Jens Baumgartner

Maria Antonietta Carillo

Kevin M Eckes

Peter Werner

Damien Faivre

Biomimetic Magnetite Formation: From Biocombinatorial Approaches to Mineralization Effects

Biological materials typically display complex morphologies and hierarchical architectures, properties that are hardly matched by synthetic materials. Understanding the biological control of mineral properties will enable the development of new synthetic approaches toward biomimetic functional materials. Here, we combine biocombinatorial approaches with a proteome homology search and in vitro mineralization assays to assess the role of biological determinants in biomimetic magnetite mineralization. Our results suggest that the identified proteins and biomimetic polypeptides influence nucleation in vitro. Even though the in vivo role cannot be directly determined from our experiments, we can rationalize the following design principles: proteins, larger complexes, or membrane components that promote nucleation in vivo are likely to expose positively charged residues to a negatively charged crystal surface. In turn, components with acidic (negatively charged) functionality are nucleation inhibitors, which stabilize an amorphous structure through the coordination of iron.

INTRODUCTION

Nature has evolved biominerals with complex morphologies and hierarchical architectures that are hardly matched by synthetic materials so far. 1 Understanding the exquisite control exerted by organisms over mineral properties might enable the exploitation of natural design principles for the development of biomimetic functional materials under physiological and environmentally friendly conditions. [START_REF] Weiner | Biomineralization: A Structural Perspective[END_REF][START_REF] Fratzl | Biomimetic Materials Research: What Can We Really Learn from Nature'S Structural Materials[END_REF][START_REF] Nudelman | Biomineralization as an Inspiration for Materials Chemistry[END_REF] However, in many biomineralizing systems it is currently unclear which of the many biological determinants are critical in controlling particular material properties or steps in their formation such as synthesis, nucleation, growth, and morphogenesis. In cases where the molecular players are known, the mechanisms by which they interact with inorganic phases have often remained elusive.

A typical strategy for the identification of involved molecules is their extraction from an organism and the characterization of biomolecules that bind an isolated mineral phase. [START_REF] Weiner | Biomineralization: A Structural Perspective[END_REF] The effect of the identified molecules on mineralization can then be studied in vitro. Examples have been reported for biogenic silica, [START_REF] Kroger | Polycationic Peptides from Diatom Biosilica That Direct Silica Nanosphere Formation[END_REF] magnetite, [START_REF] Amemiya | Controlled Formation of Magnetite Crystal by Partial Oxidation of Ferrous Hydroxide in the Presence of Recombinant Magnetotactic Bacterial Protein Mms6[END_REF][START_REF] Arakaki | A Novel Protein Tightly Bound to Bacterial Magnetite particles in Magnetospirillum magnetotacticum Strain AMB-1[END_REF][START_REF] Prozorov | Protein-Mediated Synthesis of Uniform Superparamagnetic Magnetite Nanocrystals[END_REF] and calcium carbonate. [START_REF] Politi | Asprich Mollusk Shell Protein: In Vitro Experiments Aimed at Elucidating Function in CaCO 3 Crystallization[END_REF] As an alternative synthetic approach to studying biomimetic molecular structures that interact with solids, the biocombinatorial selection of solid-binding peptides has developed into a powerful technique to identify short peptides with specific affinities for a large range of inorganic materials. [START_REF] Chen | Peptide-Based Methods for the Preparation of Nanostructured Inorganic Materials[END_REF][START_REF] Sarikaya | Materials Assembly and Formation Using Engineered Polypeptides[END_REF] Recent examples are selections for the binding of demosponge spicule silica, [START_REF] Muller | The Silicatein Propeptide Acts as Inhibitor/Modulator of Self-Organization during Spicule Axial Filament formation[END_REF] synthetic silica, [START_REF] Patwardhan | Chemistry of Aqueous Silica Nanoparticle Surfaces and the Mechanism of Selective Peptide Adsorption[END_REF] ZnO, [START_REF] Rothenstein | Isolation of ZnO-Binding 12-mer Peptides and Determination of Their Binding Epitopes by NMR Spectroscopy[END_REF] and GdO. [START_REF] Schwemmer | Peptide-Mediated Nanoengineering of Inorganic Particle Surfaces: A General Route toward Surface Functionalization via Peptide Adhesion Domains[END_REF] Because the selections can be performed under close-to-physiological conditions, the question has arisen as to whether natural and synthetic selection evolves molecules with similar characteristics and whether the biomineralizing functionality might be encoded in homologue structures for materials also found in organisms.

Here we investigated the example of the iron oxide mineral magnetite that is found in diverse organisms (bacteria, mollusks, birds, and fish) and where it serves geonavigational or mechanical purposes. Its biogenic formation is best studied in magnetotactic bacteria, which form chains of magnetic nanoparticles termed magnetosomes. [START_REF] Faivre | Magnetotactic Bacteria and Magnetosomes[END_REF] Because of their size and high monodispersity, magnetosomes are envisioned for MRI contrast agents and cancer treatment applications. [START_REF] Lisy | Fluorescent Bacterial Magnetic Nanoparticles As Bimodal Contrast Agents[END_REF] Furthermore, similarly structured synthetic magnetic nanoparticle assemblies have recently attracted much attention. [START_REF] Hu | High Yield Synthesis of Bracelet-like Hydrophilic Ni-Co Magnetic Alloy Flux-Closure Nanorings[END_REF][START_REF] Lu | Biogenic and Biomimetic Magnetic Nanosized Assemblies[END_REF][START_REF] Gao | Self-Assembled Platinum Nanochain Networks Driven by Induced Magnetic Dipoles[END_REF] Simple magnetotactic organisms have turned into a model system for iron oxide biomineralization because the genomes of several strains have been sequenced [START_REF] Jogler | Genetics, and Cell Biology of Magnetosome Formation[END_REF] and because molecular techniques have been developed for their genetic manipulation. [START_REF] Schultheiss | Development of a Genetic System for Magnetospirillum gryphiswaldense[END_REF][START_REF] Matsunaga | Gene Transfer in Magnetic Bacteria: Transposon Mutagenesis and Cloning of Genomic DNA Fragments Required for Magnetosome Synthesis[END_REF] In particular, a whole set of deletion mutants has been studied in Magnetospirillum strains, with phenotypes ranging from size and morphology changes to the complete disappearance of biomineralization. [START_REF] Murat | Comprehensive Genetic Dissection of the Magnetosome Gene Island Reveals the Step-Wise Assembly of a Prokaryotic organelle[END_REF] It has been shown that about 20 genes are sufficient to restore magnetite formation in cells deficient of the whole magnetosome island, the gene cluster responsible for magnetite biomineralization. [START_REF] Lohße | Functional Analysis of the Magnetosome Island in Magnetospirillum gryphiswaldense: The mamAB Operon Is Sufficient for Magnetite Biomineralization[END_REF][START_REF] Murat | The Magnetosome Membrane Protein, MmsF, Is a Major Regulator of Magnetite Biomineralization in Magnetospirillum magneticum AMB-1[END_REF] The encoded Mam, Mms, and Mtx proteins are therefore good potential candidates for comparison with synthetically selected molecules and subsequent in vitro mineralization studies. Furthermore, biocombinatorial peptide selection studies on magnetite have been reported earlier, which provide a basis for such a comparison (Figure 1). Using the biocombinatorial techniques of cell surface and phage display, Brown et al. and Barbas et al. had independently shown that polycationic polypeptides attach to magnetite or possibly to the very similar maghemite crystal surfaces. [START_REF] Brown | Engineered Iron Oxide-Adhesion Mutants of the Escherichia-Coli Phage-Lambda Receptor[END_REF][START_REF] Barbas | Direct Selection of Antibodies That Coordinate Metals from Semisynthetic Combinatorial Libraries[END_REF] In this work, our idea is not to use phage display directly for the direct assessment of 12 amino acid sequences on mineralization but rather to provide an alternative route toward the identification of putative biomineralizing proteins without the need for in vivo mutant generation. We thus combine biocombinatorial approaches with a proteome homology search and assess in vitro the role of the identified proteins and associated biomimetic polypeptides in the mineralization of magnetite. Our results suggest that the macromolecules indeed influence nucleation in vitro.

EXPERIMENTAL SECTION

2.1. Phage Display. The Ph.D.-12 Phage Display Peptide Library (New England Biolabs) with approximately 2.7 × 10 9 random 12-mer peptide sequences was used for selections. Two independent selections were performed on magnetite powder. In a first experiment, ≤10 mg magnetite (Sigma-Aldrich, ≤5 μm particle size) was exposed to ∼4 × 10 10 phages in 1 mL of Tris-buffered saline (TBS) and 0.1% Tween-20 (TBST, pH 7.5) for 30 min. The dispersed magnetite with bound phages was trapped on a magnetic flow-through column (MACS, Miltenyi Biotec) and washed 10 times with 1 mL of TBST. Bound phages were eluted with a low-pH buffer (0.2 M glycine-HCl, pH 2.2), neutralized with 1 M Tris-HCl (pH 9.1), and amplified by the infection of E. coli ER2738 in liquid culture. After 4.5 h, growth phages were isolated and concentrated by the removal of the bacterial cells through centrifugation and repeated precipitation with an aqueous PEG-8000 (20% w/v)/NaCl (2.5 M) solution. The amplified phages were used for the next round of selection on the mineral. In subsequent rounds, the surfactant concentration was raised by 0.1%. Individual phage clones were picked for sequencing after three and six panning rounds. Phage DNA was isolated with the M13 phage DNA isolation kit (Qiagen), sequenced (Eurofins MWG Operon), and translated into the encoded peptide. In a second experiment, the incubation time was reduced to 10 min to select for binders with a faster binding rate k on . The surfactant concentration was kept constant at 0.5% in both the incubation and washing phases to increase the initial stringency. Magnetite was kept in a 1.5 mL reaction tube, washed repeatedly (10 times) by precipitation with a magnet, resuspended in 1 mL of TBST, and transferred to a new reaction tube to prevent the potential selection of tube-material-binding peptides. Individual phages were picked and amplified for sequencing after four and six rounds of panning.

2.2. Peptide Characterization and Sequence Similarity Search. Sequence characteristics were determined using the ExPasy ProtParam tool. [START_REF] Gasteiger | Protein Identification and Analysis Tools on the ExPASy Server[END_REF] Peptides were compared to the M. magneticum (AMB-1), M. marinus (MC-1), M. magnetotacticum (MS-1), M. gryphiswaldense (MSR-1), and D. magneticus (RS-1) proteomes using the BLASTP 2.2.28+ tool on http://blast.ncbi.nlm.nih.gov/ 30 using the default parameters (word size, 3; BLOSUM62 matrix; conditional compositional score matrix adjustment; gap costs, existence 11/ extension 1; without low complexity filtering). Proteins for precipitation experiments were chosen on the basis of the lowest e values obtained for proteins unique to magnetotactic bacteria (MSR-1 MamJ, e = 1.1; MSR-1 MtxA, e = 0.12).

2.3. Proteins. Genes mamJ, mtxA, and mtxA Δ1-24 were amplified from M. gryphiswaldense MSR-1 genomic DNA by PCR (oligonucleotides in Table S1, produced by MWG Operon) using KOD polymerase (Novagen), purified, and ligated into a pET-51b(+)Ek/ LIC vector (Novagen). mtxA Δ1-24 refers to the DNA sequence encoding for the MtxA Δ1-24 protein without the leading N-terminal 24 amino acids, which were identified as a signal peptide. MamJ was expressed in E. coli Rosetta 2 (DE3) (Novagen). MtxA and Mtx Δ1-24 were expressed in E. coli BL21 (DE3) (Novagen). Precultures were grown in 5 mL of LB medium plus 100 μg mL -1 ampicillin overnight at 37 °C with 250 rpm stirring. Culture batches of 250 mL of autoinducing ZYM-5052 medium supplied with 100 μg mL -1 ampicillin were inoculated with 0.1% of the preculture and grown for 24 h at 30 °C with 250 rpm stirring. [START_REF] Studier | Protein Production by Auto-Induction in High-Density Shaking Cultures[END_REF] Cells were pelleted by centrifugation (4 °C, 4000 rpm, 15 min) and stored at -80 °C. Thawed cells were resuspended in 10 mL of Strep-Tactin wash buffer (Merck) supplied with 1 mg mL -1 lysozyme and 1 mM PMSF. After 30 min of incubation on ice, cells were lysed by sonication (10 × 15 s burst with 15 s pauses). The cell lysate was cleared by centrifugation (21 000 rpm, 4 °C, 45 min) and applied to a 5 mL bed Strep-Tactin SuperFlow agarose column (IBA). Washing and elution at 4 °C followed the manufacturer's protocols using the respective buffers. Fractions were analyzed by SDS-PAGE using Coomassie-stained 4-20% precast linear gradient polyacrylamide gels (Bio-Rad). If the SDS-PAGE indicated impurities, then pooled His-tagged proteins were subjected to a second round of affinity chromatography on Ni-IDA matrix columns (Macherey-Nagel) at 4 °C. Generally proteins were stored at 4 °C in the respective elution buffers. The protein identity was verified by ESI-MS fingerprinting after trypsine digestion. Before magnetite coprecipitation experiments, protein solutions were dialyzed against Milli-Q water for 24 h with three solvent exchanges using ServaPor dialysis tubing with a 12-14 kDa molecular weight cutoff. Protein concentrations were determined by UV absorption at 280 nm on an Implen NanoPhotometer. Molecular weights and extinction coefficients were calculated on the basis of the sequence of the overexpressed proteins using the ProtParam tool on the ExPasy server. [START_REF] Gasteiger | Protein Identification and Analysis Tools on the ExPASy Server[END_REF] MamJ has a molecular weight of 48 481 Da and absorbs 40 115 M -1 cm -1 . MtxA Δ1-24 has a mass of 34 774 Da and absorbs 33 920 M -1 cm -1 . Concentrations were adjusted to 1 mg mL -1 by dilution with Milli-Q water or concentration on U-tube concentrator (30 kDa molecular weight cutoff, Novagen) tubes by centrifugation at 4 °C.

2.4. Precipitation Experiments. Reactions were performed with a computer-controlled titration system (Metrohm AG) consisting of a 776 Dosimat dosing device with an 806 exchange unit (1 mL dosing cylinder) and a 719 Titrino titration device with an 806 exchange unit (5 mL dosing cylinder). Microloader tips (Eppendorf) were used as inlets into the reaction vessel. Reactions were performed in a 50 mL vessel with a thermostated jacket kept at constant temperature (25.0 ± 0.1 °C) by a thermostat (Lauda M3). Solutions were stirred at 2050-2250 min -1 with a mechanical stirrer. The pH was measured using a Biotrode pH meter (Metrohm). All experiments were performed under a nitrogen atmosphere. Ten milliliters minus the additive volume of deionized water was initially set to the pH of interest with NaOH. In case of precipitation in the presence of an additive, the additive was then supplied and the pH was reset. Magnetite precipitation was initiated by the addition of an iron chloride solution (Fe 3+ /Fe 2+ = 2/1) to the reactor at a rate of 1 μL min -1 . The pH of the solution in the reaction vessel was simultaneously kept constant (ΔpH ±0.1) by the addition of sodium hydroxide.

2.5. Transmission Electron Microscopy. Particles were adsorbed from aqueous suspensions to carbon film Cu TEM grids for 15 min. After the removal of the liquid with Kimwipe paper, grids were washed with a drop of Milli-Q water to remove residual salt precipitates. Standard imaging performed on a Zeiss EM 912 Omega at an acceleration voltage (U) of 120 kV. High-resolution imaging was performed on (i) a Jeol JEM 4010 transmission electron microscope (U = 400 kV) and on (ii) an FEI Titan 80/300 scanning transmission electron microscope (U = 300 kV) equipped with a probe corrector, an EDX detector, and an EELS spectrometer.

2.6. X-ray Diffraction. Precipitates were studied by synchrotron wide-angle X-ray diffraction at the μ-Spot beamline, BESSY II, Berlin. Samples were dried on a Kapton thin film (Breitlander GmbH) clamped on a custom-made sample holder. The beam size was set to 100 μm with an energy of 15 keV (λ = 0.82656 Å) defined by a Si(111) double-crystal monochromator. Diffraction data was acquired on a 3072 pixel × 3072 pixel MarMosaic 225 CCD camera (Mar USA) with a 73.242 μm pixel size. For data analysis, the beam center and the detector tilt were determined and corrected for using the respective routine of the Fit2D software. [START_REF] Hammersley | The FIT2D Home Page[END_REF] Peaks were fitted after baseline correction by a pseudo-Voigt function. Mean particle diameters were estimated under the neglect of strain-induced broadening with the Scherrer equation.

RESULTS AND DISCUSSION

We first compared the published magnetite-adhering peptide sequences (RRTVKHHN, RRSRHH, RSKRGR, RSKKMR, and RFKRVRDR) to the proteomes of the well-studied magnetotactic bacterial strains Magnetospirillum magneticum AMB-1, Magnetococcus marinus MC-1, Magnetospirillum magnetotacticum MS-1, Magnetospirillum gryphiswaldense MSR-1, and Desulfovibrio magneticus RS-1. [START_REF] Brown | Engineered Iron Oxide-Adhesion Mutants of the Escherichia-Coli Phage-Lambda Receptor[END_REF][START_REF] Barbas | Direct Selection of Antibodies That Coordinate Metals from Semisynthetic Combinatorial Libraries[END_REF] However, none of the sequences are similar to Mam-, Mms-, or Mtx-type proteins. We therefore performed new sequence pannings on magnetite particles using a randomized 12-mer phage display library to generate additional sequences (Table S2). In contrast to the earlier reported results, sequence alignments revealed neither a common motif among the retrieved peptides nor clear characteristics regarding amino acid composition, the isoelectric point (pI = 7.86 ± 2.00) or the hydrophobicity (average hydropathy = -0.77 ± 0.68 [START_REF] Kyte | A Simple Method for Displaying the Hydropathic Character of a Protein[END_REF] ). We compared the resulting sequences by alignment with the proteomes of the aforementioned magnetotactic bacterial strains because our goal was to identify new putative biomineralizing macromolecules within the proteome of magnetotactic bacteria. Because of the short length of the peptides as compared to that of typical proteins, alignments yield a large number of random hits when searching entire genomes. Most sequences are dissimilar or only weakly similar to magnetosomal proteins. However, two sequences (out of 27) stood out because each one represented the best respective hits (lowest e values) within the search space encompassing the complete AMB-1, MC-1, MS-1, MSR-1, and RS-1 proteomes and could be attributed to proteins MamJ and MtxA of MSR-1 with known or hypothesized magnetosome or magnetotaxis functionality (Tables S3 andS4). We then overexpressed both proteins in E. coli for in vitro studies. The leading 24 N-terminal amino acids of MtxA represent a membrane translocation signal peptide (Figures S1-S3) that renders the protein insoluble when overexpressed in E. coli. Therefore, we used MtxA Δ1-24 without a signal peptide for in vitro assays.

We studied the influence of the selected proteins (MamJ and MtxA Δ1-24 ) as well as the two peptide polymers poly-L-arginine (polyR) and poly-L-glutamic acid (polyE) on magnetite formation. Apart from structural differences, the proteins/ polymers differ primarily in the availability of differently charged groups that can interact with different iron or iron (oxyhydr)oxide species. Acid residues in MamJ and polyE provide binding moieties for cationic Fe II/III , whereas the cationic guanidinium group of polyR is able to interact electrostatically with (in alkaline solution) negatively charged iron (oxyhydr)oxide crystal surfaces. The presence of the additives has a strong effect on the phase, crystallinity, particle size, morphology, and aggregation of the precipitates, in agreement with the interactions with soluble or solid iron species that occur either prior to or after the nucleation of the magnetite phase (Figures 234).

3.1. Polycationic Stabilization of Magnetite Nanoparticles by Poly-L-Arginine. polyR serves as a proxy for a potential polycationic biomolecular structure as inferred from the earlier literature reports by Barbas et al. and Brown et al. [START_REF] Brown | Engineered Iron Oxide-Adhesion Mutants of the Escherichia-Coli Phage-Lambda Receptor[END_REF][START_REF] Barbas | Direct Selection of Antibodies That Coordinate Metals from Semisynthetic Combinatorial Libraries[END_REF] Coprecipitation of ferrous and ferric iron (at Fe 3+ /Fe 2+ = 2/1) under alkaline conditions at pH ≥9 yields crystalline magnetite with grain sizes dependent on the alkalinity. [START_REF] Baumgartner | Nucleation and Growth of Magnetite from Solution[END_REF][START_REF] Baumgartner | Formation of Magnetite Nanoparticles at Low Temperature: From Superparamagnetic to Stable Single Domain Particles[END_REF] Under these conditions, polyR affects the size, morphology, and aggregation behavior of the formed magnetite nanoparticles: in its presence, we obtained monodisperse, stable single-domainsized nanoparticles of 35 ± 5 nm (Figure 3) that assemble to chain structures in solution (up to several micrometers; Figures 2A andS4-S6 and a video). Despite their irregular morphology, particles are mostly single-crystalline (Figure 2C). The nucleation and colloidal stabilization effects of polyR, leading to particle chain formation in vitro, are similar to the colloidal stabilization by magnetosome compartimentalization in the bacteria in vivo. This compartimentalization is provided by a lipid membrane containing diverse transmembrane proteins of yet mostly unknown functions. Interestingly, the lipid composition of the magnetosomes is dominated by phosphatidylethanolamines. [START_REF] Grunberg | Biochemical and Proteomic Analysis of the Magnetosome Membrane in Magnetospirillum gryphiswaldense[END_REF] Such lipid layers therefore expose mainly positively charged amines toward the intracellular magnetite crystals in line with our observation of a polyR-induced colloidal stabilization effect.

3.2. Polyanionic Inhibition of Magnetite Nucleation by MamJ and Poly-L-glutamic Acid. In contrast, MamJ and polyE (which resembles the polyanionic domain found in MamJ [START_REF] Scheffel | An Acidic Protein Aligns Magnetosomes along a Filamentous Structure in Magnetotactic Bacteria[END_REF][START_REF] Scheffel | The Acidic Repetitive Domain of the Magnetospirillum gryphiswaldense MamJ Protein Displays Hypervariability but Is Not Required for Magnetosome Chain Assembly[END_REF] ) strongly affect the phase of the formed precipitates by the inhibition of magnetite nucleation with increasing additive concentration: in both cases, we obtained either amorphous gels or crystalline phases other than magnetite. Only at low additive concentrations (0.01 mg mL -1 ), magnetite could be obtained (Figure 4). At pH 11, in the presence of 0.01 mg mL -1 MamJ, diffraction patterns are consistent with an extremely small nanosized magnetite (Figure 5, line features are extremely broadened magnetite peaks, intensity increase toward low q). At pH 9, we obtained unidentifiable mixtures (Figure 4, peaks at q = 19.13, 26.66, 32.57, 34.03, 41.16, and 45.42 nm -1 ; d = 0.33, 0.24, 0.19, 0.18, 0.15, and 0.14 nm). With 0.1 mg mL -1 MamJ, we obtained a poorly ordered pattern at pH 9 (Figure 4, possibly ferrihydrite) and at pH 11 (Figure 5) a pattern with two distinct peaks at q = 15.51 nm -1 (d = 0.41 nm) and q = 25.25 nm -1 (d = 0.25 nm), which are consistent with goethite (α-FeOOH). Accordingly, in the presence of 0.1 mg mL -1 polyE we obtained no crystalline material within 1 h: at pH 9, we obtained an orange amorphous gel-like precipitate by centrifugation from a clear solution, whereas at pH 11 no pellet could be formed even by centrifugation. At low concentration (0.01 mg mL -1 ), polyE has no strong impact on the formed phase (Figure 4).

MamJ is known to be involved in the magnetosome chain assembly and has been suggested to serve as an anchor to the MamK filament in the cell. [START_REF] Scheffel | An Acidic Protein Aligns Magnetosomes along a Filamentous Structure in Magnetotactic Bacteria[END_REF] The anchoring function is provided exclusively by the N-and C-terminal domains without the involvement of a repetitive central sequence stretch rich in glutamic acid. [START_REF] Scheffel | The Acidic Repetitive Domain of the Magnetospirillum gryphiswaldense MamJ Protein Displays Hypervariability but Is Not Required for Magnetosome Chain Assembly[END_REF] The role of this polyanionic domain has remained unclear but was initially suspected to take part in biomineralization through the binding and accumulation of iron. The found consensus sequence PVA-LVNR can be located twice within this repetitive stretch of unknown function (Table S3; MamJ 83-90 and MamJ 171-178 ). However, the inhibitory function with respect to magnetite mineralization in vitro is inconsistent with the formation of the iron oxide both in and without the presence of the protein in vivo, [START_REF] Scheffel | An Acidic Protein Aligns Magnetosomes along a Filamentous Structure in Magnetotactic Bacteria[END_REF][START_REF] Scheffel | The Acidic Repetitive Domain of the Magnetospirillum gryphiswaldense MamJ Protein Displays Hypervariability but Is Not Required for Magnetosome Chain Assembly[END_REF] unless a yet unknown regulatory function of mineral formation is required for magnetosome chain formation in the bacteria. The strong effects of the synthetic peptide polymer polyE on magnetite nucleation inhibition suggest that if such an inhibitor exists in the bacteria, it will likely act by iron binding through acidic moieties.

3.3. Weak Influence on Magnetite Mineralization by MtxA Δ1-24 . Finally, MtxA Δ1-24 had only a minor influence on the phase of the formed precipitates: in the presence of 0.1 mg mL -1 MtxA Δ1-24 at pH 9, we obtained only an amorphous/ poorly crystalline material with similar orange gel-like properties, whereas under all other conditions we obtained mixtures of magnetite and amorphous gels (Figure 2G).

MtxA was identified in or attached to the magnetosome membrane. [START_REF] Tanaka | Origin of Magnetosome Membrane: Proteomic Analysis of Magnetosome Membrane and Comparison with Cytoplasmic Membrane[END_REF] It has been suggested to play role in the magnetotaxis because of its gene location in an operon encoding signal transduction genes. [START_REF] Richter | Comparative Genome Analysis of Four Magnetotactic Bacteria Reveals a Complex Set of Group-Specific Genes Implicated in Magnetosome Biomineralization and Function[END_REF] However, no experimental evidence has shown the implication of the protein in such a mechanism so far. Our in vitro assays suggest that despite the possibility that the protein can bind to magnetite it will likely have no effect on the crystallization of the mineral in vivo.

3.4. Magnetite Particle Growth in the Presence of Additives. Growth experiments over several hours at pH 9, which without additives resembles the kinetics observed in the bacteria in vivo, [START_REF] Baumgartner | Formation of Magnetite Nanoparticles at Low Temperature: From Superparamagnetic to Stable Single Domain Particles[END_REF] indicate that generally the initial magnetite grain size becomes smaller with increasing additive concentration; however, the crystal growth rate after nucleation appears to be largely unaffected (Figure 6 and Table S5). This is consistent with an effect of ionic strength on the surface tension that in turn affects the nucleus size at the formation threshold. [START_REF] Baumgartner | Nucleation and Growth of Magnetite from Solution[END_REF][START_REF] Vayssieres | Size Tailoring or Magnetite Particles Formed by Aqueous Precipitation: An Example of thermodynamic Stability of Nanometric Oxide Particles[END_REF] Furthermore, the nucleation is retarded in the presence of all investigated additives except polyR, in line with an interaction of these proteins/polymers with soluble iron species.

3.5. Implications for Biomineral Formation. Both thermodynamic and kinetic arguments can be used to explain our observations of nucleation inhibition or phase stabilization. One can argue thermodynamically that the interaction with a charged additive will influence the surface energy of a nucleating particle. A positively charged polymer in interaction with the negatively charged magnetite particle will lower the surface tension and thereby facilitate nucleation. Accordingly, the interaction between equally charged polymers and particle surfaces would be energetically unfavorable with resulting higher surface tension and eventually nucleation inhibition. Kinetically, one can argue that in the inhibited case the interaction between a positively charged ionic iron precursor and an anionic polymer is likely more rapid than the formation of the crystalline iron oxide solid, therefore entrapping iron in a polymer-induced amorphous precursor state. [START_REF] Gower | Biomimetic Model Systems for Investigating the Amorphous Precursor Pathway and Its Role in Biomineralization[END_REF] Generally, synthetic polyelectrolytes by virtue of their electrostatics find extensive use as nucleation and crystal growth modifiers as well as in the stabilization or flocculation of colloidal suspensions. [START_REF] Mortimer | Synthetic PolyelectrolytesA Review[END_REF] Equally, nature has evolved highly charged proteins (and other biomolecules) that can perform similar functions in vivo. In the context of biomineralization, this applies to proteins involved in the phase selection and growth modification of calcium carbonates and phosphates, silicate, and possibly iron oxides as investigated here. Similar to the effects observed here for the iron precipitates, the charge of interacting polyelectrolytic proteins determines the fate of the precursor species by stabilization or destabilization. For example, polyanionic aspartic acid-rich proteins play a presumably fundamental role in the stabilization of amorphous calcium carbonate (ACC) in mollusk shells. [START_REF] Politi | Asprich Mollusk Shell Protein: In Vitro Experiments Aimed at Elucidating Function in CaCO 3 Crystallization[END_REF] Whereas earlystage calcium carbonate precipitates possess slightly positive zeta potentials (for equimolar calcium and carbonate mixtures) facilitating the binding of polyanionic proteins, [START_REF] Chibowski | Time Dependent Changes in Zeta Potential of Freshly Precipitated Calcium Carbonate[END_REF] silica possesses negative surface charge throughout physiologically accessible pH ranges, favoring interaction with polycations. [START_REF] Behrens | The Charge of Glass and Silica Surfaces[END_REF] Thus, polycationic biopolymers have been shown to be involved in biomineralization by diatoms where they mediate the formation of SiO 2 . [START_REF] Kroger | Polycationic Peptides from Diatom Biosilica That Direct Silica Nanosphere Formation[END_REF] Although studying the mineralization role of proteins in vitro represents a promising alternative because deletion mutants are difficult to obtain and because their mechanistic role can be inferred more easily, most of the studies have focused on only one such magnetosomal protein (Mms6) in vitro so far. [START_REF] Arakaki | A Novel Protein Tightly Bound to Bacterial Magnetite particles in Magnetospirillum magnetotacticum Strain AMB-1[END_REF][START_REF] Prozorov | Protein-Mediated Synthesis of Uniform Superparamagnetic Magnetite Nanocrystals[END_REF][START_REF] Galloway | Magnetic Bacterial Protein Mms6 Controls Morphology, Crystallinity and Magnetism of Cobalt-Doped Magnetite Nanoparticles in Vitro[END_REF] The Mms6 protein and even its 26 amino acids C-terminal peptide are supposed to impact the size of magnetite nanoparticles, although controversial results have been obtained so far. Mms6 was initially chosen because it was shown to be tightly bound to the magnetite mineral. Recently, the putative mechanistic role of MamP has also been inferred from an in vitro study. [START_REF] Siponen | Structural Insight into Magnetochrome-Mediated Magnetite Biomineralization[END_REF] Although the in vivo effect of MamP had been recognized earlier, [START_REF] Murat | Comprehensive Genetic Dissection of the Magnetosome Gene Island Reveals the Step-Wise Assembly of a Prokaryotic organelle[END_REF] only the recent combination of protein structural and chemical reaction studies could assume its role of redox control in the oxidation of Fe(II). [START_REF] Siponen | Structural Insight into Magnetochrome-Mediated Magnetite Biomineralization[END_REF]

CONCLUSIONS

Although biocombinatorial approaches and in vitro mineralization assays alone cannot assume the role of proteins (or other biomolecules) in biomineralization in vivo, they can point

■ AUTHOR INFORMATION

Corresponding Author *Fax: (+) 49 331 567 9402. E-mail: damien.faivre@mpikg.mpg. de.

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

We thank W. Schulze for ESI-MS peptide fingerprinting and C. Li and S. Siegel for support at the μ-Spot beamline, BESSY II. This research was supported in D.F.'s laboratory by the Max Planck Society, the DFG (SPP 1420, FA835/2-1; 1569 FA835/

Figure 1 .

 1 Figure 1. Schematic method representation. A comparison of peptide sequences obtained by phage display and magnetosomal proteins affords proteins and peptides of interest for further study in Fe precipitation experiments. Depending on the additive characteristics, mineralization can be influenced to yield amorphous gels and magnetite in aggregates or self-assembled particle chains.

Figure 2 .

 2 Figure 2. Precipitation products in the presence of peptide polymer and protein additives. (A-C) Magnetite particles formed in the presence of poly-L-arginine. (Insets in C) FFTs of particles indexed as magnetite. (D) SDS-PAGE of MamJ. (E) Precipitation product in the presence of MamJ. (Inset in E) Electron diffraction reveals only amorphous scattering. (F) SDS-PAGE of MtxA Δ1-24 . (G) Precipitation product in the presence of MtxA Δ1-24 . (Inset in G) Electron diffraction shows diffraction consistent with magnetite.

Figure 3 .

 3 Figure 3. Particle size distribution of magnetite particles formed in the presence of polyR.

Figure 4 .

 4 Figure 4. X-ray diffraction patterns of precipitations formed at pH 9 without additive and in the presence of protein and peptide polymers.

Figure 5 .

 5 Figure 5. X-ray diffraction patterns of precipitates obtained at pH 11 from ferrous and ferric iron chloride solution mixtures without and in the presence of protein or polypeptide additives.

Figure 6 .

 6 Figure 6. Growth kinetics of magnetite nanoparticles formed in the presence of (a) MamJ, (b) MtxA Δ1-24 , (c) polyE, and (d) polyR and control data. Error bars represent the standard deviation from three independent reactions.


Thomas Paillet
email: thomas.paillet@inrae.fr

Eric Dugat-Bony
email: eric.dugat-bony@inrae.fr

Bacteriophage ecology of fermented foods: anything new under the sun?

Keywords: fermented foods, bacteriophages, microbial ecology, population dynamics

Bacteriophage ecology has raised an increasing attention over the last few years, thanks to the improvement and contributions of microscopy, comparative genomics and viral metagenomics methods. Fermented foods host dense and diverse microbial communities and, therefore, represent an ideal biotope for bacteriophages. If their occurrence in such environments has been demonstrated decades ago, data highlighting their impact on mixed communities and their ecological roles are scarce when compared to other microbial ecosystems. This review summarizes most recent knowledge into the bacteriophage diversity of fermented foods and stress evidences suggesting the impact of these entities on the dynamics of food microbial communities. The main ecological roles played by bacteriophages in microbial ecosystems are also addressed. Understanding the impact of bacteriophages in fermented foods will further help in designing adapted microbial consortia and thus providing a better control of the food fermentations.

Introduction

Fermented foods are widely consumed worldwide and encompass diverse types of products including fermented dairy products, meats, fishes, cereals, legumes, vegetables, seeds, roots and also alcoholic and non-alcoholic beverages [START_REF] Tamang | Fermented foods in a global age: East meets West[END_REF]. Initially developed with the objective of extending the raw products shelf-life, they are currently also appreciated for their typical sensory properties, nutritional value and potential health benefits [START_REF] Marco | Health benefits of fermented foods: microbiota and beyond[END_REF][START_REF] Hugenholtz | Traditional biotechnology for new foods and beverages[END_REF]. Food fermentation relies on the conversion of substrates present in the raw material (e.g. sugars, proteins, lipids) into simpler products such as lactic acid, acetic acid, alcohol, carbon dioxide, ammonia and free fatty acids, through the specific activity of microorganisms.

Fermented foods are dynamic microbial ecosystems, where the succession of several microbial groups occur in a short time, usually from days to weeks or months. This period varies according to the nutrients available in the raw material, abiotic parameters such as temperature, humidity, oxygen and osmotic pressure, and microbial interactions as well [START_REF] Smid | Microbe-microbe interactions in mixed culture food fermentations[END_REF].

Huge efforts have been performed by the scientific community during the past decades to describe the composition and functioning of food microbial communities, helped by the recent development of several meta-omics tools [START_REF] Walsh | Translating Omics to Food Microbiology[END_REF]. It is now well-established that microbial communities of fermented foods are generally dense (e.g. >10 9 cells per gram of cheese [START_REF] Irlinger | Cheese rind microbial communities: diversity, composition and origin[END_REF]) and have a low diversity when compared to other microbial ecosystems such as soils [START_REF] Fierer | Metagenomic and Small-Subunit rRNA Analyses Reveal the Genetic Diversity of Bacteria, Archaea, Fungi, and Viruses in Soil[END_REF] or oceans [START_REF] Breitbart | Genomic analysis of uncultured marine viral communities[END_REF]. Bacteria from diverse phyla, e.g. Firmicutes (which includes lactic acid bacteria), Actinobacteria, Proteobacteria and Bacteroidetes, yeasts and filamentous fungi constitute the main microbial groups involved in food fermentation [START_REF] Tamang | Review: Diversity of Microorganisms in Global Fermented Foods and Beverages[END_REF]. Through this fermentative activity, they contribute to the quality of the food products in several ways, i.e. by preventing the development of pathogens, modifying the texture, releasing aroma compounds or degrading anti-nutritional factors.

Bacteriophages (phages), viruses that infect bacterial cells, are considered as key ecological drivers in the functioning of microbial ecosystems [START_REF] Koskella | Bacteria-phage coevolution as a driver of ecological and evolutionary processes in microbial communities[END_REF]. Indeed, phage-bacterial interactions can potentially affect the balance between the different functional groups of microbes and then (re-)shape microbial communities. In the human gut, where phage particles probably do not outnumber bacteria, phages could play a transitory role in the homeostasis or the evolution of the microbiota [START_REF] Sausset | New insights into intestinal phages[END_REF]*. In food fermentations, although the presence of bacteriophages has been demonstrated around one century ago [START_REF] Whitehead | The Occurrence of Bacteriophage in Cultures of Lactic Streptococci: A Preliminary Note[END_REF], only few examples of deep inventory of their diversity are available and the literature about their ecological role is even rarer.

The present review summarizes the current knowledge on the occurrence and diversity of bacteriophages in fermented foods, especially in light of the recent discoveries resulting from the analysis of viral metagenomics data, and discuss the possible ecological roles played by these biological entities in the context of this peculiar ecosystem which, ultimately, may impact the success of the fermentation process.

Current knowledge on bacteriophages' occurrence and diversity in fermented foods

Several experimental approaches have been used for enumerating bacteriophages in microbial ecosystems and exploring their diversity. Some of them require the isolation of phages, which implies as first steps to identify and cultivate susceptible bacterial hosts, while others can be applied directly on environmental samples (Figure 1).

Culture-dependent approaches

The presence of bacteriophages in fermented foods was frequently investigated by culturedependent approaches. In practice, the main technic consists in collecting phages from a liquid sample obtained from the food product, or from a suspected reservoir in the manufacturing environment, and putting them into contact with a sensitive bacterial strain on a double-layer agar plate (for a detailed protocol, see [START_REF] Kropinski | Enumeration of Bacteriophages by Double Agar Overlay Plaque Assay[END_REF]). It is also possible to directly enumerate phages to determine their initial level in food products [START_REF] Cordero-Bueso | Bacteriophages as an Up-and-Coming Alternative to the Use of Sulfur Dioxide in Winemaking[END_REF], or to enrich them before isolation to enhance the probability of recovery [START_REF] Cheng | Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages[END_REF].

Once isolated, phages can be further identified and characterized by applying a variety of downstream analysis. The historical classification of bacterial viruses is based on electron microscopy (EM) observations [START_REF]Virus taxonomy: classification and nomenclature of viruses: ninth report of the International Committee on Taxonomy of Viruses[END_REF], and it is now progressively moving to a molecular classification. In Philippe et al., the authors used EM for the morphological characterization of phage GC1 isolated from wine musts [START_REF] Philippe | Bacteriophage GC1, a Novel Tectivirus Infecting Gluconobacter Cerinus, an Acetic Acid Bacterium Associated with Wine-Making[END_REF]. They observed tail-less icosahedral particles, meaning that this phage did not belong to Caudovirales order. A further characterisation of its genomic properties determined that GC1 belongs to the Tectiviridae family.

In most recent articles, genome sequencing and comparative genomics were also applied to phages isolated from fermented foods. De Melo et al., made comparative genomics on 18

Brevibacterium aurantiacum phages isolated from cheese [START_REF] Melo | DNA tandem repeats contribute to the genetic diversity of Brevibacterium aurantiacum phages[END_REF]**. They were classified into 7 distinct genomic groups based on the number of DNA tandem repeats (TRs) in each genome.

They also found that 85% of phages in databases possessed such TRs. Similarly, Cheng et al., classified 7 Propionibacterium freudenreichii phages into two clusters, based on the nucleotide identity and coverage percentage between each genomes [START_REF] Cheng | Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages[END_REF].

In most cases, such a culture-dependent approach also gives clues as to whether the isolated phage is temperate or virulent, by confronting the aspect of plaques (turbid for temperate, and usually clear for virulent [START_REF] Levine | Mutations in the temperate phage P22 and lysogeny in Salmonella[END_REF]) and the genetic analysis of the complete genome (which usually contains an integrase when the phage is temperate).

As summarized in Table 1, both virulent and temperate phages were isolated from many fermented foods. However, surprisingly, no filamentous phages were retrieved from this type of products to date. Most of them are Siphoviridae and Myoviridae but Podoviridae and Tectiviridae were also isolated occasionally [START_REF] Cordero-Bueso | Bacteriophages as an Up-and-Coming Alternative to the Use of Sulfur Dioxide in Winemaking[END_REF][START_REF] Philippe | Bacteriophage GC1, a Novel Tectivirus Infecting Gluconobacter Cerinus, an Acetic Acid Bacterium Associated with Wine-Making[END_REF][START_REF] Kleppen | Novel Podoviridae Family Bacteriophage Infecting Weissella cibaria Isolated from Kimchi[END_REF]. The hosts are usually members of the dominant species present in the corresponding food, such as Leuconostoc mesenteroides in Sauerkraut [START_REF] Lu | Sequence Analysis of Leuconostoc mesenteroides Bacteriophage Φ1-A4 Isolated from an Industrial Vegetable Fermentation[END_REF] or Oenococcus oeni in Wine [START_REF] Costantini | Phage-host interactions analysis of newly characterized Oenococcus oeni bacteriophages: Implications for malolactic fermentation in wine[END_REF]. For the particular case of cheese, where an abundant literature regarding phages infecting Lactic Acid Bacteria (LAB) starter cultures is available, as reviewed previously [START_REF] Garneau | Bacteriophages of lactic acid bacteria and their impact on milk fermentations[END_REF][START_REF] Brüssow | Phages of Dairy Bacteria[END_REF], only a few studies described the isolation of phages infecting other important bacteria such as Propionibacterium freudenreichii [START_REF] Cheng | Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages[END_REF], the bacteria responsible for the production of holes in Emmental-type cheese, Brevibacterium aurantiacum [START_REF] Melo | DNA tandem repeats contribute to the genetic diversity of Brevibacterium aurantiacum phages[END_REF], a surface ripening culture used in many cheese varieties and Enterococcus faecalis [START_REF] Del Rio | Enterococcus faecalis Bacteriophage 156 Is an Effective Biotechnological Tool for Reducing the Presence of Tyramine and Putrescine in an Experimental Cheese Model[END_REF][START_REF] Ladero | Q69 (an E. faecalis-Infecting Bacteriophage) As a Biocontrol Agent for Reducing Tyramine in Dairy Products[END_REF]*. However, in the perspective of understanding the bacteriophage ecology in fermented foods, single phage isolation suffer from the major limitation of the culture-dependent approach that is the need of a susceptible host. By definition such approach cannot accurately reflect neither the bacterial and viral diversity present in fermented foods, nor their relative abundance. It is however very efficient from a technological point of view, for example in the case where a phage is suspected to be responsible of a fermentation failure and needs to be quickly identified.

Direct detection approaches

An alternative is to detect and/or quantify phages directly in food samples or in a viral fraction extracted from food samples without a priori on the bacterial host(s). Several techniques are available to count or observe viruses directly in complex samples [START_REF] Kaletta | A rigorous assessment and comparison of enumeration methods for environmental viruses[END_REF], such as flow cytometry [START_REF] Melo | Assessment of Sep1virus interaction with stationary cultures by transcriptional and flow cytometry studies[END_REF], epifluorescence microscopy [START_REF] Vy | Use of epifluorescence microscopy to assess the effectiveness of phage P100 in controlling Listeria monocytogenes biofilms on stainless steel surfaces[END_REF], nanoparticle tracking analysis [START_REF] Carr | Nanoparticle Tracking Analysis for the rapid and direct visualisation, sizing and counting of viruses, phage and VLPs in suspension[END_REF], interferometric light microscopy [START_REF] Roose-Amsaleg | Utilization of interferometric light microscopy for the rapid analysis of virus abundance in a river[END_REF] and electron microscopy including Scanning Electron Microscopy (SEM) [START_REF] Linderoth | The Filamentous Phage pIV Multimer Visualized by Scanning Transmission Electron Microscopy[END_REF], Transmission Electron Microscopy (TEM) [START_REF] Jebri | Total coliphages removal by activated sludge process and their morphological diversity by transmission electron microscopy[END_REF] and cryo Electron Microscopy (cryo-EM) [START_REF] Chaïb | Isolation and CryoTEM of Phages Infecting Bacterial Wine Spoilers[END_REF].

Only few studies used such direct approaches to characterize the phage communities of fermented foods. Dugat-Bony et al., used interferometric light microscopy to determine the phage concentration on the surface of three cheese varieties and found that it ranged from 1×10 9 to 4×10 10 particles per gram at least [START_REF] Dugat-Bony | Viral metagenomic analysis of the cheese surface: A comparative study of rapid procedures for extracting viral particles[END_REF]*. They also observed different morphotypes on Epoisses cheese with TEM, giving first indications about the complexity of phage communities present on the cheese surface.

As a complement to their metagenomic study, Park et al., used TEM on samples from shrimps, sauerkraut and kimchi, after a cesium chloride (CsCl) density gradient purification step [START_REF] Park | Metagenomic analysis of the viral communities in fermented foods[END_REF]. They were able to characterize various phages morphologies, mainly Sipho-and Myoviridae.

To summarize, in the context of fermented foods, direct detection approaches can provide rapidly important ecological information such as total phage's concentration and rough elements about their diversity. However, since many bacteriophages can share similar morphological traits, techniques offering more precision are desirable to properly describe the composition of phage communities in fermented foods.

New contributions of whole metagenome sequencing and viral metagenomics

Two main categories of metagenomics approaches can be used to detect viral signals from environmental samples.

First, whole metagenome sequencing, which generates simultaneously sequences from both microbial cells and viruses, can be used to identify abundant phages present in microbial ecosystems using dedicated bioinformatic tools [START_REF] Roux | VirSorter: mining viral signal from microbial genomic data[END_REF][START_REF] Zheng | Mining, analyzing, and integrating viral signals from metagenomic data[END_REF]. Regarding fermented foods, only two articles refer to the use of such approach for studying the composition of phage communities. In kimchi [START_REF] Jung | Metagenomic Analysis of Kimchi, a Traditional Korean Fermented Food[END_REF], the authors identified four putative phage contigs with sequence similarity with the genome of LAB-phages. In kinema, a fermented soybean product, the viral community was dominated by phages infecting Bacillus species. Their identity was further confirmed by taxonomic analysis, and they all had one or several wellcharacterized host(s) [START_REF] Kumar | Metagenomic Insights Into the Taxonomic and Functional Features of Kinema, a Traditional Fermented Soybean Product of Sikkim Himalaya[END_REF].

Second, viral metagenomics, or "viromics", consist in extracting and sequencing the genomic material of the viral community selectively purified from an environmental sample (e.g. food products, natural environment, host-associated) [START_REF] Edwards | Viral metagenomics[END_REF]. This approach is designed to obtain a deep overview of the composition of the phage community present in a given ecosystem.

Generally, only dsDNA phages are sequenced, letting a grey area regarding RNA and ssDNA phages, whose quantities and diversity are probably underestimated thus far [START_REF] Kauffman | A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria[END_REF]. In natural ecosystems such as ocean, or soils, numerous viral metagenomic studies targeting phages have been carried out (respectively, [START_REF] Brum | Patterns and ecological drivers of ocean viral communities[END_REF] and [START_REF] Adriaenssens | Metagenomic analysis of the viral community in Namib Desert hypoliths: Metaviromics of Namib hypoliths[END_REF][START_REF] Scola | Namib Desert Soil Microbial Community Diversity, Assembly, and Function Along a Natural Xeric Gradient[END_REF]). However, they are scarce when it comes to fermented foods or fermented beverages, as reviewed in [START_REF] Ledormand | Phage community involvement in fermented beverages: an open door to technological advances?[END_REF]. Park et al. firstly analysed the metavirome of fermented shrimp, kimchi and sauerkraut [START_REF] Park | Metagenomic analysis of the viral communities in fermented foods[END_REF].

Viral particles were recovered and concentrated from the food samples by using filtration and ultracentrifugation prior to viral DNA extraction and pyrosequencing. The results revealed an important diversity in phage sequences, most of which showing no significant hits in public databases, and 6 to 27 contigs >5 Kb per food sample were assembled. Major discrepancies were observed between predicted hosts and the actual bacterial diversity detected in kimchi and fermented shrimp, reflecting the fact that viral genomes of these fermented foods were poorly represented in public databases at the time of the study, so that host predictions probably failed. On the contrary, phage host predictions in sauerkraut were reliable, thanks to the availability of several phage genomes isolated in previous work [START_REF] Lu | Sequence Analysis of Leuconostoc mesenteroides Bacteriophage Φ1-A4 Isolated from an Industrial Vegetable Fermentation[END_REF].

The viral community from ten samples representative of Korean and Chinese kimchi was also investigated [START_REF] Jung | Viral community predicts the geographical origin of fermented vegetable foods more precisely than bacterial community[END_REF]. Viral concentrates were obtained through filtration and Polyethylene Glycol (PEG) concentration, and DNA was sequenced providing several thousands of contigs >500 bp per sample, and revealing a very high diversity. The phage host prediction was consistent with the bacterial diversity and, interestingly, viral community profiling was found to outperform bacterial community profiling for predicting the geographical origin of kimchi.

Recently, the first metavirome of the cheese surface was described [START_REF] Dugat-Bony | Viral metagenomic analysis of the cheese surface: A comparative study of rapid procedures for extracting viral particles[END_REF] using Epoisses cheese as an example. The viral fraction was obtained according to an optimized protocol involving filtration, PEG concentration and chloroform treatment. DNA sequences were assembled into 124 viral contigs from 2.5 to 122 kb, highlighting the presence of an unexpected viral diversity in this ecosystem. The authors were able to predict a bacterial host for the most abundant ones, e.g. Glutamicibacter, Lactococcus, Psychrobacter, Vibrio, Leuconostoc and Halomonas, which were previously detected as dominant bacterial genera in Epoisses cheese [START_REF] Dugat-Bony | Highlighting the microbial diversity of 12 French cheese varieties[END_REF].

Overall, available data suggest that fermented foods host dense and complex phage communities, at least as diverse as bacterial ones. However, despite the growing descriptive data available regarding bacteriophages occurrence and diversity in fermented foods, there is still a gap before demonstrating that these entities play an ecological role in this environment and can impact food fermentations.

Relationships between viral and bacterial dynamics

The first step would be to determine if a relationship exists between the levels of viral and bacterial populations during the fermentation cycle. For fermented foods, few studies demonstrated such correlation.

In kimchi [START_REF] Jung | Metagenomic Analysis of Kimchi, a Traditional Korean Fermented Food[END_REF], the monitoring of the composition of bacterial and phage populations during 29 days of fermentation was achieved by using a whole metagenome sequencing approach.

The relative abundance of the four putative phage contigs identified increased during the fermentation cycle, reaching ~7% of the total metagenomics sequences after 25 days. This large number of sequences was correlated to the decrease of bacterial population observed after 25 days suggesting that bacteriophages may influence the microbial community dynamics in this product.

Recently, Kong and Park used a culture media made from sterilized supernatant of dongchimi kimchi (a watery kimchi made from radish, green onions, garlic, ginger and salt) to perform co-culture of the main LAB species involved in the fermentation of this product, i.e. Leuconostoc citreum, L. mesenteroides and Weissella cibaria [START_REF] Kong | Effect of Bacteriophages on Viability and Growth of Co-cultivated Weissella and Leuconostoc in Kimchi Fermentation[END_REF]*. They used different combinations of strains, sensitive to phages or not, and followed the dynamic of both bacterial and phage populations by culture-dependent methods over 10 days. They observed a negative relationship between the abundance of phages and the viability of their hosts, independently of the pH of the medium, demonstrating the impact of bacteriophages on the succession of LAB species during dongchimi kimchi fermentation.

Finally, Erkus et al. studied an undefined complex cheese starter culture, composed of several strains of Lactococcus lactis and Leuconostoc mesenteroides grouped in eight different genetic lineages, some of which carrying active prophages [START_REF] Erkus | Multifactorial diversity sustains microbial community stability[END_REF]. In one of the described experiment, they propagated in vitro the culture daily in milk during several weeks, mimicking the back-slopping procedure used in the dairy industry, and followed temperate phage population by titration and the different bacterial lineages by qPCR. The results indicated that although some strains can undergo dramatic decrease in abundance at certain stages of propagation, due to the increase in a particular phage population in the medium, the effect on the relative abundance of the genetic lineage they belong to was very limited, ensuring an overall stability of the community structure, both regarding genes and functionalities.

To summarize, there are increasing evidences that bacteriophages affect the dynamics of food microbial communities during fermentation. The impact of phage attacks on the overall structure and function of microbial communities is thought to depend on the complexity of the studied system and microdiversity seems to play an important role in the overall stability of microbial communities in fermented foods.

Ecological roles of bacteriophages

Studies elucidating the ecological roles of bacteriophages occurring in fermented foods are scarce. Thus, this section summarizes the main roles attributed to these entities regardless of the microbial ecosystem of origin. It is likely that major results observed within natural ecosystems are transferable to fermented products.

Direct regulation of bacterial populations

Bacteriophages may have several types of behaviours toward their bacterial hosts (Box 1).

They can directly impact the population levels of their hosts in different ways and, consequently, the whole community structure.

First, phages performing a lytic cycle, which encompass professionally lytic, virulent mutant (or ex-temperate, see [START_REF] Chopin | Analysis of six prophages in Lactococcus lactis IL1403: different genetic structure of temperate and virulent phage populations[END_REF]) and temperate phages entering a lytic cycle, foster a preypredator relationship with their host. Since bacteriophages are non-motile entities, the probability of encountering a host cell and starting infection strongly depends on the host's density. In mixed microbial communities, this means that bacteriophages kill the sensitive bacterial strain(s) with the highest density, as theorized in the Kill-the-Winner [START_REF] Thingstad | Elements of a theory for the mechanisms controlling abundance, diversity, and biogeochemical role of lytic bacterial viruses in aquatic systems[END_REF] and seedbank models [START_REF] Breitbart | Here a virus, there a virus, everywhere the same virus?[END_REF][START_REF] Brum | Patterns and ecological drivers of ocean viral communities[END_REF], while having a little impact on the total microbial biomass. This direct effect also promotes bacterial diversity since several bacterial populations sharing the same ecological niche can coexist [START_REF] Shapiro | Bacteriophage predation regulates microbial abundance and diversity in a full-scale bioreactor treating industrial wastewater[END_REF] and grow in a sequential manner. Finally, it also favours the optimal utilisation of all resources present in the ecosystem, as a single dominant bacteria wouldn't have all the necessary enzymatic equipment to exploit all the available nutrients [START_REF] Rodriguez-Valera | Explaining microbial population genomics through phage predation[END_REF].

Second, temperate phages can also affect the fitness of their host when entering a lysogenic cycle. When integrated as prophages into the bacterial host genome, they can develop either a parasitic or a mutualistic interaction with their host. In complex cheese starter culture, Alexeeva et al. [START_REF] Alexeeva | Spontaneously induced prophages are abundant in a naturally evolved bacterial starter culture and deliver competitive advantage to the host[END_REF]* demonstrated the competitive advantage of Lactococcus lactis lysogens compared to their prophage-cured derivatives but the mechanism behind this observation was not identified. Similarly, Costantini et al. [START_REF] Costantini | Phage-host interactions analysis of newly characterized Oenococcus oeni bacteriophages: Implications for malolactic fermentation in wine[END_REF] observed that, among sixteen Oenococcus oeni strains used for malolactic fermentation in wine, the ones integrating one or several prophages in their genomes were more resistant to the predation by other oenophages.

Finally, bacteriophages which cannot enter a lysogenic cycle and are producing virions in a chronic fashion without cell lysis interact with their host through a parasitic relationship. In this case, the main expected effect would be a lower fitness of the host caused by the energy cost required for the bacteriophage's replication but this was not studied in the context of fermented foods to our knowledge.

In addition to the regulation of the population level of their hosts, bacteriophages can also prompt substantial modifications of the bacterial transcriptome and proteome and, hence, affect their metabolism [START_REF] Fernández | Phage or foe: an insight into the impact of viral predation on microbial communities[END_REF].

Indirect effects on non-host populations

Bacterial lysis mediated by bacteriophages is responsible for the release of organic matter into the environment. In ecology, this phenomenon is known as the "viral shunt" and it affects trophic webs by lowering bacterial biomass before it is assimilated at higher trophic levels [START_REF] Burmeister | Reference Module in Life Sciences[END_REF]*. Bacterial lysis provides an important source of free nutrients which can support and promote the growth of other microorganisms. It has thus a large impact on microbial dynamics and densities.

For example, Fazzino et al. studied the effect of virulent bacteriophages on the mutualistic interaction between Escherichia coli and Salmonella enterica [START_REF] Fazzino | Lytic bacteriophage have diverse indirect effects in a synthetic cross-feeding community[END_REF]**. In such cross-feeding model community, phages can have extensive indirect effects affecting the bacterial community dynamics. One of those indirect effect was the release of organic matter since the growth of S. enterica was stimulated by the lysis of E. coli by phage T7.

In fermented foods, such phenomenon is likely to occur. However, contrary to other natural ecosystems, the trophic chain is short and there is generally no upper trophic level above heterotrophic microorganisms. In this case, the role of the viral shunt encompasses probably only the release of cellular debris and intra-cellular compounds, providing additional resources for non-hosts species. In line with this suspected role, Kong & Park experimentally evidenced that a bacteriophage lysate of Weissella cibaria can promote the growth of L. citreum in kimchi fermentation [START_REF] Kong | Effect of Bacteriophages on Viability and Growth of Co-cultivated Weissella and Leuconostoc in Kimchi Fermentation[END_REF].

Phages as bacterial evolution drivers

Bacterial viruses represent a continuous selective pressure on bacteria, driving them to evolve by selecting antiphage defence mechanisms [START_REF] Hampton | The arms race between bacteria and their phage foes[END_REF]*. In response, phages adopt strategies to overcome host defence systems. This antagonistic co-evolution is a kind of perpetual arms race, referred to as the Red Queen dynamics [START_REF] Lythgoe | Catching the Red Queen? The advice of the Rose[END_REF]. This is due to the extremely rapid evolution and turnover of phage particles [START_REF] Stern | The phage-host arms-race: Shaping the evolution of microbes[END_REF], increasing mutation rates in their host(s) [START_REF] Paterson | Antagonistic coevolution accelerates molecular evolution[END_REF], driving neither the predator nor the prey to extinction. The most prominent outcome of this interaction is ecological speciation, resulting in a high microdiversity of both hosts and phages.

Horizontal gene transfer (HGT), which refers to the incorporation by an organism of genetic material from another organism without mating, contributes to the microbial genome evolution [START_REF] Touchon | Embracing the enemy: the diversification of microbial gene repertoires by phage-mediated horizontal gene transfer[END_REF]. Bacterial lysis due to the phage progeny release is accompanied by the release of intra-cellular components into the environment and especially pieces of DNA that could be acquired by other bacteria through natural transformation [START_REF] Keen | Novel "Superspreader" Bacteriophages Promote Horizontal Gene Transfer by Transformation[END_REF]. HGT can also happen through transduction (generalized or specialized [START_REF] Braga | Bacterial Diversification in the Light of the Interactions with Phages: The Genetic Symbionts and Their Role in Ecological Speciation[END_REF]), when a bacteriophage accidentally packages bacterial DNA and transfers it to another host.

Lysogeny has been suggested as a survival strategy when the environment contains low host densities [START_REF] Fuhrman | Marine viruses and their biogeochemical and ecological effects[END_REF]. Prophages integrated in a host bacteria are usually in a dormant state, as they don't actively replicate their genomes, while regulating bacterial genes. If some phages have evolved to insert only in highly conserved sites [START_REF] Bobay | The Adaptation of Temperate Bacteriophages to Their Host Genomes[END_REF], others as transposable phages integrate themselves randomly in the host genome, possibly inactivating genes coding for essential functions and having therefore a detrimental effect on host fitness [START_REF] Toussaint | Transposable phages, DNA reorganization and transfer[END_REF]. Some phages are also able to display "active lysogeny", acting as regulatory switches, as they turn off the gene by integrating in its sequence [START_REF] Feiner | A new perspective on lysogeny: prophages as active regulatory switches of bacteria[END_REF].

Phages can stay in lysogenic cycle for several generations. However, they are not totally inactive since the bacterial cell expresses moron genes (for "more DNA") that are not necessary for the phage cycle, and expressed during lysogeny [START_REF] Brüssow | Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion[END_REF][START_REF] Tsao | Phage Morons Play an Important Role in Pseudomonas aeruginosa Phenotypes[END_REF]. A well-known example is the stx gene coding for shigatoxin, which is acquired by bacterial strains through lysogenic conversion, meaning the acquisition of a stx-positive prophage [START_REF] Davis | A satellite phage-encoded antirepressor induces repressor aggregation and cholera toxin gene transfer[END_REF]. These morons are far from being all characterized, but they probably enhance bacterial fitness and expand the environmental niche of the host [START_REF] Taylor | Chapter One -The Diverse Impacts of Phage Morons on Bacterial Fitness and Virulence[END_REF].

Conclusions

Fermented foods represent undoubtedly suitable ecosystems for the development of bacteriophages. The isolation of a large set of bacteriophages and the first viral metagenomics data shed light into the viral diversity of this type of products. However, the question of the impact of this diversity on the composition and functioning of microbial communities still remains underexplored.

The continuous improvement of sequencing techniques, with in particular the first applications of long-read sequencing for the description of metaviromes [START_REF] Beaulaurier | Assembly-free single-molecule sequencing recovers complete virus genomes from natural microbial communities[END_REF]**, makes it possible to envisage both the description and the monitoring of the composition of viral communities in fermented foods more easily and in a more comprehensive way. Furthermore, synthetic microbial ecosystems offer new perspectives for investigating individual to ecosystem level microbial interactions [START_REF] Shetty | Reconstructing functional networks in the human intestinal tract using synthetic microbiomes[END_REF]. The viral dimension of food microbial ecosystems should therefore be considered for the future design of synthetic ecology experiments with the objective of characterizing the role of bacteriophages in fermented foods.

The exploitation of this knowledge should help the food industry facing numerous challenges such as controlling phage contamination to reduce the risk of fermentation failure. Main applications are the biocontrol of pathogens [START_REF] Haddad | Efficacy of two Staphylococcus aureus phage cocktails in cheese production[END_REF] and spoilage bacteria [START_REF] Del Rio | Enterococcus faecalis Bacteriophage 156 Is an Effective Biotechnological Tool for Reducing the Presence of Tyramine and Putrescine in an Experimental Cheese Model[END_REF]* but new developments could lead to finely modulate the composition of microbial communities in order to reach the desired technological properties in fermented products. *For cheese, no example describing the isolation of phages infecting LAB starter culture is listed since the literature on such phages is extremely abundant and already reviewed [START_REF] Garneau | Bacteriophages of lactic acid bacteria and their impact on milk fermentations[END_REF][START_REF] Brüssow | Phages of Dairy Bacteria[END_REF]. Professionally lytic: Phage that is both obligately lytic and not recently descended from a temperate ancestor Virulent mutant (ex-temperate): Clear temperate phage mutant that can form plaques even on lysogens (i.e. bacteria hosting a prophage) formed by the phage wild-type parent Temperate phage: Description of a phage that is able to display lysogenic cycles (under the form of a prophage) or lytic cycle according to specific environmental and host parameters.

Figure captions

Filamentous phage: Phage whose productive infection is chronic (may or may not be a temperate phage)

Figure 1 :

 1 Figure 1: General methods for the study of bacteriophages in fermented foods.

Table 1 .

 1 Selected examples describing the isolation of phages directly from fermented foods. 378

	Fermented food	References	Phage families	Type	Host
		[15]	7 Siphoviridae	Virulent	Propionibacterium freudenreichii
	Cheese*	[18]	16 Siphoviridae	Virulent	Brevibacterium aurantiacum
		[25]	1 Myoviridae	Virulent	Enterococcus faecalis
		[26]	1 Siphoviridae	Virulent	Enterococcus faecalis
		[79]	1 Siphoviridae	Virulent	Lactobacillus plantarum
					Lactobacillus brevis,
	Fermented Cucumber	[80]	3 Siphoviridae and 3 Myoviridae	N.D	Lactobacillus plantarum, Weissella paramesenteroides,
					Weissella cibaria
		[81]	1 Siphoviridae	Virulent	Pediococcus sp.
		[82]	N.D	Virulent and temperate	Bacillus subtilis
	Fermented	[83]	16 Siphoviridae	N.D	Bacillus cereus
	Soybean	[84]	1 Myoviridae	N.D	Bacillus cereus
		[85]	1 Siphoviridae and 1 Myoviridae	N.D	Pediococcus halophilus
	Kefir	[86]	2 Siphoviridae	N.D	Lactobacillus plantarum
		[20]	1 Podoviridae	N.D	Weissella cibaria
	Kimchi	[49]	N.D	N.D	Weissella cibaria, Leuconostoc citreum
		[87]	1 Siphoviridae	N.D	Lactobacillus plantarum
	Salami	[88]	2 Podoviridae	N.D	Staphylococcus carnosus
		[89]	3 Siphoviridae and 3 Myoviridae	N.D	Leuconostoc fallax
					Leuconostoc
					pseudomesenteroides,
					Leuconostoc
					mesenteroides,
	Sauerkraut	[90]	5 Siphoviridae and 3 Myoviridae	N.D	Leuconostoc citreum, Leuconostoc fallax,
					Weissella sp.,
					Lactobacillus
					plantarum,
					Lactobacillus brevis
		[21]	1 Siphoviridae	Virulent	Leuconostoc mesenteroides

Acknowledgements T.P. is the recipient of a doctoral fellowship from the French Ministry of Higher Education, Research and Innovation (MESRI) and the MICA department of the French National Research Institute for Agriculture, Food and Environment (INRAE). E.D-B is member of the knowledge hub SYSTEMIC which received funding from several national research funding parties in a joint action of JPI HDHL, JPI-OCEANS and FACCE-JPI launched under the ERA-NET ERA-HDHL (n° 696295). The authors thank Marie-Agnès Petit (INRAE) for her critical review of the manuscript.

Declaration of interest. None.

Tables

Authors declare no conflict of interest.

Conflict of interest


Thomas Paillet

Julien Lossouarn

Marie-Agnès Petit

Eric Dugat-Bony

Isolation and characterization of phage-host couples from Epoisses cheese rind

Cheese surface host complex microbiota, comprising fungi, bacteria and bacteriophages (phages). The latter are known to have a substantial impact on the dynamics of microbial ecosystems. However, to date, ecological roles of phages were mainly studied in natural ecosystems and in the human gut. The hypothesis of this work is that phages affect the microbial succession observed on the cheese surface during the ripening process. In order to explore this question, isolation and characterization steps represent the first objectives.

A first overview of the microbial diversity was obtained through a shotgun metagenomic study performed on the total DNA obtained from the rind of Epoisses cheese. This allowed to identify members of the Pseudoalteromonas, Psychrobacter and Glutamicibacter genera as the dominant bacteria.

Then, several bacteria-phages couples were targeted and isolated from the rind. A few phages infecting Glutamicibacter, Psychrobacter, Brevibacterium and Leuconostoc were obtained and characterized from a morphological (transmission electron microscopy) and genetic (genome sequencing) point of views. They all seem to be virulent, and belong to different families from the Caudovirales order, namely Siphoviridae, Myoviridae and Podoviridae. Moreover, one of them have a novel genetic organization, and may potentially expand the current classification.

Overall, phages targeting main bacterial genera were isolated (except for Pseudoalteromonas), suggesting that the diversity of phages is at least as important as the bacterial diversity in cheese. This phage-bacteria collection isolated from the cheese surface will be useful for testing the hypothesis of an ecological role of phages in this ecosystem using synthetic ecology approaches. Ideally, dynamics of both phages and hosts will be monitored in real time within a cheese matrix, giving first clues of phage impact in this peculiar ecosystem. These results will be of importance for the cheese industry as they may help to reach unprecedented mastery of the ripening process.