistant and
68 sensitive populations during antibiotic stress (Lee et al., 2010). Notably, a recent study identified indole
69 production as a potential target for the increased activity of quinolones against persisters in *E. coli*
70 (Zarkan et al., 2020). Since indole appears to be beneficial for bacteria in the presence of antibiotics,
71 we addressed whether indole production is increased upon sub-MIC AG treatment in *V. cholerae* and
72 whether this can lead to improved response to lethal antibiotic concentrations.

73 Importantly, we find that indole strongly increases persistence to AGs through the action of RaiA. We
74 find that transcription from the *raiA* gene promoter is highly upregulated in the presence of indole in
75 exponential phase *V. cholerae* cells. RaiA was shown to be a ribosome associated protein, in the same
76 conditions as Rmf (ribosome modulation factor) and Hpf (hibernation promoting factor) (Maki et al.,
77 2000). The two latter factors cause dimerization of vacant 70S into inactive 100S ribosome dimers, in
78 a process called ribosome hibernation during stationary phase (Gohara and Yap, 2018), whereas RaiA
79 mostly binds to free 70S monosomes (Maki et al., 2000; Sabharwal et al., 2015). *E. coli* mutants lacking
80 these ribosome-associated factors do not show any growth defect during exponential growth, which
81 is consistent with the fact that their expression is specific to stationary phase and stress (Prossliner et
82 al., 2018). RaiA was observed to protect the 70S ribosome from degradation (Agafonov et al., 1999; Di
83 Pietro et al., 2013), and was also observed to block the binding of tRNA to the ribosomal A site in a cell
84 free translation system (Agafonov et al., 2001), and during cold shock (Vila-Sanjurjo et al., 2004). In the
85 present study, characterization of the *raiA* deletion mutant shows that RaiA is instrumental in the
86 appearance of persister cells to AGs. We propose here a new mechanism of induced persistence to
87 AGs by which RaiA positively affects the intact ribosome content of the cell, and facilitates regrowth
88 after removal of the antibiotic.

## 89 Results

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

91 Since the antibiotics ampicillin and kanamycin increase indole levels in *E. coli* (Han et al., 2011), we
92 measured indole secretion (Saint-Ruf et al., 2014) in *V. cholerae* to determine whether the
93 aminoglycoside tobramycin also impacts indole levels in this case. We found increased extracellular
94 indole concentrations in the presence of sub-MIC tobramycin (TOB 0.15 µg/ml, 20% of the MIC, **Figure
95 1**). We next addressed whether indole has an impact on the growth of *V. cholerae*, using an indole
96 concentration of 350 µM. This concentration was previously shown to be physiologically relevant in *V.
97 cholerae* and was observed to have no inhibitory effect on growth, and to complement the biofilm
98 formation defect of a Δ*tnaA* mutant deficient for indole production (Mueller et al., 2009). We found
that 350 µM indole does not affect growth in the absence of antibiotics, but improves growth in sub-MIC antibiotics tobramycin (Figure S1A).

We next addressed the effect of indole in the response to lethal concentrations of antibiotics, by measuring persister cells formation in *V. cholerae*. In order to do so, we adapted to *V. cholerae*, a protocol developed for *E. coli* (Ivan Matic and Wei-Lin Su, personal communication). Early exponential phase cultures were treated with lethal doses of antibiotics (5 to 10 times the MIC) for 20 hours. We first confirmed that cells surviving after 20 hours of antibiotic treatment and which grow upon antibiotic removal, were indeed persister cells, by performing survival curves (Figure S2A). The biphasic profiles of the killing curves we obtained was consistent with the formation of persister cells after 5 hours (Brauner et al., 2016). Furthermore, these cells were not resistant to the antibiotic, as no growth was observed when streaked on antibiotic containing plates (not shown). We thus carried on with the quantification of persisters at 20 hours of antibiotic treatment. We found that *V. cholerae* cultures grown in the presence of indole yielded higher numbers of persister cells to the AGs TOB and gentamicin (GEN) (Figure 2AB), as previously observed for *E. coli* treated with kanamycin and ofloxacin (Vega et al., 2012), but we observed no effect for persistence to carbenicillin (CRB), or trimethoprim (TMP) (Figure 2CD). Furthermore, a strain deleted for *tnaA* yielded less persisters to tobramycin than the WT strain (Figure S2B), and the effect was reversed by indole complementation, consistent with a link between indole and persistence.

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

We next addressed whether the beneficial effect of indole treatment in the presence of AGs is due to modifications in membrane potential, which could lead to decreased AG entry into the cell. AG uptake by the bacterial cell is known to be linked to the proton motive force (PMF). At high concentrations (5 mM) indole is a proton ionophore which blocks cell division by dissipating the PMF (Kralj et al., 2011), and was observed to interact with the cell membrane and change its physical structure (Mitchell, 2009). In order to measure AG entry in the bacterial cell, we used the aminoglycoside neomycin coupled to the fluorophore Cy-5, which was previously synthesized for aminoglycoside uptake studies in bacteria and demonstrated to bear the properties of aminoglycosides for uptake, mode of action and activity against Gram negative bacteria (Sabeti Azad et al., 2020), (Pierlé et al, unpublished). We found that indole at the physiological concentration of 350 µM does not affect AG entry into the bacterial cell (Figure S3A), ruling out the possibility of decreased AG entry due to modifications of PMF in the presence of indole. Moreover, the presence of 350 µM indole did not change the MIC of two aminoglycosides: tobramycin and gentamicin (Figure S3B). The beneficial effect of indole in the presence of AGs is thus not through reduced antibiotic entry.

**Indole induces RaiA (VC0706) expression.**

In order to shed light into mechanisms allowing for more efficient response to antibiotic stress upon indole treatment, we decided to study the transcriptomic changes of *V. cholerae* in response to 350 µM indole. mRNA sequencing of exponential phase cultures shows differential regulation of 260 genes shown in Table S1 and represented in Figure 3A (>2-fold change, adjusted *p* value <0.01 as in (Krin et al., 2018)). The most affected categories were respiration (31 genes upregulated), electron transfer and iron uptake (55 genes downregulated). Indole mediated protection against antibiotic killing was previously proposed to be through upregulation of efflux pumps (Blair et al., 2013; Hirakawa et al., 2005; Kobayashi et al., 2006; Lee and Lee, 2010; Nikaido et al., 2012), or through an increase in OxyR associated oxidative stress response (Vega et al., 2013). However, our RNA-seq data show no induction of oxidative stress response related genes in *V. cholerae* (*oxyR*, *soxRS*, *katG*) by indole, and rather suggest decreased expression of proteins linked to iron uptake, suggesting decreased iron and ROS levels upon indole treatment (Baharoglu et al., 2013; Mehi et al., 2014). The second most affected category belongs to translation related genes (31 genes, approximately 10% of total differentially regulated genes, with *p*<3.00E-4). Notably, one translation related gene was markedly upregulated:
raiA (VC0706, 20-fold up), together with rmf (VC1484, 3-fold up), which are both described as factors associated with inactive ribosomes in stationary phase (Agafonov et al., 1999; Di Pietro et al., 2013).
raiA expression is known to be triggered by transition to stationary phase (Maki et al., 2000), and RaiA is usually weakly expressed during exponential phase. RT-qPCR on raiA (Figure 3B) and fluorescence associated flow cytometry on cells carrying GFP fused to the raiA promoter (Figure 4A and S4AB) confirmed upregulation of raiA by indole during exponential phase, and increased expression of raiA in stationary phase. Since transcriptomic data pointed RaiA as one of the most differentially regulated genes by indole, we next decided to address the contribution of RaiA in the indole associated phenotypes in V. cholerae.

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

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

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

In order to mimic conditions of RaiA induction, we cloned it under a controlled Para promoter, which is repressed by glucose and induced by arabinose. Since the presence of different carbon sources may differentially affect growth and the response to aminoglycosides (Pierlé et al., unpublished), and also because arabinose was shown to have an impact on growth in *V. cholerae*, we compared the persistence levels of cells carrying the empty vector (p0 in Figure 5ABCDEF) or the pBAD-RaiA plasmid, in the presence of arabinose (RaiA overexpression conditions). Overexpression of RaiA strongly increased persisters formation in three aminoglycosides: TOB, GEN and NEO (Figure 5ABC), but not in three non-aminoglycoside antibiotics from different families: CRB, TMP and ciprofloxacin (CIP) (Figure SDEF), indicating that RaiA is directly and specifically involved in the persistence mechanism to aminoglycosides. Additionally, the MIC showed no difference between the raiA overexpression strain compared to the strain with empty plasmid p0 and pRaiA+ (MIC = 0.75 µg/ml for both on MH plates containing arabinose), ruling out a potential effect on resistance.
We next asked whether such increased persistence could be due to slower growth when RaiA is overexpressed. We monitored growth in conditions where 1) RaiA is not overexpressed previous to inoculation and only overexpressed during the growth curve, and 2) RaiA is previously overexpressed
in cells used for inoculation (Figure S6A). First, our results show no difference in growth rate (slope) in presence or absence of RaiA overexpression (Figure 6AD and S6B), indicating that RaiA overexpression does not cause a slow growth phenotype, which discards the hypothesis linking RaiA-mediated persistence to slow growth/dormancy. Interestingly, we observe in cells where RaiA overexpression was pre-induced, that these cells start growing faster due to a reduction in lag phase (Figure 6BC and Figure S6B). Such a reduction in lag phase is reminiscent of increased active ribosome content which allows faster resumption of growth at the exit of stationary phase (Condon et al., 1995). As a corollary, when *raiA* was deleted, the lag phase was increased (Figure 7A and Figure S6C), suggesting that RaiA levels affect inactive but "ready to use" ribosome content, which we called sleeping ribosomes, in stationary phase.

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

RaiA was previously found to be associated with the inactive ribosomes at stationary phase, in the same conditions as Rmf and Hpf factors. Rmf and Hpf are known to dimerize ribosomes into so called hibernating 100S ribosomes, whereas RaiA associates with monomeric 70S ribosomes (Gohara and Yap, 2018; Maki et al., 2000; Prossliner et al., 2018). In order to address whether Hpf/Rmf dependent ribosome hibernation also favors rapid exit from stationary phase, we performed deletion and overexpression experiments similar to what we did for RaiA. Deletion of *rmf* or *hpf* has no effect on lag phase nor growth (Figure 7A and S6C). Our results showed that in contrast to overexpression of RaiA which decreases the lag phase, overexpression of Rmf or Hpf rather increase lag phase (Figure 7B and S6C). These findings are consistent with a model where increased ribosome dimerization by Hpf/Rmf would require action of dissociation factors in order to resume growth whereas spontaneous dissociation of RaiA from inactive 70S ribosomes (Agafonov et al., 2001; Maki et al., 2000), is sufficient for growth restart. Furthermore, when we overexpressed RaiA in *Δhpf* or *Δrmf* mutants, we observed a reduction of lag phase similar to what is observed upon overexpression of RaiA in the WT strain (Figure 7DC), meaning that RaiA action is not dependent of Hpf/Rmf mediated ribosome hibernation.

In addition, we addressed the effect of hibernation factors on persistence in exponential phase. Unlike for RaiA overexpression, no increase in persistence to TOB was detected upon overexpression of Hpf or Rmf, excluding an effect of 100S ribosome dimer formation on persistence to AGs in exponential phase (Figure 5A). Unexpectedly, persistence levels even decreased upon overexpression of Hpf, suggesting that 70S-RaiA (sleeping ribosome) and 100S-Rmf/Hpf (hibernating ribosome) complexes may have opposite effects on persistence to AGs in exponentially growing bacteria. We also tested the persistence levels of *rmf* and *hpf* deletion mutants. Surprisingly again, and consistent with overexpression results, we found that deletion of *rmf* or *hpf* hibernation factors increases persistence to AGs (Figure S7). The increase of persistence due to deletion of *rmf* is dependent on the presence of *raiA*, as the double mutant *raiA rmf* shows reduced persistence compared to WT, and similar to the *raiA* mutant. This can be due to the observed increased expression of *raiA* in the absence of *rmf* (Sabharwal et al., 2015) or amplified ribosome-RaiA complex formation in the absence of *rmf*, due to decreased 100S formation (Ueta et al., 2005). Finally, it is important to note that the *hpf raiA* double mutant could not be constructed despite the use of two different strategies, implying synthetic lethality. Overall these results suggest an equilibrium between Rmf/Hpf and RaiA actions, consistent with previous literature that showed a combined role for these proteins in ribosome hibernation and antagonizing regulation of *rmf/hpf* and RaiA in V. cholerae (Sabharwal et al., 2015).

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

In order to address whether intact 70S ribosomes are protected/stored upon RaiA overexpression, we measured the ribosome contents of WT and RaiA overexpressing cells, as well as the strain deleted for
242 *raiA*, by performing 10-50% sucrose gradients on cellular extracts from 24h stationary phase cultures.
243 The profiles obtained were consistent with well described peaks for 30S and 50S subunits followed by
244 a third peak corresponding to the 70S ribosome (Ueta et al., 2013). No clear 100S peak was observed
245 in any of the experiments. Deletion of *raiA* leads to a slight increase in the proportion of dissociated
246 subunits compared to 70S ribosome. We found that the proportion of 70S ribosomes is increased
247 compared to 30S+50S dissociated subunits upon RaiA overexpression (Figure 8AB). This agrees with
248 the hypothesis that RaiA would stabilize the intact 70S ribosome and increase functional ribosome
249 pools, reducing the lag phase of the population upon growth restart.

250 **RaiA expression is under environmental control and linked to the Fur iron sensing regulon in V. cholerae**
251

252 RaiA is known to be expressed in stationary phase and upon temperature stress (Agafonov et al., 1999;
253 Agafonov et al., 2001; Maki et al., 2000; Sabharwal et al., 2015; Slamti et al., 2007). Regulation of *raiA*
254 was also described to occur through carbon catabolite control (CRP-cAMP) (Manneh-Roussel et al.,
255 2018; Shimada et al., 2013). Our transcriptomic data suggests that indole affects genes from the Fur
256 regulon (Table S1). Fur responds to iron levels and is generally known to be a repressor of iron/heme
257 transport genes (such as *hutA*, *hutXW*, *tonB*, *fbpA*, *viuB*, among others) and also activates a small
258 number of genes (namely the *napABC* operon and *menB* in V. *cholerae*) (Mey et al., 2005). The V.
259 *cholerae* *raiA* gene promoter, appears to carry sequences similar to Fur boxes described in V. *cholerae*
260 (Davies et al., 2011). In order to shed light in the means by which indole induces expression from the
261 *raiA* promoter in exponential phase, we constructed deletion mutants for *fur*, *crp* and for the stationary
262 phase sigma factor *rpoS*. We used our *PraiA-gfp* transcriptional fusion to measure expression in the
263 presence and absence of indole in the mutants compared to wild type strain.

264 As expected, in WT *V. cholerae*, fluorescence was triggered by indole at 3 hours of culture which
265 corresponds to early exponential phase (OD 0.2 to 0.3), and accumulated in stationary phase (Figure
266 **4A and S4AB**). Deletion of *crp* strongly decreases *raiA* expression (25x in exponential phase, **Figure 4B
267 and S4AB**), confirming that CRP is a prevailing activator of *V. cholerae raiA*. On the other hand, deletion
268 of *rpoS* had no major effect on *V. cholerae raiA* expression (1.25x decrease). In the *Δfur* strain, *raiA*
269 expression was increased 2-fold (**Figure 3B and S4AB**), suggesting Fur dependent repression of the *raiA*
270 promoter. No major effect on *raiA* promoter was observed upon treatment with iron, possibly because
271 iron is already in excess levels for the cells during growth. Strikingly, treatment with dipyridyl (DP), an
272 iron chelator which mimics conditions of iron starvation strongly induced fluorescence (**Figure 4A**). To
273 confirm that these effects were specific to the *raiA* promoter, we introduced constitutively expressed
274 *gfp* using the same plasmid vector in the strains WT, *Δcrp* and *Δfur* and observed no effect on
275 fluorescence (Figure S8). This was also the case upon indole treatment of the WT strain. DP appears to
276 decrease fluorescence for constitutive promoter, while increasing it 4x from *raiA* promoter (Figure 3B).
277 Overall, these data confirm that decreased fluorescence in *Δcrp* and increased fluorescence in *Δfur* or
278 upon indole and DP treatments are specific to an effect on transcription from the *raiA* promoter.

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

282 **The effect of RaiA overexpression is conserved in the Gram-negative pathogen *Pseudomonas aeruginosa***.
283

284 We next asked whether RaiA could have a similar function in other Gram-negative pathogens such as
285 *Pseudomonas aeruginosa*, an organism associated with antibiotic resistance and persistence (Koeva et
286 al., 2017; Spoering and Lewis, 2001) and of high concern regarding resistant infections. *P. aeruginosa*
287 RaiA exhibits 37% protein identity with RaiA from *V. cholerae*. We overexpressed *V. cholerae* RaiA in
288 *P. aeruginosa* and assessed lag phase and persistence, as we performed for *V. cholerae*. We found that
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upon RaiA overexpression, lag phase is also decreased in *P. aeruginosa* (Figure 9A and S6D), and strikingly, persistence to tobramycin is increased (Figure 9B). These results show that RaiA-protected sleeping ribosomes can be involved in persistent infections by various pathogenic bacteria.

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## Discussion

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We show here that indole is produced upon sub-MIC aminoglycoside treatment in *V. cholerae* and at physiological concentrations, and increases appearance of persister cells in lethal concentrations of AGs. We find that such increase in persistence occurs through an inducible mechanism involving RaiA (previously called pY or yfiA in *E. coli*, and *vrp* in *V. cholerae*). While indole improved growth in the presence of sub-MIC antibiotics seems to be nonspecific to an antibiotic class and independent of RaiA, increased persistence involving RaiA is specific to AGs in exponentially growing bacteria. Since RaiA is regulated by several environmental cues and signaling molecules, our findings highlight a new, inducible mechanism of persistence, based on increased protection of ribosomes during stress, rather than slowdown of the metabolism.

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*In vitro* characterization of RaiA in *E. coli*, has previously shown association with ribosomes during cold shock and stationary phase, but not during growth at 37°C, suggesting that binding of RaiA is prompted by stress (Agafonov et al., 2001). Based on crystal structures, RaiA was suggested to arrest translation (Vila-Sanjurjo et al., 2004). However, our results in *V. cholerae* do not support such a role, since there is no impact of RaiA overexpression on growth, and rather point to a protective effect of RaiA under ribosomal stress caused by AG treatment.

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Alternatively, since RaiA is able to stabilize the 70S ribosome monomers against dissociation *in vitro* (Agafonov et al., 1999; Di Pietro et al., 2013), it was proposed to constitute a pool of inactive 70S ribosomes preserved from degradation in bacteria (Giuliodori, 2016; Giuliodori et al., 2007; Gualerzi et al., 2011).

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*In vivo* effects of RaiA on bacterial phenotypes are less well described. A protective effect of RaiA during stress, such as starvation, was previously observed in the Gram-positive species *Mycobacterium tuberculosis* (Li et al., 2018) and *Lactococcus lactis* (Puri et al., 2014). Yet, the mechanisms remained enigmatic. Our results show a role of RaiA on survival to antibiotic stress in Gram-negative pathogens. One known mechanism of protection of non-translating ribosomes is ribosome hibernation. The ribosome hibernation factors, Rmf (ribosome modulation factor) and Hpf (hibernation promoting factor) (Maki et al., 2000), dimerize 70S ribosomes (monosomes) into 100S hibernating ribosome dimers. The importance of ribosome hibernation in stress survival is well established in various bacteria (McKay and Portnoy, 2015; Tkachenko et al., 2017), as 100S dimers are less susceptible to degradation by RNases (Feaga et al., 2020; Prossliner et al., 2018; Prossliner et al., 2021; Wada et al., 2000; Yamagishi et al., 1993). Ribosome hibernation factors were even proposed as potential new targets for antibiotics (Matzov et al., 2019). However, in some cases, 70S particles appear to be more robust during heating than 100S dimers which dissociate into 30S and 50S subunits more rapidly (Niven, 2004).

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RaiA was previously identified as bound to the ribosome together with Rmf and Hpf, but its role in relation with ribosome hibernation is unclear. In *V. cholerae*, RaiA shows a synergistic effect with Hpf for survival to starvation (Sabharwal et al., 2015), and we observe a synthetic lethal phenotype for the deletion of *raiA* and *hpf*. Despite such apparent synergy with hibernation factors, RaiA was shown to inactivate 70S ribosomes without forming 100S dimers (Polikanov et al., 2012; Ueta et al., 2005). RaiA thus appears to act in a process different than ribosome hibernation, maybe by blocking breaking down of ribosomes into 30S and 50S subunits by ribosome recycling factors (Rrf/EF-G) (Agafonov et al., 1999; Janosi et al., 1996).

In *E. coli*, RaiA can even prevent 100S dimer formation by Hpf and Rmf (Maki et al., 2000; Ueta et al., 2005). According to cryo-EM data, RaiA can compete with Rmf for ribosome binding, hence shifting the ribosome content from a Rmf-mediated dimeric inactive form to a RaiA-bound monomeric inactive form (Franken et al., 2017). Recent work has identified RaiA as a player in persister formation together with hibernation factors, through a dormancy related mechanism linked to stringent response/ppGpp
339 (Song and Wood, 2020; Wood and Song, 2020). In these studies, persister formation was assessed on
340 near stationary cell cultures (OD 0.8), where these factors are highly expressed, and persistence is
341 largely due to dimerization of inactive ribosomes, leading to a general dormancy state which also
342 favors persister formation to other antibiotics like ampicillin and ciprofloxacin. In contrast, the present
343 study shows in exponentially growing cells and without nutrient limitation, a specific mechanism
344 involving the induction of RaiA, triggering persistence specifically to aminoglycosides (and not to other
345 antibiotics), without affecting growth (Figure 10A). The mechanism we describe is also different than
346 the ppGpp-independent persistence caused in slow growing bacteria (Pontes and Groisman, 2019),
347 since increased RaiA does not affect growth rate in our study. In this regard, the RaiA-dependent
348 persistence mechanism observed in the present study is not a dormancy-like or slow growth
349 mechanism, unlike the increased persistence triggered by ppGpp to antibiotics from various families,
350 in stationary phase. Moreover, no effect of hibernation factors Rmf/Hpf is observed, suggesting
351 different persistence mechanisms than ribosome dormancy formation in growing cells. Recent studies
352 also show the existence of mechanisms triggering persistence independently of ppGpp or hibernation
353 factors in bacteria (Hossain et al., 2021; Wood et al., 2021). We show here that RaiA is one player
354 enhancing persister formation in the Gram-negative bacterium V. cholerae.

355 While RaiA, Hpf and Rmf are rapidly released from ribosomes when normal growth conditions are
356 restored (Agafonov et al., 2001; Maki et al., 2000), ribosome reactivation necessitates dissociation of
357 hibernating 100S ribosome dimers into monomers by HflX and other factors (Basu and Yap, 2017),
358 whereas no dissociation factor is needed for the reactivation of the RaiA inactivated 70S ribosome.
359 There may thus be an interplay and equilibrium between hibernating 100S-Hpf/Rmf and "sleeping"
360 70S-RaiA forms.

361 Such synergy or antagonism between inactive 70S and 100S ribosome pools can however depend on
362 the nature of the stress and the bacterial species. We show here that persistence to aminoglycosides
363 is better achieved in the presence of RaiA (70S), rather than hibernation factors (100S) in V. cholerae.
364 Here, we propose that increased production of RaiA (e.g. upon stress) leads to preservation of non-
365 translating ribosomes in a pool of inactive and intact 70S "sleeping" ribosomes. In that scenario,
366 bacteria can keep growing in the absence of the antibiotic, since RaiA will not bind to actively
367 translating ribosomes. Upon stress affecting translation (e.g. aminoglycosides), ribosomes may stall or
368 stop, which would lead to the dissociation from the mRNA. If RaiA is induced, we propose that such
369 non-translating 70S ribosomes will be bound and protected by RaiA, and avoid death upon antibiotic
370 treatment, not by entering a dormant state, but by inducing protection of ribosomes. We propose that,
371 in contrast to hibernating ribosomes which need recycling factors to dissociate into 70S monosomes,
372 such sleeping ribosomes can be rapidly reactivated upon stress relief through spontaneous dissociation
373 of RaiA from the intact 70S monosome (Agafonov et al., 2001; Maki et al., 2000), conferring an
374 advantage for stress survival (Figure 10AB).

375 In line with this, a recent study showed that the greater the ribosome content of the cell, the faster
376 persister cells resuscitate (Kim et al., 2018). In parallel, protein synthesis was shown to be necessary
377 for persister cell formation in V. cholerae (Paranjape and Shashidhar, 2019), consistent with the fact
378 that we see no effect of RaiA overexpression on growth rates, thus on protein synthesis during growth.
379 Our results do not exclude however another role for RaiA on translating ribosomes.

380 Increased RaiA levels thus allow higher persistence, but what is controlling RaiA expression? RaiA is
381 expressed in stationary phase and during cold shock (Agafonov et al., 1999; Agafonov et al., 2001; Maki
382 et al., 2000; Sabharwal et al., 2015). Other known factors are the heat shock in V. cholerae (Slamti et
383 al., 2007), stringent response (Prossliner et al., 2018), envelope stress and the carbon catabolite
384 response (Manneh-Roussel et al., 2018; Shimada et al., 2013). We additionally show that raiA
385 expression is linked to iron levels and responds to Fur regulation. Interestingly, iron associates with
386 ribosomes (Bray et al., 2018) and a link between iron and modulation of ribosome function during
387 stress has been described (Zinskie et al., 2018). Iron control of RaiA may thus allow protection of
388 ribosomes from iron related damage. Altogether, RaiA appears to be part of the bacterial response to
389 environmental stress.

390 Results of the present study constitute a link between bacterial signaling, ribosome protection and
391 persistence. Moreover, since RaiA action appears to be conserved in various pathogens (Gram-
392 negatives and positives, such as S. aureus and M. tuberculosis), it may be one factor involved in the
393 failure of treatment in persistent infections. It would be interesting to ask now whether RaiA can be
394 used as an early indicator of persistence, which would allow isolation of persisters within a
395 heterogeneous population and further studies using single cell approaches.
396

397 **Limitation of Study**
398 This study shows that indole is secreted under sub-MIC aminoglycosides treatment and increases raiA
399 expression. It also shows that indole is implicated in raiA-dependent persister formation under lethal
400 aminoglycosides treatment. However, we do not address whether indole is also produced under lethal
401 treatment because it is challenging to quantify indole production or tnaA expression on dying cells.
402 Development of this method in our lab could be the purpose of a future work.

403 In addition, raiA-dependent persister formation is shown here to be specific to aminoglycosides
404 (bactericidal), which target translation. Bacteriostatic antibiotics such as chloramphenicol or
405 tetracycline also target the ribosome, but persistence assays are usually not performed using these
406 antibiotics. Our study does not exclude a role for raiA protection on sleeping in the response to such
407 translation targeting antibiotics.
408

408
409 **Author Contributions**
410 Experiments were designed by Z.B. Experiments were conducted by M.L., E. K., C.K. and Z. B., and
411 results were interpreted by Z.B. and M.L. RNA-seq library preparation, sequencing and statistical
412 analysis was performed by O. S., H.V. and J-Y. C. The manuscript was written and reviewed by Z.B., M.L.
413 and D.M. All authors read and approved the final manuscript.

414 **Declaration of Interests**

415 The authors declare no competing interests.

416 Acknowledgements

417 We are thankful to Micheline Fromont-Racine for her valuable help with the experiments for ribosome
418 content analysis by sucrose density gradients. We thank Ivan Matic, Wei-Lin Su and Sébastien Fleurier
419 for helpful discussions and Ivan Matic for critical reading of the manuscript. We also thank Sebastian
420 Aguilar Pierlé for advice for Neo-Cy5 uptake experiments. We thank Louna Fruchard for help with
421 persistence assays. This work was supported by the Institut Pasteur, the Centre National de la
422 Recherche Scientifique (CNRS-UMR 3525), the Fondation pour la Recherche Médicale (FRM Grant No.
423 DBF20160635736), ANR Unibac (ANR-17-CE13-0010-01) and Institut Pasteur grant PTR 245-19.
424

425 Main Figure Titles and Legends

426 **Figure 1.** Indole is produced during growth in sub-MIC tobramycin and induces *raiA*. Measure of
427 extracellular indole concentrations of bacterial cultures grown overnight in rich medium MOPS
428 (Teknova EZ rich defined medium) with and without tobramycin at 0.10 µg/ml (TOB 0.10) or 0.15 µg/ml
429 (TOB 0.15) using the Kovacs reagent (Saint-Ruf et al., 2014). Δ*tnaA* strain was used as negative control
430 without tobramycin. Experiments were performed in triplicates, and statistical analysis was performed
431 (***: *p*<0.001). Error bars represent standard deviation.

432 **Figure 2.** Modulation of persistence of exponential phase WT and ΔraiA V. cholerae by indole. A to
433 D: Early exponential phase of wild-type (WT) and ΔraiA V. cholerae cultures were treated with lethal
434 doses of the specified antibiotics for 20 hours. The y axis represents survival, as the number of CFU
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growing after antibiotic treatment and removal divided by the total number of CFU at time zero (before antibiotic treatment). Bars represent geometric means and error bars represent geometric standard deviation. Tobramycin (TOB): 10 µg/ml, gentamicin (GEN): 5 µg/ml, carbenicillin (CRB): 100 µg/ml, indole (IND): 350 µM. Experiments were performed 3 to 6 times, and statistical analysis was performed (*: *p* < 0.05; **: *p* < 0.01).

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

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

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

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**Figure 6.** RaiA influences lag phase upon growth restart after the stationary phase. Growth is measured on a TECAN plate reader. The curves of wild type *V. cholerae* with the empty plasmid are compared with plasmid carrying *raiA* under inducible promoter in MH media containing either glucose or arabinose. The promoter is repressed using glucose (GLC) and induced using arabinose (ARA). Growth was performed as specified, with RaiA expression repressed or induced in the overnight culture used for inoculum (overnight culture supplemented or not with ARA; indicated as *during pre-cultures*), or/and with RaiA expression induced or not during growth in the microplate reader (growth media supplemented or not with ARA in the microplate; indicated as *during sub-cultures*) : **A.** GLC/ARA, **B.** ARA/GLC, **C.** ARA/ARA, **D.** GLC/GLC. A scheme of the experimental setup is found on Figure S5A. Experiments were performed in triplicates and geometric means are represented. Error bars represent the geometric standard deviation. See also **Figure S6B**.

**Figure 7.** The effect of RaiA on lag phase is independent of ribosome hibernation factors Rmf/Hpf. **A.** Growth in MH media of WT and mutant strains. **B.** Growth of *V. cholerae* overexpressing hibernation factors.
factors or empty plasmid in MH media containing arabinose (ARA/ARA). CD: Growth of hibernation factor mutants Δrmf and Δhpf overexpressing RaiA in MH media containing arabinose (ARA/ARA). Experiments were performed in triplicates and geometric means are represented. Error bars represent the geometric standard deviation. See also **Figure S6C**.

**Figure 8.** RaiA levels influence stationary phase 70S ribosome content relative to 50S+30S subunits in *V. cholerae*. **A.** Cellular extracts of 24 hours cultures (ΔraiA and WT *V. cholerae* carrying the p0/pBAD-RaiA vector, in MH media containing spectinomycin and arabinose) were separated on 10-50% sucrose density gradient. Ribosomal RNA content was measured at OD 260 nm using a spectrometer coupled to a pump and time on X axis represents samples from less dense (upper fragments, smaller complexes) to denser (bottom of the tube, heavier complexes). Lysis was performed in the presence of 10 mM MgCl₂. Cell debris eluting before 550 seconds are not shown. Graphs are normalized to total OD 260 nm =1 for each sample. Mean values are indicated within histogram bars. Error bars represent standard deviation. **B.** Percentage of 70S ribosomes over total ribosome subunits (70S)/(70S+50S+30S). Error bars represent standard deviation (**: p<0.01).

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

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

## STAR METHODS

## RESOURCE AVAILABILITY

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

526 **Materials availability**

527 This study did not generate new unique reagents.

528 **Data and code availability**

529 RNA-seq data are deposited at GEO: GSE182561

530 **EXPERIMENTAL MODEL AND SUBJECT DETAILS**

531 **Bacterial Strains and Plasmids**

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

536 **Media and Growth Conditions**

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

542 **METHOD DETAILS**

543 **Persistence tests.** Persistence tests were performed on early exponential phase cultures. In order to
544 clear the culture from previously non-growing cells that could potentially be present from the
545 stationary phase inoculum, we performed a two-step dilution protocol, before antibiotic treatment.
546 For overexpression experiments, glucose 1% was added to the overnight cultures to repress the pBAD
547 promoter. Overnight *V. cholerae* cultures were first diluted 1000x in 4 ml fresh Mueller-Hinton (MH)
548 medium, without indole (with the antibiotic allowing to maintain the plasmid and incubated at 37°C
549 with shaking. For overexpression experiments, arabinose 0.2% was added at this first dilution step to
550 the fresh MH media to induce the pBAD promoter. When the OD 620 nm reached ~0.2, cultures were
551 diluted 1000x a second time, in order to clear them from non-growing cells, in Erlenmeyers containing
552 25 ml fresh MH medium, without or with indole at 350 µM (with the antibiotic allowing to maintain
553 the plasmid, when the strain carried a plasmid), and were allowed to grow at 37°C. For overexpression
554 experiments, arabinose 0.2% was added again at this second dilution step to the fresh MH media to
555 induce the pBAD promoter. When cultures reached an OD 620 nm between 0.25 and 0.3 (early
556 exponential phase), appropriate dilutions were plated on MH plates to determine the total number of
557 CFUs in time zero untreated cultures. Note that for *V. cholerae*, it was important to treat cultures at
558 the precise OD 620 nm 0.25-0.3, as persistence levels seem to be particularly sensitive to growth phase
559 in this species, where they decline in stationary phase, and because we wanted to avoid any stationary
560 phase protein expression such as *raiA* or *rpoS* at later growth. 5 ml of cultures were collected into 50
561 ml Falcon tubes and treated with lethal doses of desired antibiotics (5-10 times the MIC: tobramycin
562 10 µg/ml, gentamicin 5 µg/ml, neomycin 30 µg/ml, carbenicillin 100 µg/ml, ciprofloxacin 0.025 µg/ml,
563 trimethoprim 50 µg/ml) for 20 hours at 37°C with shaking in order to guarantee oxygenation. The same
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protocol was used for persistence assays on *P. aeruginosa*. Appropriate dilutions were then plated on MH agar without antibiotics and proportion of growing CFUs were calculated by doing a ratio with total CFUs at time zero. Experiments were performed 3 to 6 times.

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**Quantification of extracellular indole concentrations.** Extracellular indole concentration was measured on bacterial cultures grown overnight with and without antibiotics using the Kovacs reagent (Saint-Ruf et al., 2014). First, we established an indole concentration standard curve using 1 ml culture medium (without bacteria) supplemented with indole 0 to 1000 µM (100 µM steps). After adding 500 µl KOVACS reagent, 100 µl of the top layer of the reaction was mixed with 800 µl isoamyl-HCl and OD was read at 570 nm. For indole measurement on bacterial samples, 1 ml of culture was subjected at the same protocol. Measured indole concentration was normalized to the bacterial dry mass based on the assumption that for an OD 600 nm = 1 bacterial dry mass is 0.3 mg/ml (Soini et al., 2008). We detected no indole production in MH medium in any condition. In order to quantify secreted indole in the presence and absence of sub-MIC antibiotics, we thus used the defined rich MOPS transparent medium (Teknova EZ rich defined medium), where MIC TOB is 0.75 µg/ml. All the following experiments (persistence, growth) were conducted in MH, as it allows to study the impact of defined indole concentrations added to the media.

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**Quantification of fluorescent neomycin uptake** was performed as described (Pierlé et al, manuscript in revision, (Okuda, 2015; Sabeti Azad et al., 2020)). Neo-cy5 is an aminoglycoside coupled to the fluorophore Cy5, and has been shown to be active against Gram- bacteria (Okuda, 2015; Sabeti Azad et al., 2020). Briefly, overnight cultures were diluted 100-fold in rich MOPS (Teknova EZ rich defined medium). When the bacterial strains reached an OD 620 nm of 0.25, they were incubated with 0.4 µM of Cy5 labeled Neomycin for 15 minutes at 37°C. 10 µl of the incubated culture were then used for flow cytometry, diluting them in 250 µl of PBS before reading fluorescence. WT *V. cholerae*, was incubated simultaneously without Neo-Cy5 as a negative control. Flow cytometry experiments were performed as described (Baharoglu et al., 2010) and repeated at least 3 times. For each experiment, 100,000 events were counted on the Miltenyi MACSquant device.

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

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**RNA-seq.** Overnight cultures of the O1 biovar El Tor N16961 *hapR+ V. cholerae* strain were diluted 100x and grown in triplicate in MH medium until an OD 620 nm = ~0.4 with or without 350 µM indole. Sample collection, total RNA extraction, library preparation, sequencing and analysis were performed as previously described (Krin et al., 2018).

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**raiA qRT-PCR.** Total RNA was extracted and purified from exponential phase cultures in MH in presence or absence of indole, as previously described (Krin et al., 2018). Reverse transcription (RT) was performed on 100 ng total RNA using SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen). Quantitative PCR was performed on 2 µl RT sample diluted 10-fold using SYBR Green PCR Master Mix (APPLIED) and QuantStudio 6. Quantification was performed using standard range. Expression values were normalized against *gyrA* as previously described in *V. cholerae* (Liu et al., 2010; Lo Scrudato and Blokesch, 2012).

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**Quantification of raiA expression by fluorescent flow cytometry using a gfp fusion.** gfp was amplified by PCR using primers carrying the raiA promoter region and cloned into pTOPO-TA cloning vector. The PraiA-gfp fragment was then extracted using EcoRI and cloned into the low copy plasmid pSC101 (1 to 5 copies per cell). The plasmid was introduced into desired strains, and fluorescence was measured on
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indicated conditions, by counting 100,000 cells on the Miltenyi MACSquant device. Likewise, the control plasmid Pc-gfp (constitutive) was constructed using primers ZIP513/ZIP200 and similarly cloned in pSC101.

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**Growth curves.** Overnight cultures were diluted 100x in fresh medium, on 96 well plates. Each well contained 200 µl. For overexpression lag experiments, glucose 1% was added to the media to repress the pBAD promoter while arabinose 0.2% was added to the media to induce the pBAD promoter, and same growth conditions (glucose or arabinose) were compared in order to avoid noise due to the effects of arabinose on growth and cells shape in V. cholerae (Espinosa et al., 2020). Spectinomycin was also added during each experiment to maintain the plasmid Plates were incubated with shaking on TECAN plate reader device at 37°C, OD 620 nm was measured every 15 minutes.

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**Preparation of cell lysate for the analysis of ribosome content.** The protocol was adapted from (Qin and Fredrick, 2013). Since we used stationary phase cultures instead of exponential phase, presence of polysomes is not expected. 10 ml of 20 hours cultures were centrifuged in ice cold 50 ml Falcon tubes for 15 minutes at 5000 rpm at 4°C. Pellets were resuspended in 500 µl lysis buffer (10 mM Tris-HCl, pH 8, 10 mM MgCl₂, Lysozyme 1 mg/ml, protease inhibitor), transferred in ice cold 1.5 ml tubes and incubated with 12 µl Ribolock RNase inhibitor (Thermo scientific) and DNasel (5 U/ml) at 4°C for 15 minutes. Cell lysis was performed through 3 cycles of flash-freezing in dry ice and thawing in a water bath at 4°C. 15 µl of 10% sodium deoxycholate were added and cell lysate was obtained after centrifugation at 10,000 rpm for 10 minutes at 4°C. The pellet containing cell debris was discarded. Lysate was kept at -80°C until sucrose gradient ultracentrifugation.

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

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

641

**Supplemental Data**

642

**Table S1. Differentially expressed genes upon indole treatment. Related to Figure 3.**

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**Table S2. Strains and plasmids. Related to STAR Methods.**

644

**Supplementary figures legends.**

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**Figure S1.** Indole improves growth of *V. cholerae* in the presence of tobramycin. A. WT. B. ΔraiA. Growth is measured on a TECAN plate reader. IND: indole 350 µM. TOB: tobramycin sub-MIC (0.6 µg/ml). Experiments were performed in triplicates and geometric means are represented. Error bars represent the geometric standard deviation. Related to **Figure 1**.
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**Figure S2. Persistence and the effect of indole.** **A.** Kinetics of survival of WT *V. cholerae* to tobramycin 10 µg/ml in MH media. Time zero corresponds to the total number of CFU before addition of antibiotics, to an early exponential phase culture (OD 620 nm 0.25-0.3), as described in the methods section. The proportion of surviving cells is calculated after plating and counting growing colonies, and is represented for each time point. Curves represent geometric means of at least 3 replicates for each time point and error bars represent geometric standard deviation. **B.** Persistence of *V. cholerae* WT and Δ*tnaA* mutants in exponential phase (in LB instead of MH, to allow indole production, see methods) after 20 hours treatment with specified antibiotics. Tobramycin (TOB): 10 µg/ml, carbenicillin (CRB): 100 µg/ml. Related to **Figure 2**.

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

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

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

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**Figure S6. Influence of RaiA on the length of lag phase upon growth restart after the stationary phase.** **A.** Experimental set-up. Repression was achieved with 1% glucose (GLC), induction was achieved with 0.2% arabinose (ARA). **B.** Lag time (minutes) defined as time to reach OD 620 nm 0.15 in WT *V. cholerae* carrying empty vector (p0) or pBAD-RaiA vector (see ABCD in Figure 6). **C.** Lag time (in minutes) defined as time to reach OD 620 nm 0.15 in *V. cholerae* ΔraiA, Δrmf and Δhpf and WT *V. cholerae* carrying empty vector (p0) or hibernation factors (pRaiA, pRmf, pHpf) (Figure 7AB) **D.** Lag time (in minutes) defined as time to reach OD 620 nm 0.15 in WT *P. aeruginosa* (Figure 9A) carrying empty vector (p0) or pBAD-RaiA vector. Experiments were performed in triplicates and statistical analysis was performed (**: *p*<0.01; ***: *p*<0.001; ****: *p*<0.0001; ns: not significant). Related to **Figure 6**, **7AB** and **9A**.

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**Figure S7. Influence of RaiA, Rmf and Hpf on persistence.** Persistence of *V. cholerae* WT and mutants in early exponential phase, after 20 hours treatment with 10 µg/ml tobramycin (TOB) in MH media.

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693 Serial dilutions are spotted on plates for estimation of survival. Spot assays were performed at least 3 times. Related to **Figure 7**

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

701

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914 Vila-Sanjurjo, A., Schuwirth, B.S., Hau, C.W., and Cate, J.H. (2004). Structural basis for the control of translation initiation during stress. Nature structural & molecular biology 11, 1054-1059.

916 Wada, A., Mikkola, R., Kurland, C.G., and Ishihama, A. (2000). Growth phase-coupled changes of the ribosome profile in natural isolates and laboratory strains of Escherichia coli. J Bacteriol 182, 2893-2899.

919 Wang, D., Ding, X., and Rather, P.N. (2001). Indole can act as an extracellular signal in Escherichia coli. J Bacteriol 183, 4210-4216.

921 Wood, T.K., and Song, S. (2020). Forming and waking dormant cells: The ppGpp ribosome dimerization persister model. Biofilm 2, 100018.

923 Wood, W.N., Mohler, K., Rinehart, J., and Ibba, M. (2021). Deacylated tRNA Accumulation Is a Trigger for Bacterial Antibiotic Persistence Independent of the Stringent Response. mBio, e0113221.

925 Yamagishi, M., Matsushima, H., Wada, A., Sakagami, M., Fujita, N., and Ishihama, A. (1993).

926 Regulation of the Escherichia coli rmf gene encoding the ribosome modulation factor: growth phase- and growth rate-dependent control. EMBO J 12, 625-630.

928 Yanofsky, C., Horn, V., and Gollnick, P. (1991). Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli. J Bacteriol 173, 6009-6017.

930 Zarkan, A., Matuszewska, M., Trigg, S.B., Zhang, M., Belgami, D., Croft, C., Liu, J., El-Ouisi, S., Greenhalgh, J., Duboff, J.S., et al. (2020). Inhibition of indole production increases the activity of quinolone antibiotics against E. coli persisters. Scientific reports 10, 11742.

933 Zinskie, J.A., Ghosh, A., Trainor, B.M., Shedlovskiy, D., Pestov, D.G., and Shcherbik, N. (2018). Iron-dependent cleavage of ribosomal RNA during oxidative stress in the yeast Saccharomyces cerevisiae. J Biol Chem 293, 14237-14248.

936

937
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

TOB 0.15
(20% MIC)

TOB
0.10

ΔtnaA
A

TOB

Survival

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

CRB

C

Survival

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

B

GEN

Survival

<figure><img src="image_6.png" /></figure>

D

TMP

Survival

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

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

B

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

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

B

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

TOB

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

B

GEN

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

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

C

NEO

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

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

D

CRB

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

E

CIP

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

F

TMP

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

<figure><img src="image_20.png" /><figcaption>RaiA overexpressed during sub-cultures only (GLC/ARA)</figcaption></figure>

B

<figure><img src="image_21.png" /><figcaption>RaiA overexpressed during pre-cultures only (ARA/GLC)</figcaption></figure>

C

<figure><img src="image_22.png" /><figcaption>RaiA overexpressed during pre and sub-cultures (ARA/ARA)</figcaption></figure>

D

<figure><img src="image_23.png" /><figcaption>RaiA never overexpressed (GLC/GLC)</figcaption></figure>
A

Growth of mutants

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

B

Hibernation factors overexpressed during pre and sub-cultures
(ARA/ARA)

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

C

D

RaiA overexpressed during pre and sub-cultures (ARA/ARA)

RaiA overexpressed during pre and sub-cultures (ARA/ARA)

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

<figure><img src="image_27.png" /></figure>
<figure><img src="image_28.png" /><figcaption>B</figcaption></figure>

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

<figure><img src="image_30.png" /><figcaption>RaiA overexpressed before and during growth curves

*P. aeruginosa*</figcaption></figure>

B

<figure><img src="image_31.png" /></figure>
## A Persistence

<figure><img src="image_32.png" /></figure>

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

## B Lag period

Translating
ribosomes

De novo
ribosome
synthesis

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

Conditions of growth arrest
or non lethal translation stress

<figure><img src="image_35.png" /></figure>

Stat phase

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

RaiA-bound 70S pool

Sleeping ribosomes

Non-translating
ribosomes

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

Dissociation
Degradation

Faster
restart

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

70S

70S

70S

Slower restart

Conditions of growth restart
<figure><img src="image_39.png" /><figcaption>**Figure S1.** Indole improves growth of *V. cholerae* in the presence of tobramycin. A. WT. B. ΔraiA. Growth is measured on a TECAN plate reader. IND: indole 350 µM. TOB: tobramycin sub-MIC (0.6 µg/ml). Experiments were performed in triplicates and geometric means are represented. Error bars represent the geometric standard deviation. Related to **Figure 1**.</figcaption></figure>
**Figure S2. Persistence and the effect of indole.** A. Kinetics of survival of WT *V. cholerae* to tobramycin 10 µg/ml in MH media. Time zero corresponds to the total number of CFU before addition of antibiotics, to an early exponential phase culture (OD 620 nm 0.25-0.3), as described in the methods section. The proportion of surviving cells is calculated after plating and counting growing colonies, and is represented for each time point. Curves represent geometric means of at least 3 replicates for each time point and error bars represent geometric standard deviation. B. Persistence of *V. cholerae* WT and *ΔtnaA* mutants in exponential phase (in LB instead of MH, to allow indole production, see methods) after 20 hours treatment with specified antibiotics. Tobramycin (TOB): 10 µg/ml, carbenicillin (CRB): 100 µg/ml. Related to **Figure 2**.

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

<figure><img src="image_41.png" /></figure>
**Figure S3.** Indole does not apparently influence aminoglycoside entry and resistance in *V. cholerae* WT and *ΔraiA*. **A.** Intracellular level of neomycin coupled to the fluorophore Cy5 measured by fluorescence associated flow cytometry. Error bars represent standard deviation. **B.** Minimal inhibitory concentrations of tobramycin (TOB) and gentamicin (GEN) measured using *etests* in *V. cholerae*, in the absence (MH) and presence of indole (IND), and indicated in µg/ml by a numeral on each image. Related to **Figure 2**.

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

<figure><img src="image_43.png" /></figure>
**Figure S4. Fluorescence detection from the raiA promoter-gfp fusion by flow cytometry.**
Representative acquisitions are shown. Each plot represents one experiment. Each experiment was performed at least 3 times and data and statistical significance are shown in the histograms in the main manuscript. A: histogram curves: GFP fluorescence is represented in the x-axis (FITC channel), the y-axis represents the number of events corresponding to the number of cells, normalized to height (same number of total cells for both conditions). B: dot plots: Bacterial cells' size and shape (rugosity). FSC: forward scatter. SSC: size scatter. The SSC (size scatter) vs FSC (forward scatter) graphs show the distribution of cells by size and shape and no major difference in cell size/shape is observed between the WT and mutants with or without indole. Related to **Figure 4**.

A

<figure><img src="image_44.png" /><figcaption>**B** Cell shape and size</figcaption></figure>

<figure><img src="image_45.png" /></figure>
**Figure S5. RaiA is involved in persistence in V. cholerae and E. coli.** A. Increase of surviving colonies (persisters) after 20 hours of treatment with tobramycin (TOB) 10 µg/ml (ratio over WT surviving colonies) in V. cholerae. Error bars represent geometric standard deviation B. Persistence of E. coli WT and *raiA* mutant, at late exponential phase (OD 620nm 0.5), after 20 hours treatment with 10 µg/ml tobramycin (TOB) in MH media. Serial dilutions are spotted on plates for estimation of survival. Spot assays were performed at least 3 times. Related to **Figure 2**.

<figure><img src="image_46.png" /></figure>
**Figure S6. Influence of RaiA on the length of lag phase upon growth restart after the stationary phase.**

**A.** Experimental set-up. Repression was achieved with 1% glucose (GLC), induction was achieved with 0.2% arabinose (ARA). **B.** Lag time (minutes) defined as time to reach OD 620 nm 0.15 in WT *V. cholerae* carrying empty vector (p0) or pBAD-RaiA vector (see ABCD in Figure 6). **C.** Lag time (in minutes) defined as time to reach OD 620 nm 0.15 in *V. cholerae* carrying empty vector (p0) or hibernation factors (pRaiA, pRmf, pHpf) (Figure 7AB) **D.** Lag time (in minutes) defined as time to reach OD 620 nm 0.15 in WT *P. aeruginosa* (Figure 9A) carrying empty vector (p0) or pBAD-RaiA vector. Experiments were performed in triplicates and statistical analysis was performed (**: *p*<0.01; **: *p*<0.001; ****: *p*<0.0001; ns: not significant). Related to **Figure 6**, **7AB** and **9A**.

A

<figure><img src="image_47.png" /></figure>

B

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

C

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

D

<figure><img src="image_50.png" /></figure>
<figure><img src="image_51.png" /><figcaption>**Figure S7. Influence of RaiA, Rmf and Hpf on persistence.** Persistence of *V. cholerae* WT and mutants in early exponential phase, after 20 hours treatment with 10 µg/ml tobramycin (TOB) in MH media. Serial dilutions are spotted on plates for estimation of survival. Spot assays were performed at least 3 times. Related to **Figure 7**.</figcaption></figure>
**Figure S8. Expression of *gfp* from constitutive promoter (Pc) in conditions where expression from the *raiA* promoter is up or down-regulated.** Fluorescence quantification of GFP expression from constitutive promoter by flow cytometry in MH media in exponential phase, in WT and indicated *V. cholerae* deletion mutants. IND: indole (350 µM), DP: 2,2'-Dipyridyl (500 µM). The Y axis represents fluorescence ratio of the mutant over wild type (WT) strain. Error bars represent standard deviation. Related to **Figure 4**.

<figure><img src="image_52.png" /></figure>
<table><caption>Table S1: Genes differentially regulated by indole. Related to Figure 3.</caption><thead><tr><th>Id</th><th>norm MH-A</th><th>norm MH-B</th><th>norm MH-C</th><th>norm IND-A</th><th>norm IND-B</th><th>norm IND-C</th><th>Fold change</th><th>p value</th><th>adj p value</th></tr></thead><tbody><tr><td>UP</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VC0706</td><td>3903</td><td>4103</td><td>3710</td><td>80171</td><td>68404</td><td>14508<br>6</td><td>20,1</td><td>8,0E-03</td><td>8,9E-03</td></tr><tr><td>VC1484</td><td>120</td><td>111</td><td>142</td><td>570</td><td>232</td><td>501</td><td>3,1</td><td>2,6E-05</td><td>1,3E-01</td></tr><tr><td>Motility - Chemotaxis</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VC0410</td><td>1779</td><td>1337</td><td>1477</td><td>3761</td><td>3910</td><td>3890</td><td><strong>2,5</strong></td><td>1,7E-14</td><td>8,0E-03</td></tr><tr><td>VC1049</td><td>413</td><td>473</td><td>450</td><td>2398</td><td>2145</td><td>2322</td><td><strong>4,9</strong></td><td>1,1E-38</td><td>9,0E-03</td></tr><tr><td>VC2370</td><td>316</td><td>270</td><td>299</td><td>1469</td><td>1694</td><td>1286</td><td><strong>4,8</strong></td><td>2,6E-35</td><td>1,0E-02</td></tr><tr><td>VC0414</td><td>3748</td><td>3638</td><td>2441</td><td>7061</td><td>7532</td><td>6276</td><td><strong>2,1</strong></td><td>1,4E-06</td><td>1,9E-02</td></tr><tr><td>VC0413</td><td>658</td><td>597</td><td>376</td><td>1239</td><td>1302</td><td>1053</td><td><strong>2,1</strong></td><td>1,2E-05</td><td>3,0E-02</td></tr><tr><td>VC0411</td><td>1939</td><td>1418</td><td>1067</td><td>3411</td><td>3912</td><td>2746</td><td><strong>2,2</strong></td><td>1,6E-05</td><td>3,9E-02</td></tr><tr><td>VCA0536</td><td>508</td><td>934</td><td>598</td><td>1748</td><td>2438</td><td>2062</td><td><strong>2,9</strong></td><td>6,5E-09</td><td>4,6E-02</td></tr><tr><td>VCA0954</td><td>1750</td><td>2718</td><td>1721</td><td>3886</td><td>5756</td><td>4105</td><td><strong>2,1</strong></td><td>2,7E-04</td><td>5,9E-02</td></tr><tr><td>VC0409</td><td>9371</td><td>6775</td><td>5433</td><td>26356</td><td>19176</td><td>30086</td><td><strong>3,3</strong></td><td>3,7E-09</td><td>6,3E-02</td></tr><tr><td>VC0827</td><td>1152</td><td>1207</td><td>1001</td><td>3000</td><td>7845</td><td>2896</td><td><strong>3,6</strong></td><td>4,4E-07</td><td>1,4E-01</td></tr><tr><td>VC0826</td><td>1967</td><td>1966</td><td>1336</td><td>5009</td><td>13957</td><td>5414</td><td><strong>4,0</strong></td><td>4,7E-08</td><td>1,5E-01</td></tr><tr><td>VC1763</td><td>428</td><td>1057</td><td>267</td><td>1554</td><td>1847</td><td>1495</td><td><strong>2,5</strong></td><td>3,3E-03</td><td>2,0E-01</td></tr><tr><td>VC0820</td><td>369</td><td>623</td><td>278</td><td>1133</td><td>751</td><td>2000</td><td><strong>2,6</strong></td><td>1,5E-03</td><td>2,2E-01</td></tr><tr><td>Respiration - energy</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VC1115</td><td>663</td><td>546</td><td>508</td><td>6006</td><td>6126</td><td>5482</td><td><strong>9,8</strong></td><td>6,1E-84</td><td>7,9E-03</td></tr><tr><td>VC0966</td><td>7486</td><td>7229</td><td>6500</td><td>15713</td><td>18048</td><td>21828</td><td><strong>2,6</strong></td><td>2,3E-14</td><td>1,1E-02</td></tr><tr><td>VC2371</td><td>163</td><td>157</td><td>107</td><td>1820</td><td>1907</td><td>1910</td><td><strong>12,1</strong></td><td>6,9E-67</td><td>1,3E-02</td></tr><tr><td>VC0477</td><td>4301</td><td>4841</td><td>4095</td><td>27291</td><td>30017</td><td>20290</td><td><strong>5,6</strong></td><td>4,2E-39</td><td>1,5E-02</td></tr><tr><td>VCA0511</td><td>174</td><td>249</td><td>197</td><td>1325</td><td>1518</td><td>1124</td><td><strong>6,0</strong></td><td>4,6E-35</td><td>1,8E-02</td></tr><tr><td>VCA0665</td><td>329</td><td>253</td><td>320</td><td>9542</td><td>8962</td><td>8507</td><td><strong>27,0</strong></td><td>5,2E-122</td><td>1,8E-02</td></tr><tr><td>VCA0665</td><td>329</td><td>253</td><td>320</td><td>9542</td><td>8962</td><td>8507</td><td><strong>27,0</strong></td><td>5,2E-122</td><td>1,8E-02</td></tr><tr><td>VC1951</td><td>347</td><td>334</td><td>242</td><td>5920</td><td>6998</td><td>5337</td><td><strong>17,9</strong></td><td>1,2E-87</td><td>2,0E-02</td></tr><tr><td>VC1114</td><td>430</td><td>337</td><td>365</td><td>1584</td><td>1201</td><td>1615</td><td><strong>3,7</strong></td><td>1,7E-17</td><td>2,3E-02</td></tr><tr><td>VC0356</td><td>583</td><td>766</td><td>592</td><td>1725</td><td>2375</td><td>1675</td><td><strong>2,9</strong></td><td>7,9E-12</td><td>2,4E-02</td></tr><tr><td>VC2145</td><td>931</td><td>952</td><td>989</td><td>7482</td><td>9974</td><td>5936</td><td><strong>7,6</strong></td><td>1,1E-42</td><td>2,4E-02</td></tr><tr><td>VCA0563</td><td>3532</td><td>3857</td><td>3297</td><td>10041</td><td>12683</td><td>8503</td><td><strong>2,8</strong></td><td>3,1E-11</td><td>2,7E-02</td></tr><tr><td>VC2368</td><td>8558</td><td>1112<br>4</td><td>8314</td><td>25789</td><td>18044</td><td>26995</td><td><strong>2,4</strong></td><td>4,1E-08</td><td>2,9E-02</td></tr><tr><td>VC0278</td><td>512</td><td>493</td><td>641</td><td>1311</td><td>1930</td><td>1207</td><td><strong>2,6</strong></td><td>1,2E-08</td><td>3,1E-02</td></tr><tr><td>VC0485</td><td>8659</td><td>1108<br>5</td><td>11206</td><td>35439</td><td>45566</td><td>26147</td><td><strong>3,3</strong></td><td>4,0E-13</td><td>3,2E-02</td></tr><tr><td>VCA0564</td><td>4137</td><td>4464</td><td>3746</td><td>10730</td><td>12961</td><td>8158</td><td><strong>2,5</strong></td><td>8,6E-08</td><td>3,4E-02</td></tr><tr><td>VC2656</td><td>941</td><td>1355</td><td>964</td><td>33710</td><td>22990</td><td>28100</td><td><strong>22,5</strong></td><td>2,0E-76</td><td>4,1E-02</td></tr><tr><td>VC2646</td><td>2110</td><td>1858</td><td>1643</td><td>4701</td><td>6486</td><td>4176</td><td><strong>2,6</strong></td><td>1,7E-07</td><td>4,6E-02</td></tr><tr><td>VC1687</td><td>61</td><td>69</td><td>112</td><td>1751</td><td>1301</td><td>1331</td><td><strong>15,5</strong></td><td>6,7E-48</td><td>5,0E-02</td></tr></tbody></table>
<table><tbody><tr><td>VC1866</td><td>7913</td><td>1267</td><td>7185</td><td>81168</td><td>10134</td><td>9</td><td>63711</td><td><strong>7,9</strong></td><td>6,8E-31</td><td>5,3E-02</td></tr><tr><td>VC2657</td><td>195</td><td>285</td><td>147</td><td>10748</td><td>7717</td><td>8506</td><td><strong>33,9</strong></td><td>1,1E-70</td><td>7,0E-02</td></tr><tr><td>VC1516</td><td>769</td><td>1005</td><td>666</td><td>2063</td><td>981</td><td>2137</td><td><strong>2,0</strong></td><td>2,6E-03</td><td>7,9E-02</td></tr><tr><td>VC2699</td><td>895</td><td>1117</td><td>1191</td><td>5749</td><td>3169</td><td>5283</td><td><strong>4,0</strong></td><td>1,1E-09</td><td>9,3E-02</td></tr><tr><td>VCA0675</td><td>293</td><td>225</td><td>186</td><td>1644</td><td>816</td><td>2194</td><td><strong>5,7</strong></td><td>1,1E-14</td><td>9,5E-02</td></tr><tr><td>VC0965</td><td>7652</td><td>1711</td><td>3</td><td>10909</td><td>25906</td><td>35487</td><td>21426</td><td><strong>2,2</strong></td><td>1,2E-03</td><td>9,7E-02</td></tr><tr><td>VC1513</td><td>979</td><td>1330</td><td>598</td><td>4220</td><td>2083</td><td>4053</td><td><strong>3,2</strong></td><td>9,9E-07</td><td>1,0E-01</td></tr><tr><td>VC2447</td><td>2591</td><td>3603</td><td>1</td><td>27345</td><td>51499</td><td>10514</td><td>0</td><td>37661</td><td><strong>2,0</strong></td><td>7,0E-03</td></tr><tr><td>VCA0680</td><td>583</td><td>386</td><td>577</td><td>2435</td><td>870</td><td>2782</td><td><strong>3,5</strong></td><td>1,5E-06</td><td>1,3E-01</td></tr><tr><td>VCA0679</td><td>626</td><td>383</td><td>350</td><td>2412</td><td>805</td><td>2866</td><td><strong>3,8</strong></td><td>9,4E-07</td><td>1,6E-01</td></tr><tr><td>VCA0678</td><td>1612</td><td>1152</td><td>1434</td><td>5851</td><td>1605</td><td>7354</td><td><strong>3,0</strong></td><td>1,3E-04</td><td>1,9E-01</td></tr><tr><td>Sugar</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VC2000</td><td>3313</td><td>3852</td><td>3</td><td>32617</td><td>94393</td><td>96880</td><td>78059</td><td><strong>2,5</strong></td><td>2,7E-15</td><td>9,0E-03</td></tr><tr><td>VC0374</td><td>3016</td><td>2691</td><td>2733</td><td>9064</td><td>9875</td><td>7214</td><td><strong>3,0</strong></td><td>2,5E-18</td><td>1,2E-02</td></tr><tr><td>VC1973</td><td>609</td><td>640</td><td>690</td><td>6655</td><td>6173</td><td>5216</td><td><strong>8,8</strong></td><td>4,0E-64</td><td>1,3E-02</td></tr><tr><td>VC1972</td><td>229</td><td>204</td><td>255</td><td>2990</td><td>2618</td><td>2426</td><td><strong>10,7</strong></td><td>3,5E-58</td><td>2,0E-02</td></tr><tr><td>VC1971</td><td>280</td><td>208</td><td>250</td><td>3269</td><td>2917</td><td>2594</td><td><strong>11,0</strong></td><td>1,4E-58</td><td>2,2E-02</td></tr><tr><td>VC0487</td><td>2056</td><td>1941</td><td>1773</td><td>12473</td><td>9768</td><td>9035</td><td><strong>5,1</strong></td><td>6,3E-27</td><td>2,8E-02</td></tr><tr><td>VCA0011</td><td>487</td><td>642</td><td>554</td><td>2564</td><td>2618</td><td>2403</td><td><strong>4,3</strong></td><td>9,8E-20</td><td>2,9E-02</td></tr><tr><td>VC2689</td><td>3011</td><td>2941</td><td>4636</td><td>23493</td><td>19880</td><td>18705</td><td><strong>5,4</strong></td><td>1,3E-24</td><td>3,9E-02</td></tr><tr><td>VC2001</td><td>1039</td><td>618</td><td>617</td><td>2290</td><td>3235</td><td>1950</td><td><strong>3,1</strong></td><td>8,4E-09</td><td>5,9E-02</td></tr><tr><td>VCA0516</td><td>258</td><td>256</td><td>239</td><td>1777</td><td>2151</td><td>1542</td><td><strong>6,5</strong></td><td>7,7E-22</td><td>6,0E-02</td></tr><tr><td>VCA0013</td><td>493</td><td>1132</td><td>476</td><td>2028</td><td>1287</td><td>1755</td><td><strong>2,2</strong></td><td>7,3E-03</td><td>1,7E-01</td></tr><tr><td>RRR</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VCA0521</td><td>1439</td><td>1451</td><td>801</td><td>3686</td><td>3125</td><td>3167</td><td><strong>2,6</strong></td><td>3,1E-07</td><td>4,4E-02</td></tr><tr><td>VCA0520</td><td>694</td><td>723</td><td>482</td><td>1449</td><td>1115</td><td>1411</td><td><strong>2,0</strong></td><td>3,1E-05</td><td>2,9E-02</td></tr><tr><td>VC2386</td><td>4774</td><td>8304</td><td>2645</td><td>8736</td><td>6458</td><td>24643</td><td><strong>2,2</strong></td><td>3,3E-02</td><td>3,6E-01</td></tr><tr><td>Metabol</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VC0336</td><td>7124</td><td>8934</td><td>9210</td><td>19907</td><td>24075</td><td>16926</td><td><strong>2,4</strong></td><td>2,8E-09</td><td>1,8E-02</td></tr><tr><td>VC0026</td><td>268</td><td>258</td><td>311</td><td>1582</td><td>1606</td><td>1262</td><td><strong>5,0</strong></td><td>1,6E-27</td><td>2,1E-02</td></tr><tr><td>VCA0235</td><td>3581</td><td>3566</td><td>3392</td><td>8268</td><td>5703</td><td>8159</td><td><strong>2,0</strong></td><td>5,8E-05</td><td>3,3E-02</td></tr><tr><td>VCA0875</td><td>2063</td><td>2616</td><td>1307</td><td>5759</td><td>4746</td><td>4221</td><td><strong>2,4</strong></td><td>7,0E-06</td><td>4,4E-02</td></tr><tr><td>VC2698</td><td>2245</td><td>3759</td><td>3380</td><td>7765</td><td>5269</td><td>7196</td><td><strong>2,1</strong></td><td>6,6E-04</td><td>5,7E-02</td></tr><tr><td>VC2373</td><td>1415</td><td>690</td><td>1058</td><td>2458</td><td>3824</td><td>2252</td><td><strong>2,5</strong></td><td>6,8E-05</td><td>8,7E-02</td></tr><tr><td>VC2361</td><td>4108</td><td>2170</td><td>3027</td><td>19721</td><td>15393</td><td>32664</td><td><strong>6,1</strong></td><td>1,1E-13</td><td>1,3E-01</td></tr><tr><td>Nucleotide</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VCA0564</td><td>4137</td><td>4464</td><td>3746</td><td>10730</td><td>12961</td><td>8158</td><td><strong>2,5</strong></td><td>8,6E-08</td><td>3,4E-02</td></tr><tr><td>VC1034</td><td>470</td><td>448</td><td>562</td><td>1528</td><td>1273</td><td>1225</td><td><strong>2,7</strong></td><td>5,9E-13</td><td>1,2E-02</td></tr><tr><td>regulators</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VC0486</td><td>1203</td><td>1242</td><td>753</td><td>11275</td><td>8624</td><td>10176</td><td><strong>8,5</strong></td><td>1,0E-37</td><td>3,6E-02</td></tr><tr><td>VC1719</td><td>1362</td><td>981</td><td>674</td><td>5308</td><td>5146</td><td>6057</td><td><strong>5,1</strong></td><td>4,1E-20</td><td>4,3E-02</td></tr><tr><td>VCA0562</td><td>824</td><td>717</td><td>348</td><td>1268</td><td>1261</td><td>1628</td><td><strong>2,1</strong></td><td>9,6E-04</td><td>7,1E-02</td></tr></tbody></table>
<table><tbody><tr><td>VC1919</td><td>1270<br>4</td><td>1288<br>3</td><td>7383</td><td>28846</td><td>15905</td><td>39769</td><td><strong>2,4</strong></td><td>1,1E-03</td><td>1,2E-01</td></tr><tr><td>VC2366</td><td>483</td><td>775</td><td>287</td><td>1133</td><td>1549</td><td>946</td><td><strong>2,2</strong></td><td>3,7E-03</td><td>1,2E-01</td></tr><tr><td>csrD</td><td>5813<br>1</td><td>4493<br>3</td><td>26252</td><td>10280<br>2</td><td>52169</td><td>15791<br>5</td><td><strong>2,2</strong></td><td>1,0E-02</td><td>1,8E-01</td></tr><tr><td>Membrane</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VC1315</td><td>54</td><td>49</td><td>49</td><td>1059</td><td>987</td><td>1071</td><td><strong>18,6</strong></td><td>6,1E-84</td><td>1,0E-08</td></tr><tr><td>VC2484</td><td>2791</td><td>2555</td><td>2045</td><td>8396</td><td>7987</td><td>7622</td><td><strong>3,2</strong></td><td>4,9E-20</td><td>1,1E-02</td></tr><tr><td>VC2485</td><td>2791</td><td>2555</td><td>2045</td><td>8396</td><td>7987</td><td>7622</td><td><strong>3,2</strong></td><td>4,9E-21</td><td>1,1E-02</td></tr><tr><td>VCA0591</td><td>531</td><td>462</td><td>342</td><td>1184</td><td>1285</td><td>1183</td><td><strong>2,7</strong></td><td>9,1E-12</td><td>1,6E-02</td></tr><tr><td>VC0976</td><td>1443</td><td>1713</td><td>1027</td><td>3220</td><td>2738</td><td>3167</td><td><strong>2,1</strong></td><td>1,4E-05</td><td>3,1E-02</td></tr><tr><td>VC2149</td><td>145</td><td>110</td><td>144</td><td>2554</td><td>1821</td><td>2827</td><td><strong>15,4</strong></td><td>6,3E-47</td><td>5,7E-02</td></tr><tr><td>VC0654</td><td>606</td><td>731</td><td>365</td><td>2927</td><td>5686</td><td>3163</td><td><strong>5,9</strong></td><td>8,2E-14</td><td>1,1E-01</td></tr><tr><td>VCA0628</td><td>680</td><td>1842</td><td>822</td><td>2839</td><td>1462</td><td>4368</td><td><strong>2,2</strong></td><td>1,7E-02</td><td>2,9E-01</td></tr><tr><td>Others</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VC0652</td><td>74</td><td>71</td><td>98</td><td>1343</td><td>1362</td><td>1262</td><td><strong>15,1</strong></td><td>3,0E-86</td><td>1,0E-08</td></tr><tr><td>VC0428</td><td>523</td><td>576</td><td>695</td><td>4516</td><td>4519</td><td>4760</td><td><strong>7,3</strong></td><td>1,6E-49</td><td>1,6E-02</td></tr><tr><td>VC1892</td><td>819</td><td>567</td><td>543</td><td>1182</td><td>2075</td><td>1046</td><td><strong>2,1</strong></td><td>5,6E-04</td><td>7,2E-02</td></tr><tr><td>VC0037</td><td>801</td><td>555</td><td>621</td><td>1749</td><td>1301</td><td>2937</td><td><strong>2,8</strong></td><td>6,0E-06</td><td>8,9E-02</td></tr><tr><td>VC1765</td><td>1538</td><td>4879</td><td>1100</td><td>7066</td><td>10157</td><td>6196</td><td><strong>2,6</strong></td><td>3,1E-03</td><td>2,9E-01</td></tr><tr><td>Hypothetical</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VCA0718</td><td>329</td><td>301</td><td>343</td><td>1989</td><td>1854</td><td>1881</td><td><strong>5,7</strong></td><td>6,9E-63</td><td>1,0E-08</td></tr><tr><td>VC1865</td><td>741</td><td>620</td><td>752</td><td>15085</td><td>13445</td><td>13011</td><td><strong>18,5</strong></td><td>7,9E-143</td><td>7,1E-03</td></tr><tr><td>VC1871</td><td>630</td><td>784</td><td>729</td><td>5093</td><td>6873</td><td>5480</td><td><strong>7,7</strong></td><td>7,7E-58</td><td>1,3E-02</td></tr><tr><td>VC1077</td><td>1496</td><td>1106</td><td>1197</td><td>2628</td><td>2923</td><td>2918</td><td><strong>2,2</strong></td><td>1,1E-08</td><td>1,4E-02</td></tr><tr><td>VCA0236</td><td>299</td><td>343</td><td>418</td><td>1039</td><td>1012</td><td>978</td><td><strong>2,8</strong></td><td>2,1E-12</td><td>1,5E-02</td></tr><tr><td>VCA1013</td><td>41</td><td>54</td><td>72</td><td>1544</td><td>916</td><td>1414</td><td><strong>19,5</strong></td><td>9,5E-54</td><td>5,0E-02</td></tr><tr><td>VCA0741</td><td>8417</td><td>7150</td><td>6445</td><td>35536</td><td>27576</td><td>52687</td><td><strong>4,8</strong></td><td>2,1E-17</td><td>5,5E-02</td></tr><tr><td>VCA0919</td><td>1107</td><td>806</td><td>959</td><td>3604</td><td>2763</td><td>4638</td><td><strong>3,6</strong></td><td>4,7E-11</td><td>5,5E-02</td></tr><tr><td>VC0871</td><td>209</td><td>193</td><td>259</td><td>1657</td><td>1019</td><td>2475</td><td><strong>6,8</strong></td><td>3,3E-19</td><td>8,0E-02</td></tr><tr><td>VC1853</td><td>2696</td><td>2439</td><td>1227</td><td>5889</td><td>3740</td><td>7203</td><td><strong>2,4</strong></td><td>3,1E-04</td><td>9,6E-02</td></tr><tr><td>VC2035</td><td>272</td><td>247</td><td>652</td><td>883</td><td>1154</td><td>1004</td><td><strong>2,4</strong></td><td>4,9E-04</td><td>1,1E-01</td></tr><tr><td>VCA0367</td><td>870</td><td>2025</td><td>677</td><td>2495</td><td>2608</td><td>3104</td><td><strong>2,1</strong></td><td>6,6E-03</td><td>1,5E-01</td></tr><tr><td>VCA0547</td><td>1134</td><td>821</td><td>2003</td><td>3510</td><td>1518</td><td>3735</td><td><strong>2,0</strong></td><td>2,3E-02</td><td>1,9E-01</td></tr><tr><td>VC1770</td><td>338</td><td>770</td><td>245</td><td>1049</td><td>1704</td><td>933</td><td><strong>2,4</strong></td><td>5,3E-03</td><td>2,1E-01</td></tr><tr><td>VC2221</td><td>97</td><td>191</td><td>99</td><td>3971</td><td>1113</td><td>3984</td><td><strong>15,1</strong></td><td>9,8E-21</td><td>2,4E-01</td></tr><tr><td>VCA0283</td><td>721</td><td>1944</td><td>390</td><td>2479</td><td>2047</td><td>3333</td><td><strong>2,2</strong></td><td>1,7E-02</td><td>2,6E-01</td></tr><tr><td>VC1764</td><td>769</td><td>2351</td><td>507</td><td>4476</td><td>4878</td><td>3997</td><td><strong>3,0</strong></td><td>5,2E-04</td><td>2,8E-01</td></tr><tr><td>DOWN</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Electron transfer - Ion</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VC2415</td><td>6690</td><td>8047</td><td>8130</td><td>1766</td><td>1466</td><td>1837</td><td>0,2</td><td>3,9E-40</td><td>6,4E-38</td></tr><tr><td>VCA0228</td><td>703</td><td>914</td><td>818</td><td>60</td><td>32</td><td>108</td><td>0,1</td><td>8,9E-25</td><td>7,7E-23</td></tr><tr><td>VC1265</td><td>1746</td><td>1306</td><td>1376</td><td>237</td><td>112</td><td>243</td><td>0,2</td><td>8,6E-24</td><td>6,8E-22</td></tr><tr><td>VCA0227</td><td>2696</td><td>3888</td><td>2205</td><td>333</td><td>155</td><td>375</td><td>0,1</td><td>4,4E-22</td><td>3,3E-20</td></tr></tbody></table>
<table>
  <tr>
    <td>VCA0230</td>
    <td>883</td>
    <td>1120</td>
    <td>957</td>
    <td>128</td>
    <td>53</td>
    <td>173</td>
    <td>0,1</td>
    <td>5,5E-19</td>
    <td>3,1E-17</td>
  </tr>
  <tr>
    <td>VCA0229</td>
    <td>545</td>
    <td>636</td>
    <td>685</td>
    <td>68</td>
    <td>15</td>
    <td>72</td>
    <td>0,1</td>
    <td>6,8E-19</td>
    <td>3,7E-17</td>
  </tr>
  <tr>
    <td>VC2559</td>
    <td>1531</td>
    <td>1298</td>
    <td>1238</td>
    <td>326</td>
    <td>158</td>
    <td>308</td>
    <td>0,2</td>
    <td>1,3E-16</td>
    <td>6,1E-15</td>
  </tr>
  <tr>
    <td>VC2414</td>
    <td>2065</td>
    <td>2573</td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td>2,5E-16</td>
    <td>1,2E-14</td>
  </tr>
  <tr>
    <td>VC1434</td>
    <td>5799</td>
    <td>4818</td>
    <td>6784</td>
    <td>1544</td>
    <td>2021</td>
    <td>1387</td>
    <td>0,3</td>
    <td>1,6E-15</td>
    <td>6,9E-14</td>
  </tr>
  <tr>
    <td>VC1544</td>
    <td>860</td>
    <td>841</td>
    <td>702</td>
    <td>211</td>
    <td>92</td>
    <td>200</td>
    <td>0,2</td>
    <td>5,8E-15</td>
    <td>2,3E-13</td>
  </tr>
  <tr>
    <td>VC1266</td>
    <td>992</td>
    <td>730</td>
    <td>899</td>
    <td>180</td>
    <td>81</td>
    <td>218</td>
    <td>0,2</td>
    <td>2,8E-14</td>
    <td>1,1E-12</td>
  </tr>
  <tr>
    <td>VC1548</td>
    <td>828</td>
    <td>977</td>
    <td>762</td>
    <td>200</td>
    <td>71</td>
    <td>185</td>
    <td>0,2</td>
    <td>3,5E-14</td>
    <td>1,3E-12</td>
  </tr>
  <tr>
    <td>VC1168</td>
    <td>2427</td>
    <td>1657</td>
    <td>3276</td>
    <td>600</td>
    <td>422</td>
    <td>657</td>
    <td>0,2</td>
    <td>4,4E-14</td>
    <td>1,6E-12</td>
  </tr>
  <tr>
    <td>VC2560</td>
    <td>938</td>
    <td>837</td>
    <td>656</td>
    <td>178</td>
    <td>57</td>
    <td>151</td>
    <td>0,2</td>
    <td>1,6E-13</td>
    <td>5,6E-12</td>
  </tr>
  <tr>
    <td>VC0575</td>
    <td>3481</td>
    <td>4499</td>
    <td>4787</td>
    <td>1741</td>
    <td>1310</td>
    <td>1531</td>
    <td>0,4</td>
    <td>1,7E-13</td>
    <td>5,9E-12</td>
  </tr>
  <tr>
    <td>VC0540</td>
    <td>1091</td>
    <td>965</td>
    <td>938</td>
    <td>257</td>
    <td>204</td>
    <td>349</td>
    <td>0,3</td>
    <td>1,9E-13</td>
    <td>6,4E-12</td>
  </tr>
  <tr>
    <td>VC1890</td>
    <td>4135</td>
    <td>4112</td>
    <td>4697</td>
    <td>2193</td>
    <td>1882</td>
    <td>2160</td>
    <td>0,5</td>
    <td>2,8E-13</td>
    <td>9,2E-12</td>
  </tr>
  <tr>
    <td>VC1267</td>
    <td>1006</td>
    <td>697</td>
    <td>951</td>
    <td>208</td>
    <td>95</td>
    <td>240</td>
    <td>0,2</td>
    <td>1,2E-12</td>
    <td>3,6E-11</td>
  </tr>
  <tr>
    <td>VC1625</td>
    <td>6073</td>
    <td>5424</td>
    <td>5715</td>
    <td>1808</td>
    <td>1036</td>
    <td>2011</td>
    <td>0,3</td>
    <td>1,3E-12</td>
    <td>3,8E-11</td>
  </tr>
  <tr>
    <td>VC1547</td>
    <td>1186</td>
    <td>1193</td>
    <td>907</td>
    <td>270</td>
    <td>101</td>
    <td>276</td>
    <td>0,2</td>
    <td>2,0E-12</td>
    <td>5,9E-11</td>
  </tr>
  <tr>
    <td>VC1623</td>
    <td>2356</td>
    <td>1972</td>
    <td>1754</td>
    <td>913</td>
    <td>895</td>
    <td>853</td>
    <td>0,4</td>
    <td>2,2E-12</td>
    <td>6,6E-11</td>
  </tr>
  <tr>
    <td>VC0608</td>
    <td>1724</td>
    <td>3895</td>
    <td>2700</td>
    <td>607</td>
    <td>375</td>
    <td>613</td>
    <td>0,2</td>
    <td>2,0E-11</td>
    <td>5,5E-10</td>
  </tr>
  <tr>
    <td>VC1543</td>
    <td>1647</td>
    <td>1544</td>
    <td>1204</td>
    <td>468</td>
    <td>228</td>
    <td>484</td>
    <td>0,3</td>
    <td>2,7E-11</td>
    <td>7,1E-10</td>
  </tr>
  <tr>
    <td>VC1264</td>
    <td>3449</td>
    <td>2931</td>
    <td>2531</td>
    <td>663</td>
    <td>257</td>
    <td>830</td>
    <td>0,2</td>
    <td>3,0E-11</td>
    <td>7,8E-10</td>
  </tr>
  <tr>
    <td>VC0541</td>
    <td>2020</td>
    <td>1993</td>
    <td>2192</td>
    <td>759</td>
    <td>645</td>
    <td>969</td>
    <td>0,4</td>
    <td>6,9E-11</td>
    <td>1,7E-09</td>
  </tr>
  <tr>
    <td>VC0627</td>
    <td>1608</td>
    <td>1342</td>
    <td>1589</td>
    <td>479</td>
    <td>458</td>
    <td>718</td>
    <td>0,4</td>
    <td>3,9E-10</td>
    <td>8,7E-09</td>
  </tr>
  <tr>
    <td>VC2413</td>
    <td>1118</td>
    <td>1287</td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td></td>
    <td>4</td>
    <td>6</td>
    <td>15480</td>
    <td>4976</td>
    <td>2760</td>
    <td>5137</td>
    <td>0,3</td>
    <td>4,8E-10</td>
    <td>1,0E-08</td>
  </tr>
  <tr>
    <td>VC0574</td>
    <td>4963</td>
    <td>6302</td>
    <td>6116</td>
    <td>2629</td>
    <td>1626</td>
    <td>2374</td>
    <td>0,4</td>
    <td>5,1E-10</td>
    <td>1,1E-08</td>
  </tr>
  <tr>
    <td>VCA0779</td>
    <td>1014</td>
    <td>1043</td>
    <td>1299</td>
    <td>573</td>
    <td>473</td>
    <td>544</td>
    <td>0,5</td>
    <td>6,7E-10</td>
    <td>1,4E-08</td>
  </tr>
  <tr>
    <td></td>
    <td>1872</td>
    <td>1856</td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td>VC1425</td>
    <td>8</td>
    <td>8</td>
    <td>11818</td>
    <td>6031</td>
    <td>6511</td>
    <td>6308</td>
    <td>0,4</td>
    <td>2,6E-09</td>
    <td>4,9E-08</td>
  </tr>
  <tr>
    <td>VC1190</td>
    <td>3535</td>
    <td>3491</td>
    <td>3519</td>
    <td>1105</td>
    <td>534</td>
    <td>1290</td>
    <td>0,3</td>
    <td>4,7E-09</td>
    <td>8,7E-08</td>
  </tr>
  <tr>
    <td>VC0539</td>
    <td>816</td>
    <td>759</td>
    <td>978</td>
    <td>299</td>
    <td>125</td>
    <td>291</td>
    <td>0,3</td>
    <td>5,5E-09</td>
    <td>1,0E-07</td>
  </tr>
  <tr>
    <td>VC2031</td>
    <td>1195</td>
    <td>956</td>
    <td>1683</td>
    <td>516</td>
    <td>398</td>
    <td>518</td>
    <td>0,4</td>
    <td>6,3E-09</td>
    <td>1,1E-07</td>
  </tr>
  <tr>
    <td>VC0538</td>
    <td>2463</td>
    <td>3016</td>
    <td>3026</td>
    <td>969</td>
    <td>418</td>
    <td>946</td>
    <td>0,3</td>
    <td>6,6E-09</td>
    <td>1,2E-07</td>
  </tr>
  <tr>
    <td>VC2044</td>
    <td>4468</td>
    <td>3773</td>
    <td>4970</td>
    <td>2081</td>
    <td>1443</td>
    <td>2069</td>
    <td>0,4</td>
    <td>6,9E-09</td>
    <td>1,2E-07</td>
  </tr>
  <tr>
    <td>VC1624</td>
    <td>2450</td>
    <td>2405</td>
    <td>2567</td>
    <td>1013</td>
    <td>552</td>
    <td>1133</td>
    <td>0,4</td>
    <td>3,1E-08</td>
    <td>5,0E-07</td>
  </tr>
  <tr>
    <td>VC0384</td>
    <td>2708</td>
    <td>2991</td>
    <td>1808</td>
    <td>989</td>
    <td>444</td>
    <td>818</td>
    <td>0,3</td>
    <td>5,1E-08</td>
    <td>8,1E-07</td>
  </tr>
  <tr>
    <td>VC1350</td>
    <td>1809</td>
    <td>1972</td>
    <td>2095</td>
    <td>700</td>
    <td>899</td>
    <td>926</td>
    <td>0,4</td>
    <td>6,0E-08</td>
    <td>9,2E-07</td>
  </tr>
  <tr>
    <td>VC2099</td>
    <td>4134</td>
    <td>4299</td>
    <td>6134</td>
    <td>2367</td>
    <td>2308</td>
    <td>2392</td>
    <td>0,5</td>
    <td>1,8E-07</td>
    <td>2,5E-06</td>
  </tr>
  <tr>
    <td>VC1299</td>
    <td>2194</td>
    <td>1529</td>
    <td>1452</td>
    <td>766</td>
    <td>807</td>
    <td>883</td>
    <td>0,5</td>
    <td>3,3E-06</td>
    <td>3,5E-05</td>
  </tr>
  <tr>
    <td>VC2389</td>
    <td>1233</td>
    <td>1044</td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
    <td></td>
  </tr>
  <tr>
    <td></td>
    <td>5</td>
    <td>6</td>
    <td>14700</td>
    <td>5767</td>
    <td>2538</td>
    <td>5581</td>
    <td>0,4</td>
    <td>3,7E-06</td>
    <td>3,8E-05</td>
  </tr>
  <tr>
    <td>VC0168</td>
    <td>4710</td>
    <td>4663</td>
    <td>3918</td>
    <td>2115</td>
    <td>1538</td>
    <td>2593</td>
    <td>0,5</td>
    <td>5,0E-06</td>
    <td>5,0E-05</td>
  </tr>
  <tr>
    <td>VC1439</td>
    <td>3658</td>
    <td>4680</td>
    <td>4161</td>
    <td>2062</td>
    <td>1186</td>
    <td>2295</td>
    <td>0,5</td>
    <td>5,5E-06</td>
    <td>5,5E-05</td>
  </tr>
  <tr>
    <td>VC0385</td>
    <td>2632</td>
    <td>2744</td>
    <td>1443</td>
    <td>910</td>
    <td>465</td>
    <td>974</td>
    <td>0,4</td>
    <td>6,3E-06</td>
    <td>6,3E-05</td>
  </tr>
  <tr>
    <td>VC0386</td>
    <td>1716</td>
    <td>1717</td>
    <td>944</td>
    <td>651</td>
    <td>435</td>
    <td>748</td>
    <td>0,4</td>
    <td>2,9E-05</td>
    <td>2,5E-04</td>
  </tr>
  <tr>
    <td>VCA0554</td>
    <td>3640</td>
    <td>1975</td>
    <td>4155</td>
    <td>1213</td>
    <td>845</td>
    <td>1601</td>
    <td>0,4</td>
    <td>3,1E-05</td>
    <td>2,6E-04</td>
  </tr>
</table>
<table><tbody><tr><td>VC0731</td><td>9328</td><td>1866</td><td>16354</td><td>5035</td><td>7322</td><td>4748</td><td>0,4</td><td>7,7E-05</td><td>5,8E-04</td></tr><tr><td>VC0034</td><td>1465</td><td>2168</td><td>3551</td><td>936</td><td>1054</td><td>1174</td><td>0,5</td><td>1,2E-04</td><td>8,2E-04</td></tr><tr><td>VC0573</td><td>4031</td><td>5106</td><td>3159</td><td>2130</td><td>1225</td><td>2439</td><td>0,5</td><td>1,4E-04</td><td>9,8E-04</td></tr><tr><td>VC1440</td><td>803</td><td>1073</td><td>578</td><td>467</td><td>268</td><td>424</td><td>0,5</td><td>1,7E-04</td><td>1,1E-03</td></tr><tr><td>VCA0907</td><td>1430</td><td>2408</td><td>1943</td><td>1139</td><td>688</td><td>868</td><td>0,5</td><td>2,0E-04</td><td>1,3E-03</td></tr><tr><td>VC2089</td><td>762</td><td>1693</td><td>896</td><td>567</td><td>307</td><td>554</td><td>0,5</td><td>2,2E-04</td><td>1,4E-03</td></tr><tr><td>VC1441</td><td>3956</td><td>4982</td><td>2505</td><td>2010</td><td>1104</td><td>2188</td><td>0,5</td><td>3,7E-04</td><td>2,2E-03</td></tr><tr><td>VCA0496</td><td>1389</td><td>942</td><td>2071</td><td>426</td><td>1108</td><td>411</td><td>0,5</td><td>4,2E-03</td><td>1,6E-02</td></tr><tr><td>Sugar metabolism</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VCA0896</td><td>3304</td><td>3007</td><td>3497</td><td>889</td><td>693</td><td>872</td><td>0,3</td><td>1,6E-32</td><td>1,8E-30</td></tr><tr><td>VC1126</td><td>7293</td><td>1025</td><td>7992</td><td>2766</td><td>2625</td><td>2583</td><td>0,3</td><td>2,4E-18</td><td>1,2E-16</td></tr><tr><td>VCA0657</td><td>685</td><td>529</td><td>732</td><td>221</td><td>201</td><td>201</td><td>0,3</td><td>7,2E-14</td><td>2,5E-12</td></tr><tr><td>VC1004</td><td>6034</td><td>4770</td><td>6826</td><td>1934</td><td>1231</td><td>1837</td><td>0,3</td><td>4,2E-13</td><td>1,4E-11</td></tr><tr><td>VC2024</td><td>3794</td><td>3293</td><td>4602</td><td>1688</td><td>1965</td><td>1679</td><td>0,5</td><td>4,2E-09</td><td>8,0E-08</td></tr><tr><td>VCA0897</td><td>3467</td><td>2804</td><td>1622</td><td>845</td><td>579</td><td>943</td><td>0,3</td><td>4,9E-09</td><td>8,9E-08</td></tr><tr><td>VCA0898</td><td>5767</td><td>4943</td><td>6369</td><td>2709</td><td>1746</td><td>2587</td><td>0,4</td><td>7,2E-09</td><td>1,3E-07</td></tr><tr><td>VC0910</td><td>3175</td><td>3395</td><td>3392</td><td>1601</td><td>1326</td><td>1539</td><td>0,5</td><td>1,1E-05</td><td>1,0E-04</td></tr><tr><td>VC0911</td><td>4479</td><td>3736</td><td>4242</td><td>1881</td><td>2225</td><td>1776</td><td>0,5</td><td>2,5E-05</td><td>2,2E-04</td></tr><tr><td>VC2480</td><td>1448</td><td>1607</td><td>2584</td><td>982</td><td>741</td><td>885</td><td>0,5</td><td>5,1E-05</td><td>4,0E-04</td></tr><tr><td>VCA1060</td><td>2171</td><td>771</td><td>1674</td><td>633</td><td>563</td><td>664</td><td>0,4</td><td>1,4E-04</td><td>9,5E-04</td></tr><tr><td>VC2183</td><td>5155</td><td>4222</td><td>6751</td><td>2904</td><td>1660</td><td>3070</td><td>0,5</td><td>2,8E-04</td><td>1,7E-03</td></tr><tr><td>Aminoacid metabolism</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VC1658</td><td>2116</td><td>1124</td><td>2478</td><td>185</td><td>194</td><td>252</td><td>0,1</td><td>8,2E-26</td><td>7,4E-24</td></tr><tr><td>VC0008</td><td>724</td><td>505</td><td>822</td><td>117</td><td>118</td><td>140</td><td>0,2</td><td>1,2E-20</td><td>7,9E-19</td></tr><tr><td>VC0009</td><td>679</td><td>385</td><td>742</td><td>88</td><td>83</td><td>99</td><td>0,2</td><td>4,2E-20</td><td>2,6E-18</td></tr><tr><td>VC0472</td><td>6447</td><td>7377</td><td>9080</td><td>2458</td><td>1906</td><td>2154</td><td>0,3</td><td>3,0E-19</td><td>1,7E-17</td></tr><tr><td>VC1312</td><td>2358</td><td>2539</td><td>3364</td><td>624</td><td>800</td><td>669</td><td>0,3</td><td>1,9E-18</td><td>9,8E-17</td></tr><tr><td>VC0010</td><td>3040</td><td>2440</td><td>3330</td><td>550</td><td>251</td><td>670</td><td>0,2</td><td>2,0E-14</td><td>7,7E-13</td></tr><tr><td>VC0880</td><td>758</td><td>657</td><td>1076</td><td>241</td><td>122</td><td>270</td><td>0,3</td><td>2,2E-10</td><td>5,2E-09</td></tr><tr><td>VCA1073</td><td>509</td><td>701</td><td>623</td><td>250</td><td>182</td><td>291</td><td>0,4</td><td>3,3E-09</td><td>6,3E-08</td></tr><tr><td>VC0743</td><td>6022</td><td>4953</td><td>6483</td><td>2669</td><td>1673</td><td>2698</td><td>0,4</td><td>7,6E-08</td><td>1,1E-06</td></tr><tr><td>VC1995</td><td>1199</td><td>1731</td><td>2309</td><td>639</td><td>415</td><td>724</td><td>0,4</td><td>8,9E-08</td><td>1,3E-06</td></tr><tr><td>VCA0815</td><td>3885</td><td>3098</td><td>3980</td><td>1836</td><td>1216</td><td>1630</td><td>0,4</td><td>1,7E-07</td><td>2,4E-06</td></tr><tr><td>VC0907</td><td>596</td><td>504</td><td>737</td><td>298</td><td>182</td><td>300</td><td>0,4</td><td>2,0E-06</td><td>2,3E-05</td></tr><tr><td>VC0941</td><td>1007</td><td>7756</td><td>8318</td><td>4801</td><td>2914</td><td>4531</td><td>0,5</td><td>2,3E-06</td><td>2,6E-05</td></tr><tr><td>VC2682</td><td>2219</td><td>1588</td><td>1853</td><td>772</td><td>900</td><td>1076</td><td>0,5</td><td>5,2E-06</td><td>5,2E-05</td></tr><tr><td>VC0947</td><td>1067</td><td>1309</td><td>9872</td><td>4646</td><td>6635</td><td>4962</td><td>0,5</td><td>1,5E-05</td><td>1,4E-04</td></tr><tr><td>VCA0772</td><td>607</td><td>666</td><td>968</td><td>364</td><td>202</td><td>431</td><td>0,5</td><td>8,6E-05</td><td>6,4E-04</td></tr><tr><td>VC0275</td><td>3965</td><td>3080</td><td>4200</td><td>1403</td><td>423</td><td>1669</td><td>0,4</td><td>1,0E-04</td><td>7,2E-04</td></tr><tr><td>VC2485</td><td>1370</td><td>3351</td><td>2132</td><td>1019</td><td>971</td><td>914</td><td>0,5</td><td>1,1E-04</td><td>8,0E-04</td></tr><tr><td>VCA0088</td><td>2895</td><td>3412</td><td>2191</td><td>1471</td><td>647</td><td>1654</td><td>0,5</td><td>3,7E-04</td><td>2,2E-03</td></tr><tr><td>VC0662</td><td>1746</td><td>1104</td><td>2625</td><td>973</td><td>890</td><td>798</td><td>0,5</td><td>3,8E-04</td><td>2,2E-03</td></tr></tbody></table>
<table>
  <tbody>
    <tr>
      <td>VC0576</td>
      <td>3097</td>
      <td>3766</td>
      <td>9997</td>
      <td>1913</td>
      <td>2839</td>
      <td>1831</td>
      <td>0,4</td>
      <td>7,3E-04</td>
      <td>3,9E-03</td>
    </tr>
    <tr>
      <td>VCA0076</td>
      <td>970</td>
      <td>1503</td>
      <td>1188</td>
      <td>541</td>
      <td>146</td>
      <td>612</td>
      <td>0,4</td>
      <td>9,4E-04</td>
      <td>4,8E-03</td>
    </tr>
    <tr>
      <td>VC1492</td>
      <td>2786</td>
      <td>6743</td>
      <td>2369</td>
      <td>1909</td>
      <td>1011</td>
      <td>2088</td>
      <td>0,5</td>
      <td>1,5E-03</td>
      <td>6,9E-03</td>
    </tr>
    <tr>
      <td>VC2746</td>
      <td>1921</td>
      <td>2904</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>Nucleotide<br>metabolism</td>
      <td>0</td>
      <td>5</td>
      <td>38036</td>
      <td>10763</td>
      <td>19348</td>
      <td>10475</td>
      <td>0,5</td>
      <td>2,3E-03</td>
      <td>9,8E-03</td>
    </tr>
    <tr>
      <td>VC0276</td>
      <td>5517</td>
      <td>4824</td>
      <td>5808</td>
      <td>1963</td>
      <td>752</td>
      <td>2223</td>
      <td>0,3</td>
      <td>3,1E-06</td>
      <td>3,3E-05</td>
    </tr>
    <tr>
      <td>VC0767</td>
      <td>8972</td>
      <td>9299</td>
      <td>12691</td>
      <td>4208</td>
      <td>4262</td>
      <td>4502</td>
      <td>0,4</td>
      <td>2,4E-10</td>
      <td>5,7E-09</td>
    </tr>
    <tr>
      <td>VC0768</td>
      <td>1763</td>
      <td>1423</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td></td>
      <td>6</td>
      <td>7</td>
      <td>15989</td>
      <td>5714</td>
      <td>3303</td>
      <td>5613</td>
      <td>0,3</td>
      <td>1,3E-09</td>
      <td>2,7E-08</td>
    </tr>
    <tr>
      <td></td>
      <td>1698</td>
      <td>1660</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>VC0986</td>
      <td>0</td>
      <td>9</td>
      <td>14315</td>
      <td>7288</td>
      <td>6361</td>
      <td>7172</td>
      <td>0,4</td>
      <td>9,9E-09</td>
      <td>1,7E-07</td>
    </tr>
    <tr>
      <td>VC1228</td>
      <td>2458</td>
      <td>2040</td>
      <td>2902</td>
      <td>784</td>
      <td>365</td>
      <td>922</td>
      <td>0,3</td>
      <td>1,6E-07</td>
      <td>2,2E-06</td>
    </tr>
    <tr>
      <td>VC1721</td>
      <td>2424</td>
      <td>2857</td>
      <td>3565</td>
      <td>1020</td>
      <td>1480</td>
      <td>1120</td>
      <td>0,4</td>
      <td>3,7E-08</td>
      <td>6,0E-07</td>
    </tr>
    <tr>
      <td>VC2171</td>
      <td>1886</td>
      <td>1374</td>
      <td>2592</td>
      <td>1049</td>
      <td>478</td>
      <td>1191</td>
      <td>0,5</td>
      <td>3,2E-03</td>
      <td>1,3E-02</td>
    </tr>
    <tr>
      <td>VC2227</td>
      <td>7683</td>
      <td>7434</td>
      <td>9542</td>
      <td>1542</td>
      <td>600</td>
      <td>1786</td>
      <td>0,2</td>
      <td>3,4E-12</td>
      <td>9,6E-11</td>
    </tr>
    <tr>
      <td>VC2258</td>
      <td>5189</td>
      <td>4353</td>
      <td>6703</td>
      <td>2373</td>
      <td>3004</td>
      <td>1852</td>
      <td>0,5</td>
      <td>1,3E-06</td>
      <td>1,6E-05</td>
    </tr>
    <tr>
      <td>VC2277</td>
      <td>6821</td>
      <td>6135</td>
      <td>7227</td>
      <td>1701</td>
      <td>1908</td>
      <td>1914</td>
      <td>0,3</td>
      <td>1,0E-18</td>
      <td>5,3E-17</td>
    </tr>
    <tr>
      <td>VC2352</td>
      <td>8009</td>
      <td>5520</td>
      <td>8212</td>
      <td>3958</td>
      <td>2318</td>
      <td>4075</td>
      <td>0,5</td>
      <td>3,7E-04</td>
      <td>2,2E-03</td>
    </tr>
    <tr>
      <td>VC2712</td>
      <td>6475</td>
      <td>3650</td>
      <td>5923</td>
      <td>2192</td>
      <td>772</td>
      <td>2789</td>
      <td>0,4</td>
      <td>7,2E-04</td>
      <td>3,8E-03</td>
    </tr>
    <tr>
      <td>VCA0607</td>
      <td>1153</td>
      <td>603</td>
      <td>1425</td>
      <td>331</td>
      <td>773</td>
      <td>334</td>
      <td>0,5</td>
      <td>3,7E-03</td>
      <td>1,5E-02</td>
    </tr>
    <tr>
      <td>VC1365</td>
      <td>972</td>
      <td>1192</td>
      <td>1204</td>
      <td>492</td>
      <td>581</td>
      <td>539</td>
      <td>0,5</td>
      <td>5,0E-07</td>
      <td>6,5E-06</td>
    </tr>
    <tr>
      <td>Translation</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>VC0218</td>
      <td>5714</td>
      <td>3623</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td></td>
      <td>1</td>
      <td>0</td>
      <td>62003</td>
      <td>25376</td>
      <td>23118</td>
      <td>26376</td>
      <td>0,5</td>
      <td>6,6E-06</td>
      <td>6,5E-05</td>
    </tr>
    <tr>
      <td>VC0291</td>
      <td>1045</td>
      <td>1172</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td></td>
      <td>6</td>
      <td>6</td>
      <td>12645</td>
      <td>5607</td>
      <td>4153</td>
      <td>5227</td>
      <td>0,4</td>
      <td>6,1E-11</td>
      <td>1,5E-09</td>
    </tr>
    <tr>
      <td>VC0359</td>
      <td>6218</td>
      <td>4594</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td></td>
      <td>6</td>
      <td>2</td>
      <td>57114</td>
      <td>27307</td>
      <td>22476</td>
      <td>29864</td>
      <td>0,5</td>
      <td>1,1E-05</td>
      <td>1,0E-04</td>
    </tr>
    <tr>
      <td>VC0360</td>
      <td>6543</td>
      <td>4661</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td></td>
      <td>5</td>
      <td>3</td>
      <td>78079</td>
      <td>26811</td>
      <td>25570</td>
      <td>27739</td>
      <td>0,4</td>
      <td>4,5E-07</td>
      <td>6,0E-06</td>
    </tr>
    <tr>
      <td>VC0361</td>
      <td>2930</td>
      <td>3845</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td></td>
      <td>4</td>
      <td>5</td>
      <td>33303</td>
      <td>17321</td>
      <td>16307</td>
      <td>16634</td>
      <td>0,5</td>
      <td>4,4E-10</td>
      <td>9,8E-09</td>
    </tr>
    <tr>
      <td>VC0369</td>
      <td>2466</td>
      <td>2120</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td></td>
      <td>5</td>
      <td>0</td>
      <td>59251</td>
      <td>15852</td>
      <td>6855</td>
      <td>16988</td>
      <td>0,4</td>
      <td>1,8E-03</td>
      <td>8,1E-03</td>
    </tr>
    <tr>
      <td>VC0379</td>
      <td>793</td>
      <td>970</td>
      <td>1720</td>
      <td>567</td>
      <td>485</td>
      <td>531</td>
      <td>0,5</td>
      <td>4,7E-05</td>
      <td>3,8E-04</td>
    </tr>
    <tr>
      <td></td>
      <td>1594</td>
      <td>1975</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>VC0520</td>
      <td>2</td>
      <td>4</td>
      <td>24705</td>
      <td>6763</td>
      <td>8291</td>
      <td>6571</td>
      <td>0,4</td>
      <td>9,5E-09</td>
      <td>1,6E-07</td>
    </tr>
    <tr>
      <td></td>
      <td>5502</td>
      <td>7240</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>VC0570</td>
      <td>6</td>
      <td>5</td>
      <td>87623</td>
      <td>29769</td>
      <td>23007</td>
      <td>32471</td>
      <td>0,4</td>
      <td>1,1E-08</td>
      <td>1,9E-07</td>
    </tr>
    <tr>
      <td>VC0571</td>
      <td>2053</td>
      <td>2730</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td></td>
      <td>8</td>
      <td>7</td>
      <td>71337</td>
      <td>12432</td>
      <td>9502</td>
      <td>11204</td>
      <td>0,3</td>
      <td>2,1E-06</td>
      <td>2,4E-05</td>
    </tr>
    <tr>
      <td>VC0644</td>
      <td>1515</td>
      <td>1015</td>
      <td>1370</td>
      <td>552</td>
      <td>434</td>
      <td>509</td>
      <td>0,4</td>
      <td>1,3E-09</td>
      <td>2,7E-08</td>
    </tr>
    <tr>
      <td>VC0645</td>
      <td>2212</td>
      <td>1491</td>
      <td>2617</td>
      <td>645</td>
      <td>503</td>
      <td>659</td>
      <td>0,3</td>
      <td>2,2E-12</td>
      <td>6,5E-11</td>
    </tr>
    <tr>
      <td></td>
      <td>7442</td>
      <td>4550</td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
      <td></td>
    </tr>
    <tr>
      <td>VC0646</td>
      <td>5</td>
      <td>8</td>
      <td>52075</td>
      <td>24930</td>
      <td>23318</td>
      <td>30660</td>
      <td>0,5</td>
      <td>1,7E-05</td>
      <td>1,5E-04</td>
    </tr>
    <tr>
      <td>VC0659</td>
      <td>4553</td>
      <td>4864</td>
      <td>6873</td>
      <td>1701</td>
      <td>1920</td>
      <td>1491</td>
      <td>0,3</td>
      <td>3,0E-14</td>
      <td>1,1E-12</td>
    </tr>
    <tr>
      <td>VC1209</td>
      <td>8390</td>
      <td>9867</td>
      <td>9418</td>
      <td>4451</td>
      <td>4396</td>
      <td>4138</td>
      <td>0,5</td>
      <td>6,6E-11</td>
      <td>1,7E-09</td>
    </tr>
    <tr>
      <td>VC1219</td>
      <td>1824</td>
      <td>1726</td>
      <td>2168</td>
      <td>991</td>
      <td>864</td>
      <td>970</td>
      <td>0,5</td>
      <td>1,6E-10</td>
      <td>3,8E-09</td>
    </tr>
    <tr>
      <td>VC1220</td>
      <td>4783</td>
      <td>4380</td>
      <td>6074</td>
      <td>2824</td>
      <td>2175</td>
      <td>2437</td>
      <td>0,5</td>
      <td>1,4E-07</td>
      <td>1,9E-06</td>
    </tr>
  </tbody>
</table>
<table><tbody><tr><td>VC1297</td><td>5980</td><td>5708</td><td>9049</td><td>2891</td><td>2950</td><td>3392</td><td>0,5</td><td>5,1E-08</td><td>8,1E-07</td></tr><tr><td>VC1640</td><td>4267</td><td>2758</td><td>6</td><td>45907</td><td>10871</td><td>10746</td><td>14654</td><td>0,3</td><td>8,3E-09</td><td>1,5E-07</td></tr><tr><td>VC1923</td><td>1463</td><td>3008</td><td>8</td><td>14378</td><td>9167</td><td>11082</td><td>8555</td><td>0,5</td><td>7,1E-04</td><td>3,8E-03</td></tr><tr><td>VC2025</td><td>1022</td><td>7808</td><td>14105</td><td>3020</td><td>3736</td><td>2634</td><td>0,3</td><td>1,5E-09</td><td>3,0E-08</td></tr><tr><td>VC2026</td><td>6945</td><td>5267</td><td>6</td><td>55871</td><td>21480</td><td>25695</td><td>22013</td><td>0,4</td><td>1,2E-07</td><td>1,7E-06</td></tr><tr><td>VC2214</td><td>6055</td><td>7838</td><td>8737</td><td>3254</td><td>3998</td><td>2586</td><td>0,5</td><td>1,1E-06</td><td>1,4E-05</td></tr><tr><td>VC2259</td><td>3475</td><td>4409</td><td>0</td><td>37821</td><td>18907</td><td>21812</td><td>17310</td><td>0,5</td><td>4,8E-07</td><td>6,2E-06</td></tr><tr><td>VC2342</td><td>6617</td><td>8699</td><td>4</td><td>10061</td><td>6</td><td>45347</td><td>39129</td><td>0,5</td><td>5,5E-08</td><td>8,5E-07</td></tr><tr><td>VC2461</td><td>2014</td><td>1761</td><td>2572</td><td>814</td><td>837</td><td>695</td><td>0,4</td><td>9,0E-13</td><td>2,9E-11</td></tr><tr><td>VC2579</td><td>8782</td><td>2325</td><td>7</td><td>18636</td><td>7681</td><td>5267</td><td>7624</td><td>0,4</td><td>7,6E-05</td><td>5,7E-04</td></tr><tr><td>VC2583</td><td>8239</td><td>2080</td><td>4</td><td>14710</td><td>7616</td><td>6339</td><td>6607</td><td>0,5</td><td>2,7E-04</td><td>1,7E-03</td></tr><tr><td>VC2620</td><td>1574</td><td>1292</td><td>1490</td><td>785</td><td>527</td><td>762</td><td>0,5</td><td>2,2E-05</td><td>1,9E-04</td></tr><tr><td>VC2664</td><td>4009</td><td>5269</td><td>4052</td><td>1503</td><td>1695</td><td>1142</td><td>0,3</td><td>4,5E-14</td><td>1,6E-12</td></tr><tr><td>Others</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>CSR2</td><td>3606</td><td>1920</td><td>2667</td><td>508</td><td>802</td><td>551</td><td>0,3</td><td>9,0E-11</td><td>2,2E-09</td></tr><tr><td>VIBCH10294</td><td>454</td><td>271</td><td>1091</td><td>284</td><td>121</td><td>291</td><td>0,4</td><td>5,8E-03</td><td>2,1E-02</td></tr><tr><td>VC0033</td><td>661</td><td>587</td><td>1321</td><td>282</td><td>403</td><td>265</td><td>0,4</td><td>1,3E-05</td><td>1,2E-04</td></tr><tr><td>VC1003</td><td>4970</td><td>3770</td><td>3744</td><td>1838</td><td>1149</td><td>2060</td><td>0,4</td><td>8,1E-07</td><td>1,0E-05</td></tr><tr><td>VC1490</td><td>685</td><td>850</td><td>1294</td><td>330</td><td>344</td><td>399</td><td>0,4</td><td>1,4E-06</td><td>1,6E-05</td></tr><tr><td>VC1208</td><td>3478</td><td>4196</td><td>5582</td><td>2153</td><td>1811</td><td>1986</td><td>0,5</td><td>1,4E-07</td><td>2,0E-06</td></tr><tr><td>VC2460</td><td>3572</td><td>3075</td><td>3149</td><td>1084</td><td>1300</td><td>1026</td><td>0,4</td><td>2,8E-20</td><td>1,8E-18</td></tr><tr><td>VC0290</td><td>7761</td><td>7686</td><td>8352</td><td>2994</td><td>2610</td><td>3228</td><td>0,4</td><td>5,3E-16</td><td>2,4E-14</td></tr><tr><td>VC2459</td><td>960</td><td>791</td><td>658</td><td>428</td><td>353</td><td>392</td><td>0,5</td><td>8,0E-08</td><td>1,2E-06</td></tr><tr><td>VC2647</td><td>6843</td><td>4552</td><td>3914</td><td>1936</td><td>3109</td><td>2207</td><td>0,5</td><td>1,8E-04</td><td>1,2E-03</td></tr><tr><td>VC0749</td><td>1248</td><td>1409</td><td>1596</td><td>739</td><td>601</td><td>674</td><td>0,5</td><td>3,0E-10</td><td>6,7E-09</td></tr><tr><td>VC0766</td><td>1307</td><td>1242</td><td>3902</td><td>984</td><td>737</td><td>1256</td><td>0,5</td><td>6,3E-03</td><td>2,2E-02</td></tr><tr><td>Membrane - transporters</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VC1577</td><td>6497</td><td>2204</td><td>6096</td><td>403</td><td>1657</td><td>363</td><td>0,2</td><td>1,4E-06</td><td>1,7E-05</td></tr><tr><td>VC1195</td><td>2511</td><td>3007</td><td>2599</td><td>1076</td><td>1138</td><td>1086</td><td>0,4</td><td>4,1E-14</td><td>1,5E-12</td></tr><tr><td>VC1962</td><td>1002</td><td>693</td><td>894</td><td>237</td><td>323</td><td>230</td><td>0,3</td><td>3,0E-11</td><td>7,8E-10</td></tr><tr><td>VC1043</td><td>2014</td><td>3465</td><td>8</td><td>22588</td><td>7960</td><td>8029</td><td>9141</td><td>0,3</td><td>5,1E-09</td><td>9,3E-08</td></tr><tr><td>VCA0862</td><td>718</td><td>539</td><td>837</td><td>239</td><td>239</td><td>338</td><td>0,4</td><td>4,9E-08</td><td>7,8E-07</td></tr><tr><td>VC1655</td><td>1156</td><td>809</td><td>1960</td><td>372</td><td>425</td><td>333</td><td>0,3</td><td>6,9E-09</td><td>1,2E-07</td></tr><tr><td>VC1409</td><td>885</td><td>311</td><td>759</td><td>79</td><td>245</td><td>73</td><td>0,3</td><td>1,1E-06</td><td>1,3E-05</td></tr><tr><td>VC1329</td><td>2576</td><td>1202</td><td>2399</td><td>450</td><td>273</td><td>641</td><td>0,3</td><td>5,4E-09</td><td>9,9E-08</td></tr><tr><td>VC2305</td><td>5931</td><td>3437</td><td>9844</td><td>1711</td><td>2267</td><td>2010</td><td>0,3</td><td>1,1E-06</td><td>1,4E-05</td></tr><tr><td>VCA0554</td><td>3640</td><td>1975</td><td>4155</td><td>1213</td><td>845</td><td>1601</td><td>0,4</td><td>3,1E-05</td><td>2,6E-04</td></tr><tr><td>VC2278</td><td>7821</td><td>6452</td><td>9648</td><td>2026</td><td>1781</td><td>2313</td><td>0,3</td><td>3,4E-18</td><td>1,7E-16</td></tr><tr><td>VC1319</td><td>3625</td><td>988</td><td>2722</td><td>474</td><td>1989</td><td>485</td><td>0,5</td><td>1,6E-02</td><td>4,9E-02</td></tr><tr><td>VCA1071</td><td>777</td><td>918</td><td>739</td><td>333</td><td>197</td><td>313</td><td>0,4</td><td>1,5E-11</td><td>4,0E-10</td></tr></tbody></table>
<table><tbody><tr><td>VC1669</td><td>764</td><td>292</td><td>1161</td><td>137</td><td>223</td><td>135</td><td>0,3</td><td>1,4E-07</td><td>1,9E-06</td></tr><tr><td>VC0156</td><td>1303</td><td>1233</td><td>11471</td><td>2758</td><td>7063</td><td>2800</td><td>0,4</td><td>6,7E-05</td><td>5,1E-04</td></tr><tr><td>VC0475</td><td>1465</td><td>834</td><td>1238</td><td>579</td><td>468</td><td>527</td><td>0,5</td><td>1,5E-06</td><td>1,8E-05</td></tr><tr><td>Hypothetical</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VC0268</td><td>1534</td><td>1816</td><td>2463</td><td>739</td><td>1253</td><td>701</td><td>0,5</td><td>5,3E-04</td><td>3,0E-03</td></tr><tr><td>VC0519</td><td>6739</td><td>7459</td><td>56109</td><td>23575</td><td>31849</td><td>20968</td><td>0,4</td><td>1,0E-07</td><td>1,5E-06</td></tr><tr><td>VC0714</td><td>3388</td><td>1594</td><td>2591</td><td>663</td><td>2020</td><td>811</td><td>0,5</td><td>8,5E-03</td><td>2,9E-02</td></tr><tr><td>VC1052</td><td>1431</td><td>1646</td><td>2032</td><td>291</td><td>395</td><td>315</td><td>0,2</td><td>7,1E-20</td><td>4,3E-18</td></tr><tr><td>VC1058</td><td>762</td><td>936</td><td>1507</td><td>368</td><td>638</td><td>343</td><td>0,5</td><td>1,0E-03</td><td>5,2E-03</td></tr><tr><td>VC1074</td><td>639</td><td>609</td><td>1692</td><td>286</td><td>317</td><td>303</td><td>0,3</td><td>2,5E-06</td><td>2,8E-05</td></tr><tr><td>VC1317</td><td>4580</td><td>788</td><td>3907</td><td>213</td><td>1306</td><td>301</td><td>0,4</td><td>6,8E-03</td><td>2,4E-02</td></tr><tr><td>VC1574</td><td>1767</td><td>647</td><td>1134</td><td>366</td><td>505</td><td>432</td><td>0,4</td><td>7,6E-06</td><td>7,3E-05</td></tr><tr><td>VC1575</td><td>786</td><td>269</td><td>1065</td><td>134</td><td>207</td><td>119</td><td>0,3</td><td>1,0E-07</td><td>1,5E-06</td></tr><tr><td>VC1576</td><td>2727</td><td>1008</td><td>1653</td><td>330</td><td>786</td><td>345</td><td>0,3</td><td>2,5E-06</td><td>2,8E-05</td></tr><tr><td>VC1578</td><td>3800</td><td>1491</td><td>1767</td><td>278</td><td>1236</td><td>270</td><td>0,3</td><td>2,3E-04</td><td>1,5E-03</td></tr><tr><td>VC1832</td><td>1010</td><td>861</td><td>1883</td><td>390</td><td>605</td><td>473</td><td>0,4</td><td>4,6E-05</td><td>3,7E-04</td></tr><tr><td>VC1941</td><td>3896</td><td>3191</td><td>4799</td><td>1591</td><td>1861</td><td>1619</td><td>0,4</td><td>1,5E-08</td><td>2,5E-07</td></tr><tr><td>VC2093</td><td>364</td><td>468</td><td>980</td><td>286</td><td>316</td><td>262</td><td>0,5</td><td>1,2E-03</td><td>6,0E-03</td></tr><tr><td>VC2443</td><td>1378</td><td>1147</td><td>2022</td><td>611</td><td>768</td><td>809</td><td>0,5</td><td>4,3E-05</td><td>3,5E-04</td></tr><tr><td>VC2472</td><td>1677</td><td>1930</td><td>2799</td><td>671</td><td>813</td><td>648</td><td>0,4</td><td>9,3E-10</td><td>1,9E-08</td></tr><tr><td>VC2706</td><td>1479</td><td>1237</td><td>19055</td><td>2652</td><td>2846</td><td>3038</td><td>0,2</td><td>2,8E-28</td><td>2,8E-26</td></tr><tr><td>VCA0026</td><td>2034</td><td>2585</td><td>2667</td><td>1005</td><td>1465</td><td>984</td><td>0,5</td><td>7,5E-05</td><td>5,6E-04</td></tr><tr><td>VCA0921</td><td>4725</td><td>4659</td><td>8190</td><td>1557</td><td>2134</td><td>1754</td><td>0,3</td><td>8,6E-09</td><td>1,5E-07</td></tr><tr><td>Metabol</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>VC0522</td><td>4113</td><td>2988</td><td>3708</td><td>1695</td><td>2094</td><td>1487</td><td>0,5</td><td>8,2E-07</td><td>1,0E-05</td></tr><tr><td>VC1942</td><td>1302</td><td>1679</td><td>3645</td><td>440</td><td>438</td><td>455</td><td>0,2</td><td>2,0E-11</td><td>5,3E-10</td></tr><tr><td>VC0869</td><td>1473</td><td>1274</td><td>18061</td><td>3007</td><td>1208</td><td>3242</td><td>0,2</td><td>2,7E-12</td><td>7,7E-11</td></tr><tr><td>VC1040</td><td>977</td><td>1202</td><td>1356</td><td>697</td><td>434</td><td>627</td><td>0,5</td><td>8,6E-06</td><td>8,3E-05</td></tr><tr><td>VCA0150</td><td>2635</td><td>1976</td><td>2642</td><td>1116</td><td>702</td><td>1170</td><td>0,4</td><td>1,4E-05</td><td>1,3E-04</td></tr><tr><td>VCA0614</td><td>2154</td><td>1520</td><td>2118</td><td>759</td><td>457</td><td>842</td><td>0,4</td><td>1,2E-07</td><td>1,7E-06</td></tr><tr><td>VCA0558</td><td>2076</td><td>607</td><td>4525</td><td>607</td><td>1460</td><td>718</td><td>0,5</td><td>1,0E-02</td><td>3,4E-02</td></tr><tr><td>VCA0496</td><td>1389</td><td>942</td><td>2071</td><td>426</td><td>1108</td><td>411</td><td>0,5</td><td>4,2E-03</td><td>1,6E-02</td></tr><tr><td>VC0992</td><td>1408</td><td>1336</td><td>1672</td><td>627</td><td>435</td><td>786</td><td>0,4</td><td>1,1E-07</td><td>1,6E-06</td></tr><tr><td>VCA0843</td><td>1854</td><td>2880</td><td>1583</td><td>839</td><td>200</td><td>997</td><td>0,4</td><td>4,7E-04</td><td>2,7E-03</td></tr><tr><td>VC2545</td><td>1017</td><td>1401</td><td>15506</td><td>3639</td><td>4482</td><td>3767</td><td>0,3</td><td>1,8E-10</td><td>4,4E-09</td></tr><tr><td>VC0745</td><td>1524</td><td>1570</td><td>17276</td><td>6155</td><td>7574</td><td>7144</td><td>0,4</td><td>3,3E-08</td><td>5,3E-07</td></tr><tr><td>VCA0863</td><td>2047</td><td>1494</td><td>2025</td><td>616</td><td>561</td><td>814</td><td>0,4</td><td>3,9E-11</td><td>1,0E-09</td></tr><tr><td>VCA0661</td><td>794</td><td>886</td><td>1062</td><td>483</td><td>357</td><td>513</td><td>0,5</td><td>2,9E-06</td><td>3,1E-05</td></tr><tr><td>VC0051</td><td>4018</td><td>2862</td><td>4106</td><td>1016</td><td>374</td><td>1138</td><td>0,3</td><td>5,6E-08</td><td>8,7E-07</td></tr><tr><td>VC0052</td><td>2282</td><td>2082</td><td>3109</td><td>621</td><td>263</td><td>597</td><td>0,2</td><td>8,0E-12</td><td>2,2E-10</td></tr><tr><td>VC2226</td><td>1185</td><td>1260</td><td>16080</td><td>2538</td><td>1208</td><td>2848</td><td>0,2</td><td>3,7E-15</td><td>1,5E-13</td></tr></tbody></table>
<table><tr><td>VC1625</td><td>6073</td><td>5424</td><td>5715</td><td>1808</td><td>1036</td><td>2011</td><td>0,3</td><td>1,3E-12</td><td>3,8E-11</td></tr><tr><td>VC1738</td><td>8039</td><td>9711</td><td>16080</td><td>6283</td><td>5005</td><td>4652</td><td>0,5</td><td>4,8E-05</td><td>3,8E-04</td></tr><tr><td>VC2256</td><td>2868</td><td>2130</td><td>4467</td><td>1588</td><td>1450</td><td>1314</td><td>0,5</td><td>2,5E-05</td><td>2,1E-04</td></tr></table>
<table><caption>**Table S2: Strains and plasmids.** Related to STAR Methods.</caption><thead><tr><th>Strain</th><th>Strain number</th><th>Construction</th></tr></thead><tbody><tr><td><strong>Vibrio cholerae</strong></td><td></td><td></td></tr><tr><td>N16961 wt strain</td><td></td><td></td></tr><tr><td>N16961 hapR+ wt strain</td><td>F606</td><td>Gift from Melanie Blokesch</td></tr><tr><td>ΔraiA (VC0706)</td><td>J251</td><td>PCR amplification of 500 bp up and down regions of VC0706 using primers ZIP381/384 and ZIP382/383. PCR amplification of aadA7 conferring spectinomycin resistance on pAM34 using ZB47/48. PCR assembly of the VCA0706::aad7 fragment using ZIP383/384 and allelic exchange by natural transformation, as described previously (Val et al., 2012, Negro et al., 2019)</td></tr><tr><td>Δrmf (VC1484)</td><td>L557</td><td>allelic exchange by integration and excision of conjugative suicide plasmid pMP7 L045, replacing the gene with <i>frt::aph::frt</i> as described previously (Val et al., 2012, Negro et al., 2019)</td></tr><tr><td>Δhpf (VC2530)</td><td>M566</td><td>allelic exchange by integration and excision of conjugative suicide plasmid pMP7 M472, replacing the gene with <i>frt::aph::frt</i> as described previously (Val et al., 2012, Negro et al., 2019)</td></tr><tr><td>ΔraiA Δrmf</td><td>L790</td><td>allelic exchange by integration and excision of conjugative suicide plasmid pMP7 L045 in J251, replacing the gene with <i>frt::aph::frt</i> as described previously (Val et al., 2012, Negro et al., 2019)</td></tr><tr><td>Δcrp</td><td>9950</td><td>allelic exchange by integration and excision of conjugative suicide plasmid pMP7 8348, replacing the gene with aadA7 (Val et al., 2012, Negro et al., 2019)</td></tr><tr><td>Δfur</td><td>N541</td><td>allelic exchange by integration and excision of conjugative suicide plasmid pMP7 N450, replacing the gene with <i>frt::aph::frt</i> as described previously (Val et al., 2012, Negro et al., 2019)</td></tr><tr><td>ΔrpoS</td><td>A321</td><td>Baharoglu et al., 2013</td></tr><tr><td>ΔtnaA (VC0161)</td><td>J253</td><td>PCR amplification of 500 bp up and down regions of VC0161 using primers ZIP93/94 and ZIP95/96. PCR amplification of aadA7 conferring spectinomycin resistance on pAM34 using ZIP97/98. PCR assembly of the VCA0161::spec fragment using ZIP93/96 and allelic exchange by natural transformation, as described previously (Val et al., 2012, Negro et al., 2019)</td></tr><tr><td><strong>Escherichia coli</strong></td><td></td><td></td></tr><tr><td>MG1655 wt strain</td><td></td><td>laboratory collection</td></tr><tr><td>ΔraiA</td><td>J237</td><td>P1 transduction from KEIO strain JW2578-1</td></tr><tr><td><strong>Pseudomonas aeruginosa PAO1</strong></td><td></td><td>laboratory collection</td></tr><tr><td><strong>Plasmids</strong></td><td></td><td></td></tr><tr><td>pBAD43</td><td></td><td>spectinomycin resistant. Carries Para promoter repressed by glucose 1% and induced by arabinose 0.2%. (Guzman, 1995)</td></tr><tr><td>pBAD43-RaiA+</td><td>L907</td><td>raiA (VC0706) amplified using primers raiAeco/raiAxba and cloned under Pbad using 5' EcoRI and 3' XbaI restriction sites</td></tr><tr><td>pBAD43-Hpf+</td><td>M834</td><td>hpf (VC2530) amplified using primers hpfeco/hpfxba and cloned under Pbad promoter using 5' EcoRI and 3' XbaI restriction sites</td></tr><tr><td>pBAD43-Rmf+</td><td>M831</td><td>rmf (VC1484) amplified using primers rmfeco/rmf ska and cloned under Pbad using 5' EcoRI and 3' XbaI restriction sites</td></tr></tbody></table>
<table><tr><td>pSC101-PraiA-gfp</td><td>L707</td><td>raiA promoter region (500 bp) was amplified using primers ZIP383/446 and gfp was amplified using primers zip443/444. 2 fragments were assembled by PCR assembly using primers ZIP383/ZIP443, so that gfp is fused to the raiA promoter at the ATG start codon of raiA. The assembled fragment was cloned into pTOPO and extracted using EcoRI and cloned into pSC101 low copy plasmid (carbenicillin resistant).</td></tr><tr><td>pSC101-Pc-gfp</td><td>N110</td><td>gfp was amplified using primers carrying the promoter zip513/200. The fragment was cloned into pTOPO and extracted using EcoRI and cloned into pSC101 low copy plasmid (carbenicillin resistant).</td></tr><tr><td>pMP7-Δhpf::kan</td><td>M472</td><td>gibson assembly using primers MV450/451 for the amplification of pMP7 vector, primers VC2530hpf5bis/7 and VC2530hpf6bis/8bis for up and down regions of the gene, and primers MV268/269 on pKD4 plasmid for the resistance gene (frt::kan::frt).</td></tr><tr><td>pMP7-Δrmf::kan</td><td>L045</td><td>gibson assembly using primers MV450/451 for the amplification of pMP7 vector, primers VC1484rmf5/7 and VC1484rmf6/8 for up and down regions of the gene, and primers MV268/269 on pKD4 plasmid for the resistance gene (frt::kan::frt).</td></tr><tr><td>pMP7-Δcrp::aad7</td><td>8348</td><td>Baharoglu et al., 2012</td></tr><tr><td>pMP7-Δfur::kan</td><td>N450</td><td>gibson assembly using primers MV450/451 for the amplification of pMP7 vector, primers VC2106fur5/7 and VC2106fur9/8 for up and down regions of the gene, and primers MV268/269 on pKD4 plasmid for the resistance gene (frt::kan::frt).</td></tr><tr><td colspan="3"><strong>Primers</strong></td></tr><tr><td>raiAecoRI</td><td></td><td>ggaattcaccATGAAAATCAACATCACTGGTAA</td></tr><tr><td>raiAxba</td><td></td><td>gctctagaTTATTCCACTTCTTCGCTCAG</td></tr><tr><td>hpfeco</td><td></td><td>ggaattcaccATGCAAATCAACATTCAAGGCC</td></tr><tr><td>hpfxda</td><td></td><td>gctctagaTTAATGACTACTTAGCTTTTCTTT</td></tr><tr><td>rmfeco</td><td></td><td>ggaattcaccATGAAGAGACAAAAGCGTGAT</td></tr><tr><td>rmfxda</td><td></td><td>gctctagaTTATTTGCAGAGACCAGAAAGTTT</td></tr><tr><td>ZB47</td><td></td><td>CCCGTTCCATACAGAAGCTGGGCGAACAAACGATGCTCGC</td></tr><tr><td>ZB48</td><td></td><td>GACATTATTTGCCGACTACCTTGGTGATCTCGCCTTTCACG</td></tr><tr><td>ZIP381</td><td></td><td>GCGAGCATCGTTTGTTCGCCCAGCTTCTGTATGGAACGGGTAAATA GAATGATGGGAGATAGCGC</td></tr><tr><td>ZIP382</td><td></td><td>CGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCAATGC TTTTTCCTCTGTGTCATCCCTTATGG</td></tr><tr><td>ZIP383</td><td></td><td>AACCTATCCAGATACCGAAGCGGC</td></tr><tr><td>ZIP384</td><td></td><td>GTTTTGCAGATAGGATTGCTGAAGC</td></tr><tr><td>ZIP93</td><td></td><td>GTCGAATGCCAAACGAAAGCGGAAAATACC</td></tr><tr><td>ZIP94</td><td></td><td>GCGAGCATCGTTTGTTCGCCCAGCTTCTGTATGGAACGGGTACGGT ATTGAAAAAGTCAGCCTGC</td></tr><tr><td>ZIP95</td><td></td><td>CGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCGCTGG GTATTCCTAAAAAATTAAAATATAAAGTCATGC</td></tr><tr><td>ZIP96</td><td></td><td>CACAACAACTCTAGTATCTGGTTTACCCTCG</td></tr><tr><td>ZIP97</td><td></td><td>GGCAGCCTATGCAGGCTGCACTTTTTCAATACCGTCCCGTTCCATAC AGAAGCTGGGCGAACAAACGATGCTCGC</td></tr><tr><td>ZIP98</td><td></td><td>ATGACTTTATATTTTAATTTTTAGGAATACCCAGCGACATTATTTGCC GACTACCTTGGTGATCTCGCCTTTCACG</td></tr><tr><td>ZIP443</td><td></td><td>TTATTTGTATAGTTCATCCATGCCATGTGTAATCCCAGC</td></tr><tr><td>ZIP444</td><td></td><td>ATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCC</td></tr><tr><td>ZIP446</td><td></td><td>GGACAACTCCAGTGAAAAGTTCTTCTCCTTTACGCATAATGCTTTTTC CTCTGTGTCATCCCTTATGG</td></tr><tr><td>VC2530hpf5bis</td><td></td><td>CTATTATTTAAACTCTTTCCgccgatggtgctcaacgatg</td></tr></table>
<table><tr><td>VC2530hpf7</td><td>CTACACAATCGCTCAAGACGTGagactttccttctctagtttagg</td></tr><tr><td>VC2530hpf6bis</td><td>TACGTAGAATGTATCAGACTcttcacactgcaatagcacg</td></tr><tr><td>VC2530hpf8bis</td><td>CTAATTCCCATGTCAGCCGTGCGCCAGATCGCCGAAGCTA</td></tr><tr><td>VC1484rmf5</td><td>CTATTATTTAAACTCTTTCCggcttggttgtagatgatc</td></tr><tr><td>VC1484rmf7</td><td>CTACACAATCGCTCAAGACGTGagttctgtcctcatacggtta</td></tr><tr><td>VC1484rmf6</td><td>TACGTAGAATGTATCAGACTgttgatgatggatcgcattacc</td></tr><tr><td>VC1484rmf8</td><td>CTAATTCCCATGTCAGCCGTttcccatacaacaagcttga</td></tr><tr><td>VC2106fur5</td><td>CTATTATTTAAACTCTTTCCAAGCGGATGCGAACTTCGC</td></tr><tr><td>VC2106fur7</td><td>CTACACAATCGCTCAAGACGTGATACTTTCCTGTTGATGTTCTGC</td></tr><tr><td>VC2106fur8</td><td>CTAATTCCCATGTCAGCCGTGCTCACAAGCCGAAGAAATAA</td></tr><tr><td>VC2106fur9</td><td>TACGTAGAATGTATCAGACTccacaaatcgatcagtttatgg</td></tr><tr><td>ZIP200</td><td>TATCAAGCTTATTTGTATAGTTCATCCATGCC</td></tr><tr><td>ZIP513</td><td>GAG CTG TTG ACA ATT AAT CAT CCG GCT CGT ATA ATG TGT GGA ATT GTG AGC GGA TAA CAA TTT CAC ACA GGA AAC ACA TAT GCG TAA AGG AGA AGA AC</td></tr></table>