Odile Tresse
email: odile.tresse@oniris-nantes.fr

Ramila C Rodrigues

Nabila Haddad

Didier Chevret

Jean-Michel Cappelier

Comparison of Proteomics Profiles of Campylobacter jejuni Strain Bf under Microaerobic and Aerobic Conditions

Keywords: foodborne pathogen, Campylobacter jejuni, proteomics, aerotolerance, oxidative stress AAC, aerobically acclimated cells, AC, aerobic conditions, BB, bromophenol blue, BSA, bovine serum albumin, GBS, Guillian-Barré Syndrome, MAC, microaerobic conditions, MFS, Miller Fisher Syndrome, PCA, Principal Component Analysis, PQ, paraquat, ROS, reactive oxygen species, TCA, tricarboxylic acid

Campylobacter jejuni accounts for one of the leading causes of foodborne bacterial enteritis in humans. Despite being considered an obligate microaerobic microorganism, C. jejuni is regularly exposed to oxidative stress. However, its adaptive strategies to survive the atmospheric oxygen level during transmission to humans remain unclear. Recently, the clinical C. jejuni strain Bf was singled out for its unexpected ability to grow under ambient atmosphere. Here, we aimed to understand better the biological mechanisms underlying its atypical aerotolerance trait using two-dimensional protein electrophoresis, gene expression, and enzymatic activities. Forty-seven proteins were identified with a significantly different abundance between cultivation under microaerobic and aerobic conditions. The over-expressed proteins in aerobiosis belonged mainly to the oxidative stress response, enzymes of the tricarboxylic acid cycle, iron uptake, and regulation, and amino acid uptake when compared to microaerobic conditions. The higher abundance of proteins related to oxidative stress was correlated to dramatically higher transcript levels of the corresponding encoding genes in aerobic conditions compared to microaerobic conditions. In addition, a higher catalase-equivalent activity in strain Bf was observed. Despite the restricted catabolic capacities of C. jejuni, this study reveals that strain Bf is equipped to withstand oxidative stress. This ability could contribute to emergence and persistence of particular strains of C. jejuni throughout food processing or macrophage attack during human infection.

INTRODUCTION

Campylobacter, a Gram-negative spiral-shaped bacterium, is currently recognized as the main cause of foodborne gastroenteritis in humans in developed countries [START_REF] Epps | Foodborne Campylobacter: infections, metabolism, pathogenesis and reservoirs[END_REF][START_REF] Gölz | Relevance of Campylobacter to public health-The need for a one health approach[END_REF]. In 2014, this microorganism was responsible for more than 236,000 confirmed campylobacteriosis cases in Europe, with an incidence of 71 per 100,000 people (EFSA and ECDC, 2016). This incidence increased by 9.6% compared with 2013. Of these reported cases, 81.8% were related to C. jejuni, 7.1% to C. coli, 0.13% to C. lari, 0.09% to C. fetus and 0.07% to C. upsaliensis (EFSA and ECDC, 2016). C. jejuni infection induces acute gastroenteritis and is marked by fever, diarrhea, and intense abdominal pains. The infectious dose was estimated to be as low as 500-800 bacteria [START_REF] Robinson | Infective dose of Campylobacter jejuni in milk[END_REF][START_REF] Young | Campylobacter jejuni: molecular biology and pathogenesis[END_REF]. In addition, some patients can develop post-infection complications including Guillian Barré Syndrome (GBS) and its variant form Miller Fisher Syndrome (MFS), a chronic and potentially fatal form of paralysis [START_REF] Nachamkin | Chronic effects of Campylobacter infection[END_REF][START_REF] Kuwabara | Does Campylobacter jejuni infection elicit axonal or demyelinating Guillain-Barre syndrome, or both?[END_REF][START_REF] Huizinga | Innate Immunity to Campylobacter jejuni in Guillain-Barre Syndrome[END_REF].

Overall, as an obligate microaerobic microorganism, C. jejuni requires 5-10% oxygen for its optimal growth and cannot grow under atmospheric oxygen levels [START_REF] Kaakoush | Potential role of thiol:disulfide oxidoreductases in the pathogenesis of Helicobacter pylori[END_REF][START_REF] Macé | Influence of measurement and control of microaerobic gaseous atmospheres in methods for Campylobacter growth studies[END_REF]. However, it is regularly exposed to oxidative stress during its life cycle. During respiration, the reduction of dioxygen to H 2 O is accompanied by the production of a variety of toxic intermediate products called reactive oxygen species (ROS) including the superoxide anion radical (O •- 2 ), hydrogen peroxide (H 2 O 2 ) and the hydroxyl radical ( • OH), which can cause irreversible damage to nucleic acids, proteins, and lipids [START_REF] Cabiscol | Oxidative stress in bacteria and protein damage by reactive oxygen species[END_REF]. To detoxify ROS, C. jejuni produces the main oxidative stress defense enzymes: superoxide dismutase (SodB), catalase (KatA), and alkyl hydroperoxide reductase (AhpC) [START_REF] Pesci | Genetic, enzymatic, and pathogenic studies of the iron superoxide dismutase of Campylobacter jejuni[END_REF][START_REF] Grant | Molecular characterization of KatA from Campylobacter jejuni and generation of a catalase-deficient mutant of Campylobacter coli by interspecific alleic exchange[END_REF][START_REF] Baillon | An iron-regulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter jejuni[END_REF][START_REF] Atack | Oxidative stress in Campylobacter jejuni: responses, resistance and regulation[END_REF]. In comparison with other enteric bacteria, C. jejuni appears to have very limited capacity to regulate gene expression in response to environmental stresses, especially oxidative stress [START_REF] Park | The physiology of Campylobacter species and its relevance to their role as foodborne pathogens[END_REF]. The key regulators of the stress defense systems described in other enteric bacteria are absent in this pathogen. For instance, SoxRS, OxyR, and the sigma factor RpoS, for general stress-defense gene regulation under hostile environmental conditions, are not present in the C. jejuni genome [START_REF] Parkhill | The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences[END_REF]. Despite the lack of common bacterial stress responses and the fragility of this organism faced with atmospheric oxygen concentrations, it is surprising that C. jejuni is the major foodborne bacterial causative agent of gastrointestinal disorders in humans. Its adaptive strategies to survive the atmospheric oxygen level during transmission to humans remain unclear.

The investigation of C. jejuni ability to grow in vitro under atmospheres with high oxygen tension has not been conducted in depth, although its ability to tolerate and adapt to oxygen has often been reported [START_REF] Jones | Campylobacter jejuni adapts to aerobic metabolism in the environment[END_REF][START_REF] Fields | Campylobacter jejuni CsrA mediates oxidative stress responses, biofilm formation, and host cell invasion[END_REF][START_REF] Sulaeman | Enhanced Adhesion of Campylobacter jejuni to abiotic surfaces is mediated by membrane proteins in oxygen-enriched conditions[END_REF][START_REF] Kassem | The impairment of methylmenaquinol:fumarate reductase affects hydrogen peroxide susceptibility and accumulation in Campylobacter jejuni[END_REF][START_REF] Rodrigues | Description of Campylobacter jejuni Bf, an atypical aero-tolerant strain[END_REF][START_REF] Turonova | Biofilm spatial organization by the emerging pathogen Campylobacter jejuni: comparison between NCTC 11168 and 81-176 strains under microaerobic and oxygen-enriched conditions[END_REF]. In our laboratory, we have isolated an atypical clinical C. jejuni strain (Bf) characterized by its ability to grow under aerobic conditions [START_REF] Rodrigues | Description of Campylobacter jejuni Bf, an atypical aero-tolerant strain[END_REF]. This was correlated to a lower susceptibility to compounds generating superoxide and peroxide stresses [START_REF] Rodrigues | Description of Campylobacter jejuni Bf, an atypical aero-tolerant strain[END_REF]. The first analyses of the genome of Bf did not reveal any deletion/insertion, SNPs, or organization of the genes already described to be involved in the sub-system of oxygen detoxification in other C. jejuni strains [START_REF] Bronnec | Draft genome sequence of Campylobacter jejuni Bf, an atypical strain able to grow under Aerobiosis[END_REF].

In this study, the adaptation of Bf to aerobic conditions was investigated by comparing two-dimensional (2-D) profiles of multiplying cells in microaerobic conditions and aerobic conditions. The gene expression of differentially regulated proteins involved in the sub-system of oxygen detoxification and the H 2 O 2 breakdown enzymatic activity were then compared between cells grown in microaerobic conditions, cells grown in aerobic conditions, or cells acclimated to aerobic conditions to identify the underlying molecular mechanisms contributing to the atypicity of this strain.

MATERIALS AND METHODS

Bacterial Strains and Growth Conditions

C. jejuni Bf and C. jejuni NCTC 11168 were stored at -80 • C in brain and heart infusion medium (BHI) with sterile glycerol at 20%. Preparation of C. jejuni cells was performed as previously described in [START_REF] Rodrigues | Description of Campylobacter jejuni Bf, an atypical aero-tolerant strain[END_REF] with the following modifications. Before each experiment, the strains were grown on Karmali agar plates (Oxoid, France) at 42 • C for 48 h (h) in microaerobic conditions (MAC) generated using gas replacement jars operated by a MACSmics gassing system (BioMérieux, France) with a gas blend composed of 5% O 2 , 10% CO 2 and 85% N 2 and four filled/flushed cycles at -50 kPa.

To obtain cells of C. jejuni Bf cultured in aerobic conditions (AC) (ambient atmosphere), cells were first subcultured in microaerobic conditions at 42 • C on Karmali agar for 48 h and then harvested and resuspended in peptone saline solution. An absorbance at 600 nm (OD 600nm ) of 0.03 (≈ 10 6 CFU.mL -1 ) was used to standardize cell suspension. The bacterial suspension was then inoculated onto Karmali agar plates and incubated under aerobic conditions for 24 h at 42 • C. The aerobically acclimated cells (AAC) were prepared as described before by [START_REF] Rodrigues | Description of Campylobacter jejuni Bf, an atypical aero-tolerant strain[END_REF]. Briefly, C. jejuni Bf was first subcultured under microaerobic conditions onto Karmali plates for 48 h. Colonies were then harvested and subcultured three times successively on Karmali agar plates followed by incubation at 42 • C for 24 h under aerobic conditions.

Protein Extraction and Two-Dimensional Gel Electrophoresis (2-DE)

Proteins of harvested cells of C. jejuni Bf were extracted according to [START_REF] Bièche | Dynamic proteome changes in Campylobacter jejuni 81-176 after high pressure shock and subsequent recovery[END_REF]. Briefly, after resuspension in 200 mM glycine solution, followed by 100 mM Tris-HCl pH 7.0 and 10 mM Tris-HCl pH 7.0 solutions, cells were lysed by ultrasonic treatment at 25 kHz. Ultracentrifugation at 188,000 × g was conducted to separate cytosoluble proteins from membrane fractions. Then, purified proteins were quantified using the Micro BCA TM Protein Assay Kit (Perbio-Science, France) and stored at -80 • C.

Concentration of 30 µg of proteins to reach a final volume of 15 ± 5 µL was performed using the Concentrator 5301 (Eppendorf, Le Pecq, France) at room temperature. Then, each sample was resuspended in 185 µL of rehydration buffer as described previously [START_REF] Haddad | Long-term survival of Campylobacter jejuni at low temperatures is dependent on polynucleotide phosphorylase activity[END_REF][START_REF] Bièche | Dynamic proteome changes in Campylobacter jejuni 81-176 after high pressure shock and subsequent recovery[END_REF] and rehydrated using a Protean IEF cell system at 20 • C (Bio-Rad, France) overnight in the system with a 50 V current and 11 cm pI 4-7 IPG strip (Bio-Rad, France) or pI 6-11 IPG strip (GE Healthcare Life Sciences, France). Isoelectrofocusing (IEF) was performed as follows: from 0 V to 250 V for 3 h, from 250 V to 4500 V for 3 h and at 4500 V until 42,000 Vh was reached and at 500 V for 20 h maximum. Finally, strips were equilibrated for 2 × 20 min in 1.5 mL of buffer 1 and 2, respectively (buffer 1: 6 M urea, 2% SDS, 0.05 M Tris-HCl (pH 8.8), 30% glycerol, and a 0.02% bromophenol blue, supplemented with 2% DTT; buffer 2: 6 M urea, 2% SDS, 0.05 M Tris-HCl (pH 8.8), 30% glycerol, and 0.02% bromophenol blue, supplemented with 4% iodoacetamide (Bio-Rad, France).

The second dimension was performed using a precast polyacrylamide gel gradient (4-15%) (Bio-Rad, France) covered with 1% low-melting point agarose (Bio-Rad, USA) and run at 20 mA/gel at 14 • C using the Criterion TM Dodeca TM Cell (Bio-Rad, France) until the migration of bromophenol blue reached the bottom of the gels. Proteins in gels were silver-stained according to [START_REF] Garnier | Adaptation to cold and proteomic responses of the psychrotrophic biopreservative Lactococcus piscium strain CNCM I-4031[END_REF] to reveal the cytoplasmic proteome map and scanned with a GS-800 densitometer (Bio-Rad, France) operated with the QuantityOne R software (Bio-Rad, France) at a resolution of 42.3 µm.

Gel Image and Statistical Treatment

Comparisons between gels (detection, quantification, alignment, spot matching, and statistical analyses) were carried out using Progenesis Samespots R 4.0 software (NonLinear Dynamics, Newcastle upon Tyne, UK). Statistical analysis of protein abundance was carried out on six gels for each condition, obtained from three independent cultures and two technical replicates for each independent culture. Principal Component Analysis (PCA) was performed to highlight differences between the conditions tested. Normalization of the spots was based on the abundance ratio, which was selected in the algorithm developed by Progenesis SameSpots R . Differences between matched spot intensities were statistically confirmed by calculating an ANOVA (at a 5% significance level). P-values were refined by the q-value (at 5% significance level) to discard any false positives as described before [START_REF] Bièche | Dynamic proteome changes in Campylobacter jejuni 81-176 after high pressure shock and subsequent recovery[END_REF]. The q-value calculates the false discovery rate (FDR) for any spot selected with a p-value < 0.05 (Table S1). The power threshold used was 80% (P > 0.8) which is considered to be relevant for 3 biological replicates and 3 technical replicates for each condition and only fold of at least 1.5 was defined as significant.

Protein Identification by LC-MS/MS

When spots of interest were located, 2-DE gels were run again using 700 µg of protein, and stained with BioSafe colloidal Coomassie blue (Bio-Rad, France). Spots of interest were excised for further LC-MS/MS analysis using PAPPSO platform facilities (http://pappso.inra.fr). In-gel digestion was performed according to a standard trypsinolysis protocol. Gel plugs were first washed twice with 25 mM ammonium bicarbonate-50% acetonitrile.

Proteins were reduced and alkylated prior to digestion. 100 µl of 10 mM DTT was added and the sample was incubated for 1 h at 55 • C. The samples were allowed to cool and the supernatant was removed before 100 µL of 55 mM iodoacetamide was added. Gel pieces were washed further with 25 mM NH 4 CO 3 and dehydrated in acetonitrile. Digestion was conducted for 6 h at 37 • C with 100 ng of modified trypsin dissolved in 10 µL of 25 mM NH 4 CO 3 . For further LC-MS/MS analysis, peptides of the digestion mix were extracted from the gel with 50%

(v/v) acetonitrile, 0.5% trifluoroacetic acid in water, and then with pure acetonitrile. Both peptide extracts were pooled, dried in a vacuum speed concentrator, and suspended in 25 µL of 2% (v/v) acetonitrile, 0.08% (v/v) trifluoroacetic acid in water. LC-MS/MS analysis was achieved on an Ultimate 3000 LC system (Dionex, Voisins-le-Bretonneux, France) linked to an LTQ-Orbitrap Discovery mass spectrometer (Thermo, USA) via a nanoelectrospray ion source. Samples (4 µL) were loaded at 20 µL/min onto a pre-column cartridge (stationary phase: C18 PepMap 100, 5 µm; column: 100 µm inner diameter, 1 cm in length; Thermo, France) and desalted with 2% acetonitrile -0.1% formic acid (buffer A). After 4 min, the pre-column was connected to the separating nano-column Pepmap C18 (0.075 × 15 cm, 100 Å, 3 µm; Thermo, France) and a linear gradient was started from 2 to 36% of buffer B (80% acetonitrile, 0.1% formic acid) at 450 nl/min over 12 min. The doubly and triply charged precursor ions were subjected to MS/MS fragmentation with a 1 min exclusion window, and with classic peptide fragmentation parameters (Qz = 0.22, activation time = 50 ms, collision energy = 35%). Proteins were identified by querying MS/MS data against the Campylobacter jejuni subsp. jejuni strain NCTC 11168 protein database (Uniprot, 2015.03.31) along with an inhouse contaminant database, using the X!Tandem software (X! tandem CYCLONE (2011.12.01.1), http://www.thegpm.org) with the following parameters: one missed trypsin cleavage, alkylation of cysteine and conditional oxidation of methionine precursor and fragment ion set to 10 ppm and 0.5 Da, respectively. A refined search was added with similar parameters, except that the semi-tryptic peptides, and the possibly N-terminal acetylated proteins were included. All the peptides that matched an E-value lower than 0.05 were parsed with an in-house program (http://http:/PAPPSO.inra.fr/bioinformatique.html).

Proteins identified by at least two unique peptides and a log (E-value) lower than -2.6 were considered to be validated.

The KEGG pathway database of C. jejuni NCTC 11168 (http://www.genome.jp/kegg/) was used to infer the function and cell location of the identified proteins (Table S1).

RNA Extraction and Quantitative RT-PCR

Cell cultures of C. jejuni NCTC 11168 and C. jejuni Bf strains were grown in microaerobic conditions and C. jejuni Bf was cultured in aerobic conditions and aerobically acclimated as described above. Identical number of cell subculturing for all conditions was performed. Bacterial suspension were immerged in RNA Protect Reagent (Qiagen, Hilden) and centrifuged at 3300 × g for 6 min at 4 • C, then resuspended in 1 mL of Extract-All (Eurobio, France) and 0.2 mL of chloroform. After treating samples with DNase, the quality, and quantity of RNA were checked using a spectrometer NanoDrop (NanoDrop R 2000, Thermo Scientific) and then adjusted to 20 ng/µl. Samples of RNA were submitted to reverse transcription using RevertAid H Minus First-Strand cDNA synthesis kit (Euromedex) and random hexamer primers (Eurobio) according to the manufacturer's instructions and [START_REF] Bièche | Dynamic proteome changes in Campylobacter jejuni 81-176 after high pressure shock and subsequent recovery[END_REF] with the following modifications: DNA removal was checked by PCR using 341F/758R primers and the gene expression of katA, sodB, ahpC, tpx, trxB, and rrs was assessed using specific primers

→3 ′ 341 F CCTACGGGAGGCACGAG 758 R CTACCAGGGTATCTAATCC ahpC F TTCGTACGGCTGGAGATAAG ahpC R GTGAAAGAACAGGCGAAGAG katA F CAAACAGCTATGATAATAGCC katA R GGAGCATATCTTTGTGCTACG sodB F CTTCAAACGCAGCTACACCA sodB R CCCAGTTAATATGAGCATAGAA tpx F GCCAGTTACAATGGTGCTGA tpx R TTTGCCACAAAATCACTTGC trxB F AGCCCAAGTTATGGATGGAA trxB R TTTGGAGCTGAGCCTGT rrs F AAGGGCCATGATGACTTGACG rrs R AGCGCAACCCACGTATTTAG
(Table 1). The rrs gene was used as the endogenous control as described previously [START_REF] Li | Rapid kinetic microassay for catalase activity[END_REF][START_REF] Hyytiäinen | Importance of RNA stabilization: evaluation of ansB, ggt, and rpoA transcripts in microaerophilic Campylobacter jejuni 81-176[END_REF]. The composition of the PCR mix was as follows: 5.0 µL of sample, reverse primer (1 µM), forward primer (1 µM), and 12.5 µL of SYBR Green I Master Mix. The amplification program included an initial denaturing step at 95 • C (10 min), followed by 40 cycles of 95 • C (15 s) and 60 • C (1 min). A negative control (without cDNA) was included in each run.

The relative quantification of the specific gene expression was calculated according to the 2 -Ct method. The experiments were performed in triplicate from three independent cultures. Significant differences were determined using Student's t-test comparisons at a 0.05 significance level.

Enzymatic Activity Assays

Catalase-equivalent activity (CEA) was determined according to the methodology proposed by [START_REF] Li | Rapid kinetic microassay for catalase activity[END_REF], with the following modifications: sensitivity and accuracy of this method was evaluated using a commercial Bovine liver catalase (Sigma C-30, France) as a quality control. and quantified according to the Bradford method using bovine serum albumin (BSA) as a standard (DC Protein Assay kit, Bio-Rad, France). CEA was determined from 10 µg of total protein and the results were expressed as units per mg of total protein. Data were computerized and statistically significant differences were calculated using Student's t-test. A P-value of less than 0.05 was considered statistically significant.

Alternatively, the H 2 O 2 breakdown activity was verified using a positive catalase test (ID color Catalase; BioMérieux, France) in bacterial suspensions containing approximately 10 8 CFU.mL -1 of C. jejuni NCTC 11168 and Bf strains.

RESULTS

Proteome Variations of C. jejuni Bf after Exposure to Aerobic Conditions

Protein abundance influenced by aerobic conditions compared to microaerobic conditions in C. jejuni Bf was examined using 2-D gel electrophoresis. Proteins were resolved for pI ranges 4-7 and 6-11 (Figure 1). Overall, 47 predominant spots with a significant variation were identified (Figure 1,Table 2). Of these, 37 were detected in pI range 4-7 and 10 in pI range 6-11. Some proteins identified from several spots were considered as isoforms. Altogether, identification of 28 unique non-redundant proteins were obtained and classified into categories according to their biological function using the KEGG: Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/) (Table 2). Based on their function, these 28 proteins, expressed differently in aerobic conditions, could be divided into 11 metabolic groups.

The main proteins found at higher abundance in aerobic conditions are involved in stress responses (AhpC, KatA, Tpx, TrxB, LuxS, GroL, Dps, ClpB, and DnaK) and carbohydrate metabolism, particularly the tricarboxylic acid (TCA) cycle (FumC, FrdA, Cj1287c, GltA, AcnB, OorA). Proteins involved in processes such as membrane transport (p19, Cj1380, CfbpA, LivK), energy metabolism (AtpD), amino acid biosynthesis (IlvH), and signal transduction (Cj0448c) were also more abundant under aerobic conditions. In contrast, proteins related to fatty acid biosynthesis (FabF), membrane transport (PstS), lipopolysaccharide biosynthesis (HldD), amino acid biosynthesis (CysM), biosynthesis of secondary metabolites (MetK), and energy metabolism through the TCA cycle (AcnB) were less abundant under aerobic conditions. An overview of enzymes differently expressed in TCA cycle and ROS detoxification during multiplication of Bf under aerobic conditions is summarized in Figure 4.

Transcript Levels of the Specific Genes Involved in ROS Detoxification in C. jejuni in Aerobic Conditions

AhpC, KatA, Tpx, and TrxB proteins were selected to investigate their expression patterns further at the transcriptional level. Although SodB was not detected among the more abundant proteins in aerobic conditions using 2-D electrophoresis, the transcript level of SodB was also evaluated as this protein is important for the cellular detoxification process in C. jejuni. The strain NCTC 11168 grown in microaerobic conditions was used as a reference strain. The transcript levels of the genes involved in ROS detoxification in C. jejuni Bf were evaluated in optimal growth conditions (microaerobiosis), in aerobic conditions and after aerobic acclimation. The data revealed an increase in ahpC, katA, tpx, and trxB transcript levels in C. jejuni Bf cells cultured in aerobic conditions or aerobically acclimated when compared to the relative transcript level of the reference strain C. jejuni NCTC 11168 (P < 0.05) (Figures 2A-D). These results indicate that the transcript level of these genes followed the same trend as the proteomic abundance changes in aerobic conditions. The expression of sodB also increased significantly in cells incubated in aerobic conditions (63.3-fold) and aerobically acclimated (142.1-fold) (Figure 2E).

Effect of Aerobic Conditions on CEA in C. jejuni NCTC 11168 and C. jejuni Bf

H 2 O 2 breakdown activity was quantified using the method based on the decrease in OD 240nm expressed as CEA. The sensitivity and accuracy of the method were assessed using known concentrations of bovine liver catalase for the calibration curve. The linear rate of H 2 O 2 decomposition was obtained in the presence of catalase levels ranging from 0.05 U to 2.00 U (Figure 3A). The curve showed a linear slope of 0.96 (Figure 3B) indicating that the method provides an accurate estimate of the activity of catalase within the range of analysis. Cellular extracts obtained from the exponential culture of C. jejuni Bf grown in microaerobic conditions, in aerobic conditions or after acclimation to aerobic conditions, produced CEA corresponding to 125.93, 132.46, and 197.82 units per mg of protein, respectively, which was higher than the transcript level basis of C. jejuni NCTC 11168 in microaerobic conditions (20.81 units per mg of protein) (Figure 3A). In addition, these data are in agreement with the visual observations of gas production by Bf mediated by the breakdown of H 2 O 2 to water and dioxygen gas (Figure 3B).

DISCUSSION

We have previously demonstrated the atypical behavior of C. jejuni Bf strain with its ability to grow under an ambient gaseous atmosphere unlike other strains of C. jejuni [START_REF] Rodrigues | Description of Campylobacter jejuni Bf, an atypical aero-tolerant strain[END_REF]. In addition, this strain showed a higher survival rate in the presence of ROS-promoting agents (paraquat (PQ) and H 2 O 2 ) compared to the reference strain NCTC 11168 [START_REF] Rodrigues | Description of Campylobacter jejuni Bf, an atypical aero-tolerant strain[END_REF]. The genome sequence of C. jejuni Bf is being analyzed and has not yet revealed a Normalized spots were compared for their abundance between aerobic and microaerobic conditions. Significant difference is indicated for each spot. b Positive values correspond to the fold of higher abundance in aerobic conditions and negative values correspond to the fold of lower abundance in aerobic conditions for each spot.

C Each protein was identified with a mass tolerance <20 ppm and at least two peptides. d No. of MP/TP (Pc): Number of matched/total peptides (Protein coverage).

any major genetic traits related to its aerotolerance when compared to other genomes of C. jejuni (AN: FCEZ01000001-FCEZ01000095).

In this study, we focused on the cellular factors involved in the phenotypic response of Bf in aerobiosis. Comparison of the 2-D electrophoretic profiles between growth in aerobiosis and microaerobiosis revealed more abundant proteins involved in the oxidative stress response in C. jejuni including AhpC, KatA, Tpx, TrxB, LuxS, and Dps [START_REF] Baillon | An iron-regulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter jejuni[END_REF][START_REF] Storz | Oxidative stress[END_REF][START_REF] Chelikani | Diversity of structures and properties among catalases[END_REF][START_REF] Atack | Oxidative stress in Campylobacter jejuni: responses, resistance and regulation[END_REF]. C. jejuni possesses eight major proteins involved in ROS detoxification: AhpC, SodB, KatA, Tpx, Bcp (thiol peroxidase), Dps (bacterioferritin), MsrA/B, and Cj1386 (ankyrin-containing protein involved in heme trafficking to catalase) [START_REF] Pesci | Genetic, enzymatic, and pathogenic studies of the iron superoxide dismutase of Campylobacter jejuni[END_REF][START_REF] Grant | Molecular characterization of KatA from Campylobacter jejuni and generation of a catalase-deficient mutant of Campylobacter coli by interspecific alleic exchange[END_REF][START_REF] Baillon | An iron-regulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter jejuni[END_REF][START_REF] Purdy | Generation of a superoxide dismutase (SOD)-deficient mutant of Campylobacter coli: evidence for the significance of SOD in Campylobacter survival and colonization[END_REF][START_REF] Ishikawa | The iron-binding protein Dps confers hydrogen peroxide stress resistance to Campylobacter jejuni[END_REF][START_REF] Atack | The Campylobacter jejuni thiol peroxidases tpx and bcp both contribute to aerotolerance and peroxide-mediated stress resistance but have distinct substrate specificities[END_REF][START_REF] Atack | Oxidative stress in Campylobacter jejuni: responses, resistance and regulation[END_REF]. In addition, TrxB, LuxS, and Dps have been suggested as contributing to the oxidative stress response [START_REF] Zhao | Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of E. coli[END_REF][START_REF] Ishikawa | The iron-binding protein Dps confers hydrogen peroxide stress resistance to Campylobacter jejuni[END_REF][START_REF] He | Analysis of AI-2/LuxSdependent transcription in Campylobacter jejuni strain 81-176[END_REF]. The enzyme AhpC, encoded by cj0334 in NCTC 11168, is important for aerotolerance maintenance and organic peroxide stress resistance in C. jejuni [START_REF] Baillon | An iron-regulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter jejuni[END_REF]. In E. coli, Ahp is composed of two subunits: the catalytic subunit AhpC and the flavoprotein AhpF, which recycles AhpC [START_REF] Poole | AhpF and other NADH:peroxiredoxin oxidoreductases, homologues of low Mr thioredoxin reductase[END_REF]. In the C. jejuni genome, a homologs gene to ahpF could not be identified [START_REF] Parkhill | The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences[END_REF]; however, the function of AhpF was suggested as being mediated by TrxB, like in its close relative H. pylori [START_REF] Baker | Essential thioredoxin-dependent peroxiredoxin system from Helicobacter pylori: genetic and kinetic characterization[END_REF]. Thus, the concomitant overexpression of AhpC and TrxB in the strain Bf in aerobiosis is not surprising. Defense against H 2 O 2 cytotoxicity in C. jejuni is also achieved by KatA through the breakdown of two molecules of H 2 O 2 to H 2 O and O 2 [START_REF] Storz | Oxidative stress[END_REF][START_REF] Grant | Co-ordination of pathogenicity island expression by the BipA GTPase in enteropathogenic Escherichia coli (EPEC)[END_REF]. The peroxiredoxin thiol peroxidase (Tpx), encoded by cj0779 in NCTC 11168, confers resistance to organic hydroperoxides. The synthesis of Tpx enzyme is induced when C. jejuni is exposed to atmospheric concentrations of oxygen, and probably constitutes the primary defense against oxidative stress [START_REF] Atack | The Campylobacter jejuni thiol peroxidases tpx and bcp both contribute to aerotolerance and peroxide-mediated stress resistance but have distinct substrate specificities[END_REF]. Tpx, together with bacterioferritin co-migratory protein (Bcp), are considered the main defense enzymes against aerobic conditions in C. jejuni [START_REF] Atack | The Campylobacter jejuni thiol peroxidases tpx and bcp both contribute to aerotolerance and peroxide-mediated stress resistance but have distinct substrate specificities[END_REF]. The overexpression of AhpC, TrxB and KatA and their concomitant higher levels of transcripts in C. jejuni Bf in aerobiosis indicate that none of these three detoxification enzyme systems seem to be predominant to ensure the multiplication of this strain in atmospheric conditions. LuxS is described as a cellular factor playing a role in both signaling and the activated methyl cycle [START_REF] Pereira | AI-2-mediated signalling in bacteria[END_REF]. With the lower sensitivity of the isogenic luxS mutant of C. jejuni 81-176 to H 2 O 2 , [START_REF] He | Analysis of AI-2/LuxSdependent transcription in Campylobacter jejuni strain 81-176[END_REF] also observed a lower transcription of the ahpC and tpx genes. Our study confirmed the importance of luxS in the sustainable growth under aerobiosis of C. jejuni Bf. Dps is a protein that can bind non-specifically to DNA, which confers protection against stress caused by peroxides [START_REF] Almirón | A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli[END_REF][START_REF] Andrews | Bacterial iron homeostasis[END_REF]. In addition, Dps is considered an iron storage protein (40 iron atoms per monomer) that can release iron in order to cope with iron deficiency in the environment [START_REF] Andrews | Bacterial iron homeostasis[END_REF]. In fact, interactions between iron and oxygen reduction residues lead to the accumulation of • OH which causes widespread damage to DNA, proteins and membranes [START_REF] Zhao | Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of E. coli[END_REF]. Therefore, Dps is able to minimize the effect of iron with oxidative stress to ensure survival and prevent cell death [START_REF] Ishikawa | The iron-binding protein Dps confers hydrogen peroxide stress resistance to Campylobacter jejuni[END_REF]. At the regulation level, iron homeostasis is achieved by controlling the absorption, metabolism and storage of iron in C. jejuni [START_REF] Van Vliet | Campylobacter jejuni contains two fur homologs: characterization of ironresponsive regulation of peroxide stress defense genes by the PerR repressor[END_REF]. The expression of katA, ahpC and sodB in C. jejuni is also iron-dependent. katA and ahpC genes are both regulated by the iron-dependent transcriptional regulators FurR and PerR [START_REF] Baillon | An iron-regulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter jejuni[END_REF][START_REF] Van Vliet | Campylobacter jejuni contains two fur homologs: characterization of ironresponsive regulation of peroxide stress defense genes by the PerR repressor[END_REF]. KatA and SodB also require iron or iron cofactors for their catalytic activity [START_REF] Pesci | Genetic, enzymatic, and pathogenic studies of the iron superoxide dismutase of Campylobacter jejuni[END_REF][START_REF] Miller | Pumping iron: mechanisms for iron uptake by Campylobacter[END_REF]. Consequently, the higher abundance of Dps in aerobiosis could be correlated with the protection of C. jejuni against oxidative stress. This assumption is reinforced by the higher abundance of the protein CfbpA in aerobiosis, a periplasmic iron-uptake protein.

Transcriptional analyses indicated up-regulated transcript levels of the genes katA, ahpC, sodB, tpx, and trxB in C. jejuni Bf in aerobiosis and in aerobically acclimated cells when compared to the reference strain C. jejuni NCTC 11168. Interestingly, the transcript level was not significantly different between NCTC 11168 and Bf for all proteins in microaerobic conditions except for katA. The greater abundance of protein KatA and the simultaneously higher transcript level of its encoding gene in Bf in microaerobic conditions were confirmed by a higher CEA. This could contribute to explain the greater potential of strain Bf to adapt to aerobic conditions. This is confirmed with the increased transcript and protein levels of enzymes involved in ROS scavenging in aerobic conditions and in aerobically acclimated cells. Furthermore, the gene encoding SodB, another enzyme crucial for ROS scavenging, showed a higher transcript level in aerobiosis than in microaerobiosis. SodB is thought to provide the first line of defense against the toxic effects of reactive oxygen derivatives. This enzyme is responsible for the conversion of superoxide anions (O •- 2 ) to H 2 O 2 and O 2 , thus playing a protective role against the effects of oxidative stress, as shown by several studies [START_REF] Pesci | Genetic, enzymatic, and pathogenic studies of the iron superoxide dismutase of Campylobacter jejuni[END_REF][START_REF] Purdy | Generation of a superoxide dismutase (SOD)-deficient mutant of Campylobacter coli: evidence for the significance of SOD in Campylobacter survival and colonization[END_REF][START_REF] Palyada | Characterization of the oxidative stress stimulon and PerR regulon of Campylobacter jejuni[END_REF]. SodB was previously resolved on our pI range 4-7 electrophoretic gels of C. jejuni cytosoluble proteins [START_REF] Bièche | Dynamic proteome changes in Campylobacter jejuni 81-176 after high pressure shock and subsequent recovery[END_REF]. The absence of detection of a higher abundance of SodB in our electrophoretic protein fingerprinting in aerobic conditions could be explained by the absence of a variation in SodB in these conditions or by a variation below the threshold of our statistical model. Altogether, these data indicate that the sub-system of oxygen detoxification is enhanced during the multiplication of strain Bf in aerobic conditions.

Concomitantly, several proteins involved in carbohydrate metabolism through the TCA cycle were more abundant in C. jejuni Bf under aerobic conditions. This essential metabolic pathway for all oxidative organisms provides precursors for anabolic processes and reducing factors such as NADH and FADH 2 , leading to generation of energy [START_REF] Mailloux | The tricarboxylic acid cycle, an ancient metabolic network with a novel twist[END_REF]. C. jejuni is unable to use many common carbohydrates as carbon sources [START_REF] Parkhill | The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences[END_REF]. So this asaccharolytic microorganism depends heavily on the TCA cycle for its carbon source through gluconeogenesis [START_REF] Velayudhan | Analysis of gluconeogenic and anaplerotic enzymes in Campylobacter jejuni: an essential role for phosphoenolpyruvate carboxykinase[END_REF]. It can uptake several TCA cycle intermediates and use them directly as nutrient sources [START_REF] Stahl | Nutrient acquisition and metabolism by Campylobacter jejuni[END_REF]. The TCA cycle of C. jejuni is composed of Fe-S cluster enzymes which is not common in microaerophiles or aerophiles. Oxidation of (Fe-S) cluster of proteins has been shown to be implicated in oxidative stress susceptibility [START_REF] Atack | Oxidative stress in Campylobacter jejuni: responses, resistance and regulation[END_REF]. The Oor enzyme, which contains oxygen labile (4Fe-4S) centers, was recently demonstrated to be rapidly inactivated after exposure of cells to aerobiosis [START_REF] Kendall | Hemerythrins in the microaerophilic bacterium Campylobacter jejuni help protect key iron-sulphur cluster enzymes from oxidative damage[END_REF]. This oxygen lability was suggested as a major contributor to the microaerophily of C. jejuni [START_REF] Kendall | Hemerythrins in the microaerophilic bacterium Campylobacter jejuni help protect key iron-sulphur cluster enzymes from oxidative damage[END_REF]. The higher abundance of OorA in Bf under aerobic conditions tends to indicate that this protein is functional in atmospheric conditions. However, further analyses are required to determine the activity of Oor in Bf under aerobiosis. The greater abundance of most enzymes involved in the TCA cycle in Bf under aerobic conditions (Figure 4) indicates that this strain requires more carbon sources and energy in these conditions than in microaerobiosis, even though its growth rate in aerobic conditions is not higher than in microaerobic conditions [START_REF] Rodrigues | Description of Campylobacter jejuni Bf, an atypical aero-tolerant strain[END_REF].

The replenishment of TCA cycle intermediates of this microorganism under aerobic conditions could be ensured by proteins involved in amino acid uptake detected in C. jejuni Bf under aerobic conditions, such as Peb1A and LivK. C. jejuni takes up glutamate and aspartate using an ABC transporter system encoded by the peb locus (cj0919c-cj0922c) [START_REF] Hofreuter | Defining the metabolic requirements for the growth and colonization capacity of Campylobacter jejuni[END_REF]. Some studies have described the periplasmic permease Peb1A as a glutamate-and aspartate-binding protein (Leon-Kempis [START_REF] Mdel | The Campylobacter jejuni PEB1a adhesin is an aspartate/glutamate-binding protein of an ABC transporter essential for microaerobic growth on dicarboxylic amino acids[END_REF][START_REF] Müller | A Bacterial virulence factor with a dual role as an adhesin and a solutebinding protein: the crystal structure at 1.5 A resolution of the PEB1a protein from the food-borne human pathogen Campylobacter jejuni[END_REF]. Aspartate is considered as an efficient carbon and energy source for C. jejuni as it is used to feed TCA cycle intermediates by deamination to fumarate catalyzed by the aspartate ammonia lyase AspA [START_REF] Guccione | Amino acid-dependent growth of Campylobacter jejuni: key roles for aspartase (AspA) under microaerobic and oxygen-limited conditions and identification of AspB (Cj0762), essential for growth on glutamate[END_REF][START_REF] Novik | Identification of Campylobacter jejuni genes involved in its interaction with epithelial cells[END_REF]. Glutamate can be transformed in glutamine by glutamine synthetase GlnA (Cj0699c) or also used as substrate of the aspartate:glutamate transaminase AspB (Cj0762c) to produce aspartate and α-ketoglutarate from oxaloacetate and glutamate [START_REF] Guccione | Amino acid-dependent growth of Campylobacter jejuni: key roles for aspartase (AspA) under microaerobic and oxygen-limited conditions and identification of AspB (Cj0762), essential for growth on glutamate[END_REF]. Moreover, glutamate catabolism and the utilization of aspartate are crucial for the metabolic fitness and growth of C. jejuni [START_REF] Guccione | Amino acid-dependent growth of Campylobacter jejuni: key roles for aspartase (AspA) under microaerobic and oxygen-limited conditions and identification of AspB (Cj0762), essential for growth on glutamate[END_REF][START_REF] Hofreuter | Defining the metabolic requirements for the growth and colonization capacity of Campylobacter jejuni[END_REF]. The branched-chain amino acid ABC transporter system LIV (leucine, isoleucine, valine) of C. jejuni is composed of six genes encoding for two periplasmic binding proteins (LivJ and LivK), the permeases (LivH and LivM), and the cytoplasmic ATPases (LivG and LivF). Although these amino acids are not directly associated with C. jejuni growth [START_REF] Guccione | Amino acid-dependent growth of Campylobacter jejuni: key roles for aspartase (AspA) under microaerobic and oxygen-limited conditions and identification of AspB (Cj0762), essential for growth on glutamate[END_REF][START_REF] Hofreuter | Metabolic diversity in Campylobacter jejuni enhances specific tissue colonization[END_REF], their degradation results in the synthesis of succinyl-CoA present in the TCA cycle. The higher abundance of LivK, Peb1 and IlvH tend to show that amino acid uptake and biosynthesis is important for the growth of Bf in aerobisosis.

Taken together, the atypical aerotolerance of C. jejuni Bf seems to result from multifactorial events ensuring an adaptive response to oxidative stress and a functional TCA cycle in aerobic conditions.

CONCLUSION

Using a combined proteomic, transcriptionnal and enzymatic approach, we pinpointed the major pathways induced in the atypical strain Bf for its growth in aerobic conditions. Our findings provide new insights into the cellular mechanisms underlying the aerotolerance of C. jejuni Bf with the induction of an oxidative stress response, a modulation of energy production via the TCA cycle, iron uptake, amino acid uptake and factors involved in the sub-system of oxygen detoxification. This study could contribute to better understand the obligate microaerophily of C. jejuni and in this particular strain Bf its atypical aerotolerance. Further studies are required to explore this ability widely among C. jejuni strains in order to understand better the survival and contamination routes of C. jejuni.

FIGURE 1

 1 FIGURE 1 | 2-DE electrophoretic profiles of C. jejuni Bf in aerobic (A,C) and microaerobic (B,D) conditions. A total of 30 µg proteins was separated on IPG strips with pI 4-7 (A,B) and pI 6-11 (C,D) followed by a 4-15% gradient SDS-PAGE. Arrows indicate over-expressed proteins. Two replicates from each three independent protein extraction were performed for each condition.

FIGURE 2 |

 2 FIGURE 2 | Transcript levels of ahpC (A), katA (B), tpx (C), trxB (D) and sodB (E) genes in C. jejuni NCTC 11168 and C. jejuni Bf in microaerobic conditions (MAC), aerobic conditions (AC) and after acclimation to aerobic conditions (AAC). Transcript levels were measured by RT-qPCR and calculated using the critical threshold ( CT ) method with rrs gene as the internal control. Results were normalized to the gene transcription of the reference strain C. jejuni NCTC 11168 in microaerobic conditions. Error bars represents the standard deviation of three independent experiments. Significant differences, indicated by a different letter, were determined using the Student's t-test comparisons with a 5% confidence level.

FIGURE 3 |

 3 FIGURE 3 | Catalase-equivalent activity of C. jejuni Bf in different gaseous conditions. H 2 O 2 cleavage was first assessed over time in the presence of calibrated quantities of bovine liver catalase and a correlation was calculated between the rate of decrease in absorbance of H 2 O 2 and bovine liver catalase activity (y = 0.0153x-0.0026; R 2 = 0.97). (A) Catalase-equivalent activity of C. jejuni Bf in microaerobic conditions (MAC), in aerobic conditions (AC) and after acclimation to aerobic conditions (AAC), C. jejuni 11168 was used as a control; (B) Bubble production of C. jejuni Bf in MAC, AC, and ACC mediated by the breakdown of H 2 O 2 to water and dioxygen gas. C. jejuni 11168 was used as a control. Plotted points are the mean measurements from three independent experiments. Significant differences, indicated by a different letter, were determined using the Student's t-test comparisons at 5% confidence level.

FIGURE 4 |

 4 FIGURE 4 | Main metabolic pathways required by C. jejuni Bf in aerobic conditions. The arrows indicate the differential abundance of enzymes when Bf is cultivated under aerobic conditions as compared to cells grown in microaerobiosis.

TABLE 1 |

 1 Primers used in this study.

	Primers	Sequences 5 ′

TABLE 2 |

 2 Identification of proteins predominantly affected in C. jejuni Bf under aerobic conditions.

	Protein ID	Name	Gene	ANOVA a	Fold b	Theoretical MW	Log	No. of MP/TP
				(p-value)		(Da)/pI	E-value c	(Pc) d
	CELL PROCESS
	Detoxification
	tr|Q0PBH5	Alkyl hydroperoxide reductase	AhpC	6.27E-03	+2.0	21.9/5.66	-110.26	25/75 (84%)
				2.03E-02	+1.8		-82.37	13/171 (65%)
				6.01E-03	+2.5		-88.39	22/46 (57%)
	sp|Q59296	Catalase	KatA	1.08E-02	+1.7	58.29/7.74	-102.42	22/53 (46%)
	sp|Q9PPE0	Probable thiol peroxidase	Tpx	7.25E-03	+1.7	18.40/5.13	-63.49	8/22 (47%)
	tr|Q0PBZ1	Thioredoxin reductase	TrxB	3.92E-02	+1.5	33/5.60	-92.07	11/60 (57%)
				3.55E-03	+2.8		-65.10	9/22 (37%)
	Chaperones, chaperonins, starvation
	sp|O69289	60 kDa chaperonin	GroL	7.39E-03	-1.8	57.79/5.02	-279.73	40/187 (65%)
				1.42E-02	+2.0		-33.35	6/10 (15%)
				1.07E-02	+2.1		-7.86	2/3 (6%)
				5.46E-03	+1.5		-211.64	31/50 (52%)
				4.42E-02	-2.2		-186.05	34/51 (58%)
				2.99E-02	-1.7		-227.73	34/89 (59%)
	sp|Q0P891	DNA protection during starvation	Dps	2.56E-04	+2.9	17.1/5.55	-13.89	4/4 (34%)
		protein
	sp|Q9PI02	Chaperone protein	ClpB	7.25E-03	+1.7	95.40/5.47	-110.17	27/46 (34%)
		ClpB		1.40E-02	+1.7		-62.96	16/20 (22%)
	sp|O69298	Chaperone protein	DnaK	2.46E-02	+1.6	67.30/4.97	-270.16	48/85 (50%)
		DnaK
	Transport/binding proteins
	tr|Q0P7X0	Periplasmic protein	p19	1.15E-03	+1.5	19.6/4.97	-97.57	16/70 (64%)
				1.05E-02	-1.5		-39.82	5/77 (50%)
	tr|Q0P8K8	Putative periplasmic	Cj1406c	4.13E-02	+2.3	24.9/5.89	-17.77	8/9 (39%)
		protein
	tr|Q0P8M9	Peb1A major cell-binding factor	Peb1A	6.10E-03	+1.5		-46.86	13/14 (45%)
	tr|Q0PAQ4	Putative periplasmic phosphate	PstS	1.58E-04	-2.3	36/5.76	-111.37	20/70 (58%)
		binding protein
	tr|Q0PBW4	Putative iron-uptake ABC	CfbpA	4.08E-03	+2.1	37.4/8.97	-111.73	20/86 (57%)
		transport system periplasmic
		iron-binding protein
	tr|Q0P9N4	Branched-chain amino-acid ABC	LivK	1.95E-02	+2.0	39.59/8.70	-86.36	11/17 (35%)
		transport system periplasmic		3.76E-02	+2.2		-86.38	11/35%
		binding protein
	SMALL MOLECULE METABOLISM
	Energy metabolism -tricarboxylic acid cycle
	sp|O69294	Fumarate hydratase	FumC	1.39E-03	+1.9	50.59/6.12	-138.94	27/109 (51%)
				1.61E-03	+1.7		-156.55	30/48 (53%)
	tr|Q0PBA1	Fumarate reductase flavoprotein	FrdA	6.03E-04	+2.2	73.59/6.36	-136.96	27/71 (44%)
		subunit		2.32E-04	+2.2		-121.49	25/42 (43%)
	tr|Q0P8X0	Malate oxidoreductase	Mez	1.82E-03	+2.4	43.90/5.78	-94.02	16/49 (40%)
	tr|Q0P7U8	Citrate synthase	GltA	1.49E-04	+3.1	47.90/6.47	-80.33	17/42 (48%)
	tr|Q0PA55	Aconitate hydratase 2	AcnB	6.73E-03	-1.6	92.59/6.04	-144.40	27/82 (41%)
				1.97E-02	-1.6		-196.23	32/117 (46%)
				2.30E-02	-1.5		-222.33	37/142 (50%)
	tr|Q0PAY0	2-oxoglutarate:acceptor	OorA	5.26E-03	+1.8	40.90/6.12	-74.90	17/37 (42%)
		oxidoreductase
								(Continued)

TABLE 2

 2

	| Continued
	Protein ID	Name	Gene	ANOVA a	Fold b	Theoretical MW	Log	No. of MP/TP
				(p-value)		(Da)/pI	E-value c	(Pc) d
	ATP-proton motive force
	sp|Q0PC30	ATP synthase subunit beta	AtpD	1.77E-02	+1.5	50.70/4.97	-137.76	25/127 (59%)
	Lipid metabolism; fatty acid biosynthesis
	tr|Q0PB68	3-oxoacyl-[acyl-carrier-protein]	FabF	3.54E-02	-1.5	42.59/5.64	-180.60	29/103 (58%)
		synthase 2		1.60E-02	-1.5		-74.51	12/146 (53%)
	Lipopolysaccharide biosynthesis
	tr|Q0P9A5	ADP-L-glycero-D-manno-	HldD	3.59E-04	-1.7	35.90/6.12	-138.21	23/73 (69%)
		heptose-6-epimerase
	CENTRAL INTERMEDIARY METABOLISM
	Amino acid biosynthesis
	sp|P71128	Cysteine synthase B	CysM	6.28E-04	-1.8	32.30/6.34	-82.93	16/67 (57%)
				6.72E-03	-2.2		-19.81	3/21 (21%)
	tr|Q0PAU2	Acetolactate synthase small	IlvH	1.48E-02	+1.8	17.3/6.75	-26.11	8/10 (52%)
		subunit
	Biosynthesis of secondary metabolites
	tr|Q0P9F8	S-adenosylmethionine	MetK	4.04E-02	-1.6	43.7/5.45	-103.70	19/63 (56%)
		synthetase
				6.50E-03	-1.9		-126.01	25/86 (55%)
				3.21E-02	-1.9		-142.72	27/102 (59%)
	BROAD REGULATORY FUNCTIONS
	Signal transduction
	tr|Q0PB65	Putative MCP-type signal	Cj0448c	3.17E-02	+1.6	40.40/5.58	-80.75	15/55 (47%)
		transduction protein
	Synthesis of autoinducer 2
	sp|Q9PN97	S-ribosylhomocysteine lyase	LuxS	7.25E-03	+1.7	18.10/6.19	-38.61	7/16 (50%)

Frontiers in Microbiology | www.frontiersin.org

October 2016 | Volume 7 | Article 1596

ACKNOWLEDGMENTS

Ramila Cristiane Rodrigues was supported by the Brazilian government thanks to the program "Ciências sem Fronteiras" and CNPq (Brazil; 201727/2012-9). Molecular biology was performed at the PFBM of Oniris.

AUTHOR CONTRIBUTIONS

OT conceived the study. OT and NH and JC designed the study. RR performed the experimental work. DC performed the protein identification. RR prepared the manuscript and OT, NH, and JC contributed to the final manuscript.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.

2016.01596

Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Rodrigues, Haddad, Chevret, Cappelier and Tresse. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.