David Picard

Inbar Fijalkow

Second Order Model Deviations of Local Gabor Features for Texture Classification

In this paper, we tackle the problem of texture classification with a local approach based on measuring second order deviations with respect to a dictionary of characteristic patterns. At each pixel, we extract local signal properties thanks to several Gabor filters that are aggregated on a small support region. Then, we compute a dictionary of such features that serves as a universal model. The texture signature is the deviation of second order statistics between its local features and the universal model. Experiments are made on two sets of photographic paper textures, and show the soundness of the approach.

I. INTRODUCTION

In this paper, we are interested in the identification of photographic paper textures sharing physical properties (same sheet, same package, same manufacturer), based solely on visual content [START_REF] Messier | Automated surface texture classification of inkjet and photographic media[END_REF]. The identification of photographic paper is an important challenge for conservation science, since the aging of old photographs depends strongly upon the material it was printed of. Since these photographs are of high value, nondestructive identification techniques have to be used. In our case, the identification is performed on high resolution pictures of the paper, using digital image processing techniques.

Usually, texture identification systems are designed to discriminate between a wide variety of textures such as wood, marble, cloth, etc. In our case, images from different papers are visually very similar, and the distinction is made on tiny variations. As such, usuall texture recognition approaches are not well adapted to this specific problem.

To solve the problem, we propose an approach based on Gabor filters, which have been widely used in texture discrimination [START_REF] Turner | Texture discrimination by gabor functions[END_REF]. However, such characteristics are often computed on the whole image and are not enough to discriminate between the fine categories of photographic paper textures. We argue these textures are composed of several distinctive patterns (or textons), each having its own frequency characteristics. To capture such patterns, we propose to compute a universal dictionary of local responses of a Gabor filter bank on all the available images. Then, we compute the second order moments of the local Gabor responses of an image with respect to the universal dictionary, which amounts to map the image on several Riemannian manifolds (of symmetric positive definite matrices S ++ ). The distance between textures is computed using a geodesic distance on S ++ , allowing to take into account the specific topology of the proposed representation space.

The remaining of this paper is as follows: First we present a quick overview of texture recognition methods as well as related work. Then, we present our method in section III. We perform experiments in section IV before we conclude.

II. RELATED WORK

Texture recognition has been a major topic of interest in the image processing community for several decades. Early methods focused on characterizing the statistical distributions of specific frequency band of the Fourier domain [START_REF] Turner | Texture discrimination by gabor functions[END_REF], [START_REF] Duvernoy | Optical digital processing of directional terrain textures invar-iant under translation, rotation, and change of scale[END_REF]. in [START_REF] Davis | Polarograms -a new tool for image texture analysis[END_REF], the authors used polarograms to obtain rotation invariant statistical features. In [START_REF] Eichmann | Topologically invariant texture descriptors[END_REF], the authors proposed to used Hough transforms to describe the line structure of the texture.

All these global approaches have been outperformed by local approaches that extract characteristic information on a small neighborhood of the image. In [START_REF] Varma | A statistical approach to material classification using image patch exemplars[END_REF], the authors proposed for example to use local image patches quantized into a texton codebook. In [START_REF] Ojala | Multiresolution gray-scale and rotation invariant texture classification with local binary patterns[END_REF], the difference of intensities in the neighborhood of a pixel is encoded into a binary code that can be rotation invariant. the resulting Local Binary Pattern (LBP) is shown to have very good performances in texture recognition, although it encodes image properties at a small local scale. This descriptor has been extended to encode more information, like in Complete LBP [START_REF] Guo | A completed modeling of local binary pattern operator for texture classification[END_REF] or in the Pattern of Oriented Edge Magnitude [START_REF] Vu | Enhanced patterns of oriented edge magnitudes for face recognition and image matching[END_REF].

Even more recently, visual dictionaries have been use to perform texture recognition [START_REF] Lazebnik | A sparse texture representation using local affine regions[END_REF]. In these approaches, a set of local features is extracted from the images and quantized so as to produce a dictionary of prototypical descriptors. The image is represented by computing moments of occurrences of the dictionary entries. This approach has been very popular in computer vision to perform object recognition [START_REF] Sivic | Video google: A text retrieval approach to object matching in videos[END_REF]. Given a set of local descriptors (such as LBP), the Bag of Word method consist in assigning each descriptor to its nearest entry in the dictinnary, and computing the histogram of such assignments. This roughly corresponds to computing the zero order moment of the image with respect to the dictionary. Several authors extended this approach by computing higher order moments [START_REF] Jégou | Aggregating local descriptors into a compact image representation[END_REF], [START_REF] Perronnin | Improving the fisher kernel for large-scale image classification[END_REF], [START_REF] Picard | Efficient image signatures and similarities using tensor products of local descriptors[END_REF]. In particular, second order moments have shown very good performances in both image retrieval [START_REF] Negrel | Web scale image retrieval using compact tensor aggregation of visual descriptors[END_REF]and image classification [START_REF]Using spatial pyramids with compacted vlat for image categorization[END_REF].

In this paper, with take advantage of these second order dictionary approaches, while also taking inspiration from the filter banks methods. In particular, we propose to compute local descriptors using Gabor filters similar to that of the VZ approach [START_REF] Varma | A statistical approach to material classification using image patch exemplars[END_REF], while computing second order moments not unlike [START_REF] Perronnin | Improving the fisher kernel for large-scale image classification[END_REF] and [START_REF] Picard | Efficient image signatures and similarities using tensor products of local descriptors[END_REF].

A. Local descriptors

We describe each pixel of the image by the responses to a set of Gabor filters g(θ, f ) [START_REF] Gabor | Theory of communication. part 1: The analysis of information[END_REF], taken at m different orientations θ and n different frequencies f :

g(θ, f ) = 1 2πσ 2 exp - x 2 θ + γ 2 y 2 θ 2σ 2 cos 2πf x θ + π 2 (1)
with

x θ = x cos(θ) + y sin(θ), y θ = -x sin(θ) + y cos(θ),
and γ = π/m. To allow better robustness to translation, we compute the modulus of such responses at any pixel p:

r θ,f (p) = |g θ,f I(p)| (2)
To better account for the local properties, the responses are then aggregated in a neighborhood around the pixel p by convolution with a uniform filter u to form the descriptor d(p):

d(p) = [u r θ,f (p)] θ,f (3)
Each pixel of the image is thus described by a feature vector of dimension mn, with m being the number of orientations and n the number of frequencies. The pixels are then sorted by decreasing norm of their feature vectors d and subsequently normalized to have unit 2 norm.

B. Texture features

Starting from a sampling of the feature vectors of all the images, we compute a dictionary of characteristic patterns. The feature space is modeled as a Gaussian Mixture Model trained on the subset of the feature vectors with highest norm (before normalization), using the EM algorithm.

The images are represented by computing second order moments with respect to the model obtained previously. For each Gaussian of the model, we compute the covariance matrix Γ I,c of all feature vectors d(p), p ∈ I of image I, weighted by their respective likelihood h c (x) to model component c:

Γ I,c = p∈I h c (d(p))(d(p) -µ I,c )(d(p) -µ I,c ) p∈I h c (d(p)) (4)
with

µ I,c = p∈I h c (d(p))d(p) x h c (d(p)) . ( 5
)
Each image is thus represented by an ordered set of positive definite matrices. These matrices correspond to the specificity (or deviation) of the distribution of local Gabor features with respect to a universal model, as measured by their second order moments.

C. Similarity measure

The computation of the covariance matrix for each component induces a mapping of the images into the space of symmetric positive definite matrices S ++ :

ψ c :R M ×N → S ++ (6) I → Γ I,c (7)
To compute the distance between any 2 images I 1 and I 2 , we consider the concatenation of the spaces induced by each component of the Gaussian mixture. The square distance in the concatenated space is then simply the sum of the square distances in the spaces induced by each component:

dist(I 1 , I 2 ) = c dist S++ (Γ I1,c , Γ I2,c ) (8)
To take into account the topology of S ++ , we propose to use the Log-Euclidean distance instead of the Euclidean (Frobenius) distance:

d(I 1 , I 2 ) 2 = c log(Γ I1,c ) -log(Γ I2,c ) 2 F (9)
The Log-Euclidean distance has nice properties: it is a metric and corresponds to a geodesic distance on S ++ [START_REF] Jayasumana | Kernel methods on the riemannian manifold of symmetric positive definite matrices[END_REF].

The incentives to use the Log-Euclidean distance are the following: We consider the eigen-decomposition of a matrix Γ I,c . Each of its eigenvectors corresponds to a specific combination of the filters in the bank, while the related eigenvalue encodes the contribution (or energy) of this combination. When using the Euclidean distance, the direction of the eigenvectors (i.e., the weights of the filters for each combination) and the contribution are considered equivalently. When using the Log-Euclidean distance, the eigenvalues relative differences are shrunk, whereas the directions remain. We claim that the direction is more important than the contribution, since slight variations in direction lead to textures based on different pattern, while variations on the contributions lead textures based on the same pattern, albeit in different proportions. Using the Log-Euclidean distance, the eigenvalues are processed through a logarithm, which lower their effect on the total distance. Using this distance, the changes in the direction of the eigenvectors are better taken into account.

IV. EXPERIMENTS

We carried out experiments on two datasets of photographic paper textures named inkjet and b&w [START_REF] Messier | Automated surface texture classification of inkjet and photographic media[END_REF]. Each of these datasets contains 120 images grouped in 9 classes based on their physical properties (same sheet, same package, same manufacturer, etc). Each class contains 10 images, and the last 30 images are distractors and do not belong to any class. Images of representatives textures from these datasets are shown in Figure 1. As we can see, the textures look already similar, even though they are all drawn from different classes.

We compute local descriptors using a bank of 8 Gabor filters using 4 orientations and 2 scales. These filters are shown in Figure 2. The signatures are computed using a Gaussian mixture model with 8 components.

To evaluate our method, we compute the pairwise distance matrices of each dataset. We perform the computation with the Euclidean distance and the Log-Euclidean distance. On Figure 3, we show the corresponding distance matrices, as well as a reference distance matrix provided by the expert P. Messier. As we can see, Using the Log-Euclidean distance allows to obtain results more consistent with the groundtruth.

We also compared our results with the best distance matrices from [START_REF] Picard | Photographic paper texture classification using model deviation of local visual descriptors[END_REF]. As we can see, the results from [START_REF] Picard | Photographic paper texture classification using model deviation of local visual descriptors[END_REF] are very close to the groundtruth matrices. However, they tend to be too discriminative (very diagonal structure), especially with the textures from the distractors class (last 30 row/columns). The distance obtained by our proposed method seems to better take into account the true distance to the distractor textures.

V. CONCLUSION

In this paper, we presented a new method to perform texture identification, primarily designed to handle the recognition of photographic paper textures. Our approach is divided in three steps. First, we compute and sum the response to a bank of Gabor filter, leading to a set of local descriptors. Then, we model the space of local descriptors with a Gaussian mixture model, and we compute the covariance matrix of the descriptors of each image, weighted by their likely hood with respect to each component of the model. Finally, we compare the matrices of different textures using the sum of componentwise Log-Euclidean distances to better take into account the topology of the space of covariance matrices. We show in the experiments that this approach has promising results on this challenging task.

  (a) Images from the bw dataset. (b) Images from the inkjet dataset.

Fig. 1 .

 1 Fig. 1. Images extracted from the texture datasets. The first row contains images from the bw dataset, while the second row contains images from the inkjet dataset. Each image is from a different category.

Fig. 2 .

 2 Fig. 2. Gabor filter bank used in this paper.

Fig. 3 .

 3 Fig. 3. Distance matrices for the inkjet dataset (left) and the BW dataset (right). Dark colors indicate small distances. The last row is taken from [19].

ACKNOWLEDGMENT

The authors would like to thank P. Messier and C.R. Johnson Jr for the access to the data basis and related discussions, and A. Klein and P. Abry for their interactions on this work.


Renee Kern

Abderrahim Malki

Jad Abdallah

Jihen Tagourti

Gilbert Richarme
email: richarme@ccr.jussieu.fr

Renée Kern

ESCHERICHIA COLI HDEB IS AN ACID-STRESS CHAPERONE

Escherichia coli HdeB is an acid-stress chaperone.

INTRODUCTION

In their natural habitats, bacteria are constantly under assault from a wide array of environmental stresses, including UV, heat, oxidative, osmotic and pH stresses [START_REF] De Angelis | Environmental stress responses in Lactobacillus: a review[END_REF]. One of the most frequently encountered is acid stress [START_REF] Foster | Escherichia coli acid resistance: tales of an amateur acidophile[END_REF]. Enterobacteria, when travelling through the gastrointestinal tract encounter an extremely low pH; facultative intracellular pathogens tolerate episodes of low pH within macrophage phagolysosomes, and fermentative bacteria excrete acidic fermentation products that trigger an endogeneous acid stress [START_REF] De Angelis | Environmental stress responses in Lactobacillus: a review[END_REF][START_REF] Lim | Identification of stress-inducible proteins in Lactobacillus delbrueckii subsp. bulgaricus[END_REF].

In response to acid stress, several mechanisms regulate the homeostasis of bacterial pH. Many bacteria, including E. coli, possess aminoacid (glutamate, arginine, or lysine) decarboxylase systems, each of which consists of a cytoplasmic decarboxylase which converts its substrate to a related amine (γ-aminobutyric acid, agmatine or cadaverine, respectively), and an antiporter which exchanges the imported amino acid for the cytoplasmic amine produced [START_REF] De Angelis | Environmental stress responses in Lactobacillus: a review[END_REF][START_REF] Foster | Escherichia coli acid resistance: tales of an amateur acidophile[END_REF][START_REF] Hersh | A glutamate-dependent acid resistance gene in Escherichia coli[END_REF][START_REF] Richard | Escherichia coli glutamate-and arginine-dependent acid resistance systems increase internal pH and reverse transmembrane potential[END_REF][START_REF] Small | Acid stress, anaerobiosis and gadCB: lessons from Lactococcus lactis and Escherichia coli[END_REF]. These systems consume one cytoplasmic proton during amino acid decarboxylation, and one extracytoplasmic proton during protonation of the exported amine, thus leading to cytoplasmic and periplasmic alkalinization. In several bacteria, including fermentative bacteria such as Lactococcus lactis, the proton-translocating F 1 F 0 ATPase can export protons as a consequence of ATP hydrolysis [START_REF] Tucker | Gene expression profiling of the pH response in Escherichia coli[END_REF]. Further protection against acid stress is obtained by decreasing the permeability of the inner and outer membranes to protons ; several inner and outer membrane proteins are overexpressed in response to acid stress, including the cyclopropane fatty acyl phospholipid synthase, OmpC, the LPS biosynthesis enzyme YfbF and the outer membrane lipoprotein Slp [START_REF] Tucker | Gene expression profiling of the pH response in Escherichia coli[END_REF]. Furthermore, bacteria can reverse their cytoplasmic membrane potential to an inside-positive potential that slows the influx of protons into the cell [START_REF] Foster | Escherichia coli acid resistance: tales of an amateur acidophile[END_REF][START_REF] Richard | Escherichia coli glutamate-and arginine-dependent acid resistance systems increase internal pH and reverse transmembrane potential[END_REF]. They can also reorient their metabolism towards pathways that decrease proton production or increase amine production with a consequent alkalinization (underexpression of catabolic sugar enzymes, such as those of the maltose regulon [START_REF] Yohannes | pH-dependent catabolic protein expression during anaerobic growth of Escherichia coli K-12[END_REF] decreases the production of organic acids, and overexpression of urease increases ammonia production in Helicobacter pylori [START_REF] Booth | Sensing and adapting to acid stress[END_REF][START_REF] Ferrero | Construction of isogenic ureasenegative mutants of Helicobacter pylori by allelic exchange[END_REF]). Moreover, several protective proteins may be induced upon acid stress, including the DnaK and GroEL chaperone machines [START_REF] Lim | Identification of stress-inducible proteins in Lactobacillus delbrueckii subsp. bulgaricus[END_REF] and several DNA repair enzymes [START_REF] Thompson | Isolation of the Helicobacter pylori recA gene and involvement of the recA region in resistance to low pH[END_REF].

The acid resistance mechanisms of the periplasm are not as well understood as those of the cytoplasm.

Periplasmic proteins are probably more vulnerable than cytoplasmic proteins to acid stress, due to the relative permeability of the outer membrane porins to molecules smaller than 600 Da [START_REF] Koebnik | Structure and function of bacterial outer membrane proteins: barrels in a nutshell[END_REF][START_REF] Schirmer | General and specific porins from bacterial outer membranes[END_REF].

Recently, a periplasmic chaperone, HdeA, which supports acid resistance in E. coli, was discovered [START_REF] Gajiwala | HdeA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria[END_REF][START_REF] Hong | Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation[END_REF]. HdeA exhibits chaperone-like activity at strong acidic pH only (below pH 3), preventing the acidinduced aggregation of bacterial periplasmic extracts, and of several model substrate proteins such as alcohol dehydrogenase (ADH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the periplasmic ribose receptor [START_REF] Hong | Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation[END_REF].

In this study, we report the cloning, purification and characterization of HdeB as a novel acid-stress chaperone, and we show that, whereas HdeA is more efficient than HdeB in protein solubilization at pH 2, HdeB is more efficient than HdeA at pH 3. Thus, E. coli possesses two acid-stress chaperones, HdeA and HdeB, which are encoded by the acid-stress operon hdeAB, and prevent the aggregation of periplasmic proteins at acidic pH.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditons. The E. coli BL21 (DE3) strain (Novagen, USA) was used for the transformation of the new expression vector constructs containing the genes encoding for HdeB and HdeA, respectively. We generated the genes coding for HdeB and HdeA by amplifying the hdeB and hdeA genes from E. coli genomic DNA (from strain MG1655) by PCR using the forward primers (5'GGTGGTTGCTCTTCACATATGAATATTTCATCTCTC-3' for hdeB, and 5'GGTGGTTGCTCTTCACATATGAAAAAAGTATTAGGC-3' for hdeA) containing a Nde1 site and the reverse primers (5'GGTGGTCTGGGATCCTCATTAATTCGGCAAGTCATT-3' for hdeB, and 5'GGTGGTCTGGAATCCTCATCATTACATATCTTTCTYTAAT-3' for hdeA) containing a BamH1 site. The whole hdeAB operon was generated by PCR using the hdeA forward primer and the hdeB reverse primer. The PCR was done in a volume of 50 µl, in the presence of 0.5 µg of template DNA, 1 µg of forward and reverse primers and 5 units of Taq DNA polymerase (Applied Biosystems), for 35 cycles (1 min at 94°C, 2 min at 58°C, 2 min at 72°C°) followed by a final cycle (1 min at 94°C, 2 min at 58°C, 10 min at 72°C). The resulting products were digested with Nde1 and BamH1, ligated to the pET-21a (Novagen, USA) Nde1 and BamH1 backbone fragment, and transformed into strain BL21 (DE3). The sequences of the cloned genes were confirmed by DNA sequencing (not shown).

Construction of the hdeA and hdeB mutants. We achieved the one-step inactivation of the hdeA and hdeB genes using the phage λ recombination (Red) system (4). The strategy is analogous to the PCRbased gene deletion method in yeast, except that we used E. coli cells carrying an easily curable, low copy number plasmid pSC101 (replication temperature-sensitive derivative)-based plasmid pKD46 carrying the λ Red recombinase genes expressing the Red system. The basic strategy was to replace a chromosomal sequence with a selectable antibiotic resistance gene that is generated by PCR by using primers with 50-nt extensions homologous to the adjacent upstream or downstream flanking regions of the target gene and 20-nt 3' end for the amplification of the kanamycin (kan) resistance gene. This replacement was achieved by λ Red-mediated recombination in these flanking homologies (4). Strain BW25113 (lacI q rrnB3 ∆lacZ4787 hsdR514 ∆(araBAD)567 ∆(rhaBAD)568 rph-1) was used as the parent for construction of the hdeA and hdeB mutants. The construction of the hdeA and hdeB disruption mutants is part of the systematic construction of E. coli single gene deletion mutants [START_REF] Baba | Construction of Escherichia coli K-12 in frame, single-gene knock out mutants, the Keio collection[END_REF].

The amounts of HdeA and HdeB in the hdeA-and hdeB-deficient mutants, and in these mutants complemented with pBAD vectors (see below) were evaluated by 1D-or 2D-gel electrophoresis of osmotic shock fluids prepared from these strains.

Construction of pBAD vectors. Construction of pBAD vectors for in vivo expression of HdeA and

HdeB in the hdeA-and hdeB-deficient strains was done as follows. The hdeA gene, the hdeB gene and the hdeAB operon were excised from the pET-21a-hdeA, pET-21a-hdeB and pET-21a-hdeAB vectors using Xba I and Hind III, and ligated to the pBAD33 Xba I and Hind III backbone fragment (pBAD33 is a pACYC184-derived vector containing the P BAD promoter of the araBAD operon and the gene encoding the regulator of this promoter, araC) [START_REF] Guzman | Tight regulation, modulation, and high-level expression by vectors containing the arabinose P BAD promoter[END_REF]. The pBAD vectors were induced with 0.05% arabinose.

Preparation of bacterial extracts, and purification of HdeA and HdeB. The HdeA and HdeB overproducing strains BL21 (DE3) pET-21a-hdeA and BL21 (DE3) pET-21a-hdeB were grown at 37°C in 1 liter of Luria-Bertani medium [START_REF] Nossal | The release of enzymes by osmotic shock from Escherichia coli in exponential phase[END_REF] supplemented with ampicillin (50 µg/ml) to an OD 600 = 0.5. HdeA and HdeB overexpression was induced with 1 mM IPTG and growth was continued for 3 hours. Cells were harvested by centrifugation at 4°C. The cell pellets were resuspended in 10 ml of 30 mM Tris pH 8.0, 20 mM NaCl, 1 mM DTT, 0.5 mM EDTA, and bacterial periplasmic extracts were prepared according to the osmotic shock procedure described in [START_REF] Higgins | Periplasmic protein associated with the oligopeptide permeases of Salmonella typhimurium and Escherichia coli[END_REF]. HdeA and HdeB were each loaded onto a DEAE -Sephacel column equilibrated in 30 mM Tris pH 8.0, 1 mM DTT at 20°C, and eluted with a linear gradient of 0 -0.5 M NaCl in the same buffer. Fractions were analyzed by SDSpolyacrylamide gel electrophoresis and quantified by the Bradford assay. Purified fractions of HdeB and HdeA were each loaded onto a hydroxyapatite column (Bio-Gel HTP from BioRad), equilibrated at 20°C in 30 mM Tris pH 7.5, 1 mM DTT and eluted with a linear gradient of 0 -50 mM sodium phosphate pH 7.5 in the same buffer. HdeA and HdeB were stored at -70°C. For experiments at acidic pH, HdeA and HdeB were equilibrated in distilled water by gel permeation on a Bio-Gel P10 column equilibrated in water (BioRad). We performed electrophoresis according to Laemmli, using 16% polyacrylamide gels (Bio-Rad) with Coomassie blue staining [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. All the experiments showing polyacrylamide gels (Figure 2345) have been repeated at least three times, leading to similar results.

We used NIH-Image 1.62 software (rsb.info.nih.gov/nih-image/Default/html) to quantify protein bands on polyacrylamide gels. Chaperone assays. The chaperone activity of HdeA and HdeB was assayed by incubating periplasmic extracts or model substrate proteins for 60 min at 25°C in the presence of HdeA and/or HdeB at acidic pH, and monitoring the appearance of the proteins in the 15,000 x g pellet or supernatant (protein were analyzed by SDS-PAGE) [START_REF] Hong | Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation[END_REF]. Sulfate was used as the anion in order to achieve effective aggregation of substrate proteins at low pH values [START_REF] Goto | Conformational states of beta-lactamase: molten-globule states at acidic and alkaline pH with high salt[END_REF][START_REF] Hong | Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation[END_REF]. were used as molecular weight standards. For experiments at pH 7.5, the column was equilibrated in 20 mM Tris, pH 7.5, 100 mM NaCl at 20°C, loaded with 20 µl of protein (2.4 mg/ml) and eluted at a flow rate of 0.5 ml/min. For experiments at pH 3 and pH 2, the column was equilibrated with 150 mM Na 2 SO 4 adjusted to these pHs with sulfuric acid. Proteins were detected by their absorbance at 280 nm. When both proteins were loaded onto the column, they were detected by SDS-PAGE (on a 18% acrylamide gel).

Mass spectrometric identification of

ANS binding assay to

Acid stress sensitivity of the hdeA and hdeB mutants, and of these mutants complemented by plasmids pBAD33-hdeA, pBAD33-hdeB and pBAD33-hdeAB. Mutant and parental strains were grown in LB medium containing 0.05% arabinose for 24 hours at 37°C with aeration, in the presence of the required antibiotics. They were diluted to 10 6 bacteria per ml, and incubated for 90 min in acidified LB medium (pH 2 or pH 3) at 37°C under aeration [START_REF] Gajiwala | HdeA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria[END_REF]. A control was done at pH 7. Survival patterns were determined by plating bacteria overnight on LB agar plates at pH 7.

Reagents. Restriction enzymes were from Invitrogen, and the plasmid extraction kit was from Qiagen. ADH (from rabbit muscle, 36 kDa) and GAPDH (from Saccharomyces cerevisiaeae, 36 kDa), were from Sigma, and OppA was purified as described in Richarme and Caldas [START_REF] Richarme | Chaperone properties of the bacterial periplasmic substratebinding proteins[END_REF]. All other chemicals were from Sigma and were reagent grade.

RESULTS

Expression and purification of HdeA and HdeB. The BL21 (DE3) strains, transformed with the recombinant expression vector pET-21a-hdeB was induced for 3 hours with 1 mM IPTG, and its periplasmic extract was prepared by a modification (13) of the osmotic shock procedure of Nossal and Heppel [START_REF] Nossal | The release of enzymes by osmotic shock from Escherichia coli in exponential phase[END_REF]. The HdeB overproducing strain accumulates a periplasmic protein migrating in SDS-PAGE with an apparent molecular weight of 10 kDa, which matches the expected HdeB molecular weight of 9 kDa (after processing of the signal sequence) (Figure 1, lane 2). The overexpressed protein represents 29 % in mass of the proteins in the induced periplasmic extract. HdeB was purified, as described in "Materials and Methods", by two chromatographic steps on a DEAE-Sephacel column and a hydroxyapatite column, (Figure 1, lane 3), and its identity was confirmed by mass spectrometry (not shown). We purified HdeA from strain pET-21a-hdeA, using the same osmotic shock procedure and chromatographic steps as for HdeB (Figure 1, lane 4), and confirmed its identity by mass spectrometry (not shown).

Acid-induced aggregation of periplasmic extracts from the hdeA and hdeB mutants. Periplasmic protein extracts are relatively resistant to acid-induced aggregation [START_REF] Liu | Periplasmic proteins of Escherichia coli are highly resistant to aggregation: reappraisal for roles of molecular chaperones in periplasm[END_REF], and the acid-stress chaperone HdeA contributes to this resistance [START_REF] Gajiwala | HdeA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria[END_REF][START_REF] Hong | Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation[END_REF]. We investigated the acid-induced aggregation of periplasmic extracts from the hdeA and hdeB mutants and their parent, by analyzing pellets and supernatants after an acidic treatment at pH 2 or pH 3 for 60 min. The wild-type extract displays a moderate protein aggregation at acidic pH (Figure 2A). In contrast, a massive protein aggregation is observed at both pHs in periplasmic extracts from the hdeA mutant (as reported previously ( 14)), and from the hdeB mutant. Conversely, a lower quantity of proteins from the two mutants (as compared to their parent) remains in the supernatant at these acidic pHs (Figure 2B). For both mutants, protein aggregation is more extensive at pH 2 than at pH 3, and for the hdeA periplasmic extract than for the hdeB extract. Furthermore, the proteins that appear to aggregate in periplasmic extracts from both mutants are similar. A fraction of HdeA (characterized by N-terminal sequencing) is found in the pellets at pH 2 (slightly above the 7 kDa marker in Figure 3A, except in the HdeA-deficient extract). At pH 7, there is no significant protein aggregation, suggesting that HdeA and HdeB do not function as neutral pH chaperones.. The two mutants display a strong protein aggregation at both pH 2 and pH 3, but HdeA and HdeB are mainly involved in periplasmic protein solubilization, at pH 2 and pH 3, respectively (see below). We checked the expression level of HdeA and HdeB in each mutant. Our 2D-gel electrophoresis analysis (performed as described in ( 17)) of periplasmic extracts from the hdeA and hdeB mutants showed that HdeA and HdeB are both undetectable in the hdeA mutant (which consequently behaves like a hdeAB mutant), and that HdeB is undetectable in the hdeB mutant, while HdeA is expressed at around 24% of the wild-type level (not shown), The null expression of the two chaperones in the hdeA mutant explains its aggregation phenotype at pH 2 and pH 3. The aggregation phenotype of the hdeB mutant cannot be explained by its low HdeA level, since complementation of the mutant by the P BAD HdeA expression plasmid (the complemented strain expresses HdeA at a level that is 148% of that of the wild-type) does not significantly modify its aggregation behaviour (not shown).

HdeA is more efficient than HdeB in preventing periplasmic protein aggregation at pH 2.

Periplasmic extracts from the parental strain and from the hdeA and hdeB mutants, either alone or supplemented with purified HdeA, HdeB, or both, were incubated at pH 2 for 60 min, and their 15,000

x g pellets and supernatants were analyzed. As reported above, protein aggregation at pH 2 increases in extracts from both mutants. The addition of HdeA decreases the aggregation of the hdeA (Figure 3A) and hdeB (not shown) extracts, whereas the addition of HdeB is much less efficient. HdeA and HdeB, together, are no more efficient than HdeA on its own. For the different periplasmic extracts, a high quantity of protein in the supernatant fraction (Figure 3B) corresponds to a low quantity in the pellet (Figure 3A), and vice versa. Most of HdeA and HdeB remains in the supernatant. We quantified the aggregated proteins in the hdeB mutant extract and in its wild-type control using NIH 1.62 software (Figure 3C). The profile of the HdeB-supplemented extract is only slightly different from that of the unsupplemented extract, whereas the profile of the HdeA-supplemented extract approaches that of the parental strain extract. These results are consistent with the hypothesis that HdeA is the main chaperone involved in protein solubilization at pH 2. The important role of HdeA in preventing protein aggregation at pH 2 has already been discussed [START_REF] Gajiwala | HdeA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria[END_REF][START_REF] Hong | Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation[END_REF].

HdeB is more efficient than HdeA in preventing periplasmic protein aggregation at pH 3.

Periplasmic extracts from the parental strain and from the hdeA and hdeB mutants, either alone or supplemented with HdeA, HdeB, or both, were incubated at pH 3 for 60 min, and their 15,000 x g pellet and supernatant were analyzed. As reported above, there is a strong protein aggregation in extracts from both mutants. HdeA decreases the aggregation of the hdeA (Figure 4A) and hdeB (not shown) extracts, but HdeB is much more efficient than HdeA. HdeA and HdeB, together, are no more efficient than HdeB on its own. For the different periplasmic extracts, a high quantity of protein in the supernatant fraction (Figure 4B) corresponds to a low quantity in the pellet, and vice versa. We quantified the aggregated proteins in the hdeA mutant extract and in its wild-type control from Figure 4A using NIH 1.62 software (Figure 4C). The protein profiles of the HdeA-and HdeB-supplemented extracts reflect the greater efficiency of HdeB in solubilizing periplasmic proteins at pH 3. These results are consistent with the hypothesis that HdeB is the main chaperone for periplasmic protein solubilization at pH 3, and clarify the poor efficiency of the HdeA chaperone at pH 3 (14, this study).

These results, together with those presented in the preceding paragraph, suggest that, to achieve the solubization of periplasmic extracts (the natural substrate of HdeA and HdeB) the task is divided between the two chaperones, HdeA being involved in protein solubilization mainly at pH 2, and HdeB mainly at pH 3.

Solubilization of model substrate proteins at pH 2 and pH 3.

We examined the aggregationsuppressing function of HdeA and HdeB, using two usual substrates of chaperone activity assays, alcohol dehydrogenase (ADH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the periplasmic oligopeptide receptor OppA. Protein aggregation was monitored by analysis of 15,000 x g supernatants and pellets, after incubation of these proteins at acidic pHs in the absence or presence of chaperones. In the absence of chaperones, GAPDH remains in the supernatant at pH 7 and 4 (not shown), but not at pH 3 and 2 (Figure 5A). At pH 2, HdeA increases the fraction of soluble GAPDH from 12% to 81%, whereas HdeB has only a weak effect, and HdeA and HdeB together are no more efficient than HdeA on its own. At pH 3, HdeA and HdeB, separately, increase the fraction of soluble GAPDH from 1% to 12% and 8%, respectively, whereas HdeA and HdeB together solubilize up to 55% of GAPDH, suggesting that a synergy occurs between the two chaperones for the solubilization of this substrate protein to take place. For each experiment, the amounts of GAPDH found in the supernatant and the pellet are additive (in all the following experiments, the initial amount of each substrate protein added was quantitatively recovered in its supernatant and pellet fractions).

Experiments similar to that shown in Figure 6A were performed with ADH and OppA. For ADH solubilization, HdeA, but not HdeB, was efficient at pH 2 (98% solubilization); at pH 3, HdeA or HdeB, alone, were relatively inefficient (less than 10% solubilization) whereas HdeA and HdeB together solubilized up to 68% of the protein (Figure 5B). For OppA, HdeA solubilized 95% and 52% of the protein at pH 2 and pH 3, respectively, whereas HdeB solubilized 14% and 25% of OppA; the addition of both proteins at the same time was not significantly more efficient than HdeA alone (Figure 5C). These results are consistent with the hypothesis that HdeA makes a major contribution for the solubilization of model proteins at pH 2, that the efficiency of each chaperone is variable for protein solubilization at pH 3, and a synergy between the two chaperones occurs to enable the solubilization of the two cytoplasmic proteins tested at pH 3 (ADH and GAPDH) to take place.

HdeB exposes surfaces that are less hydrophobic than those of HdeA at acidic pHs. It is generally believed that molecular chaperones interact with their unfolded substrate proteins via hydrophobic interactions. HdeA exposes hydrophobic surfaces at acidic pH, but not at neutral pH, in accordance with the appearrance of its chaperone activity at acidic pHs [START_REF] Hong | Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation[END_REF]. We measured the fluorescence emission of the hydrophobic probe ANS, in the presence of HdeA or HdeB, at different pHs. As shown in Figure 6, ANS binding to HdeA and HdeB is negligible at pH 8, and increases at pH 3 and pH 2 (ANS fluorescence is stronger at pH 2 than at pH 3, and stronger with HdeA than with HdeB). Furthermore, the blue shift of ANS fluorescence is similar for both chaperones at pH 2 (around 30 nm), but lower for HdeB (11 nm) than for HdeA (35 nm) at pH 3. This suggests that the surface of HdeB is less hydrophobic than that of HdeA, especially at pH 3. Such a difference in the exposed hydrophobicity of these two chaperones might explain their different chaperone properties described above, and the combination of both chaperones, under certain circumstances, might be required for the optimal handling of unfolded proteins.

Oligomeric forms of HdeB at neutral and acidic pHs. HdeB was analyzed by size-exclusion chromatography on a SW G-2000 HPLC column, as described in Materials and Methods. At pH 7.5, HdeB (loaded at 2.4 mg/ml) migrates as a dimer with an apparent molecular weight of around 16,000 Da (Figure 7), whereas at both pH 3 (Figure 7) and pH 2 (not shown), it migrates as a monomer (9,000 Da). At pH 7.5, HdeA (loaded at 2.4 mg/ml) migrates as a dimer (as previously reported ( 14)) with an apparent molecular weight of around 17,000 Da (not shown), whereas at pH 3 it migrates as a mixture of monomers and dimers (with an apparent molecular weight of around 14,000 Da) and at pH 2 as a monomer (10,000 Da) (not shown), as reported previously [START_REF] Hong | Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation[END_REF]. Thus, like HdeA, HdeB dissociates from dimers to monomers at acidic pHs, but, whereas the dissociation of HdeB is complete at pH 3, that of HdeA is not. This difference might explain why HdeB is a more efficient chaperone than HdeA at pH 3.

We also checked whether HdeA and HdeB form a complex at pH 3. When a mixture of HdeA and HdeB (2.4 mg/ml, each) was loaded onto the SW G-2000 HPLC column equilibrated at pH 3, HdeA migrated (as it does when alone) as a mixture of monomers and dimers with an apparent molecular weight of around 14,000 Da,, and HdeB migrated (as it does when alone) as a monomer of around 9,000 Da (not shown).

Acid stress sensitivity of the hdeA and hdeB mutants and of these mutants complemented by pBAD33-hdeA, pBAD33-hdeB and pBAD33-hdeAB. Cultures of the mutants, and of their parent were incubated for 90 min at 37°C at either pH 2 or pH 3 (a control was done at pH 7), as described under "Materials and Methods", and their survival patterns were observed on LB plates at pH 7. The hdeA mutant, which contains neither HdeA nor HdeB (see above), displays 4% and 3% survival rates at pH 3 and pH 2, respectively. The HdeB mutant, which contains a reduced amount of HdeA (24% of the wild-type level (see above)) and no HdeB displays 8% and 7% survival rates at pH 3 and pH 2, respectively. (Figure 8). This suggests that, both genes are required for resistance of E. coli to these acidic pHs (the involvement of hdeA in E. coli's resistance at pH 2 has already been reported ( 9)).

However, since the hdeA mutant expresses neither HdeA nor HdeB, and since the hdeB mutant expresses 24% of the wild-type level of HdeA, in order to determine the respective contribution of each chaperone in acid-stress resistance, we complemented these mutants with pBAD vectors expressing HdeA or (and) HdeB under the control of the arabinose P BAD promoter.

The viability of the hdeA mutant complemented with pBAD33-hdeA (this strain contains HdeA (140% of the wild-type level), but no HdeB) increased to 12% (a 3-fold increase) and 18% (a six-fold increase) after the pH 3 and pH 2 stresses, respectively. This suggests that HdeA alone affords some protection in vivo against acid stresses at these pHs.

The viability of the hdeA mutant complemented with pBAD33-hdeB (this strain contains HdeB (175% of the wild-type level) but no HdeA) increased to 16% (a 4-fold increase) and 11% (a 4-fold increase) after the pH 3 and pH 2 stresses, respectively. This suggests that HdeB alone affords a moderate protection of bacteria against acid stresses at these pHs.

The viability of the hdeB mutant complemented with pBAD33-hdeA (this strain contains HdeA (148% of the wild-type level) but no HdeB) increased to 9% and 16% after the pH 3 and pH 2 stresses, respectively. These viabilities are not very different (especially at pH 3) from those of the unsupplemented hdeB mutant (8% and 7%, respectively). Importantly, this suggests that the low viability of the uncomplemented hdeB mutant is not a consequence of its reduced HdeA level (25% of the level of the parental strain) but results indeed from an HdeB deficiency.

The viability of the hdeB mutant expressing the hdeB plasmid (this strain contains HdeA (28% of the wild-type level) and 125% of the wild-type HdeB level) increased to 28% and 18% after the pH 3 and pH 2 stresses, respectively.

The viabilities of the hdeA and hdeB mutants expressing the hdeAB plasmid attained 50-75% of the wild-type level, suggesting that the P BAD expression system satisfyingly complements the strains deficient in acid-stress chaperones.

In short, these viability experiments suggest that both HdeA and HdeB are required for an optimal protection of bacteria against acid stress in vivo, at either pH 3 or pH 2 (especially since the hdeA-and the hdeB-deficient strains display a lower viability when complemented with a single chaperone than when complemented with both chaperones).

DISCUSSION

We have cloned, overexpressed, purified and characterized HdeB as a novel acid-stress chaperone.

HdeB was found in the periplasm in a soluble form. It was purified by osmotic shock, followed by two chromatographic steps on DEAE-Sephacel and hydroxyapatite columns, and identified by MALDI-ToF mass spectrometry. We purified HdeA in parallel in order to compare the two chaperones.

A periplasmic extract from the hdeB mutant aggregates at pH 2 and pH 3, like an extract from the hdeA mutant [START_REF] Gajiwala | HdeA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria[END_REF]. The aggregation defect of the hdeB mutant does not result from the lower expression (24% of the wild-type level) of HdeA since complementation of this mutant by the HdeA expression plasmid did not rescue its aggregation defect. Thus, HdeB is important for the solubility of the bacterial periplasm at acidic pH.

At pH 2, HdeA is the main chaperone involved in the in vitro solubilization of periplasmic extracts and of the model substrate proteins used in our study (the chaperone properties of HdeA at a pH around 2 have already been described by others [START_REF] Gajiwala | HdeA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria[END_REF][START_REF] Hong | Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation[END_REF]). HdeB is much less efficient than HdeA in solubilizing periplasmic extracts at pH 2, and its best performance at this pH was the 25% solubilization of OppA. Furthermore, HdeB did not increase the efficiency of HdeA at pH 2.

At pH 3, HdeB solubilizes periplasmic extracts more efficiently than HdeA. It can prevent the aggregation of periplasmic extracts in the absence of HdeA, and thus functions as an acid-stress chaperone by itself. HdeA or HdeB, separately, are relatively inefficient in solubilizing GAPDH and ADH (less than 10% of these proteins were solubilized), and there is a functional synergy between them (leading to 52% and 68% solubilization of GAPDH and ADH, respectively). The occurrence of a synergy between HdeA and HdeB seems to depend on the particular substrate protein used (the synergy observed for the solubilization of the cytoplasmic proteins ADH and GAPDH is not obvious for that of OppA), or on chaperone / substrate ratios (at lower chaperone concentrations, we also observed some synergy between HdeA and HdeB for the solubilization of periplasmic extracts at pH 3, unpublished results). Like HdeA, HdeB displays more hydrophobic surfaces at acidic pHs than at neutral pH, as judged from ANS fluorescence spectra. The surface of HdeB, however, appears less hydrophobic than that of HdeA, and such a difference might explain their respective roles in the renaturation of unfolded proteins. Like HdeA, HdeB dissociates from dimers at neutral pH to monomers at acidic pHs. The dissociation of HdeB into monomers, however, is complete at pH 3 whereas that of HdeA is not. The easier dissociation of HdeB into monomers possibly explains its better chaperone properties at pH 3. Although HdeA and HdeB appear to cooperate in the solubilizaton of several protein substrates at pH 3, we could not detect the formation of a stable heterodimer between the two chaperones at this pH.

An hdeB-deficient strain, like an hdeA-deficient strain [START_REF] Gajiwala | HdeA, a periplasmic protein that supports acid resistance in pathogenic enteric bacteria[END_REF], displays an increased sensitivity to acid stress at pH 2 and pH 3 (and its sensitivity to acid stress is not a consequence of its reduced HdeA level), suggesting that HdeB, like HdeA, is involved in resistance to acid stress. In vivo, there seems to be a requirement for both chaperones for an optimal resistance of bacteria to acid stresses at either pH 3 or pH 2, in accordance with their co-expression from the same operon. This contrasts somewhat with results obtained in vitro suggesting that HdeA is more efficient at pH 2 and HdeB at pH 3. In fact, there are many differences between the in vivo and in vitro situations, including differences in protein concentrations and in the physico-chemical properties of the medium.

HdeA and HdeB are general chaperones (like DnaK, GroEL, small Hsps and periplasmic chaperones that also function as general chaperones [START_REF] Bukau | The Hsp70 and Hsp60 chaperone machines[END_REF][START_REF] Duguay | Quality control in the bacterial periplasm[END_REF]), since they reduce the aggregation of many different periplasmic proteins. Furthermore, whereas HdeA and HdeB display a different pH specificity in vitro (pH 2 for HdeA, pH 3 for HdeB), they solubilize roughly the same proteins, since the profile of aggregated proteins at pH 2 in the presence of HdeA is similar to that of aggregated proteins at pH 3 in the presence of HdeB. Finally, the hdeA and hdeB mutants do not present a protein aggregation phenotype at neutral pH, suggesting that HdeA and HdeB are not involved in protein solubilization at neutral pH (this has previously been reported for HdeA [START_REF] Hong | Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation[END_REF]). Computational and structural experiments will allow us to understand further the different specificities of these two chaperones which have been designed by enterobacteria to prevent periplasmic protein aggregation at acidic pH.

ACKNOWLEDGEMENTS. The authors thank Dr. Hirotada Mori (Nara Institute of Sciences and Technology, Nara, Japan) for the construction of the hdeA-and hdeB-disrupted strain, Dr. Teresa Caldas for her help during the early course of this work, Jean-Jacques Montagne for mass spectrometry analysis, Catherine Dubucs for 2D-gel electrophoresis, Antonia Kropfinger for correction of the English language, and Myriam Barre for her help in preparation of the manuscript. (light grey) or pH 2 (dark grey) for 90 min at 37°C, as described in "Materials and Methods", and survival patterns were determined by plating bacteria overnight on LB agar plates at pH 7. The results are the average ± SEM of three experiments. The colony counts of the mutants were normalized to those of the parental strain. All the strains displayed similar viabilities at pH 7.

LEGENDS TO FIGURES

  HdeA and HdeB. Excised HdeA and HdeB gel bands were ingel digested with mass spectrometry grade trypsin (Roche). Mass spectra were recorded in the positive ion reflection mode of a matrix -assisted laser desorption ionization-time of flight (MALDI-ToF) Voyager DE PRO (Applied Biosystems). The petide masses obtained were searched against the E. coli database (PIR, National Biomedical Research Foundation) with the Mascot engine available online (www.matrixscience.com).

  HdeA and HdeB. Using a Kontron SFM 25 fluorescence spectrophotometer, we monitored the binding of ANS (100 µM) to HdeA or HdeB (7 µM) by measuring the increase in fluorescence intensity of ANS at 25°C upon its binding to protein. Samples were excited at 395 nm, with emission being recorded between 400 and 600 nm. Fluorescence readings were made for triplicate samples. Oligomeric forms of HdeA and HdeB. The molecular weight of HdeA and HdeB was determined by filtration of the proteins on a TSK G-2000-SW HPLC gel permeation column (Hewlett Packard). Blue dextran (2 MDa), yellow dextran (20,000 Da), cytochrome C (12,500 Da) and vitamin B12 (1382 Da)

Figure 1 .

 1 Figure 1. Purification of HdeA and HdeB. Protein samples were separated by dodecylsulfate polyacrylamide gels (16%), and stained with Coomassie brillant blue. Lane 1, periplasmic extract from uninduced strain BL21(DE3), pET-21a-hdeB; lane 2, periplasmic extract from BL21(DE3), pET-21a -hdeB induced for 3 hours with IPTG; lane3, 5 µg of purified HdeB; lane 4, 5 µg of purified HdeA. The positions of molecular weight markers are indicated on the left.

Figure 2 .

 2 Figure 2. Acid-induced aggregation of periplasmic extracts. SDS-PAGE analysis of the pellet (A) and supernatant (B) fractions of periplasmic extracts (20 µg each) from the hdeA mutant, hdeB mutant and their parent, after an acid treatment at pH 2 or at pH 3 for 60 min (a control was made at pH 7).

Figure 3 .

 3 Figure 3. Prevention of periplasmic protein aggregation at pH 2. SDS-PAGE analysis of the pellet (A) and supernatant (B) fractions of periplasmic extracts (20 µg) from the hdeA mutant and its parental strain, after incubation for 60 min at pH 2, either alone or in the presence of purified (15 µg each) HdeA, HdeB or both (7.5 µg each), C) Densitometric scan of the protein distribution of periplasmic extracts from the wild-type strain, and the hdeB mutant supplemented with HdeA or HdeB, as indicated. We checked that the intensities of protein bands (excluding the HdeA/B bands) in pellets and supernatants add up to similar amounts in all cases.

Figure 4 .

 4 Figure 4. Prevention of periplasmic protein aggregation at pH 3. A) SDS-PAGE analysis of pellet (A) and supernatant (B) fractions of periplasmic extracts (20 µg) from the hdeA mutant and its parental strain, after incubation for 60 min at pH 3, either alone or in the presence of purified (15 µg each) HdeA, HdeB or both (7.5 µg each), B) Densitometric scan of protein distribution of periplasmic extracts from the wild-type strain and the hdeA mutant supplemented with HdeA or HdeB, as indicated. We checked that the intensities of protein bands (excluding the HdeA/B bands) in pellets and supernatants add up to similar amounts in all cases.

Figure 5 .

 5 Figure 5. Solubilization of GAPDH, ADH and OppA at acidic pH. A) SDS-PAGE analysis ofthe supernatant and pellet fractions of GAPDH (10 µg) that was subjected to treatment for 60 min at pH 3 or 2, either alone or in the presence of (5 µg each) HdeA, HdeB, or both. B) SDS-PAGE analysis of the supernatant fractions of ADH (10 µg) that was subjected to treatment for 60 min at pH 3 or 2, either alone or in the presence of (5 µg each) HdeA, HdeB, or both. C) SDS-PAGE analysis of the supernatant fractions of OppA (10 µg) that was subjected to treatment for 60 min at pH 3 or 2, either alone or in the presence of (5 µg each) HdeA, HdeB, or both. The molecular weights (monomeric form) of HdeA, HdeB, GAPDH, ADH and OppA are 10 kDa, 9 kDa, 36 kDa, 36 kDa and 61 kDa, respectively.

Figure 6 .

 6 Figure 6. ANS fluorescence of HdeB and HdeA at neutral and acidic pHs. The intensity of ANS fluorescence (100 µM, in H 2 SO 4 solution of pH 2 or pH 3, or in 10 mM Tris pH 8) in the presence of 7 µM HdeA or 7 µM HdeB was measured after excitation at 395 nm.

Figure 7 .

 7 Figure 7. Oligomeric forms of HdeB at neutral and acidic pHs. For experiments at pH 7.5, the column was equilibrated in 20 mM Tris, pH 7.5, 100 mM NaCl at 20°C, loaded with 20 µl of HdeB (2.4 mg/ml) and eluted at a flow rate of 0.5 ml/min. For experiments at pH 3 and pH 2, the column was equilibrated with 150 mM Na 2 SO 4 adjusted to these pHs with sulfuric acid, and loaded with 20 µl of HdeB or (and) HdeA (2.4 mg/ml each), equilibrated at the pH of the column). Proteins were detected by their absorbance at 280 nm. Blue dextran (2 MDa), yellow dextran (20,000 Da), cytochrome C (12,500 Da) and vitamin B12 (1382 Da) were used as molecular weight standards.

Figure 8 .

 8 Figure 8. Acid stress sensitivity of the hdeA and hdeB mutants, and of these mutants complemented with plasmids pBAD33-hdeA, pBAD33-hdeB and pBAD33-hdeAB. The mutants, either uncomplemented (0) or complemented with pBAD33-hdeA (A), pBAD33-hdeB (B) or pBAD33-hdeAB (AB) and their parent were subjected to acid stress in LB medium adjusted to pH 3


Hélène Chaumeil
email: helene.chaumeil@uha.fr

Patrice Jacques

Vincent Diemer

Didier Le Nouën

Christiane Carré

A 13 C NMR Study of pyridinium phenoxide series with

Keywords: C NMR, torsion, quinone, zwitterion, steric hindrance

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

Introduction

One representative phenolate betaine dye still holds the largest solvatochromism observed and defines the famed ET(30) polarity parameter. [START_REF] Reichardt | Solvents and Solvent effects in Organic Chemistry[END_REF] Depending on their structures, solvatochromic phenolate betaines present positive or negative behaviour. Even more interesting, reversal behaviour can occur, a fact which at first sight violates the Onsager's relations at the basis of solvatochromism. The most studied example of such behaviour is the Brooker merocyanine. [START_REF] Brooker | [END_REF]3 A very recent publication presents an unified view of this problematics. 4 It should be added that solvatochromism of phenolate betaine dyes is not easily computed by modern theoretical methods. 5 Moreover, they have generally pronounced zwitterionic character and as such possess large ground state dipole moments, large quadratic hyperpolarisabilities which opens promising applications for non linear optics, in fields such as optical data storage or photonic integrated circuits. 6,7 We have recently synthesized two series of five pyridinium phenoxides 1a-e and 2a-e (scheme 1) with different torsion angles between the two phenyl rings. 8,9 This was achieved by increasing the steric hindrance through the introduction -at the ortho, ortho' position of the intercyclic bond -of alkyl groups R1, R2 (at meta position of the phenolate functionality for 1a-e) or R3, R4 (at meta position of the pyridinium ring for 2a-e) with different sizes.

It should be highlighted here that the N, O positions in phenol betaines can influence the contribution or lack of mesomeric form in the molecule. In the present case, the two structures have to be considered (quinones [Q1], [Q2] and zwitterions [Z1], [Z2]) (scheme 2).

Moreover, in the full acceptation of the word zwitterionic, the charges should be completely localized which is not true for 1a-e or 2a-e. Anyhow, the compounds 1a-e and 2a-e will be termed further zwitterions as usually in the literature, keeping in mind that [Q1], [Q2] and [Z1], [Z2] are limiting structures.

The present paper aims at delimiting the influence of the progressive twisting on both the [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C chemical shifts of 1a-e and 2a-e, the deuteration of 1a-e and the polarity of the solvent on the ground state structure, using [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C NMR data obtained in two representative deuterated solvents: i) polar without specific interaction D6-DMSO ii) polar and highly hydrogen bonding CD3OD.

N O CH 3 R 3 R 4 3' 2' 1' 6' 5'
[Q 2 ] Quinone O N N O [Z 2 ] Zwitterion CH 3 CH 3 [Q 2 ] Quinone O N R 3 R 4 N O R 3 R 4 [Z 2 ] Zwitterion CH 3 CH 3 R 1 R 2 R 2 R 1
Scheme 2: Limiting forms of compounds studied.

Experimental section

13

C NMR (100.6 MHz) spectra were measured with a Bruker Avance series 400 spectrometer. Chemical shifts are reported in ppm relative to SiMe4. The synthesis of all compounds were already published. 8,9 Procedure for deuteration studies on 1a-d: 2 mL of a solution of 1a-d (0.9 x 10 -2 M) and KOH (10 -1 M) was prepared. [START_REF] Reichardt | Solvents and Solvent effects in Organic Chemistry[END_REF] H NMR spectrum of this solution is recorded after 45 s and then every 10 min over 5 h.

Results and discussion

Investigation of the chemical/physical properties of zwitterions is generally impeded by three difficulties which can only be partially bypassed. First of all, the acid-base equilibrium existing between the zwitterion and the corresponding phenol, obtained by a rapid protonation in nonanhydrous protic solvents, is mostly suppressed by addition of appropriate base in orded to maintain exclusively the zwitterionic form. 9 Second, a possible aggregation effects due to the inherent strong dipole moment can be attenuated by using low concentrations satisfying the Beer-Lambert law. Finally, the deceasing solubility of zwitterions in low polar solvents is the main drawback when looking at the solvent effects on their photophysical properties and/or in view of technical applications. Unfortunately, reliable [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C NMR spectra were obtained only in CD3OD and D6-DMSO with the experimental conditions satisfying as possible the above mentioned problems.

1-13 C NMR data of 2a-e

The [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C NMR data of 2a-e recorded in CD3OD and in D6-DMSO are reported in table 1. The increasing torsion in the series 2a-e changes significantly the chemical shifts of both parts of the molecules. 7 Concerning the phenoxide ring, the most affected atom is the C(4') linked to the oxygen atom. A shielding of 10 ppm is observed which is typical of an increase of negative charge on the oxygen atom. 10.11 Then, the torsion seems to favour the zwitterionic form (see further).

Even more large shift is observed for the atoms C(3), C(5) localized on the pyridinium ring: 32 ppm. However, it is not possible to rely directly these changes to the increasing torsion. Indeed, the synthetic strategy used presently to alter the geometry of the molecules, the introduction of alkyl substituents R3, R4 would concomitantly likely introduce electronic effects in the corresponding ring. For example, it is well known from studies on photoinduced electron transfer that inserting alkyl groups into an aryl ring is an elegant way for varying oxidation/reduction potentials. 12 Therefore, it is necessary to disentangle the contributions due to the R3, R4 substituents from those due to the geometry. At this end, the electronic effects of the R3 , R4 substituents on the pyridinium part are evaluated from increment constants obtained experimentally from the pyridinium iodide 3a-e both in CD3OD and in D6-DMSO (table 2). The contribution of alkyl groups on the only pyridine rings are actually reported in literature, [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] but to our best knowledge no data are available for groups anchored on pyridinium salts.

It can be seen that the introduction of alkyl groups more and more voluminous at position 3 (or 3 and 5), leading to more and more important interplanar angle, results in a strong increase of the C(3) chemical shift (11.7 ppm, 17.3 ppm, 21.5 ppm for R = Me, Et, i-Pr) or of the C(3) and C(5) chemical shift (10.8 ppm for R5 = R6 = Me). The other atoms are not significantly affected (table 2). It should be noted that addition of the effects of substituents at C3 and C5 from the spectrum of 3b leads to increment values very close to those deduced from the spectrum of 3c. Therefore, calculating increments corresponding to the effects of two ethyls or two iso-propyls can be classically done, without large approximation, by simple addition of the values obtained from the spectra of 3d and 3e. Now, it is possible to get the chemical shifts of the pyridinium by subtracting the corresponding increment constants from the experimental data. However, it seems reasonable to assume that the electronic contribution of R3, R4 substituents does not affect the chemical shifts of carbon of phenoxide ring. The results gathered in table 2 indicate very slight differences between the increment constants in CD3OD and those calculated in D6-DMSO. Consequently, we use the value obtained in CD3OD to calculate the corrected chemical shifts of 2e in D6-DMSO, knowing that the [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C NMR spectrum of 3-isopropyl pyridinium iodide was only recorded in CD3OD. It is now instructive to report the data corrected from the electronic effects due to the R3, R4 substituents, data that can be directly attributed to the different geometries (table 3). Quite evidently, we need to discuss only the new data concerning the pyridinium ring. Two points are worth consideration, underlining the usefulness of the chemical shift correction. It can be seen from table 3 that C(3), C(5) are still significantly affected by the torsion, but at a less extent than before correction for R3, R4 substituent effects (table 1): Δδ2a-e= -10.5 ppm in CD3OD (-8.7 ppm in D6-DMSO) instead of -31.7 ppm (-29.9 ppm in D6-DMSO). On the opposite, Δδ2a-e for C (4) increases in absolute value from -7.1 ppm in CD3OD (-7.6 ppm in D6-DMSO) to -10.9 ppm (-11.4 ppm in D6-DMSO) and moreover the C(4) atom presents now the largest variation.

Table 1: Chemical shifts of 2a-e (ppm) in CD3OD and in D6-DMSO.

Influence of the torsion on the chemical structure of 2a-e

It is still an exciting challenge to evaluate the percentage of the quinone [Q] and the zwitterrionic [Z] forms in the real structure of such molecules. In order to get further insight in the question, we have studied the [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C NMR spectra of the compounds 4, 5 (table 4).

These two compounds are well representative of the two limit structures [Q2], [Z2] of the phenoxide part of the series 2a-e. Note that the investigation of the [Q]  [Z] mesomery is easier when looking at the phenoxide moieties than at the pyridinium: the negative charge is strongly localized on the oxygen atom whereas the positive one is delocalized on the pyridinium ring. From table 3 and illustrated on figure 1, it is clear that the δ13C(4') of the five compounds studied 2a-e are flanked by the C(4') limit values of 4 [Q] and 5 [Z] (table 4). Moreover, C(3') (respectively C(5')) have values flanked by the corresponding limiting values of model compounds 4 and 5. However, it is not well satisfied by atoms C(2') (respectively C(6')) and even more less by C(1'), likely because these atoms are nearer to the pyridinium ring for which we have not the corresponding model compounds. Indeed, it can be inferred a valuable advice from the most sensitive atom C(4') on the phenoxide moiety. It can be concluded that compound 2a has a quinone like structure whereas 2e presents a zwitterionic structure. Another set of data confirms this result: the chemical shift of proton C(7) switches from 3.88 ppm for 2a to 4.32 ppm for 2e in CD3OD and from 3.77 ppm to 4.15 ppm in D6-DMSO (table 5) as a result of a better localisation of the positive charge on nitrogen (zwitterion form). Such a behaviour has been already described for methylene protons adjacent to pyridinium nitrogen. [START_REF] Peixoto | [END_REF]15 Thus, the torsion impacts severely the structure of the compounds by increasing the charge separation between the two rings. The important conclusion is that the torsion is a key parameter for modulating the physical properties of molecules which is well in line with the results obtained in the study of the hyperpolarisabilities of compounds 2a-d.

Influence of the solvents

As soon mentioned, the poor solubility in low polarity solvents of the compounds 2a-e excludes a detailed investigation of the solvent effect on [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C NMR. However, it is tempting to comment the data obtained in D6-DMSO, a solvent with a polarity comparable to CD3OD, but free from specific interactions with the solute (hydrogen bonding). Here again, the carbon C(4') furnishes the most useful information. The increasing torsion induces the same evolution from the quinone structure towards the zwitterionic structure, as in the case of deuterated methanol as solvent. However, the change is significantly lower for D6-DMSO (Δδ2a-e C(4') = 6.1 ppm) than for CD3OD (9.9 ppm). This is not surprising since methanol is well-known to interact strongly with the oxygen atom bearing a negative charge through an hydrogen bond, favouring the [Z] form. Interestingly, we can note on figure 2 that the solvent effect D6-DMSO  CD3OD shows a reversal with increasing steric hindrance. Compared to D6-DMSO, the hydroxylic solvent promotes the [Q] form of 2a and on the contrary [Z] form of 2e. This confirms again that the screening effect of the two tert-butyl groups vanishes as the interplanar angle increases, that is as the negative charge on the oxygen atom increases.

Despites the unfortunate lack of data in other solvents (recording the NMR spectra in CDCl3 would require an unrealistic time for accumulation), this nearly two fold increase of Δδ2a-e for atom C(4') when changing D6-DMSO to CD3OD is indicative of the important role of solvatation. This aspect should be also considered when choosing an appropriate medium for application development in non linear optics.

Another point of interest is the comparison of the data for the non hindered compound 2a with the related molecule M.O.E.D. (table 6). In both solvents, the [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C shifts are nearly the same which underlines the similarity of these two compounds, except for C(3') as expected due to the tert-butyl substitution in 2a. Moreover, all the 13 C shifts increase when changing D6-DMSO to CD3OD, except for C(2'), C(4') of 2a. Indeed, it is instructive to focus once more on the evolution of atom C(4'). The observed opposite variation could be explained as follows. It is well-known that polarity and hydrogen bonding favour the zwitterionic form of pyridinium phenoxides. [START_REF] Reichardt | Solvents and Solvent effects in Organic Chemistry[END_REF] Considering M.O.E.D., the large decrease (3.3 ppm) is due to the strong hydrogen bonding with "free" negative charged oxygen atom, thus favouring the zwitterionic form. On the contrary, compound 2a presents a slight increase (1.1 ppm), indicating that the quinone form is now favoured in CD3OD compared to D6-DMSO. This is likely due to the fact that its oxygen atom is to some extend screened by the two tert-butyl groups from hydrogen bonding. As a result, DMSO with larger dielectric constant and dipole moment than CH3OH is more polar than methanol towards 2a.

4-13 C NMR data of 1a-e

Unlike compounds 2a-e, [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C NMR spectra of 1a-e were only recorded in CD3OD. The data gathered in table 7 highlight a significant electronic effect of alkyl groups R1 and R2 anchored at meta position of the phenolate functionality on the chemical shifts of carbon corresponding to the phenoxide ring. As expected, C(2') and C(6') are the most affected. Conversely, these bulky groups should have no significant influence on the chemical shifts of carbon of pyridinium ring, given the distance. The progressive and significant deshielding observed for C(3), C(5) and C(4) may only be due to the torsion induced by alkyl groups of increasing size.

In the presence of potassium hydroxide, the [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C NMR experiments recorded for 1a-e exhibit a progressive deuteration of C( 2) and C (6). In order to quantitatively study the kinetics of deuteration, the decreasing of the signal of the protons linked on C(2) and C( 6) is monitored by [START_REF] Reichardt | Solvents and Solvent effects in Organic Chemistry[END_REF] H NMR. Whereas the speed of deuteration is first order, half life times, as well as constant rates, have been determined for compounds 1a-d. Acceleration of deuteration is clearly observed as the interplanar angle increases. The comparison of the deuteration of 1a with this of 1d (k = 10 -5 s -1 and k = 30.2 10 -5 s -1 ) indicates the acceleration of the labelling by a factor of thirty.

Table 7: [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C NMR shifts (ppm) in CD3OD of 1a-e.

It has been seen above that increase of interplanar angle promotes the zwitterionic form. Its structure is related to those of para-substituted pyridinium salts which deuteration of some of them has been described. [16][17][18][19] In each case, the parasubstituted salts was heated in D2O in the presence or not of NEt3 and led to H-D exchange at C(2) and C(6). This ortho selectivity is in accord with the mechanism of electrophilic substitution. The easy labelling of our compounds can be explained as follows: Potassium hydroxide added into the NMR tube in order to avoid rapid protonation of zwitterions in CD3OD, gives high concentration of OD - and allows a rapid H-D exchange at room temperature. On the contrary, the slightly twisted pyridinium phenolates, in a quinone limiting form are less reactive than the former at C(2) and C (6). As a consequence, the deuteration is slower.

Conclusion

The [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C NMR study enlightens on the key role of the torsion in tuning the structure of pyridinium phenoxides. The more important the twist of pyridinium phenolate is, the more the limiting zwitterionic form is approached. Hopefully, it can be inferred that, at 90° torsion, the two rings are completely decoupled with the consequence that charge separation is optimum.

Varying the torsion in this type of molecules is therefore a powerful way (more promising than, for instance, changing the acceptor/donor combination in push-pull systems) to get efficient compounds in view of nonlinear optic applications.

A

  13C NMR Study of pyridinium phenoxide series with increasing sterical hindrance reveals the dramatic influence of torsion on their structure Hélène Chaumeil, Patrice Jacques, Vincent Diemer, Didier Le Nouen, Christiane Carré To cite this version: Hélène Chaumeil, Patrice Jacques, Vincent Diemer, Didier Le Nouen, Christiane Carré. A 13C NMR Study of pyridinium phenoxide series with increasing sterical hindrance reveals the dramatic influence of torsion on their structure. Journal of Molecular Structure, 2011, 1002 (1-3), pp.70-75. 10.1016/j.molstruc.2011.06.049. hal-01151415

2 Scheme 1 :

 21 Scheme 1: Compounds studied.

a

  : R 5 = R 6 = H b : R 5 = H, R 6 = Me c : R 5 = R 6 = Me d : R 5 = H, R 6 = Et e : R 5 = H, R 6 = i-Pr 3a-e R 6

Figure 1 :

 1 Figure 1: 13 C shifts in CD3OD of the phenoxide part. [Q], [Z] are limiting values of the model compounds 4 and 5 (see text). The numbers of the corresponding atoms are put between brackets.

Figure 2 :

 2 Figure 2: Evolution of the 13 C NMR shifts as a function of the electron density on the oxygen atom (4') for the series 2a-e compared to this on the atom (4') for the compounds 4 [Q] and 5 [Z] recorded in CD3OD.

Table 2 :

 2 Determination of the increments in CD3OD and D6-DMSO by introducing alkyl groups at position 3 of the pyridinium iodide.

	Zwitterion C(2)	C(3)	C(4)	C(5)	C(6)	C(7)	C(1')	C(2'), C(6')	C(3'), C(5')	C(4')
		In CD3OD ([KOH] = 10 -1 M and [2a-e] = 10 -2 M)
	2a	142.3	117.5	154.6	117.5 142.3	36.2	111.7	126.0	142.2	180.8
	2b	144.7	132.3	159.1	124.0 140.6	36.2	114.9	128.1	141.2	177.1
	2c	142.9	136.9	162.8	136.9 142.9	36.2	114.3	126.7	140.2	172.7
	2d	142.5	144.0	162.9	144.0 142.5	36.1	114.7	125.4	140.5	171.5
	2e	141.3	149.2	161.7	149.2 141.3	36.1	114.7	125.3	140.4	170.9
		In D6-DMSO ([tetramethylguanidine] = 10 -1 M and [2a-e] = 10 -2 M)
	2a	140.6	114.6	150.1	114.6 140.6	43.6	107.1	124.1	139.6	179.7
	2b	142.4	127.0	153.5	119.6 138.3	44.0	109.0	126.4	138.5	177.4
	2c	140.8	131.3	158.0	131.3 140.8	45.0	106.0	126.8	136.0	175.0
	2d	140.5	138.6	158.6	138.6 140.5	45.6	106.1	125.6	136.2	174.2
	2e	139.7	144.5	157.7	144.5 139.7	46.1	105.7	125.4	135.8	173.6
		3a	3b			3c		3d			3e
		13 C	13 C	incre-	13 C	incre-	13 C	incre-		13 C	incre-
		NMR	NMR	ments	NMR	ments	NMR	ments	NMR	ments
		In CD3OD
	C(2)	146.9	146.5	-0.4	143.7	-3.2	145.9	-1		145.3	-1.6
	C(3)	129.2	140.9	11.7	140.0	10.8	146.5	17.3		150.7	21.5
	C(4)	146.6	146.9	0.3	147.5	0.9	145.9	-0.7		144.7	-1.9
	C(5)	129.2	128.5	-0.7	140.0	10.8	128.7	-0.5		128.9	-0.3
	C(6)	146.9	143.9	-3	143.7	-3.2	144.1	-2.8		145.3	-1.6
	C(7)	49.0	49.1	0.1	48.7	-0.3	49.2	0.2		49.2	0.2
		In D6-DMSO
	C(2)	145.4	144.9	-0.5	142.2	-3.2	143.8	-1.6
	C(3)	127.6	138.1	10.5	137.3	9.7	144.4	16.8
	C(4)	145.0	145.2	0.1	145.6	0.6	144.3	-0.7
	C(5)	127.6	126.9	-0.7	137.3	9.7	127.1	-0.5
	C(6)	145.4	142.7	-2.7	142.2	-3.2	142.8	-2.8
	C(7)	48.1	47.6	-0.5	47.4	-0.7	47.7	-0.4

Table 3 :

 3 [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C NMR shifts of 2a-e (ppm), in CD3OD and D6-DMSO adjusted by means of increments reported in Table2.

Table 4 :

 4 [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C NMR shifts of compounds 3 and 4 in CD3OD.

Table 5 :

 5 [START_REF] Reichardt | Solvents and Solvent effects in Organic Chemistry[END_REF] H NMR shifts (ppm) of proton on C(7) of 2a-e.

  CDCl3 has been added in order to enhance the solubility of the diquinone.

					O
		* number correspond to those used for 2a-e
	Compound			C(1)	C(2)	C(3)	C(4)	C(1')	C(2')	C(3')	C(4')
	4, [Q]	CD3OD/CDCl3 70/30	±	187.5	151.3	127.2	137.2	137.2	127.2	151.3	187.5
	5, [Z]	CD3OD, KOH (10 -2 M)	126.3	129.5	127.2	146	130.0	124.1	139.8	160.5
						CD3OD D6-DMSO
				2a		3.88	3.77
				2b		4.06	3.87
				2c		4.19	3.99
				2d		4.27	/
				2e		4.32	4.15

±

Table 6 :

 6 [START_REF] Pretsch | Spectral Data for Structure Determination of Organic Compounds[END_REF] C NMR shifts (ppm) of M.O.E.D., the Brooker's merocyanine (from10 ) and 2a (this work).

Acknowledgments

The authors are indebted to the Région Alsace and the Programme Interreg III A Rhenaphotonics for their financial supports. We would like to gratefully acknowledge Dr. Albert Defoin for his valuable help in guiding this research work and for numerous fruitful comments.