Sébastien Bontemps-Gallo

Edwige Madec

Jean-Marie Lacroix
email: jean-marie.lacroix@univ-lille1.fr

The inactivation of pecS restores the virulence of mutant devoid of Osmoregulated Periplasmic Glucans (OPGs) in the phytopathogen bacteria Dickeya dadantii

Keywords: Osmoregulated periplasmic glucans, plant pathogen, D. dadantii, PecS, regulation

Dickeya dadantii is a phytopathogen Enterobacterium that causes soft rot disease in a wide range of plant species. The maceration, apparent symptoms of the disease, is the result of the synthesis and secretion of a set of plant cell wall degrading enzymes but many additional factors are required for full virulence. Among them, osmoregulated periplasmic glucans (OPGs) and the PecS transcriptional regulator are one of these essential virulence factors. Several cellular functions are controlled by both OPGs and PecS. Strains devoid of OPGs display a pleiotropic phenotype including total loss of virulence, loss of motility and severe reduction in plant cell wall degrading enzymes synthesis. PecS is one of the major regulator of virulence in D. dadantii acting mainly as a repressor. It repress various cellular functions including virulence, motility and plant cell wall degrading enzymes synthesis. In this paper, we show that inactivation of the pecS gene restored virulence in a D. dadantii strain devoid of OPGs indicating that PecS cannot be derepressed in strains devoid of OPGs.

Introduction

Osmoregulated periplasmic glucans (OPGs) are a family of periplasmic oligosaccharides found in the envelope of most Proteobacteria. Their two common features are that glucose is the sole constitutive sugar and their abundance in the periplasm increases as the osmolarity of the medium decreases [START_REF] Bohin | Osmoregulation in the periplasm[END_REF]. In Enterobacteria and related bacteria, both products of the opgGH operon are required for synthesis of the glucose backbone of OPGs. opgG or opgH mutant strains are completely devoid of OPGs and exhibit a reduced or non-virulent phenotype indicating that these glucans belong to the common virulence factors of many zoo-pathogens and phytopathogens [START_REF] Loubens | Homology between a genetic locus (mdoA) involved in the osmoregulated biosynthesis of periplasmic glucans in Escherichia coli and a genetic locus (hrpM) controlling pathogenicity of Pseudomonas syringae[END_REF][START_REF] Mahajan-Miklos | Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa-Caenorhabditis elegans pathogenesis model[END_REF][START_REF] Page | Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity[END_REF].

Dickeya dadantii is a phytopathogen Enterobacterium that causes soft rot disease in a wide range of plant species. This pathogen devastates economically important crops in storage facilities or in growing plants [START_REF] Glasner | Genome sequence of the plant-pathogenic bacterium Dickeya dadantii 3937[END_REF]. The maceration, apparent symptoms of the disease, is the result of the synthesis and secretion of a set of plant cell wall degrading enzymes (PCWDE) particularly pectinases [START_REF] Barras | Extracellular Enzymes and Pathogenesis of Soft-Rot Erwinia[END_REF][START_REF] Collmer | The Role of Pectic Enzymes in Plant Pathogenesis[END_REF]) but many additional factors are required for full virulence. Expression of these factors during pathogenesis is strictly controlled by a complex regulatory network required for adaptation resulting in bacterial response to host injury after entry into the host. Particularly, bacteria must respond to acid stress upon penetration in host cells, oxidative stress during invasion and osmotic stress during maceration [START_REF] Plessis | New ABA-hypersensitive Arabidopsis mutants are affected in loci mediating responses to water deficit and Dickeya dadantii infection[END_REF].

In D. dadantii, mutants devoid of OPGs show a pleiotropic phenotype including a loss of motility, a decreased synthesis and secretion of PCWDE linked to a reduced synthesis of the Out system, an increased exopolysaccharide synthesis, an induction of general stress response and a complete loss of virulence on potato tubers or chicory leaves [START_REF] Bouchart | Proteomic analysis of a non-virulent mutant of the phytopathogenic bacterium Erwinia chrysanthemi deficient in osmoregulated periplasmic glucans: change in protein expression is not restricted to the envelope, but affects general metabolism[END_REF][START_REF] Page | Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity[END_REF]. In strains of D. dadantii devoid of OPGs, inactivation of the RcsCDB phosphorelay regulatory system restored PCWDE synthesis and secretion, motility, normal exopolysaccharide synthesis and virulence in potato tubers indicating that OPGs control the activation level of this phosphorelay [START_REF] Bontemps-Gallo | Concentration of osmoregulated periplasmic glucans (OPGs) modulates the activation level of the RcsCD RcsB phosphorelay in the phytopathogen bacteria Dickeya dadantii[END_REF][START_REF] Bouchart | The virulence of a Dickeya dadantii 3937 mutant devoid of osmoregulated periplasmic glucans is restored by inactivation of the RcsCD-RcsB phosphorelay[END_REF]. Relationship between this phosphorelay and OPGs highlight the role of these glucans as intermediate in the perception of environmental changes since phosphorelays are the main systems used by bacteria to sense and adapt to environmental variations [START_REF] Clarke | The Rcs phosphorelay: more than just a two-component pathway[END_REF]. Repression of the activation level of the RcsCDB phosphorelay by OPGs is not sufficient to explain the pleiotropic phenotype of strains devoid of OPGs since virulence restoration was observed only on potato tubers but not on chicory leaves. This suggests relationship between OPGs and additional regulatory proteins linked to virulence.

Methods

Bacterial strains, media and growth conditions

Bacterial strains are described in Table 1. Bacteria were grown at 30°C in lysogeny broth (LB) [START_REF] Bertani | Lysogeny at Mid-Twentieth Century: P1, P2, and OtherExperimental Systems[END_REF], or in minimal medium M63 supplemented with glycerol as a carbon source at a concentration of 2 g.l -1 [START_REF] Miller | A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria[END_REF] or with PGA as a inducer of pectate-lyase synthesis at a concentration of 4 g.l -1 [START_REF] Surgey | The Erwinia chrysanthemi pecT gene regulates pectinase gene expression[END_REF]. Solid media were obtained by adding agar at 15 g.l -1 .

Motility tests were made on LB plates containing agar at 0.4 g.l -1 . 10 7 bacteria in 5µL were spotted onto the plate, incubated at 30°C and swim diameters were measured after 30 hours of incubation.

Antibiotics in media were used at following concentrations: kanamycin, 25 µg.ml -1 (D. dadantii) or 50 µg.ml -1 (E. coli) chloramphenicol, 25 µg.ml -1 (D. dadantii); spectinomycin, 50 µg.ml -1 (D. dadantii), gentamycin, 2 µg.ml -1 (D. dadantii) tetracycline 12.5µg.ml -1 (D. dadantii) and ampicillin 50 µg.ml -1 (E.

coli).

Construction of transcriptional fusions.

For construction of the pecS::uidA and pecM::uidA, coding sequence of the uidA gene was amplified by PCR (Gus-SphI-F forward CACGCATGCATTCCGGACCAGTATTATTATC and Gus-HindIII-R reverse

Transduction and Conjugation.

Construction of strains were performed by transferring genes from one strain of D. dadantii to another by generalized transduction with phage ΦEC2 according to [START_REF] Resibois | Phi-EC2, a new generalized transducing phage of Erwinia chrysanthemi[END_REF].

Plasmids were introduced in D. dadantii by conjugation. The fusions were integrated into the D.

dadantii chromosome by transposition events selected in the presence of the appropriate antibiotic.

Determination of enzyme activities

β-glucuronidase assays were performed on crude extracts obtained from bacteria disrupted by sonication 2 x 20 s (Sonifier cell disruptor B-30, Branson, 70% duty cycle, 7 microtip limit, Hold time, continuous, appropriate probe). β-glucuronidase activity was determined by spectrometric monitoring the hydrolysis of PNPU (4-nitrophenylβ-D-glucuronide) at 405 nm.

The protein concentration was determined by the Bradford assay with bovine serum albumin as a standard [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF].

Assays of pectate-lyase

Strains were grown overnight in M63 supplemented with glycerol with or without the inducer PGA.

Cultures were centrifuged at 2500g and supernatants were used for pectate-lyase activity assays.

Pectate-lyase activity was determined by spectrometric monitoring the degradation of PGA to unsaturated products at 235 nm [START_REF] Moran | Extracellular and intracellular polygalacturonic acid transeliminases of Erwinia carotovora[END_REF]) at 37°C in the PL buffer (Tris-HCl 50 mM pH 8,5, CaCl2 0,1 mM, PGA 0,05%)..

Quantification of blue pigment indigoidine production

Strains were grown overnight in M63 medium supplemented with glycerol . The culture was centrifuge at 3000g to harvest the cells. After centrifugation, the bacterial pellet was suspended in 20% of the initial culture volume of dimethylformamide (99%) to extract the blue pigment. Cells were discarded after centrifugation at 3000g and the supernatant was used to quantify the blue pigment. The quantity of the blue pigment was measured in a spectrophotometer at 615 nm.

Susceptibility to oxidative stress and antimicrobial peptides.

The susceptibility to oxidative stress (H2O2) and to antimicrobial peptides (polymixin B) was previously described [START_REF] Haque | SlyA, a MarR family transcriptional regulator, is essential for virulence in Dickeya dadantii 3937[END_REF]. H2O2at 6 mM or polymixin B at 1 mg.ml -1 was added to cells grown until stationary-phase in M63 glycerol medium. After different times of incubation, aliquot of bacteria were taken up, and the survival were determined by numeration of CFU after plating serial dilutions on LB plates.

Pathogenicity test.

Chicory leaves and potato tubers were inoculated as previously described [START_REF] Page | Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity[END_REF].

Briefly, bacteria from an overnight culture in LB medium were recovered by centrifugation and diluted in physiological water. After wounding, 10 7 bacteria were inoculated and incubated in a dew chamber until 48h at 28°C.

To measure the severity of the disease on potato tubers, the whole maceration was weighted. To determine the number of bacteria within infected plants, whole maceration of potato tubers or chicory leaves was suspended in 10 ml of sterile water. Number of CFU was determined after plating serial dilutions on LB plates.

Statistical Analysis.

For statistical analyses, XLstat software (Addinsoft) was used. Data were analysed by paired Wilcoxon Mann Whitney test, a value of p<0.05 was considered significant.

Results

The inactivation of the pecS gene in strains devoid of OPGs restores the virulence on potato tubers and on chicory leaves

Relationship between the pecS gene, encoding one of the main repressors of virulence of D. dadantii (see introduction) and OPGs was tested by construction by transduction and analysis of a double mutant strain opgG pecS (see methods). The virulence of the wild-type, the opgG, the pecS and the opgG pecS double mutant strains, were assessed by measuring the severity of the disease for 48h following inoculation of 10 7 bacteria on potato tubers and on chicory leaves. On potato tubers, virulence was observed after 24h, and 48h of incubation. For the wild-type strain, growth occurred from 7 10 8 cells and 0.5g of maceration after 24h incubation until 3 10 9 cells and 1.5g of maceration after 48h of incubation (Fig. 1a). As expected, no maceration nor growth of bacteria were observed for the opgG mutant strain on potato tubers (Fig. 1b). For the single mutant strain pecS, maceration amount and bacterial growth were increased by a factor of 1.7 after 24h incubation as compared to the wild-type strain (Fig. 1c). After 48h of incubation, growth was 3.4 higher for the pecS strain as compared to the wild-type strain whereas maceration increases by a factor of 2.2 as compared to the wild-type strain (Fig. 1c). Virulence was restored for the opgG pecS double mutant strain as compared to the opgG strain. Maceration amount and bacterial growth were similar to those observed for the wild-type strain at 24h and 48h (Fig. 1d). On chicory leaves, virulence was observed after 20h, 24h, 30h, 40h and 48h of incubation. For the wild-type strain, maceration area increased as the incubation time increased (Fig. 2). As expected, no maceration occurred for the opgG mutant strain (Fig. 2). The maceration area was severely increased for the pecS single mutant strain as compared to the wild-type strain at each time tested (Fig. 2). Virulence was restored in the opgG pecS double mutant strain as compared to the opgG strain. The maceration area was similar for the opgG pecS double mutant and for the wild-type strains whatever the incubation time suggesting that colonization speed of chicory leaves was similar for both strains (Fig. 2). These results strongly suggest that in strains devoid of OPGs, repression by the PecS negative transcriptional regulator cannot be resumed.

Synthesis and secretion of pectinases is restored in the opgG pecS double mutant strain

Pectinases are essential factors required for full virulence (see introduction). Synthesis and secretion of these exoenzymes are cellular functions strictly controlled directly by many transcriptional regulators particularly by PecS and indirectly by OPGs. The global pectate-lyase activity was quantified after growth with or without the well known inducer polygalacturonate (PGA). As expected, in the wild-type strain, pectinase activity was 3 time higher after growth in medium containing PGA. In the opgG single mutant strain, pectinase activity remained inducible by PGA since activity was 3 time higher after growth in medium containing PGA but pectinase activities were decreased by a factor of 2 as compared to the wild-type strain (Fig. 3) as previously described [START_REF] Page | Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity[END_REF]. For the pecS single mutant strain, pectinase activity remained inducible by PGA since activity was 3 time higher after growth in medium containing PGA but pectinase activity was increased by a factor of 2 as compared to the wild-type strain (Fig. 3). For the opgG pecS double mutant strain, pectinase activity was restored as compared to the opgG single mutant strain.

Pectinase activity remained inducible by PGA since activity was two time higher after growth in medium containing PGA. When grown in medium containing PGA, pectinase activity of this double mutant strain was similar to the pectinase activity measured in the wild-type strain. When grown in medium without PGA, pectinase activity of this double mutant strain was 1.5 time higher than the pectinase activity measured in the wild-type strain (Fig. 3). These results suggest that restoration of pectinase synthesis and secretion are required for restoration of virulence in strains devoid of OPGs and one can imagine that restoration of pectinase synthesis and secretion may be sufficient to restore virulence in strains devoid of OPGs.

Restoration of pectinase activity is sufficient to restore virulence of the opgG strain in potato tubers but not in chicory leaves

To test this hypothesis, inactivation of the kdgR and the pecT genes was performed, introduced by transduction in an opgG mutant strain and pectinase and virulence activity were assessed in the conditions described for the pecS and derivative mutants strains (see before). The KdgR transcriptional regulator is known to repress only the synthesis of pectinases [START_REF] Reverchon | Characterization of kdgR, a gene of Erwinia chrysanthemi that regulates pectin degradation[END_REF]. This repression is likely to be suppressed in planta or when PGA was added to the medium resulting in the accumulation in bacteria of the 2-keto-3-deoxygluconate (KDG) a pectin degradation product and in planta inducer of KdgR [START_REF] Nasser | Purification and functional characterization of the KdgR protein, a major repressor of pectinolysis genes of Erwinia chrysanthemi[END_REF]. The PecT transcriptional regulator is also known to repress the synthesis of pectinases [START_REF] Surgey | The Erwinia chrysanthemi pecT gene regulates pectinase gene expression[END_REF]. In the kdgR single mutant strain, pectinases were constitutively expressed since pectinase activities were similar after growth with or without PGA added and was similar to the pectinase activity observed for the wild-type strain grown with PGA added (Fig. 3) [START_REF] Nasser | Inducing properties of analogs of 2-keto-3-deoxygluconate on the expression of pectinase genes of Erwinia chrysanthemi[END_REF]. For the opgG kdgR double mutant strain, pectinase activity was restored as compared to the opgG mutant strain. Pectinase activities were constitutively expressed since pectinase activities were similar after growth with or without PGA added, to a level similar to the pectinase activity observed in the wild-type strain grown with PGA added (Fig. 3). Virulence was assessed on potato tubers. For the single mutant strains kdgR, maceration amount and bacterial growth were increased by a factor of 4 and 3.4 after 24h and 48h

incubation respectively as compared to the wild-type strain (Fig. 1e). For the double mutant strain opgG kdgR, virulence was restored as compared to the opgG strain but maceration amount and bacterial growth were decreased by a factor of 1.7 and 1.4 after 24h and 48h incubation respectively as compared to the wild-type strain (Fig. 1f). On chicory leaves, maceration occurred to a similar level for the single mutant kdgR strain and the wild-type strain while no maceration occurred for the opgG kdgR double mutant strain (Fig. 2). For the pecT single mutant strain, pectinase activity remained inducible by PGA since activity was 3 time higher after growth in medium containing PGA but pectinase activity was increased by a factor of 2 as compared to the wild-type strain (Fig. 3). For the opgG pecT double mutant strain, pectinase activity was restored as compared to the opgG single mutant strain without PGA added but was no longer inducible by PGA (Fig. 3). For the single mutant strain pecT, maceration amount and bacterial growth were increased by a factor of 3.6 and 2 after 24h and 48h incubation respectively as compared to the wild-type strain (Fig. 1g). For the double mutant strain opgG pecT, virulence was restored as compared to the opgG strain but maceration amount and bacterial growth were decreased by a factor of 25 and 25 after 24h and 48h incubation respectively as compared to the wild-type strain (Fig. 1h). On chicory leaves, maceration occurred to a similar level for the single mutant pecT strain and the wild-type strain while no maceration occurred for the opgG pecT double mutant strain (Fig. 2). Secreted pectinases were extracted and concentrated from a supernatant of wild-type culture (see methods). The opgG mutant and the wildtype strains were incubated with these purified pectinases on potato tubers (Fig. 1 i,j). Virulence was restored in the opgG strain grown with pectinases added as compared to the same mutant strain grown without pectinase. In contrast, no virulence was observed when an opgG mutant strain was incubated with purified pectinases on chicory leaves since maceration occurred but growth of bacteria never resumed (data not shown). Taken together, these results indicated that restoration of pectinase synthesis is sufficient to restore virulence of strains devoid of OPGs on potato tubers but that additional characters must be restored to restore virulence on chicory leaves.

Motility is not restored in the opgG pecS double mutant strain

OPGs and PecS control motility which is required for full virulence and was thought to be required for extension of the disease throughout the plant. The sigma factor  28 , encoded by the fliA gene, is required for expression of the flagella apparatus genes including the fliC gene encoding flagellin.

Expression of the fliA gene is repressed by PecS and activated by FlhC2D2 [START_REF] Hommais | PecS is a global regulator of the symptomatic phase in the phytopathogenic bacterium Erwinia chrysanthemi 3937[END_REF] encoded by the master operon flhDC requiring OPGs for expression [START_REF] Bontemps-Gallo | Concentration of osmoregulated periplasmic glucans (OPGs) modulates the activation level of the RcsCD RcsB phosphorelay in the phytopathogen bacteria Dickeya dadantii[END_REF].

Thus, restoration of motility may be one of the factors required for restoration of virulence in strains devoid of OPGs. Motility was measured on soft agar plates. The opgG single mutant strain showed a severe decrease in motility as compared to the wild-type strain while the pecS single mutant strain showed a slight increase in motility (Fig. 4a). Surprisingly, the opgG pecS double mutant strain was completely non motile. Expression of the flhD-uidA and the fliC-uidA gene fusions in the same strains correlated with these results. No expression of both genes was observed for the opgG pecS double mutant strain while a decrease of expression was observed for the opgG single mutant strain as compared to the wild-type strain (Fig. 4b,c). No difference of expression for the flhD gene was observed for the pecS single mutant strain as compared to the wild-type strain confirming that the flhDC operon is not regulated by PecS while an important increase of the fliC expression was observed as compared to the wild-type strain confirming that regulator role of PecS in flagella synthesis (Fig. 4b,c). These data indicated that motility is not required for virulence in our conditions.

Potato tubers are unable to eliminate the opgG strain.

One can imagine that one of the most important differences between potato tubers and chicory leaves submitted to bacterial pathogens injury resulted in the more important capability of chicory leaves to induce defense mechanisms against these bacterial pathogens (see introduction). Thus, we decided to compare survival of the opgG strain inoculated into potato tubers and chicory leaves with wild-type strain as a control. 10 7 bacteria were inoculated into potato tubers and chicory leaves.

Bacteria were recovered after 24h and 48h, of incubation and numeration was performed on LB plates. On potato tubers, no growth occurred but bacterial amount remained constant whatever the incubation time (Fig. 5a). On chicory leaves, no growth occurred but bacterial amount decreased regularly over time. In addition, death of the entire bacterial inoculum was observed in less than 72h (fig. 5a). As a control, the same experiments were performed on the wild-type strain. As expected, bacterial amount increased regularly over time in both vegetable (Fig. 5b). These results strongly suggested that the opgG strain is impaired in resistance against defence of plant host and that potato tubers are unable to induce an effective defence mecanism if any against an opgG strain.

The resistance to major stresses remained unaffected in the opgG pecS double mutant strain

The essential resistance mechanisms required by D. dadantii to fight against defense generated by plant hosts are resistance against acid, oxidative molecules, osmotic stress and antimicrobial peptides [START_REF] Plessis | New ABA-hypersensitive Arabidopsis mutants are affected in loci mediating responses to water deficit and Dickeya dadantii infection[END_REF].

Resistance of the wild-type strain, the single opgG and pecS mutant strains and the opgG pecS double mutant strain was compared in these various conditions of stress.

Resistance to osmotic stress, encountered by bacteria during maceration, was monitored by comparison of growth rate between the different mutant strains in 63 media of increasing osmolarity: half strength, full strength, full strength with 0.1M NaCl and full strength with 0.2M NaCl. For each osmolarity tested, no significant growth rate difference was observed between the different strains ranging from 0,40 h -1 , 0,69 h -1 , 0,70 h -1 and 0,24 h -1 at increased osmolarity respectively.

Resistance to acid stress, encountered by bacteria during penetration in the host plant, was monitored by comparison of growth rate between the different mutants strains in 63 media of increasing pH: pH5, pH6, pH7 and pH8. For each pH tested, no significant growth rate difference was observed between the different strains ranging from 0,61 h -1 , 0,68 h -1 , 0,70 h -1 and 0,69 h -1 at pH 5, 6, 7 and 8 respectively.

Resistance of plants hosts are also associated to secretion of antimicrobial peptides.. Resistance of the wild-type strain, the opgG and pecS single mutant strains and the opgG pecS double mutant strains was followed by numeration of the survival bacteria in the presence of 1 mg/mL of polymixin B during 1h. No significant difference in survival was observed for the different strains tested.

These data indicated that resistance to the tested stresses cannot explain the restoration of virulence observed in chicory leaves for the pecS opgG double mutant strain.

The resistance to the oxidative stress is increased in the opgG strain but not in the opgG pecS strain Finally, the oxidative stress was tested. In the pecS strains, the indABC operon, encoding the products catalysing the synthesis of the blue pigment indigoidine, is up-regulated [START_REF] Reverchon | Characterization of indigoidine biosynthetic genes in Erwinia chrysanthemi and role of this blue pigment in pathogenicity[END_REF]. This pigment is known to be one of the responses to the oxidative stress induced by the host plant during invasion. The amount of indigoidine secreted by the wild-type strain, the opgG and pecS single mutant strains and the opgG pecS double mutant strain was quantified (See Methods) (Fig. 6a). No detectable indigoidine was measured for the wild-type and the opgG mutant strains. As expected, a high level of indigoidine was secreted by the pecS mutant as well as the double opgG pecS mutant (Fig. 6a). Expression of an indA-uidA transcriptional fusion was measured after introduction in the same strains and correlated with indigoidine secretion. In the wild-type strain and the opgG single mutant strain, the indA gene expression level was similarly low (Fig. 6b). In the pecS single mutant and the opgG pecS double mutant strains, the indA gene expression level was similar and 10 times higher than in the wild-type strain (Fig. 6b).

Thus, the virulence of the pecS opgG double mutant strain in planta may be explained by a restoration of the defences against oxidative stress induced by the plant. To test this hypothesis, the resistance to the ROS (Reactive Oxygen Species), generating the oxidative stress, was tested by measuring the hydrogen peroxide resistance of the four strains. Resistance of the wild-type strain, the opgG and pecS single mutant strains and the opgG pecS double mutant strain different strains was followed by numeration of the survival bacteria in the presence of 6 mM H2O2 during 1h (Fig. 6c). The survival percentage of the wild-type strain, the single pecS and the double opgG pecS mutants decreased rapidly to reach 20% of the initial population after 20 min. of incubation then stabilize for the pecS and the opgG pecS strains while the survival reach to 5% of the initial population for the wild-type strain. Surprisingly, the opgG mutant was more resistant since 40% of the initial population was still alive after 60 min. of incubation. These data indicated that despite the increased synthesis of indigoidine of the opgG pecS double mutant strain and a ROS resistance increased as compared to the wild-type strain, this double mutant strain remains less resistant than the opgG single mutant strain. Thus, restoration of oxidative stress cannot explain restoration of virulence of the opgG pecS double mutant strain in chicory leaves.

Increased synthesis of exopolysaccharides is responsible for increased resistance to ROS in opgG strain.

It was recently shown that in the symbiotic bacterium Sinorhizobium meliloti, exopolysaccharides had a protective effect against ROS [START_REF] Lehman | Exopolysaccharides from Sinorhizobium meliloti can Protect Against H2O2-Dependent Damage[END_REF] and exopolysaccharides are much more abundant around bacteria in opg strains as compared to the wild-type strain [START_REF] Page | Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity[END_REF].

Second, in Escherichia coli, it was shown that the closing of the TCA cycle generated ROS synthesis within bacteria thus inducing the ROS stress response [START_REF] Kohanski | Mistranslation of membrane proteins and two-component system activation trigger antibiotic-mediated cell death[END_REF] and in a previous proteomic analysis [START_REF] Bouchart | Proteomic analysis of a non-virulent mutant of the phytopathogenic bacterium Erwinia chrysanthemi deficient in osmoregulated periplasmic glucans: change in protein expression is not restricted to the envelope, but affects general metabolism[END_REF], it was shown that in an opgG strain, increasing energy level was required as compared to a wild-type strain and resulted in a closed tricarboxylic acid (TCA) cycle. To test these hypothesis, an opgG eps double mutant strain was constructed by transduction. Exopolysaccharides are synthesized by the eps operon products [START_REF] Condemine | The PecT repressor coregulates synthesis of exopolysaccharides and virulence factors in Erwinia chrysanthemi[END_REF]. While the opgG strain displays a mucoid phenotype, the eps opgG strain restored a non mucoid phenotype. Resistance to ROS was performed as described before. The survival percentage of the single eps and the double opgG eps mutants decreased rapidly to reach 15% of the initial population after 20 min. of incubation then stabilize for the eps strain while the survival reach to 10% of the initial population for the opgG eps double mutant strain after 60 min. of incubation (Fig. 6c). Thus, the ROS resistance decreased severely in the opgG eps double mutant strain as compared to the opgG single mutant strain. These data indicated that the ROS resistance of the opg negative strains is the result of the increased exopolysaccharide synthesis and secretion. One can imagine that increased exopolysaccharides synthesis and secretion are one of the reasons of the full survival of opgG strains in potato tubers and the 72h survival on chicory leaves. Thus, we decided to compare survival of the opgG eps strain inoculated into potato tubers and chicory leaves with the opgG strain. 10 7 bacteria were inoculated into potato tubers and chicory leaves. Bacteria were recovered after 24h and 48h, of incubation and numeration was performed on LB plates. On potato tubers, no growth occurred but bacterial amount remained constant whatever the incubation time as observed for the opgG strain (Fig. 5c). On chicory leaves, no growth occurred but bacterial amount decreased regularly over time. In addition, death of the entire bacterial inoculum occurred more rapidly for the opgG eps strain (48h, Fig. 5c) than in the opgG strain (72h, Fig. 5 a). As a control, the same experiments were performed on the eps mutant strain. Bacterial amount increased regularly over time in both vegetable (Fig. 5d) but to a less extend than observed for the wild-type strain (Fig. 5b). These data strongly suggest that exopolysaccharides display a protective but insufficient effect for opg negative strains in chicory leaves.

Discussion

In this paper, we describe the relationship between the transcriptional regulator PecS and OPGs.

Inactivation of the pecS gene leads to the restoration of virulence in an opgG strain both on potato tubers and on chicory leaves. In contrast, the other known suppressor of the Opg phenotype, i.e. the rcsC gene [START_REF] Bouchart | The virulence of a Dickeya dadantii 3937 mutant devoid of osmoregulated periplasmic glucans is restored by inactivation of the RcsCD-RcsB phosphorelay[END_REF], the kdgR and the pecT genes (this study) allow restoration only on potato tubers. All these suppressors restore pectinase synthesis and secretion and data presented here showed that this restoration is sufficient to restore virulence in potato tubers in strains devoid of OPGs. This result could be explained by the incapability of this reserve tissue to fight efficiently against the bacterial pathogen. This hypothesis was strengthen by the observation that strains devoid of OPGs do not grow but survive for days in potato tubers. Other kind of plant tissues such as leaves possessed several defence mechanisms to respond to bacterial injury. One can imagine that in strains devoid of OPGs, response to stresses caused by plant tissues in response to bacterial pathogen entry are severely impaired. This hypothesis was strengthen by the observation that strains devoid of OPGs die within 72 hours in chicory leaves.

Surprisingly, response and resistance to major stresses known to be encountered by bacteria in planta (changes in the pH and the osmolarity of the environment, antimicrobial peptide or ROS secretion) were neither decreased in the opgG single mutant strain nor increased in the opgG pecS double mutant strain. On the contrary, in the opgG strain, ROS resistance was increased as compared to the wild-type strain. We showed that this is the result of the protective effect of exopolysaccharides against ROS as observed recently in the symbiotic bacterium Sinorhizobium meliloti, [START_REF] Lehman | Exopolysaccharides from Sinorhizobium meliloti can Protect Against H2O2-Dependent Damage[END_REF] because exopolysaccharides are much more abundant around bacteria in opg strains as compared to the wild-type strain [START_REF] Page | Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity[END_REF]. Data presented here strongly suggest that still unknown additional virulence factors are required for restoration of virulence in plant for the opgG pecS mutant strain. Data also strongly suggested that these virulence factors are repressed by PecS. The role of OPGs remains unclear and is probably indirect. In a previous study, relationship between OPGs and the phosphorelay RcsCDB was demonstrated [START_REF] Bouchart | The virulence of a Dickeya dadantii 3937 mutant devoid of osmoregulated periplasmic glucans is restored by inactivation of the RcsCD-RcsB phosphorelay[END_REF]. Direct interaction between the RcsC and/or RcsD sensor proteins and OPGs cannot be excluded since periplasmic domain of the both proteins may interact with these periplasmic glucans. In contrast, despite the fact that in an opgG strain, the transcriptional regulator PecS cannot be derepressed, the relationship between the cytoplasmic PecS protein and periplasmic glucans is probably indirect. It remains very surprising that the loss of such a simple oligosaccharide leads to such an important deregulation of several cellular pathways, two of them resulting from the absence of derepression of the PecS regulator and the constitutive activation of the RcsCDB phosphorelay. The result is a severe alteration in the interactions with their environment of these opg negative strains. This leads to a global disorder of the internal control systems of the bacteria that we can qualify of "bacterial autism". or with pectate-lyases added (j) , the pecS (c), the ogpG pecS (d), the kdgR (e), the opgG kdgR (f), the pecT (g) and the opgG pecT (h)strains inoculated on potato tubers. 10 7 bacteria were inoculated into holes on potato tubers. Weight of macerated tissue (bars with the scale on the right) and number of CFU within macerated tissue (curves with the scale on the left) were determined after 24h and 48h of incubation at 28°C. Pectate-lyase enzymes were purified from D. dadantii grown in vitro. The results reported are an average of three independent experiments. for the wild-type, the opgG, the pecS mutants the opgG pecS, the eps and the opgG eps strains (c).

Secreted indigoidine was assayed by measuring optical density at 615 nm (see Methods). The indigoidine quantity is expressed as the OD615nm/OD610nm. For expression of the indA-uidA gene fusion, bacteria were grown until mid-log phase and broken by sonication. The β-glucuronidase activity was measured with PNPU as a substrate. Specific activity is expressed as the change in optical density at 410 nm per min and per mg of protein. For resistance to H2O2, bacteria were grown until mid-log phase. The culture was incubated with 6 mM H2O2 and the survival were numerated. The survival rate was expressed as the number of CFU counted at each time/the number of CFU before addition of H2O2. The asterisks indicated that a significant difference exists only between the value of the opgG strain and the values of the other strains. Results are an average of three independent experiments.

  CACAAAGCTTATCCGCCGCTCACAATTCCAC primers). The uidA DNA fragment was digested by SphI and HindIII and cloned into pUC18Not digested by the same enzymes to give pUC18Not-uidA.Promoter and the beginning of the coding sequences of the pecS and pecM genes were amplified by PCR (pecSfusF forward CACTCTAGACAGGGAGCGTAAAAAGCGA and pecSfusR reverse ). The pecS and pecM DNA fragments were digested by XbaI and SphI and cloned into pUC18Not-uidA digested by the same enzymes to give pNFW518 and pNFW520 respectively. Both plasmids were digested by NotI and cloned in pUTminiTn5 to give pNFW519 and pNFW521 respectively. These two last plasmids were introduced into D. dadantii by conjugation.

Figure legends Fig. 1 .

 legends1 Figure legends

Fig. 2 .

 2 Fig.2. Time course of symptoms development for the wild-type, the opgG, the pecS mutants, the opgG pecS , the kdgR, the opgG kdgR, the pecT and the opgG pecT strains inoculated into chicory leaves. Virulence was monitored for 2 days.

Fig. 3 .

 3 Fig.3. Pectate-lyase activity of the wild-type, the opgG, the pecS, the opgG pecS, the kdgR the opgG kdgR, the pecT and the opgG pecT strains. Cultures were grown in minimal medium overnight with (black bars) or without (white bars) the inducer polygalacturonate (PGA). Cells were pelleted and the pectate-lyases activity in the culture supernatant was determined (see Methods). The results reported are an average of four independent experiments.

Fig. 4 .

 4 Fig. 4. Motility (a), flhD gene expression (b), and fliC (c) gene expression of the wild-type, the opgG, the pecS mutants, and the opgG pecS strains. For the expression of the flhD::uidA and fliC::uidA gene fusions, bacteria were grown until mid-log phase and broken by sonication. The β-glucuronidase activity was measured with PNPU as a substrate. Specific activity was expressed as the change in optical density at 405 nm per min and per mg of protein. Results are the average of three independent experiments.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Survival of the opgG (a), the wild-type (b), the opgG eps (c) and the eps (d) strains in potato tubers and on chicory leaves. 10 7 bacteria were inoculated into holes on potato tubers or into chicory leaves. The number of bacteria was counted each 24h for 72h. Results are the average of four independent experiments.

Table 1 1

 1

	Strain or plasmid	Relevant description/genotype a	Source or reference
		Dickeya dadantii
	EC 3937	wild-type strain	Laboratory collection
	A2148	pecT::Cml	G. Condemine
	A2575	eps::lacZ-Tet	Condemine et al., 1999
	A 3953	pecS::MudIIPR13-Cml	Reverchon et al., 1994
	A 3954	indA::uidA-Kan	Reverchon et al., 1994
	A 3956	pecS::MudIIPR13-Cml indA::uidA-Kan	Hommais et al., 2008
	A 4115	kdgR::Spe	G. Condemine
	A 5009	fliC::uidA-Kan	Hommais et al., 2008
	NFB	opgG::uidA-Kan	Laboratory collection
	NFB	opgG::Cml	Laboratory collection
	NFB	flhD::uidA-Kan	Bouchart et al., 2010
	NFB	opgG::FRT	Laboratory collection
	NFB	opgG::uidA-Kan, pecS::MudIIPR13-Cml	This work
	NFB	opgG::Cml indA::uidA-Kan	This work
	NFB	opgG::FRT pecS::MudIIPR13-Cml indA::uidA-Kan This work
	NFB	opgG::FRT pecS::MudIIPR13-Cml flhD::uidA-Kan	This work
	NFB	pecS::MudIIPR13-Cml flhD::uidA-Kan	This work
	NFB	opgG::Cml kdgR::Spe	This work
	NFB	opgG::Cml flhD::uidA-Kan	Bontemps-Gallo et al., 2013
	NFB	pecT::Cml opgG::uidA-Kan	This work
	NFB	fliC::uidA-Kan opgG::Cml	This work
	NFB	fliC::uidA-Kan opgG::FRT pecS:: MudIIPR13-Cml	This work
	NFB	fliC::uidA-Kan pecS:: MudIIPR13-Cml	This work
	NFB7527	eps::lacZ-Tet opgG::uidA-Kan	This work
	NFB	pecS::uidA-Kan	This work
	NFB	pecS::uidA-Kan opgG::Cml	This work
	NFB	pecM::uidA-Kan	This work
	NFB	pecM::uidA-Kan	This work
		Escherichia coli
	Top 10 F'	F'[lacI q Tn10] mcrA, Δ(mrr-hsdRMS-mcrBC)	Invitrogen
		80lacZΔM15, ΔlacX74, deoR, nupG, recA1,
		araD139, Δ(ara-leu)7697, galU, galK,
		rpsL,endA1
	S17-1λpir	recA, thi, pro, hsdR-M+, RP4-2-Tet::Mu-	Simon et al., 1989
		Kan::Tn7,pir
		Plasmids
	pUC18Not	AmpR	Yanisch-Perron et al., 1995
	pUC18Not-uidA	AmpR	This work
	pUTminiTn5-Kan Mini-Tn5-Kan, oriR6K, KanR, AmpR	De Lorenzo et al., 1990

The transcriptional regulatory protein PecS, acting mainly as a repressor, is a complex global regulator of major importance for the virulence of D. dadantii. As it was observed for the RcsCDB phosphorelay, several cellular functions are controlled by both this regulator and OPGs. Among the numerous genes of the PecS regulon are found PCWDE genes, oxidative stress response genes such as the antioxidant blue pigment indigoidine, and motility genes[START_REF] Hommais | PecS is a global regulator of the symptomatic phase in the phytopathogenic bacterium Erwinia chrysanthemi 3937[END_REF][START_REF] Rouanet | Definition of a consensus DNAbinding site for PecS, a global regulator of virulence gene expression in Erwinia chrysanthemi and identification of new members of the PecS regulon[END_REF]. Actually, no inducer is known to inactivate the PecS transcriptional repressor.In this study, we investigate the relationship between OPGs and the PecS regulator. Inactivation of this regulator restores virulence in potato tubers but also in chicory leaves in strain devoid of OPGs. This suggests a constitutive repression state of the PecS transcriptional regulator in strains devoid of OPGs. In addition, we showed that restoration of PCWDE secretion and synthesis was sufficient to explain restoration of virulence of strains devoid of OPGs on potato tubers but not on chicory leaves.

Acknowledgements

The 'A strains' were kindly providing by Dr. G. Condemine and Dr. W. Nasser (CNRS UMR 5240, University of Lyon 1). The authors thank Martine Ratajczak for the providing of potato tubers. This work was supported by Grants from the Centre National de la Recherche Scientifique (CNRS), the Université de Lille 1 and the Ministère de l'Enseignement Supérieur et de la Recherche. SBG was funded by doctoral fellowship of the Lille 1 University.


Edwige Madec

Sébastien Bontemps-Gallo

Jean-Marie Lacroix
email: jean-marie.lacroix@univ-lille1.fr

Increased phosphorylation of the RcsB regulator of the RcsCDB phosphorelay in strains of Dickeya dadantii devoid of osmoregulated periplasmic glucans revealed by Phos-tag TM gel analysis

Keywords: Osmoregulated periplasmic glucans, RcsCDB phosphorelay, Dickeya dadantii, phosphorylation

Osmoregulated periplasmic glucans (OPGs) are general constituents of many Proteobacteria. OPGs are important factors required for full virulence in many pathogens including Dickeya dadantii. D. dadantii causes the soft-rot disease on a wide range of plant species. The total loss of virulence and motility of opg-negative strains of D. dadantii is linked to the constitutive activation of the RcsCDB phosphorelay deduced from expression analysis of genes of the RcsCDB regulon. Here, the in vivo level of constitutive activation of the RcsCDB phosphorelay opg-negative strain was demonstrated by direct analysis of the phosphorylation level of the RcsB regulator protein by using a Phos-tag TM retardation gel approach and was correlated with the phenotype and the expression of gene of motility.

Introduction

Osmoregulated periplasmic glucans (OPGs) are oligosaccharides found in the envelope of most Proteobacteria [START_REF] Bohin | Osmoregulation in the périplasme[END_REF]. The backbone is synthesized by the two products of the opgGH operon and consists of β,1-2 linked glucose units to which the branches are attached by β,1-6 linkages. These glucans belong to the common virulence factors of many Proteobacteria zoopathogens and phytopathogens such as Pseudomonas aeruginosa [START_REF] Mahajan-Miklos | Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa-Caenorhabditis elegans pathogenesis model[END_REF], Brucella abortus [START_REF] Arellano-Reynoso | Cyclic beta-1,2-glucan is a Brucella virulence factor required for intracellular survival[END_REF] or Dickeya dadantii (Page et al., 2001).

Dickeya dadantii is a phytopathogen Enterobacteria that causes soft rot disease in a wide range of plant species [START_REF] Perombelon | Potato diseases caused by soft-rot erwinias: an overview of pathogenesis[END_REF]. This opportunistic pathogen devastates economically important crops in storage facilities or in growing plants. In D. dadantii, opgG or opgH mutant, completely devoid of OPGs, show a pleiotropic phenotype including loss of motility (Page et al., 2001), induction of a general stress response (Bouchart et al., 2007) and complete loss of virulence on potato tubers and chicory leaves (Page et al., 2001). All these phenotypes suggest an impairment in the perception of the environment. This hypothesis was confirmed by the restoration of motility and virulence on potato tubers by inactivation of the RcsCDB phosphorelay regulatory system in strains devoid of OPGs.

Phosphorelays (also called two component systems) are the key of gene expression plasticity in response to environmental variations. The RcsC sensor has both kinase and phosphatase activities [START_REF] Clarke | The Rcs phosphorelay: more than just a two-component pathway[END_REF]. Under various stimuli, the transmembrane RcsC sensor activates, its kinase activity increased, RcsC autophosphorylates and transfers its phosphate group to its cognate cytoplasmic response regulator, RcsB, via the intermediate transmembrane protein RcsD. In turn, RcsB regulates the expression of numerous target genes affecting various cellular processes including repression of virulence and motility (Cano et al., 2002, Majdalani and[START_REF] Majdalani | The Rcs phosphorelay: a complex signal transduction system[END_REF]. In contrast, when the RcsC sensor phosphatase activates and dephosphorylates the RcsB regulator, virulence and motility occurs.

Loss of OPGs leads to a constitutive activation of the RcsCDB phosphorelay system.

Restoration of virulence and motility was obtained in the opgG by secondary mutations decreasing the activation level of the RcsCDB phosphorelay. This was the case for the opgG rcsC2, the opgG rcsC or the opgG rcsBD56N double mutant strains. In these strains, the secondary mutation encoded a mutated RcsC2 protein with an increased phosphatase activity, no RcsC protein or a non phosphorylatable RcsBD56N protein respectively [START_REF] Bouchart | The virulence of a Dickeya dadantii 3937 mutant devoid of osmoregulated periplasmic glucans is restored by inactivation of the RcsCD-RcsB phosphorelay[END_REF][START_REF] Bontemps-Gallo | Concentration of osmoregulated periplasmic glucans (OPGs) modulates the activation level of the RcsCD RcsB phosphorelay in the phytopathogen bacteria Dickeya dadantii[END_REF]. The phosphorylation of the RcsBD56N protein is impossible since its phosphorylatable aspartic acid residue at position 56 was changed in an asparagine residue. These conclusions about the activation level of the RcsCDB phosphorelay system in the wild-type and the different mutant strains were classically deduced from expression analysis of genes regulated by this phosphorelay.

In the present study, we showed the variation of activation level of the RcsCDB phosphorelay system in different genetic background by direct observation of the RcsB phosphorylated and non phosphorylated protein forms separated with a Phos-tag TM acrylamide gel retardation approach(ref phos tag). We demonstrated that the loss of OPGs severely increases the phosphorylation level of the RcsB protein and this constitutive phosphorylation repress motility .

Materials and Methods

Bacterial strains, media and growth conditions

Bacterial strains and plasmids are described in Table 1. Bacteria were grown in Lysogeny broth (LB) [START_REF] Bertani | Lysogeny at mid-twentieth century: P1, P2, and other experimental systems[END_REF] at 30°C (D. dadantii) or 37°C (Escherichia coli). Antibiotics were used at the following concentrations for D. dadantii: gentamicin at 2 µg.ml -1 , chloramphenicol at 12.5 µg ml -1 , and kanamycin at 25 µg ml -1 , spectinomycin at 50 µg ml -1 .

Ampicillin was used at 50 µg ml -1 for E. coli.

Plasmid constructs and protein purification

Plasmid used for the expression of the His tagged-RcsB, pNF410, was previously described in [START_REF] Bontemps-Gallo | Concentration of osmoregulated periplasmic glucans (OPGs) modulates the activation level of the RcsCD RcsB phosphorelay in the phytopathogen bacteria Dickeya dadantii[END_REF] For the construction of the His tagged-RcsBD56N, a DNA fragment encoding the mutated rcsBD56N gene was amplified by PCR using pNFW374 as template, with the rcsBacf (CACCATGAGCAATCTAAACGTAATTATTGCAG) and rcsBacr (CGCCGGCAAGAGCATTACTC) primers, cloned into the His-tag expression vector pET100/D-Topo ® (Invitrogen Life Technologies) to give pNFW491. The His-tagged RcsB and RcsBD56N proteins were expressed in E. coli BL21(DE3) and purified according to [START_REF] Bontemps-Gallo | Concentration of osmoregulated periplasmic glucans (OPGs) modulates the activation level of the RcsCD RcsB phosphorelay in the phytopathogen bacteria Dickeya dadantii[END_REF]

Production of the phosphoramidate

The phosphoramidate (PA) used to phosphorylate the RcsB Protein in vitro was synthesized by reaction of phosphoryl chloride with ammonium as described by Sheridan [START_REF] Sheridan | Phosphoramidic acid and its salts[END_REF].

Analysis of RcsB phosphorylation in vitro

Phosphorylation reactions of purified RcsB or RcsBD56N were performed with 5 µg of proteins in 50 mM Tris-HCl, pH7.5, 100 mM NaCl, 10 mM MgCl2 and 2 mM βmercaptoethanol. PA was added to a final concentration of 15 mM to initiate the reaction.

After 0, 15 or 30 minutes of incubation at room temperature, the reactions were stopped by addition of SDS-PAGE loading buffer (final concentration: 50 mM Tris-HCl pH6.8, 2% (w/v) SDS, 10% (w/v) glycerol, 20 mM DTT, 0.02% bromophenol blue). The mixtures were resolved using phosphoprotein affinity gel electrophoresis as described in [START_REF] Barbieri | Universally applicable methods for monitoring response regulator aspartate phosphorylation both in vitro and in vivo using Phos-tag TM -based reagents[END_REF]

Analysis of RcsB phosphorylation in vivo

At mid-log phase, equivalent of 1 ml of 0.15 OD620nm of D. datantii cells was harvested by centrifugation and pellets were immediately lysed with 12.7 µl of 1M formic acid, solubilised by 5 µl of 4X SDS-PAGE loading buffer and neutralized by 2.8 µl 5 N NaOH.

Samples were quickly loaded onto gels containing 35 µM Phos-tag TM acrylamide and 70 µM MnCl2 prepared and run as described before. After 10 min wash with transfer buffer (25 mM Tris and 192 mM glycine) supplied with 1 mM EDTA, followed by 10 min wash with transfer buffer without EDTA, gels were transferred to nitrocellulose membranes using a

Trans-Blot ® Turbo TM Blotting system (Bio-Rad) with a pre-programmed protocol (2.5 A, up to 25 V, 7 min). Western blotting against RcsB were performed as described in Bontemps-Gallo et al., 2013. When required, purified RcsB or RcsBD56N protein (2.5 µg) were phosphorylated by a 30 min incubation at room temperature with PA, as described previously. RcsB or phosphorylated RcsB proteins were added to bacteria prior lysis.

2.6 Quantification of the phosphorylated RcsB protein amount.

Phosphorylated RcsB and RcsB were quantified by determination of the area intensity of each band with the software Quantity One (Bio Rad) after staining with coomassie blue or detection by Western blot. Quantification of phosphorylated RcsB was expressed as the ratio of the phosphorylated RcsB amount divided by the sum of the RcsB and the phosphorylated RcsB amounts as described elsewhere [START_REF] Barbieri | Universally applicable methods for monitoring response regulator aspartate phosphorylation both in vitro and in vivo using Phos-tag TM -based reagents[END_REF].

Dosage fusion

….

2.8 Analyse motility nage et fliC antibodies

Results and Discussion

Phosphorylated RcsB (RcsB-P) migrate slower than RcsB

The method to observe the phosphorylated form of cytoplasmic regulators proteins is available when the phosphate group is linked to a serine, threonine or tyrosine because the linkage is relatively stable. In the case of bacterial phosphorelays, the phosphate group is linked to an aspartate residue for the cytoplasmic regulator. Unfortunately, in this last case, linkage is highly unstable and since recently, observation of thse kind of phosphorylated proteins was impossible.

In 2008, Stock's lab published a method allowing separation of the phosphorylated and the non-phosphorylated forms of phosphorelay regulators [START_REF] Barbieri | Universally applicable methods for monitoring response regulator aspartate phosphorylation both in vitro and in vivo using Phos-tag TM -based reagents[END_REF]. This method relies on Phos-tag TM : a dinuclear metal complex acts as a specific phosphatebinding agent. In a Phos-tag TM acrylamide gel, the Phos-tag TM linked to acrylamide molecules interact with the phosphorylated form of the protein which migrates slower than the non-phosphorylated protein allowing the separation of both forms of the protein.The method was first applied in vitro on RcsB since method must be adapted to each regulator (dixit Stock ref). Purified RcsB was phosphorylated in vitro by the phosphodonor phosphoramidate (PA) (Lukat et al., 1992). 5 µg of RcsB was incubated different times with or without PA (see materials and methods), samples were loaded onto a Phos-tag TM acrylamide gel and RcsB was revealed with coomassie blue after migration. Without PA added, a single band, corresponding to the non-phosphorylated RcsB form (called RcsB)

was observed (lanes 1 and 2, Fig. 1A). In contrast, a retardation of migration of 57% and 61% of the band was observed (lanes 3 and 4, Fig. 1A) when PA was added during 15 min and 30 min respectively. This strongly suggests that the shifted band corresponds to the phosphorylated RcsB form (called RcsB-P) but indicating that in our conditions, PA was unable to phosphorylate all RcsB. The same experiment was performed with the non phosphorylatable RcsBD56N protein (lanes 5 to 8, Fig. 1A). In these lanes, a single band migrating as the single band of lanes 1 and 2 was observed. These data indicate that the lower band corresponded to RcsB, that the upper band corresponded to RcsB-P and that PA phosphorylates the proper aspartic residue of RcsB. These conditions allowed us to analyze the stability of the phosphorylation of RcsB in vivo.

Fin de la correction XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

The lysis with formic acid had no effect on the RcsB-P amount

The in vivo analysis required rapid lyse of bacteria with formic acid before loading onto the gel [START_REF] Barbieri | Universally applicable methods for monitoring response regulator aspartate phosphorylation both in vitro and in vivo using Phos-tag TM -based reagents[END_REF]. Because the phospho-aspartate bond is very unstable, it was important to check both the effect of the formic acid and the lysis of cell on the stability of RcsB and of the RcsB-P phospho-aspartate bond. To do this, 5 µg of purified RcsB or RcsB-P, phosphorylated by PA, was added to an rcsB null mutant strain of D. dadantii (NFB7279)

prior lysis with formic acid. Lysates were then loaded onto a Phos-tag TM gel. As controls, the same amount of RcsB and RcsB-P without bacteria were loaded onto the same gel. The same experiments and controls were performed with the RcsBD56N. After run, RcsB was revealed by Western blot. When the lysate of the NFB7279 strain was loaded onto the gel alone, RcsB was not detected (lane 1, Fig. 1B). A similar amount of RcsB was revealed when RcsB was added to the NFB7279 strain before lysis (lane 2, Fig. 1B) or when RcsB was loaded alone onto the gel (lane 6, Fig. 1B). The same two bands in similar amount, corresponding to RcsB (53%, lower band) and RcsB-P (47%, upper band), were revealed when RcsB phosphorylated by PA was added to the NFB7279 strain before lysis (lane 3, Fig. 1B). The same two bands in similar amount, corresponding to RcsB (52%, lower band) and RcsB-P (48%, upper band), were revealed when RcsB phosphorylated by PA was loaded alone on the gel (lane 7, Fig. 1B). The same conclusions can be drawn when RcsBD56N was used instead of RcsB except that only one band was observed corresponding to the non phosphorylated form of RcsB (lane 4, 5, 8, 9, Fig. 1B). Taken together, these results indicate that neither cell lysate nor formic acid affect the RcsB stability or the RcsB-P phosphoaspartate bond stability and that the two forms of RcsB are separated within a cell lysate.

These results allowed us to analyse the RcsB phosphorylation in vivo.

The phosphorylation of RcsB is increased in a strain devoid of OPGs

We previously showed that the RcsCDB phosphorelay was constitutively activated in an opgG strain devoid of OPGs and constitutive activation was resumed by secondary mutations reducing the activity of the RcsCDB phosphorelay (see introduction). These conclusions were deduced from expression analysis of genes regulated by the RcsCDB phosphorelay. To confirm these results, the level of in vivo phosphorylation of RcsB was performed using the Phos-tag TM approach in the same strains (Fig. 1C). As expected, no RcsB was detected in the rcsB strain (lane 7, Fig. 1C). As expected, in the rcsBD56N, only one band was observed corresponding to the RcsB form (lane 6, Fig. 1C). In the wild-type strain (lane 1, Fig. 1C), a low amount of RcsB-P (11% upper band) was observed as compared to the RcsB amount (89%, lower band). In the opgG strain (lane 2, Fig. 1C), an increased amount of RcsB-P (23%, upper band) as compared to RcsB (77%, lower band) was observed as compared to the wild-type but RcsB remained more important. In the opgG rcsC2 (lane 3, Fig. 1C), in the rcsC2 (lane 4, Fig. 1C), in the rcsC null mutant (lane 5, Fig. 1C), and in the rcsBD56N (lane 6, Fig. 1C) strains, RcsB-P cannot be detected. These data confirmed the results observed by [START_REF] Bouchart | The virulence of a Dickeya dadantii 3937 mutant devoid of osmoregulated periplasmic glucans is restored by inactivation of the RcsCD-RcsB phosphorelay[END_REF]. In addition, they showed that in wild-type cells, the RcsB-P form is present in very limited amount and that a moderate increase in RcsB-P (opgG strain) is sufficient to confer the pleiotropic phenotype observed in D. dadantii. The direct analysis of phosphorylation level of regulators of phosphorelays is of a great interest particularly for determination of the phosphorylated to non phosphorylated ratio of regulator imposible to do with analysis of gene expression but remains difficult to use systematically because adaptation of the method is required for each new phosphorelay studied is time consuming.

Acknowledgements

This word was supported by grants from the Centre National de la Recherche Scientifique This study a rcsCBD56ND is carried by a Spe r mini-Tn5 b Cml r , chloramphenicol resistance; Amp r , ampicillin resistance, Kan r , kanamycin resistance, Spe r , spectinomycin resistance, Gm r , gentamicin resistance.

(

  CNRS), from the Ministère de l'Education Nationale et de la Recherche and from the Université des Sciences et Technologies. SBG was funded by doctoral fellowship of the Lille 1 University.

Figure 1 :

 1 Figure 1: Separation of RcsB and RcsB-P by Phos-tag TM polyacrylamide gel electrophoresis and detection in vivo by Western analysis. A. Phosphorylation of RcsB in vitro. Each lane contains 5µg of either purified wild-type RcsB (lanes 1 to 4) or RcsBD56N (lanes 5 to 8) incubated in presence of 15mM PA, during 0 min (lanes 2, 6), 15 min (lanes 3, 7) and 30 min (lanes 4, 8) or in absence of PA (lanes 1, 5) before loading onto the gel. After migration, RcsB and RcsB-P proteins were visualized by coomassie-stained Phos-tag TM gel. B. Stability of RcsB and phospho-aspartate of RcsB-P during cell lysate processing. PurifiedRcsB (lane 2), RcsB-P (lane 3) or unphosphorylatable RcsBD56N (lanes 4-5) were mixed with rcsB mutant strain before treatment with formic acid as described in Materials and Methods. As controls, rcsB cell lysate (lane 1), RcsB protein (lanes 6) RcsB-P (lane 7) and RcsBD56N protein (lanes 8, 9) were also loaded on the gel. Revelation of RcsB was performed by Western blot analysis after electrophoresis on Phos-tag TM gel. Lanes were PA is noted indicated that RcsB (or RcsBD56N) was treated 30 min with PA prior to lysis.C. Phosphorylation of RcsB in vivo. Cell lysates of wild-type (lane 1), opgG (lane 2), opgG rcsC2 (lane 3), rcsC2 (lane 4), and rcsC (lane 5), rcsBD56N (lane 6) and rcsB (lane 7) mutant strains of D. dadantii were subjected to Phos-tag TM acrylamide gel electrophoresis and Western blot analysis.

  minor modifications. Briefly, Phos-tag TM acrylamide gels were composed

	of a 10% resolving solution [10% (w/v) 37.5:1 acrylamide/N,N'methylenebisacrylamide,
	375 mM Tris (pH8.8) and 0.1% (w/v) SDS, 125µM Phos-tag TM acrylamide and 250 µM
	MnCl2]	and	a	4%	stacking	solution	[4%	(w/v)	37.5:1
	acrylamide/N,N'methylenebisacrylamide, 125 mM Tris (pH6.8) and 0.1% (w/v) SDS]. The
	gels were run at 4 °C under constant voltage (150 V) with standard running buffer (0.1%
	(w/v) SDS, 25 mM Tris and 192 mM glycine) and stained with Coomassie Blue.

Table 1

 1 StrainGenotype a, b Source or Reference

	Dickeya dadantii
	EC3937	Wild-type


Fabien Blas

Florence Ansart

Philippe Lours

Jean-Pierre Bonino

Sandrine Duluard
email: duluard@chimie.ups-tlse.fr

Vanessa Vidal

Lisa Pin

Guillaume Pujol

Luiza Bonin

Processing thermal barrier coatings via sol-gel route: crack network control and durability

Keywords:

Introduction

Thermal barrier coatings (TBC) are used as thermal insulators in aircraft jet engines and more specifically as a protection for turbine blades engine. Service temperatures have been increased for some years and these temperatures are sometimes higher than the degradation temperature of the used superalloys. In these conditions, the role of TBCs is to maintain or even to increase the lifetime of such complex turbine blade systems [START_REF] Stöver | Directions of the development of thermal barrier coatings in energy applications[END_REF]. TBCs systems are made up of a single crystal nickel-based superalloy, which ensures good mechanical stability. The bondcoat, deposited onto the superalloy, plays an important role in the accommodation of lateral thermomechanical constraints. It also plays the role of a reservoir of aluminium allowing the formation of a socalled Thermal Grown Oxide (TGO) on its surface. Finally, the topcoat is a protective ceramic layer of yttria-stabilized zirconia. This ceramic has the great advantage of having a low thermal conductivity, which allows to enhance the achievable thermal gradient up to 1 °C per μm.

Nowadays, two processes are conventionally used in industry to elaborate TBCs. The first is the Electron Beam Physical Vapour Deposition leading typically to a columnar structure coating [START_REF] Lugscheider | Ceramic thermal barrier coatings deposited with the electron beam-physical vapour deposition technique[END_REF]. This technique is used to coat parts subjected to high thermomechanical constraints since lateral movement of column remains possible to sustain in-service stresses. The second process is the Air Plasma Spraying which generates a lamellar structure parallel to the substrate [START_REF] Wortman | Thermal barrier coatings for gas turbine use[END_REF][START_REF] Nusair Khan | Manipulation of air plasma spraying parameters for the production of ceramic coatings[END_REF] and shows particularly good thermal insulation properties. The control of vertical and horizontal cracks development in Air Plasma Sprayed thermal barrier coatings have been studied in order to improve thermomechanical stress accommodation [START_REF] Karger | Atmospheric plasma sprayed thermal barrier coatings with high segmentation crack densities: spraying process, microstructure and thermal cycling behavior[END_REF].

One of the key-issue is to develop an innovative low-cost process to elaborate thermal barrier coatings with equivalent or better performances than that of thermal barrier coatings deposited using the above standard industrial processes. To tackle this problem, several methods can be investigated such as electrodeposition [START_REF] Put | Microstructural engineering of functionally graded materials by electrophoretic deposition[END_REF] or sol-gel route, a technique which has been developed over the last few years for TBC. Fenech et al. prepared YSZ:RE (Rare Earth RE = La, Sm, Er) by sol-gel route and demonstrated the impact of doping level on the particle size [START_REF] Fenech | Sol-gel processing and characterization of (RE-Y)-zirconia powders for thermal barrier coatings[END_REF]. Sniezewski et al. optimized the manufacturing route by changing the formulation to avoid the presence of aggregates [START_REF] Sniezewski | Sol-gel thermal barrier coatings: optimization of the manufacturing route and durability under cyclic oxidation[END_REF], and improved

The complete methodology is the following: i) Identification of new dispersants & formulation of the dispersions ii) Processing of the TBC iii) Spray-coating reinforcement step if applicable iv) Comparison with the reference material (made with C213 dispersant): cracks network morphologies and cyclic oxidation performances The substrates used for depositing YSZ are discs of 25 mm ( ± 5) in diameter made of AM1 Ni-based superalloys covered with (Ni,Pt)Al bond-coats. They were provided by SAFRAN Group. Samples were sandblasted with corundum 220 meshes at 2.5 bars for 10 s. Then, they were pre-treated using acetone cleaning followed by an ultrasonic cleaning in 1-propanol. Substrates were then pre-oxidized at 950 °C for 2 h at 5 × 10 -4 mbar P O2 synthetic air to favour the growth of a thin, dense and adherent alpha alumina layer [START_REF] Monceau | Effects of bond-coat preoxidation and surface finish on isothermal and cyclic oxidation, high temperature corrosion and thermal shock resistance of TBC systems[END_REF]. This Thermal Grown Oxide (TGO) thickness was < 1 μm after pre-oxidation.

Materials and methods

Elaboration of the

Sol-gel deposition process

To elaborate TBCs, the first step consists in preparing an aerogel YSZ powder by the sol-gel route from zirconium (IV) propoxide Zr(OPr) 4 (Aldrich) and yttrium (III) nitrate hexahydrate (Acros Organics) [START_REF] Shane | Sol-gel synthesis of zirconia barrier coatings[END_REF]. Acetylacetone (AcAc) is used as a complexing agent to reduce the hydrolysis rate of zirconium alkoxide [START_REF] Viazzi | Synthesis by sol-gel route and characterization of yttria stabilized zirconia coatings for thermal barrier applications[END_REF], and the solvent is 1-propanol. Molar ratios between AcAc and Zr(OPr) [START_REF] Nusair Khan | Manipulation of air plasma spraying parameters for the production of ceramic coatings[END_REF] and between H 2 O and Zr (OPr) 4 are respectively 0.8 and 9.5. Zirconium concentration is kept constant at 0.5 mol•L -1 . Then, supercritical drying of the propanol at 270 °C for 1 h is performed so as to obtain an aerogel microstructure. To complete the processing, a heat treatment at 700 °C is carried out to remove organics. Finally, grinding for 1 h at 350 round per minute is required to obtain a YSZ powder at 9,7% mol YO 1.5 with submicronic aggregates constituted large particles (20-30 nm).

In each case, the protocol is the same either for the C213 (a phosphate ester, CECA), the Hypermer KD1 (polyester polyamine condensation polymer, CRODA) and the KD2 or the PVP (polyvinylpyrrolidone, Mw = 3500 g/mol, BASF) dispersants. Only the weight percentage of dispersant is different. Indeed, a previous rheological study showed that 10 wt% (compared to the weight of the solvent) for Beycostat C213, 15 wt% for KD1, 10 wt% for KD2 and 1 wt% for PVP were the best conditions. The required amount of dispersant is dissolved in 1-propanol and a 20 wt% of a YSZ sol with Acac/Zr (OPr) 4 = 1.2, H 2 O/Zr(OPr) 4 = 9.5 and Zr(OPr)4 concentration of 0.5 mol•L -1 is added. Each composite sol is then loaded with 50 wt% of aerogel powder. The criteria, here, is the obtention of a stable dispersion, which is assessed by the absence of a hysteresis in the evolution of the viscosity with increasing and decreasing shear rate. Then, the superalloy substrates are dipped into the slurry and withdrawn at a controlled rate (250 mm•min -1 ) to shape the coatings. Following each dip, the layer is dried at 50 °C for 5 min. These operations are repeated until the required TBC thickness is reached (about 150 μm). Specimens are sintered at 1100 C for 2 h using heating and cooling rates of 50 °C/ h to generate a controlled micro-cracks network and release thermomechanical constraints due to sintering.

The reinforcement step consists in spraying a sol with x wt% of YSZ commercial powder (TOSOH 8Y), 20 wt% of the YSZ sol, 4%wt ultrapure water and propanol. The sequence is a six steps process consisting of two steps with x = 20 wt% and four steps with x = 10 wt%. The slurry is sprayed at the vertical on top of the TBC coating, and the pressure and displacement rate of the spray nozzle are controlled. The sample is dried for 5 min between each spraying step.

Choice of a new dispersant

The reference dispersant is a commercial product: the Beycostat C213, which is a phosphoric ester composed of 50% monoester and 50% diester. This dispersant presents a combination of electrostatic and steric repulsion forces. To investigate substitutes to Beycostat C213, several dispersants with similar dispersion mechanism (i.e. electrostatic and steric hindrance) were investigated. Hypermer KD1 (polyester polyamine condensation polymer), Hypermer KD2, polyvinylpyrrolidone PVP M w 3500 and PVP M w 55,000 were considered, due to the presence of groups with similar functions as, for example in PVP, a pyrrolidone cycle as anchoring group and polymeric chains for steric hindrance. Their ability to dissolve and to process stable dispersions in the alcohol based media were also taken into account. In order to preserve a similar protocol, starting precursors used for the the durability under cyclic oxidation as well as under controlled temperature gradient cycling [START_REF] Sniezewski | Oxidation and spallation of FeCrAl alloys and thermal barrier coatings: in situ investigation under controlled temperature gradient[END_REF]. The works of Pin et al. were dedicated to the control of the crack network formation [START_REF] Pin | Processing, repairing and cyclic oxidation behaviour of sol-gel thermal barrier coatings[END_REF] and reinforcement by dip-coating or spray-coating [START_REF] Pin | Reinforced sol-gel thermal barrier coatings and their cyclic oxidation life[END_REF]. The latest results obtained are very promising [START_REF] Pin | Optimized sol-gel thermal barrier coatings for long term cyclic oxidation life[END_REF] since TBCs elaborated by the sol-gel route achieved 1485 cycles under cyclic oxidation (1 h at 1100 °C and 5 min of cooling) with no detrimental damage and no extensive spallation.

The key experimental technique to monitor possible spallation and address overall lifetime of thermal barrier coatings is undoubtedly the cyclic oxidation test. Indeed, at the beginning of the development of TBCs elaborated by the sol gel route, preliminary results obtained using this technique were well below expectation (lifetime lower than 40 cycles). The commonly considered specification, is a number of cycles to failure of 500 or higher (corresponding to < 20% of the surface spalled off). The result for lifetime being unsatisfactory, some investigations, in previous works [START_REF] Pin | Optimized sol-gel thermal barrier coatings for long term cyclic oxidation life[END_REF] focussed on the filling of additional ceramics in the bottom of the initially formed cracks to further reinforce the (micro-cracks) network and/or to modify the scale of this microcracked network. So, as presented by Pin et al. reinforcement by spray coating was successfully performed. This technique consists in spraying a fluid sol to partially fill in and consequently reinforce the micro-crack network. Nevertheless these promising results can only be reached if a double step process is carried out: i) deposit and thermal treatment of the TBC and generation of a primary cracks network and ii) reinforcement of the micro-cracks network by partially filling with additional ceramics within the cracks so as to reduce the size of the secondary cracks network. This two-step processing route is obviously not optimized in terms of manufacturing time and ease of preparation considerations. This is the reason why the challenge of the study presented in this paper is to process in only one step new thermal barrier coatings by investigating a new dispersant able to directly generate a primary finer cracks network, stiff enough to satisfactorily sustain high number of oxidation cycles.

Regarding the elaboration step of the coating, an initial sequence consists in covering the superalloy with an yttria-stabilized zirconia composite sol by successive dip-coatings at a controlled speed. In this study, for the sol formulation, several dispersants are used in the composite medium. The phosphate ester Beycostat C213 dispersant (CECA), previously used to process thermal barrier coatings [START_REF] Pin | Optimized sol-gel thermal barrier coatings for long term cyclic oxidation life[END_REF] and now subject to environmental restrictions, is the reference. The Polyvinylpyrrolidone (PVP) compound (BASF) with M w = 3500 g/mol, as well as commercial Hypermer KD1 and KD2 (Croda) dispersants often used in a broad range of technical applications (cosmetics, adhesives, inks, textiles and fibers…), are chosen as alternative dispersants.

Then, a heat treatment is required to generate and control a microcrack network. For the durability test, samples are submitted to cyclic oxidation with the following parameters for each cycle: exposure at 1100 °C for 1 h followed by 5 min cooling. The evolution of the microcrack network is then recorded by charge coupled device (CCD) camera upon each cooling sequence. formulation of the composite sol (previously described) remained unchanged: 1-propanol, acetylacetone, ultra-pure water, zirconium propoxide, yttrium nitrate hexahydrate. The criterion to define the optimal percentages for each dispersant (based on their ability to disperse), is relative to their rheological properties (Fig. 1). The chosen dispersant must have a viscosity approaching that of the C213 dispersant which is the case of all the selected dispersant (10-25 mPa•s at 100 s -1 ). Another parameter to take into account is the stability of the suspension, that was evaluated from the hysteresis area between the values of viscosities taken with increasing shear rates and those taken with decreasing shear rates (the lowest the hysteresis the highest the stability).

Following this preliminary step, the chosen dispersant as a function of the viscosity criterion, was PVP 3500 M w corresponding to 1% wt because of the lack of hysteresis, which means that the composite sol is stable. The absence of hysteresis is also a signature of the lack of large aggregates that could be broken at high shear rates, thus smaller aggregation is expected in PVP dispersion as compared to C213 dispersions. PVP dispersant is made up of a long carbon chain and a vinyl group on the ramifications. These groups allow a steric hindrance in the solution that causes the particles to repel each other. Note that PVP percentage is ten times lower than for the C213 (10%wt) so that the particle volume fraction is higher for PVP based material as compared to C213 ones. According to [START_REF] Kendall | Agglomerate strength[END_REF][START_REF] Birk-Braun | Generation of strength in a drying film: How fracture toughness depends on dispersion properties[END_REF] works, the increase in particle volume fraction will have a huge impact on material fracture toughness since elastic modulus and energy release for the creation of crack surface depend on the particle volume fraction to the fourth power. In order to evaluate such effect PVP and C213 were selected for developing the thermal barrier coatings investigated in this paper.

Characterizations techniques

Rheological measurements are performed at room temperature (20 °C) with an ANTON PAAR Physica MCR 301 viscosimeter. The samples surface is characterized by optical microscopy using a numerical device KEYENCE VHX-1000 to compare the global and general surface characteristics of microstructures and cracked networks obtained from each dispersant. Then, an image analysis using APHELION (ADCIS) software is performed to determine the density of micro-cracks, the mean length and mean width of cracks as well as the mean size of cells.

Surface morphological characteristics have also been analysed through a micro-topography approach (ALTISURF 520-Altimet Device) by evaluating the roughness parameters and extracting the cracks profiles, giving parameters such as depth, amplitude… (Altimap software). The surface profile of samples was measured using contactless surface microtopography so as to determine various roughness (R a , R sk ) and morphological parameters (cracks shape and depth).

Thermal barrier coatings were cyclically oxidized within a dedicated instrumented rig allowing to monitor, using various types of optical means, the evolution of the sample surface during the cooling phases [START_REF] Sniezewski | Oxidation and spallation of FeCrAl alloys and thermal barrier coatings: in situ investigation under controlled temperature gradient[END_REF]. Typical cycles include 5 min heating, 1 h holding at high temperature and 5 min cooling down to room temperature.

Results and discussion

Cracks parameters for C213 and PVP based thermal barriers

After elaboration, TBCs are (i) characterized using image analysis of optical micrographs at an adequate magnification revealing the different individual cells and (ii) compared in terms of morphological characteristics (Fig. 2). On Fig. 2.i, optical micrographs of the sample surface show the initial microstructure. Then, by using image analysis (Fig. 2.ii and 2.iii), different microstructural parameters were evaluated: the density of cracks, the mean size (area) of the cells (A in μm 2 ), the mean length and the mean width of cracks (in μm). Results from the image analysis of Fig. 2.iii are detailed on Fig. 3.

In Fig. 3.a, the surface fraction of cell is plotted versus the equivalent diameter (D = (4A/Π) 0.5 ) of the cells, assumed, as a first approximation, to be disc-shaped.

The arithmetic average of roughness values (Ra), the skewness (Rsk) and the maximum peak-to-valley values (PV) were obtained from the characteristic surface profiles presented in Fig. 4.

Cracks geometry

A preliminary analysis clearly shows two different micro-crack networks, depending on the dispersant used. It is firstly noted that the crack mean density is roughly similar for both dispersants, with 14% of micro-cracks for C213 and 18% for PVP. The overall quasi-bell curve of the fraction of cells depending on the cell diameter shows for C213 a maximum for a diameter of 170 μm with a large distribution range. On the contrary in the case of PVP dispersing agent, cracks are smaller and their size distribution is notably narrower. Note that the maximum density obtained in the two cases are different: for the TBC PVP , the equivalent average diameter of cells is around 100 μm for a maximum cell density of 35% while for the TBC C213 , the equivalent average diameter is 170 μm for a maximum cell density of 13%. In addition, the distribution for the TBC PVP is narrower than for the TBC C213 showing that in the case of TBC PVP , the cell size and consequently the microstructure is both finer and more homogeneous. Fig. 3.b gives information on the width of the cracks. The two distributions differ in terms of their overall shape. Indeed, the experimental values with PVP correspond to a relatively tight distribution, centred on an average width of 18 μm while for the TBC C213 , various peaks can be observed including a principal one centred on 28 μm.

As well, regarding the length of cracks (Fig. 3.c), the distribution shows several maxima, with a main contribution for a length of 75 μm for thermal barriers obtained from suspensions using the C 213 dispersant. The cracks width distribution (Fig. 3.b) is more homogeneous with a satisfactory symmetry around the well-defined peak shown at 28 μm.

The finer and more homogeneous micro-crack networks observed when PVP is used can be related both to the quantity of dispersing agent incorporated (1% PVP against 10% C213) and to the narrower distribution of particles size in PVP dispersion (no hysteresis in viscosity vs. shear rates curves). Indeed, following heat treatment, the organic matter from the dispersing agent which is degraded will be much reduced in the case of PVP and will generate less material shrinkage. Moreover due to the finer microstructure with smaller defects and to the reduced volume of dispersant, the particle volume fraction is higher in PVP materials. As already explained, these conditions are expected to increase the toughness of such materials. This provides here an experimental evidence, that changing the dispersing agent has a direct effect on the micro-cracks network formed. Indeed, for roughly the same TBC thickness (approximately 150 μm), the dimensional parameters of cells and cracks are different. Fig. 3.c shows the distribution of the fraction of crack versus the average crack length for the two dispersing agents. The distributions are similar even if the plot for the C213 is still more irregular. The change of the dispersing agent does not have any significant influence on the average length of cracks.

Topology of the cracked surface

The characteristic topographic profiles are presented in Fig. 4 for one typical profile (five profiles were measured for each samples). Considering the profiles, the thermal barrier coatings prepared with the Note that an overthickness is brought up on the sample filled by spray-coating. Of course, this global overthickness can include a preferential deposition on the sharpest areas of the coating inducing a significant increase of the Peak-to-Valley height parameter (from 45 to 90 μm). This overthickness is then in the range [30-40 μm]. The question is now to evaluate the impact of this microstructure on the lifetime of the reinforced TBC PVP . The difference in lifetime between unfilled and filled PVP samples is not very significant. First, no spallation is observed up to 1100 cycles except for the edge effects due to the dip-coating process [START_REF] Blas | La voie sol-gel pour la mise en oeuvre de barrières thermiques aéronautiques[END_REF]. The variation is much less pronounced than in the case of C213 formulations due to the finer morphology of the PVP coating.

As compared to EB-PVD coatings, sustaining typically around 1000-1200 cycles, when cyclically oxidized in similar conditions, very good performances is achieved with sol-gel coatings. Indeed lifetime of the thermal barriers, reaching > 1000 cycles for the TBC obtained with PVP, confirms that the use of PVP dispersant can beneficially replace the Beycostat C213 as dispersing agent as well as filling agent for cracks, if necessary. However, it is shown that the reinforcement of the micro-crack network is no more required in this case, the gain in lifetime being non-significant. Indeed, the process implemented in only one step is efficient enough to promote the development of reliable and competitive sol-gel thermal barrier coatings. They remain without spallation for > 1000 oxidation cycles, in perfect agreement with required lifetime expected by engine makers.

Conclusion and perspectives

In this paper, the use of PVP as dispersant was validated in the formulation of sol-gel TBC. Effectively, it was shown that thermal barriers elaborated using PVP dispersions are made from more stable dispersions (no hysteresis in rheological curves) with lower volume fraction of dispersants (1% for PVP based materials versus 10% for C213 ones). This leads, in PVP materials, to smaller particle sizes (less aggregation in the dispersion) and higher particle volume fraction (smaller quantities of dispersant). Both contributions are known to increase material toughness i.e. material resistance to fracture. After heat treatment the cracks development is different within PVP and C213 materials with smaller cracks width and depth for PVP. During cyclic PVP dispersant exhibits narrower and less deep cracks than that formulated and shaped with C213 dispersant.

This study confirms the observation made earlier, that there is a much smoother surface topography when PVP is used. The average surface roughness (Ra) of C213 based material is twice as much as PVP ones (7 μm versus 3.5 μm). Peak-to-valley height is smaller in the case of PVP, which means that the cracks are much less deep than in the case of a material prepared with C213 (117 μm). Moreover as already discussed the distribution of cracks is more regular in the case of PVP.

Cyclic oxidation

As previously revealed, there is a significant difference in the microstructure obtained from each formulation using the two dispersants (C213 and PVP). Consequently, the impact of these microstructural parameters on the lifetime of TBC upon cyclic oxidation has to be addressed.

During cyclic oxidation, the lifetimes of the thermal barriers elaborated with C213 and PVP dispersant are highly different. Indeed for reaching 20% surface spallation, in agreement with the current industrial specifications, sample (a), with C213 dispersant, sustains < 300 cycles while sample (b), with PVP, exhibits 1160 cycles. The expected behaviour with slower crack propagation kinetic for PVP based materials is observed.

Effect of reinforcement by spray coating

It has been shown in a previous study [START_REF] Pin | Reinforced sol-gel thermal barrier coatings and their cyclic oxidation life[END_REF][START_REF] Pin | Optimized sol-gel thermal barrier coatings for long term cyclic oxidation life[END_REF], that reinforced TBC C213 (where the network of micro-cracks was filled by spraycoating), showed a promising lifetime upon cyclic oxidation. Actually, such TBC exhibited a large number of cycles to spallation, typically 1100 cycles and up to 1485 cycles, indicating that the spray-coating reinforcement is likely to enhance significantly lifetimes.

As a consequence, it is therefore of primary importance to address the effect of filling cracks by spray-coating on PVP samples, in order to check whether the cyclic oxidation lifetime is improved as well in this case.

The surface micrographs of the non-filled up sample i.e. PVP-NC (a) and the characteristic profiles of the filled up one i.e. PVP-SC (b) before and after spray-coating treatment are shown in Fig. 5.

5mm

Len t h oxidation tests, cracks propagation proceeds via cracks widening and deepening across the material. As expected the kinetic of cracks propagation is slower for PVP based materials and lifetime of TBC is longer (> 1000 cycles) than in the case of the TBC with C213 dispersant (< 300 cycles). Whereas reinforcement is mandatory with C213 dispersant to obtain lifetime > 1000 cycles, such reinforcement is not necessary for PVP dispersant. Then the use of PVP dispersant represents a superior alternative to the material based on C213 dispersant. Thermal barrier coatings with > 1000 oxidation cycles are achieved with a cost-effective simple deposition process.

Fig. 1 .

 1 Fig. 1. Evolution of the viscosity versus the shear rate for each dispersant.

Fig. 2 .

 2 Fig. 2. 3D optical micrograph, binary image and cell distribution of a) C213 dispersant; b) PVP dispersant i)3D optical micrograph; ii) binary image; iii) cell distribution of the surface of TBC.

Fig. 3 .

 3 Fig. 3. Geometrical parameters of cells and cracks as a function of the dispersant.

Fig. 4 .

 4 Fig. 4. Characteristic profiles for a) C213; b) PVP.

4 4. 5

 45 Lengt~mm

Fig. 5 .

 5 Fig. 5. Surface micrographs and characteristic profiles of the thermal barriers unfilled (TBC PVP-NC) and with the spray coating reinforcement step (TBC PVP-SC).

Longueur - 5 .

 5 001 mm pt _ 92.26 µm Echelle -150 µm llmax cracks de pth = 90 µm 0

Acknowledgments

This work was supported by the Ecole Doctorale Aéronautique Astronautique (ED467) (France).