Prashant P Damke

Louisa Celma

Sumedha Kondekar

Anne Marie

Di Guilmi

Stephanie Marsin

Jordane Depagne

Xavier Veaute

Pierre Legrand

Hélène Walbott

Julien Vercruyssen

Stéphanie Marsin

Jordane Dépagne

Raphaël Guérois

Sophie Quevillon-Cheruel
email: sophie.quevillon-cheruel@i2bc.paris-saclay.fr

J Pablo Radicella
email: pablo.radicella@cea.fr

ComF is a key mediator in single-stranded DNA transport and handling during natural transformation

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

Introduction

Bacterial populations display an amazing capacity to adapt to changes in their environment.

In pathogens, this is reflected in the generation of variants able to colonise new hosts, the propagation of virulence factors or the acquisition antibiotics resistance. A key mechanism in the propagation of those traits is horizontal gene transfer (HGT). There are three major mechanisms of HGT in bacteria: conjugation, phage transduction and natural transformation (NT). Although NT has been documented in at least 80 bacterial species [START_REF] Johnston | Bacterial transformation: distribution, shared mechanisms and divergent control[END_REF] , many aspects of the underlying mechanisms and players remain to be unveiled. Unlike the other pathways of HGT, NT only requires proteins coded by the recipient cell. It relies on the presence in naturally competent bacteria of a sophisticated apparatus capable of capturing DNA present in the environment and integrating it into their chromosome [START_REF] Dubnau | Mechanisms of DNA Uptake by Naturally Competent Bacteria[END_REF] .

In both gram-positive and -negative species, NT can be divided in four distinct steps [START_REF] Dubnau | Mechanisms of DNA Uptake by Naturally Competent Bacteria[END_REF] .

It is initiated by the capture of exogenous double-stranded DNA (dsDNA) molecule at the surface of the cell where it binds to macromolecular complexes. During the uptake step, dsDNA is imported from the surface and into the periplasm (defined here as the compartments between the outer and inner membranes in gram-negative bacteria and between the cell wall and the membrane in gram-positive bacteria [START_REF] Dubnau | Mechanisms of DNA Uptake by Naturally Competent Bacteria[END_REF] ). In the periplasm, the incoming DNA is directed to the inner cell membrane for its transport into the cytoplasm as single-stranded DNA (ssDNA). There, it is handled to the recombination machinery leading eventually to its incorporation into the chromosome, the last step of the process.

At each step of NT specialised proteins are required. Some of them are common to most species studied, but others are species-specific. While type IV pseudo-pili components have been shown to mediate the binding of the DNA to the cell surface in Streptococcus pneumoniae [START_REF] Laurenceau | A Type IV Pilus Mediates DNA Binding during Natural Transformation in Streptococcus pneumoniae[END_REF] , this observation cannot be generalised to all competent bacteria. In Bacillus subtilis, wall teichoic acids, but not the pseudo-pilus, are involved in the initial binding of the DNA [START_REF] Briley | The secretion ATPase ComGA is required for the binding and transport of transforming DNA[END_REF][START_REF] Mirouze | Antibiotic sensitivity reveals that wall teichoic acids mediate DNA binding during competence in Bacillus subtilis[END_REF] . Actually, in most cases the molecules responsible for DNA capture are still unidentified. Helicobacter pylori, a species characterised for its high capacity for NT, does not harbour genes coding for type IV (pseudo-)pili. As for the uptake step, in nearly all the naturally transformable bacteria it is mediated by a type IV pseudo-pilus [START_REF] Dubnau | Mechanisms of DNA Uptake by Naturally Competent Bacteria[END_REF][START_REF] Seitz | DNA-uptake machinery of naturally competent Vibrio cholerae[END_REF] , with the exception of H. pylori that uses a type IV secretion system to pull the DNA into the periplasm [START_REF] Hofreuter | Natural competence for DNA transformation in Helicobacter pylori: identification and genetic characterization of the comB locus[END_REF][START_REF] Karnholz | Functional and topological characterization of novel components of the comB DNA transformation competence system in Helicobacter pylori[END_REF][START_REF] Stingl | Composite system mediates two-step DNA uptake into Helicobacter pylori[END_REF] . To complete this step, the conserved DNA receptor ComEA, a periplasmic or membrane-associated protein in gram-negative or gram-positive bacteria, respectively, is required. Here again, H. pylori constitutes an exception, since a unique protein, ComH, takes this role [START_REF] Damke | Identification of the periplasmic DNA receptor for natural transformation of Helicobacter pylori[END_REF] . The transport step is carried out by the membrane channel ComEC [START_REF] Seitz | DNA-uptake machinery of naturally competent Vibrio cholerae[END_REF][START_REF] Stingl | Composite system mediates two-step DNA uptake into Helicobacter pylori[END_REF][START_REF] Draskovic | Biogenesis of a putative channel protein, ComEC, required for DNA uptake: membrane topology, oligomerization and formation of disulphide bonds[END_REF][START_REF] Corbinais | Following transforming DNA in Helicobacter pylori from uptake to expression[END_REF][START_REF] Pimentel | Evolution of the Natural Transformation Protein, ComEC, in Bacteria[END_REF] . ComEC is present and required for NT in all naturally transformable bacteria. Finally, the handing of the incoming DNA to the recombination machinery requires DprA [START_REF] Mortier-Barriere | A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA[END_REF] , another transformation-specific protein with orthologues in all naturally transformable species.

Other proteins essential for NT have been identified by genetics approaches, but their role is still unknown. One of them, ComF(C), is present in all naturally competent bacteria. The requirement of this protein for transformation was discovered almost 30 years ago [START_REF] Larson | Donor DNA processing is blocked by a mutation in the com101A locus of Haemophilus influenzae[END_REF][START_REF] Londono-Vallejo | comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP-dependent RNA/DNA helicases[END_REF] . The B. subtilis ComF locus consists of an operon harbouring three open reading frames coding for the proteins ComFA, ComFB and ComFC. All three are required for competence [START_REF] Londono-Vallejo | comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP-dependent RNA/DNA helicases[END_REF][START_REF] Sysoeva | Structural characterization of the late competence protein ComFB from Bacillus subtilis[END_REF] . ComFA, which appears to be present in all naturally transformable Gram-positive bacteria but has not been identified in Gram-negative species, is a DNA-dependent ATPase [START_REF] Diallo | Bacterial transformation: ComFA is a DNA-dependent ATPase that forms complexes with ComFC and DprA[END_REF][START_REF] Londoño-Vallejo | Membrane association and role in DNA uptake of the Bacillus subtilis PriA anaiogue ComF1[END_REF][START_REF] Chilton | A conserved metal binding motif in the Bacillus subtilis competence protein ComFA enhances transformation[END_REF] . A regulatory function has been proposed for ComFB [START_REF] Sysoeva | Structural characterization of the late competence protein ComFB from Bacillus subtilis[END_REF] . In the case of ComFC, its function remains unknown despite being the only one of the three for which orthologues have been identified and described as essential for competence in all naturally transformable species studied.

Here, we characterised the ComF(C), herein ComF, from H. pylori and investigated its role in NT. Through the determination of its 3D structure we found that ComF harbours phosphoribosyl transferase (PRT) and Zn-finger domains, both essential for transformation.

We show that in the absence of ComF, not only the transport of the transforming DNA (tDNA) into the cytoplasm is blocked but also its integration into the bacterial chromosome is impaired when the DNA is directly delivered to the cytoplasm. These phenotypes, together with the observations of ComF association with the inner cell membrane and its capacity to bind ssDNA suggest a model in which ComF role provides a link between the transport and recombination steps during NT.

Results and discussion

ComF participates in DNA transport through the inner membrane. Despite the critical role of natural transformation in bacterial evolution and in the propagation of virulence and antibiotic resistances, many aspects of the transforming DNA uptake and processing remain poorly understood. A notorious example is the role of the ComF protein, which, while its gene was identified almost 30 years ago as essential for competence [START_REF] Larson | Donor DNA processing is blocked by a mutation in the com101A locus of Haemophilus influenzae[END_REF][START_REF] Londono-Vallejo | comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP-dependent RNA/DNA helicases[END_REF] and conserved in all naturally transformable species studied so far, remains unknown. A transposon mutagenesis screen originally identified hp1473 (comF), a comFC orthologue, as a gene essential for natural transformation in H. pylori [START_REF] Chang | Identification of genes associated with natural competence in Helicobacter pylori by transposon shuttle random mutagenesis[END_REF] . In the present study we undertook the characterisation of this protein and its function by a combination of approaches to (i) define the step(s) at which the protein acts during NT, (ii) determine its 3D structure and (iii) analyse its biochemical properties.

To confirm the effect of comF inactivation on H. pylori NT, a hp1473 null mutant was generated by insertion of a non-polar cassette and NT frequencies were determined using genomic DNA from a streptomycin resistant strain as transforming DNA. The absence of ComF led to almost a four log decrease in the transformation efficiency when compared to the wildtype strain (Fig. 1a). This phenotype, although less severe than that induced by inactivation of comB, comEC or recA, was similar to that observed for DcomH and DdprA strains. Wild-type levels of transformation were restored by the re-expression of comF gene introduced with its own promoter at the rdxA locus (Fig. 1b), ruling out polar effects of the deletion.

We then sought to define at which stage(s) during the transformation process ComF is required. Deletion of comF did not affect the uptake step as illustrated by the presence of transforming DNA foci (Fig. 2a andb). Consistently with the cytoplasmic localisation of ComF and the two-steps model for DNA uptake [START_REF] Stingl | Composite system mediates two-step DNA uptake into Helicobacter pylori[END_REF][START_REF] Kruger | Two steps away from novelty--principles of bacterial DNA uptake[END_REF] , the wild-type levels of DNA foci in DcomF strains indicated that ComF is not required for the exogenous DNA capture and uptake into the periplasm. The same conclusion was reached by monitoring by PCR the presence of tDNA in DcomF mutant strains of V. cholerae [START_REF] Seitz | DNA-uptake machinery of naturally competent Vibrio cholerae[END_REF] . However, when the persistence of the foci was monitored, we observed that in the DcomF strain foci are detected for longer times than in the wild type (Fig. 2b andc), similar to what was observed in a comEC mutant [START_REF] Corbinais | Following transforming DNA in Helicobacter pylori from uptake to expression[END_REF] . This suggested that ComF is needed for an efficient transport of the incoming DNA through the inner membrane. Consistently, when the kinetics of fluorescent DNA internalisation were followed in living bacteria [START_REF] Corbinais | Following transforming DNA in Helicobacter pylori from uptake to expression[END_REF] , we observed that in the DcomF mutant, as it is the case in the DcomEC one, the transforming DNA could not be detected as entering into the cytoplasm (Fig. 2d and Supplementary Movies 1-3). These observations are very similar to those described in DcomEC strains [START_REF] Stingl | Composite system mediates two-step DNA uptake into Helicobacter pylori[END_REF][START_REF] Corbinais | Following transforming DNA in Helicobacter pylori from uptake to expression[END_REF] , suggesting that ComEC and ComF act at the same NT step to mediate DNA transport across the inner membrane. Taken together, these results indicate that ComF participates in the passage of the transforming DNA into the cytosol.

ComF is associated with the inner membrane. A role of ComF in the tDNA translocation from the periplasm to the cytoplasm supposes a connection of the protein with the membrane. To explore such a possibility, we developed an antibody against ComF. Initially we were not able to detect the protein by immunoblot with this antibody, but by skipping the boiling step, a specific, albeit weak, signal was detectable in the wild-type strain extract (Supplementary Figure 1). We then fractionated the extracts into soluble and membrane fractions and we observed that ComF was associated with the membrane compartment (Fig. 3a). Further fractionation showed that ComF co-purified with the inner membrane fraction (Fig. 3b). To obtain a better signal, ComF fused to a FLAG tag (ComF-FLAG) was ectopically expressed from the ureA promoter. ComF-FLAG complementation of the comF deletion was less efficient, but could still support high levels of transformation (Fig. 1b). ComF-FLAG was also present in the membrane fraction, although it could also be found in the soluble fraction probably due to its overexpression (Fig. 3c).

The association of ComF with the membrane is consistent with the role of the protein facilitating the transport of the exogenous DNA through the inner cell membrane. The link to the membrane could be direct or mediated by either another protein or the incoming DNA. A candidate for coupling ComF to the membrane in Gram-positive bacteria could be ComFA. In B. subtilis ComFA was shown to be a membrane protein [START_REF] Londoño-Vallejo | Membrane association and role in DNA uptake of the Bacillus subtilis PriA anaiogue ComF1[END_REF] . Furthermore, it was shown that its S. pneumoniae orthologue interacts with ComF(C) [START_REF] Diallo | Bacterial transformation: ComFA is a DNA-dependent ATPase that forms complexes with ComFC and DprA[END_REF] . However, no orthologue of ComFA has been so far found in Gram-negative naturally transformable bacteria where ComF could interact with a functional, but yet to be identified, homologue of ComFA. Alternatively, ComF targeting to the membrane could be mediated by a completely unrelated protein. Finally, although unlikely in view of the lack of a membrane anchoring domain, ComF could be itself binding to the periphery of membranes.

ComF is required for tDNA handling within the cytoplasm. While the experiments described above demonstrated a role of ComF in the internalisation of the transforming DNA, they do not rule out its involvement in downstream steps of the natural transformation process. In order to test this possibility, the internalisation step, impaired in the DcomF mutant, needs to be bypassed. The transforming ssDNA was therefore delivered to the cytoplasm by electroporation. While ssDNA is a poor substrate for NT [START_REF] Levine | Plastic cells and populations: DNA substrate characteristics in Helicobacter pylori transformation define a flexible but conservative system for genomic variation[END_REF] , electroporation with a 75-mer ssDNA carrying a streptomycin resistance marker allowed transformation of mutants deficient in the uptake (DcomB2) and internalisation (DcomEC) steps [START_REF] Damke | Identification of the periplasmic DNA receptor for natural transformation of Helicobacter pylori[END_REF] . However, after electroporatin with the same ssDNA, either much less or no streptomycin resistant transformants was observed for mutants affecting the homologous recombination process (DdprA, DrecA) (Fig. 4a). When a DcomF mutant was electroporated with the same ssDNA, the level of streptomycin resistant recombinants was similar to that obtained with a DdprA strain. Similar results were obtained with a longer substrate (139-mer also carrying the mutation conferring streptomycin resistance) (Supplementary Figure 2). The reduced efficiency of a DcomF mutant in transformation by electroporation with single-stranded DNA suggests that ComF is involved in NT steps downstream of the transport through the inner membrane.

To further explore this role of ComF in the handling of the tDNA in the cytoplasm, we purified ComF and analysed its capacity to bind DNA by electrophoretic mobility shift assays (EMSA). ComF formed discrete nucleoprotein complexes with a 62-mer single-stranded DNA (ssDNA) in a concentration-dependent manner while no binding to the corresponding dsDNA was detectable (Fig. 4b). ComF bound single-stranded oligonucleotides with relatively high affinity (half-maximal binding concentration of 300 nM). This marked preference for ssDNA is consistent with the fact that during NT the incoming DNA enters the cytoplasm as ssDNA [START_REF] Mejean | DNA processing during entry in transformation of Streptococcus pneumoniae[END_REF] .

The failure to bypass the transformation defect of mutant DcomF by electroporation with ssDNA together with the capacity of ComF to bind ssDNA, indicate that ComF is likely to be implicated in the steps leading to the formation of the recombination substrate within the cytoplasm. ComF could, together with DprA and RecA [START_REF] Bergé | Transformation of Streptococcus pneumoniae relies on DprA-and RecA-dependent protection of incoming DNA single strands[END_REF] , participate in the protection of the incoming DNA from degradation. Interestingly, ComF from Campilobacter jejuni and ComF(C) from S. pneumoniae, both required for natural transformation [START_REF] Diallo | Bacterial transformation: ComFA is a DNA-dependent ATPase that forms complexes with ComFC and DprA[END_REF][START_REF] Wiesner | Natural transformation of Campylobacter jejuni requires components of a type II secretion system[END_REF] , interact with DprA [START_REF] Diallo | Bacterial transformation: ComFA is a DNA-dependent ATPase that forms complexes with ComFC and DprA[END_REF][START_REF] Parrish | A proteome-wide protein interaction map for Campylobacter jejuni[END_REF] .

Since DprA plays a critical role in the loading of the recombinase to the transforming DNA [START_REF] Mortier-Barriere | A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA[END_REF] , it is tempting to speculate that ComF binds the tDNA emerging from ComEC into the cytoplasm and targets it to DprA to protect it from degradation and allow further processing by the recombination machinery.

ComF harbours zinc-finger and PRT domains. Despite its conservation amongst naturally competent bacteria (Supplementary Figure 3), no structural data on ComF(C) proteins is available. The determination of its 3D structure has been elusive. After unsuccessful crystallisation attempts with the isolated protein we generated a gene fusion between the full-length comF gene and an artificial αRep binder coding sequence selected from a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats in order to help cristallisation [START_REF] Guellouz | Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep)[END_REF][START_REF] Valerio-Lepiniec | The αrep artificial repeat protein scaffold: A new tool for crystallization and live cell applications[END_REF] . Since structural homology predictions using HHpred (toolkit.tuebingen.mpg.de/hhpred/) [START_REF] Zimmermann | A Completely Reimplemented MPI Bioinformatics Toolkit with a New HHpred Server at its Core[END_REF] suggested that the C-terminal domain of ComF (residues 53 to 188) harbours a putative nucleoside binding site characteristic of phosphoribosyl transferases (PRTases) belonging to the PRT family (PurF, PDB number 6CZF-A, probability 99.15%), crystals were grown in the presence of 5-phospho-α-D-ribosyl 1pyrophosphate (PRPP). Diffracting crystals were obtained with the purified fusion protein and the 3D structure of full-length ComF in complex with PRPP was solved at 2.56 Å resolution (Table 1, Materials and Methods) [START_REF] Chevrel | Alpha repeat proteins (αRep) as expression and crystallization helpers[END_REF] . The fusion (αRep: residues 1-229, linker: residues 230-236, ComF: residues 237-427) is present in four copies in the asymmetric unit, organised in two domain-swapped dimers: the αRep of one fusion covers the ComF of the other one (Fig. 5a). The 3D structure obtained confirmed the presence of two predicted distinct domains in ComF.

The presence of the αRep impeded to conclude from the crystal structure on the possibility of ComF adopting higher order quaternary structures. Most PRT proteins form dimers [START_REF] Sinha | The PRT protein family[END_REF] . This, together with the dimers described for the S. pneumoniae ComFC protein [START_REF] Diallo | Bacterial transformation: ComFA is a DNA-dependent ATPase that forms complexes with ComFC and DprA[END_REF] , prompted us to explore by bacterial two hybrid assays (BacTH) whether the H. pylori orthologue could also interact with itself. This was indeed the case (Fig. 5b). To better define the interaction mode, BacTH assays using the separate CTD (residues 26 -191) and NTD (residues 1 -25) domains were performed. While no signal above background was obtained when the individual domains were tested for the formation of homodimers, a strong interaction between the PRT and the Zn-finger domains was revealed, suggesting that HpComF could form head to tail dimers (Fig. 5b).

The zinc-finger domain is required for ComF function. The small NTD of ComF (residues M1-D21), which is part of a larger domain corresponding to the additional and variable "hood" domain of the PRTase family [START_REF] Sinha | The PRT protein family[END_REF] , is a 4-Cys Zn-finger (in grey in Fig. 5c and Supplementary Figure 4a). The Zn-finger is connected by a five amino acid linker to the rest of the hood domain (residues L22-T54), structured into a small sheet of two β strands followed by a kinked α helix.

A Zn 2+ ion is liganded into the protein via the four cysteine residues (C3, C6, C15 and C18 in pink in Supplementary Figure 4a) which are highly conserved within the ComF(C) family (Supplementary Figure 3).

To explore the role of the zinc-finger, we expressed from either the rdxA or the ureA loci a ComF version in which cysteines 15 and 18 were replaced by serine (ComF-C15S,C18S), and tested its capacity to complement the transformation phenotype of a DcomF strain.

Unlike the wild-type ComF, the mutant protein could not restore natural transformation (Fig. 1b). Furthermore, mutation of the two cysteines hindered the integration into the bacterial chromosome of a ssDNA delivered by electroporation into the cytosol (Fig. 4a). Zinc-finger domains are most often found in proteins known to bind DNA or RNA [START_REF] Pabo | Transcription factors: Structural families and principles of DNA recognition[END_REF] . In particular, 4-Cys zinc-fingers are present in ribosomal proteins or in enzymes involved in DNA replication, recombination and transcription [START_REF] Leon | Zinc fingers: DNA binding and protein-protein interactions[END_REF][START_REF] Laity | Zinc finger proteins: New insights into structural and functional diversity[END_REF] . Unfortunately, attempts to purify ComF versions either mutated in cysteines 15 and 18 or deleted of the zinc-finger domain were unsuccessful, preventing further exploration of its function at the biochemical level. There are, however, several examples of zinc-finger domains that do not participate in nucleic acid binding, but are involved in protein-protein interactions [START_REF] Crossley | Self-association of the erythroid transcription factor GATA-1 mediated by its zinc finger domains[END_REF][START_REF] Sun | Zinc finger-mediated protein interactions modulate lkaros activity, a molecular control of lymphocyte development[END_REF] . Interestingly, this is the case for RadA, a DNA helicase implicated in NT of Gram-positive bacteria. While RadA mutated in its 4-Cys domain is still able to bind DNA and to carry out its ATPase and helicase activities, it cannot interact with RecA, thus limiting its D-loop unwinding capacity [START_REF] Marie | Bacterial RadA is a DnaB-Type helicase interacting with RecA to promote bidirectional D-loop extension[END_REF] .

The closest structural homologue of the ComF zinc-finger is the zinc-finger domain of RecR, a recombination protein (RMSD of 0.8 Å for 22 aligned residues, PDB number 4O6O [START_REF] Tang | RecOR complex including RecR N-N dimer and RecO monomer displays a high affinity for ssDNA[END_REF] or PDB number 5Z2V [START_REF] Che | Crystal structure of RecR, a member of the RecFOR DNA-repair pathway, from Pseudomonas aeruginosa PAO1[END_REF] ). While the role of the RecR zinc-finger remains to be determined [START_REF] Ayora | RecR is a zinc metalloprotein from Bacillus subtilis 168[END_REF][START_REF] Lee | Ring-shaped architecture of RecR: Implications for its role in homologous recombinational DNA repair[END_REF] , it has been suggested that it has a structural role in protein folding [START_REF] Lee | Ring-shaped architecture of RecR: Implications for its role in homologous recombinational DNA repair[END_REF] . Supporting this possibility, as in our case, the authors were unable to produce soluble forms of E. coli or H. pylori RecR mutated in its zinc-finger cysteines. While it is tempting to speculate that ComF zinc-finger is involved in the binding of the transforming DNA, we cannot rule out a role of this domain in the interaction of the protein with other NT partners. Further studies are required to define its precise role.

The PRPP binding domain in necessary for ComF function. The ComF CTD (residues L55-D190, the last E191 is not defined in the electron density) shares the common core of the amidophosphoribosyl transferase type 1 fold (RMSD between 2.6 and 3 Å for 100 to 130 aligned residues, PDB number 5ZGO [START_REF] Arco | Structural and functional characterization of thermostable biocatalysts for the synthesis of 6-aminopurine nucleoside-5ʺ-monophospate analogues[END_REF] as an example). A central parallel β sheet characteristic of the PRTase core domains is present (β strands 183 AIA 185 , 153 YFLLD 157 , 85 LYGIA 89 and 113 LKP 115 , in yellow in Fig. 5c), extended by the two β strands of the NTD ( 27 KVRVL 31 and 34 VSVYS [START_REF] Marie | Bacterial RadA is a DnaB-Type helicase interacting with RecA to promote bidirectional D-loop extension[END_REF] , in grey in Fig. 5c). The three Mg•PRPP-binding loops of the family are present, providing a large hydrogen bonds network with the PRPP (in green in Fig. 5c and Supplementary Figures 3 and4b). An electron density that can correspond to an Mg 2+ ion is present close to the PRPP. The "PRPP loop" carries the canonical 157 DDIITTGTTL 166 active site signature allowing the binding of the ribose-5-phosphate group of the PRPP (Supplementary Figures 3 and4b). The most variable "PPi loop" (A 89 to H 100 ) allowing the binding of the PPi group of the PRPP is slightly longer than the standard four amino acids loops. The "flexible loop" (L 120 to T 144 ) closes the pocket of the binding site occupied in our structure by the PRPP (red sticks in Fig. 5c). The presence of all three loops (Supplementary Figures 3) is considered the signature of the PRT family [START_REF] Sinha | The PRT protein family[END_REF] .

The PRPP-binding domain, present in a large variety of proteins, is known to bind small molecules such as nucleotides or NMPs [START_REF] Sinha | The PRT protein family[END_REF][START_REF] Schramm | Phosphoribosyltransferase Mechanisms and Roles in Nucleic Acid Metabolism[END_REF] . We performed differential scanning fluorimetry/thermal shift assays to detect interactions of purified H. pylori His6-ComF with various potential ligands. Fig. 6a shows that the wild-type protein exhibited a Tm of around 46 o C. In the presence of AMP the fluorescence maxima observed for the wild-type protein was shifted by +9 o C, suggesting the stabilisation of the protein through binding of the nucleotide.

Albeit to a lesser extent, ADP addition to ComF also resulted in an increase in melting temperature (Supplementary Table 1). No effect was observed with the triphosphate nucleotide. To confirm that the nucleotide binding was through the PRPP-binding domain, we purified a mutant version of the protein where the threonine165 present in the conserved 155 LLDDIITTGTTL 166 motif was replaced by an alanine. ComF T165A had a melting temperature close to that of the wild-type, indicating that the amino acid replacement did not affect significantly the structure of the protein. However, the addition of the nucleotides had a very modest effect on the thermal stability of the protein (Fig. 6b and Supplementary Table 1), confirming the role of the conserved threonine in ligand binding.

To assess the relevance of ComF nucleotide binding capacity in the function of the protein during NT, we expressed in a DcomF strain either ComF T165A from the rdxA locus and its own promoter or ComF-FLAG T165A at the urea locus. While ComF T165A restored to a certain extent the transformation capacity, the recombinant frequency was 43-fold lower than that obtained with the wild-type protein expressed in the same conditions (Fig. 1). In the case of the strain expressing ComF-FLAG T165A, the expression of the protein was unable to complement the transformation phenotype of the ΔcomF strain (Fig. 1b).

While the presence of a zinc-finger is consistent with an involvement of ComF in the handling of the transforming DNA, the fact that the protein belongs to the PRT family is surprising. [START_REF] Sinha | The PRT protein family[END_REF] . The majority of PRT proteins are enzymes that catalyse the displacement of pyrophosphate from PRPP by a nitrogen-containing nucleophile [START_REF] Sinha | The PRT protein family[END_REF] . While there are other PRTases, those belonging to the PRT family are involved in nucleotide synthesis and salvage pathways. Our results (Supplementary Table 1) show that ComF PRT domain is capable of binding not only PRPP but also monophosphorylated nucleotides and, albeit with less affinity, nucleotide di-phosphates. The physiological ligand remains, however, to be determined.

In a few PRT proteins the PRTase capacity to bind PRPP or the nucleotide substrate has been co-opted for regulatory functions as described for two Bacillus subtilis regulators of gene expression. PurR binds to DNA operator sequences to repress the expression of purine genes.

Binding of PRPP lowers its affinity for the DNA, triggering expression [START_REF] Weng | Identification of the Bacillus subtilis pur operon repressor[END_REF] . PyrR binds regulatory regions of pyrimidine genes transcripts attenuating their expression. Its affinity for the mRNA is regulated by UMP [START_REF] Swttzer | Regulation of the Bacillus subtilis Pyrimidine Biosynthetic Operon by Transcriptional Attenuation: Control of Gene Expression by an mRNA-Binding Protein[END_REF] . We thus asked if the T165A mutation affected ComF affinity for the ssDNA. Even though this substitution abolished the interaction with nucleotides (Supplementary Table 1) it did not significantly affect the binding of ssDNA (Fig. 6c and Supplementary Figure 5).

The experiments presented here do not allow to conclude on whether ComF PRT domain provides a PRTase activity or a regulatory function. An intriguing hypothesis is that the deoxyribomononucleotides released by the degradation of the non-transforming strand [START_REF] Dubnau | Fate of transforming DNA following uptake by competent Bacillus subtilis. IV. The endwise attachment and uptake of transforming DNA[END_REF] might regulate ComF capacity to bind DNA. Although ComF T165A is not affected in its DNA affinity (Fig. 6c), it is possible that binding to the wild-type protein of a so far unidentified nucleotide results in a reduced affinity for the transforming DNA. It is worth noting that the T165A mutant, while completely impaired in nucleotide binding (Fig. 6b and Supplementary Table 1), can still partially rescue the transformation phenotype of a DcomF mutant (Figure 1b), suggesting that nucleotide binding to ComF could provide a fine-tuning mechanism of the transformation process. Such a scenario would be consistent with the recently proposed hypothesis that ComF provides a link between transformation and metabolism [START_REF] De Santis | ComEB protein is dispensable for the transformation but must be translated for the optimal synthesis of comEC[END_REF] .

Conclusion

In this study, using H. pylori as a model, we sought to unveil the role of ComF, one of the most conserved proteins involved in horizontal gene transfer through NT. Despite the discovery of its essentiality for competence over 30 years ago, the understanding of where and how this protein participates in NT remained elusive. We showed here that ComF is required for at least two different steps in NT. First, ComF facilitates the transport of the tDNA through the cell membrane. Consistent with this finding we found that the protein localises to the inner membrane. Secondly, ComF, which we show has affinity for ssDNA, is involved in the handling of the DNA within the cytosol. We therefore propose that ComF provides a link between these two distinct steps during NT. Our structural studies demonstrated that ComF is composed of two conserved domains, both essential for its in vivo activity: a 4-Cys zinc-finger domain and a PRPP-binding domain. While several details of ComF mechanism of action remain to be elucidated, the data presented here shed light on the role of this protein critical for NT in all naturally competent bacteria. 2. Cultures were grown under microaerophilic conditions (5% O2, 10% CO2, using the MAC-MIC system from AES Chemunex) at 37 °C. Blood agar base medium (BAB) supplemented with 10% defibrillated horse blood (AES) was used for plate cultures. Liquid cultures were grown in brain heart infusion media (BHI) supplemented with 10% defibrillated and de-complemented fetal bovine serum (Invitrogen, Carlsbad, CA, USA) with constant shaking (180 rpm). Antibiotic mix containing polymyxin B (0.155 mg/ml), vancomycin (6.25 mg/ml), trimethoprim (3.125 mg/ml), and amphotericin B (1.25 mg/ml) was added to both plate and liquid cultures.

Methods

H. pylori cultures. H. pylori strains are listed in Supplementary Table

Additional antibiotics were added as required: kanamycin (20 μg/ml), apramycin (12.5 μg/ml), and chloramphenicol (8 μg/ml) streptomycin (10 μg/ml) as required.

Construction of gene variants in H. pylori. All oligonucleotides and plasmids used in this work

are listed in Supplementary Tables 3 and4. H. pylori 26695 gene sequences were obtained from the annotated complete genome sequence of 26695 deposited at http://genolist.pasteur.fr/PyloriGene/. Gene/locus specific primers (listed in Supplementary Table 3) were used to amplify region of interest by PCR, and fragments were joined together by either classical restriction-ligation method or using sequence-and ligation-independent cloning (SLIC). Different protein tags and mutations in the genes were introduced using SLIC or site directed mutagenesis, respectively. All the plasmids generated were verified by DNA sequencing (listed in Supplementary Table 4). The knock-out /Knock-in cassette were introduced into H. pylori by natural transformation. Their correct integration in H. pylori genome was confirmed by PCR using locus and gene specific primers. Verified strains (Table S2) were stored at -80 °C in BHI media supplemented with 12.5 % glycerol. The details of different constructions generated in this study are given below.

Construction of hp1473 null mutants in H. pylori.

To generate hp1473 locus disrupted by a non-polar chloramphenicol cassette (hp1473::Cm), hp1473 locus was amplified using gene specific primers (hp1473F and hp1473R) and ligated to blunt pjET1.2 vector to generate pJet1.2-hp1473. PCR fragments generated by amplification of this plasmid (using primers 1473 inverse F and 1473 inverse R), and non-polar chloramphenicol (using primers KpnI-Cm-for and BamHI-Cm-rev) resistance cassette were digested using KpnI and BamHI and then ligated to generate the knockout cassette p978 (pJet1.2-hp1473::Cm).

To generate hp1473 locus disrupted by a non-polar apramycin cassette (hp1473::apramycin), hp1473 locus was amplified using primers Op853 and Op854 and ligated to pjET1.2 vector amplified using Op855-Op856 to generate pJet1.2-hp1473. The PCR fragments generated by amplification of pJet1.2-hp1473 (using primers Op859 and Op860) and a non-polar apramycin resistance cassette (using primers Op857 and Op858) were ligated using SLIC to generate p1699.

Ectopic expression of hp1473 variants. hp1473 locus (+ 152 bp upstream sequence) was amplified using Op5 and Op6 and inserted in rdXA::km cassette present in the plasmid p1175 (amplified using Op3 and Op4) using SLIC to generate plasmid p1176 . T165A and C15SC18S mutations in the hp1473 coding region were introduced by PCR using mutagenic primers (op13+ Op14 and Op302 + Op303 respectively). The native hp1473 locus was disrupted using p978. For expression using the urea promoter hp1473 loci, wild-type or containing T165A or C15SC18S point mutations were amplified using Op611 and Op612 (containing the sequence for Flag tag) was ligated with p1088 (amplified using Op613 and Op614) containing the Promoter-UreA-Cm cassette using SLIC to generate p1672, p1674 and p1676 respectively. These plasmids were used to transform H. pylori followed by disruption of native hp1473 locus was using p1699.

Determination of transformation frequencies. Natural transformation frequencies were determined as described [START_REF] Damke | The nuclease activities of both the Smr domain and an additional LDLK motif are required for an efficient antirecombination function of Helicobacter pyloriMutS2[END_REF] . Briefly, total chromosomal DNA (200 ng) from a streptomycin resistant but otherwise isogenic strain was incubated overnight with exponentially growing H. pylori cells (optical density of 4.0 at 600 nm), on solid medium. Next day, serial dilutions of H. pylori were spread on plates with and without streptomycin (10 μg/ml). Transformation frequencies after electroporation were determined as described [START_REF] Damke | Identification of the periplasmic DNA receptor for natural transformation of Helicobacter pylori[END_REF] . Briefly, electrocompetent cells were prepared by treating H. pylori cells (optical density of 10 OD/ml at 600 nm) with ice-cold Glycerol 15 % + Sucrose 9 %. 50 µl of electro-competent cells mixed with 1 µg of 139-mer or 75-mer-ssDNA (Supplementary Table 3) carrying A128G mutation in the hp1197 gene were electroporated at 2.5 kV cm -1 and 25 µF. The cells were mixed with 100 µl BHI, and 50 µl cells were spotted on BAB plates. Next day, serial dilutions of H. pylori cells were plated on plates with or without streptomycin (10 μg/ml). The transformation frequencies were calculated as the number of streptomycin resistance colonies per recipient colony-forming unit. P values were calculated using the Mann-Whitney U test on GraphPad Prism software.

Fluorescence microscopy experiments.

Microscopy experiments were performed as described earlier [START_REF] Damke | Identification of the periplasmic DNA receptor for natural transformation of Helicobacter pylori[END_REF][START_REF] Corbinais | Following transforming DNA in Helicobacter pylori from uptake to expression[END_REF] . Fluorescent dsDNA (408 bp) was prepared by amplification of hp1197 locus from 26695 gDNA (100 ng) using primers 1197-5' and 1197-3' (0.5 μM each), 250 μM of dNTP mix, 5 Units of ExTaq enzyme (Takara) supplemented with 10 μM of ATTO-550-aminoallyl-dUTP (Jena bioscience). PCR elongation was performed at 72 °C (2 min per kb) and the amplified products were purified by illustra GFX purification kit (GE Healthcare Little Chalfont, UK).

Exponentially growing H. pylori cells were incubated with fluorescent DNA (200 ng) for 7 min at 37 °C, the unbound DNA was washed the bacteria were re-suspended in BHI, covered with low melting agarose (1.4 %) supplemented with 10 % fetal bovine serum and were observed under live conditions [gas mixture (10 % CO2, 3 % O2), humidity (90 %)] at 37 °C for 3 h. Alternatively, the bacteria were fixed with 4 % formaldehyde (90 mins at 4 °C) followed by quenching with 100 mM Glycine. All the images were captured with 60X objective using inverted Nikon A1R confocal laser scanning microscope system. The images were processed and analyzed using NIS-element software (Nikon Corp., Tokyo, Japan) and ImageJ software.

The percentage of bacteria with DNA foci was calculated as the number of bacteria with DNA foci over total number of bacteria counted in at least two independent biological replicates.

To monitor the time dependent stability DNA foci, the total number of bacteria with DNA foci at t=15 mins were considered as 100%. The volumes of internalised DNA in GFP expressing bacteria were estimated by 3-D image analysis performed using Volocity software (Perkin Elmer, Waltham, USA). Subcellular fractionation. Subcellular fractions of exponentially growing H. pylori were collected by differential centrifugation and detergent mediated solubilisation as described earlier [START_REF] Damke | Identification of the periplasmic DNA receptor for natural transformation of Helicobacter pylori[END_REF] . Briefly, 100 ml cell pellet was re-suspended in buffer A (10 mM Tris-HCl, pH 7.5, 1mM DTT, 1X protease inhibitor cocktail) followed by lysis by sonication. Total extracts were centrifuged 14,000 rpm for 15 minutes. The supernatant containing the soluble fraction was collected after ultracentrifugation at 45,000 rpm for 45 min of the total extract. The pellet containing the membrane fractions was re-suspended in buffer B (10 mM Tris-HCl, pH 7.5, 1mM DTT, 1X protease inhibitor cocktail, 1% N-Lauroylsarcosine). The supernatant containing the inner membrane fractions were collected after ultracentrifugation at 45,000 rpm for 45 min. The presence of the proteins in the various fractions was monitored by immunoblotting.

Western blots. The fractionation samples were resolved on a 15% SDS-PAGE, transferred on a nitrocellulose membrane. The membrane was blocked with 2% BSA prepared in PBST (1X PBS + 0.03% Tween 20). Blots were probed with either a mouse monoclonal anti-Flag antibody (1: 5000 dilution, Sigma Aldrich), rabbit anti-MotB antibody (1: 2500 dilution) (kind gift from Dr. Ivo Boneca, Pasteur Institute) or rabbit anti-HpComF antibody from our laboratory collection. The blots were then probed with Advansta fluorescently labeled secondary antibodies IR700 and IR800 respectively. The imaging was done using Odissey Clx imaging system. E. coli cultures. Escherichia coli strains used for cloning, protein overexpression and purification were cultured in Luria-Bertani (LB) broth, or LB agar plates supplemented with the required antibiotics [ampicillin (100 μg/ml), kanamycin (50 μg/ml), apramycin (50 μg/ml), or chloramphenicol (34 μg/ml)].

Protein samples preparation. Cloning of comF (Hp1473) coding region was performed using genomic DNA from H. pylori strain 26695 as template for PCR. Six histidine codons were added at the 5' end during the PCR process. The fragment was inserted into the NdeI-XhoI sites of pET21a vector (Novagen). Site directed mutagenesis was performed using the resulting pET21:ComF-6His plasmid as a template and non-overlapping oligonucleotides phosphorylated in 5' (Eurofins), to construct the HpComF T165A mutant. The fusion of comF with an αRep protein (named B2), for its structural study, is described in [START_REF] Chevrel | Alpha repeat proteins (αRep) as expression and crystallization helpers[END_REF] .

Expression of ComF or its mutant forms in BL21(DE3) Gold strain was performed in 800 ml 2xYT o/n at 37°C after induction with 0.5 mM IPTG (Sigma). Cells were harvested by centrifugation, resuspended in buffer 500 mM NaCl, 20 mM Tris-HCl (pH 7.5), 5% glycerol for ComF-6His constructs, or in buffer 1 M NaCl, 100 mM Tris-HCl (pH 8), 100 µM TCEP for B2-ComF-6His (SeMet labelled according to the protocol described in [START_REF] Quevillon-Cheruel | Cloning, production, and purification of proteins for a mediumscale structural genomics project[END_REF] and stored at -20°C. Cell lysis was completed by sonication (probe-tip sonicator Branson). After centrifugation at 20,000 g and 8°C for 30 min, the proteins were purified on a Ni-NTA column (Qiagen Inc.), eluted with imidazole. ComF-6His and B2-ComF-6His were then desalted up to 100 mM NaCl and loaded respectively onto a Heparin and a MonoQ column (Amersham Pharmacia Biotech) and eluted with a gradient of NaCl (from 100 mM to 1 M). The proteins were desalted up to 200 mM NaCl and concentrated using Vivaspin 5,000 or 30,000 nominal molecular weight limit cut-off centrifugal concentrators (Sartorius), respectively, aliquoted, flash frozen in liquid nitrogen and stored at -80°C, or dialyzed in a 50% (vol/vol) glycerol buffer for storage at -20°C.

Electrophoretic mobility shift assay. DNA biding assays were performed by incubating indicated concentrations of proteins with fixed concentrations of Cy5 labelled DNA (Supplementary Table 3) in binding buffer (10 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM DTT, 0.1 µg/µl BSA) in cold room for 30 min. The nucleoprotein complexes were separated using native TBE-PAGE (6%). The gels were visualized by using Typhoon. The depletion in substrate DNA was quantified using ImageJ by considering DNA without protein as 100%.

Crystal structure determination. SeMet modified B2-HpComF-6His (12.5 mg/ml) was incubated with PRPP (3 mM) and MgCl2 (5 mM) at 4°C. Crystals were grown in hanging drops by mixing the protein with reservoir solution in a 1:1 ratio. Crystals appeared after 5 days at 4°C in 0.2 M Tri-potassium citrate + 18% PEG 3350. Glycerol cryo-protected crystals (two steps at 15 and 30%) were flash frozen in liquid nitrogen.

Diffraction data and refinement statistics are given in Table 1. Crystallographic data were collected at the selenium peak wavelength on the PROXIMA-2A from Synchrotron SOLEIL (Saint-Aubin, France) and processed with XDS 51 through XDSME (https://githubcom/legrandp/xdsme). Diffraction anisotropy was corrected using the STARANISO server (http://staraniso.globalphasing.org). The structure was solved by the SAD phasing method at 2.5 Å resolution using SHELX C/D 52 to locate the 12 heavy atom sites, PHASER [START_REF] Mccoy | Phaser crystallographic software[END_REF] to determine the initial phases and PARROT [START_REF] Cowtan | Recent developments in classical density modification[END_REF] to improve the phases by density modification, through the CCP4 program suite [START_REF] Winn | Overview of the CCP4 suite and current developments[END_REF] . The construction of the model was initiated using Buccaneer [START_REF] Cowtan | Fitting molecular fragments into electron density[END_REF] and refined with the BUSTER using TLS and NCS restraints [START_REF] Smart | Exploiting structure similarity in refinement: Automated NCS and targetstructure restraints in BUSTER[END_REF] . The model was corrected and completed using COOT [START_REF] Emsley | Features and development of Coot[END_REF] . The presence of a Zn 2+ ion in the B2-HpComF structure was demonstrated by an energy scan performed on the crystals at the beamline (energy peak at 9.664 keV). Exploration of the 3D structures was performed using the following tools: Dali server [START_REF] Holm | Dali server: Conservation mapping in 3D[END_REF] , I-TASSER [START_REF] Zhang | I-TASSER server for protein 3D structure prediction[END_REF] and SWISS-MODEL servers [START_REF] Arnold | The SWISS-MODEL workspace: A web-based environment for protein structure homology modelling[END_REF] and PyMOL Molecular Graphics System (http://www.pymol.org).

Bacterial Two-Hybrid assays. The Bacterial Two-Hybrid test was used to probe the interactions between proteins [START_REF] Karimova | A bacterial two-hybrid system based on a reconstituted signal transduction pathway[END_REF] . The full-length ComF encoding sequence was fused to T18, at the C-terminal and N-terminal ends, T18-ComF (pUT18C vector) and ComF-T18 (pUT18 vector), respectively. The same strategy has been used for ZnF and PRPP, the N-terminal and C-terminal domains of ComF, respectively. Plasmids encoding T25-ComF, ComF-T25 and T25-PRPP were constructed using the pKT25 and pKNT25 vectors.

Plasmids encoding T18 and T25 fusion proteins were co-transformed in E. coli strain BTH101 and transformants were selected in Luria-Bertani agar plates containing kanamycin and ampicillin at 30˚C. Colonies were then spotted on plates containing kanamycin, ampicillin, IPTG and X-gal, incubated at 30°C and stored at RT to follow the appearance and evolution of the blue colour.

Differential

  Scanning Fluorimetry/Thermal shift assay. Purified protein (10.5 µg) was incubated with different analytes in reaction buffer (20 mM Tris-Cl, pH 7.5, 200 mM NaCl, 5X Sypro Orange). The temperature of the reaction mixture was raised from 25 o C to 95 o C. Shift in the fluorescence due to binding of the Sypro-Orange dye as the hydrophobic patches of the protein were exposed due to denaturation of the protein was recorded. The fluorescence maxima observed was used to calculate the approximate melting temperature of the protein in native conditions and in presence of the analyte.

Fig. 1 :

 1 Fig. 1: ComF is essential for genetic transformation of H. pylori. (a) Natural transformation frequencies for indicated H. pylori strains. (b) Complementation of the DcomF strain. Natural transformation frequencies for indicated H. pylori strains were determined using isogenic streptomycin resistant total genomic DNA as donor. Bars correspond to the mean and standard deviation from at least three independent biological replicates. ns, not significant (P > 0.05); **P < 0.01, ***P < 0.001 and ****P < 0.0001. P values were calculated using the Mann-Whitney U test on GraphPad Prism software.

Fig. 2 :

 2 Fig. 2: ComF supports translocation of tDNA across the cytoplasmic membrane. (a) Percentage of cells with fluorescent DNA foci for indicated H. pylori strains. Bars correspond to the average and standard deviation from at least two independent biological experiments. (b) Time course of DNA foci for indicated H. pylori strains. Z maximum projections of merged images of ATTO-550 (red channel) and differential interference contrast (DIC) are presented. (c) Stability of DNA foci displayed by H. pylori strains. Data points correspond to the mean and standard deviation from at least two independent experiments except for the ΔcomEC strain. (d) Internalization kinetics of fluorescent DNA in indicated H. pylori strains. GFP expressing bacteria displaying fluorescent DNA foci were followed for 3 h by confocal microscopy in live conditions (Supplementary Movies 1-3). The mean +-SEM volumes of DNA internalized were measured by 3D-analysis of individual bacterial cells for wild-type (n=26), ΔcomEC (n=25), ΔcomF (n=148) strains. At least two independent experiments were performed for each strain except for ΔcomEC. P-values calculated using Kruskal-Wallis statistics indicate that ΔcomEC (p<0.0001), ΔdprA (p<0.0050), and ΔcomF (p=0.0003) curves are significantly different from the wild-type curve.

Fig. 3 :

 3 Fig. 3: ComF is a membrane-associated protein. (a) and (b) Localisation of ComF in wild-type H. pylori strain. (c) Localisation of overexpressed ComF-FLAG. The inner membrane protein MotB was used as marker for the fractionation experiments. T: total extract, S: soluble fraction, M: membrane. B: boiled samples, NB: not boiled samples.

Fig. 4 :Fig. 5 :Fig. 6 :

 456 Fig. 4: ComF binds single-stranded DNA and promotes its chromosomal integration (a) Transformation frequencies after electroporation with a chemically synthesized singlestranded DNA (75 -mer) coding for streptomycin resistance as donor DNA. Bars correspond to the average and standard deviation from at least two independent biological experiments. (b) Selective affinity of ComF for single-stranded DNA. Indicated concentrations of His6-ComFC were incubated with Cy5 labelled single-or double-stranded DNA substrate. The nucleoprotein complexes were resolved by native-PAGE.

  Figure 1

Table 1 . Data Collection and Structure Refinement Statistics

 1

	Data collection	PRPP-bound B2-HpComF ¤
	Space group	P1
	Unit cell parameters	a=58Å b=88Å c=123Å
		a=80° ß=76° γ=76°
	Wavelength (Å)	0.979
	Resolution range (Å) †	46.5-2.5 (2.6-2.5)
	Before STARANISO
	Measured/Unique reflections †	895493/77488 (57841/5398)
	Completeness (%) †	98.4 (92.6)
	Anomalous completeness (%) †	95.0 (84.8)
	I/σ(I) †	15.7 (0.6)
	After STARANISO
	Measured/Unique reflections †	752919/63154 (44297/3158)
	Completeness (%) †	93.5 (94.2)
	Anomalous completeness (%) †	90.6 (92.7)
	I/σ(I) †	19.2 (1.6)
	Redundancy †	11.9 (14.0)
	Anomalous redundancy †	6.1 (7.1)
	CC1/2 †	0.999 (0.387)
	CCano †	0.715 (0.403)
	|DANO|/σ(DANO)	1.404 (0.814)
	Rmerge (%) †	8.8 (140.1)
	Rpim (%) †	2.7 (38.3)
	SAD phasing
	Number of sites	12
	Overall FOM	0.39
	Refinement
	Resolution range (Å) †	46.5-2.5 (2.6-2.5)
	Number of work/test reflections	63118/3156
	R/Rfree (%) †	22.1/24.5 (25.6/27.2)
	Geometry statistics
	Number of atoms
	Protein	12672
	Ligand/ion	184
	Water	282
	r.m.s. deviations from ideal values
	Bond lengths (Å)	0.007
	Bond angles (°)	0.87
	Average B-factor (Å 2 )
	From atoms	69.74
	From Wilson plot	79.46
	Ramachandran plot
	Favoured (%)	97
	Allowed (%)	3
	Outliers (%)	0

Acknowledgements

We thank the beamline staff for assistance and advice during data collections at Synchrotron SOLEIL (Saint-Aubin, France; beamline Proxima 2). We thank Christopher Corbinais and Mariano Prado-Acosta for the construction of the initial comF mutants. This work has benefited from the I2BC Macromolecular interactions measurements and Crystallization Platforms. Financial support for this work was provided by the Indo-French Centre for Promotion of Advanced research (CEFIPRA) grant 5203-5 (JPR, PPD), Agence Nationale de la Recherche grant ANR-19-CE12-0003-01 (JPR), French Infrastructure for Integrated Structural Biology (FRISBI) grant ANR-10-INSB-05-01 (SQC, RG), Région Ile de France grant DIM1Health (JPR, PPD), Commissariat à l'Energie Atomique (JPR, AMDG, XV, JD), Centre National de la Recherche Scientifique (SQC, SM), Enhanced Eurotalents fellowship programme (CEA/EU) (PPD) and Collectivité Régionale de Martinique (LC)

Data availability

The atomic coordinates and structure factors of B2-HpComF have been deposited at the Brookhaven Protein Data Bank under the accession number 7P0H.

All the other data are available in the main text or the supplementary materials.

Author contributions

Conceptualisation


M Fiore

• O Vermeersch

• M Forte

• G Casalis

• C François

O Vermeersch
email: olivier.vermeersch@onera.fr

Characterization of a highly efficient chevron-shaped anti-contamination device

Introduction

Reducing the extent of turbulent boundary layer on aircraft is of considerable interest because it lowers friction drag, fuel consumption and its related pollutant emission. More precisely, progress has been made to maintain as far as possible a laminar flow around tail planes and wings of commercial aircraft. These efforts can be annihilated by leading-edge contamination which can occur on swept wings. Turbulence convected along the fuselage propagates along the attachment line and spreads over the wing surface leading to the transition of the boundary layer. Theoretical developments (see [START_REF] Görtler | Dreidimensionale instabilität der ebenen staupunktströmung gegenüber wirbelartigen störungen. Fifty years of boundary layer research[END_REF][START_REF] Pfenninger | Flow phenomena at the leading edge of swept wings[END_REF]Poll 1978) have led to the definition of a quantity called «Reynolds bar» and denoted R to establish if contamina- tion process is likely to occur depending on the value of this quantity (threshold). In standard flight conditions, R for commercial aircraft is often higher than the critical threshold and the leading-edge contamination may happen. In order to address this issue, Gaster (1965) designed a passive device able to stop turbulence coming from wind tunnel wall that simulated the fuselage. This small fairing, located close to the wing root, was made in such a way that contaminating turbulent boundary layer was brought to face with a stagnation point, while a «clean» laminar boundary layer developed downstream. This «Gaster bump» or anticontamination device (ACD) was able to prevent leadingedge contamination up to R values higher than the criti- cal threshold in smooth condition R = 250, but the gain remained modest.

In 1967, after some wind tunnel experiments, Gaster reported a maintenance of a laminar flow downstream this bump along the attachment line up to R = 420. More recent in-flight experiments on Falcon 50 and 900 (see [START_REF] Arnal | Control of laminar turbulent transition for skin friction drag reduction[END_REF] have shown similar results for a standard Gaster Abstract This paper is devoted to the characterization of an optimized chevron-shaped anti-contamination device (ACD). This device can prevent efficiently the propagation of turbulence from the fuselage along the attachment line (hypothetical streamline that spreads the flow going to suction side and the one going to pressure side) of swept wings and enables the development of a new laminar boundary layer downstream. More specifically, the aim is to prevent boundary-layer transition along the attachment line by a contamination process. This process is characterized by the typical Reynolds number R and the associated Poll's crite- rion. Thus, ACD efficiency will be expressed in terms of R values. Some experiments performed on a new numeri- cally optimized ACD have shown its ability to prevent leading-edge contamination up to R values close to the natural transition process of the laminar boundary layer along the attachment line. The corresponding stability analysis of the laminar boundary layer is made using the Görtler-Hämmerlin stability approach. The study is completed with the different transition processes that can occur downstream the attachment line, around the airfoil, especially with crossflow analysis. bump set-up on wings allowing to maintain laminarity up to R values between 350 and 400. These values showed an improvement relatively to the contamination criterion without ACD (R = 250). However, values of R on lifting sur- faces of civil aircraft range from 400 to 700 near the wing root. Therefore, a clear improvement in ACD efficiency had to be achieved for civil aircraft applications. Krier and Sucipto (2009) and Krier et al. (2009) were the first to propose an arrow-shaped ACD in the framework of the SUPERTRAC European project (SUPersonic TRANsition Control). The ACD design by Krier et al was made of a cylindrical nose prolonged by streamlined legs in order to alter as less as possible the flow in the attachment line region. This shape was successfully tested in a supersonic wind tunnel for two Mach numbers M = 1.7 and M = 2.7. This ACD was able to delay contamination up to R = 330 at M = 1.7 and R = 400 at M = 2.7. After these experiments, Euler computations have shown the possibility of such devices to delay contamination for lower speeds and thus their potential application in transonic commercial aircraft.

Since several decades, ONERA has acquired a strong expertise in the design and testing of ACDs (see [START_REF] Arnal | Control of attachment line contamination. In: 2nd European forum on laminar flow technology, Bordeaux Bippes H[END_REF][START_REF] Gasparian | Experimental study and transition mechanisms modeling on swept wing, with application to laminar flow control[END_REF]) that has led to the development of a very efficient ACD experimentally tested in 2014 and detailed through this article. These research activities have started in 2010 in the framework of the Joint Technology Initiative Clean Sky Project and have enabled the development of an optimized chevron-shaped ACD specifically designed for low-speed conditions.

Section 2 will introduce general knowledge about flow characteristics related to a swept wing with wall junction. This basis will enable further developments on leading-edge contamination characterization. When this phenomenon can be avoided, for example, by using an ACD, the laminar boundary layer that develops downstream can naturally become unstable. This second attachment line transition process will also be addressed. The third section will be dedicated to ACD specifications and development performed by ONERA in order to optimize its ability to prevent leadingedge contamination. Then, the experimental set-up will be introduced in Sect. 4. Finally, results related to this test campaign will be detailed, especially concerning destabilizing process characteristics and ACD performance validation.

Mean flow and transition processes around swept wings

Mean flow

This section gives a general overview about swept-wing flow characteristics. Mean flow along the attachment line of a swept wing (sweep angle ϕ) is the three-dimensional swept stagnation flow. It consists of the superposition of the [START_REF] Hiemenz | Die grenzschicht an einem in den gleichförmigen flüssigkeitsstrom eingetauchten geraden kreiszylinder[END_REF] plane stagnation flow [(x, y) with x chord- wise direction and y wall normal direction] and a transverse uniform velocity in the span direction Z (see Fig. 1). The coordinate x will be used as the curvilinear abscissa around the airfoil with the origin x = 0 on the attachment line.

The coordinate y is the distance from the wall in its normal direction. Under the assumption of an infinite swept wing (∂/∂Z = 0), velocity components in the boundary layer (U, V, W) in the (x, y, Z) spatial directions are formulated using self-similarity functions (f, g) defined as:

where (U e , V e , W e ) are the velocity components at the edge of the boundary layer in the (x, y, Z) directions. W ∞ = Q ∞ sin(ϕ) is the far-field velocity component in the span direction Z. k and ν represent respectively a strain rate and the kinematic viscosity of the fluid. Self-similarity functions are resolved from the following ordinary differential equations obtained for incompressible flows:

with boundary conditions imposed at the wall (η = 0) and in far-field direction (η → +∞): Based on the latter Falkner-Skan-Cooke self-similarity functions (see Fig. 2), Reynolds number from displacement, momentum thicknesses (R δ 1 , R θ ) and shape factor (H) can then be deduced:

(1a) U e (x, y) = kx V e (x, y) = -ky W e (x, y) = W ∞ (1b) U U e = f ′ (η) V V e = -f (η) W W e = g ′ (η) (1c) with η = y � and � = ν k (2a) f ′′ + f ′′ f -f ′2 + 1 = 0 (2b) g ′′ + g ′ f = 0 (3a) f (0) = 0 f ′ (0) = 0 g(0) = 0 g ′ (0) = 0 (3b) lim η→+∞ f ′ (η) = 1 lim η→+∞ g ′ (η) = 1
We will see in the next paragraph that latter quantities can be formulated more simply using R.

In laminar-turbulent transition process along the attachment line of a swept wing, mainly two transition processes may occur: either a leading-edge contamination (see Sect. 2.2) that can be linked to a bypass transition, or when contamination can be prevented (by an ACD for example), the new laminar boundary layer that develops downstream can also become turbulent due to a natural transition process involving Görtler-Hämmerlin

(4a) R δ 1 = W e δ 1 ν with δ 1 = � δ 0 (1 -g ′ (η)) dη (4b) R θ = W e θ ν with θ = � δ 0 g ′ (η)(1 -g ′ (η)) dη (4c) H = δ 1 θ
(GH) instabilities (see Fig. 3). In the first case, transition along the attachment line is driven by non linear interactions with the fuselage turbulent boundary layer, while the second process can be described using a linear stability approach (see Sect. 2.3).

Leading-edge contamination

Many experiments concerning leading-edge contamination phenomenon have been carried out and have highlighted a limited number of relevant parameters. These parameters are gathered in the quantity R that can be formulated as:

At low speed, [START_REF] Pfenninger | Flow phenomena at the leading edge of swept wings[END_REF] and Poll (1978) have developed a simple criterion (referred as Poll's criterion) based on the value of R to establish if leading-edge con- tamination is likely to occur:

If R ≤ 250, the bursts of turbulence convected along the attachment line are damped and vanish as they travel along the attachment line.

For R ≥ 250, bursts are self-sustaining. They grow and overlap so that the attachment line boundary layer becomes fully turbulent. [START_REF] Poll | Boundary layer transition on the windward face of space shuttle during re-entry[END_REF] extended this criterion to high-speed flows by introducing a modified length scale η * and a modified Reynolds number R * which have the same definition than η and R, except that ν is replaced by ν * . The latter quan- tity is the kinematic viscosity computed at a reference temperature T * which can be estimated from an empirical relationship:

where T e is the temperature at the boundary-layer edge, T w is the wall temperature, and T aw is the adiabatic wall (recovery) temperature. A = 0.1 and B = 0.6 are empiri- cal constants commonly used. Correlation of existing data suggested that contamination is likely to occur for R * = 245 ± 35.

Reynolds numbers for displacement and momentum thicknesses can then be expressed using R by: Poll's criterion threshold can then be defined in terms of momentum thickness by R θ,crit = 101.

(5) R = W e � ν (6) T * = T e + A(T w -T e ) + B(T aw -T e ) (7a) R δ 1 = 1.026 R (7b) R θ = 0.404 R (7c) H = 2.54

Natural transition

When contamination phenomenon can be avoided (see Fig. 3), different processes all related to natural boundarylayer instabilities can lead to transition in different locations of the wing surface. Even if each of these different processes exhibit particularities that will be depicted in this article, some common main steps can be identified. The first step is the receptivity phase. Free-stream turbulence and noise, vibrations or small roughness elements can «enter» the laminar boundary layer and begin to excite its eigenmodes.1 Then, during the linear amplification phase, eigenmodes that take the form of periodic waves can grow or decay following a linear mechanism. Finally, some amplified modes evolve nonlinearly, so that turbulent spots appear and lead to transition. The first natural transition process that may occur when contamination can be avoided is a boundary-layer destabilization along the attachment line. As we will see, this destabilization referred as Görtler-Hämmerlin instability will be studied using the threedimensional swept stagnation flow introduced previously. This approach will be of central interest for ACD efficiency characterization.

When the boundary layer can be maintained laminar along the attachment line, it can become unstable around the airfoil in chordwise direction. Depending on the location, pressure gradient can have a destabilizing effect on the boundary layer. Near the leading edge where the flow is strongly accelerated (negative pressure gradient), the crossflow mean-velocity profile (W) inside the boundary layer is highly unstable. It generates crossflow instabilities with a wave-number vector making a nearly orthogonal angle with respect to the free-stream direction. This leads to the formation of co-rotating vortices, the so-called crossflow instabilities. In particular, steady modes are strongly amplified and are suspected to be the dominant modes in a low free-stream turbulent environment (e.g. in flight). Downstream, in region of decelerated flow (positive pressure gradient), the streamwise mean-velocity profile (U) is unstable. Some waves, similar to two-dimensional Tollmien-Schlichting waves, are amplified with a wave-number vector nearly aligned with the free-stream direction and trigger the transition. The possibility of designing laminar wings is thus given by the ability to control these different destabilizing phenomena.

A common theoretical framework can be adopted to study the growth and destabilization of these boundarylayer modes. In the linear stability theory that will be applied, only the linear amplification step (second phase) is considered to estimate the transition location. Transition will then be given for a certain amplification value A/A 0 of a wave. A (respectively A 0 ) is the amplitude of the considered mode at a given abscissa (respectively at a reference abscissa). Wave amplification is obtained from the classical Orr-Sommerfeld theory (1908). For incompressible flow, velocity field and pressure (∧) are decomposed into a non-dimensional base flow (-) and disturbances (′):

where u, v, w, are the wave amplitudes in the x, y and Z directions and π the pressure fluctuation amplitude. α = α r + iα i ∈ C, β and ω ∈ R are, respectively, the wave numbers along x, Z and the angular frequency. This formalism is adapted to the destabilization of the flow around the airfoil in the x direction (Tollmien-Schlicht- ing and crossflow waves). Considering this approach as starting point, Görtler and Hämmerlin (1955) developed a stability theory for attachment line application using a particular mathematical form for the base flow and disturbance quantities:

where β = β r + iβ i ∈ C.
The main difference with the Orr-Sommerfeld approach is the x dependence of u ′ simi- larly to its correlated external velocity U e . For a consid- ered wave (defined as its real angular frequency ω) and a fixed R, the stability theory gives access to the corre- sponding amplified Görtler-Hämmerlin mode. When β i is negative, according to the formulation (9b), this wave is amplified in spanwise direction and can lead to the natural transition (NT on Fig. 3).

From this attachment line stability analysis, the neutral curve (iso β i = 0) that separates stable from unstable domains can be drawn in a (R, ω) diagram (see Fig. 4). When leading-edge contamination can be prevented, it can be noticed that the attachment line laminar boundary layer becomes unstable for R ≥ 582.

(8a) ∧ U, ∧ V , ∧ W , ∧ P t = (U, V , W , P) t + u ′ , v ′ , w ′ , π t (8b)     u ′ v ′ w ′ π     =     u(y) v(y) w(y) π(y)     exp[i(αx + βZ -ωt)] (9a) U = x f ′ R V = -f R W = g ′ (9b)     u ′ v ′ w ′ π     =     x u(y) v(y) w(y) π(y)     exp[i(βZ -ωt)]

ACD design

Some prerequisites have to be fulfilled to guarantee the ACD efficiency (see [START_REF] Arnal | Control of attachment line contamination. In: 2nd European forum on laminar flow technology, Bordeaux Bippes H[END_REF]. The anti-contamination process is based on a stagnation point generated by the ACD that stops the turbulent boundary layer from the fuselage and enables the downstream development of a new laminar boundary layer. To be fully efficient, the ACD has to be high enough so that the stagnation point would be located above the incoming fuselage turbulent boundary layer. Besides, the pressure distribution downstream the ACD has to allow a region of attached laminar flow. It was noticed that an increase in the radius at the top corner of the ACD and a humped shape could improve its efficiency. For aeronautic applications, the ACD has to tolerate some variations in angle of attack and sideslip angle that may occur during cruise. Finally, the ACD must have a very little impact on total drag in order to keep the potential benefits of a laminar boundary layer. In 2010, a numerical model of the ACD mounted on a swept wing has been achieved using CATIA software. This approach enabled a parametric study of the ACD where some geometric parameters were varied: dimensions, angles and shape while keeping its ability to be put on the same swept-wing leading edge. Structured meshes were then generated using an ANSYS software. Three-dimensional Reynolds-Averaged Navier-Stokes (RANS) computations were carried out using the ONERA elsA code with future corresponding experimental conditions P i0 = 101,325 Pa , T i0 = 288 K and Q ∞ = 20 m s -1 (R = 375). Efforts were made to enable the development of a boundary layer without separation on and around the ACD, especially by studying the wall streamlines (see Fig. 5). After a selection of the most promising shapes, it was decided to perform new numerical computations using laminar-turbulent transition criteria (AHD-C1, Arnal and Habiballah 1984) with an extended velocity range up to Q ∞ = 50 m s -1 (R = 590). This led to a new optimized shape that was tested in 2014. A sketch of this ACD is depicted in Fig. 6 with the most relevant geometrical parameters. Their values, made dimensionless by the total length L of this ACD, are gathered in Table 1. During the numerical optimization process, 20 shapes have been designed and analysed through RANS computations.

In Table 1, the last column provides the ranges in which the key geometrical parameters have been varied in. The associated test campaign, dedicated to confirm this promising ACD shape, is detailed in the next paragraph.

Experimental test campaign

Wind tunnel and model

The experimental campaign has been performed in the F2 wind tunnel located at the ONERA Le Fauga-Mauzac A first ACD-free configuration has enabled the validation of the experimental set-up by checking the Poll's criterion (threshold of contamination R = 250). Additionally, a configuration with ACD and roughness (tripping device) has been investigated to check whether turbulence was selfsustaining for R values higher than 250. The corresponding velocities range from 5 to 60 m s -1 .

In the second configuration, the DTP-A model was equipped with the optimized shape ACD. For this configuration, a wide range of velocity values (from 10 to 90 m s -1 ) has been covered with a low velocity step so as to properly capture Görtler-Hämmerlin and crossflow instabilities (related to natural instability).

Metrology

Wall pressure and hot-film measurements were performed as well as infrared visualizations (see Fig. 7). Wall pressure measurements were achieved using 53 taps located on 3 lines orthogonal to the leading edge and distributed symmetrically from the attachment line:

1. 40 % span: 27 taps (PP1), 2. 60 % span: 13 taps (PP2), 3. 73.5 % span: 13 taps (PP3), Hot-film measurements (HF) were also performed in order to detect the nature of the boundary layer (laminar, transitional or turbulent). Dantec 55R47 hot-films were conditioned with a home-made constant temperature anemometer. Whatever the configuration of the model, hot-films HF1 to HF5 were all flush-mounted along the attachment line in order to be as non-intrusive as possible for the flow (configuration set on the detailed model view, Fig. 7). For the configuration with ACD, the removable part of the smooth leading edge was replaced by the block with the ACD plus two additional hot-films (HF5′, HF6) which were simply stuck on the model (detached block on Fig. 7). The hot-film 6 was located in a region where crossflow instabilities are alleged to occur (x = 70 mm). The corresponding hot-film positions are given in Table 2. After AC coupling, the data acquisition system was composed of anti-aliasing low-pass filters (10 kHz) and amplifiers (gain 20 or 100). Meas- urements were made at a 25 kHz sampling rate. The test section was also equipped with an infrared camera (IRC 900) set above the wind tunnel ceiling. The heat flux between the surface and the flow is more important with a turbulent flow than a laminar one. Areas in the model where the flow is turbulent lead to a quicker temperature change. By cooling or heating punctually the flow in the wind tunnel, transition position can then be identified. An example of infrared visualization is given in Fig. 12 where lightest area represents a laminar boundary layer.

For both configurations, the only varying parameter was the wind tunnel velocity. Based on experimental measurements, the quantity R will be estimated as a function of the velocity. Next paragraph is devoted to R estimation using an intermedi- ate quantity called effective sweep angle and denoted ϕ eff .

Experimental results

Effective sweep angle and R determination

ϕ eff makes reference to the sweep angle ϕ that should be taken in inviscid fluid solution in order to fit as close as possible to the experimental pressure distribution on the attachment line. Due to viscous and set-up effects, stall and horse-shoe vortices, ϕ eff is usually different from the geometrical one. The effective sweep angle ϕ eff is cho- sen in order to recover the value of the interpolated pressure coefficient C p,interp from measurements on the attach- ment line (θ = 0 see Fig. 8). The link between ϕ eff and C p,interp (θ = 0) is given in Eq. ( 11). We first start with the classical relationship giving the theoretical expression of C p for a swept cylinder as a function of ϕ and θ:

where p ∞ and ρ ∞ are, respectively, the inflow pressure and density. Fitted experimental pressure coefficient around θ ≈ 0 is then used to evaluate ϕ eff :

The evolution of the effective sweep angle in spanwise direction is illustrated in Fig. 9. Measurements (symbols) have been plotted at locations corresponding to the wall pressure taps (PP1, PP2, PP3) for three values of the inflow velocity Q ∞ . The geometrical sweep angle (ϕ geom = 50 • ) is repre- sented by the horizontal dashed line. The difference between effective and geometrical angles varies from 6 • to 8.5 • and (10)

C p (θ) = p(θ) -p ∞ 1 2 ρ ∞ Q 2 ∞ = cos 2 (ϕ) 1 -4 sin 2 (θ) (11a) C p,interp (θ = 0) = cos 2 (ϕ eff ) (11b) ϕ eff = cos -1 C p,interp (θ = 0)
increases with the inflow velocity. This demonstrates that wall effects are significant. Figure 8 depicts a typical distribution of pressure coefficient around the leading edge. Measurements (circles) were made at Z/2R = 2.55 (PP1) for an inflow velocity Q ∞ = 75 m s -1 . Two additional curves have been plotted. The dashed curve corresponds to C p evolu- tion provided by Eq. ( 10) using the geometrical sweep angle ϕ geom , whereas the full line stands for C p values obtained with the effective sweep angle ϕ eff = 57.5 • . In the vicinity of the attachment line, measurements match with the ϕ eff curve, but further downstream, the geometrical angle gives best results. Rigorous characterization of effective sweep angle is of prime importance to provide accurate values of R. Indeed, under the assumption of a cylindrical leading edge (U e = 2U ∞ x/R), it can be demonstrated that R (defined in Eq. 5) becomes a function of ϕ eff which has been estimated previously:

where R is the radius of the cylindrical leading edge. Leading edge of the experimental model being cylindrical, this approach has been applied to determine corresponding R

(12) R = Q ∞ R sin(ϕ eff ) tan(ϕ eff ) 2ν
ϕ ef f ( o ) Experiment (Q ∞ = 30 m.s -1 ) Experiment (Q ∞ = 40 m.s -1 ) Experiment (Q ∞ = 60 m.s -1
) Fig. 9 ϕ eff along span for different free-stream velocities, ϕ geo = 50 • values for each pressure measurement section. According to experimental results, ϕ eff is likely to increase slightly along span (see Fig. 9) so that R increases correspondingly.

It can be noticed that this approach for R estimation can be extended to all airfoil considering that any leading edge can be approached by its osculating circle.

Validation of Poll's criterion

Contamination occurrence and transition locations are achieved using hot-film measurements. Contamination and transition from a laminar to a turbulent boundary layer lead to a strong increase in the recorded RMS values of the hotfilm signal. Hot-films being spread all along the attachment line, transition location can thus be estimated. As mentioned previously, an ACD-free configuration has been used in order to check the compliance of Poll's criterion. Figure 10 shows high RMS values for HF1 that can be explained by the fact that this hot-film is down enough to be permanently under the influence of the turbulent boundary layer which develops on the wind tunnel floor. This boundary layer has been measured at the entry of the test section during previous campaigns and has a maximum thickness of 100 mm for an upstream velocity of 12 m s -1 . The HF1 signal will be used as a reference for RMS values related to turbulent boundary-layer flow. Elsewhere, for hot-films 2 to 5 located higher on the attachment line, Fig. 10 shows a strong increase in RMS values around R = 250-280 which means that contamination has just occurred. This clearly shows a good agreement of Poll's criterion with the current experimental set-up.

Another way to demonstrate that our set-up was compliant with the Poll's criterion was to destabilize the new laminar boundary layer on the ACD surface using a small roughness element (Zigzag tape 700 µm thick). An infrared visualization has been performed to highlight the nature of the flow downstream the roughness at values of R close to 250.

For values of R slightly lower than 250 (R = 237), a laminar region can be observed (represented by a lighter central area) downstream the roughness and low RMS values for the hot-film located in this wake (Fig. 11 left). For values of R slightly higher than 250 (R = 263), a uniform colour density area downstream the roughness is observed and high RMS values indicate a whole turbulent area (see Fig. 11 right). Isolated roughness criteria 2 have been applied and tend to show that the roughness is not high enough to be responsible for the transition to turbulence observed.

Hot-film measurements on this smooth configuration and the observations done with the small roughness element confirm Poll's criterion about the critical value of R and are likely to show the self-sustainability of turbulence at values higher than this threshold.

Validation of ACD efficiency

The second configuration aims at evaluating the efficiency of the ACD to prevent leading-edge contamination. Low RMS values are recorded for the hot-films distributed along the attachment line downstream the ACD (see Fig. 13, HF2 to HF4) around R ≃ 250, whereas RMS values were already high at this same R in smooth condition (ACD-free configuration).

Furthermore, RMS values remain low up to R ≃ 580 for hot-films HF3 and HF4. Along the attachment line, without contamination, the linear stability theory developed in Sect. 2.3 established that natural transition could occur as soon as R = 582 (Görtler-Hämmerlin approach). This makes possible to conclude that the ACD fully plays its role allowing the downstream development of a new laminar boundary layer until the inherent natural destabilization which represents the maximal value that can be reached without using any other stabilizing devices (wall suction for example).

This ACD even confirms its ability to prevent leadingedge contamination at R values higher than 580. Indeed, according to the infrared visualization presented in Fig. 12 with a transition witness (Zigzag tape 400 µm thick), the boundary layer downstream the ACD is still laminar at R = 650 showing that no turbulent structure has come across the ACD. This observation is confirmed on Fig. 13 for the closest hot-film HF5′ that still exhibits low RMS values at this R. It can be noticed that HF5′ and HF6 record low RMS values related to a laminar boundary layer at values of R higher than the Görtler-Hämmerlin critical R . These two hot-films being close to the ACD, the spa- tial amplification has to be enhanced (higher velocities, i.e. higher values of R) so that instabilities reach a criti- cal threshold and trigger the transition. HF6 is transitional at lower values than HF5′ due to a crossflow transition as 2 van [START_REF] Van Driest | Boundary layer transition at supersonic speeds. Three-dimensional roughness effects (spheres)[END_REF] and von Doenhoff and Braslow (1961) criteria. explained in the following section. Relatively to the best existing ACDs and Gaster bumps that were efficient up to R ≃ 450, this ACD shows a significant improvement in the anti-contamination ability.

A characterization of natural leading edge instability observed in this ACD configuration is proposed in the next section and will make possible to confirm that transition process observed for HF2 to HF4 at R ≃ 580 is due to a natural transition.

Natural transition process downstream the ACD

Görtler-Hämmerlin transition process

Time-resolved hot-film measurements performed just before transition onset enable to perform some spectral analyses. Frequencies of amplified waves have been estimated by extracting the amplitude spectrum of the signal using a Fast Fourier Transform (FFT). In order to define the link between these amplified frequencies and Görtler-Hämmerlin instabilities, spectra have been compared to the stability approach described in Sect. 2.3.

Referring to the form of a perturbation introduced in Eq. (9b), the amplitude A (norm) of a perturbation can be written as:

where q ′ = u ′ , v ′ , w ′ , π ′ and q = xu, v, w, π

Using exponential properties, this leads to:

Assuming a reference amplitude A 0 as known, amplifica- tion can be written as: Under the assumption of an infinite wing (∂/∂Z = 0), the amplification can be directly written as:

(13) A =� q ′ �= 1 √ 2 � q(x, y) � exp(-β i Z) (14) dA A = -β i Z (15) A A 0 = exp -β i dZ
where Z 0 is a reference position and A 0 the amplitude of the disturbances at this location. For sake of simplicity, it will be considered in the following that Z 0 = 0 and corresponds to the nose of the ACD. For any experimental hot-film spectrum where R is known, ω = 2π f is made varying to meet the interval of experimental frequency band [0, 12,500 Hz].

For each couple (R, ω), the stability approach gives the imaginary part β i of Görtler-Hämmerlin mode. The posi- tion Z of the hot-film being known, A/A 0 = exp(-β i Z) can be calculated for each frequency. It remains that experimental spectrum is expressed in terms of amplitude (A), whereas the numerical one is expressed in terms of amplification (A/A 0 ). However, hot-films were not calibrated during the experimental test campaign and had just been used to qualitatively analyse the state of the boundary layer. This means that HF signal cannot be employed to quantify amplitude of instabilities in the laminar region of the boundary layer. As a consequence, in order to compare stability theory to measurements, numerical results have been rescaled to fit with the maximum value of the voltage fluctuation.

Agreement in amplified frequencies between experimental and stability approach is good (see Figs. 14,15). This tends to confirm that the destabilization of the flow is due to natural transition process involving Görtler-Hämmerlin instabilities.

In laminar-turbulent transition studies, a common expectation is the ability to determine or predict transition location. To this end, [START_REF] Smith | Transition, pressure gradient and stability theory. Technical report ES-26388[END_REF] as well as van Ingen (1956) developed a semi-empirical method, the so-called e N method, that links the transition triggering to a critical value of the N factor. This approach makes the assumption that natural transition occurs quickly after the nonlinear phase. The N factor corresponds, for a fixed R, to (16)

A A 0 = exp(-β i Z -Z 0 )
the most amplified wave A/A 0 expressed in terms of natural logarithm:

According to this expression, if the value of N at transition is known, it makes possible to estimate the transition position along span (Z). Depending on the inflow velocity, a hot-film signal may be laminar (for a given value of R lam ) or turbulent (for a higher value of R turb ), R cr being between these two values. Associated -(β i ) max is estimated using the stability approach. It is then possible to give a range for the critical N factor associated with Görtler-Hämmerlin instability along the attachment line and corresponding to transition onset.

The N factor range is estimated using HF2 and HF5. Experimental results (see Table 3) give a possible interval for the N T factor of [7.2, 10] for hot-film 2 and [7.1, 7.8] for hot-film 5.

Crossflow transition process

In the experiment, hot-film 6 has been set up slightly shifted from the attachment line, in region where crossflow instabilities are alleged to occur (x = 70 mm). Spectra have been extracted from the temporal signal of HF6 in order to study the frequency of instabilities developing inside the boundary layer at this chordwise location. The aim is to compare these frequencies to the one provided by the linear stability theory. The chordwise evolution of N factors for distinct frequencies and an upstream velocity Q ∞ = 50 m s -1 is represented in Fig. 16. It demonstrates that unsteady crossflow modes are more amplified than their stationary counterpart. At HF6 location (represented by the vertical dashed line), the N factor is N = 3.6 for the stationary CF mode, while it reaches N = 7.2 for f = 2 Travelling modes are generated by a combined effect of external turbulence fluctuations and wall roughness elements. Bippes (1999) has shown that stationary and travelling modes should be in the same order of magnitude for Tu ≃ 0.1 %. In the current experiment, the turbulence level is close to this value so that a possible competition between steady/unsteady modes may exist. This is shown by the experimental spectrum for Q ∞ = 50 m s -1 (full line in Fig. 17) which demonstrates that unsteady instabilities propagate inside the boundary layer. It has been compared to the numerical spectrum (dashed line in Fig. 17) obtained from linear stability theory. Numerical levels have been scaled in order to fit with the measurements. Wave amplification given by stability analysis is in good agreement with experimental one, despite a wider numerical range of amplified frequencies.

(17a) N = max ln A A 0 = -β i,max dZ (17b) = -β i,max Z
Crossflow vortices are amplified at low frequency and correspond to what is commonly admitted for this type of flow destabilization. Transition onset results from a competition between steady/unsteady modes depending on the receptivity and amplification phases. Crossflow transition line is visible in Fig. 12 6 Conclusion

This paper deals with attachment line transition processes that may occur on real swept wings of commercial aircraft and specially the contamination issue. In this framework, ONERA developed an entire procedure to design a highefficiency anti-contamination devices (ACDs) which can be fitted on various realistic applications. This goes from the parametrized conception of the shape to the validation of the anti-contamination efficiency using both numerical and experimental approaches. optimized design of a chevron-shaped ACD resulting from this procedure has been tested in a wind tunnel and its efficiency is assessed. This ACD has shown its ability to prevent leading-edge contamination up to values of R close to the natural transition pro- cess, i.e. the maximal value that can be expected without additional stabilizing process. The ability of this ACD to let the development of a laminar boundary layer downstream has made possible to characterize natural instabilities along the attachment line, preferential amplified frequencies and N factor of transition. This optimized shape ACD is an important step to deal with laminar maintenance of boundary layer along the attachment line of commercial aircraft evolving at high R values.

It corresponds to the first necessary condition to the maintenance of a laminar boundary layer as far as possible around aircraft airfoil where further downstream other mechanisms can lead to turbulence, especially crossflow vortices and Tollmien-Schlichting destabilizing waves. R values of common commercial aircraft being usually higher than the natural stability one, a promising way to maintain a laminar flow along the leading edge at higher R would be to couple this ACD with a suction system along the attachment line that could delay the natural transition. Contamination process is then prevented by the ACD, and natural transition process can be moved forward thanks to suction delaying the transition at higher R.

Fig. 1

 1 Fig. 1 Swept-wing flow characteristics

Fig. 2

 2 Fig. 2 Falkner-Skan-Cooke solutions

Fig. 3

 3 Fig. 3 Possible path to turbulence on a swept wing for a non-contaminated configuration (with efficient ACD). NT natural transition

Fig. 4 Fig. 5

 45 Fig. 4 iso-β i curves from the Görtler-Hämmerlin stability analysis

Fig. 7

 7 Fig. 7 Experimental set-up

Fig. 8

 8 Fig. 8 Experimental and inviscid fluid solution for wall pressure coefficient (PP1) at Q ∞ = 75 m s -1 , C p,interp (θ = 0) = 0.31

Fig. 10

 10 Fig. 10 Experimental hot-film RMS values as a function of R for the reference configuration (without ACD)

Fig. 13

 13 Fig. 13 Experimental hot-film RMS values along R for the ACD configuration

Fig. 14 Fig. 15

 1415 Fig.14Experimental versus stability approach for HF5′ at Q ∞ = 52.5 m s -1 , R = 621

Fig. 16 Fig. 17

 1617 Fig. 16 Spatial evolution of crossflow instability N factors for various frequencies: f = 0, 1, 2, 3 (KHz) for Q ∞ = 50 m s -1 . The vertical dashed line corresponds to HF6 location

Table 1

 1 Geometrical parameters of the optimized ACD shape (dimensionless with the total length L)

			Optimized	Optimization
			ACD	Span
	Total length	L	1	[0.65, 1]
	Chordwise width	l	0.25	[0.22, 0.25]
	Front height	h	0.058	[0.0145, 0.058]
	Apex half angle	a	35 •	[35 • , 50 • ]
	Top front edge radius	r	0.0145	[0.00145, 0.0145]

Table 3

 3 Görtler-Hämmerlin critical N factor values around transition onset Hot-film 2 (Z = 0.910) Hot-film 5 (Z = 0.535)

	R lam -R turb	682-707	721-732
	-β i,max N	7.9, 11.0 [7.2, 10]	13.3, 14.6 [7.1, 7.8]

Here, it is assumed that the external disturbance level is raher low so that the transition is not driven by the so-called bypass mechanism, see[START_REF] Morkovin | Transition in open flow systems -a reassessment[END_REF].

Acknowledgments

Part of this work has been undertaken within the Joint Technology Initiative «JTI Clean Sky» and specifically in the framework of the Smart Fixed Wing Aircraft Integrated Technology Demonstrator (SFWA-ITD Contract No. CSJU-GAM-SFWA-2008-001) financed by the 7th Framework Program of European Commission. The authors would like to thank the partners of the project for their collaboration and fruitful discussions. The authors are grateful to ONERA/GMT F2 wind tunnel team J.M. Deluc, T. Joret and P. Loiret for their technical support during the experimental campaign in F2 wind tunnel. The authors would also like to warmly thank F. Ternoy and G. Outtier who manufactured the ACD. Special thanks are given to D. Arnal and A. Séraudie who initiated this study and performed the first wind tunnel campaign in 2010 (they well deserved their retirement).