Nadege Gouignard

Eric Theveneau
email: eric.theveneau@univ-tlse3.fr

Jean-Pierre Saint-Jeannet

Dynamic expression of MMP28 during cranial morphogenesis

Keywords: MMP, embryogenesis, cranial placodes, neural crest, Xenopus

Matrix metalloproteinases (MMP) are a large family of proteases comprising 24 members in vertebrates. They are well known for their extracellular matrix remodelling activity. MMP28 is the latest member of the family to be discovered. It is a secreted MMP involved in wound healing, immune system maturation, cell survival and migration. MMP28 is also expressed during embryogenesis in human and mouse. Here we describe the detailed expression profile of MMP28 in Xenopus laevis embryos. We show that MMP28 is expressed maternally and accumulates at neurula and tailbud stages specifically in the cranial placode territories adjacent to migrating neural crest cells. As a secreted MMP, MMP28 may be required in neural crestplacode interactions.

Background

Matrix metalloproteinases form a large family of proteases regrouping 23 members in human and 24 in Xenopus laevis [START_REF] Woessner | MMPs and TIMPs-An Historical Perspective[END_REF]. MMPs are translated as zymogens, or pro-enzymes, where the N-terminal part of the protein, called pro-domain, blocks the catalytic domain. This prodomain can either change conformation (a process known as allosteric activation) or be removed by proteolytic cleavage to ensure the activity of the protein [START_REF] Ra | Control of matrix metalloproteinase catalytic activity[END_REF]. MMPs are Zn 2+ -dependent proteases mostly known for their extracellular matrix remodelling activity but they are also capable of cleaving a wide range of substrates including growth factors and their cognate receptors, adhesion molecules and chemokines [START_REF] Rodríguez | Matrix metalloproteinases: What do they not do? New substrates and biological roles identified by murine models and proteomics[END_REF]. For decades, MMPs have been studied in the context of cardiovascular diseases, rheumatoid arthritis and neurological disorders. MMPs are also of prime interest in cancer since MMPs are present in virtually all human cancers and most members of the MMP family have been found to be dysregulated in human cancers [START_REF] Cathcart | Targeting matrix metalloproteinases in cancer: Bringing new life to old ideas[END_REF].

MMPs are essential for key cellular processes from extracellular matrix regulation to cell migration and invasion to cell proliferation and apoptosis. Therefore, they are important in adults for wound healing, angiogenesis, tissues homeostasis and immunity. In addition, MMPs are expressed during embryogenesis and are involved throughout development [START_REF] Cui | Biochemical and Biological Attributes of Matrix Metalloproteinases[END_REF]. Indeed, about half of all MMP family members are expressed during Xenopus laevis embryogenesis (see Table 1).

MMP28 was first identified in human and is the latest member of the family to be discovered [START_REF] Lohi | Epilysin, a novel human matrix metalloproteinase (MMP-28) expressed in testis and keratinocytes and in response to injury[END_REF][START_REF] Marchenko | MMP-28, a new human matrix metalloproteinase with an unusual cysteine-switch sequence is widely expressed in tumors[END_REF]. MMP28 is a soluble MMP with endopeptidase activity and the ability to degrade casein, N-CAM and Nogo-A proteins [START_REF] Werner | Neural MMP-28 expression precedes myelination during development and peripheral nerve repair. Developmental dynamics : an official publication of the American Association of Anatomists[END_REF]. MMP28 is involved in nerve repair and wound healing, immune system maturation, cell migration and invasion [START_REF] Saarialho-Kere | Epilysin (MMP-28) expression is associated with cell proliferation during epithelial repair[END_REF][START_REF] Reno | Effect of in vitro mechanical compression on Epilysin (matrix metalloproteinase-28) expression in hypertrophic scars[END_REF]. MMP28 was detected by PCR in human foetal tissues including skeletal muscle, lung, kidney and brain, and by in situ hybridization in mouse heart, central nervous system and peripheral nervous system from stage E.10 onward [START_REF] Marchenko | MMP-28, a new human matrix metalloproteinase with an unusual cysteine-switch sequence is widely expressed in tumors[END_REF][START_REF] Werner | Neural MMP-28 expression precedes myelination during development and peripheral nerve repair. Developmental dynamics : an official publication of the American Association of Anatomists[END_REF][START_REF] Illman | Promoter characterization of the human and mouse epilysin (MMP-28) genes[END_REF]. In early Xenopus embryogenesis, MMP28 was first identified in a comparative microarray screen performed on dissected neural border explants to identify neural crest (NC)-specific transcripts [START_REF] Plouhinec | Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers[END_REF]. NC cells arise from the neural plate border alongside the cranial placodes. They give rise to a wide variety of derivatives including smooth muscles, melanocytes, craniofacial bones and cartilages, and together with placode cells, all the peripheral nervous system of the head [START_REF] Steventon | Differential requirements of BMP and Wnt signalling during gastrulation and neurulation define two steps in neural crest induction[END_REF]. So far MMP2, 11, 13, 14 and 16 were described as being expressed by the NC or by tissues surrounding the NC (see Table1). However, a detailed expression profile of MMP28 during Xenopus development is still lacking.

In this study, we used qPCR analyses, single and double in situ hybridization to assess the expression of MMP28 during Xenopus laevis embryogenesis. We found that MMP28 has a well-defined expression domain in the pre-placodal region at early neurula stage. During NC cell migration, MMP28 is then detected in some placodes and NC subpopulations. Given that NC and placodes interact to form the cephalic peripheral nervous system [START_REF] Steventon | Differential requirements of BMP and Wnt signalling during gastrulation and neurulation define two steps in neural crest induction[END_REF] and that MMP28 is secreted, we propose that MMP28 may play a role in mediating these interactions to direct cranial morphogenesis.

Materials and Methods

Xenopus manipulation and in vitro fertilization

All experiments were performed in accordance with the guidelines of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and were approved by the Institutional Animal Care and Use Committee of New York University (animal protocol #IA16-00052). Female Xenopus laevis were injected with 500 to 1000 Units of chorionic gonadotrophin and kept overnight at 18°C. For fertilization, a suspension of minced testis was added to the oocytes collected in a petri dish in 0.1X Normal Amphibian Medium (NAM): NaCl (110mM), KCl (2mM), Ca(CO3)2 (1mM), MgSO4 (1mM), EDTA (0.1mM), NaHCO3 (1mM), Sodium Phosphate (2mM). Embryos were staged according to Nieuwkoop and Faber, 1967. 5'-TGGCCAACTGACCATGTAAA-3', Six1_fdw: 5'-CTGGAGAGCCACCAGTTCTC-3', Six1_rev:

5'-AGTGGTCTCCCCCTCAGTTT-3' ODC_fdw: 5'-ACATGGCATTCTCCCTGAAG-3', ODC_rev:

5'-TGGTCCCAAGGCTAAAGTTG-3'. All primer pairs were validated using the standard curve method. Data from embryos collected from three different females were normalized to ODC using the ΔCt method( 14) and expressed as mean with standard deviation error bars.

In situ hybridization and histology

Embryos were fixed for 1h at room temperature in MEMFA (0.1 M MOPS pH 7.42 mM EGTA pH 7.0, 1 mM MgSO4, 3.7 % (wt:vol) Formaldehyde) and stored in 100% methanol.

Embryos were then rehydrated in methanol solutions of decreasing concentrations, and processed for single or double in situ hybridization as previously described [START_REF]Whole-Mount In Situ Hybridization of Xenopus Embryos[END_REF]. The following probes were used: Xl-Snai2 ( 16), Xl-MMP28 (this study), Xl-Sox10 ( 17), Xl-Six1 ( 18), Xl-Foxi4.1 [START_REF] Schlosser | Molecular anatomy of placode development in Xenopus laevis[END_REF], Xl-Sox2 [START_REF] Mizuseki | Xenopus Zic-related-1 and Sox-2, two factors induced by chordin, have distinct activities in the initiation of neural induction[END_REF] and Xl-Dmrta1 [START_REF] Huang | The doublesex-related gene, XDmrt4, is required for neurogenesis in the olfactory system[END_REF]. Each staining was performed at least three times on batches of embryos collected from different females. For histology, stained embryos were embedded in Paraplast+, sectioned (12 µm) on an Olympus rotary microtome, counter stained with Eosin and mounted in Permount.

Results

Xenopus has two homologous copies [START_REF] Session | Genome evolution in the allotetraploid frog Xenopus laevis[END_REF] of MMP28 gene on chromosome 2L (MMP28.L) and 2S (MMP28.S), which are composed of 8 exons each and encode a 1977 bp and 1916 bp mRNAs, respectively. Both mRNA sequences share 84.71% identity, and 92.9%

identity for the open reading frame. MMP28.L and MMP28.S code for proteins of 496 and 497 amino acids, respectively, with a predicted molecular weight of 57 kDa. Both protein sequences share 91.15% identity and 97.58% similarity. As the sequences share high identity, we designed primers and probes for in situ hybridization that detect both MMP28.L and MMP28.S.

We assessed the temporal expression of MMP28 by relative qPCR at different stages of Xenopus development from stage 4 (8-cell stage) to tail bud stage (Figure 1, blue bars), and compared its expression profile to that of Twist (Figure 1, light grey bars), and Sox10 (Figure 1, white bars), two markers of cephalic NC [START_REF] Aoki | Sox10 regulates the development of neural crest-derived melanocytes in Xenopus[END_REF][START_REF] Hopwood | A Xenopus mRNA related to Drosophila twist is expressed in response to induction in the mesoderm and the neural crest[END_REF] and Six1, a marker for cranial placodes [START_REF] Pandur | Xenopus Six1 gene is expressed in neurogenic cranial placodes and maintained in the differentiating lateral lines[END_REF] (Figure 1, black bars). MMP28 is maternally expressed (stage 4) although at relatively low levels, similar to Six1, while Twist and Sox10 do not appear to have a maternal contribution.

After the mid-blastula transition the expression of embryonic genes is initiated. MMP28 expression levels remain relatively low at stage 11 and stage 12.5 similar to Sox10, while both Twist and Six1 expression increase significantly during that period until stage 15 when they both transiently reach a plateau of expression. MMP28 shows a marked increased expression at stage We next performed in situ hybridization to characterize the spatial expression of MMP28 during Xenopus development. MMP28 is not detected at gastrula stage (Figure 2B,C). MMP28

transcripts first accumulate at detectable levels at the end of gastrulation (NF stage 12.5), in the form of a thin horseshoe shape domain at the anterior part of the embryo, which represents the neural plate border (Figure 2 E), the region that gives rise to both cranial placodes and NC cells.

A control sense probe confirmed the specificity of this expression (Figure 2D). At stage 14/15, MMP28 expression is more defined into two bilateral domains on either side of the neural plate (Figure 2F-H). This staining strongly suggests a placodal expression since the neural fold is devoid of staining. At this stage, MMP28 is also detected in a more discrete domain along the border of the neural plate (Figure 2H and I', arrowhead). To substantiate these observations, expression was confirmed using a control sense probe (Figure 2M).

In order to identify more precisely which tissues express MMP28, we performed in situ hybridization at stage 15 and 25 for MMP28 and genes expressed in similar regions of the ectoderm, including Sox2 (neural plate and cranial placodes), Snai2/Slug (NC), Dmrt1a

(olfactory placodes), Foxi4.1 (epibranchial placodes) and Sox10 (migrating NC). The probes were used alone or in combination as indicated (Figure 3). Spatially, there is no overlap between MMP28 and Snai2, as Snai2 expression domain (Figure 3A,A') is medial to that of MMP28 (Figure 3G, G' and B, B'). Sox2 is expressed in two regions, the neural plate (Figure 3C, arrow) and lateral placodes (Figure 3C and C', arrowhead). MMP28-positive territory overlaps exclusively with Sox2 placodal expression domain (Figure 3 D,D'). Since cranial placodes are a broad area giving rise to different derivatives, we also compared MMP28 to Dmrta1, a gene restricted to the most anterior placodal region, the prospective olfactory placodes (Figure 3E,E').

We found that MMP28 and Dmrta1 are expressed in adjacent but non-overlapping domains (Figure 3F, F'). At stage 25, Foxi4.1 is strongly expressed in the epibranchial placodes and sensory layer of the ectoderm (Figure 3J, arrowheads) but not in the NC streams (Figure 3J, arrows). In contrast, Sox10 is expressed strongly in all NC streams (Figure 3, arrows) and excluded from the branchial ectoderm. This histological analysis indicates that MMP28 is not expressed in the posterior NC streams (Figure 3L, arrows) but shows clear expression in the epibranchial placodes and sensory layer of the ectoderm (Figure 3L, arrowheads).

Our results indicate that, at neurula stage, MMP28 is not expressed in the NC but is confined to the cranial placodes territory overlapping with Sox2 (Figure 3 H) and that its expression is maintained in the epibranchial placodes and sensory layer of the ectoderm at tail bud stage (Figure 3L).

Discussion

Here we report the developmental expression of MMP28, a member of the matrix metalloproteinases family, in Xenopus. Our results showed that MMP28 is expressed maternally even though faintly and is upregulated at the end of gastrulation (St. 12.5) at which point it becomes detectable by in situ hybridization. We observed that MMP28 is a secreted MMP uniquely expressed at the neurula stage in the pre-placodal region adjacent to the prospective NC territory.

We showed that MMP28 expression is limited to the neural plate border at early neurula stage, then to the lateral placodes. This placodal expression is sustained in the epibranchial placodes at tail bud stage. In addition, MMP28 expression is detected along the migratory pathway of the first NC stream. As indicated earlier, MMP28 was first isolated in a screen for genes activated in the NC tissue at neurula stage [START_REF] Plouhinec | Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers[END_REF]. However, as discussed by the authors of this study, because the microarray-based approach was performed on manually dissected embryonic tissues at different stages, potential contamination by surrounding tissues could not be excluded. In fact, the authors reported that MMP28 transcripts were detected at the edge of the NC domain, not the NC per se [START_REF] Plouhinec | Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers[END_REF]. At neurula stage, our double in situ hybridization show that MMP28-positive signal does not overlap with the NC marker Snai2/Slug, but co-localizes with the lateral placodal domain of Sox2. While we have detected expression in the medial NC, overall MMP28 expression is primarily confined to the cranial placodes at early neurula stages.

The fact that MMP28 is strongly expressed in placodes is very interesting and unique.

MMP1, 2, 3, 7, 9, 11, 13, 14, 15, 18, 20 and 24 are expressed during Xenopus embryogenesis (Table 1). Almost half of them are expressed by the NC or surrounding tissues and their derivatives, but none of them has been described in placode cells at the stages studied here.

MMP28 expression profile over time is comparable to that of Six1. However, the MMP28positive territory only represents a subdomain of Six1 [START_REF] Pandur | Xenopus Six1 gene is expressed in neurogenic cranial placodes and maintained in the differentiating lateral lines[END_REF]. Notably, MMP28 was not detected in the most anterior placodal domain giving rise to olfactory placodes. By contrast, MMP28 expression is maintained in the epibranchial placodes that originate from the lateral placodes (Sox2-positive territory) and later is detected in between migratory NC streams.

NC cells and cranial placodes cooperate to form the cephalic peripheral nervous system.

NC cells give rise to glial cells while neurons have a dual origin coming from either NC, placodes or both depending on the cranial ganglion of interest [START_REF] Theveneau | Collective cell migration of the cephalic neural crest: The art of integrating information[END_REF]. Interestingly, NC and placode interaction involved in peripheral nervous system patterning starts during NC migration.

In Xenopus, placodal cells are the source of Stromal cell-derived factor 1 (Sdf1/Cxcl12) a key regulator of NC cell migration [START_REF] Theveneau | Collective chemotaxis requires contact-dependent cell polarity[END_REF]. Sdf1 promotes NC cell motility, which in the context of the constraints of the developing head leads to migration of NC cells towards the placodes [START_REF] Bajanca | In vivo topology converts competition for cell-matrix adhesion into directional migration[END_REF].

However, physical interactions between the two cell types leads to a repulsion. This short range repulsion mid-range attraction system, coined "chase-and-run", leads to a sustained directional migration of both NC and placode cells towards the ventral regions of the face [START_REF] Theveneau | Chase-and-run between adjacent cell populations promotes directional collective migration[END_REF]. Placodal cells are less motile than NC cells and are progressively pushed aside while NC cells migrate ventrally. This leads to a pattern of accumulation of placode cells in between NC streams [START_REF] Theveneau | Chase-and-run between adjacent cell populations promotes directional collective migration[END_REF][START_REF] Szabó | Neural crest streaming as an emergent property of tissue interactions during morphogenesis[END_REF]. Therefore interfering with NC migration leads to defects in placode patterning and this relationship is conserved in vertebrates [START_REF] Theveneau | Chase-and-run between adjacent cell populations promotes directional collective migration[END_REF][START_REF] Culbertson | Chondrogenic and Gliogenic Subpopulations of Neural Crest Play Distinct Roles during the Assembly of Epibranchial Ganglia[END_REF][START_REF] Freter | Cranial neural crest cells form corridors prefiguring sensory neuroblast migration[END_REF][START_REF] Golding | Defects in pathfinding by cranial neural crest cells in mice lacking the neuregulin receptor ErbB4[END_REF]. The expression of MMP28, a secreted MMP, in the placodes prior to the onset for NC migration raises the possibility that this enzyme might be required in proper NC-placode interactions.

Conclusion

Overall our data indicate that the onset of MMP28 expression in placodes occurs after the NC and pre-placodal domains have been specified at the neural plate border. MMP28 expression is excluded from the anterior-most placodes and persists in epibranchial placodes strongly suggesting a role for this molecule in NC-placode interactions at early stages of the cranial peripheral nervous system formation.

qPCR

  Embryos were collected by quick-freeze into liquid nitrogen. Total RNAs from batches of 3 to 5 embryos were extracted with the RNeasy Micro Kit (Qiagen, Valencia CA). Relative quantitative PCR was performed on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Foster City, CA) using Power SYBR™ Green RNA-to-CT 1-Step Kit according to manufacturer instructions and the following primers: Twist1qPCR_fdw: 5'-

  stage 15 embryos were sectioned to visualize MMP28 expression in more detail (Figure2H, H', H'' and H'''). MMP28 expression is localized in the deep ectoderm layers consistent with cranial placodes expression (Figure2H''' and I', arrow). By contrast, the thin line of expression along the neural plate is localized in the most superficial ectoderm layer (Figure2I' and H''', arrowhead), which presumably correspond to the medial NC[START_REF] Spokony | The transcription factor Sox9 is required for cranial neural crest development in <em>Xenopus</em&gt[END_REF][START_REF] Sadaghiani | Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy[END_REF]. At stage 17, the expression is broader and outlines the prospective eyes (Figure2J, arrowhead). At stage 23, during NC cells migration, MMP28 expression seems to be restricted to discrete domains around the eyes and in territories posterior to that region, likely to correspond to the epibranchial placodes (Figure2L, L'). At stage 25, strong MMP28 expression is detected in the first stream of NC as well as what appears to be the third and fourth NC streams (Figure2N, arrows). The specificity of MMP28

Figure 1 :

 1 Figure 1: Expression of MMP28 assessed by qPCR. Relative expression of Six1 (black), Twist (light grey), Sox10 (white), MMP28 (blue) during Xenopus embryogenesis from stage 4 to stage 27. Results were generated using Ct method with the house-keeping gene ODC as reference and expressed as mean of at least three independent biological samples, error bars correspond to standard deviation. For representation purposes, relative expression values results were multiplied by 1000.

Figure 2 :

 2 Figure 2: Developmental expression of MMP28 by in situ hybridization. ISH for MMP28 in lateral view (B, C, I, I', L, M, N), anterior view (D-E, J, L'), dorsal view (F-H) and transversally sectioned embryos (H' and H''). A and K: schematic representation of placodes (dark grey), neural crest (light grey) and neural plate (grey). (B-C) At St 11 MMP28 is not detected. (D-E) MMP28 is first detected at the anterior neural plate border at St 12.5. (F-J) Neurula stage embryos show expression in the lateral placodes (arrow) and medial NC (arrowhead). (H'-H''') Transversal sections as indicated in H, placodes (arrow), medial NC (arrowhead). (L-L') Expression in the epibranchial placodes (arrow heads). (M-N) At tail bud stage MMP28 expression is strongly detected in the first NC stream (arrows) and epibranchial

Figure 3 :

 3 Figure 3: Comparative analysis of MMP28, Snai2, Sox2, Dmrta1, Foxi4.1 and Sox10 expression by single and double in situ hybridization. A-H: Xenopus embryos at stage 15 (lateral view, anterior to right, dorsal to top) after single in situ hybridization for Snai2 (A-A'), Sox2 (C-C'), Dmrta1 (E-E') and MMP28 (G-G'), or double in situ hybridization for MMP28/Snai2 (B-B'), MMP28/Sox2 (D-D') and MMP28/Dmrta1 (F-F'). Summary of the expression domains of these genes (H). Doted lines represent expression

  14, which is maintained until stage 18. Later in development, MMP28 expression progressively increases up to stage 27, the last stage examined in this study. Sox10 expression increases strongly between stages 15 and 23 before declining at stage 27. Twist reaches its higher expression level at stage 18 without any further increase later in development. Six1 expression decreases significantly at stage 18 and at later stage progressively increases in a similar manner as MMP28. These data indicate that MMP28 expression is strongly upregulated at the neurula stage (stage 14) and the escalation of Twist and Six1 expressions precedes that of MMP28 and Sox10.

Acknowledgment: The authors would like to thank Ms. Allison Williams for excellent technical assistance.

Funding: This work was supported by a grant from the National Institutes of Health to J-P.S-J.

(R01-DE25806), a pilot grant from the NYU CSCB which was established by NIH to N.G.

(1P30DE020754) and funding from the Fondation pour la Recherche Medicale (FRMAJE201224), the Midi-Pyrenees regional council (grant 13053025) and the CNRS to E.T.


Chongqi Yu

Romain Schira

Harald Brune

Bernd Von Issendorff

Franck Rabilloud

Wolfgang Harbich

Optical properties of size selected neutral Ag clusters: electronic shell stuctures and the surface plasmon resonance †

We present optical absorption spectra from the ultraviolet to the visible for size selected neutral Ag n clusters (n = 5 -120) embedded in solid Ne. We compare the spectra to time-dependent density functional calculations (TDDFT) that address the influence of the Ne matrix. With increasing size, several highly correlated electron excitations gradually develop into a single surface plasmon. Its energy is situated between 3.9 and 4.1 eV and varies with size according to the spherical electronic shell model. The plasmon energy is highest for clusters with atom numbers fully filling states with the lowest radial quantum number (e.g. 1s, 1p, 1d ...). TDDFT calculations for clusters with several candidate geometrical structures embedded in Ne show excellent agreement with the experimental data, demonstrating that the absorption bands depend only weakly on the exact structure of the cluster

Introduction

Small noble metal clusters and nano-particles have particular optical properties related to the energetically localized surface plasmon which determines the optical absorption spectrum in the UVvisible wavelength range. These properties make them interesting for bio-labelling, nanophotonics, light energy harvesting, sensing, electronics, and catalysis [1][2][3][4][5] . The investigation and fundamental understanding of the optical properties of these small metal clusters are key for the development of applications. The optical properties of very large metal particles in the nanometer size range can be characterized to a very good approximation in the framework of classical electromagnetic theory using the dielectric function of the bulk material 2,6 . On the other hand, very small metal clusters containing less than 10 atoms exhibit molecular optical properties and therefore have to be treated with detailed quantum chemical methods like TDDFT [START_REF] Lecoultre | [END_REF][8][9][10][11] . In addition, these calculations have to take the cluster structure into account since for this size range it has a significant influence on the optical properties. While these two size ranges have been extensively studied, data on the intermediate non-scalable 12,13 size regime is scarce, mainly because this regime is experimentally and theoretically much more challenging. At the nanoscale, the well known surface plasmon resonances, classically understood as a collective response of valence electrons, are characterized by an absorption band whose shape depends on the symmetry of the particle 14,15 . Its center wavelength and width changes as a function of size 6 , smoothly for larger particles, more dramatically for the very small ones.

Silver clusters are particularly interesting since their 4d and 5s electrons are sufficiently far in energy to enable a fundamental understanding of their respective roles, at the same time, they are sufficiently close to enable mutual interactions. Specifically, the 4d electrons quench the oscillator strengths by screening the 5s-electrons and get partially involved in excitations. A recent paper 16 shows the center of the plasmon absorption (for small clusters the center of the several discrete absorption lines) as a function of cluster size. Large particles show a monotonous redshift of the plasmon energy with increasing cluster size, which is fairly well understood since their dielectric function is close to that of bulk. Very small particles (up to 39 atoms), which have been measured on mass selected neutral clusters, clearly do not follow the trend of a monotonous redshift for increasing size but show shell closing and multi peak absorption spectra 17,18 . The intermediate size regime (40 -120 atoms) is not covered by measurements on size selected neutral clusters and this is the gap we are filling with this paper.

We present optical absorption measurements on neutral, size selected silver clusters embedded in a solid neon matrix at 6 K. These spectra are recorded on monodisperse cluster ensembles for sizes from 1 to 120 atoms and provide a unique way to investigate the evolution of the plasmon energy with size. TDDFT calculations are compared with the experimental results.

Experimental and theoretical methods

Experiment

The experimental setup couples a sputter gas aggregation cluster source 19 to a newly designed cluster ion beam line which allows for single mass selection 20 . Clusters are injected in a custom built conical octupole 21 , guided by a radio-frequency coupled quadrupole, and mass-selected by a high-mass quadrupole (extranuclear). Cluster ions up to 16000 amu can be selected with an electronically controllable mass resolution. The cluster cations are deposited with a kinetic energy of 10 eV, and coadsorbed with Neon forming the matrix, on a super polished aluminium mirror held at 6 K. The cluster ions are neutralized after deposition by an electron cloud formed in front of the matrix. The excess energy from the neutralization process is coupled into the rare gas support avoiding fragmentation. The matrix has a thickness of 50 µm and the ratio of clusters to rare gas atoms is typically 10 -4 which ensures that mutual interactions between clusters can be neglected while still yielding sufficient signal.

The deposition time varies between 120 and 240 min, depending on the cluster size. Optical absorption measurements are performed in transmission through the 2 mm long matrix. The transmitted light is collected with an optical fiber of 400 µm core diameter and analyzed by an optical spectrometer coupled to a liquid-nitrogen-cooled charge coupled device. Comparing the intensity of the light passing through a matrix containing clusters with a reference spectrum recorded for a pure neon matrix yields the absorption spectrum according to Beer's law. The optical setup covers the UV-visible wavelength range between 250 nm and 1 µm.

Calculation method

Very few theoretical studies have considered the effects of the rare-gas matrix on the optical response of metal clusters 11,20,22,23 . Here our simulations considered clusters both in gas phase and embedded in a Ne matrix. TDDFT calculations have been performed using range-separated hybrid density functionals, namely ωB97x 24 for Ag 20 , Ag 35 , Ag 58 and Ag 92 , and LC-M06L(µ = 0.33) 25,26 for Ag 55 . A relativistic effective core potential (RECP) was used, so that only 19 valence electrons per atom were treated explicitly, together with the corresponding Gaussian basis set 27,28 . The geometrical structures of Ag 35 , Ag 55 , Ag 58 and Ag 92 were taken from a study by Chen et al. 29 using a genetic algorithm with an embedded atom method potential, while we took for Ag 20 the ground state structure of C s symmetry obtained with DFT 8 . Of course we cannot be sure that these structures are indeed the lowest energy ones, but our tests show that the spectra characterized by a plasmon-like band are only weakly dependent on the exact geometrical cluster structure as long as the shape is somewhat spherical. The above structures were optimized within our calculations before setting off to determine their absorption spectra.

Calculations on clusters embedded in a neon matrix have been performed in two steps: first we optimized the structure of the cluster Ag n Ne 100 , in which the silver cluster is surrounded by 100 Ne atoms in an amorphous arrangement, and placed in a dielectric medium characterized by the dielectric function of solid neon (ε = 1.5) 6 . The structure optimisation was performed using the ωB97xD functional which includes empirical dispersion 30 . Then the absorption spectra were calculated with the hybrid functional within the solvent reaction field. All calculations were performed with the Gaussian09 suite of programs [START_REF] Frisch | Gaussian 09 Revision D.01[END_REF] .

Results and discussion

Optical absorption spectra of Ag 1-120

Figure 1 presents the measured absorption spectra in an energy range from 2 to 6 eV for Ag n (n = 1, 2, 5, 8, 11, 20, 21, 35, 55, 58, 84, 92, 120) clusters embedded in solid neon at 6 K. The spectra for Ag n (n = 1, 2, 5) are taken from ref. [START_REF] Lecoultre | [END_REF] and are given here for completeness. The spectrum for Ag 300 has been measured (non size selected) in a direct absorption measurement in the gas phase 40 . Spectra have been normalized to cluster density and to the number of atoms per cluster. The experimental data are shown in red. The blue lines are multi-gaussian fits with variable energy, intensity, and line-width. The respective positions and intensities are represented as vertical grey bars. We show in addition the calculated bulk limit, i.e., the optical spectrum in the dipolar approximation using the dielectric function of bulk silver [START_REF] Johnson | [END_REF] . This spectrum has been redshifted by 0.17 eV in order to account for the dielectric shift of the Ne matrix 33 . Table 1 lists energy, intensity, and line-width for all fitted transitions. The central absorption energies of these clusters are around 4 eV, which is a typical plasmon energy for small silver clusters in the gas phase. Besides, we can see that Ag 20 , Ag 55 , and Ag 92 show narrow absorption widths of around 0.3 eV full width at half-maximum (FWHM), while the absorption profiles for Ag 35 , Ag 58 , Ag 84 , and Ag 120 are with 0.5 eV of FWHM significantly wider.

In order to compare to absorption spectra of clusters in the gas phase, the spectra of Fig. 1 have to be blue-shifted to remove the dielectric matrix shift. This shift was found to be 0.17 eV for large cluster sizes down to Ag 11 33 . To further scrutinize the validity of this blueshift, we compare in Fig. 2 the TDDFT absorption spectra of Ag n clusters in a Neon matrix, blue-shifted by 0.17 eV, with those obtained in the gas phase for n = 20, 58, 92. The very good agreement between both calculations shows that the Ne matrix does not change the overall shape of the absorption features and that the value of 0.17 eV is correct at least in the size range of n = 20 -92. In our calculations, this shift is not validated for smaller particles, we rather find that the confinement compensates the dielectric effect for smaller particles such as Ag 8 .

optical absorption [a.

Comparison with TDDFT calculations

Figure 3 shows calculated spectra for the gas phase in comparison with experimental ones that were blue-shifted by 0.17 ences. For Ag 20 the T d structure is characterized by one strong electronic transition at around 4 eV, while the experimental spectrum clearly is composed of multiple transitions (see Table 1) which together account for the total width of the absorption peak. Hence, this comparison with experiment clearly invalidates the assumption of T d structures, in agreement with electron diffraction measurements on charged clusters 34 . Very recently, Erhart et al. 35 have calculated the spectrum of the icosahedral Ag 55 in the real-time TDDFT approach. Their main band centered at ∼ 4.2 eV is in good agreement with our experimental result.

Mean absorption energies and polarizabilities

An interesting question is the size dependence of the surface plasmon energy which can be well defined by the single symmetric absorption peak for larger particles supposed to be spherical in shape. This situation is more complicated for smaller clusters whose shape deviates from the sphere with a splitting of the plasmon resonance in multiple peaks. A further complication arises from the perturbation of this resonance due to the dielectric influence of the substrate for supported clusters (inhomogenous dielectric environment) and embedded clusters (homogenous dielectric environment). Most experiments for larger clusters are, because of technical difficulties restricted to an ensemble of sizes which makes the attribution of an absorption profile to a specific size difficult. Exceptions are the recent electron energy loss measurements recorded with transmission electron microscopy on single Ag clusters supported on carbon grids from Scholl et al. 15 and on silicon nitride by Raza et al. 38 . Figure 4 shows the plasmon energy against the inverse particle diameter 1/D. The data of Refs. 15,37,39 have been shifted to obtain the resonance energies in vacuum. Please note, here we use 0.25 eV instead of 0.29 eV 16 to blue shift the plasmon energy from argon matrix to vacuum according to the matrix effects 33 . Our present results for Ag n in Ne matrices coincide perfectly with the older data for Ag n in Ar. This shows again that matrix effects 33 will only weakly influence the optical absorption of embedded clusters except for the red shift and justifies again the constant redshift of 0.17 eV in Neon for all measured cluster sizes to extract gas phase energies.

Our results show a higher plasmon energy compared to Ref. 39 . We attribute this to the fact that the silver clusters used in that reference are not mass-selected. The larger clusters have larger signal than the smaller ones, so that averaging over a finite size distribution, and presenting this average as being caused by a single cluster, leads to systematic deviation to lower plasmon energies. This conclusion is consistent with the theoretical result of Ref. 41 .

Interestingly, we find that silver clusters with atom numbers of 8, 18, 34, 58, 92 show a localised maximum value of the plasmon energy. According to the well-known shell model 42 , these numbers correspond to the fully filled states of 1S, 1P, 1D, 1F, 1G and 1H. Our DFT calculations confirm such a filling. Indeed the electronic structure clearly shows the shell filling in which the s valence electrons are found to be distributed in delocalized orbitals in the following energy sequence of electronic shell model 1S 2 , 1P 6 , 1D 10 , 2S 2 , 1F 14 , 2P 6 , 1G 18 , 2D 10 , 3S 2 , 1H 22 , etc. As an illustration, the density of states (DOS) together with representative Kohn-Sham orbitals are given in Fig. 5 for Ag 55 . All molecular orbitals can be found in the supporting information, together with those of Ag 20 and Ag 92 . For all clusters, we can easily distinguish the orbitals associated to S, P, D, F, G shells, even if some shells are splitting into subshells due to the lower symmetry with respect to the K h group for a perfect sphere. The static polarizability of the clusters can be calculated from the experimental optical spectra as 6 :

α = Ne 2 m e • 1 ω 2 = Ne 2 ε 0 m e • ∞ 0 1 ω 2 • σ (ω)dω ∞ 0 σ (ω)dω . ( 1
)
where N is the total number of electrons in the cluster, ω the absorption energy, and σ the absorption cross section. The polarizability divided by the number of atoms is shown in Fig. 6, together with data for smaller clusters from earlier measurements 17,18 . We find values from 0.7 -1.0. Local minima are observed for the shell closings, corresponding to the stiffest clusters. An exception makes Ag 55 which is the geometrical shell closing of the icosahedron (I h group) rather than 58 which is the filling of the 1G state. Static polarizabilities can also be extracted from the DFT calculations. We show in Fig. 6 the DFT values calculated at ωB97x/def2TZVP 43 level using the geometrical structures proposed by Chen et al. 29 . To obtain values per atom as in experiment we calculate α(Ag) per atomic volume of 7.45 Å 3 . They show good agreement with experiment, even if the calculated values for small cluster sizes are slightly above the experimental ones. The minima at n = 8, 18, 34, 55, and 92 are well reproduced. It is worth noticing that the experimental values obtained with the formula (1) integrated up to 5 eV are in good agreement with the exact calculated values because the static polarizability results mainly from valence electrons. Our data manifest that the clusters with fully filled states have higher plasmon energies and lower static polarizabilies than clusters with partial filled states. A similar finding has been reported by Puska et al. 44 in DFT calculations for simple metal clusters (Li, Al). Relating the optical resonance ω 1 to the static polarizability through the equation 6 :

ω 1 = Ne 2 m e α . ( 2
)
allows us to conclude that the higher plasmon energies for magic numbers reflect small polarizabilities.

Conclusions

We present optical absorption spectra of mass selected Ag n (n = 5, 20, 35, 55, 58, 84, 92, 120) clusters embedded in a solid neon matrix. This extends the size range to fill the gap in the center position of plasmon resonances of neutral silver clusters. All spectra, although showing a plasmon like absorption profile, are composed of multiple transitions. TDDFT calculations are in excellent agreement with the experimental spectra. The plasmon energies lie between 3.9 -4.1 eV with an absorption width (FWHM) between 0.3 -0.5 eV depending on the shape of the cluster. Plasmon energies and static polarizabilities are structured and clearly show shell effects where the silver clusters with atom numbers fully filling the states with the lowest quantum level of ν = 1 show a localized maximum value of the plasmon energy.

a

  Institute of Physics, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland b Université Claude Bernard Lyon 1, CNRS, Institut Lumière Matière, F-69622 Villeurbanne, France c Université Fakultät für Physik, Universität Freiburg, Germany † Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/b000000x/ ‡ Additional footnotes to the title and authors can be included e.g. 'Present address:' or 'These authors contributed equally to this work' as above using the symbols: ‡, §, and ¶. Please place the appropriate symbol next to the author's name and include a \footnotetext entry in the the correct place in the list.

2 Fig. 1 Fig. 2

 212 Fig. 1 Measured optical absorption spectra (red) of monodisperse Ag n (n = 1 -120) in Ne at 6 K. Blue lines are multi-peak Gaussian fits, grey

Fig. 3

 3 photon energy[eV]

  It seems therefore plausible to assume a less symmetric structure, like the present calculated structure of C s symmetry, as the lowest lying ground state for Ag 20 . Similarly, the T d symmetry for Ag 35 , Ag 84 and Ag 120 leads to rather narrow plasmon widths, while the experimental spectra of the corresponding sizes are broad compared to the ones of Ag 55 and Ag 92 . An additional difference to the experimental data is the larger redshift of the plasmon peak with size of about 0.6 eV in the calculations of T d Ag n clusters.

Fig. 4

 4 Fig. 4 Center of the surface plasmon resonance of silver particles in vacuum as a function of the inverse cluster diameter 1/D.37 ; 39 ; 15 ; 38 ; 40 ; • 32 ; Present work;

Fig. 5

 5 Fig. 5 Density of states of Ag + 55 together with the representative Kohn-Sham molecular orbitals aligned above the DOS peaks. Blue and red refer to positive and negative sign of the molecular orbitals. More molecular orbitals (together with those of Ag 20 and Ag 92 ) are given in the supporting information.

Fig. 6

 6 Fig. 6 Experimental and theoretical results of the polarizability of silver clusters as a function of the cluster size. Ag n embedded in argon 18 ; Ag n embedded in neon (present work); • DFT (present work).

J o u r

  n a l N a me , [ y e a r ] , [ v o l . ] , 1-8 | 5

1SFigure S2 :

 S2 Figure S2: Density of states of Ag 55 together with the representative Kohn-Sham molecular orbitals. Blue and red refer to positive and negative sign of the molecular orbitals. The orbitals 1S and 1P are located inside the d band.

Figure S3 :

 S3 Figure S3: Density of states of Ag 92 together with the representative Kohn-Sham molecular orbitals. Blue and red refer to positive and negative sign of the molecular orbitals. The orbitals 1S, 1P, 1D, and 2S are located inside the d band.

Table 1

 1 37 ; 39 ; 15 ; 38 ; 40 ; • 32 ; Present work; Energies, intensities and widths of plasmons and transitions contributing to the absorption spectra. this work; • 7 ; * 37 ; 32 .

	Ag n	Plasmon		Abs. peak 1		Abs. peak 2		Abs. peak 3		Abs. peak 4
		En.	FWHM	En.	Int.	FWHM	En.	Int.	FWHM	En.	Int.	FWHM	En.	Int.	FWHM
		(eV)	(eV)	(eV)	(a.u.)	(eV)	(eV)	(a.u.)	(eV)	(eV)	(a.u.)	(eV)	(eV)	(a.u.)	(eV)
	•5	3.4	-	3.26	2.86	0.05	3.3	2.86	0.06	3.70	3.29	0.07	3.83	0.49	0.10
	•8	3.93	-	3.15	0.65	0.11	3.66	2.56	0.04	3.98	2.91	0.06	4.03	2.97	0.06
	* 11	3.7	-	3.54	0.36	1.54	3.68	0.67	0.05	4.19	0.19	0.20	4.25	0.82	0.09
	20	3.83	0.316	3.68	1.10	0.19	3.79	1.20	0.17	3.87	1.26	0.24	-	-	-
	* 21	3.75	-	3.31	0.14	0.16	3.83	0.81	0.21	-	-	-	-	-	-
	35	3.84	0.485	3.70	2.37	0.33	3.83	0.96	0.21	3.94	1.50	0.18	4.04	0.80	0.19
	55	3.9	0.320	3.60	0.12	0.17	3.87	1.56	0.25	4.04	0.48	0.20	-	-	-
	58	3.93	0.417	3.64	0.59	0.37	3.94	1.44	0.32	-	-	-	-	-	-
	84	3.77	0.504	3.62	2.32	0.34	3.76	1.73	0.28	3.93	2.43	0.31	-	-	-
	92	3.8	0.248	3.62	0.07	0.22	3.77	1.08	0.28	-	-	-	-	-	-
	120	3.73	0.530	3.44	0.20	0.16	3.57	0.25	0.18	3.69	0.28	0.23	3.87	0.37	0.42
	bulk	3.14	0.07	3.14	0.63	0.07	-	-	-	-	-	-	-	-	-

| 1-8 J o u r n a l N a me , [ y e a r ] , [ v o l . ] ,

1-8 J o u r n a l N a me , [ y e a r ] , [ v o l . ] ,

| 1-8

Acknowledgements

We acknowledge support from the Swiss National Science Foundation (Grant 200020_149388). RS and FR thank the GENCI-IDRIS (Grant i2015086864) center for generous allocation of computational time.

Conflicts of interest

There are no conflicts to declare'.

Supporting Information

Optical properties of size selected neutral Ag clusters: electronic shell stuctures and the surface plasmon resonance DFT calculations have been performed using the exchange and correlation functional ωB97x 1 and a relativistic effective core potential (RECP), so that only 19 electrons per atom were treated explicitly, together with the corresponding Gaussian basis set 2,3 . The geometrical structures of Ag n have been taken from a study by Chen et al. 4 using a genetic algorithm with an embedded atom method potential, except that of Ag 20 which has been taken as the ground state structure of C s symmetry obtained with DFT 5 . Calculations have been performed with the Gaussian09 suite of programs 6 .

The three figures S1, S2, S3 give the density of states (DOS) together with the Kohn-Sham orbitals of valence orbitals for Ag 20 , Ag 55 and Ag 92 . The s-type valence electrons are found to be distributed in the following energy sequence of electronic shell model 1S 2 , 1P 6 , 1D 10 , 2S 2 , 1F 14 , 2P 6 , 1G 18 , 2D 10 , 3S 2 , 1H 22 . We can easily distinguish the orbitals associated to S, P, D, F, G supershells. Some shells are splitting into subshells due to the lower symmetry with respect to the K h group for a perfect sphere, and the atomic structure.


domain: chim sde sdv

text:
Zeineb Bouhlel

Alexandre A Arnold

Dror E Warschawski

Karine Lemarchand

Réjean Tremblay

Isabelle Marcotte
email: marcotte.isabelle@uqam.ca

Labelling strategy and membrane characterization of marine bacteria Vibrio splendidus by in vivo 2 H NMR

Keywords: In-cell NMR, deuterium, isotopic labelling, membrane fluidity, fatty acids, magic-angle spinning CHAPS,3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate, cyC17:0, cyclopropaneoctanoic acid-2-hydroxyl acid, cyC19:0, cyclopropaneoctanoic acid-2-octyl acid, CMC, critical micelle concentration, d 31 -PA, deuterated palmitic acid, DF, degree of freedom, DPC, dodecylphosphocholine, F, F-statistic, FA, fatty acid, FAME, fatty acid methyl ester

Vibrio splendidus is a marine bacterium often considered as a threat in aquaculture hatcheries where it is responsible of mass mortality events, notably of bivalves larvae. This bacterium is highly adapted to dynamic salty ecosystems where it has become an opportunistic and resistant species. In an attempt to characterize their membranes as a first and necessary step toward studying bacterial interactions with diverse molecules, we establish a labelling protocol for in vivo 2 H solid-state nuclear magnetic resonance (SS-NMR) analysis of V. splendidus. 2 H SS-NMR is a useful tool to study the organization and dynamics of phospholipids at the molecular level, and its application to intact bacteria is further advantageous as it allows probing acyl chains in their natural environment and study membrane interactions. However, due to the complexity of metabolic pathways and biological systems, isotopic labelling can be challenging. In this study, we show that V. splendidus can be labelled using deuterated palmitic acid, and demonstrate the importance of surfactant choice in the labelling protocol. Moreover, we assess the impact of lipid deuteration on the general fitness of the bacteria, as well as the ratio of saturated-to-unsaturated fatty acid chains and its impact on the membrane properties. We further characterize the evolution of V. splendidus membrane fluidity during different growth stages and relate it to fatty acid chain composition. Our results show a superior membrane fluidity during the stationary growth phase compared to the exponential growth phase under labelling conditions -an information to take into account for future in vivo SS-NMR studies. Our lipid deuteration protocol optimized for V. splendidus is likely applicable other microorganisms for in vivo NMR studies.

INTRODUCTION

Vibrio species are Gram-negative bacteria widely spread in coastal marine and estuarine waters and sediments [1][2][3][4]. They might also be found in aquatic animal tissues, causing serious pathologies leading in some cases to economic loss in aquaculture industry [5][6][7][8]. To date, most research on marine Vibrio species concerned their effects on aquatic organisms acting on immunological process [8][9][10][11][12][13][14]. As a matter of fact, a common trait of all members of the Vibrio genus is their opportunistic nature, which allows them to benefit from the collapse of the immune system of cultured organisms in specific conditions to become virulent [14,15]. Investigating the virulence/response of these species requires considering numerous biotic and abiotic factors where the physiological responses can remain elusive. For instance, V. splendidus are different from their Vibrio congeners by their high genetic variability between strains, where each strain can have a different opportunistic and/or virulence pattern [8,15,16].

Very few researches have so far focused on the membranes of environmental bacteria compared to human pathogens, albeit the importance of the external cell envelopes in biological events. Marine bacteria are of great interest for membrane properties investigation considering their high resistance phenotypes and high adaptability to a large scale of salinity and temperature, attributable to their permanent exposure to changing ecosystems [1,3,4,11,[17][18][19][20][START_REF] Soto | Salinity and temperature effects on physiological responses of Vibrio fischeri from diverse ecological niches[END_REF]. Moreover, marine and coastal environments constitute a dynamic platform for water mixing, thus containing a plethora of molecules such as pollutants, aquatic bioactive components, organic or chemical toxins and industrial antibiotics [START_REF] Phillips | The use of biological indicator organisms to monitor trace metal pollution in marine and estuarine environments-a review[END_REF][START_REF] Olsen | Pollutant-particle associations and dynamics in coastal marine environments: a review[END_REF][START_REF] Nixon | Coastal marine eutrophication: a definition, social causes, and future concerns[END_REF][START_REF] Baquero | Antibiotics and antibiotic resistance in water environments[END_REF][START_REF] Kümmerer | Antibiotics in the aquatic environment-a review-part I[END_REF][START_REF] Riedel | Molecular signatures of biogeochemical transformations in dissolved organic matter from ten world rivers[END_REF]. The cell envelope of indigenous bacteria in such environments represents the first barrier encountered by external molecules which could either cross the membrane and ultimately intracellular sites, or directly affect their structural components [START_REF] Hancock | The bacterial outer membrane as a drug barrier[END_REF][START_REF] Shai | Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by α-helical antimicrobial and cell non-selective membrane-lytic peptides[END_REF]. Acquiring knowledge on Vibrio membrane structure and fluidity would help tackling physiological processes inside the cell and gain a better understanding of the interaction mechanisms of these marine bacteria with molecules in their environment. This should contribute to improve investigations of larval infections and therapeutic treatments in aquaculture.

The objective of this work was to establish a protocol to deuterate the lipid acyl chains in V. splendidus membranes, to enable the in vivo 2 H solid-state nuclear magnetic resonance (SS-NMR) study of this marine bacterium. More specifically we used the indigenous 7SHRW strain isolated from the St. Lawrence Gulf sediments (Canada) which can cause significant mortalities to blue mussels and scallops at larval stages [START_REF] Mateo | Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: changes in cell structure, numbers and adherence[END_REF][START_REF] Turcotte | Prophylactic effect of Haslea ostrearia culture supernatant containing the pigment marennine to stabilize bivalve hatchery production[END_REF], and has the advantage to be easily cultured in laboratory conditions. Like other marine bacteria, very few research on membrane phospholipids of Vibrio sp. are available [START_REF] Oliver | Extractable lipids of gram-negative marine bacteria: phospholipid composition[END_REF][START_REF] Oliver | Lipid composition of a psychrophilic marine Vibrio sp. during starvation-induced morphogenesis[END_REF][START_REF] Byers | Elongation of exogenous fatty acids by the bioluminescent bacterium Vibrio harveyi[END_REF][START_REF] Brown | Regulation of fatty acid metabolism by FadR is essential for Vibrio vulnificus to cause infection of mice[END_REF][START_REF] Zhang | Transcriptional regulation in bacterial membrane lipid synthesis[END_REF] and studies on V. splendidus in this regard are even more sparse. To the best of our knowledge, only one study describes membrane phospholipids of V. splendidus, using a strain living in deep anoxic sediments [START_REF] Freese | Gammaproteobacteria as a possible source of eicosapentaenoic acid in anoxic intertidal sediments[END_REF].

Deuterium SS-NMR is an excellent tool to investigate the structure and dynamics of membrane lipids at the molecular level [START_REF] Davis | The description of membrane lipid conformation, order and dynamics by 2H-NMR[END_REF][START_REF] Li | Applications of nuclear magnetic resonance in lipid analyses: An emerging powerful tool for lipidomics studies[END_REF][START_REF] Seelig | Deuterium magnetic resonance: theory and application to lipid membranes[END_REF]. Traditionally applied to model systems, the emerging field of in vivo SS-NMR now enables the study of intact living organisms such as bacteria, microalgae, and small invertebrates [START_REF] Chauton | HR MAS ¹H NMR spectroscopy analysis of marine microalgal whole cells[END_REF][START_REF] Arnold | Identification of lipid and saccharide constituents of whole microalgal cells by 13 C solidstate NMR[END_REF][START_REF] Simpson | Perspective: in vivo NMR-a potentially powerful tool for environmental research[END_REF][START_REF] Warnet | In-cell solid-state NMR: an emerging technique for the study of biological membranes[END_REF][START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF]. Warnet et al. showed that magic-angle spinning (MAS) can be used in 2 H in-cell SS-NMR studies to shorten the acquisition time by a factor of 10 while simultaneously maintaining spectral sensitivity, thus favoring the in vivo character of the experiment [START_REF] Warnet | A 2 H magic-angle spinning solid-state NMR characterisation of lipid membranes in intact bacteria[END_REF]. However, 2 H NMR requires isotopic labelling which can be challenging in biological organisms such as V. splendidus. Notably, deuteration labelling protocols of bacteria phospholipids require the use of surfactants [START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF][START_REF] Pius | 2 H solid-state nuclear magnetic resonance investigation of whole Escherichia coli interacting with antimicrobial peptide MSI-78[END_REF][START_REF] Tardy-Laporte | A 2 H solid-state NMR study of the effect of antimicrobial agents on intact Escherichia coli without mutating[END_REF] to micellize deuterated fatty acids prior to their uptake by the bacteria and use in the phospholipids biosynthesis. These surfactants can possibly affect the membrane. Moreover, the natural saturated/unsaturated lipid ratio in the bacterial membrane should be preserved. A careful optimization of the deuteration protocol is thus necessary to ensure genuine in vivo characterization of these microorganisms.

In this work we propose for the first time a protocol for the deuteration of lipids in a model marine bacterium, V. splendidus. To do so, we assessed the effect of different surfactants on the cell growth. We also studied the effect of palmitic and oleic acid on the lipid profile and membrane fluidity by in vivo 2 H SS-NMR as a function of cell growth stage. The labelling strategy developed here has the potential to be amenable to the in vivo NMR investigations of a variety of marine and terrestrial bacteria.

MATERIALS AND METHODS

Materials

Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), Brij35, oleic (OA) and deuterated palmitic (d 31 -PA) acids, deuterium-depleted water, as well as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and fatty acid methyl ester (FAME) mix C4-C24 were all purchased from Sigma Aldrich (Oakville, Canada). Dodecylphosphocholine (DPC) and octylglucopyranoside (OG) were obtained from Avanti Polar Lipids (Alabaster, AL). Polyethylene glycol sorbitan monolaurate (Tween® 20) was purchased from BioShop Canada Inc. (Burlington, Canada), whereas LB (Lysogeny broth) Broth Miller was obtained from BioBasic Inc. (Markham, Canada).

Bacterial growth and 2 H labelling protocol

Vibrio splendidus strain 7SHRW were isolated from sediments of Hillsborough River, Prince Edward Island (Gulf of St. Lawrence, Canada) [START_REF] Mateo | Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: changes in cell structure, numbers and adherence[END_REF]. Cell culture was initiated by adding 100 µl of a frozen cell stock solution (in 40% glycerol at -80°C in 10 mL LB medium (NaCl 10g/L, tryptone 10 g/L, yeast extracts 5 g/L), and incubated at 24.5°C (± 0.5°C) on a rotary shaker (INFORS HT Multitron Pro, USA) operating at 100 rpm. After 3 days of growth, bacteria were transferred into 250 mL Erlenmeyer flasks containing 100 mL of LB 1X (initial OD 600nm adjusted to ≈ 0.02) and 1 mL was transferred into a 24-well plate to monitor the growth kinetic with a multiple plate reader (Infinite M200 TECAN, Männedorf, Switzerland). Plates were conditioned to 24.5°C (± 0.5°C) and to an agitation of 87 rpm before measuring the absorbance at 600 nm every 30 min during 48 hours. For each treatment, 3 to 4 wells were used. For isotopic labelling, V. splendidus were grown in the same conditions as described above but in an LB medium enriched with deuterated palmitic acid (d 31 -PA). Prior to each use, the Vibrio strain was grown for two days and its purity was verified on LB-agar. The initial bacteria concentration was adjusted to be the same between replicates, i.e., an optical density (OD 600nm ) of 0.02 at 600 nm wavelengths.

Initially, different commonly used surfactants were tested to micellize palmitic acid (0.3 mM) in culture media, and bacterial growths were monitored. Surfactants were used above their critical micelle concentration (CMC): Brij35 (0.1 mM), Tween20 (0.06 mM), Triton-X (0.4 mM), OG (20 mM), CHAPS (6 mM), and DPC (1.5 mM). The lipid deuteration procedure was optimized as follows: LB culture medium was supplemented with a mixture of d 31 -PA (0.3 mM) and Tween-20 (0.14 mM), heated at 85°C, and the corresponding solution was flash-frozen and heated again several times until the complete dissolution of d 31 -PA crystals. Protonated OA was added in the same proportions (0.3 mM) during bacteria inoculation, to mitigate potential unbalance of the saturated/unsaturated fatty acid (SFA/UFA) ratio within the membrane [START_REF] Warnet | A 2 H magic-angle spinning solid-state NMR characterisation of lipid membranes in intact bacteria[END_REF] . Potential effect of the 2 H labelling was verified by comparing the growth of bacteria in deuterated and nondeuterated media. The specific growth rate (µ) was estimated from the slope regression of ln (OD 600nm ) as a function of elapsed time [START_REF] Nedwell | Influence of temperature on growth rate and competition between two psychrotolerant Antarctic bacteria: low temperature diminishes affinity for substrate uptake[END_REF]

Fatty acid profile analysis

Fatty acid (FA) profiles were analyzed using gas chromatography combined to mass spectrometry (GC-MS) to obtain the proportion of each FA (deuterated or protonated) in ng.mg -1 , and were expressed as relative concentration (weight % relative to total FA contents) as described by Tardy-Laporte et al. [START_REF] Tardy-Laporte | A 2 H solid-state NMR study of the effect of antimicrobial agents on intact Escherichia coli without mutating[END_REF]. Briefly, starting from three pools of 30 to 60 mg of dry-freezed bacteria, total lipids were extracted using dichloromethane/methanol (2:1 CH 2 Cl 2 /MeOH v/v) and 0.88% KCl solution in a Potter glass homogenizer. Neutral and polar lipids were separated by elution through a silica gel column (30×5 mm) hydrated with 6% water. Polar lipids were transesterified using 2 mL of H 2 SO 4 (2% in MeOH) and 0.8 mL of toluene. Final extracts were diluted in hexane solution and adjusted to a volume of 0.5 mL before GC-MS analysis (Agilent technology-7890A, Santa Clara, CA, USA). FA analyses were performed in parallel on a FAME mix which was used as a standard.

Sample preparation for 2 H SS-NMR analysis and viability assays

Freshly collected bacterial cells were centrifuged at 4000 rpm for 10 min to remove the culture medium. Pellets were then suspended in a saline sterile rinsing solution (9‰ NaCl) to remove any residual FAs and detergent molecules, and centrifuged again at 3800 rpm for 5 min. Rinsing was carried out at least 3 times, twice with saline solution prepared with nanopure water, and a final wash with saline solution prepared with deuterium-depleted water. The final pellet was used to fill a 4-mm zirconium oxide rotor, which corresponds to approximately 90 mg of hydrated bacteria. NMR experiments were performed on deuterated bacteria harvested at three different growth times (mid-log, early stationary phase and late stationary phase) and prepared in triplicate.

Following NMR experiments V. splendidus viability was determined using MTT reduction assays [START_REF] Wang | An improved 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) reduction assay for evaluating the viability of Escherichia coli cells[END_REF]. Cell suspensions from NMR samples were diluted in 5 replicates to a final OD 600nm of 0.1 each, and were mixed with MTT solution (5mg/ml) to a ratio of MTT/cell suspension of 1:10 (v/v). The mixture was incubated in Eppendorf tubes for 20 minutes at 25°C with open caps until the formation of formazan crystals. Preparations were centrifuged (10.000g ×1min.) and the crystal pellets were dissolved in dimethylsulfoxide and incubated at room temperature for 15 minutes. Optical density was measured at 550 nm and cell viability of bacteria was expressed in relative percentage compared to freshly harvested bacteria before rinse. MTT assays were performed on triplicates of cultures corresponding to the NMR samples.

In vivo 2 H SS-NMR experiments and moment analysis

All 2 H solid-state NMR experiments were performed at 25°C on a Bruker Avance HD III wide Bore 600 MHz spectrometer (Billerica, MA, USA) using a double-resonance magic angle spinning (MAS) probe tuned to 92.1 MHz. Sample spinning frequency was set to 10 kHz. Typically, spectra were acquired using a Hahn Echo pulse sequence with the following operating conditions: 5 µs 90° pulses separated by an echo delay of 100 µs and a recycle time of 0.5 s. Each spectrum was obtained in approximately 43 min of acquisition time, corresponding to 4096 scans. A total of 32k points were acquired for a spectral width of 500 kHz. Spectra were Fourier transformed after application of a 50 Hz exponential line broadening and zero filling to 64k points.

Spectral moment analysis was performed using MestRenova software V6.0 (Mestrelab Research, Santiago de Compostela, Spain) and a macro developed by Pierre Audet (Université Laval). The second moment (M 2 ) was calculated as described in equation 1 [START_REF] Warnet | A 2 H magic-angle spinning solid-state NMR characterisation of lipid membranes in intact bacteria[END_REF]

: 𝑁 2 𝐴 𝑁 ∞ 𝑁=0 ∑ 𝐴 𝑁 ∞ 𝑁=0 = 4𝜋 2 𝜈 𝑄 2 5 ⟨𝑆 𝐶𝐷 2 ⟩ (1)
where ω r is the spinning frequency, N the side band number, and A N the area of each sideband obtained by spectral integration, S 2 CD is the mean square order parameter, and Q is the quadrupolar splitting constant equal to 168 kHz for a C-D bond in acyl chains [START_REF] Burnett | Deuteron quadrupole coupling constants in three solid deuterated paraffin hydrocarbons: C2D6, C4D10, C6D14[END_REF].

RESULTS

Optimized 2 H labelling of V. splendidus for in vivo SS-NMR

The enzymatic machinery of bacteria energetically favours the incorporation of exogenous FAs into phospholipids [START_REF] Marcotte | 2 H Solid-State NMR Study of Peptide-Membrane Interactions in Intact Bacteria[END_REF], thus enabling the incorporation of deuterated PA chains exclusively in the membranes. We based our 2 H labelling procedure of V. splendidus on a previously published protocol for another Gram(-) bacterium, Escherichia coli, which involved micellization of FAs by DPC in the growth medium to facilitate the FA uptake. However, since the presence of surfactants could be harmful to bacteria, and since another surfactant (Brij-58) we tested a series of detergents to identify the best suited for optimal bacterial lipid deuteration. Therefore, Tween-20, Brij-35, DPC, Triton-X, CHAPS and OG were assessed. To do so, we first monitored bacterial growth in different culture media when mixing protonated palmitic acid (PA) with these non-anionic surfactants above their CMC. Figure 1 shows that Tween-20 is the less harmful detergent for the lipid deuteration of V. splendidus, and that a high concentration of bacteria is measured even at a detergent concentration of 0.25 mM, i.e., well above Tween-20's CMC (0.06 mM). A concentration of 0.14 mM of Tween-20 was thus employed for the rest of the study because it allows complete solubilization of PA (concentration of 0.3 mM) without affecting the bacterial culture.

The deuteration of V. splendidus membrane lipids was then verified by 2 H SS-NMR. The second spectral moment M 2 was calculated to assess the membrane fluidity. When specific quadrupolar splittings cannot be measured, M 2 values are good reporter of the spectral distribution which reflects the lipid phases -the greater the M 2 value, the greater the lipid ordering. Figure 2B-C shows that the deuteration protocol used herein was successful as a good signal-to-noise ratio is observed in vivo by 2 H SS-NMR when bacteria are sampled in the exponential growth phase. The importance of the surfactant-mediated micellization step can be seen in Figure 2A which shows that when d 31 -PA is used without Tween-20, no side bands can be detected (i.e., there is no labelling). Figure 2C

Fatty acid composition as a function of cell growth and labelling conditions

Since the labelling protocol was successful, the membrane FA profile was monitored in order to quantify the assimilation of exogenous FAs at different growth stages and assess the potential impact of 2 H labelling on the membrane. Lipid analyses were thus performed on labelled and with and without OA. Cell growth data are presented in the "Supplementary material" section.

Figure 3 shows that the incorporation of d 31 -PA is indeed successful and that 77% of the PA acyl chains in the membrane are deuterated during the mid-log phase for V. splendidus grown in the presence of d 31 -PA (amounting to 44% of all FAs). The deuteration level of PA was maintained as high as 69% (31% of all fatty acids, numerical values are reported in Supplementary material, Table SI1) when OA was added in the growth medium, in spite of the high incorporation of OA into the membranes lipids.

Depending on the growth regimes, V. splendidus FA profile reveals more than 15 different FA chains with three major and recurring components: palmitoleic acid (C16:1), PA (C16), and OA (C18:1) (Fig. 3). Our results (Fig. 3A) indicate that deuteration with d 31 -PA respects the native composition of V. splendidus. In addition, exogenous OA was highly incorporated during the exponential phase, at the expense of palmitoleic acid -the most naturally abundant UFA in V. splendidus membranes. The choice to enrich the culture with OA as an UFA was to balance as much as possible the incorporation of saturated d 31 -PA and to allow comparison with previously published deuteration protocols for other bacteria such as E. coli [START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF][START_REF] Warnet | A 2 H magic-angle spinning solid-state NMR characterisation of lipid membranes in intact bacteria[END_REF][START_REF] Pius | 2 H solid-state nuclear magnetic resonance investigation of whole Escherichia coli interacting with antimicrobial peptide MSI-78[END_REF]. It also enabled exploring the adaptability of V. splendidus to integrate exogenous FAs and to modulate its FA chain fluidity accordingly.

Indeed bacteria responded to the enrichment with both exogenous FAs by including them in their lipid profile, more strikingly during the exponential phase, without affecting much the proportions of the other FAs (Fig. 3B). Permutational multivariate analysis of variance (Permanova) on Bray-Curtis matrices (Primer 7.0.13) of FAs composition of cell culture from different treatments and growth phases reveal significant interaction between both factors (DF = 2 and 22, Pseudo-F = 4.05, p = 0.005). Pair-wise Permanova comparison test indicates more specifically where differences were observed. Interestingly, the FA compositions were similar to that of the native bacteria membranes during the plateau phase (Fig. 3A), suggesting an adaptation of the bacteria to the growth medium (p = 0.089). In contrast, growing bacteria with only d 31 -PA leads to a more altered FA profile (p = 0.002). For instance, the major FA chains for cyclopropane FAs (cyC17:0 and cyC19:0) were highly synthetized under this regime (see

Supplementary material).

Under labelling conditions, both with and without OA, the SFA/UFA ratios were higher in the exponential phase. However, the ratio was reversed in the plateau phase due to increased Figure 3 Saturated/unsaturated fatty acid ratios were calculated from FA content expressed in mol% per total FA (See S.I). Growth temperature was 25°C for all cultures. cyC19:0). It should also be noted that the SFA/UFA ratio of bacteria labelled in the presence of OA and during early stationary phase (SFA/UFA = 0.66 ± 0.05) was close to that of unlabelled cultures which was about 0.59 ± 0.05 and 0.60 ± 0.01 during mid-log and early stationary phases, respectively.

Membrane fluidity characterization by SS-NMR

The 2 H SS-NMR spectra obtained for the labelled bacteria (Figure 4) show that membrane rigidity varies with cell division. Under the same growth conditions (d 31 -PA with OA) and in a timeframe of 5 hours, a reduction was observed in the sideband intensity and number.

Accordingly, the M 2 values dropped by half going from the mid-log to the stationary phase.

These results are consistent with the decreased SFA/UFA ratio which reveal an increase in membrane fluidity as a function of growth time under the same growth conditions. Figure 4: 2 H MAS (10 kHz) SS-NMR spectra of intact V. splendidus harvested at three different cell growth times: (A) after 15 h in the mid-log phase (OD 600nm ≈ 0.3), (B) after 22 h (±2h) at the beginning of the stationary stage (OD 600nm ≈0.5), and (C) after 30 h at advanced stationary phase (OD 600nm ≈ 0.5). Bacteria were labelled with d 31 -PA in presence of OA. Average second spectral moments M 2 are indicated (10 9 s -2 ).

DISCUSSION

2 H labelling optimization for in vivo study of membranes by 2 H SS-NMR

Deuterium labelling of lipid chains is a useful approach to explore changes occurring in biological membranes using SS-NMR. However, when studying living organisms, the deuteration protocol could be a disturbing factor to physiological pathways, or induce stress.

Previously published work has established the feasibility and pertinence of bacterial membrane deuteration on human pathogens E. coli and B. subtilis [START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF][START_REF] Warnet | A 2 H magic-angle spinning solid-state NMR characterisation of lipid membranes in intact bacteria[END_REF][START_REF] Pius | 2 H solid-state nuclear magnetic resonance investigation of whole Escherichia coli interacting with antimicrobial peptide MSI-78[END_REF][START_REF] Tardy-Laporte | A 2 H solid-state NMR study of the effect of antimicrobial agents on intact Escherichia coli without mutating[END_REF][START_REF] Davis | Deuterium magnetic resonance study of the gel and liquid crystalline phases of dipalmitoyl phosphatidylcholine[END_REF]. Here, a marine bacterium, V. splendidus, is deuterated for the first time while minimizing disturbances on the microorganism. Although V. splendidus and previously characterized E. coli [START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF][START_REF] Warnet | A 2 H magic-angle spinning solid-state NMR characterisation of lipid membranes in intact bacteria[END_REF] are both Gram(-) bacteria, differences in 2 H SS-NMR spectra between these two species were expected, as they may be related to species-specific biological requirements [START_REF] Farmer | The family Vibrionaceae, in The prokaryotes[END_REF][START_REF] Gauthier-Clerc | In vivo exposure of Mytilus edulis to living enteric bacteria: a threat for immune competency?[END_REF].

The capacity of the bacteria to grow under our labelling conditions was first verified. While deuteration of molecules such as FAs does not affect their structure and thermodynamical Figure 4: 2 H MAS (10 kHz) SS-NMR spectra of intact V. splendidus harvested at three different cell growth times: (A) after 15 h in the mid-log phase (OD 600nm ≈ 0.3), (B) after 22 h (±2h) at the beginning of the stationary stage (OD 600nm ≈0.5), and (C) after 30 h at advanced stationary phase (OD 600nm ≈ 0.5). Bacteria were labelled with d 31 -PA in presence of OA. Average second spectral moments M 2 are indicated (10 9 s -2 ). stability, surfactants used to facilitate the deuterated FA incorporation into the bacteria [START_REF] Dal Molin | 13 C-isotope labelling for the facilitated NMR analysis of a complex dynamic chemical system[END_REF], could have a detrimental effect since detergents are commonly used for biochemical applications such as extraction of membrane proteins. Therefore, the physiological state of the bacteria during labelling procedure was monitored using growth kinetics, and our results showed that Tween-20 had the lowest impact. This result is in agreement with Schuck et al. who compared sensitivities of diverse cells toward a set of detergents and showed that Tween-20 was always the least effective in provoking membrane resistance, and the best at preserving membrane integrity [START_REF] Schuck | Resistance of cell membranes to different detergents[END_REF].

Tween-20 has a low CMC, does not affect protein activity, and is rarely used for cell lysis or protein extraction, but rather as a gentle washing agent. [START_REF] Schuck | Resistance of cell membranes to different detergents[END_REF][START_REF] Johnson | Detergents: Triton X-100, tween-20, and more[END_REF]. On the other hand, although harmless for the first 10 hours of culture, the zwitterionic detergent DPC used to deuterate E. coli and B. subtilis lipids showed a hampering action on V. splendidus at longer exposure time, which could be explained by its ability to break protein-lipid and lipid-lipid associations [START_REF] Johnson | Detergents: Triton X-100, tween-20, and more[END_REF][START_REF] Le Maire | Interaction of membrane proteins and lipids with solubilizing detergents[END_REF].

De novo synthesis of FAs is the most energetically expensive mechanism in phospholipid synthesis for bacteria membrane [START_REF] Zhang | Transcriptional regulation in bacterial membrane lipid synthesis[END_REF]. Because they do not have specialized functional cellular compartments, prokaryotic organisms rely on multiple regulation tools to control their phospholipid composition according to the niche in which they live [START_REF] Byers | Elongation of exogenous fatty acids by the bioluminescent bacterium Vibrio harveyi[END_REF][START_REF] Zhang | Transcriptional regulation in bacterial membrane lipid synthesis[END_REF][START_REF] Freese | Gammaproteobacteria as a possible source of eicosapentaenoic acid in anoxic intertidal sediments[END_REF][START_REF] Shibuya | Metabolic regulation and biological functions of phospholipids in Escherichia coli[END_REF][START_REF] Barton | Cellular Growth and Reproduction[END_REF]. Overall, bacteria tend to sustain their membrane homeostasis through adaptation mechanisms by maintaining zwitterionic/anionic, protein/lipid [START_REF] Shibuya | Metabolic regulation and biological functions of phospholipids in Escherichia coli[END_REF] or FA chain ratios [START_REF] Silbert | Fatty acid replacements in a fatty acid auxotroph of Escherichia coli[END_REF]. The SFA/UFA ratio is one of the most used reporters of membrane fluidity [START_REF] Beney | Influence of the fluidity of the membrane on the response of microorganisms to environmental stresses[END_REF][START_REF] Mykytczuk | Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress[END_REF][START_REF] Yoon | Membrane fluidity-related adaptive response mechanisms of foodborne bacterial pathogens under environmental stresses[END_REF] as well as bacterial adaptive strategies [START_REF] Beney | Influence of the fluidity of the membrane on the response of microorganisms to environmental stresses[END_REF][START_REF] Mykytczuk | Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress[END_REF][START_REF] Yoon | Membrane fluidity-related adaptive response mechanisms of foodborne bacterial pathogens under environmental stresses[END_REF][START_REF] Rock | Pathways for the incorporation of exogenous fatty acids into phosphatidylethanolamine in Escherichia coli[END_REF][START_REF] Denich | Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes[END_REF]. Our results showed that when PA was added in the growth medium of V. splendidus without OA, bacteria adapted by synthesizing more cyclic fatty acids (cyC17:0 and cyC19:0) that restored membrane fluidity. Similarly, when OA was also added and despite the fact that OA is not the major UFA in V. splendidus, this FA was highly integrated in the exponential phase, resulting in a FA profile similar to that of unlabelled bacteria. The SFA/UFA ratios determined here confirm the need to enrich the medium with UFAs during labelling with d 31 -PA in order to avoid extreme shifts from the natural lipid composition (Fig. 3), in agreement with previous work carried out on E. coli [START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF][START_REF] Warnet | A 2 H magic-angle spinning solid-state NMR characterisation of lipid membranes in intact bacteria[END_REF][START_REF] Pius | 2 H solid-state nuclear magnetic resonance investigation of whole Escherichia coli interacting with antimicrobial peptide MSI-78[END_REF].

In short, our lipid deuteration protocol on V. splendidus allowed a similar or better labelling rate as compared to previous work on bacteria. Lipid profile analyses showed 77% deuteration of C16:0 acyl chains at mid-log when d 31 -PA was added to the growth medium, while a 69% labelling was determined when equal proportions of d 31 -PA and OA were employed. For both protocols (with and without OA), the deuteration level is thus higher than what was reported for E. coli, i.e., 60-69% for bacteria grown in the presence of d 31 -PA [START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF][START_REF] Warnet | A 2 H magic-angle spinning solid-state NMR characterisation of lipid membranes in intact bacteria[END_REF]. Except for minor differences in the FA incorporation process [START_REF] Byers | Elongation of exogenous fatty acids by the bioluminescent bacterium Vibrio harveyi[END_REF] and a different culture time lapse, the lipid metabolism of Vibrio species is very similar to E. coli's since they are both Gram(-) bacteria with similar transcriptional regulatory genes for FA metabolism and phospholipid composition [START_REF] Byers | Elongation of exogenous fatty acids by the bioluminescent bacterium Vibrio harveyi[END_REF][START_REF] Zhang | Transcriptional regulation in bacterial membrane lipid synthesis[END_REF][START_REF] Silbert | Fatty acid replacements in a fatty acid auxotroph of Escherichia coli[END_REF][START_REF] Rock | Pathways for the incorporation of exogenous fatty acids into phosphatidylethanolamine in Escherichia coli[END_REF].

Membrane evolution during bacterial growth

Our results showed that the membrane fluidity varied with bacterial growth during the labelling process. Indeed bacteria experience the fastest growth rate in the exponential phase during which they produce membrane lipids [START_REF] Barton | Cellular Growth and Reproduction[END_REF]. Once lipids are synthesized, the lipid profile remains the same until the stationary phase is reached, i.e., when nutrients become limited, DNA replication, protein synthesis and respiration are reduced, and cell lysis begins [START_REF] Carty | Lipid synthesis during the Escherichia coli cell cycle[END_REF]. Previous in vivo NMR studies have been carried out on bacteria sampled at the mid-log growth stage [START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF][START_REF] Warnet | A 2 H magic-angle spinning solid-state NMR characterisation of lipid membranes in intact bacteria[END_REF][START_REF] Pius | 2 H solid-state nuclear magnetic resonance investigation of whole Escherichia coli interacting with antimicrobial peptide MSI-78[END_REF][START_REF] Tardy-Laporte | A 2 H solid-state NMR study of the effect of antimicrobial agents on intact Escherichia coli without mutating[END_REF] which provides good NMR signal-to-noise ratio. However, our results revealed that samples obtained at later stages, especially the late exponential stage, provide additional advantages. Indeed GC-MS analyses showed that exogenous PA and OA were highly integrated in the membrane at mid-log phase during which they become the most abundant FA chains in the membrane (Figure 3). In subsequent growth stages, PA and OA diminished to reach more "native" proportions (i.e., those measured in the control samples), either by being converted into other lipid chains or by triggering the biosynthesis of other FAs. This is consistent with other investigations on Vibrio species, showing that exogenous FAs are incorporated by direct transacylation or after incorporation into the biosynthesis pool [START_REF] Byers | Elongation of exogenous fatty acids by the bioluminescent bacterium Vibrio harveyi[END_REF]. Additionally the SFA/UFA ratio, which decreased as a function of growth time, reaching that of unlabelled bacteria (especially when both PA and OA were added to the culture medium), validate the evolution of the FA profile. The 2 H SS-NMR spectra also reveal a decrease in membrane fluidity as a function of V. splendidus growth stages when d 31 -PA, and to a lesser extent when equal proportions of OA and d 31 -PA, are added to the growth medium (Figure 4). In the exponential phase, a more rigid membrane was observed, close to a gel phase (M 2 above 20×10 9 s -2 ), whereas in later stages the M 2 value was closer to that of a fluid phase membrane (M 2 below 10×10 9 s -2 ) [START_REF] Warnet | A 2 H magic-angle spinning solid-state NMR characterisation of lipid membranes in intact bacteria[END_REF]. Since the FA composition and SFA/UFA ratio were closer to their "native" values in the late exponential phase, the lower M 2 values would thus report with biological membranes in the fluid phase [START_REF] Morein | Wild-type Escherichia coli cells regulate the membrane lipid composition in a window between gel and non-lamellar structures[END_REF].

The contribution of signal from free d 31 -PA cannot explain the change in lipid profile during cell growth. Indeed Figure 2A shows no 2 H SS-NMR signal after rinsing. Additionally, free d 31 -PA is insoluble in water and would lead to a very broad 2 H SS-NMR spectrum with M 2 above 70×10 9 s -2 (data not shown), which is incompatible with our results. Moreover, the 2 H SS-NMR spectrum of d 31 -PA crystals would not vary with growth time. In light of these results, the increase in membrane fluidity with cell growth time would be explained by a gradual adaptation of the microorganism metabolism upon isotopic labelling to cope with possible stress and disturbance of metabolic pathways [START_REF] Beney | Influence of the fluidity of the membrane on the response of microorganisms to environmental stresses[END_REF][START_REF] Mykytczuk | Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress[END_REF][START_REF] Yoon | Membrane fluidity-related adaptive response mechanisms of foodborne bacterial pathogens under environmental stresses[END_REF] due to chemical isotopic labelling. In most cases, stress is known to lead to an increase in membrane rigidity, although the opposite pattern could happen in the case of marine prokaryotic organisms, and specifically for Vibrio species [START_REF] Oliver | Lipid composition of a psychrophilic marine Vibrio sp. during starvation-induced morphogenesis[END_REF][START_REF] Yoon | Membrane fluidity-related adaptive response mechanisms of foodborne bacterial pathogens under environmental stresses[END_REF].

The increased fluidity due to increased UFA proportions, and notably cyclopropanes FAs observed when PA was added in the growth medium, seems to prove such environmental stress [START_REF] Yoon | Membrane fluidity-related adaptive response mechanisms of foodborne bacterial pathogens under environmental stresses[END_REF][START_REF] O'leary | The fatty acids of bacteria[END_REF]. However, when both PA and OA were present in the medium, cyclopropane FA proportions were inverted (Table SI1), suggesting that stress was reduced [START_REF] O'leary | The fatty acids of bacteria[END_REF]. Similarly, the superimposed growth curves (see Fig. SI1) indicate that V. splendidus metabolism kinetics under isotopic labelling was not affected by labelling stress. Therefore, the FAs turnover would be fast and all exogenous d 31 -PA transferred into the membrane lipids at the mid-log phase. Yet, being long and saturated, they would induce large M 2 values. In the late exponential phase, bacteria would have had enough time to regulate their metabolism to the presence of exogenous FAs in the growth medium, generally through adapting their enzymatic machinery [START_REF] Barton | Cellular Growth and Reproduction[END_REF], and have either converted into or synthesized new UFAs in order to reduce the membrane rigidity, and reach their initial membrane state, as observed by the reduced M 2 values.

CONCLUSION AND PERSPECTIVES

Setting out labelling procedures is an important step towards developing effective and reliable tools for in vivo NMR studies. Here, membranes of intact V. splendidus were characterized for the first time by SS-NMR, as a model of marine Gram(-) bacteria. This work provides a better understanding of changes in bacterial membrane properties as a function of growth time and conditions that have never been studied so far on any biological system by in vivo NMR. Overall, the present study should help in the design of efficient labelling protocols of bacteria, which takes into account not only the technical spectroscopy requirements for analyses (such as signal-tonoise ratio) but also biological considerations. This 2 H labelling protocol is useful for 2 H SS-NMR, but it could be useful for neutron diffraction, and could also be transposed to the labelling of cell membranes by other isotopes such as 13 C. Additionally, by characterizing the membrane of V. splendidus, a potential virulent bacterium for marine organisms, this work paves the way towards studying the interactions of its membrane with exogenous molecules in the aquatic environment.

  also shows that supplementing the culture medium with oleic acid (OA) in addition to d 31 -PA leads to a reduction in side bands intensities, indicating an increase in membrane fluidity further demonstrated by the decrease in M 2 value. These findings suggest that both unsaturated (OA) and saturated (d 31 -PA) FAs were integrated by the cell. The in vivo NMR conditions during V. splendidus analysis were confirmed by estimating the bacterial viability, which was 95 ± 5 %.

Figure 1 :

 1 Figure 1: Effect of different surfactants on V. splendidus growth. Control (a) experiment using the growth medium, and was compared to the following detergents at their CMC: Tween-20 (b), Brij-35 (c), DPC (d), Triton-X (e), CHAPS (f) and OG (g). Tween-20 was also tested at 0.25 mM (h).

Figure 2 :

 2 Figure 2 : In vivo 2 H MAS (10 kHz) SS-NMR spectra of V. splendidus harvested in the mid-log phase. Control experiment corresponding to bacteria labelled with d 31 -PA without detergent (A), bacteria labelled with d 31 -PA in the presence of Tween-20 (0.14 mM) without (B), and with (C) OA (1:1). Spectra are normalized according to the central peak. Average second spectral moments M 2 are indicated (10 9 s -2 ).

ACKNOWLEDGMENTS

This work was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada (grant 326750-2013 to I.M. and grant 299100 to R.T.) and the Centre National de la Recherche Scientifique (UMR 7099 to D.E.W.). Z.B. would like to acknowledge the Ressources Aquatiques Québec (RAQ) research network (RS-171172) for the award of scholarships and Pierre Audet for sharing the MestRenova macro. I.M. and R.T. are members of the RAQ.

COMPETING INTERESTS

No competing interests declared.

AUTHOR CONTRIBUTIONS

Z.B. designed and conducted all sampling, data collection and analysis, and wrote the first draft of the manuscript. D.E.W. and A.A. contributed to the data analysis and writing of the manuscript. K.L. provided the initial Vibrio strain culture and participated to the revision of the manuscript. R.T. and I.M. designed and supervised the research, contributed to the data analysis and writing of the manuscript.