Katayoun Kazemzadeh

Mahmoud Hajj Chehade

Gautier Hourdoir

Camille Dorothée Brunet

Yvan Caspar

Laurent Loiseau

Frederic Barras

Fabien Pierrel

Ludovic Pelosi
email: ludovic.pelosi@univ-grenoble-alpes.fr

The biosynthetic pathway of ubiquinone contributes to pathogenicity of Francisella

Keywords: ubiquinone biosynthesis, coenzyme Q, quinone, aerobic respiration, Francisella tularensis, Francisella novicida Francisella novicida, Francisella tularensis, ubiquinone biosynthesis, respiratory chain, metabolism, pathogenicity

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Francisella tularensis is a Gram-negative, strictly aerobic, facultative intracellular pathogen responsible for tularemia. Infection can occur by inhalation, ingestion, transmission from arthropod vectors or exposure to infected animals [START_REF] Sjostedt | Special topic on Francisella tularensis and tularemia[END_REF]. After its entry into macrophages, the bacteria are sequestered into phagosomes and prevent further endosomal maturation.

Francisella cells then disrupt the phagosome and are released into the cytosol in which they rapidly proliferate [START_REF] Wallet | Francisella Inflammasomes: Integrated responses to a cytosolic stealth bacterium[END_REF]. Eventually, the infected cells undergo apoptosis or pyroptosis, and the progeny bacteria are released to initiate new rounds of infection [START_REF] Wallet | Francisella Inflammasomes: Integrated responses to a cytosolic stealth bacterium[END_REF]. Currently, there is no suitable vaccine against tularemia and due to its extreme infectivity and high virulence, F. tularensis species have been classified as a biothreat agent [START_REF] Oyston | Tularaemia: bioterrorism defence renews interest in Francisella tularensis[END_REF]. The genus Francisella includes the three species: F. tularensis, F. novicida and F. philomiragia [START_REF] Sjostedt | Tularemia: history, epidemiology, pathogen physiology, and clinical manifestations[END_REF]. Moreover, F. tularensis is further divided into the subspecies tularensis (Type A strains) and holarctica (Type B strains), which are the most virulent strains responsible for human disease, whereas F. philomiragia and F. novicida are avirulent in healthy humans [START_REF] Sjostedt | Tularemia: history, epidemiology, pathogen physiology, and clinical manifestations[END_REF]. A F. novicida type strain U112 is commonly used as a surrogate for Francisella tularensis in virulence studies using animal models [START_REF] Kingry | Comparative review of Francisella tularensis and Francisella novicida[END_REF].

The development of genome-scale genetic methods allowed the identification of hundreds of genes participating to variable extents to Francisella virulence [START_REF] Jones | Uncovering the components of the Francisella tularensis virulence stealth strategy[END_REF]. However, the specific contribution of only a limited number of these genes was demonstrated at the molecular level. Although an important proportion of the identified genes are related to metabolic functions, the relationship between metabolism and the life cycle of Francisella is still poorly understood. However, global analysis of genes essential for the growth in culture of F. novicida U112 [START_REF] Gallagher | A comprehensive transposon mutant library of Francisella novicida, a bioweapon surrogate[END_REF] and more recently of that of F. tularensis ssp tularensis Schu S4 [START_REF] Ireland | Global analysis of genes essential for Francisella tularensis Schu S4 growth in vitro and for fitness during competitive infection of fischer 344 rats[END_REF] highlighted the involvement of several ubiquitous pathways found in proteobacteria. Among the most significant are the folate pathway, the heme synthesis pathway, the methylerythritol phosphate pathway involved in isoprenoid synthesis, the chorismate pathway and the ubiquinone (UQ) synthesis pathway, on which this work is focused.

Isoprenoid quinones are conserved in most respiratory and photosynthetic organisms and function primarily as electron and proton carriers in the electron-transfer chains.

Quinones are composed of a polar redox-active head group linked to a lipid side chain, which varies in both length and degree of saturation [START_REF] Nowicka | Occurrence, biosynthesis and function of isoprenoid quinones[END_REF]. Proteobacteria contain two main types of quinone, i.e. benzoquinones and naphthoquinones, represented by UQ (or coenzyme Q) and menaquinone (MK)/ demethylmenaquinone (DMK), respectively [START_REF] Nowicka | Occurrence, biosynthesis and function of isoprenoid quinones[END_REF]. UQ is the major electron carrier used for the reduction of dioxygen by various cytochrome oxidases, whereas MK and DMK function predominantly in anaerobic respiratory chains [START_REF] Nowicka | Occurrence, biosynthesis and function of isoprenoid quinones[END_REF]. However, as demonstrated recently in Pseudomonas aeruginosa, UQ can also be produced and used as a main respiratory quinone under anaerobic conditions [START_REF] Vo | The O 2 -independent pathway of ubiquinone biosynthesis is essential for denitrification in Pseudomonas aeruginosa[END_REF]. Besides its role in bioenergetics, UQ was also reported to be involved in gene regulation, oxidative stress, virulence and resistance to antibiotics [START_REF] Soballe | Microbial ubiquinones: multiple roles in respiration, gene regulation and oxidative stress management[END_REF][START_REF] Aussel | Biosynthesis and physiology of coenzyme Q in bacteria[END_REF]. More recently, new functions for UQ in bacteria were discovered such as its requirement for Escherichia coli to grow on medium containing longchain fatty acids as a carbon source [START_REF] Agrawal | A genome-wide screen in Escherichia coli reveals that ubiquinone is a key antioxidant for metabolism of long-chain fatty acids[END_REF]. UQ biosynthesis in aerobic conditions has been widely studied in E. coli [START_REF] Abby | Advances in bacterial pathways for the biosynthesis of ubiquinone[END_REF]. The classical UQ biosynthetic pathway requires twelve proteins (UbiA to UbiK and UbiX). UbiC catalyzes the first committed step in the biosynthesis of UQ, the conversion of chorismate to the 4-hydroxybenzoate (4HB) precursor. Then, UbiA, UbiD to UbiI and UbiX catalyze the prenylation, decarboxylation, hydroxylations and methylations of the phenyl ring of the 4HB to synthesize UQ. In addition, UbiB and UbiK are accessory proteins while UbiJ is involved in the assembly and/or the stability of the aerobic Ubicomplex, which was recently characterized in E. coli [START_REF] Hajj Chehade | A soluble metabolon synthesizes the isoprenoid lipid ubiquinone[END_REF]. This latter is able to synthesize UQ also under anoxic conditions and we identified three proteins, UbiU, UbiV and UbiT, which are required for UQ biosynthesis only under anoxic conditions [START_REF] Pelosi | Ubiquinone biosynthesis over the entire O 2 range: characterization of a conserved O 2 -independent pathway[END_REF].

Here, we show that UQ 8 is the major quinone of F. novicida U112. We identified candidate Ubi-proteins in F. novicida U112 and validated their functions by heterologous complementation in E. coli mutant strains. Our results show that UQ biosynthesis in Francisella spp is mostly similar to that of E. coli, with the notable absence of UbiX and UbiD for the decarboxylation step. Genetic and chemical inactivation of UQ biosynthesis thanks to a transposon mutant and to a new inhibitor (3-amino-4-hydroxybenzoic acid), respectively, demonstrated that UQ 8 is crucial for the growth of F. novicida in respiratory media and that UQ deficiency impairs the pathogenicity of F. novicida against Galleria mellonella. Altogether, our results shed light on the role of UQ in the life cycle of Francisella and show that UQ contributes to its pathogenicity.

Results

UQ 8 is the major quinone of F. novicida.

The quinone content of F. novicida grown under ambient air at 37°C in Chamberlain media supplemented with either glucose (fermentative medium) or succinate (respiratory medium) as the only carbon source was determined and compared with that of E. coli MG1655 grown in the same fermentative medium. In the electrochromatograms of lipid extracts from F. novicida, a single peak was observed around 8.3 min, the same retention time as UQ 8 in E. coli extracts (Fig. 1A). Note that in these analyses, UQ 10 was used as an internal standard, which was added to the samples. MS analysis of the major peak in F. novicida extracts showed a predominant ammonium adduct (M + NH 4

+

) with an m/z ratio of 744.5, together with minor adducts, such as Na + (749.7) and H + (727.8) (Fig. 1B). These masses identify UQ 8 (monoisotopic mass, 726.5) as the major quinone produced by F. novicida.

Interestingly, the carbon source in the culture media did not greatly affect the UQ 8 content (Fig. 1A). The F. novicida extracts did not contain any naphthoquinones, unlike E. coli, which showed predominantly demethylmenaquinone (DMK 8 ) eluting around 12 min. The absence of detectable levels of naphthoquinones in F. novicida lipid extracts (Fig. 1A) is in agreement with the absence of menaquinone biosynthesis (Men or futalosine) encoding genes in its genome. Together, our results establish that E. coli and F. novicida share UQ 8 as a main quinone in aerobic conditions.

Identification of Ubi-proteins in the Francisella spp.

To identify candidate Ubi-proteins in F. novicida, UbiX, and UbiA to UbiK from E. coli MG1655 were screened for homologs in the protein sequence data set, available at MicroScope (www.genoscope.cns.fr/agc/microscope), using the BLASTP software. As listed in Table S1, this analysis identified eight homologous proteins in F. novicida, i.e. UbiA to UbiC, UbiE, UbiG to UbiI and UbiK, called hereafter UbiA Fn to UbiC Fn , UbiE Fn , UbiG Fn to UbiI Fn and UbiK Fn , respectively. Genes ubiA Fn and ubiC Fn in one hand and genes ubiI Fn and ubiH Fn in another hand present an organization similar to the ubiC-ubiA and the ubiH-ubiI operons from E. coli, respectively [START_REF] Aussel | Biosynthesis and physiology of coenzyme Q in bacteria[END_REF]. As reported previously for Pseudomonas aeruginosa [START_REF] Stenmark | A new member of the family of di-iron carboxylate proteins. Coq7 (clk-1), a membranebound hydroxylase involved in ubiquinone biosynthesis[END_REF] and Xanthomonas campestris [START_REF] Zhou | Biosynthesis of coenzyme Q in the Phytopathogen xanthomonas campestris via a yeast-like pathway[END_REF], F. novicida possesses a Coq7 hydroxylase, which is a functional homolog of the UbiF protein found in E. coli and other species [START_REF] Pelosi | Evolution of ubiquinone biosynthesis: multiple proteobacterial enzymes with various regioselectivities to catalyze three contiguous aromatic hydroxylation reactions[END_REF]. The detection of an homolog for E. coli UbiJ required less restrictive Blast parameters. We noticed that the gene coding for the putative UbiJ candidate FTN_0460, hereafter called ubiJ Fn , lies between ubiE Fn and ubiB Fn , an organization similar to the ubiE-ubiJ-ubiB operon from E. coli [START_REF] Poon | Identification of Escherichia coli ubiB, a gene required for the first monooxygenase step in ubiquinone biosynthesis[END_REF].

UbiJ Fn has 21% amino-acid identity with UbiJ from E. coli and both proteins contain a sterol carrier protein 2 domain in their N-terminal regions (http://pfam.xfam.org/) [START_REF] Aussel | ubiJ, a new gene required for aerobic growth and proliferation in macrophage, is involved in coenzyme Q biosynthesis in Escherichia coli and Salmonella enterica serovar Typhimurium[END_REF]. The same Ubi-proteins were identified in the highly virulent strain F. tularensis ssp tularensis Schu S4 (Table S1).

Homologs of UbiD and UbiX

were not yet identified and the counterparts of these two proteins in Francisella spp remain to be determined. The work is in progress in our laboratory. Under anaerobic conditions, E. coli still synthesizes UQ, and we recently identified three genes, which we called ubiT, ubiU, and ubiV, as essential for this process [START_REF] Pelosi | Ubiquinone biosynthesis over the entire O 2 range: characterization of a conserved O 2 -independent pathway[END_REF]. Homologs of ubiT, ubiU, and ubiV, which participate to the O 2 -independent UQ biosynthetic pathway, were not identified in the screened Francisella genomes (Table S1), in agreement with the strictly aerobic metabolism of Francisella spp. In overall, these data show that the O 2 -dependent UQ biosynthetic pathways in F. novicida, in F. tularensis and in E. coli are related, the major difference being the absence of UbiX-UbiD for the decarboxylation step (Fig. 2).

Functional characterization of Ubi Fn -proteins in E. coli.

To test whether the candidate Ubi-proteins identified in F. novicida were indeed involved in UQ biosynthesis, we assessed their capacity to functionally complement E. coli strains in which the UQ protein-encoding genes were inactivated (ubiAc, ubiBc, ubiCc, ubiEc, ubiFc, ubiGc, ubiHc, ubiIc, ubiJ and ubiKc, see Table S2). We assessed the quinone content and the capacity to grow on solid minimal medium containing fermentable (glucose) or respiratory (succinate) carbon sources. E. coli ubiAc, ubiBc, ubiGc, ubiHc and ubiJ transformed with empty vector are unable to synthesize UQ 8 (Fig. 3A) and are thus unable to grow on a respiratory medium (Fig. 3C). In contrast, their growth on a fermentative medium is not affected (Fig. 3C). Except for the ubiAc mutant strain, in which the prenylation reaction of the 4HB is impaired, most mutants accumulate an early intermediate corresponding to octaprenylphenol (OPP) (Fig. 2 and3A). E. coli ubiEc and ubiFc cells accumulate C2-demethyl-C6-demethoxy-UQ 8 (DDMQ 8 ) and C6-demethoxy-UQ 8 (DMQ 8 ), which are the substrates of UbiE and UbiF, respectively (Fig. 2 and3A). We found that UbiA Fn , UbiB Fn , UbiE Fn , Coq7 Fn and UbiG Fn restored growth of E. coli ubiAc, ubiBc, ubiEc, ubiFc and ubiGc cells on respiratory medium (Fig. 3C) and allowed for UQ 8 biosynthesis in LB medium to 96, 26, 7, 49 and 38% of the level of UQ 8 present in the WT cells, respectively (Fig. 3A and4A). Concomitantly, OPP content decreased and Coq7 Fn abolished the accumulation of DMQ 8 in ubiFc cells (Fig. 3A). As we previously reported, E. coli ubiIc and ubiKc cells displayed a strong decrease in UQ 8 [START_REF] Hajj Chehade | ubiI, a new gene in Escherichia coli coenzyme Q biosynthesis, is involved in aerobic C5-hydroxylation[END_REF][START_REF] Loiseau | The UbiK protein is an accessory factor necessary for bacterial ubiquinone (UQ) biosynthesis and forms a complex with the UQ biogenesis factor UbiJ[END_REF], but the residual UQ 8 content was sufficient to support growth on succinate (Fig. 3A and3C). Similar results were obtained with ubiCc cells grown in minimal M9 medium (Fig. 3B and3C), which had to be used instead of LB since the later contains 4HB that restores normal UQ 8 content in ubiCc (data not shown). In all three strains, the expression of the corresponding Ubiproteins, UbiC Fn , UbiI Fn and UbiK Fn , increased significantly the UQ 8 content (Fig. 4A and4B). Since the increase obtained in ubiIc cells was moderate (from 25 to 40%), we further confirmed the ability of UbiI Fn to catalyze C5-hydroxylation by using an E. coli ubiIcubiF strain. This deletion mutant lacks C5-and C6-hydroxylation activities and consequently accumulates 3-octaprenyl-4-hydroxyphenol (4HP 8 ) [START_REF] Hajj Chehade | ubiI, a new gene in Escherichia coli coenzyme Q biosynthesis, is involved in aerobic C5-hydroxylation[END_REF]. We found that UbiI Fn was able to restore DMQ 8 biosynthesis in E. coli ubiIcubiF cells (Fig. S1), i.e. to catalyze C5hydroxylation, concomitantly to a strong decrease of 4HP 8 . Taken together, all these results confirm unambiguously that UbiA Fn , UbiB Fn , UbiC Fn , Coq7 Fn , UbiE Fn , UbiG Fn , UbiI Fn and UbiK Fn are the functional counterpart of the E. coli Ubi-proteins and we propose that they compose the biosynthetic pathway of UQ 8 in F. novicida. Only two proteins, UbiJ Fn and UbiH Fn did not complement the E. coli ubiJ and ubiHc (Fig. 3A, 3C and4A). The low percentage of identity between UbiJ and UbiH from E. coli and their homologs in F. novicida (21 and 27%, respectively) could explain these results (Table S1). UQ 8 biosynthesis is essential for the growth of F. novicida in respiratory medium.

To evaluate the physiological importance of UQ for F. novicida, we screened ubigenes in the F. novicida transposon (Tn) mutant library available at the Manoil Laboratory [START_REF] Gallagher | A comprehensive transposon mutant library of Francisella novicida, a bioweapon surrogate[END_REF]. Only the Tn mutant of ubiC Fn (called hereafter Tn-ubiC Fn ) was available in the library, and we compared this mutant strain to its isogenic control strain U112 (Table S2). Recall that UbiC catalyzes the first committed step in the biosynthesis of UQ, i.e. the conversion of chorismate to 4HB (Fig. 2). First, we showed that the growth of Tn-ubiC Fn cells under ambient air in respiratory Chamberlain medium was severely impaired compared to the WT (Fig. 5A). In contrast, the growth of F. novicida in fermentative medium was less affected (Fig. 5B). In parallel, UQ 8 content was strongly lowered in Tn-ubiC Fn cells from 166 to 7 pmol/mg cells in fermentative medium and from 134 to 11 pmol/mg cells in respiratory medium (Fig. 5C). As expected, addition of 4HB to the culture rescued the growth of Tn-ubiC Fn in respiratory medium and increased the UQ 8 content to WT levels (Fig. 5A and5C).

Taken together, these results show the overall requirement of UQ 8 for the growth of F. novicida especially in respiratory medium.

3A4HB inhibits UQ 8 biosynthesis and impairs the growth of F. novicida in respiratory medium.

Besides genetic inactivation of the UQ pathway, we were interested in the possibility to decrease UQ levels by chemical inhibition. Since we had found UQ to be particularly important for growth of F. novicida in respiratory medium (Fig. 5A), we screened for compounds that could inhibit growth in such medium. We tested several compounds: 3amino-4-hydroxybenzoic acid (3A4HB), 4-amino-benzoic acid (pABA), 4-amino-2-methoxybenzoic acid (pA2MBA) and 4-amino-3-methoxy-benzoic acid (pA3MBA). All these molecules are analogs of 4HB, the native precursor of UQ (Fig. S2A). We observed that bacterial growth was slightly affected in respiratory medium in presence of pABA and pA2MBA, while pA3MBA inhibited growth both in fermentative and respiratory media (Fig. S2B-C). Interestingly, 3A4HB strongly impaired bacterial growth in respiratory medium, while inhibition was milder in fermentative medium (Fig. S2B-C). Based on these results, we followed up on this compound.

We then examined how and to what extent 3A4HB could affect UQ 8 biosynthesis in F. novicida. Bacteria were cultured under ambient air in fermentative Chamberlain medium supplemented with 3A4HB (from 10 µM to 1 mM, final concentration). Endogenous UQ 8 content was measured in the bacterial cells and compared to a control condition in which only DMSO was added. Figure 6A shows that the UQ 8 content decreased with increasing concentrations of 3A4HB in the medium, with 0.5 mM yielding to a 90% decrease of the UQ 8 content. Concomitantly, we confirmed that growth of F. novicida in presence of 1 mM 3A4HB was strongly impaired in respiratory medium (Fig. 6B), but less so in a fermentative medium (Fig. 6C). Control experiments showed that addition of 4HB to the growth medium counteracted the negative effect of 3A4HB, both in terms of UQ 8 biosynthesis and bacterial growth (Fig. 6A to 6C).

Treatment with 3A4HB caused the accumulation of a redox compound that eluted at 6.5 min (compound X on Fig. 6A). MS analysis of this peak showed a predominant proton adduct (M + H + ) with an m/z ratio of 682.6, together with a minor sodium adduct (M + Na + ) with an m/z ratio of 704.6 (Fig. 6D). Both species are compatible with a monoisotopic mass of 681.7 g.mol -1 , which could correspond to that of 2-octaprenyl-3-methyl-6-amino-1,4benzoquinone (Fig. 6E). According to the sequence of reactions proposed in Figure 2, the formation of compound X would result from prenylation of 3A4HB, decarboxylation and hydroxylation at C1 and then methylation at C3. Thus, 3-octaprenyl-2-methyl-5-amino-1,4benzoquinone seems to be the "dead-end" product of the UQ 8 pathway in F. novicida cells treated with 3A4HB. Collectively, these results demonstrate unequivocally that 3A4HB acts as a competitive inhibitor of UQ 8 biosynthesis and affects particularly the respiratory metabolism of F. novicida. UQ 8 is involved in the pathogenesis of F. novicida in the later steps of the infection.

We evaluated the importance of UQ in the pathogenicity of F. novicida by studying the Tn-ubiC Fn mutant. To assess the overall virulence of the Tn-ubiC Fn strain in a whole organism, we used the wax moth (G. mellonella) infection model, which was previously used in studies of human pathogenic and closely related opportunistic and non-pathogenic Francisella spp, such as F. novicida [START_REF] Thelaus | Galleria mellonella Reveals niche differences between highly pathogenic and closely related strains of Francisella spp[END_REF][START_REF] Aperis | Galleria mellonella as a model host to study infection by the Francisella tularensis live vaccine strain[END_REF][START_REF] Propst | Francisella philomiragia Infection and lethality in mammalian tissue culture cell models, Galleria mellonella, and BALB/c mice[END_REF][START_REF] Brodmann | Type VI secretion system and its effectors PdpC, PdpD and OpiA contribute to Francisella virulence in Galleria mellonella larvae[END_REF]. We monitored the survival of larvae infected with the Tn-ubiC Fn strain or with the isogenic strain U112 as control. When the larvae are turning grey/black and no movement of the larval legs can be observed, they are considered dead (Fig. 7A). The Tn-ubiC Fn strain was found to be statistically much less virulent than the wild type, but was nevertheless still capable to kill Galleria larvae (Fig. 7B). This result suggests that UQ 8 is involved in the virulence potential of F. novicida in G. mellonella. To better understand the role of UQ 8 in the different stages of infection in G. mellonella, the pathogenicity of the isogenic control strain pre-treated with 1 mM 3A4HB was studied in order to mimic a UQ 8 acute deficiency. Recall that this treatment causes a 90% decrease of the UQ 8 content (Fig. 6A) but the inhibition should be alleviated over the infection cycle in the larvae where 3A4HB is not present. The pretreatment with 3A4HB has no effect on the capacity of F. novicida to kill Galleria larvae (Fig. 7C), suggesting that UQ 8 does not contribute to the virulence of Francisella novicida in the early steps of the infection but more likely in later ones. This result contrasts with that obtained with the Tn-ubiC Fn strain, which represents a chronic deficiency of UQ 8 .

Discussion

The chemical analysis performed in this paper established that UQ 8 is the major isoprenoid quinone synthesized by F. novicida. In two representative Francisella genomes, we identified homologs for nine of the twelve genes, which are currently known to contribute to UQ biosynthesis in E. coli under aerobic conditions. We confirmed the function of seven of the nine homologs by heterologous complementation of E. coli Δubi mutants. From these results, we show again that E. coli is a good model to study the function of most exogenous ubi-genes [START_REF] Pelosi | Evolution of ubiquinone biosynthesis: multiple proteobacterial enzymes with various regioselectivities to catalyze three contiguous aromatic hydroxylation reactions[END_REF]. We could not confirm the function of UbiH Fn and UbiJ Fn , but the fact that ubiH Fn and ubiJ Fn show the same genetic organization as in E. coli (a ubiI-ubiH operon and a ubiE-ubiJ-ubiB operon) strongly supports the implication of these genes in the UQ biosynthetic pathway. Interestingly, both proteins are part of the Ubi-complex in E. coli [START_REF] Hajj Chehade | A soluble metabolon synthesizes the isoprenoid lipid ubiquinone[END_REF].

We hypothesize that the low identity of UbiH Fn and UbiJ Fn with their E. coli homologs (25%) might impair their assembly within the E. coli Ubi-complex and thus compromise our in vivo complementation assays. Another possibility relates to the proposed implication in UQ biosynthesis of a non-coding RNA partially overlapping the ORF of UbiJ from E. coli [START_REF] Tang | Differential quantitative proteomics reveals the functional difference of two yigP locus products, UbiJ and EsrE[END_REF].

We note that the expression of UbiJ from X. campestris was also unable to complement an E. coli ΔubiJ strain [START_REF] Zhou | Biosynthesis of coenzyme Q in the Phytopathogen xanthomonas campestris via a yeast-like pathway[END_REF]. Francisella spp shares with P. aeruginosa and X. campestris a yeast Coq7 protein homolog, which catalyzes the C6-hydroxylation as UbiF from E. coli [START_REF] Stenmark | A new member of the family of di-iron carboxylate proteins. Coq7 (clk-1), a membranebound hydroxylase involved in ubiquinone biosynthesis[END_REF][START_REF] Zhou | Biosynthesis of coenzyme Q in the Phytopathogen xanthomonas campestris via a yeast-like pathway[END_REF][START_REF] Jiang | Coenzyme Q biosynthesis in the biopesticide Shenqinmycin-producing Pseudomonas aeruginosa strain M18[END_REF]. As we demonstrated previously, the Coq7 proteins are found in all three subclasses, alpha-, beta-and gamma-proteobacteria. In contrast, homologs of UbiF proteins are limited to the gamma-proteobacteria [START_REF] Pelosi | Evolution of ubiquinone biosynthesis: multiple proteobacterial enzymes with various regioselectivities to catalyze three contiguous aromatic hydroxylation reactions[END_REF]. Our analysis disclosed also the presence in Francisella spp of UbiI and UbiH homologous proteins, which catalyze C5-and C1-hydroxylation in E. coli, respectively [START_REF] Hajj Chehade | ubiI, a new gene in Escherichia coli coenzyme Q biosynthesis, is involved in aerobic C5-hydroxylation[END_REF][START_REF] Nakahigashi | Isolation and characterization of a light-sensitive mutant of Escherichia coli K-12 with a mutation in a gene that is required for the biosynthesis of ubiquinone[END_REF]. Consequently, we propose that both E. coli and Francisella spp share a UQ biosynthetic pathway involving three hydroxylases, i.e. UbiI, UbiH and UbiF in E. coli or UbiI, UbiH and Coq7 in Francisella spp. Several studies highlighted that the enzymes involved in multiple steps of the UQ biosynthetic pathway vary between bacterial species [START_REF] Abby | Advances in bacterial pathways for the biosynthesis of ubiquinone[END_REF], like for the hydroxylation steps [START_REF] Pelosi | Evolution of ubiquinone biosynthesis: multiple proteobacterial enzymes with various regioselectivities to catalyze three contiguous aromatic hydroxylation reactions[END_REF], or for the production of 4HB from chorismate by UbiC or XanB2 proteins [START_REF] Zhou | The diffusible factor synthase XanB2 is a bifunctional chorismatase that links the shikimate pathway to ubiquinone and xanthomonadins biosynthetic pathways[END_REF]. The decarboxylation step involves UbiD and UbiX in E. coli [START_REF] Marshall | The UbiX-UbiD system: The biosynthesis and use of prenylated flavin (prFMN)[END_REF], but we could not identify homologs in Francisella genomes. A candidate gene ubiZ was proposed based on its co-localization with ubiE and ubiB in the genomes of Acinetobacter spp and Psychrobacter sp. PRwf-1, which are also devoid of homologs of UbiD and UbiX [START_REF] Ravcheev | Genomic analysis of the human gut microbiome suggests novel enzymes involved in quinone biosynthesis[END_REF]. However, ubiZ was not confirmed functionally and this gene is not conserved in Francisella genomes. We demonstrated that UbiI proteins from F. novicida and from E. coli shared the same function, i.e. the catalysis of the first hydroxylation of the OPP, which is the product of the decarboxylation step in E. coli (Fig. 2). Consequently, we propose that the decarboxylation step occurring in F. novicida also precedes the first hydroxylation of the OPP. Collectively, these data suggest the existence of another decarboxylation system operating in UQ biosynthesis in Francisella spp and potentially in other bacteria lacking ubiX and ubiD [START_REF] Esposti | A journey across genomes uncovers the origin of ubiquinone in cyanobacteria[END_REF].

To assess the essentiality of the UQ biosynthetic pathway in the respiratory metabolism of F. novicida, two different approaches were carried out. First, we showed that a transposon mutation of ubiC Fn gene, which decreases 4HB synthesis, impaired the growth of F. novicida mainly in respiratory medium. Interestingly, among all the ubi-gene identified in Francisella genomes, only ubiC was mutated in large-scale studies [START_REF] Gallagher | A comprehensive transposon mutant library of Francisella novicida, a bioweapon surrogate[END_REF][START_REF] Ireland | Global analysis of genes essential for Francisella tularensis Schu S4 growth in vitro and for fitness during competitive infection of fischer 344 rats[END_REF]. This supports that the other ubi genes are essential for the viability of Francisella spp and strengthens the idea that UQ is key for the development of these bacteria. We noted that the mutation of the ubiC gene affects more severely F. novicida than E. coli for growth in respiratory media, despite both mutants producing comparable amounts of UQ (~7-8% compared to the WT) (Fig. 4B and 5C). As E. coli synthesizes naphthoquinones but F. novicida does not, we propose that the milder phenotype of the E. coli ubiC mutant results from naphthoquinones participating to aerobic respiration, as previously suggested [START_REF] Sharma | On the function of the various quinone species in Escherichia coli[END_REF]. Second, we tested the effect of structural analogs of 4HB and we showed that 3A4HB impaired the growth of F. novicida mainly in respiratory medium in agreement with a strong decrease of UQ 8 biosynthesis. We demonstrated that 3A4HB competes with endogenous 4HB and progresses through several steps of the UQ biosynthetic pathway to form the redox compound X that we propose to be 3octaprenyl-2-methyl-5-amino-1,4-benzoquinone. As the Tn-ubiC Fn strain and the control strain U112 treated with 1 mM 3A4HB yielded both to a 90% decrease of the UQ 8 content and presented a strong impairment of the growth in a respiratory medium (Fig. 5 and6), we propose that compound X would not be used as a quinone in the respiratory chain of F. novicida.

We noted that homologs of ubiT, ubiU, and ubiV, which belongs to the O 2independent UQ biosynthetic pathway characterized in E. coli and P. aeruginosa [START_REF] Vo | The O 2 -independent pathway of ubiquinone biosynthesis is essential for denitrification in Pseudomonas aeruginosa[END_REF][START_REF] Pelosi | Ubiquinone biosynthesis over the entire O 2 range: characterization of a conserved O 2 -independent pathway[END_REF], were not identified in the screened genomes of Francisella spp. This result is in agreement with the strictly aerobic metabolism of these bacteria. Indeed, tricarboxylic acid (TCA) cycle and the UQ-dependent electron-transfer chain, leading to efficient oxidative phosphorylation, take place in Francisella spp [START_REF] Ziveri | Importance of metabolic adaptations in francisella pathogenesis[END_REF]. A possible link between stress defense and the TCA cycle was previously suggested in Francisella pathogenesis [START_REF] Dieppedale | Possible links between stress defense and the tricarboxylic acid (TCA) cycle in Francisella pathogenesis[END_REF]. Unfortunately, the contribution of UQ and the electron-transfer chain to virulence has not been well documented to date in Francisella spp. Using G. mellonella as infection model at the scale of an entire organism, we demonstrated through the study of the Tn-ubiC Fn mutant and the isogenic control strain pretreated with 3A4HB, that UQ 8 contributes to the virulence of F. novicida and more likely in the later steps of the infection, during which the bacteria undergo extensive replication [START_REF] Chong | The Francisella intracellular life cycle: toward molecular mechanisms of intracellular survival and proliferation[END_REF]. Such a notion support the view that, as other facultative intracellular bacteria, Francisella spp are able to use several substrates in order to grow in various environments, such as macrophages. Glycerol via the gluconeogenesis and amino acids were identified as main sources of carbon during intracellular replication of Francisella spp in the host cells [START_REF] Ziveri | Importance of metabolic adaptations in francisella pathogenesis[END_REF][START_REF] Brissac | Gluconeogenesis, an essential metabolic pathway for pathogenic Francisella[END_REF].

However, glycerol requires UQ to be efficiently metabolized via the ubiquitous enzyme GlpD [START_REF] Austin | Nucleotide sequence of the glpD gene encoding aerobic sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12[END_REF] and amino acids degradation is closely linked to the TCA cycle, which produces reducing equivalents in Francisella spp [START_REF] Ziveri | Importance of metabolic adaptations in francisella pathogenesis[END_REF]. Besides its requirement for bioenergetics, UQ might also contribute to the antioxidant capacity of Francisella since it was shown to be a potent lipid soluble antioxidant in E. coli [START_REF] Soballe | Ubiquinone limits oxidative stress in Escherichia coli[END_REF]. During its intracellular life, Francisella is exposed to oxidative stress. Indeed, as a defense mechanism for the clearance of phagocytosed microorganisms, both macrophages and neutrophils produce reactive oxygen species, which in turn trigger bacterial killing by causing damage to macromolecules [START_REF] Kinkead | Multifaceted effects of Francisella tularensis on human neutrophil function and lifespan[END_REF][START_REF] Steiner | Protective role for macrophages in respiratory Francisella tularensis Infection[END_REF]. We propose that the reduced content in UQ in the Tn-ubiC Fn mutant could therefore affect F. novicida's oxidative defense. This hypothesis is in good agreement with recent data showing that reduced expression of UbiC Fn decreases the resistance of F. novicida to oxidative stress [START_REF] Felix | Structural and functional analysis of the Francisella lysine decarboxylase as a key actor in oxidative stress resistance[END_REF].

In a similar way, we showed previously that UbiE, UbiJ and UbiB proteins were needed in Salmonella enterica serovar Typhimurium intracellular proliferation in macrophages [START_REF] Aussel | ubiJ, a new gene required for aerobic growth and proliferation in macrophage, is involved in coenzyme Q biosynthesis in Escherichia coli and Salmonella enterica serovar Typhimurium[END_REF].

Collectively, all these data assign a role for Ubi-proteins in bacterial intracellular proliferation and, more generally, highlight the importance of UQ production for bacterial virulence.

Materials and methods

Bacterial strains and growth conditions

All bacterial strains used in this study are listed in Table S2. F. novicida U112 was obtained from the Centre National de Référence des Francisella, CHU Grenoble-Alpes, France. The transposon mutant Tn-ubiC Fn in the F. novicida U112 strain was obtained from the Manoil Laboratory, Department of Genome Science, University of Washington [START_REF] Gallagher | A comprehensive transposon mutant library of Francisella novicida, a bioweapon surrogate[END_REF]. Both strains were grown on Polyvitex enriched chocolate agar plates (PVX-CHA, bioMérieux, Marcy L'Etoile, France) incubated at 37°C for 48-72 h. Liquid cultures were carried out at 37°C with rotary shaking at 200 rpm in Chamberlain medium (45) supplemented with either glucose or succinate (0.4% (wt/vol) final concentration) as the only carbon source. For growth studies, overnight cultures were used to inoculate a 96-well plate to obtain a starting optical density at 600 nm (OD600) of around 0.1 and further incubated under shaking at 37°C. Changes in OD 600 were monitored every 10 min for 40 h using the Infinite 200 PRO microplate reader (Tecan, Lyon, France). When required, the medium was supplemented with 4HB in DMSO at 50-100 µM final concentration, pABA, pA2MBA and pA3MBA in DMSO at 1 mM final concentration or 3A4HB at 10 µM-1 mM, final concentration. For CFU counting, bacteria were suspended in PBS and cell suspensions were serially diluted in PBS.

For each sample, 100 µL of at least four different dilutions were plated on PVX-CHA plates and incubated for 72 h at 37°C, and CFU were counted using a Scan 100 Interscience.

The E. coli ΔubiA and ΔubiJ mutants were constructed as described previously [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[END_REF]. Briefly, the ubiA::cat and ubiJ::cat mutation was generated in a one-step inactivation of the ubiA and ubiJ genes. A DNA fragment containing the cat gene flanked with the 5' and 3' regions of the ubiA and ubiJ genes was PCR amplified using pKD3 as a template and oligonucleotides 5'-wannerubiA/3'-wannerubiA and 5'-wannerubiJ/3'-wannerubiJ, respectively (Table S3). The ΔubiB mutant was generated as follows: the cat gene was inserted in ubiB gene between the two sites NruI at 842 and 1004 pb. Then, ubiB::cat was PCR amplified using oligonucleotides 5'-xbaIubiB/3'-xbaIubiB (Table S3). Strain BW25113, carrying the pKD46 plasmid, was transformed by electroporation with the amplified fragments and Cat r colonies were selected. The replacement of chromosomal ubi by the cat gene was verified by PCR amplification in the Cat r clones. E. coli K-12 strains JW5713 and JW2226 from the Keio Collection (47) were used as donors in transduction experiments to construct the ΔubiC::kan and ΔubiG::kan mutants of E. coli MG1655 strains. The ΔubiA, ΔubiB, ΔubiC, ΔubiE, ΔubiG and ΔubiK strains were cured with pCP20 to yield ΔubiAc, ΔubiBc, ΔubiCc, ΔubiEc, ΔubiGc and ΔubiKc strains, respectively (Table S2). E. coli strains (K12, MG1655 or Top10) were grown on lysogeny broth (LB)-rich medium or in M9 minimal medium (supplemented with glucose or succinate, 0.4% (wt/vol) final concentration) at 37°C. Ampicillin (100 µg/ml), kanamycin (50 µg/ml), chloramphenicol (35 µg/ml) and IPTG (100 µM) were added when needed.

Cloning, plasmid construction, and complementation assays.

The plasmids and the primers used in this study are listed in Tables S2 andS3 (supplemental material), respectively. All the plasmids produced in this work were verified by DNA sequencing (GATC Biotech, Konstanz, Germany). The FTN_0385 (ubiA Fn ), FTN_0459

(ubiB Fn ), FTN_0386 (ubiC Fn ), FTN_0461 (ubiE Fn ), FTN_1146 (coq7 Fn ), FTN_0321 (ubiG Fn ), FTN_1237 (ubiH Fn ), FTN_1236 (ubiI Fn ), FTN_0460 (ubiJ Fn ) and FTN_1666 (ubiK Fn ) inserts were obtained by PCR amplification using the F. novicida U112 genome as template and the oligonucleotides described in Table S3. Inserts were EcoRI-BamHI or EcoRI-HindIII digested and inserted into EcoRI-BamHI-or EcoRI-HindIII-digested pTrc99a plasmids, respectively, yielding the pubiA Fn , pubiB Fn , pubiC Fn , pubiE Fn , pcoq7 Fn , pubiG Fn , pubiH Fn , pubiI Fn , pubiJ Fn and pubiK Fn plasmids (Table S3). The plasmids were transformed into E. coli MG1655 strains with mutation of the ubiA, ubiB, ubiC, ubiE, ubiF, ubiG, ubiH, ubiI, ubiJ and ubiK genes (single and double mutations, see Table S2), and complementation of the UQ 8 biosynthetic defect was assessed by both measuring the quinone content and plating serial dilutions onto solid M9 minimal medium supplemented with glucose or succinate (0.4% (wt/vol) final concentration) as the only carbon sources and overnight growth at 37°C. Expression of the Ubi-proteins was induced by addition of IPTG to a final concentration of 100 µM.

Lipid extractions and quinone analysis

Cultures (5 ml under ambient air) were cooled down on ice 30 min before centrifugation at 3200 X g at 4 °C for 10 min. Cell pellets were washed in 1 ml ice-cold PBS and transferred to preweighted 1.5 mL Eppendorf tubes. After centrifugation at 12,000 g at 4

°C for 1 min, the supernatant was discarded, the cell-wet weight was determined (~5-30 mg)

and pellets were stored at -20°C. Quinone extraction from cell pellets was performed as previously described [START_REF] Hajj Chehade | ubiI, a new gene in Escherichia coli coenzyme Q biosynthesis, is involved in aerobic C5-hydroxylation[END_REF]. Lipid extracts corresponding to MS detection were quantified with a standard curve of UQ 10 as previously described [START_REF] Hajj Chehade | ubiI, a new gene in Escherichia coli coenzyme Q biosynthesis, is involved in aerobic C5-hydroxylation[END_REF].

Infections of G. mellonella larvae

Larvae of the wax moth G. mellonella were purchased from Lombri'carraz SARL, Mery, France. Healthy and uniformly white larvae measuring around 3 cm were selected for infection. The bacteria were grown over night to an OD 600 of around three. Culture medium was removed by centrifugation and bacteria were diluted in PBS to 10 Each group of G. mellonella (n=20) was injected with around 10 6 CFU/larva and PBS injection was used as control. n.s. (no significant), P = 0.6670 by Log-rank (Mantel-Cox) test.

  4 s; OPP (M + NH4 + ), 656.0-657, 5-9 min, scan time of 0.4 s; compound X (M + H + ), m/z 682-683, 5-10 min, scan time of 0.4 s. MS spectra were recorded between m/z 600 and 900 with a scan time of 0.3 s. ECD and MS peak areas were corrected for sample loss during extraction on the basis of the recovery of the UQ 10 internal standard and then were normalized to cell wet weight. The peaks of UQ 8 obtained with electrochemical detection or

Figure 4 .

 4 Figure 4. Quantification of cellular UQ 8 contents of ubi E. coli mutant strains expressing the Ubi Fn -proteins. The ubi E. coli mutant strains transformed with pTrc99a (vec) or pTrc99a encompassing the ubi Fn -genes were grown over night at 37°C in LB medium (A) or in M9 minimal medium (B) with 0.4% (w/v) glucose as the sole carbon source.Expression of the Ubi Fn -proteins was described in the legend of the Figure3. Quantifications are expressed as the percentages of the control value, which corresponds to the UQ 8 content of the wild-type strain (n = 3). ****, P < 0.0001 and **, P < 0.005 by unpaired Student's t test.

Figure 5 .

 5 Figure 5. UQ 8 is essential for the growth of F. novicida in respiratory medium. F. novicida (F.n.) and transposon mutant of ubiC Fn (Tn-ubiC Fn ) were grown aerobically in

Figure 6 .

 6 Figure 6. Effect of 3A4HB on UQ 8 biosynthesis and growth of F. novicida. (A) HPLC-ECD analysis of lipid extracts from 1 mg of F. novicida cells grown aerobically in Chamberlain medium with 0.4% (w/v) glucose as the sole carbon source and in the presence of different concentration of 3A4HB solubilized in DMSO (a = DMSO; b = 0.01 mM; c = 0.1 mM; d = 0.25 mM; e = 0.5 mM; f = 1 mM; g = 1 mM 3A4HB + 1 mM 4HB). The chromatograms are representative of three independent experiments. The peaks corresponding to UQ 8 and the UQ 10 standard, are indicated. Compound X eluting at 6.5 min is marked. Growth curves for F. novicida cultured under aerobic conditions in Chamberlain medium with 0.4% (w/v) either succinate (B) or glucose (C) as the sole carbon source and in the presence of either DMSO (control), 1 mM 3A4HB or 1 mM 3A4HB + 100 µM 4HB. The growth for each condition (average of sixtiplicate growth curves) was followed as the change in the absorbance at 600 nm in a Tecan plate reader. (D) Mass spectrum of compound X eluting from extracts of F. novicida grown in the Chamberlain media with 1 mM of 3A4HB. H + and Na + adducts corresponding to this molecule were indicated. (E) Proposed structure of compound X in its oxidized form.

Figure 7 :

 7 Figure 7: UQ 8 contributes to the later steps of the infection of F. novicida in G. mellonella. (A) The larvae are turning grey/black when infected. (B) Survival curve of G. mellonella infected with either F. novicida (F.n.) or the transposon mutant of ubiC Fn (Tn-ubiC Fn ). ****, P < 0.0001 by Log-rank (Mantel-Cox) test. (C) Survival curve of G. mellonella infected with F. novicida (F.n.) pre-treated or not with 3A4HB (1 mM final concentration).

Proposed UQ 8 biosynthetic pathway in F. novicida deduced from the one characterized in E. coli.

  Corresponding protein IDs in F. novicida are indicated in parenthesis. There is no identified counterpart of UbiD and UbiX in F. novicida proteome.

	UbiF is only identified in E. coli and its functional homolog in F. novicida is a Coq7
	hydroxylase. Abbreviations used are 4HB, 4-hydroxybenzoic acid; OHB, 3-octaprenyl-4-
	hydroxybenzoic acid; OPP, octaprenylphenol; DMQ 8 , C6-demethoxy-ubiquinone 8; DDMQ 8 ,
	C1-demethyl-C6-demethoxy-ubiquinone 8; UQ 8 , ubiquinone 8.
	Figure 3.
	8 CFU/mL. Insulin
	cartridges were sterilized before filling with bacterial solutions. Larvae were injected with 10
	μL of bacterial suspensions (10 6 CFU per larva as recommended (24)) using an insulin pen or
	with 10 µL of PBS only. The precise number of bacteria transferred in injections was
	determined by spotting serial dilutions onto chocolate agar plates, and counting CFU after
	growth at 37°C for 48h. Infected larvae were placed in Petri dishes and maintained at 37°C.

Survival of larvae was monitored for 6 days by counting the number of dead larvae each day. Figure 2:

Complementation analysis of E. coli UQ 8 biosynthesis mutants with the putative Ubi-proteins from

F. novicida. The ubi E. coli mutant strains transformed with pTrc99a (vec) or pTrc99a encompassing the ubi Fn genes were grown over night at 37°C in LB medium (A) or in M9 minimal medium (B) with 0.4% (w/v) glucose as the sole carbon source. Expression of the Ubi Fn -proteins was induced by addition of IPTG to a final concentration of 100 µM. The E. coli wild-type strain MG1655 (WT) transformed with the pTrc99a empty vector was used as a control. HPLC-ECD analysis of lipid extracts from 1 mg of cells. The chromatograms are representative of three independent experiments. The peaks corresponding to OPP, DDMQ 8, DMQ 8 , UQ 8 , MK 8 , DMK 8 and the UQ 10 standard are indicated. (C) Serial dilutions were spotted onto plates containing M9 minimal medium with 0.4% (w/v) either glucose or succinate as the sole carbon source and IPTG (100 µM final concentration). The plates were incubated overnight at 37°C.

Acknowledgments

This work was supported by the Agence Nationale de la Recherche (ANR), project O2-taboo ANR-19-CE44-0014, project Emergence (TIMC-UGA), the University Grenoble Alpes (UGA) and the French Centre National de la Recherche Scientifique (CNRS). We thank Dr Patricia Renesto for constructive discussions, technical assistance and Professor Laurent Aussel for critically reading the paper.

A cohort of 20 larvae was used per condition, and the experiment was performed twice. As a control, an untreated cohort of larvae was also followed.

Contributions


Mario Santoro

Vasyl Veremeienko

Michelangelo Polisi

Riccardo Fantini

Frederico Alabarse

Rossella Arletti

Simona Quatieri

Volodymyr Svitlyk

Arie Van Der Lee

Jérôme Rouquette

Mario Santoro

Bruno Alonso

Francesco Di Renzo

Benoît Coasne

Julien Haines

Insertion and Confinement of H 2 O in Hydrophobic Siliceous Zeolites at High Pressure

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The penetration of H2O in porous materials is of great importance for chemistry, materials and Earth science. Water penetration strongly modifies the properties of materials and minerals and has implications for energy storage, aging of materials, and geological processes. Particular interest has been directed towards microporous zeolite-type materials with typical diameters between 3-8Å, for which it has been shown that high pressure can lead to a superhydrated state [START_REF] Hazen | Zeolite Molecular-Sieve 4A -Anomalous Compressibility and Volume Discontinuities at High-Pressure[END_REF][START_REF] Lee | First Structural Investigation of a Super-Hydrated Zeolite[END_REF][START_REF] Lee | Non-Framework Cation Migration and Irreversible Pressure-Induced Hydration in a Zeolite[END_REF][START_REF] Colligan | Synchrotron X-Ray Powder Diffraction and Computational Investigation of Purely Siliceous Zeolite Y under Pressure[END_REF][START_REF] Colligan | High-Pressure Neutron Diffraction Study of Superhydrated Natrolite[END_REF][START_REF] Seoung | Super-Hydrated Zeolites: Pressure-Induced Hydration in Natrolites[END_REF][START_REF] Arletti | Elastic Behavior of MFI-Type Zeolites: 1-Compressibility of Na-ZSM-5 in Penetrating and Non-Penetrating Media[END_REF][START_REF] Vezzalini | High-Pressure-Induced Structural Changes, Amorphization and Molecule Penetration in MFI Microporous Materials: A Review[END_REF][START_REF] Arletti | Pressure-Induced Water Intrusion in FER-Type Zeolites and the Influence of Extraframework Species on Structural Deformations[END_REF][START_REF] Alabarse | Mechanism of H2O Insertion and Chemical Bond Formation in AlPO4-54•xH2O at High Pressure[END_REF] . This effect strongly modifies the phase stability and the elastic properties of these materials. Hydrophobic, all silica zeolites coupled to nonwetting fluids are particularly suited for applications for the storage, absorption, and dissipation of mechanical energy for use as molecular springs, bumpers and shock absorbers [START_REF] Eroshenko | Energetics: A New Field of Applications for Hydrophobic Zeolites[END_REF][START_REF] Desbiens | Water Condensation in Hydrophobic Silicalite-1 Zeolite: A Molecular Simulation Study[END_REF][START_REF] Cailliez | Thermodynamics of Water Intrusion in Nanoporous Hydrophobic Solids[END_REF][START_REF] Karbowiak | Thermal Effects of Water Intrusion in Hydrophobic Nanoporous Materials[END_REF][START_REF] Karbowiak | New Insights in the Formation of Silanol Defects in Silicalite-1 by Water Intrusion under High Pressure[END_REF][START_REF] Bushuev | Atomistic Simulation of Water Intrusion-Extrusion in ITQ-4 (IFR) and ZSM-22 (TON): The Role of Silanol Defects[END_REF][START_REF] Tzanis | High Pressure Water Intrusion Investigation of Pure Silica 1D Channel AFI, MTW and TON-Type Zeolites[END_REF][START_REF] Tzanis | Energetic Performances of Channel and Cage-Type Zeosils[END_REF] .

One of the most studied siliceous zeolites for such applications, both from an experimental and computational point of view, is silicalite-1 with a MFI structure [START_REF] Kokotailo | Structure of Synthetic Zeolite ZSM-5[END_REF] . This material is characterized by a three dimensional pore system consisting of 5.1×5.5Å diameter straight channels along the [010] direction and intersecting 5.3×5.6Å sinusoidal channels in the ac plane. This phase is known to undergo a monoclinic to orthorhombic phase transition as a function of temperature, pressure and guest insertion [START_REF] Hay | Examination of the Monoclinic Orthorhombic Transition in Silicalite using XRD and Silicon NMR[END_REF][START_REF] Haines | Deactivation of Pressure-Induced Amorphization in Silicalite SiO2 by Insertion of Guest Species[END_REF][START_REF] Ardit | Monoclinic-Orthorhombic Phase Transition in ZSM-5 Zeolite: Spontaneous Strain Variation and Thermodynamic Properties[END_REF] . Porosimetric studies revealed that, once H2O is intruded in silicalite-1 a reversible-penetration of molecules occurs close to 100 MPa [START_REF] Eroshenko | Energetics: A New Field of Applications for Hydrophobic Zeolites[END_REF][START_REF] Tzanis | Energetic Performances of Channel and Cage-Type Zeosils[END_REF] with some hysteresis.

Superior energy storage properties have been observed for an orthorhombic 1-D siliceous zeolite TON, which has unidirectional 4.6×5.7Å elliptical channels along the [001] direction [START_REF] Barri | Structure of Theta-1, the First Unidimensional Medium-Pore High-Silica Zeolite[END_REF][START_REF] Marler | Silica-ZSM-22: Synthesis and Single Crystal Structure Refinement[END_REF][START_REF] Papiz | Structure of the Zeolite Theta-1. Redetermination using Single-Crystal Synchrotron-Radiation Data[END_REF] . Penetration by water molecules occurs at 186 MPa [START_REF] Tzanis | High Pressure Water Intrusion Investigation of Pure Silica 1D Channel AFI, MTW and TON-Type Zeolites[END_REF] . This material undergoes a pressure-induced phase transition between Cmc21 and Pbn21 orthorhombic forms [START_REF] Thibaud | High-Pressure Phase Transition, Pore Collapse, and Amorphization in the Siliceous 1D Zeolite, TON[END_REF] at close to 700 MPa. This transition occurs in non-penetrating hydrostatic pressure transmitting media, but it is also observed with a lower degree of distortion in the presence of guest atoms such as Ne or Ar [START_REF] Thibaud | Saturation, of the Siliceous Zeolite TON with Neon at High Pressure[END_REF] . This phase transition is a consequence of the collapse of the pores along opposite diagonals [START_REF] Thibaud | High-Pressure Phase Transition, Pore Collapse, and Amorphization in the Siliceous 1D Zeolite, TON[END_REF] .

The goal of the present study is to investigate the mechanism of pore filling of TON and MFI by H2O at high pressure from a structural point of view using a combination of in situ X-ray powder diffraction and Monte Carlo modelling and also from a vibrational point of view by infrared spectroscopy.

Experimental and theoretical methods

Micron-sized crystals of TON were prepared by sol-gel techniques using triethylenetetramine as the structure directing agent followed by crystallization at 170°C under hydrothermal conditions and then calcination at 600°C [START_REF] Thibaud | High-Pressure Phase Transition, Pore Collapse, and Amorphization in the Siliceous 1D Zeolite, TON[END_REF][START_REF] Di Renzo | Influence of Diffusional Barriers on the Thermal-Analysis of the Zeolite TON[END_REF][START_REF] Di Renzo | Crystallization Kinetics of Zeolite TON[END_REF] . The Si:Al ratio of the crystals was found to be 50. Chemical analysis showed that the material contained 0.41%N and 0.37%C corresponding to a residue from the calcined structure directing agent and 0.3% Na from the starting gel. The nitrogen adsorption isotherm indicated that 100% of the porosity was available. The initial unit cell parameters obtained on a Panalytical X'Pert diffractometer using Cu K radiation were: a=13.8652(1)Å, b=17.4233(3)Å, c=5.03928 (7)Å in space group Cmc21. MFI crystals were also prepared by sol-gel techniques in the presence of ammonium fluoride using fumed silica as the silica source and tetrapropylammonium bromide as the structure directing agent. Crystallization was obtained under hydrothermal conditions at 200°C for 15 days and was followed by calcination at 550°C. The resulting crystals were finely ground down to micron size under wet conditions using a

McCrone TM X-ray diffraction mill. The initial unit cell parameters obtained on a Bruker D8 Discover diffractometer using Cu K radiation were: a=20.1344(1)Å, b=19.9018(1)Å, c=13.38641(8)Å, =90.6207 (5) in the non-standard setting of the space group P21/n (P21/n11) in order to retain the same axes as the orthorhombic aristotype Pnma.

X-ray powder diffraction (=0.3738 Å) experiments were performed in a LENS membrane-type diamond anvil cell (DAC) on the ID27 High Pressure beamline at the ESRF synchrotron (Grenoble, France) using a MAR165 CCD detector. We first homogeneously filled the 300 µm diameter hole of a 70 µm thick indent in a stainless steel gasket with hydrophobic micron-sized TON or MFI powder, and the powder was just gently compacted in order not to prevent the subsequent loading of water. A ruby microsphere was added as pressure calibrant. A drop of ultrapure water was then added as pressure transmitting medium, which filled both the pores of the zeolite (confined H2O) and the residual empty space between the grains of the zeolite (bulk H2O). Pressure was measured based on the shift of the ruby R1 fluorescence line [START_REF] Mao | Calibration of the Ruby Pressure Gauge to 800 Kbar under Quasi-Hydrostatic Conditions[END_REF] . The diffraction patterns were analyzed and integrated using the Dioptas 0.4.0 program [START_REF] Prescher | Dioptas: A Program for Reduction of Two-Dimensional X-Ray Diffraction Data and Data Exploration[END_REF] . Rietveld refinements were performed using the program Fullprof [START_REF] Rodriguez-Carvajal | Magnetic Structure Determination from Powder Diffraction Using the Program Fullprof[END_REF] . Fractional atomic coordinates for the framework from previous work were used as the starting model for TON [START_REF] Thibaud | High-Pressure Phase Transition, Pore Collapse, and Amorphization in the Siliceous 1D Zeolite, TON[END_REF] . In the case of MFI, the starting fractional atomic coordinates were obtained from an x-ray diffraction study of a single-crystal synthesized under similar conditions. Soft constraints were applied to the Si-O distances, O-O distances and/or the O-Si-O angles. As it was not possible to locate the hydrogen atoms of the H2O molecules and in order to correctly account for the number of electrons associated with these H2O molecules, the scattering curve of isoelectronic neon was used in the refinements. An overall isotropic atomic displacement parameter (ADP) was used for all framework Si and O atoms and extraframework H2O molecules as no improvement was obtained using individual ADPs. Crystal structures were plotted using the program VESTA [START_REF] Momma | Vesta 3 for Three-Dimensional Visualization of Crystal, Volumetric and Morphology Data[END_REF] . IR absorption spectra on the H2O/zeolite mixtures were measured by a Bruker IFS-120 HR Fourier transform infrared spectrometer equipped with a globar lamp, a KBr beam splitter, an MCT detector, and an optical beam condenser based on ellipsoidal mirrors providing a natural beam spot size of several hundreds of microns (see Gorelli et al. [START_REF] Gorelli | The Epsilon Phase of Solid Oxygen: Evidence of an O4 Molecule Lattice[END_REF] and references therein). The typical spectral resolution was 1 cm -1 .

Monte Carlo simulations in the Grand Canonical ensemble (GCMC) were performed to determine the number and structure of water confined at room temperature in TON and in MFI zeolites. These atomscale simulations at constant chemical potential μ, volume V, and temperature T allow determining the number of adsorbed water molecules, nH2O(), per zeolite unit cell as a function of chemical potential   .

For a given  and T, the corresponding pressure P was obtained according to the data by Desbiens et al. [START_REF] Desbiens | Water Condensation in Hydrophobic Silicalite-1 Zeolite: A Molecular Simulation Study[END_REF] The pressures (or chemical potential) and unit cell parameters obtained from the experiments were used to perform these molecular simulations. A TON structure made of 2 × 2 × 5 rigid unit cells and a MFI structure made of 2 × 2 × 3 rigid unit cells were considered and periodic boundary conditions were used along the x, y, z directions to avoid finite size effects. Water was described in these atom-scale simulations using the TIP4P potential, which can be assumed to be appropriate in the pressure range up to 1 GPa. Following previous work [START_REF] Desbiens | Water Condensation in Hydrophobic Silicalite-1 Zeolite: A Molecular Simulation Study[END_REF] , interactions between water and the zeolite consisted of a Lennard-Jones potential and a Coulomb contribution (partial charges on the O and Si atoms of the pure silica zeolites are taken equal to -1e and +2e, respectively). For the Lennard-Jones potential, the cross parameters  and  were determined by combining the like-atom parameters using the Lorentz -Berthelot rules (the likeatom parameters for the O atom in zeolite are  = 0.3 nm and /kB = 93.53 K).

Results and discussion

The two zeolite powders TON and MFI were compressed in H2O up to 0.9 GPa, just below the solidification pressure of ice VI [START_REF] Mishima | Phase-Relations of Ice under Pressure[END_REF] . X-ray diffraction measurements were then performed on pressure release (Figure 1). In both cases the diffraction patterns clearly showed that both materials underwent phase transitions to their high pressure polymorphs with space groups Pbn21 for TON and Pnma for MFI. At high pressure, the relative intensities of the diffraction lines were strongly modified with respect to those observed at ambient pressure, clearly indicating the insertion of H2O. The high pressure forms were retained during the measurements on decompression, with the exception of that at ambient pressure, for which both reverted to their low-pressure forms (Cmc21 for TON and P21/n11 for MFI) with major changes in relative intensities indicative of significant extrusion of H2O.

The structure of TON was thus refined (Figures 2 and3, and CIF files in SI) using the above structural models for the high pressure and ambient pressure measurements respectively. Starting from the highpressure point at 0.90 GPa, the Pbn21 structure of empty TON was used in the initial stages of the Rietveld refinement. At the highest pressure investigated, difference Fourier maps allowed two sets of 4a Wyckoff sites (x,y,z) with an occupation of 0.89(4) to be located, giving close to 8 H2O molecules per unit cell and thus 4 in each pore. The difference between the refined occupancy and full occupation is probably due to the C, N and Na remaining in the pores after calcination and detected by chemical analysis and any disorder present in the H2O distribution in the channels. The corresponding electron density was accounted for in the refinement as in previous studies [START_REF] Thibaud | High-Pressure Phase Transition, Pore Collapse, and Amorphization in the Siliceous 1D Zeolite, TON[END_REF][START_REF] Thibaud | Saturation, of the Siliceous Zeolite TON with Neon at High Pressure[END_REF] by in this case on average 0.5 carbon atoms per unit cell.

The configuration of sites occupied by the H2O molecules is consistent with two hydrogen bonded chains per pore (Figure 4 and CIF files in SI). The structure is in good agreement with the results of the MC simulations, which give a maximum of 8.9 molecules per unit cell at the maximum pressure. The amount of H2O molecules hosted in the channels decreases upon decompression at a similar rate in the experimental data and in the MC simulations (Figure 5). At ambient pressure, refinements indicated the presence of residual H2O. The refinement allows 2.5 H2O molecules per unit cell to be located on a set of 4a Wyckoff sites (0,y,z). The amount of residual water can be related to the presence of sodium cations (0.3%) and protons needed to compensate the charge of the framework containing 2% Al and to the eventual formation of silanol groups at high pressure.

Due to the complexity of the MFI structure with a much larger unit cell and a 3D pore system, the data were not of sufficient quality to determine the positions of the H2O molecules in the pores by difference Fourier maps. H2O molecules with soft constraints set to an approximate hydrogen-bond O-O distance of 2.8 Å were placed in a periodic way in the pores based on the results of MC models (see below) to build an initial structural model. Very good fits in Rietveld refinements were obtained with this structural model (Figure 2 and CIF files in SI). This periodic structural model used for the Rietveld refinements can readily be used to account for the electron density in the pores in terms of occupation by water molecules with a maximum of 16.6 molecules/24 SiO2 at 0.86 GPa. On pressure release, some residual molecules H2O (1.4 molecules/24 SiO2) are found in the MFI channels. This amount of residual H2O irreversibly absorbed in this hydrophobic MFI is significantly lower than the H2O uptake in hydrophilic MFI (3 molecules/24 SiO2), but higher than the uptake of hydrophobic MFI (0.4 molecules/24 SiO2) in adsorption isotherm measurements [START_REF] Zhang | Adsorption of Water and Ethanol in MFI-Type Zeolites[END_REF] . This residual H2O content could be linked to the formation of silanol groups at very high pressures as found in previous work [START_REF] Karbowiak | Thermal Effects of Water Intrusion in Hydrophobic Nanoporous Materials[END_REF][START_REF] Karbowiak | New Insights in the Formation of Silanol Defects in Silicalite-1 by Water Intrusion under High Pressure[END_REF] in spite of the hydrophobic nature of calcined MFI obtained by the fluoride route, which is preserved from the creation of silanol defects during the calcination process.

The present result indicates that the concentration of silanol defects is dramatically increased by compression up to 0.9 GPa.

The experimental H2O content measured in this study are in excellent agreement with the points at low pressure obtained in previous volumetric studies (Figure 5); however, a much larger number of H2O molecules enter the pores up to the maximum pressure reached in the present study. If instead of quantifying the number of guests with respect to the number of framework atoms, the data are plotted as a function of the degree of pore filling at 0.9 GPa (Figure 5b), assuming that a maximum attainable H2O density in the pores of 1.23 g/cm 3 corresponding to that of liquid H2O at the same pressure [START_REF] Koster | Das Spezifische Volumen Des Wassers Bei Hohen Drucken Bis 600°C Und 10 Kbar*)[END_REF] , the points of both materials lie on the same curve. The water density value attained at 0.9 GPa in the case of TON is 1.09(4) g/cm [START_REF] Lee | Non-Framework Cation Migration and Irreversible Pressure-Induced Hydration in a Zeolite[END_REF] , whereas the maximum value for MFI is 1.17(6) g/cm 3 at 0.86 GPa. The lower value for TON can be related to the presence of the remaining C, N and Na atoms, which take the place of H2O molecules. The results of MC simulations (see below) confirm that the density of inserted H2O in both materials is essentially equivalent to that of liquid water at the same pressure. These results indicate that at high enough pressure, full pore filling occurs in hydrophobic zeolites. This process is reversible to a very large extent and the majority of the water is extruded on pressure release. The residual H2O content is higher for TON (32%) as compared to MFI (8%), which can be linked to Na cations and protons in the pores, which compensate the charges due to the Al cations in the framework.

In both materials the unit cell parameters (except b in TON, see below) are larger than those of the corresponding empty zeolites [START_REF] Thibaud | Saturation, of the Siliceous Zeolite TON with Neon at High Pressure[END_REF][START_REF] Haines | Topologically Ordered Amorphous Silica Obtained from the Collapsed Siliceous Zeolite, Silicalite-1-F: A Step toward "Perfect" Glasses[END_REF] compressed in non-penetrating media from 0.9 down to 0.6 GPa due to swelling of the pores induced by the presence of H2O molecules (Figures 6 and7). In the case of TON, the difference between the a and b parameters is greater due to greater distortion of the pores giving a very marked decrease in b at the phase transition. The recovered TON has essentially identical cell parameters and volume of the empty TON. The behavior of MFI is different. Upon pressure release, the unit cell remains slightly contracted, which is consistent with the residual H2O content and to models showing a slight collapse in zeolite structures in some cases when a layer of molecules is adsorbed on the inner surface of the pores [START_REF] Coasne | Enhanced Mechanical Strength of Zeolites by Adsorption of Guest Molecules[END_REF] .

The amount of adsorbed water in the MFI and TON structures was determined using MC simulations in the Grand Canonical ensemble. When normalized per number of SiO2 units in the zeolites, we find 8.00, 8.59 and 8.89 H2O per 24 SiO2 units for TON at 0.29, 0.6 and 0.9 GPa and 13.43, 15.21 and 15.95 H2O per 24 SiO2 units for MFI at 0.3, 0.67 and 0.8 GPa. As can be seen in Fig. 5, these values are in very good agreement with the experimental data. In the case of MFI, these numbers are slightly larger than those predicted by Desbiens et al. [START_REF] Desbiens | Water Condensation in Hydrophobic Silicalite-1 Zeolite: A Molecular Simulation Study[END_REF] . Typically, at 300 MPa, we find 53.7 H2O per unit cell in MFI instead of 49 H2O per unit cell in Ref. 12. Such differences can be due to the model used to describe the water molecule and its interaction with the host silica zeolitetypically, the partial charges carried by the atomic sites in water and in the zeolite but also the Lennard-Jones parameters considered. For instance, in Ref. 12, upon considering different water models, the authors observed important differences in terms of the amount of water adsorbed with values at P = 350 MPa that can vary from 47 to 52 H2O per unit cell (see Fig. 7 in Ref. 12). As for water in TON, our results are in qualitative agreement with those from Bushuev and Sastre [START_REF] Bushuev | Atomistic Simulation of Water Intrusion-Extrusion in ITQ-4 (IFR) and ZSM-22 (TON): The Role of Silanol Defects[END_REF] who found a maximum of 27 H2O molecules for a TON channel, equivalent to 4 times the unit cell along the c-axis (therefore leading to about 7 water molecules per unit cell, in fair agreement with the value of 8 H2O per unit cell found in the present work). It can be noted that in the previous study, the high-pressure phase transition in TON had not yet been discovered and was thus not considered in the calculation. Empty symbols correspond to bulk H2O intrusion data from the literature [START_REF] Tzanis | High Pressure Water Intrusion Investigation of Pure Silica 1D Channel AFI, MTW and TON-Type Zeolites[END_REF][START_REF] Trzpit | Influence of Defects on the Water Intrusion in Silicalite-1 Zeolite: Confrontation of Experimental and Molecular Simulation Results[END_REF] . Continuous lines represent the behavior of TON (orthorhombic phases I and II) and MFI (monoclinic phase) in non-penetrating pressure media [START_REF] Thibaud | High-Pressure Phase Transition, Pore Collapse, and Amorphization in the Siliceous 1D Zeolite, TON[END_REF][START_REF] Haines | Topologically Ordered Amorphous Silica Obtained from the Collapsed Siliceous Zeolite, Silicalite-1-F: A Step toward "Perfect" Glasses[END_REF] . Error bars are smaller than the symbol size. and MFI (monoclinic phase) in non-penetrating pressure media [START_REF] Thibaud | High-Pressure Phase Transition, Pore Collapse, and Amorphization in the Siliceous 1D Zeolite, TON[END_REF][START_REF] Haines | Topologically Ordered Amorphous Silica Obtained from the Collapsed Siliceous Zeolite, Silicalite-1-F: A Step toward "Perfect" Glasses[END_REF] . Error bars are smaller than the symbol size.

IR absorption measurements were performed in the H2O overtone and combination frequency region, since fundamental IR peaks of water were entirely saturated. In Figure 8, we report the IR peaks for the HOH bending + OH stretching (left) and for the double OH stretching (right) vibrational transitions, for pure H2O, H2O/TON mixtures and H2O/MFI mixtures, respectively, at a selected pressure value of 0.8

GPa. In spite of the width of the bands of several hundreds of wavenumbers, which prevents peaks of bulk water and of water confined in the zeolites to be distinguished, it is clear that the peaks corresponding V / V 0 P (GPa)

to overlapping contributions of bulk and confined water are shifted to slightly higher frequencies with respect to those of pure water. In order to provide a quantitative description of microscopic interactions probed by the confined H2O molecules, we calculated the spectral centroid of the two H2O IR peaks, that is, the average wavenumber avg calculated as avg = ∫A()d/∫A()d, where  and A() are the wavenumber and the absorbance, respectively. In Figure 9, we report the pressure shift of the spectral centroid for the two peaks. In all three systems, pure H2O and the two H2O/zeolite mixtures, the average frequency linearly decreases upon increasing pressure, which is due to the increasing H-bond at the expense of the covalent O-H bond. More importantly here, the result is that the average frequency for the two H2O/zeolites is systematically higher than that for pure H2O, by a few tens of wavenumbers at all pressures. This difference shows that indeed H-bonds in confined water are weaker than in bulk H2O. This could be due to a longer intermolecular OO distance, a O-HO angle for the confined molecules differing from 180° and/or disorder in confined water contributing to H-bond weakening by disrupting the ideal tetrahedral arrangement of H-bonds. This disorder of the confined molecules, as a matter of fact, is found to be slightly more pronounced for MFI from MC simulations than for TON. Our MC modelling results give an insight into these different effects. Figure 10 shows the distribution of hydrogen bonding angles < OH-O > and the radial pair distribution function g(r) between Ow atoms in water. These data, which were obtained by means of Grand Canonical

Monte Carlo simulations at room temperature and a pressure of 900 MPa, are shown for bulk water and for water confined in MFI and in TON. Both sets of data show that the microscopic structure of confined water differs from bulk water under the same thermodynamic conditions. However, owing to the more severe confinement in its framework, the TON zeolite leads to a more ordered water microscopic structure with more pronounced features/peaks in the angle and radial distributionscompared to the MFI structure. In particular, as shown in Figure 10, while both the orientational (angle distribution) and translational (radial distribution) orderings are affected by nanometric confinement in zeolites, the microscopic structure of water in MFI is less distorted than in TON in the sense that its departs less from that of bulk water under the same thermodynamic conditions (as can be inferred by comparing the amplitude/full width at half maximum for the different systems). These data are consistent with the data by Zhou et al. who

showed by means of molecular simulation that, in highly confined environments, even weak water-surface interactions lead to strong perturbations of the intimate water structure [START_REF] Zhou | Deconstructing the Confinement Effect Upon the Organization and Dynamics of Water in Hydrophobic Nanoporous Materials: Lessons Learned from Zeolites[END_REF] .

Confined water, which is more ordered in TON, exhibits sharper distributions than in MFI with an average angle closer to 180° corresponding to stronger H-bonds. This is in agreement with the lower IR O-H stretching frequencies for H2O/TON. An intermediate value for the average angle is obtained for bulk water. The explanation for the stronger H-bond in pure water thus has to be different, and is probably linked to the possibility to have a true tetrahedral environment without the spatial and geometrical constraints of confinement. In all three systems the average O-O distances are very similar indicating that this does not play a major role. This is consistent with the fact that the O-O distance is mostly governed by a balance between attractive Coulomb/dispersive interactions and short-range steric repulsion, which is only very weakly affected by confinement.

While the simulation data reported in the present paper only focus on thermodynamic/structural aspects, these features are of key importance to account for the rich dynamical/transport properties of water intrusion in hydrophobic media. In particular, the complex structure of water in such ultraconfined environments can be responsible for the mechanisms behaviors observed upon intrusion in all-silica zeolites such as bottleneck effects at the windows of the zeolite structure, broad scattering of residence times, etc. 45

Conclusions

The present work indicated that H2O insertion in TON and MFI zeolites is highly reversible up to 0.9

GPa and that pore filling is essentially complete at this pressure, at which the density of H2O is essentially identical to liquid water under the same conditions. The structure of inserted H2O appears more ordered in the 1D pores of TON forming two hydrogen bonded chains, whereas significantly more disorder is observed in the larger pores of the 3D network in MFI. Based on infrared spectroscopy, the hydrogen bonds are weakest in MFI and strongest in liquid water. This can be related to geometric constraints imposed by the pores and disorder, which disrupt the ideal tetrahedral network of H-bonds.

Figure 1 .

 1 Figure 1. X-ray diffraction patterns of TON and MFI in H2O on decompression (=0.3738 Å). Arrows

1 Figure 2 .

 12 Figure 2. Experimental (black), calculated (red) and difference (blue) profiles (=0.3738 Å) for the

Figure 3 .

 3 Figure 3. Refined crystal structures (left) and density maps (right) from MC modelling of H2O-filled

Figure 4 .

 4 Figure 4. Refined crystal structure of H2O-filled TON at 0.9 GPa (Pbn21) showing the chains of H2O

Figure 5 .

 5 Figure 5. Water content (left) and pore filling (right) of TON (gold symbols) and MFI (black symbols)

Figure 6 .

 6 Figure 6. Relative cell parameters of TON and MFI in H2O as a function of pressure (solid symbols).

Figure 7 .

 7 Figure 7. Relative volume (V/V0) of TON in of TON and MFI in H2O as a function of pressure (solid

Figure 8 .

 8 Figure 8. IR absorption spectra in the overtone and combination band spectral regions at a selected

Figure 9 .

 9 Figure 9. Average frequency, that is the center of mass for the frequencies of the IR overtone and

Figure 10 .

 10 Figure 10. (left) Distribution function of hydrogen bonding angles < OH-O > as obtained from Grand

ASSOCIATED CONTENT Supporting Information. Crystallographic information files (CIF) files containing the structural data for the TON-H2O system at 0.29, 0.6 and 0.9 GPa and the MFI-H2O system at 0.31, 0.67 and 0.86 GPa have been supplied as supporting information. This material is available free of charge via the Internet at http://pubs.acs.org..

ACKNOWLEDGMENT

We acknowledge funding from the Italian Ministry of Education, Universities and Research, MIUR PRIN project ZAPPING, number 2015HK93L7. We also thank the Deep Carbon Observatory (DCO) initiative under the project Physics and Chemistry of Carbon at Extreme Conditions and the Ente Cassa di Risparmio di Firenze under the project Firenze Hydrolab 2.

Converting the calculated number of water molecules per unit cell into a density of confined water is a complex task as the porous volume is an ill-defined quantity since its determination is probe-and/or technique-dependent. In the present work, we used the following approach. The porous volume for each zeolite was taken from the work by Krishna and Van Baten who used an in-silico screening of different zeolite materials including TON and MFI [START_REF] Krishna | In Silico Screening of Zeolite Membranes for CO2 Capture[END_REF] . These authors evaluated the accessible pore volume by a Connollysurface approach using He as a probe. The values given are in excellent agreement with experimental O2 and N2 adsorption data on silicalite [START_REF] Flanigen | a New Hydrophobic Crystalline Silica Molecular-Sieve[END_REF] and TON [START_REF] Di Renzo | Influence of Diffusional Barriers on the Thermal-Analysis of the Zeolite TON[END_REF] . While these values were obtained for the unstrained structures at ambient pressure conditions, we estimated the porous volume at the different pressures considered here by assuming that the porous volume Vp scales like the volume V of the sample, i.e. Vp/Vp,0 ~ V/V0. Using V0 ~ 231.4 Å 3 for TON and V0 ~ 1584.9 Å 3 for MFI. Considering the different strains estimated for the different pressure conditions, we obtained 1.06, 1.16 and 1.21 g/cm [START_REF] Lee | Non-Framework Cation Migration and Irreversible Pressure-Induced Hydration in a Zeolite[END_REF] for TON at 290, 600 and 900 MPa and 1.03, 1.18 and 1.24 g/cm [START_REF] Lee | Non-Framework Cation Migration and Irreversible Pressure-Induced Hydration in a Zeolite[END_REF] for MFI at 300, 670 and 800 MPa.

AUTHOR INFORMATION

Corresponding Author

*Julien Haines

Julien.Haines@umontpellier.fr

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interests.