Marina Cretenet

Sébastien Nouaille

Jennifer Rivière

Lucie Gouffier

Ludwig Stenz

Patrice François

Jacques-Antoine Hennekinne

Michel Piot

Marie-Bernadette Bernadette Maillard

Jacques Fauquant

Jennifer Riviere

Pascal Loubière

Yves Le Loir

Sergine Even
email: sergine.even@rennes.inra.fr

Staphylococcus aureus virulence and metabolism are dramatically affected by Lactococcus lactis in cheese matrix

Keywords: microbe:microbe interactions, environmental signal/stress responses Staphylococcus aureus, cheese, virulence, enterotoxin, ecosystem, bacterial interactions

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Summary

In complex environments such as cheeses, the lack of relevant information on the physiology and virulence expression of pathogenic bacteria and the impact of endogenous microbiota has hindered progress in risk assessment and control. Here, we investigated the behaviour of Staphylococcus aureus, a major foodborne pathogen, in a cheese matrix, either alone or in the presence of Lactococcus lactis, as a dominant species of cheese ecosystems. The dynamics of S. aureus was explored in situ by coupling a microbiological and, for the first time, a transcriptomic approach. L. lactis affected the carbohydrate and nitrogen metabolisms and the stress response of S. aureus by acidifying, proteolysing and decreasing the redox potential of the cheese matrix. Enterotoxin expression was positively or negatively modulated by both L. lactis and the cheese matrix itself, depending on the enterotoxin type. Among the main enterotoxins involved in staphylococcal food poisoning, sea expression was slightly favoured in the presence of L. Lactis, whereas a strong repression of sec4 was observed in cheese matrix, even in the absence of L. lactis, and correlated with a reduced saeRS expression.

Remarkably, the agr system was downregulated by the presence of L. lactis, in part because of the decrease in pH. This study highlights the intimate link between environment, metabolism and virulence, as illustrated by the influence of the cheese matrix context, including the presence of L. lactis, on two major virulence regulators, the agr system and saeRS.

Introduction

Staphylococcus aureus is a major human pathogen, with clinical manifestations ranging from skin infections to sepsis [START_REF] Wertheim | The role of nasal carriage in Staphylococcus aureus infections[END_REF]. S. aureus is also a major causative agent of food poisonings, due to the production of staphylococcal enterotoxins (SE) by strains contaminating foodstuffs [START_REF] Zhang | The enterotoxin D plasmid of Staphylococcus aureus encodes a second enterotoxin determinant (sej)[END_REF][START_REF] Tirado | WHO Surveillance Programme for Control of Foodborne Infections and Intoxications: Preliminary Results and Trends Across Greater Europe[END_REF][START_REF] Le Loir | Staphylococcus aureus and food poisoning[END_REF][START_REF] Ikeda | Mass Outbreak of Food Poisoning Disease Caused by Small Amounts of Staphylococcal Enterotoxins A and H[END_REF]. Although symptoms are not life-threatening or only rarely so, staphylococcal food poisoning (SFP) outbreaks remain a persistent public health problem.

Milk and milk products are often incriminated in SFP, especially in France [START_REF] De Buyser | Implication of milk and milk products in food-borne diseases in France and in different industrialised countries[END_REF][START_REF] Jorgensen | The occurrence of Staphylococcus aureus on a farm with small-scale production of raw milk cheese[END_REF][START_REF] Delbes | Staphylococcus aureus growth and enterotoxin production during the manufacture of uncooked, semihard cheese from cows' raw milk[END_REF].

The capacity of S. aureus to provoke one or another infection relies on a wide variety of virulence factors, the expression of which reportedly depends on the nature of the environment and stresses encountered. This intimate link between metabolic processes and virulence gene expression in S. aureus has been recently highlighted by Somerville and Proctor [START_REF] Somerville | At the crossroads of bacterial metabolism and virulence factor synthesis in Staphylococci[END_REF]. In recent years, functional genomics, transcriptomics and proteomics have led to significant results on virulence gene expression and regulation and on the responses of S. aureus to different stressors [START_REF] Dunman | Transcription Profiling-Based Identification of Staphylococcus aureus Genes Regulated by the agr and/or sarA Loci[END_REF][START_REF] Bischoff | Microarray-based analysis of the Staphylococcus aureus sigmaB regulon[END_REF][START_REF] Weinrick | Effect of mild acid on gene expression in Staphylococcus aureus[END_REF][START_REF] Cui | DNA Microarray-Based Identification of Genes Associated with Glycopeptide Resistance in Staphylococcus aureus[END_REF][START_REF] Chang | Global Transcriptome Analysis of Staphylococcus aureus Response to Hydrogen Peroxide[END_REF][START_REF] Pane-Farre | The sigmaB regulon in Staphylococcus aureus and its regulation[END_REF][START_REF] Rogasch | Influence of the two-component system SaeRS on global gene expression in two different Staphylococcus aureus strains[END_REF]. However these studies were conducted either in laboratory conditions or in a setting relevant to the medical context and thus are hardly transferable to complex food environments.

S. aureus growth and enterotoxin detection have been reported for years in several foodstuffs including milk products [START_REF] Vernozy-Rozand | Behaviour and enterotoxin production by Staphylococcus aureus during the manufacture and ripening of raw goats' milk lactic cheeses[END_REF][START_REF] Meyrand | Growth and enterotoxin production of Staphylococcus aureus during the manufacture and ripening of Camembert-type cheeses from raw goats' milk[END_REF][START_REF] Delbes | Staphylococcus aureus growth and enterotoxin production during the manufacture of uncooked, semihard cheese from cows' raw milk[END_REF][START_REF] Poli | Detection of Staphylococcus aureus and enterotoxin genotype diversity in Monte Veronese, a Protected Designation of Origin Italian cheese[END_REF][START_REF] Cremonesi | Detection of enterotoxigenic Staphylococcus aureus isolates in raw milk cheese[END_REF]. More recently, a few studies have investigated S. aureus behaviour in milk, allowing relevant genes in this context to be pointed out [START_REF] Lammers | Identification of Staphylococcus aureus genes expressed during growth in milk: a useful model for selection of genes important in bovine mastitis?[END_REF][START_REF] Sharer | Differential expression of the lactose transporter gene affects growth of Staphylococcus aureus in milk[END_REF][START_REF] Borezee-Durant | Dual Role of the Oligopeptide Permease Opp3 during Growth of Staphylococcus aureus in Milk[END_REF]. Nevertheless, apart 5 from these few recent and targeted studies, the physiology of S. aureus and the mechanisms governing its virulence expression are poorly understood in the milk and cheese context. In particular, our knowledge of individual enterotoxin gene expression and regulation was mainly derived from studies performed in laboratory media and in planktonic growth conditions [START_REF] Derzelle | Differential temporal expression of the staphylococcal enterotoxins genes during cell growth[END_REF]. Recently, technical bottlenecks have been overcome, allowing the measurement of gene expression, including expression of enterotoxin genes, in complex matrices such as cheeses [START_REF] Ulve | RNA extraction from cheese for analysis of in situ gene expression of Lactococcus lactis[END_REF][START_REF] Duquenne | Tool for Quantification of Staphylococcal Enterotoxin Gene Expression in Cheese[END_REF]. As a consequence of these advances, in situ approaches can now be used to investigate S. aureus physiology, including virulence expression, in realistic environmental conditions.

Several factors can affect S. aureus behaviour in cheese. In particular, the presence of other microorganisms, especially lactic acid bacteria (LAB), reportedly limits S. aureus growth and enterotoxin production. However, again, most of our knowledge relies on observations or screenings for inhibitory activities, the molecular mechanisms of which remain unknown.

LAB antagonism is a complex and multifactorial phenomenon, and various hypotheses have been advanced regarding its mechanisms, including production of inhibitory compounds (bacteriocin, hydrogen peroxide), acidification, and nutritional competition [START_REF] Barber | Effect of pH and oxygen tension on staphylococcal growth and enterotoxin formation in fermented sausage[END_REF][START_REF] Haines | Effect of selected lactic acid bacteria on growth of Staphylococcus aureus and production of enterotoxin[END_REF][START_REF] Notermans | Combined effect of water activity, pH and suboptimal temperature on growth and enterotoxin production of Staphylococcus aureus[END_REF][START_REF] Ammor | Antibacterial activity of lactic acid bacteria against spoilage and pathogenic bacteria isolated from the same meat small-scale facility: 2--Behaviour of pathogenic and spoilage bacteria in dual species biofilms including a bacteriocin-like-producing lactic acid bacteria[END_REF][START_REF] Delbes | Staphylococcus aureus growth and enterotoxin production during the manufacture of uncooked, semihard cheese from cows' raw milk[END_REF][START_REF] Otero | Inhibition of Staphylococcus aureus by H2O2producing Lactobacillus gasseri isolated from the vaginal tract of cattle[END_REF]Charlier et al., 2008a). Molecular and global approaches, including transcriptomics and proteomics, are powerful tools that have already allowed the mechanisms of action of one species of bacteria upon another to be deciphered [START_REF] Mashburn | Staphylococcus aureus serves as an iron source for Pseudomonas aeruginosa during in vivo coculture[END_REF][START_REF] Laughton | Inhibition of expression of a staphylococcal superantigen-like protein by a soluble factor from Lactobacillus reuteri[END_REF]. Recently, we reported the first transcriptomic approach regarding the interaction between S. aureus and L. lactis in a chemically defined medium (CDM) [START_REF] Even | Staphylococcus aureus virulence expression is impaired by Lactococcus lactis in mixed cultures[END_REF][START_REF] Nouaille | Transcriptomic response of Lactococcus lactis in mixed culture with Staphylococcus aureus[END_REF]. We showed that L. lactis was able to strongly affect the expression of several regulatory systems and virulence In this study, S. aureus MW2 growth was followed in cheese matrix for 7 days by coupling a microbiological and, for the first time, a transcriptomic approach. S. aureus was grown either alone or in the presence of L. Lactis in order to address the influence of this antagonistic flora.

Results show that, in cheese matrix, expression of important S. aureus virulence genes was strongly altered by both the cheese matrix itself, as an abiotic environmental component, and by the presence of L. lactis.

Results

One log inhibition of S. aureus growth by L. lactis in cheese matrix. S. aureus MW2 was grown in ultra-filtrate (UF) retentate, a cheese matrix, as previously described [START_REF] Ulve | RNA extraction from cheese for analysis of in situ gene expression of Lactococcus lactis[END_REF] to mimic the conditions encountered during the cheese-making process (i.e., immobilized growth in the colony in a dairy environment). UF retentate, the cheese matrix used for this study, is based on the MMV (Maubois-Moquot-Vassal) process and has been widely used in several cheese-making processes for soft to semi-hard cheeses [START_REF] Mistry | Application of membrane separation technology to cheese production[END_REF][START_REF] Maubois | La preparation et la mise en oeuvre des rétentats d'ultrafiltration. L'emploi du procédé MMV[END_REF]. MW2 was selected among all sequenced strains of S. aureus for its origin (community-acquired) and its content in enterotoxin genes [START_REF] Baba | Genome and virulence determinants of high virulence community-acquired MRSA[END_REF]. Two additional S. aureus field strains (kindly provided by AFSSA-lerqap) involved in SFP outbreaks were tested in similar growth conditions to validate our results: S. aureus 257D (isolated from chocolate milk and carrying the sea gene) and S. aureus 253E (isolated from cheese and carrying the sea and sed genes).

S. aureus

MW2 grew exponentially in the cheese matrix for the first 6 h of culture and entered into the stationary phase before the temperature shift at 10 h. The final population density was 2 x 10 9 cfu g -1 at 24 h and was maintained until day 7 (Fig. 1). The kinetics of S. aureus MW2 growth in mixed culture revealed an inhibition of S. aureus by L. lactis from 6 h onwards, reaching one log at 7 days (Fig. 1A). Lactose was never depleted from the medium during the 7 days of culture (Fig. 1B). In S. aureus pure culture, small amounts of acetate were quantified by day 7, whereas lactate production was not observed (Fig. 1B). pH remained unchanged. In contrast, the presence of L. lactis caused a drop in pH values down to 5 by day 7, in accordance with lactate and acetate production (Fig. 1C). Similar inhibition by L. lactis was observed on S. aureus 257D and 253E, the two field strains. Both strains grew to a final density (10 9 cfu g -1 by day 7) in pure culture in cheese matrix that was similar to that of the S. A severe decrease of the expression of genes involved in cellular machinery in the presence of L. lactis. The gene expression profiles of S. aureus in pure and mixed cultures with L. lactis in cheese matrix were analysed using microarrays and reverse transcription quantitative PCR (RT-qPCR) over 7 days. Expression of genes involved in cell division, transcription, translation, and cell envelope biosynthesis were maintained for 7 days in pure culture but were downregulated in mixed cultures (Table 1, Table S1). Indeed, the expression of genes involved in cell division (ftsH, ftsZ, ftsL) and cell envelope biosynthesis (pbp4, gpsA, MW2013) continuously decreased during the first 24 h in mixed culture but was not significantly modified in pure culture. Additionally, expression of genes involved in transcription (rho, rpoC) and translation (fus, tufA, rpmI) decreased in mixed culture at all times compared to 6 h. These results were confirmed by RT-qPCR and expanded to 7 days of culture for ftsZ and pbp4 (Table 1). In conclusion, although S. aureus entered a stationary phase in pure culture after 24 h, cells retained an active metabolic status, as revealed by the maintenance of expression of several genes related to growth, transcription and translation.

The presence of L. lactis provoked a premature arrest of S. aureus MW2 growth.

Accordingly, expression of growth-related genes decreased in mixed compared to pure culture.

A major acid stress triggered by L. lactis. S. aureus did not undergo major stresses when grown in pure culture in cheese matrix (Table 1, Table S1). Indeed, no specific stress response was revealed by the transcriptomic profile in pure culture except the induction of the SigmaB regulon, in agreement with the transition to the stationary phase. Conversely, the presence of L. lactis altered the expression of stress-related genes. In agreement with cheese matrix acidification by L. lactis, genes associated with the acid stress response showed increased expression in mixed culture, as revealed by both microarray and RT-qPCR data. The urease (ureD) pathway, as well as part of the deaminase pathway (arcC, arcR) involved in the acid stress response [START_REF] Cotter | Surviving the acid test: responses of gram-positive bacteria to low pH[END_REF], were overexpressed in mixed compared to pure culture (Table 1). Accordingly, urea was consumed more rapidly in mixed culture than in pure culture (Table S2). Urea can indeed only be consumed by S. aureus, as L. lactis does not possess a urease-encoding gene. The sodA gene, encoding a superoxide dismutase and reportedly involved in the response to oxidative stress generated by an acidic environment [START_REF] Clements | Characterization of the major superoxide dismutase of Staphylococcus aureus and its role in starvation survival, stress resistance, and pathogenicity[END_REF], was also highly upregulated in mixed culture, with a ∼4-fold increase at 24 h and 7 days compared to pure culture (Table 1). In addition, expression of members of the CtsR regulon, including the chaperone-encoding genes dnaK and clpC, as well as ctsR itself, increased in mixed culture over 7 days (Table 1), as previously reported in response to various stresses, including acid stress [START_REF] Chastanet | Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram-positive bacteria[END_REF].

Apart from the induction of acid stress-related gene expression, S. aureus did not undergo major stresses in the presence of L. lactis. Genes associated with the general stress response were underexpressed in mixed culture. Notably, genes known to be under SigmaB control (eg, asp23, MW1682 and MW0781, encoding, respectively, the alkaline shock protein 23 and two general stress protein-like proteins) were underexpressed in mixed compared to pure cultures (Table S1, Table 1). The expression of sigB itself had decreased in mixed culture by day 7 (Table 1). SigmaB activity is regulated at the protein rather than the transcriptional level [START_REF] Senn | Molecular analysis and organization of the sigmaB operon in Staphylococcus aureus[END_REF], and SigmaB response to stressful conditions is reportedly primarily controlled by an induction of rsbU transcription [START_REF] Pane-Farre | Role of RsbU in controlling SigB activity in Staphylococcus aureus following alkaline stress[END_REF]. Here, rsbU was also downregulated in mixed culture (Table S1), which may account for the reduced activity of SigmaB and thus the lower expression of the SigmaB regulon. Interestingly, genes known to be involved in the oxidative stress response (ie, trxA, encoding the thioredoxin, and katA, encoding a catalase) were down-expressed in the mixed compared to the pure culture (Table 1). Likewise, the expression of ahpF, encoding the alkyl hydroperoxide reductase subunit F, was induced in pure culture whereas it remained constant in mixed culture over 24 h (Table S1). Collectively, these data suggest that S. aureus cells sensed a lower redox potential in mixed culture, in accordance with the reducing activity of L. lactis [START_REF] Tachon | NoxE NADH Oxidase and the Electron Transport Chain Are Responsible for the Ability of Lactococcus lactis To Decrease the Redox Potential of Milk[END_REF].

In conclusion, response to acid stress seemed predominant over any other stress-specific response encountered by S. aureus during growth in mixed culture with L. lactis.

Major changes in S. aureus carbohydrate metabolism. In cheese matrix, lactose concentration decreased only slightly by day 7 in pure culture, suggesting that S. aureus did not actively metabolize lactose as a carbon source (Fig. 1B). S. aureus can grow on amino acids, particularly glutamate [START_REF] Mah | Nutritional requirements of Staphylococcus aureus S-6[END_REF][START_REF] Borezee-Durant | Dual Role of the Oligopeptide Permease Opp3 during Growth of Staphylococcus aureus in Milk[END_REF]. Predictably, the glutamate concentration strongly decreased until 24 h in pure culture (Table S2). Whether glutamate was also used as a carbon source by S. aureus in mixed culture in cheese matrix can be assumed but not proved, because in mixed culture changes in free amino acid and carbohydrates resulted from both lactococcal and staphylococcal metabolisms. Indeed, several amino acids, including glutamate, accumulated from 6 h onwards in cheese matrix in mixed culture as a result of lactococcal proteolytic activity (Table S2). Glutamate was available in mixed culture to sustain S. aureus growth.

Strikingly, the presence of L. lactis provoked a shift of the expression of several genes related to pyruvate metabolism. The expression of lctE and adh1, encoding lactate dehydrogenase and alcohol dehydrogenase, was transiently induced in pure culture, whereas it decreased from 10 h onwards in mixed culture (Table 1). On the contrary, the expression of pflA, encoding the pyruvate formate lyase, was higher in mixed culture from 24 h onwards. In the mixed culture, alsS and butA, encoding respectively the alpha acetolactate synthase and the acetoin reductase, were strongly over-expressed, at all time points in the case of alsS (Table 1). Of note, production of acetoin was detected in mixed culture (data not shown), although we cannot exclude, as previously mentioned for amino acids and carbohydrates, that it resulted from both lactococcal and staphylococcal activities. The expression of alsS is under the control of CidR, which has been shown to be induced by acetate and lactate, together with the cid and lrg operons [START_REF] Yang | Characterization of the Staphylococcus aureus CidR regulon: elucidation of a novel role for acetoin metabolism in cell death and lysis[END_REF]. In addition, we found here that cidA and, to a lesser extent, lrgA were overexpressed in mixed culture (Table 1), which correlated well with the production of lactate and acetate by L. lactis in mixed culture (Fig. 1B).

L. lactis proteolysis relieved S. aureus amino acid limitation in cheese matrix. Cheese matrices are reportedly poor in free amino acids, so the main source of amino acids is provided by proteolysis of caseins [START_REF] Savijoki | Proteolytic systems of lactic acid bacteria[END_REF]. Accordingly, a biochemical analysis revealed that very few amino acids were initially present in the UF retentate (Table S2). Most of them disappeared before 6 h in S. aureus pure culture, suggesting a free amino-acid limitation in cheese matrix similar to that previously observed in milk [START_REF] Borezee-Durant | Dual Role of the Oligopeptide Permease Opp3 during Growth of Staphylococcus aureus in Milk[END_REF]. A few amino acids had accumulated in pure culture by day 7, yet at a low level, suggesting a limited but active proteolysis of S. aureus MW2 on caseins. Urea was also used as an alternative nitrogen source after 6 h (Table S2). Urease is known to be induced in the case of nitrogen starvation in Bacillus subtilis and Corynebacterium glutamicum [START_REF] Schmid | Response to nitrogen starvation in Corynebacterium glutamicum[END_REF][START_REF] Brandenburg | Roles of PucR, G1nR, and TnrA in regulating expression of the Bacillus subtilis ure p3 promoter[END_REF][START_REF] Beckers | Molecular identification of the urea uptake system and transcriptional analysis of urea transporter-and urease-encoding genes in Corynebacterium glutamicum[END_REF].

In contrast, in mixed culture free amino acids were released and were thus available for the growth of both lactococcal and staphylococcal populations (Table S2). Amino acid accumulation is likely to result mainly from lactococcal proteolytic activity. Amino acid release might thus relieve the limitation observed in S. aureus pure culture. Indeed, the expression of relA, a gene involved in the stringent response, that is induced in the case of amino acid limitation [START_REF] Anderson | Characterization of the Staphylococcus aureus heat shock, cold shock, stringent, and SOS responses and their effects on log-phase mRNA turnover[END_REF], was significantly reduced in mixed culture (ratios 0.2 at 24 h and 0.1 at day 7). Furthermore, several genes involved in nitrogen nutrition and amino acid biosynthesis were downregulated during mixed culture. Genes encoding peptidases such as pepT and MW1694 as well as opp3, the sole oligopeptide transport system used for growth in milk [START_REF] Hiron | Only one of four oligopeptide transport systems mediates nitrogen nutrition in Staphylococcus aureus[END_REF], were downregulated in mixed culture (Table 1, Table S1). Expression of genes involved in the biosynthesis pathway of leucine (leuA) and isoleucine (ilvD) was also lower in mixed culture, especially at 24 h (Table 1). Biosynthesis of these branched-chain amino acids (BCAA: leucine, isoleucine and valine) is under the control of the CodY repressor [START_REF] Pohl | CodY in Staphylococcus aureus: a regulatory link between metabolism and virulence gene expression[END_REF]. Increased proteolytic activity in the mixed culture likely provided the necessary co-repressors (BCAA) for CodY-mediated repression of branched-chain amino acid biosynthesis. Collectively, these results suggest that, in mixed culture, lactococcal proteolysis deeply modified the expression of S. aureus genes involved in nitrogen nutrition and amino acid biosynthesis. Downregulation of the expression of S. aureus agr system in mixed culture. S. aureus virulence expression is tightly controlled by several regulatory systems, of which the agr system, sarA, saeRS and codY are key elements [START_REF] Novick | Autoinduction and signal transduction in the regulation of staphylococcal virulence[END_REF][START_REF] Bronner | Regulation of virulence determinants in Staphylococcus aureus: complexity and applications[END_REF]. A striking difference was noted in agr system expression when pure and mixed cultures were compared. In pure culture, both agrA and RNAIII (agr effector) expressions increased over time, especially at 24 h and 7 days, whereas these inductions were strongly attenuated in the presence of L. lactis (Table 1). Hence, in mixed culture, the expression of agrA and RNAIII increased slightly at 10 h but decreased afterward. As a result, the expression of RNAIII and agrA was strongly downregulated from 24h onward in mixed culture with L. lactis compared 13 to pure culture. Accordingly, expression of rot, which is normally repressed by RNAIII in the stationary phase [START_REF] Geisinger | Inhibition of rot translation by RNAIII, a key feature of agr function[END_REF], was higher in mixed culture at 7 days (Table 1).

Likewise, expression of sarA and mgrA, both known as activators of the agr system [START_REF] Chien | SarA level is a determinant of agr activation in Staphylococcus aureus[END_REF][START_REF] Ingavale | Rat/MgrA, a regulator of autolysis, is a regulator of virulence genes in Staphylococcus aureus[END_REF], were downregulated at 7 days in mixed compared to pure cultures (Table 1). The downregulation of the agr system by L. lactis was confirmed in the field strain S. aureus 257D: the expression of both agrA and RNAIII was strongly enhanced in pure culture in cheese matrix and severely diminished in mixed culture (data not shown).

No transcripts of agrA and RNAIII were quantified in S. aureus 253E, indicating that this strain was agr deficient.

Regarding virulence factors, differential expression of several genes occurred in the presence of L. lactis. Some cell wall-associated virulence factors showed enhanced expression over 24 h in S. aureus pure cultures only (e.g. fnb (MW0764) and ebpS, encoding fibrinogen and elastin-binding proteins, respectively; Table S1). Other genes were induced in both conditions but earlier in pure culture, as observed for clfA (clumping factor A) and cap8A (involved in the capsular polysaccharide synthesis), compared to mixed culture (Table 1). Additionally, ssaA (staphylococcus secretory antigen SsaA homolog) was over-expressed in pure culture from 6 h to 24 h (Table S1). Conversely, fnb (MW2421), sdrD and sdrC encoding fibrinogenbinding proteins were over-expressed in mixed culture (Table 1, Table S1). Finally, the biofilm-associated gene icaA was strongly downregulated in mixed compared to pure culture (Table 1). Few secreted virulence factors showed differential expression in pure and mixed cultures of S. aureus. The hemolysin III (MW2096)-and hemolysin (MW0802)-encoding genes showed increased expression in pure culture from 6 h to 24 h, while their expression remained constant in mixed culture during this period (Table S1). Conversely, isaB (immunodominant antigen B) expression was induced earlier in pure culture but reached a higher level in the mixed culture than in the pure culture at 7 days (14.3-fold) (Table 1). In conclusion, few virulence genes showed differential expression between pure and mixed cultures, and no general trends were observed. Strikingly, downregulation of the agr system was provoked by L. lactis, as previously observed in a chemically defined medium under environmental conditions very different from those of the cheese matrix [START_REF] Even | Staphylococcus aureus virulence expression is impaired by Lactococcus lactis in mixed cultures[END_REF].

CodY-independent inhibition of S. aureus virulence by L. lactis. Transcriptomic analysis

of S. aureus MW2 suggested an increased CodY repression in mixed culture with L. lactis.

Increased proteolytic activity in mixed culture was indeed likely to relieve the amino acid limitation that occurred in pure culture and to provide the co-repressors (valine, isoleucine and leucine) for CodY-mediated repression. Recently, it was shown that a mutation in codY was associated with an increased expression of both RNAII and RNAIII [START_REF] Majerczyk | Staphylococcus aureus CodY negatively regulates virulence gene expression[END_REF]. We thus tested whether the higher CodY activity in mixed culture resulting from L. lactis proteolysis could be responsible for the downregulation of the agr system. To address this question, a S. aureus MW2 ∆codY mutant strain was constructed and tested in pure and mixed cultures. In pure culture, a higher expression of RNAIII expression was observed in the codY mutant as previously described by Majerczyk et al. The downregulation of both agrA and RNAIII by L. lactis occurred in the codY mutant as in the wild-type strain (Fig. 2). These results indicated that the downregulation of the S. aureus agr system by L. lactis was not mediated through CodY repression and thus was probably not related to the proteolytic activity of L. lactis; however, a CodY-independent regulation pathway could not be excluded.

Acidification by L. lactis is partially involved in S. aureus virulence inhibition.

To further evaluate the mechanisms involved in inhibition of S. aureus growth and lack of induction of the agr system expression in mixed culture, the decrease in pH was dissociated from the presence of L. lactis. Glucono delta-lactone (GDL) was used to mimic acidification by L. lactis. Compared to other acids, spontaneous GDL hydrolysis into gluconic acid is slow, occurs in the cheese matrix after curd formation and mimics the kinetics of the decrease in pH induced by L. lactis (Fig. 3B). S. aureus growth was not significantly affected by the addition of GDL (Fig. 3A). We investigated the effect of acidification on the expression of the agr system in the three environments and found dissimilar effects on the transcription of RNAII and RNAIII. Expression of agrA was decreased only in mixed culture, whereas RNAIII expression was reduced by both GDL acidification and mixed culture conditions (Fig. 3C and3D). However, it must be noted that RNAIII reduction was only 4-fold in GDL conditions compared to 80-fold in mixed culture. These results demonstrate that, in cheese matrix, the decrease in pH in mixed culture could partially account for RNAIII downregulation. Finally, it should be noted that acidification directly inhibited the P3 promoter (RNAIII) independently of an effect on the autoinducing system (RNAII).

Downregulation of enterotoxin gene expression in cheese matrix.

We especially focused on the production of enterotoxins because they are essential causative agents of SFP. Of the six enterotoxins possessed by S. aureus MW2, SEA, SEC, SEG and SEH have a demonstrated emetic activity, whereas SElL and SElK are non-emetic [START_REF] Orwin | Biochemical and biological properties of Staphylococcal enterotoxin K[END_REF][START_REF] Thomas | Diversity in Staphylococcus aureus enterotoxins[END_REF]. The expression profiles of enterotoxin genes were heterogeneous with respect to time and L. lactis effect. In S. aureus pure culture in cheese matrix, sea and seh expression decreased with time (6-fold and 8-fold, respectively, between 6 h and 7 days; Table 1). In contrast, sec4 expression did not vary significantly over 7 days, and expression of seg2, sek2 and sel2 decreased only slightly with time. While seg2 and sek2 were under-expressed in mixed culture from 10 h onwards, no significant variation was observed for sec4 expression in the mixed vs the pure culture. On the contrary, both sel2 and seh genes were over-expressed in the mixed compared to the pure culture, with a maximum ratio of 17-fold for seh at 7 days 1). The expression profile of sea was similar in mixed and pure cultures, and sea expression showed a slight increase in mixed culture. Results obtained with S. aureus field strains isolated from SFP confirmed that sea expression was not affected by the presence of L. lactis LD61 and even significantly increased in S. aureus 257D. Indeed, sea expression decreased with time in both pure and mixed cultures in the field strain S. aureus 253E, without any significant effect of the presence of L. lactis. An inductive effect of L. lactis on sea expression was observed in the field strain 257D: sea expression was higher in mixed compared to pure culture at both 9 h and 7 days (data not shown).

The kinetics of enterotoxin expression in pure S. aureus culture in cheese matrix were in agreement with previously published data obtained in BHI, a rich laboratory medium, for sea, seg2 and sek [START_REF] Derzelle | Differential temporal expression of the staphylococcal enterotoxins genes during cell growth[END_REF]. Under planktonic growth conditions in BHI, expression of sec, seh and sel was enhanced after the exponential growth phase [START_REF] Derzelle | Differential temporal expression of the staphylococcal enterotoxins genes during cell growth[END_REF], whereas in the cheese matrix expression of these genes did not change or was decreased (Table 1). Enterotoxin expression was further compared in S. aureus MW2 between growth in pure culture in cheese matrix and in chemically defined medium (CDM) under planktonic conditions over 24 h, corresponding to the late stationary phase (Table 2, [START_REF] Even | Staphylococcus aureus virulence expression is impaired by Lactococcus lactis in mixed cultures[END_REF].

The influence of the cheese matrix clearly varied with the enterotoxin types. The expression profile of sea was similar in both conditions, and seg2 and sek2 expressions were slightly lower in cheese matrix compared to CDM during the exponential growth phase (2-to 3-fold at 8 h). Strikingly, the cheese matrix strongly altered both the level and dynamics of sec4, seh and sel2 expression. sec4, seh and sel2 expressions were higher in CDM compared to cheese matrix at all time-points, from 3-to 23-fold for sec4, 5-to 64-fold for seh, and from 4-to 42fold for sel2 (Table 2). Although seh expression remained constant in CDM over 24 h [START_REF] Even | Staphylococcus aureus virulence expression is impaired by Lactococcus lactis in mixed cultures[END_REF], it strongly decreased in the stationary phase (24 h) in cheese matrix (Table 1). In addition, sec4 and sel2 expression strongly increased in CDM in the stationary phase (Even et ) but remained constant in cheese matrix and even slightly decreased at 24 h (Table 1). The differences in sec4 expression in cheese matrix versus CDM were validated by immunological detection of SEC production. Production of 5.16 µg ml -1 of SEC by S. aureus MW2 was detected after 24 h in CDM, wherease no SEC was detected at 24 h in cheese matrix. In cheese matrix, SEC was detectable but not quantifiable (quantification limit: 0.021 ng ml -1 ) at 7 days.

In conclusion, these results show that both the cheese matrix itself and the presence of L. lactis modulated enterotoxin gene expression and that these modulations were strongly dependent on enterotoxin type. growth conditions in cheese matrix differ from classical laboratory conditions in at least 2 major ways. Milk is a complex nutritional matrix that undergoes several physico-chemical modifications, in a concomitant or sequential way, including transition from liquid to solid state. As a consequence, S. aureus growth in liquid medium is planktonic, whereas in cheese matrix S. aureus is immobilised and grows as colonies. In addition, cheeses contain more or less complex microbial ecosystems, including acidifying flora such as LAB that have been shown to display antagonistic properties against S. aureus. In this study, we investigated for the first time the dynamics of S. aureus growth and global gene expression in a cheese matrix for 7 days, allowing a global understanding of S. aureus physiology in this particular context, with special attention paid to the influence of L. lactis, the model LAB species.

Premature arrest of S. aureus growth in the presence of L. lactis. The overall view of the S. aureus transcriptome in mixed culture with L. lactis showed a massive downregulation of genes involved in cell division, transcription, translation and cell envelope metabolism in comparison to the pure culture. Growth of S. aureus stopped earlier in mixed culture, resulting in one log inhibition at 24 h. Then, between 24 h and 7 days, the population remained viable. S. aureus growth slowed earlier (at 6 h) in mixed culture and at a nonlimiting pH (6.3) since S. aureus can grow in a more acidic environment (Charlier et al., 2008b). Hence, acidification did not seem to be involved in the early inhibition of S. aureus growth. This was further reinforced by GDL experiments, which revealed that the decrease in pH did not affect S. aureus growth during the course of the 7-day study. However, it must be noted that the acid used to mimic acidification was not lactate and that the nature of the acid used to adjust pH in the culture medium reportedly influences the impact of pH on S. aureus growth [START_REF] Domenech | Effect of six organic acids on staphylococcal growth and enterotoxin production[END_REF]. A previously reported early inhibition of S. aureus growth by L. lactis in liquid milk was not due to acidification but rather to unidentified nutrientrelated phenomena (Charlier et al., 2008b). The early inhibition of S. aureus growth observed in the cheese matrix likely relied on similar mechanisms.

Major acid stress but no general stress. S. aureus did not undergo major stresses when cultivated alone in cheese matrix, whereas the acid stress response was major in the S. aureus transcriptomic profile in mixed culture, with induction of the expression of genes encoding detoxification enzymes (ureD, sodA), the arginine deaminase pathway (arcC, arcR) and the ctsR regulon (ctsR, dnaK, clpC). S. aureus sensed not only the decrease in pH but also the production of lactate (and acetate) itself that triggered major changes in central metabolism. Acidification of the cheese matrix by L. lactis led to transcription of S. aureus genes involved in acetoïn production (alsS and butA) instead of the gene encoding lactate dehydrogenase. The induction of alsS expression in the presence of lactate or acetate is mediated by CidR [START_REF] Yang | Characterization of the Staphylococcus aureus CidR regulon: elucidation of a novel role for acetoin metabolism in cell death and lysis[END_REF]. Concomitantly, lactate and acetate induced the expression of cid and lrg operons in mixed culture. These two operons were shown to affect S. aureus murein hydrolase activity, autolysis, viability and biofilm formation [START_REF] Rice | The Staphylococcus aureus cidAB operon: Evaluation of its role in regulation of murein hydrolase activity and penicillin tolerance[END_REF][START_REF] Rice | Acetic acid induces expression of the Staphylococcus aureus cidABC and lrgAB murein hydrolase regulator operons[END_REF][START_REF] Rice | The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus[END_REF]. In particular, lactate was shown to promote a transition of cell physiology towards a "survival" state, in which cells remained viable with a reduced metabolic activity. Apart from acidic stress, no other major stress was revealed in the S. aureus transcriptome in mixed culture. It should be noted that the presence of L. lactis relieved the amino acid limitation of S. aureus in cheese matrix. The high proteolytic activity of L. lactis in cheese matrix resulted in the release of free amino acids (especially branched-chain amino acids, co-factors of CodY) that were then available for S. aureus. In mixed culture, we observed a strong repression of leucine and isoleucine biosynthesis pathways, suggesting CodY-mediated activity [START_REF] Pohl | CodY in Staphylococcus aureus: a regulatory link between metabolism and virulence gene expression[END_REF].

S. aureus sensed a lower redox potential in mixed culture. Expression of genes belonging to various functional categories (stress, carbon metabolism and virulence) suggested the redox potential was lower in mixed culture. First, genes involved in oxidative stress response, including trxA, katA, and ahpF, were upregulated in pure compared to mixed culture. In addition, the expression of clpL, known to be under SigmaB control, was more than 7-fold higher in mixed culture than pure culture, whereas the expression of other SigmaB-dependent genes was reduced. The expression of clpL has been shown to be strongly induced in an anaerobic environment independently of any SigmaB induction [START_REF] Fuchs | Anaerobic gene expression in Staphylococcus aureus[END_REF]. In our study, lacG (lactose transporter), pflA (involved in acetate formation) as well as isaB (immunodominant antigen B) were over-expressed in mixed compared to pure cultures. lacA, another gene belonging to the lac operon, pflA and isaB were also found to be induced in anaerobic conditions by Fuchs et al. Collectively, these results suggest that S. aureus cells sensed a lower redox potential in mixed compared to pure cultures, as might be expected given that the early steps of L. lactis growth reportedly result in oxygen exhaustion and reduction of the growth medium [START_REF] Brasca | Redox potential to discriminate among species of lactic acid bacteria[END_REF][START_REF] Tachon | NoxE NADH Oxidase and the Electron Transport Chain Are Responsible for the Ability of Lactococcus lactis To Decrease the Redox Potential of Milk[END_REF].

A biofilm-like behaviour in mixed culture? In mixed culture, S. aureus responded to a more acidic and anaerobic-like (lower redox potential) environment than in pure culture. These two whose expression is induced in cheese matrix in mixed culture are associated with biofilm formation in the literature. Comparison of S. aureus transcriptomes in biofilm versus planktonic growth showed higher transcription of genes involved in the deiminase pathway (arc genes), genes coding for urease (ureA-G), and the pyruvate formate lyase (plflA) as well as a decreased expression of genes involved in nitrogen metabolism (opp3, ilvE) [START_REF] Beenken | Global gene expression in Staphylococcus aureus biofilms[END_REF][START_REF] Resch | Differential Gene Expression Profiling of Staphylococcus aureus Cultivated under Biofilm and Planktonic Conditions[END_REF]. Enhanced expression of isaB can be related to anaerobic conditions [START_REF] Fuchs | Anaerobic gene expression in Staphylococcus aureus[END_REF] and enhanced expression of sdrC to mild acidic conditions [START_REF] Weinrick | Effect of mild acid on gene expression in Staphylococcus aureus[END_REF]. Both expressions were shown to be induced in biofilm compared to planktonic conditions [START_REF] Resch | Differential Gene Expression Profiling of Staphylococcus aureus Cultivated under Biofilm and Planktonic Conditions[END_REF]. Of note, cidA was shown to contribute to biofilm adherence by affecting cell lysis and the release of genomic DNA. Extracellular DNA has been found in the biofilm matrix produced by S. aureus [START_REF] Rice | The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus[END_REF][START_REF] Mann | Modulation of eDNA release and degradation affects Staphylococcus aureus biofilm maturation[END_REF].

All these genes associated with S. aureus biofilm formation were upregulated in mixed compared to pure cultures. Nevertheless, it should be noted that this biofilm-like behaviour of S. aureus in mixed culture was ica-independent as icaA expression was strongly downregulated in mixed culture. This strong downregulation of icaA expression in mixed culture may indeed be due to higher repression by CodY [START_REF] Majerczyk | Staphylococcus aureus CodY negatively regulates virulence gene expression[END_REF].

Collectively, our data suggest that the acidic and anaerobic environment generated by L. lactis triggered the same metabolic pathways involved in biofilm formation.

Inhibition of the expression of the S. aureus agr system. In mixed culture with L. lactis, a lack of induction of the agr system (both RNAII (agrA) and RNAIII) was observed in the post-exponential growth phase. Several mechanisms and signalling pathways have been shown to influence the expression of the agr system. Notably, growth in an acid environment has been reported to affect S. aureus RNAIII expression; however, this remains a matter of [START_REF] Regassa | Glucose and nonmaintained pH decrease expression of the accessory gene regulator (agr) in Staphylococcus aureus[END_REF][START_REF] Weinrick | Effect of mild acid on gene expression in Staphylococcus aureus[END_REF][START_REF] Bore | Acid-shock responses in Staphylococcus aureus investigated by global gene expression analysis[END_REF]. Here, using GDL, we demonstrated that, in the cheese matrix context, a decrease in pH could, at least in part, account for RNAIII downregulation. The expression of agrA was not affected by the addition of GDL, showing that the decrease in pH affected RNAIII level directly and was not dependent for this effect on the autoinducing system (agrABCD). Such uncoupling between RNAII and RNAIII expression has been previously reported [START_REF] Xiong | Activation and transcriptional interaction between agr RNAII and RNAIII in Staphylococcus aureus in vitro and in an experimental endocarditis model[END_REF][START_REF] Garzoni | A global view of Staphylococcus aureus whole genome expression upon internalization in human epithelial cells[END_REF]. Interestingly, downregulation of the agr system (agrA and RNAIII) by L. lactis had been previously observed in a chemically defined medium under environmental conditions far removed from the cheese matrix context [START_REF] Even | Staphylococcus aureus virulence expression is impaired by Lactococcus lactis in mixed cultures[END_REF]. The mechanism(s) involved in agr system attenuation in CDM were not elucidated, but it was established that they are not related to acidification as pH was maintained at 6.6. Hence, although the ability of L. lactis to alter the agr system expression was not restricted to specific conditions, this ability appears to rely on multiple mechanisms acting on RNAII and or RNAIII.

In cheese matrix, one could hypothesize that lactococcal proteolysis acted on agr system expression through a CodY-mediated regulation in S. aureus. However, although CodY reportedly affects agr system expression [START_REF] Majerczyk | Staphylococcus aureus CodY negatively regulates virulence gene expression[END_REF], it did not seem to be involved here since a codY mutant of S. aureus MW2 and the wild-type strain had similar expression profiles of the agr system.

What does enterotoxin expression in cheese matrix imply for food poisoning? In foodstuff, SE production is the main risk associated with S. aureus. Strikingly, three enterotoxins of S. aureus, namely sec4, seh and sel2, were strongly downregulated in cheese matrix compared to a chemically defined medium. This observation at the gene expression level was confirmed at the protein level by quantification of SEC. Our results on sec4 expression and production were in agreement with those of previous studies, which failed to 23 detect SEC production in cheese by strains carrying sec genes and producing SEC in laboratory media [START_REF] Otero | Production of staphylococcal enterotoxins C1 and C2 and thermonuclease throughout the growth cycle[END_REF][START_REF] Otero | Behaviour of Staphylococcus aureus strains FRI 137 and FRI 361 during the manufacture and ripening of Manchego cheese[END_REF][START_REF] Poli | Detection of Staphylococcus aureus and enterotoxin genotype diversity in Monte Veronese, a Protected Designation of Origin Italian cheese[END_REF]. In contrast, sea expression was not affected in cheese matrix and the production of SEA was not inhibited, perhaps accounting for its high frequency in SFP [START_REF] Mossel | Staphylococcus aureus and Related Staphylococci in Foods -Ecology, Proliferation, Toxinogenesis, Control and Monitoring[END_REF][START_REF] Lehn | Frequency of Toxic Shock Syndrome Toxin-Producing and Enterotoxin-Producing Clinical Isolates of Staphylococcus aureus[END_REF][START_REF] Omoe | Comprehensive analysis of classical and newly described staphylococcal superantigenic toxin genes in Staphylococcus aureus isolates[END_REF].

Regulation of SE genes is poorly understood and mainly restricted to classical SE such as SEA and SEC. Expression of the sec4 gene is reported to be under a positive control by the agr system and SaeRS and to decrease in glucose-containing medium (independently of medium acidification) and high salt concentration [START_REF] Genigeorgis | Effect of sodium chloride and pH on enterotoxin C production[END_REF][START_REF] Regassa | Glucose and nonmaintained pH decrease expression of the accessory gene regulator (agr) in Staphylococcus aureus[END_REF][START_REF] Regassa | High sodium chloride concentrations inhibit staphylococcal enterotoxin C gene (sec) expression at the level of sec mRNA[END_REF][START_REF] Voyich | The SaeR/S Gene Regulatory System Is Essential for Innate Immune Evasion by Staphylococcus aureus[END_REF]. The promoter of saeRS was shown to be repressed by a high salt concentration, implying that water activity was also reduced [START_REF] Geiger | The virulence regulator Sae of Staphylococcus aureus: promoter activities and response to phagocytosis-related signals[END_REF]. Taken together, these results establish a link between water activity and expression of sec4 via SaeRS regulation. In cheeses, water activity is lower than in a liquid medium such as CDM (typical water activities of cheeses vary from 0.9 to 0.99) [START_REF] Marcos | Water activity in cheese in relation to composition, stability and safety[END_REF]. Here, water activity was estimated at 0.96 in UF retentate cheese matrix. In agreement, saeS expression was found to be lower in cheese matrix than in CDM from 8 h onward (Table 2). Reduced water activity in the cheese matrix may thus partially account for the downregulation of saeRS, which may in turn be responsible for low sec4 expression.

In addition to the effect of the cheese matrix itself on SE expression, the presence of L. lactis was able to alter SE production. Of note, the effect of L. lactis on SE expression differed between CDM and cheese matrix, highlighting again the crucial role of environmental conditions [START_REF] Even | Staphylococcus aureus virulence expression is impaired by Lactococcus lactis in mixed cultures[END_REF]. Among the main enterotoxins involved in SFP, sea expression was slightly increased in cheese matrix in the S. aureus MW2 strain as well as in S. aureus 257D, by the presence of L. lactis. This higher sea expression in the presence of L. lactis might be due to acidification, as was recently reported [START_REF] Wallin-Carlquist | Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus[END_REF]. In this study, addition of acetic acid provoked an increase of sea expression. This was related to the induction of the sea-encoding prophage and thus to an increase of intracellular sea gene copy number. The genetic context of enterotoxin genes is thus likely to play a major role in the control of enterotoxin gene expression. Enterotoxin genes are carried by different genetic determinants: sea, seg2 and sek2 are carried by the same prophage, sel2 and sec4 are located on the same genomic island and seh exists beyond the right boundary of the chromosome cassette SCCmec, carrying the methicillin resistance gene [START_REF] Baba | Genome and virulence determinants of high virulence community-acquired MRSA[END_REF]. The expression of SE genes might thus respond to various regulation mechanisms depending on their genetic support. If we are to better understand the variations observed here for SE expression, further studies will be required to unravel the impact of mixed culture conditions on S. aureus phage and mobile genetic element biology.

Conclusions.

The behaviour and dynamic response of S. aureus to L. lactis antagonism was explored through in situ gene-expression profiling in conditions mimicking the cheese-making process.

This study provides new insights into how S. aureus adapts to a changing environment and during microbial competition. It also highlights the important role of environmental conditions, thus reinforcing the requirement for in situ approaches. In particular, this study showed that SFP occurrence in the cheese ecosystem is a multilayer problem involving both S. aureus metabolism and virulence expression in response to environmental changes driven by technological processes (temperature, renneting) and microbial activity (L. lactis growth and impact on the cheese matrix). Such parameters, individually or in combination, affect S.

Experimental procedures

Bacterial strain and growth conditions. L. lactis subsp. lactis biovar diacetylactis LD61 (kindly provided by R. Perrin, Soredab, La Boissière Ecole, France) and S. aureus MW2 were used throughout this work. Two additional S. aureus strains isolated from foodstuffs involved in food poisoning and kindly provided by AFSSA-lerqap were used in complementary interaction experiments: S. aureus 257D (isolated from chocolate milk involved in SFP, contains sea), and 253E (isolated from cheese involved in SFP, contains sea, sed). S. aureus was grown in UF-cheeses in pure and mixed cultures with L. lactis LD61 as previously described [START_REF] Ulve | RNA extraction from cheese for analysis of in situ gene expression of Lactococcus lactis[END_REF]. Briefly, the UF retentate was heated to 30 °C and inoculated with S. aureus from subculture on Tryptic Soy Broth (TSB) at 10 6 cfu g -1 for assessment of the worst-case scenario in case of contamination. When added, L. lactis LD61 was inoculated at 10 6 cfu g -1 from subculture on M17. Rennet (Maxiren 180; DSM Food Specialities, Delft, The Netherlands) was added simultaneously at a concentration of 0.3 µl ml -1 . After incubation for 10 h at 30 °C, UF cheeses (20 g) were transferred to 12 °C to mimic the temperature shift encountered by S. aureus strains during the making of soft cheese. S. aureus and L. lactis populations were determined as previously described [START_REF] Nouaille | Transcriptomic response of Lactococcus lactis in mixed culture with Staphylococcus aureus[END_REF].

In order to investigate the effect of acidification on S. aureus, glucono-delta-lactone (GDL, Hansen, Arpagon, France) was added sequentially to the cheese matrix after 5 h and 10 h of growth to mimic the pH decrease in mixed culture.

Chemical analysis of UF cheeses. Determination of sugars, organic acids and free aminoacid content was performed as described previously (Cretenet et al., submitted).

Quantification of enterotoxin C production. Detection and quantification of enterotoxin C

were performed by the EU-CRL screening method (Anonymous, 2007) and confirmed by the EU-CRL confirmatory method when positive [START_REF] Hennekinne | Intralaboratory validation according to the EN ISO 16 140 standard of the Vidas SET2 detection kit for use in official controls of staphylococcal enterotoxins in milk products[END_REF]. Construction of the codY mutant strain. A codY deletion mutant of S. aureus MW2 was obtained by double crossing over as previously described by Hiron et al [START_REF] Hiron | Only one of four oligopeptide transport systems mediates nitrogen nutrition in Staphylococcus aureus[END_REF], using plasmid pCG29 constructed and described by Pohl et al [START_REF] Pohl | CodY in Staphylococcus aureus: a regulatory link between metabolism and virulence gene expression[END_REF].

RNA extraction and transcriptome analysis. RNA samples were extracted from both pure and mixed cultures at different time points (6 h, 8 h, 10 h, 24 h and 7 days) in order to monitor the dynamic response of S. aureus MW2 in cheese matrix, with and without L. lactis. Briefly, cells were separated from the cheese matrix as previously described [START_REF] Ulve | RNA extraction from cheese for analysis of in situ gene expression of Lactococcus lactis[END_REF]. RNA samples from S. aureus in pure or mixed cultures were then obtained and quantified, and their qualities were evaluated as described in Even et al [START_REF] Even | Staphylococcus aureus virulence expression is impaired by Lactococcus lactis in mixed cultures[END_REF]. Labelling and hybridization using a dedicated microarray were performed as previously described (Even et al., 2009). The expression profiles of S. aureus in pure and mixed cultures were analysed with the R software (R Development Core Team, 2007) following the protocol from Even et al. [START_REF] Even | Staphylococcus aureus virulence expression is impaired by Lactococcus lactis in mixed cultures[END_REF]. Hybridizations were carried out with a constant amount of total RNAs to maintain the efficiency and reproducibility of the labelling and hybridisation steps. In mixed cultures, RNA samples were characterized by a mixture of both lactococcal and staphylococcal cDNA, resulting in lower signals. Total RNA yielded a signal (signal /noise ratio >2) in 90% of the spots retained for statistical analysis in pure cultures, but this figure decreased to 18% at 7 days in mixed culture. The sampling time 7 days was therefore removed from our statistical analysis for insufficient quality. Statistical analyses were performed in pure and mixed cultures only until 24 h of culture. Statistical analysis was performed using the ANOVA test considering a P value and FDR lower than 0.05. Genes showing at least a 2-fold change were considered to be differentially expressed. The microarray data for S. aureus in pure and mixed cultures were deposited in the public repository database ArrayExpress under the accession numbers E-MEXP-2617 and XXXX.

All genes showing statistically significant variations of expression with time with a minimum S1. This first analysis was used to reveal trends in gene expression and to establish a list of genes, the expression of which was verified and completed by quantitative RT-qPCR over the complete duration of the experiment (including day 7).

Reverse transcription-quantitative PCR. Reverse transcription-quantitative PCR (RT-qPCR) were performed using the oligonucleotides listed in Table S3 as previously described [START_REF] Even | Staphylococcus aureus virulence expression is impaired by Lactococcus lactis in mixed cultures[END_REF], with three independent biological replicates for all experimental time points. gyrB, was used as an internal standard for normalization as previously described [START_REF] Beenken | Global gene expression in Staphylococcus aureus biofilms[END_REF][START_REF] Bore | Acid-shock responses in Staphylococcus aureus investigated by global gene expression analysis[END_REF]. gyrB was not differentially expressed over time in the microarray data in either the pure or the mixed culture. Gene expression was thus reported relative to gyrB and calculated using the following formula: (copies of each gene/copies of gyrB) X 10 in each cDNA preparation. Statistical analysis was performed using the ANOVA test considering a P value lower than 0.05 to identify genes showing significant change in expression with time. Similarly, statistical analysis was performed using the T-test considering a P value lower than 0.05 to identify, for each time point, genes showing a significant change in expression in pure versus mixed cultures.

  S. aureus, including the accessory gene regulator (agr), a key regulator of bacterial virulence.

  . Final populations of strains 257D and 253E after 7 days were 1.5-and 2log lower, respectively, in mixed culture compared to pure culture (data not shown).

  physiology, including virulence expression and regulation, has been widely studied in the past decades by either targeted or global strategies. However, most of them have been done in laboratory growth conditions or in vivo conditions relevant to human or animal infectious contexts and far removed from the context of food such as cheese matrix. S. aureus

  often associated with biofilm formation. Many of the genes

  aureus behaviour through various regulatory pathways. Deciphering the molecular mechanisms underlying S. aureus response(s) to these multifactorial and dynamic cheese matrix opens new avenues for the development of novel prevention strategies against this major foodborne pathogen.

  change in pure or mixed cultures are presented in Table

Figure legends Fig. 1 :

 legends1 Figure legends

Fig. 2 :

 2 Fig. 2: Gene expression (relative to gyrB) of agrA (RNAII) (A) and RNAIII (B) in S. aureus

Fig. 3 :

 3 Fig. 3: S. aureus population (A), pH (B) and profiles of gene expression (relative to gyrB) of

Table 1 :

 1 Expression profiles of selected genes determined by RT-qPCR. The average gene expression of three independent biological replicates 887 is expressed relative to gyrB x10. Statistically differentially expressed genes are in bold. 888

	MW2	Gene	Description of gene product	Gene expression in pure culture		Gene expression in mixed culture	Ratio of expression Mixed/Pure
	ORF	name
				6 h	8 h	10 h	24 h	7 d	6 h	8 h	10 h	24 h	7 d	6 h	8 h	10 h	24 h	7 d
	Cell division
	MW1069	ftsZ	cell division protein	68.1	60.9	59.8	40.3	74.5	57.3	53.9	28.4	16.9	14.6	0.8	0.9	0.5	0.4	0.2
	MW0604	pbp4	Penicillin-binding protein 4	2.9	1.8	1.6	1.0	3.3	4.0	4.7	2.8	1.1	0.3	1.4	2.7	1.8	1.1	0.1
	Stress response
	MW1532	dnaK	DnaK protein	40.9	26.0	26.2	15.8	19.2	55.8	43.5	60.7	132.7 438.6	1.4	1.7	2.3	8.4	22.9
	MW0477	ctsR	transcription repressor of	5.5	6.9	8.8	9.0	6.1	7.7	7.0	9.5	46.1	149.0	1.4	1.0	1.1	5.1	24.3
			stress genes
	MW2212	ureD	urease accessory protein	8.5	5.5	3.5	1.2	1.8	9.6	13.9	15.0	13.3	35.1	1.1	2.5	4.3	11.4	19.9
	MW2553	arcC	carbamate kinase	1.6	7.7	15.6	0.6	6.5	5.7	15.3	14.5	8.5	27.5	3.5	2.0	0.9	13.4	4.3
	MW2552 arcR	deaminase pathway repressor	2.3	9.0	16.6	1.2	8.3	10.0	21.5	21.4	13.0	39.0	4.4	2.4	1.3	10.8	4.7
	MW1505	sodA	superoxide dismutase	122.0 189.5 209.6 137.8 68.2	135.8 178.4 157.8 506.3 288.3	1.1	0.9	0.8	3.7	4.2
	MW1028 trxA	thioredoxin	75.3	86.6	94.9	57.7	64.0	104.0 108.0 64.5	39.3	25.5	1.4	1.2	0.7	0.7	0.4
	MW1221	katA	catalase	14.4	31.2	51.6	37.4	26.2	16.7	27.5	26.3	20.2	5.5	1.2	0.9	0.5	0.5	0.2
	MW1988 sigB	sigma factor B	33.2	32.4	33.9	31.0	59.4	26.4	43.4	33.9	31.4	12.8	0.8	1.3	1.0	1.0	0.2
	MW2108	asp23	alkaline shock protein 23	428.4 1079 1331 1013 1239	206.8 300.4 423.5 1016 794.0	0.5	0.3	0.3	1.0	0.6
	MW2482 crtN	staphyloxanthin synthase	12.7	12.5	13.7	9.6	20.3	6.2	8.0	8.8	4.4	4.7	0.5	0.6	0.6	0.5	0.2
	MW2469	clpL	ATPdependent Clp	83.0	165.2 255.2 46.9	103.7	104.7 82.3	182.4 353.2 791.7	1.3	0.5	0.7	7.5	7.6
			proteinase chain
	MW0480 clpC	ATPdependent Clp proteinase	13.5	13.4	11.7	14.7	14.0	12.7	12.3	27.2	145.9 422.1	0.9	0.9	2.3	9.9	30.2
			chain
	MW0730	clpP	ATPdependent Clp protease	40.6	41.7	41.5	32.2	45.4	59.9	57.9	42.5	34.8	21.0	1.5	1.4	1.0	1.1	0.5
	MW1290 cspA	cold shock protein A	22.2	16.5	23.3	12.4	9.4	35.7	40.2	19.0	9.4	5.4	1.6	2.4	0.8	0.8	0.6
	MW2623 cspB	cold shock protein B	238.0 147.5 130.5 25.9	45.2	248.7 214.1 131.6 64.5	43.1	1.0	1.5	1.0	2.5	1.0
	Virulence
	MW1959 hld	deltahemolysin	86.8	494.5 1104 4243 3293	94.5	233.8 285.8 164.9 189.5	1.1	0.5	0.3	3.9E-	0.1
		(RNAIII)															02
	MW2586	icaA	intercellular adhesion protein	0.2	0.5	0.9	0.1	0.3	0.04	0.04	0.01	ND	ND	0.2	0.1	5.7E-	NC	NC
			A													03
	MW2559	isaB	immunodominant antigen B	21.4	35.0	56.9	57.7	11.1	23.6	24.4	22.1	83.7	158.7	1.1	0.7	0.4	1.5	14.3

Gene name Description of gene product Gene expression in pure culture Gene expression in mixed culture Ratio of expression Mixed/Pure

	MW2
	ORF
				6 h	8 h	10 h	24 h	7 d	6 h	8 h	10 h	24 h	7 d	6 h	8 h	10 h	24 h	7 d
	MW0764 clfA	clumping factor A	74.8	123.6 206.5 147.9 144.8	52.4	74.6	80.5	337.8 409.2	0.7	0.6	0.4	2.3	2.8
	MW0516 MW0124 cap8A sdrC MW1885 sak MW1889 sea MW1937 seg2 MW1938 sek2 MW0760 sel2 MW0051 seh MW0759 sec4 Carbohydrate metabolism SerAsp rich fibrinogenbinding, capsular polysaccharide Cap8A staphylokinase enterotoxin A enterotoxin G enterotoxin K enterotoxin L enterotoxin H enterotoxin C MW2115 lacG 6phosphobetagalactosidase MW1641 pykA pyruvate kinase MW0217 lctE Llactate dehydrogenase MW0568 adh1 alcohol dehydrogenase I MW0202 pflA formate acetyltransferase enzyme MW1237 citb aconitate hydratase MW2526 mqo2 malate/quinone oxidoreductase MW1129 sucD succinylCoA synthetase MW2460 cidC pyruvate oxydase MW2462 cidA holin-like protein cidA MW0238 lrgA holin-like protein LrgA MW2132 alsS alpha-acetolactate synthase F o r P e e r R e v i e w 0.1 0.2 0.1 0.1 0.1 0.6 2.2 19.5 46.7 64.6 55.8 26.8 10.8 11.6 1.5 1.7 1.4 0.4 0.6 2.9 3.1 5.8 6.7 6.0 2.2 1.1 10.4 14.9 3.4 4.1 4.9 1.8 2.2 2.9 3.6 1.5 1.7 1.9 0.8 0.9 1.6 2.0 2.5 2.8 3.6 1.2 1.5 4.1 5.5 1.6 1.4 2.5 0.1 0.2 2.7 3.3 3.1 3.4 3.7 1.8 4.6 4.1 7.7 2.4 11.5 20.5 17.1 55.0 9.8 30.4 25.8 24.5 29.8 15.1 24.7 27.0 24.3 93.4 120.2 217.7 4.2 73.9 71.4 73.3 18.3 25.3 41.7 0.5 4.9 15.4 19.8 65.5 122.7 125.4 3.6 20.0 176.7 269.7 73.1 4.1 20.5 0.9 6.5 1.9 0.9 3.0 2.1 4.6 19.4 33.9 13.4 3.5 12.4 34.2 47.1 27.0 18.2 15.2 32.0 28.0 48.1 36.3 31.8 25.9 25.0 42.1 36.0 28.0 80.9 97.6 84.7 63.5 31.2 87.0 89.7 38.2 12.9 20.6 21.4 26.6 33.6 9.5 11.9 22.1 0.1 0.1 0.1 0.1 0.3 0.5 1.5 2.3 0.4 0.5 0.5 0.1 1.0 2.4 1.1 0.5 0.8 0.8 1.0 0.5 4.4 2.9 8.5 15.6 O n l y 2.8 3.1 66.2 13.9 0.5 1.6 5.6 3.2 0.6 0.5 0.4 0.7 2.6 3.8 1.8 4.2 1.9 2.1 7.0 23.6 28.2 77.6 3.6 8.7 1.2 3.6 28.2 86.1 46.0 7.6 23.5 38.8 25.6 18.1 78.9 50.8 4.8 3.3 0.3 0.9 48.0 87.9	4.1 0.6 1.9 1.8 0.9 1.1 1.7 1.7 1.3 4.0 1.0 0.8 0.8 2.7 1.2 0.9 1.1 0.7 3.9 5.3 3.6	14.0 0.2 1.9 2.2 0.9 1.2 1.9 2.3 2.3 2.7 1.0 0.6 0.8 2.2 0.9 1.0 0.9 0.6 14.2 2.2 11.2	32.9 0.3 0.6 1.1 0.4 0.5 0.8 0.8 1.3 0.9 1.1 0.1 0.1 0.6 0.6 0.9 0.5 1.0 19.4 1.0 15.2	37.8 1.2 1.2 2.5 0.4 0.5 2.1 12.7 1.1 0.4 1.9 0.8 2.5 7.9 1.7 0.9 0.4 3.0 79.0 2.0 89.2	30.1 0.5 2.6 2.9 0.2 0.8 2.6 17.2 0.4 0.4 3.1 0.1 0.7 4.3 0.4 1.6 0.6 1.5 12.2 1.0 20.1
	MW0100 butA	acetoin(diacetyl)reductase	9.2	13.5	20.8	24.7	14.5	12.6	15.9	23.9	48.2	193.1	1.4	1.2	1.2	2.0	13.3
	Nitrogen metabolism
	MW0705	pepT	aminotripeptidase	9.7	11.0	9.8	10.2	10.8	9.9	9.9	7.2	4.2	5.1	1.0	0.9	0.7	0.4	0.5
	MW1694		XaaHis dipeptidase homolog	2.6	3.1	3.3	2.2	2.7	3.4	4.2	3.8	1.3	0.9	1.3	1.3	1.2	0.6	0.3
	MW1584 relA	GTP pyrophosphokinase	6.0	7.5	7.7	4.1	7.6	5.6	5.2	4.5	1.0	1.0	0.9	0.7	0.6	0.2	0.1
	MW1977 ilvD	dihydroxy-acid dehydratase	11.2	9.2	4.1	25.2	2.4	46.0	4.7	2.2	0.2	1.1	4.1	0.5	0.5	7.8E-	0.4
																	03
																		44

Gene name Description of gene product Gene expression in pure culture Gene expression in mixed culture Ratio of expression Mixed/Pure

				6 h	8 h	10 h	24 h	7 d	6 h	8 h	10 h	24 h	7 d	6 h	8 h	10 h	24 h	7 d
	MW1981 leuA	2-isopropylmalate synthase	16.6	15.0	13.0	34.5	3.8	116.1 13.4	1.8	1.0	2.7	7.0	0.9	0.1	2.8E-	0.7
																	02
	MW1138 codY	transcription pleiotropic	24.0	23.8	24.3	13.7	30.1	31.9	35.6	26.8	10.4	11.3	1.3	1.5	1.1	0.8	0.4
			repressor
	Regulators
	MW1963	agrA	accessory gene regulator A	20.7	38.7	51.8	111.1 200.4	25.1	61.4	93.4	59.7	53.1	1.2	1.6	1.8	0.5	0.3
		(RNAII)
	MW0580	sarA	staphylococcal accessory	313.0 459.2 544.4 417.4 420.8	257.3 298.7 342.4 418.0 162.0	0.8	0.7	0.6	1.0	0.4
			regulator A
	MW1705 rot	transcriptional regulator	36.8	31.1	36.3	12.1	5.6	38.7	37.7	24.9	22.0	19.5	1.1	1.2	0.7	1.8	3.5
	MW0667 saeS	histidine protein kinase	20.1	18.5	11.4	5.3	27.3	19.5	28.2	11.7	3.7	1.3	1.0	1.5	1.0	0.7	0.0
	MW0648 mgrA MW2585 icaR MW1446 srrA F transcriptional regulator operon ica repressor staphylococcal respiratory response o r	37.1 8.6 27.3	26.7 13.0 25.5	27.6 19.7 28.4	17.3 10.5 11.7	20.6 3.9 17.9	48.1 10.6 29.0	32.0 13.3 31.7	18.1 9.3 15.6	12.2 12.0 21.9	9.4 3.0 9.5	1.3 1.2 1.1	1.2 1.0 1.2	0.7 0.5 0.6	0.7 1.1 1.9	0.5 0.8 0.5
			P
			e
			e r
			R
			e
				v i e
						w
						O n l
								y

Table 2 .

 2 Expression ratio of SE genes and saeS in S. aureus MW2 strain grown in CDM versus cheese matrix, as determined by RT-qPCR, using gyrB as an internal standard for normalisation. Data on CDM were adapted from Even et al.[START_REF] Even | Staphylococcus aureus virulence expression is impaired by Lactococcus lactis in mixed cultures[END_REF]. Statistically

	differentially expressed genes between CDM and cheese matrix are in bold (as determined by
	T-test considering a P value lower than 0.05).
				Ratio of expression CDM/cheese matrix
	MW2	Gene	Description of gene	6 h -	8 h -late	10 h -	24 h -
	ORF	name	product	exponential	exponential	entry into	stationary
				phase	phase	stationary	phase
						phase
	MW1889 sea	enterotoxin A	0.8	1.2	0.8	0.5
	MW1937 seg2 enterotoxin G	1.4	2.3	1.2	1.4
	MW1938 sek2	enterotoxin K	1.8	2.7	1.4	1.3
	MW0760 sel2	enterotoxin L	3.6	7.9	3.8	41.6
	MW0051 seh	enterotoxin H	5.8	10.1	4.5	64.0
	MW0759 sec4	enterotoxin C	3.3	11.6	15.1	22.8
	MW0667 saeS histidine protein	0.5	3.1	2.0	7.6
			kinase

Table S1 . Expression profiles of S. aureus MW2 genes that exhibited significant variations in pure and/or mixed cultures with L. lactis LD61 in cheese matrix.

 S1 Expression levels in pure and mixed cultures at 6, 8, 10 and 24 h, detected with the S. aureus microarray and presented as normalized cDNA/gDNA ratios. Only genes that showed significant variations in expression levels in pure and or mixed cultures over time (as determined by the ANOVA test with the criterion of an FDR of 0.05) and a degree of change of 2-fold were defined as differentially expressed. Statistically differentially expressed genes are in bold.

	MW1063 MW2304 MW1531 MW0124 MW0018	ftsL gltT dnaJ cap8A vicR	cell division protein proton/sodiumglutamate symport protein DnaJ protein capsular polysaccharide synthesis enzyme Cap5A response regulator	5.1 7.0 3.9 4.0 1.9	5.6 5.4 2.2 10.7 12.2 15.9 3.4 2.0 4.1 7.0 1.8 2.0 3.1 3.5 2.7	2.5 3.5 2.6 1.6 1.7	1.7 2.2 1.8 1.3 1.5	1.4 1.6 2.8 2.3 1.8	0.7 1.0 3.0 4.1 2.0
	MW1069 MW1694 MW1532 MW0139 MW1637	ftsZ dnaK cap8P phoP	cell division protein XaaHis dipeptidase homolog DnaK protein capsular polysaccharide synthesis enzyme Cap5P alkaline phosphatase synthesis transcriptional regulatory	5.9 0.6 5.5 1.0 1.6	4.3 0.7 5.0 0.9 3.1	5.0 0.9 4.0 1.0 3.4	5.3 1.1 4.6 0.4 3.5	3.4 1.9 3.7 0.7 1.4	2.2 1.5 2.2 0.7 1.3	1.9 1.2 4.7 0.8 1.5	1.4 0.7 7.5 1.2 1.7
	MW2 ORF DNA replication, recombination and repair MW1362 MW1168 MW0720 MW1029 MW1078 Transcription, translation and cellular division MW2045 MW0498 MW1513 MW1623 MW0502 MW0503 IGS2 MW0466 MW2007 Cell wall biosynthesis and associated protein MW2589 MW1363 MW1064 MW0604 MW0031 MW2013 Carbohydrate metabolism MW2115 MW0217 MW0568 MW1129 MW2459 MW2084 MW0163 MW0245 MW0218 MW2538 MW2175 Nitrogen metabolism MW1980 MW0705 MW2522 MW0875 MW0868 MW0871 MW2553 MW1138 Energetic metabolism MW0999 MW0588 MW2526 Vitamins, nucleotides and nucleic acid metabolism MW2536 MW2196 MW2186 MW0476 Iron metabolism MW1011 MW1834 MW0200 Stress response MW0477 MW2469 MW1136 MW1618 MW0726 MW2535 MW0356 MW1221 MW2212 MW0453 MW1991 MW2108 MW1682 MW0781 Virulence MW1963 sRNA_RNAIII MW0668 MW2418 MW2420 MW2421 MW0764 MW1369 MW2559 MW0517 MW0627 MW2589 MW1801 MW0802 MW2096 MW2344 Signal transduction/ secretion MW1775 MW2149 Transporters MW1235 MW0988 MW1507 MW1554 MW2298 MW0586 MW0605 MW2478 MW2334 MW0213 MW0879 MW1111 MW2552 Antibiotic related genes MW0623 MW1158 Others sRNA_sgrA sRNA_sbrD sRNA_45S F Gene Name Description of gene product hu DNAbinding protein II recA RecA protein uvrB exinuclease ABC subunit B uvrC excinuclease ABC subunit C tnp transposase rho transcription termination factor Rho rpoC RNA polymerase betaprime chain sigA RNA polymerase sigma factor rpmI 50S ribosomal protein L35 fus translational elongation factor G tufA translational elongation factor TU intergenic sequence 16S-23S ftsH celldivision protein F o r P e e r R e v i e w rod shape determining protein RodA icaC intercellular adhesion protein C gpsA glycerol3phosphate dehydrogenase pbpA penicillinbinding protein 1 pbp4 penicillin binding protein 4 mecA penicillin binding protein 2 prime lipoprotein precursor lacG 6phosphobetagalactosidase lctE Llactate dehydrogenase adh1 alcohol dehydrogenase I sucD succinylCoA synthetase ptsG PTS system, glucosespecific mtlA PTS system, mannitol specific glcA PTS enzyme II rbsD ribose permease PTS enzyme, maltose and glucose specific similar to Mg citrate secondary transporter glucose uptake protein homolog ilvC alphaketobetahydroxylacil reductoisomerase F o r P e e r R e v i e w pepT aminotripeptidase amino acid transporter appF oligopeptide transport system ATPbinding protein AppF homologue oppB oligopeptide transport system permease protein opp3 oligopeptide transport system ATPbinding protein OppF homologue arcC carbamate kinase codY transcription pleiotropic repressor ctaB cytochrome caa3 oxidase homologue MnhD homologue,similar to Na+/H+ antiporter subunit mqo2 malate/quinone oxidoreductase anaerobic (class III) ribonucleotide reductase small subunit chain modB probable molybdenum transport permease moaA molybdenum cofactor biosynthesis protein A nupC pyrimidine nucleoside transport protein isdB ironregulated cell wallanchored protein ferritin similar to periplasmicironbinding protein BitC F o r P e e r R e v i e w ctsR transcription repressor of stress genes clpL ATPdependent Clp proteinase chain clpQ heat shock protein HslV clpX protease trxB thioredoxine reductase cudT choline transporter ahpF alkyl hydroperoxide reductase subunit F katA catalase ureD urease accessory protein spoVG stage V sporulation protein G homologue rsbU sigmaB regulation protein asp23 alkaline shock protein 23 general stress proteinlike protein similar to general stress protein agrA accessory gene regulator A RNAIII RNAIII saeR response regulator sarH2 staphylococcal accessory regulator A homolog fnbB fibronectinbinding protein homolog fnb fibrinogenbinding protein fnb fibrinogenbinding protein ebpS elastin binding protein isaB immunodominant antigen B sdrD SerAsp rich fibrinogenbinding F o r P e e r R e v i e w secretory antigen SsaA homologue protein aur zinc metalloproteinase aureolysin perR transcription regulator Fur family homolog hemolysin hemolysin III hlgB gammahemolysin component B traP signal transduction protein secY preprotein translocase SecY subunit mscL largeconductance mechanosensitive channel Mn2+transport protein ABC transporter MreB probable transmembrane transport protein cationic transporter Na+/H+ antiporter ATPbinding cassette transporter A copA coppertransporting ATPase ABC transporter (ATP binding subunit) similar to nickel ABC transporter nickelbinding protein spx conserved hypothetical protein transcription factor fapR transcriptional regulator vraF ABC transporter ATPbinding protein similar to metallobetalactamase family protein sgrA sbrD 45S o r F o r P e e r R e v i e w MW1494 exogenous DNAbinding protein comGC MW0906 competence transcription factor P MW1619 tig prolyl isomerase e e r R e Regulators MW1446 srrA staphylococcal respiratory response protein MW1445 srrB staphylococcal respiratory response protein MW1790 yhcS two-component sensor histidine kinase v i e MW1789 yhcR two-component response regulator homolog w	Gene expression in pure culture 6 h 8 h 10 h 24 h 54.3 86.8 84.3 70.6 9.3 9.5 11.1 9.9 0.7 0.7 0.7 1.4 0.8 0.9 1.1 1.6 0.9 1.0 1.1 2.5 2.0 1.5 1.3 1.3 6.0 3.1 2.1 6.7 2.4 2.4 2.4 5.0 9.3 6.0 4.0 3.9 26.9 12.6 5.1 11.1 44.1 24.2 11.8 20.0 24.3 18.5 11.8 10.6 6.1 6.3 5.7 8.2 O n l y 4.1 14.3 25.5 106.1 2.6 Gene expression in mixed culture 6 h 8 h 10 h 24 h 53.1 24.2 20.2 10.8 4.4 2.9 2.9 1.9 0.8 0.8 0.7 0.8 1.2 1.4 1.4 1.3 1.4 1.9 2.0 2.3 1.3 1.0 0.9 0.7 3.2 1.8 1.5 1.2 1.6 1.4 1.9 2.0 6.0 3.3 3.1 2.3 13.8 5.3 3.8 1.8 23.5 11.2 8.4 3.5 13.1 7.4 6.5 3.4 5.2 3.6 3.7 1.8 3.0 4.8 2.3 0.4 1.0 1.4 1.5 0.7 0.6 0.6 0.9 3.8 2.8 2.0 2.4 2.2 1.6 1.3 1.0 2.2 1.6 1.2 0.9 1.6 1.3 1.3 1.3 4.2 2.4 1.3 2.0 2.3 1.4 1.2 0.9 0.5 0.7 1.2 1.3 0.9 1.5 1.2 1.2 4.6 3.6 1.6 2.8 3.2 1.6 1.2 1.0 0.6 1.7 3.1 4.6 1.2 1.6 1.7 1.0 5.7 8.6 12.7 0.8 2.7 1.8 0.9 0.8 1.5 1.7 3.2 1.0 1.1 1.0 0.8 0.9 13.7 17.4 16.5 19.7 6.8 4.2 2.9 1.7 2.4 0.7 0.8 0.5 0.6 0.5 0.5 0.5 1.5 2.6 2.3 3.6 1.0 0.8 1.0 1.3 1.5 0.4 0.4 0.3 0.5 0.7 0.6 0.8 0.7 1.8 1.3 0.6 0.8 0.8 0.6 0.6 0.8 0.5 0.7 0.3 0.8 0.6 0.5 0.7 3.3 2.1 1.9 1.5 1.8 1.0 0.8 0.7 1.1 0.7 0.5 0.7 1.1 1.2 1.1 1.2 5.6 6.3 1.2 5.7 5.4 0.7 0.6 0.6 O n l y 1.9 2.5 2.5 4.6 1.2 1.0 1.0 1.1 7.1 5.0 4.2 3.2 3.3 1.2 1.0 0.6 0.6 1.0 0.6 0.5 3.1 2.3 1.3 1.0 6.1 5.9 6.1 4.2 2.7 2.2 1.8 1.0 6.4 5.9 5.3 5.0 2.4 1.8 1.3 1.0 0.3 1.2 2.1 0.3 0.7 0.9 1.1 0.8 3.8 4.6 3.2 3.2 2.4 1.7 1.6 0.9 4.2 4.4 4.9 3.1 2.7 2.1 1.5 1.2 15.5 7.9 4.0 6.3 7.3 3.6 2.7 1.6 3.1 1.5 1.7 1.9 1.2 1.0 1.0 0.9 0.7 0.7 1.1 0.3 0.7 0.8 0.9 0.9 1.4 1.6 1.8 3.2 0.5 0.6 0.7 0.7 0.5 0.5 0.5 0.9 0.5 0.6 0.7 0.8 2.5 2.3 2.0 1.6 2.0 1.4 1.0 0.8 0.4 0.4 0.6 0.6 0.7 0.9 1.0 1.1 0.9 4.6 8.6 5.4 0.8 1.8 1.7 3.1 0.5 0.9 0.9 1.2 0.6 0.8 0.7 0.9 O n l y 1.2 1.6 2.0 3.3 1.2 1.1 1.3 2.8 6.5 11.9 15.5 5.3 4.0 1.9 6.5 7.2 3.7 3.9 3.2 3.3 2.8 2.0 1.8 1.3 5.5 5.0 4.0 4.6 2.3 1.5 1.5 1.6 5.7 5.5 6.0 5.8 4.0 3.1 3.2 1.6 0.3 0.4 0.5 1.1 0.7 0.8 0.6 0.6 0.6 0.7 0.9 4.5 1.0 0.9 1.0 1.1 10.4 21.7 24.5 36.2 9.9 9.6 13.8 9.1 1.2 0.7 0.6 0.4 1.1 1.2 1.6 1.3 1.4 2.3 4.1 6.5 0.9 0.9 1.3 1.5 4.1 3.5 2.8 2.9 1.6 1.1 1.0 0.7 28.3 57.4 72.3 78.6 14.7 9.1 26.6 35.7 13.3 24.2 34.6 41.5 6.6 4.4 8.4 7.8 4.2 10.9 17.0 29.1 2.2 1.8 4.6 6.6 1.4 2.3 3.3 10.8 1.2 1.5 3.0 1.5 8.2 39.2 68.5 116.7 6.5 8.3 15.1 6.3 4.5 4.3 3.9 2.6 2.4 2.0 1.5 0.7 0.2 0.3 0.5 1.1 0.5 0.7 0.8 1.0 0.5 0.3 0.2 0.2 0.5 0.5 0.5 0.5 0.5 0.3 0.2 0.2 3.4 2.8 5.4 9.5 6.0 12.7 16.2 18.3 3.4 2.8 5.4 9.5 2.1 3.5 5.8 7.0 2.1 1.8 2.8 2.3 1.3 2.7 3.7 5.8 1.2 1.1 1.1 2.3 0.2 0.2 0.2 0.4 0.4 0.6 0.6 O n l y 6.9 7.8 10.3 9.1 3.8 3.6 3.4 1.8 3.0 3.0 1.4 0.9 2.2 1.7 0.9 1.3 1.5 2.1 4.5 1.1 1.0 1.0 1.0 0.9 0.6 1.0 1.1 1.4 0.6 0.7 0.8 1.1 0.9 0.9 1.2 2.8 0.9 0.9 0.9 1.0 0.9 0.5 0.5 0.2 0.7 0.7 0.6 0.6 3.9 4.2 4.4 2.5 1.9 1.4 1.3 1.0 45.0 20.5 10.7 26.4 21.6 8.8 6.0 3.2 6.8 7.0 10.2 9.5 3.1 2.3 1.9 1.4 3.1 2.8 3.8 4.5 13.3 16.9 11.1 4.5 1.1 0.8 0.7 0.8 1.0 0.9 1.1 1.5 0.4 0.5 0.5 0.7 0.4 0.5 0.5 0.7 0.2 0.6 0.6 1.5 0.4 0.5 0.4 0.5 5.8 2.2 1.4 2.7 3.0 1.6 1.6 1.2 1.2 1.2 1.3 3.3 2.6 2.3 3.6 3.1 0.5 0.5 0.4 1.0 0.5 0.6 0.9 1.7 0.5 0.3 0.4 0.4 0.6 0.7 0.7 0.8 0.4 0.3 0.4 0.2 0.6 0.6 0.6 0.6 8.0 10.2 16.2 24.3 4.5 2.9 3.7 3.1 3.4 3.0 2.0 1.4 2.0 1.4 1.1 0.9 0.4 1.0 2.1 1.0 0.9 1.1 1.4 1.0 1.2 1.3 1.3 0.7 0.9 0.9 0.7 0.9 4.3 3.4 3.2 2.2 2.2 1.4 1.6 1.2 1.3 2.0 2.9 5.1 2.6 2.6 3.8 4.5 0.2 0.2 0.3 0.6 0.4 0.5 0.7 0.9 0.2 0.3 0.5 0.5 1.6 1.6 2.9 2.8 7.6 8.9 9.4 10.1 5.9 3.0 2.3 2.0 0.1 0.2 0.2 0.2 0.5 0.6 0.7 0.8 3.7 2.5 2.0 1.9 3.8 2.3 1.9 1.7 O n l y 3.4 3.3 2.8 2.4 2.2 1.6 1.3 1.2 1.1 0.9 0.8 0.6 1.1 1.0 1.1 1.1 0.6 0.7 0.9 2.0 0.6 0.6 0.7 0.8 0.9 0.6 0.7 0.8 0.9 0.9 1.0 1.3 1.5
			O n l
			y

Table S3 .

 S3 List of oligonucleotides used in this study

	MW2 ORF	Gene name	Description of gene product	Forward primer	Reverse primer
	MW0005 gyrB	gyrase	AATCAGCGTTAGATGTAGCAAGC GTCACGACCAGATTTTGTAGACC
	MW0051 seh	enterotoxin H	TGGTCAATATAATCACCCATTCA TCAAATCATTGCCACTATCACC
	MW0100 butA	acetoin(diacetyl)reductase	TTATCAATGCAACATCTCAAGCA CTAAATCTTGTGCGGCTACTTGT
	MW0124 cap8A	capsular polysaccharide Cap8A	GCGCTATTGTTACATTTTTCGTC	TCTTGTGCCATAAACTGAGGATT
	MW0202 pflA	formate acetyltransferase enzyme	AGGGACACTTACATTCTGTCGAA	CAAGTATCTGGATTGTGGCAATA
	MW0217 lctE	Llactate dehydrogenase	AATGAAGATGCCGTATTGACTGT GATTTCTACGACGTTGCGAATAC
	MW0238 lrgA	holin-like protein LrgA	CTGTTAAGTTAGGCGAAGTCGAA	GCTTGGCTAATGACACCTAAAGA
	MW0477 ctsR	transcription repressor of stress genes CAGCTGATTGGACCTTCTATTTC	GCTTGAATCATTTTAGCTTCACG
	MW0480 clpC	ATPdependent Clp proteinase chain	AGTTATTGCAGGATGGACAGGTA	TTTGCCCAATAACTCTCTCATGT
	MW0516 sdrC	SerAsp rich fibrinogenbinding,	GGCATGATACCAAATCGATTAAA CTTCATGACCACTTAACCCAAAA
	MW0568 adh1	alcohol dehydrogenase I	TGCTGATTTTGGTGATGTTACAG	TAGACACACGGTCTCCAATTTTT
	MW0580 sarA	staphylococcal accessory regulator A TTGCTTTGAGTTGTTATCAATGG	TTTCTCTTTGTTTTCGCTGATGT
	MW0604 pbp4	penicillin binding protein 4	ATCCAGCGTCTATGACCAAATTA TTCTTTGTTCGTCATTGTGACAG
	MW0648 mgrA	transcriptional regulator	CGAAGTCGATCAACGTGAAGTAT AGAAGAAGCTGAAGCGACTTTGT
	MW0667 saeS	histidine protein kinase	TCAATATTGCAACCATATGAGCA AGTGGCGTTCGATATTGATAAAA
	MW0705 pepT	aminotripeptidase	GATATTCCAGCAAACACTGAACC GAAGTTACCGCAACCTGTAAAGA
	MW0730 clpP	ATPdependent Clp protease	GCTGGTTTTGCGATTTATGATAC	TTGCACCAGCTGCTAATAAGAAT
	MW0759 sec4	enterotoxin C	AAACATGAAGGAAACCACTTTGA TTTGCACTTCAAAAGAAATTGTG
	MW0760 sel2	enterotoxin L	GGTTACCGCACAAGAAATAGATG TGCCGTATTCTTTACCTTTACCA
	MW0764 clfA	clumping factor A	TTACGAATCAGTTGACGAATGTG AGGCACTGAAAAACCATAATTCA
	MW1028 trxA	thioredoxin	TAAAATGATCGCTCCGGTATTAG TTTAGCTGCAGTTGATGGATTTT
	MW1069 ftsZ	cell division protein	CTTCAAATTCATCAAATGCACAA CGTCTTGTTCTTCTTGAACGTCT
	MW1129 sucD	succinylCoA synthetase	ATGCCTGGCTATATTCACAAAAA CCAACAGCTGTAGTTTGACCAAT
	MW1138 codY	transcription pleiotropic repressor	ATGGAAATCTTACGTGAGAAGCA ATGTTCAATCGCTTCTTTTTCAG
	MW1221 k atA	catalase	ATTTACGTCATATGCATGGGTTC	TTTTCAATACCTTGTTGCGTTCT
	MW1237 citb	aconitate hydratase	GCGAGGTACCATTTAAACCTTCT	AGTAATATCTCCCCCAACGTCAT
	MW1290 cspA	cold shock protein A	TCGAAGTTGAAGGAGAAAATGAC ACTTCAAACTCAACAGCTTGACC
	MW1446 srrA	staphylococcal respiratory response	ATCTTTTGAAATCCATGAAGCAA TCCATTTCAGGCAACATTAAATC
	MW1505 sodA	superoxide dismutase	TTCTGGGAGTTACTTTCACCAAA	CTGCTTTGTCAGCAAATTCTTTT
	MW1532 dnaK	DnaK protein	AAGGTTCACGTACAACACCATCT GATTGAACAGTGTTTGGGTTTGT
	MW1584 relA	GTP pyrophosphokinase	TCGAAGGCGATATAAATGGTAGA ATAACACGTATCGCCAACAAATC
	MW1641 pykA	pyruvate kinase	GTATTGGACGTGGATCAGTTGTT	ACAAACGTTTCATCGATTGAGTT
	MW1694		XaaHis dipeptidase homolog	AATTATTGAAAAAGGCGACATGA TCAGTTTAGGATCTGGTTCTCCA
	MW1705 rot	transcriptional regulator	ATTGGGAGATGTTTAGCATGAAA TTCAATCTCGCTGAAAATTGAGT
	MW1885 sak	staphylokinase	CGCAAAGATCGAAGTCACTTATT ATGCTCTGATAAATCTGGGACAA
	MW1889 sea	enterotoxin A	TAATCGATTGACCGAAGAGAAAA ATAACGTCTTGCTTGAAGATCCA
	MW1937 seg2	enterotoxin G	TACGATTTGTTTTACACCGGAAC	TCCAAATGAAAATTCTCTGCATC
	MW1938 sek2	enterotoxin K	CTACACAGGAGATGATGGGCTAC CATCCAAATGGAATTTCTCAGAC
	MW1959 hld (RNAIII) deltahemolysin	TAAGGAAGGAGTGATTTCAATGG GTGAATTTGTTCACTGTGTCGAT
	MW1963 agrA (RNAII) accessory gene regulator A	CCTCGCAACTGATAATCCTTATG	ACGAATTTCACTGCCTAATTTGA
	MW1977 ilvD	dihydroxy-acid dehydratase	GATGAAGAAAGATGGCACGTTAC GAGGGTGAATGACATCAAAGTTC
	MW1981 leuA	2-isopropylmalate synthase	CGCTTCTTTATCAATGCCATAAC	AACTTGTTGGTGTAAGCACGACT
	MW1988 sigB	sigma factor B	TTCACCTGAGCAAATTAACCAAT ATCTTCGTGATGTGATTGTCCTT
	MW2108 asp23	alkaline shock protein 23	AGACATGAAAGGTGGCTTAACTG GCTTGTTTTTCACCAACTTCAAC
	MW2115 lacG	6phosphobetagalactosidase	AGTATTCTCGTGAAACGATGGAA GCTGCATCTAAAATGGCATAATC
	MW2132 alsS	alpha-acetolactate synthase	CCAATTTACCCGTTGTAGAAACA ATTCATCACCAACTTGATTACGG
	MW2212 ureD	urease accessory protein	ATTCGATTGTCGTGTTGCTATTT	TCTGCGATATAGCTTTGGATGAT
	MW2460 cidC	pyruvate oxydase	ACAATGCAGGATGCAGATTTACT TTTTAGGATTTGTGTCGATTTGG
	MW2462 cidA	holin-like protein cidA	TGTACCGCTAACTTGGGTAGAAG	TTCGGAAGCAACATCCATAATAC
	MW2469 clpL	ATPdependent Clp proteinase chain	AAGATGCACTGATTCGACTTGAT TGTCATCATAACCGACATAACCA
	MW2482 crtN	staphyloxanthin synthase	AAGATGTTTTTACAGCGTGTGGT	AATCTGTAGGCACCGTTATACGA
	MW2526 mqo2	malate/quinone oxidoreductase	CGATGCCAAAGTTTATGGTAAAG AGCAAATGGTCCAAATAACAATG
	MW2552 arcR	deaminase pathway repressor	TTCTTGGATTGCCTAGAGAATTG	TTCATGTGTTGCTGCTCATTATC
	MW2553 arcC	carbamate kinase	AATCCAACAAGCTAAATCGAACA GATTGATTTCAGTTTCCAACCAA
	MW2559 isaB	immunodominant antigen B	AGGAAGCTGATAGCGGTATTTTT	TGTCCAACTGTTACTTCGACAAC
	MW2585 icaR	operon ica repressor	AAATCGAACTATTCAATTGATGC CAGAAAATTCCTCAGGCGTATTA
	MW2586 icaA	intercellular adhesion protein A	AAGAAGCCTGACATAAATGTGGA TCTCGTATTTGAGTGCAAGAACA
	MW2623 cspB	cold shock protein B	AATGGTTTAACGCAGAAAAAGGT TCTTCTAAAGTTTTGTAGCCATCG

Wiley-Blackwell and Society for Applied Microbiology

Acknowledgements

Spotting and hybridization were provided by the transcriptomic platform of Ouest Genopole (Rennes, France) and the transcriptomic platform IFR140 GFAS (Rennes, France), respectively. Authors are grateful to Aurélie Le Cam, Jérome Monfort, Amandine Etchevery and Régis Bouvet for their help and technical assistance during this work. We thank Christiane Wolz for providing us with plasmid pCG29. Marina Cretenet is a recipient of a CIFRE PhD fellowship from Centre National Interprofessionnel de l'Economie Laitière. This work was financially supported by the French National Research Agency (ANR) project

GenoFerment

(ANR-05-PNRA-020) and Nabab (ANR-08-ALIA-11).

F o r P e e r R e v i e w O n l y