Annabelle Fernandez

Frédéric Borges
email: bourget@nancy.inra.fr

Brigitte Gintz

Bernard Decaris

Nathalie N Leblond-Bourget

The rggC locus, with a frameshift

Keywords: Streptococcus thermophilus, oxidative stress response, transcriptional regulator, Rgg, genetic variability, frameshift mutation

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

INTRODUCTION

The genus Streptococcus is composed of numerous pathogenic species and the food grade species S. thermophilus which is used as a starter of fermentation in yogurt and cheese production. During the biotransformation process, the formation of reactive oxygen species (ROS) may occur and be deleterious to the cells. S. thermophilus is an anaerobic aerotolerant organism that can grow in presence of oxygen and survive in low concentrations of superoxide and hydroxyl radicals [START_REF] Thibessard | Hydrogen peroxide effects on Streptococcus thermophilus CNRZ368 cell viability[END_REF]. This suggests that it has developed strategies for cell detoxication and damage repair caused by ROS.

Recently, the genome sequences of two S. thermophilus strains LMG18311 and CNRZ1066 were completed [START_REF] Bolotin | Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus[END_REF]) and putative genes involved in oxidative stress defence were identified by genomic analysis [START_REF] Hols | New insights in the molecular biology and physiology of Streptococcus thermophilus revealed by comparative genomics[END_REF]. Genes in the disulfide-reducing pathway like glutathione reductase, thioredoxin or thioredoxin reductase have been detected. These genes products help to maintain the redox state of the cell and allow reducing intracellular disulfide.

S. thermophilus antioxidant defence also comprises genes encoding enzymes that detoxify oxygen or ROS. Thus, the S. thermophilus genome encodes an H2O-forming NADH oxidase, an essential enzyme for the NAD + regeneration during aerobic growth [START_REF] Higuchi | Functions of two types of NADH oxidases in energy metabolism and oxidative stress of Streptococcus mutans[END_REF]. In Streptococcus mutans, induction of H2O-forming NADH oxidase protects cells against oxygen toxicity [START_REF] Higuchi | Reduced nicotinamide adenine dinucleotide oxidase involvement in defense against oxygen toxicity of Streptococcus mutans[END_REF]. S. thermophilus also encodes a superoxide dismutase thought to catalyze superoxide radicals dismutation in O2 and H2O2. Although deprived of H2O2detoxifying enzymes like catalase, NADH-peroxidase or alkyl hydroperoxidase, S. thermophilus possess an H2O2 defence system to reduce damage induced by this compound. This system could include psaD and dpr genes. PsaD is encoding a thiol-peroxidase and Dpr is an iron-binding protein required for oxygen tolerance in S. mutans [START_REF] Yamamoto | Regulation of the intracellular free iron pool by Dpr provides oxygen tolerance to Streptococcus mutans[END_REF]). Dpr iron sequestration activity is thought to prevent the formation of toxic hydroxyl radicals via the Fenton reaction [START_REF] Niven | Iron content of Streptococcus suis and evidence for a dpr homologue[END_REF][START_REF] Yamamoto | An iron-binding protein, Dpr, from Streptococcus mutans prevents iron-dependent hydroxyl radical formation in vitro[END_REF][START_REF] Pulliainen | Molecular basis of H2O2 resistance mediated by Streptococcal Dpr. Demonstration of the functional involvement of the putative ferroxidase center by sitedirected mutagenesis in Streptococcus suis[END_REF][START_REF] Yamamoto | Regulation of the intracellular free iron pool by Dpr provides oxygen tolerance to Streptococcus mutans[END_REF][START_REF] Pulliainen | Dps/Dpr ferritin-like protein: insights into the mechanism of iron incorporation and evidence for a central role in cellular iron homeostasis in Streptococcus suis[END_REF]. Valuable information on S. thermophilus oxidative stress processes have also acquired by genetic approach using random IS mutagenesis.

Inactivation of several genes involved either in cell wall metabolism, exopolysaccharide translocation, tRNA modification, purine metabolism, or iron metabolism have been implicated in cell survival during oxidative stress [START_REF] Thibessard | Identification of Streptococcus thermophilus CNRZ368 genes involved in defense against superoxide stress[END_REF]).

Among the well characterised oxidative stress-regulators in other organisms such as OxyR, PerR, sigma(B) and OhrR, controlling the peroxide response (for reviews: [START_REF] Hecker | Non-specific, general and multiple stress resistance of growth-restricted Bacillus subtilis cells by the expression of the sigmaB regulon[END_REF][START_REF] Mongkolsuk | Regulation of inducible peroxide stress responses[END_REF] and the superoxide stress response system SoxRS [START_REF] Demple | Redox signaling and gene control in the Escherichia coli soxRS oxidative stress regulon--a review[END_REF]only perR, is present in the S. thermophilus genome.

In pathogenic streptococci, several regulators involved in oxidative stress have been described. Among these regulatory proteins is RopA, a key regulator of acid and oxidative stress tolerance, genetic competence, biofilm formation and virulence in S. mutans [START_REF] Wen | Trigger factor in Streptococcus mutans is involved in stress tolerance, competence development, and biofilm formation[END_REF].

In Streptococcus pneumoniae, the perception of O2 deprivation is associated with DNA uptake and controlled by CiaRH and MicAB two-component systems [START_REF] Echenique | Competence regulation by oxygen in Streptococcus pneumoniae: involvement of ciaRH and comCDE[END_REF][START_REF] Echenique | Competence modulation by the NADH oxidase of Streptococcus pneumoniae involves signal transduction[END_REF]. Both ropA and micAB genes have been identified in the S. thermophilus genome [START_REF] Bolotin | Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus[END_REF]). Additional to this list are the regulatory factors belonging to the Rgg family. These proteins are only found in Streptococcus and few other low-GC Grampositive genera, such as Listeria, Lactobacillus and Lactococcus. In S. pyogenes, Rgg interacts with regulatory networks to co-regulate virulence factor expression [START_REF] Chaussee | Identification of Rgg-regulated exoproteins of Streptococcus pyogenes[END_REF] and has also been implicated in oxidative and thermic stress responses as well as in the synthesis or stability of growth phase-regulated proteins associated with amino-acid metabolism [START_REF] Chaussee | Rgg regulates growth phase-dependent expression of proteins associated with secondary metabolism and stress in Streptococcus pyogenes[END_REF]).

In order to characterise S. thermophilus oxidative stress tolerance molecular basis, we previously used a random IS mutagenesis approach to produce mutants and screened them for their potential impaired tolerance to menadione (a superoxide generating compound). This strategy led us to identify 18 S. thermophilus CNRZ368 mutants [START_REF] Thibessard | Identification of Streptococcus thermophilus CNRZ368 genes involved in defense against superoxide stress[END_REF]. One of which is rggC. This gene is known to contain a frameshift mutation from the S. thermophilus genome sequence. This work is focused on rggC and attempts to determine how such a locus inactivated by a frameshift mutation is involved in oxidative stress tolerance. The rggC locus analysis was performed by engineering partial deletions of this locus and examining the ability of mutants to survive in the presence of menadione. Our results support an active role for RggC in S. thermophilus oxidative stress response.

MATERIALS AND METHODS

Bacterial strains growth conditions

The bacterial strains used in this study are listed in Table 1. Streptococcal strains including wild-type and mutant strains were cultured either in M17 [START_REF] Terzaghy | Improved medium for lactic streptococci[END_REF], or TPPY (a Tryptose, Proteose Peptone, Yeast extract containing medium) [START_REF] Bracquart | An agar medium for the differential enumeration of Streptococcus thermophilus and Lactobacillus bulgaricus in yoghurt[END_REF] or milk mediums, at 30°C or 42°C, depending on the experiments requirement. When appropriate, these media were supplemented with antibiotics at the following concentrations: erythromycin at 2 g.ml -1 , and chloramphenicol at 3 g.ml -1 . E. coli EC101 was used for cloning experiments and transformed by electroporation with various plasmids as indicated below. E. coli strains were grown on Luria-Bertani (LB) medium (Sambrook et al. 1989) and the following selective antibiotics were added, when needed: erythromycin (150 g.ml -1 ), chloramphenicol (15 g.ml -1 ) or ampicillin (100 g.ml -1 ).

DNA manipulations

Isolation of genomic or plasmid DNA, agarose gel electrophoresis, restriction enzyme digestions, Southern hybridisation, PCR, DNA ligation and E. coli transformation were performed as described in Sambrook et al. (1989). Sequencing was carried out with the DNA sequencing kit (Applied Biosystems) on an ABI Prism 377 Genetic Analyser (Applied Biosystems). Sequence data were analysed with Blast, ProDom and TIGRfam Model software.

The rggC1 and rggC2 DNA sequences have previously been deposited in GenBank under accession numbers from AY598750. The orf2 and rsp DNA sequences obtained in this work were deposited under accession number AM087201.

RNA manipulations

RNA was extracted from S. thermophilus CNRZ368overnight (OD600 = 1.6) cultures with a single guanidinium thiocyanate step and phenol-chloroform extraction by using the Tri-Reagent (Sigma). Northern blot hybridisation analysis, templates of RNA samples (30 g) were electrophoresed into a denaturing (i.e. formaldehyde) 1.1 % agarose gel and transferred to a HybondN + (Amersham pharmacia biotech) according to the manufacturer's instructions.

Digoxigenin-labelled transcripts complementary to rggC2 were used as a probe. These rggC2 transcripts were synthesised in vitro using the T3 promoter RNA polymerase system carried by the pBKS-rggC2 vector. The latter was constructed by cloning a 533 bp internal fragment of rggC2 (nucleotides 932 to 1464) in the pBKS vector. Hybridisation and detection procedures were performed according to manufacturer's instructions (Roche Molecular Biochemicals). RT-PCR experiments were carried out as previously described [START_REF] Fernandez | Characterization of oxidative stress-resistant mutants of Streptococcus thermophilus CNRZ368[END_REF]). Primers used in PCRs were purchased from MWG Biotech AG (Ebersberg) and their sequences were 5' -AGGTGACTAGAATGAAATATGG-3' for P1, 5' -TCCATTTGGCTAATTAATAGC-3' for P2, 5' -TATTGCTGCCACGAAATTGGT-3' for P3 and 5' -CACCGCCACAAGAATGACTT-3' for P4. Primers used in rggC locus sequencing were 5' -AGGAGGTGACTAGAATGAAATATGG-3' for V1 and 5' -TCCATTTGGCTAATTAATAGC-3' for V2.

Constructions of rggC 1 , rggC 2 , orf2 and rggC 8T mutants

The rggC1 gene contained a deletion of 43 % of the CDS. A stop codon (TAA) was inserted between the potential RBS of rggC1 and the start codon of rggC2 to prevent using this RBS sequence for RggC2 translation. The 5' and 3' ends of the rggC1 gene were independently amplified with the following sets of primers: 5' -CTTGCTTTGAATTCCAGGCT-3' (named I1) and 5' -TAAAGATAAAGCTTAAGATTCTC-3' for the 5' region;

5' -GGCTGATTTAAGCTTCAGCAAATT-3' and 5' -ATCATATGAATTCTTAGCAT-3', (named I2), for the 3' region (engineered restriction sites are underlined, stop codon is in boldface type). Amplified fragments were digested with HindIII and ligated to each other. The resulting fragment was amplified, digested with EcoRI and cloned into pGh9 vector to produce pGh9rggC1 plasmid. The same procedure was used to produce pGh9rggC2 and pGh9orf2Z1,2 plasmids. pGh9rggC2 carried a 44 % deletion of rggC2 and was constructed using the primers:

5' -TTATAAGAATTCAGGAGGTG-3' and 5' -TGATTTAAAGCTTCTATTGTAT-3' to amplify the 5' region; 5' -AAATGTAAAGCTTCCACGAAA-3' and 5' -AAATCCAGAATTCGATTATCC-3' for the 3' region.

The construction of the pGh9orf2Z1,2 plasmid containing 2 stop codons (TAA, TAA) in the 2 first codons of orf2 was carried out using the following set of primers:

5' -TCGAAGAATTCGTTTACG-3' and 5' -TCACCTGAAGCTTATTACATGGT-3' for the 5' region; 5' -AACCATGTAATAAGCTTCAGGTGAT-3' and 5' -TTTGATGAATTCTAGTATATGT-3' for the 3' region.

The pGh9rggC8T plasmid, carrying a thymine point deletion allowing a RggC1-C2 fusion, was constructed. To generate this mutation, the 5' and 3' regions of rgg were amplified with the following sets of primers: I1 and 5' -CTAACGCTAAAAAAAATTTGCTG-3' for the 5' region;

5' -CAAATTTTTTTTAGCGTTAGATGC-3' and I2 for the 3' region. These 2 products were mixed together, used as templates in a PCR reaction to generate a full length fragment and cloned into pGh9 vector to produce the pGh9rggC8T plasmid. This recombinant plasmid DNA was extracted and used to transformed S. thermophilus CNRZ368 cells as previously described by (O'Sullivan and Fitzgerald 1999). Allelic gene replacements were done as previously described [START_REF] Biswas | High-efficiency gene inactivation and replacement system for gram-positive bacteria[END_REF] with the following modifications: the medium used for S. thermophilus growth was M17, 3 cycles of growth were carried out with two steps, one at 30°C (permissive temperature) and another one at 42°C (restrictive temperature). The chromosomal structure of mutant alleles was checked by PCR, southern blot analysis and sequencing.

Menadione assay

S. thermophilus and its mutant derivatives were grown overnight in milk medium and diluted 100 times into TPPY liquid medium. Log-phase cultures (OD600 = 0.6) were exposed for 3h to menadione concentrations (0 to72 mM). Aliquots of the culture were then diluted in TPPY and appropriate dilutions plated on TPPY agar plates. The plates were stocked in jars containing a

GENbox anaer system (bioMérieux) providing hypoxic conditions. Colony-forming units (CFU)

were counted after 20 h of incubation and the percentage of surviving CFUs was determined by comparing viability with or without treatment. All experiments were performed independently at least three times.

RESULTS

Characterisation of the disrupted loci in osrC genome

In a previous study, the osrC mutant of S. thermophilus CNRZ368 was characterised in its resistance to menadione, a superoxide-generating agent [START_REF] Fernandez | Characterization of oxidative stress-resistant mutants of Streptococcus thermophilus CNRZ368[END_REF]. In this mutant, pGhost9::ISS1 was inserted in rggC2 region. The rggC1 ORF encodes a predicted 65 aa protein exhibiting 77% identity to the C-terminal region of S. mutans Rgg. The second ORF, rggC2 encodes a putative 232 aa protein sharing 54 % identity with the N-terminal region of S. mutans

Rgg. These two ORFs could initially have constituted a single ORF, having undergone a frameshift mutation (Fig. 1A). This mutational event would have occurred between positions 886 to 894, where a 9-mer (TTT TTT TTT) known to be genetically unstable is observed. Ribosomebinding site (RBS) consensus sequence (AAAGGAGG in Gram-positive bacteria) scan revealed a potential RBS (AGGAGG) located 8 bp upstream rggC1. No canonical RBS sequencepreceding rggC2 was detected upstream of the first putative start codon or within the CDS of rggC1. These results suggest that only RggC1 or a full fusion protein RggC1-2, could be produced, indicating a translational or transcriptional frameshifting.

Since Rgg proteins often regulate adjacent genes [START_REF] Sulavik | Rgg is a positive transcriptional regulator of the Streptococcus gordonii gtfG gene[END_REF][START_REF] Lyon | A role for trigger factor and an rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes[END_REF][START_REF] Chaussee | The rgg gene of Streptococcus pyogenes NZ131 positively influences extracellular SPE B production[END_REF][START_REF] Qi | Purification of mutacin III from group III Streptococcus mutans UA787 and genetic analyses of mutacin III biosynthesis genes[END_REF][START_REF] Neely | Role of RopB in growth phase expression of the SpeB cysteine protease of Streptococcus pyogenes[END_REF], the ORFs localised downstream of the rggC locus were analysed. RggC2 is followed by a small ORF, orf2 that encodes a putative 35 aa protein with no significant homology to any database sequences. orf2 is preceded by a canonical RBS with the sequence, AAAGGAGG suggesting it was expressed. Downstream orf2, there is an rsp gene (for radical SAM protein) that encodes a putative protein sharing 29 % identity (46% similarity) with a possible Fe-S oxidoreductase of Thermoanaerobacter tengcongensis belonging to the radical SAM protein family [START_REF] Sofia | Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods[END_REF]. A RBS sequence (GGAGG) was also found upstream rsp.

The transcriptional signals scan analysis allowed us to identify a potential promoter (TATGATATAAT), located 61 bp upstream of rggC1, and constitute an extended -10 region [START_REF] Voskuil | The -16 region of Bacillus subtilis and other gram-positive bacterial promoters[END_REF]. Moreover, a putative rho-independant terminator consisting of a perfect inverted repeat of 10 bp followed by a stretch of T's, was visualized 9 bp downstream of the orf2 stop codon. A potential promoter located upstream rsp was identified including a -10

(TGTAAT) and a -35 (TTTAGA) region with a 15 bp spacer.

Transcriptional analysis of the rggC locus

Northern blot analysis using rggC2 antisense-RNA (nucleotides 932 to 1464) as a probe was performed on RNA extracted from stationary phase cells. One transcript of approximately 1200 nucleotides was detected (Fig. 1B). Since the rggC2 gene size is 0.7 kb, this transcript observation is compatible with the polycistronic transcription of rggC2 with the neighbouring ORFs.

To better characterise this transcript, RT-PCR analyses were carried out. cDNA generated from S. thermophilus CNRZ368 RNA were used in PCRs experiments using primers P1/P2 and P3/P4 localized in rggC1/rggC2 and rggC2/orf2 respectively (Fig. 1C). In both cases, a PCR product of the expected size (310 bp and 265 bp respectively) was amplified suggesting the existence of at least one transcript including rggC1, rggC2 and orf2. The transcript size was in agreement with the transcription signals visualized on the sequence.

Involvement of the rggC locus in the oxidative stress defence

Since rggC locus exhibits operon structure, the resistance phenotype could be either the result of the inactivation of rggC2 gene or/and the result of polar effects on the rggC1 and orf2

neighbouring genes. In this study, non-polar rggC1, rggC2 and orf2 mutants were constructed via allelic replacement and their survival in presence of menadione was studied (see materials and methods). Compared to the reference strain, the rggC1 mutant displayed a 2-fold higher resistance following exposure to menadione at concentrations of 27 mM and 36 mM (Fig. 2A).

The rggC2 mutant exhibited a 2-fold decreased of surviving cells at a menadione concentration of 9 mM, 18 mM and 27 mM (Fig. 2B). Thus, a decreased survival was observed for the rggC2 mutant, while the survival of the rggC1 strain was enhanced indicating the involvement of both of these ORFs in S. thermophilus oxidative stress response. In contrast, orf2 mutant survival was the same as the reference strain (data not shown) suggesting that orf2 was not involved in this pathway.

Intraspecific polymorphism of rggC potential shift site

The analysis of the DNA sequence of the rggC locus predicted 2 ORFs: rggC1 and rggC2. A sequence of 9 T's was observed at the 3'end of rggC1. Since such mononucleotide repeats are known to exhibit high frequencies of nucleotide deletion or insertion events, the potential polymorphism of the rggC shift site was investigated among 21 strains S. thermophilus. The rggC1 gene was shown to be present in all 21 studied strains, using PCR amplification with primers V1 and V2 designed to bind the 5' part of rggC1 and the central part of rggC2, respectively. The frameshift mutation of rggC1 was analysed by sequencing the PCR products with primer V1. Four different sequences were found varying from 7 to 9 T's (Table 2). A deletion of one A upstream of the T tract in rggC from CNRZ1402, CNRZ455 and LMD9 strains was also observed. The strain ATCC1958 is characterised by the presence of 8 instead of 9 Ts.

This mutation would result in formation of an in-frame ORF, allowing potentially full-length RggC protein translation whereas in all the other cases a RggC1 protein was expected.

Involvement of RggC in oxidative stress defence of S. thermophilus CNRZ368

To determine the involvement in oxidative stress defence of a full-length RggC protein, an additional rggC8T mutant was constructed via allelic replacement. This mutant from CNRZ368 contains an eight T chromosomal sequence. Its survival in the presence of different concentrations of menadione was determined and showed to be reduced at a menadione concentration of 18 mM, 27 mM and 36 mM compared to wild-type confirming a role for rggC in S. thermophilus CNRZ368 oxidative stress response (Fig. 3).

DISCUSSION

Among the fully sequenced streptococcal genomes, that of S. thermophilus exhibits the highest proportion of pseudogenes. Indeed, 10% of the S. thermophilus genes are characterised by frameshift, nonsense mutation, deletion or truncation and are therefore considered as non functional [START_REF] Bolotin | Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus[END_REF]). Here, we examined rggC, considered in S. thermophilus as a pseudogene, and we demonstrated its role in S. thermophilus oxidative stress response.

In osrC mutant, the disruption of rggC2, with pGh9:ISS1, confers to the cells a resistance to menadione. This insertion mutant shows a polar mutation affecting the transcript stability of the rggC locus and of the neighbouring rsp gene.

To understand this phenomenon, deletions of rggC1 and rggC2 were undertaken and demonstrated that both ORFs were involved in oxidative stress response. However, these 2 mutants displayed 2 distinct phenotypes: one being resistant and the other sensitive to oxidative stress. Two alternative hypotheses can explain this. Firstly, the 2 proteins RggC1 and RggC2 could be synthesised and would be characterised by different activities. Nevertheless, the lack of a RBS sequence-preceding rggC2 is a strong argument in disfavour of this hypothesis. Secondly, the rggC2 phenotype could result from the synthesis of a full RggC protein by frameshifting as observed in translational or transcriptional slippage (for review : [START_REF] Baranov | Transcriptional slippage in bacteria: distribution in sequenced genomes and utilization in IS element gene expression[END_REF].

Taking all these data into account, both RggC1 and RggC proteins are likely to be synthesised in the wild type strain.

In CNRZ368 strain, a stretch of 9 T residues could allow a transcriptional slippage, and consequently lead to the RggC fusion protein by translation of a subpopulation of rggC transcript in the alternate reading frame. Alternatively, since RNA-DNA hybrids of 9 are described as genetically unstable they could provide RNA polymerase slippage by re-alignment of the nascent RNA chain and its DNA template [START_REF] Wagner | Transcriptional slippage occurs during elongation at runs of adenine or thymine in Escherichia coli[END_REF]. Few examples of transcriptional slippage are identified in prokaryotes. In Thermus thermophilus,  and  subunit proteins of DNA polymerase III are synthesized from dnaX gene by this mechanism. This ORF shows 9 adenine residues allowing the slippage of RNA polymerase and producing many mRNAs with additional or subtractive adenine residues [START_REF] Larsen | Nonlinearity in genetic decoding: homologous DNA replicase genes use alternatives of transcriptional slippage or translational frameshifting[END_REF]. In Shigella flexneri, the mixE gene, encoding a transcription activator, consists of 2 overlapping ORFs. A track of 9 thymine residues is observed at the site of overlapping. Transcriptional incorporation of one additional nucleotide allows synthesis of the functional MixE protein [START_REF] Penno | Frameshifting by transcriptional slippage is involved in production of MxiE, the transcription activator regulated by the activity of the type III secretion apparatus in Shigella flexneri[END_REF].

The putative RggC protein belongs to the family of Rgg-like transcriptional regulators. RggC1 displayed the typical N-terminal helix-turn-helix motif suggestive of a DNA-binding function.

Among characterised Rgg-like proteins, rgg from Streptococcus gordonii, rgg and ropB from S. pyogenes are located immediately adjacent to their regulatory target sequence [START_REF] Sulavik | Rgg is a positive transcriptional regulator of the Streptococcus gordonii gtfG gene[END_REF][START_REF] Lyon | A role for trigger factor and an rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes[END_REF][START_REF] Chaussee | The rgg gene of Streptococcus pyogenes NZ131 positively influences extracellular SPE B production[END_REF][START_REF] Qi | Purification of mutacin III from group III Streptococcus mutans UA787 and genetic analyses of mutacin III biosynthesis genes[END_REF][START_REF] Neely | Role of RopB in growth phase expression of the SpeB cysteine protease of Streptococcus pyogenes[END_REF]. Thus, the transcriptional expression of orf2 and rsp ORFs from S. thermophilus CNRZ368 was analysed by quantitative RT-PCR in rggC1, rggC2 and wild-type genetic contexts (data not shown). Partial deletion of rggC locus did not affect the expression of orf2 and rsp ORFs compared with the level seen in the wild type CNRZ368 strain. However, S. thermophilus genome contains 7 rgg genes [START_REF] Bolotin | Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus[END_REF]. It is therefore possible that one of the Rgg proteins replace RggC function in the defective strain. Moreover, it still is possible that RggC directly or indirectly regulates the transcription of other genes since deletion of rgg from S. pyogenes has been demonstrated to have genome wide effects on virulence-associated transcription [START_REF] Chaussee | Identification of Rgg-regulated exoproteins of Streptococcus pyogenes[END_REF][START_REF] Chaussee | Rgg influences the expression of multiple regulatory loci to coregulate virulence factor expression in Streptococcus pyogenes[END_REF].

Analysis of 21 S. thermophilus rgg sequences revealed a polymorphism of the 9 T residue region. Repeats of a single or several nucleotides, named microsatellite DNA, are involved in numerous phase variation events that could be defined as a high-frequency and reversible mechanisms of gene-switching (for review: [START_REF] Hallet | Playing Dr Jekyll and Mr Hyde: combined mechanisms of phase variation in bacteria[END_REF]). This inter-strain length polymorphism supports evidence for the phase variability of the rggC locus. Some cases of phase variation have been identified in the Streptococcus genus, allowing the control of transcriptional or translational expression [START_REF] Lukomski | Identification and characterization of a second extracellular collagen-like protein made by group A Streptococcus: control of production at the level of translation[END_REF][START_REF] Puopolo | Upstream short sequence repeats regulate expression of the alpha C protein of group B Streptococcus[END_REF]. A phase variation event at the rggC locus could influence a potential programmed frameshifting by inactivation.

In 20/21 cases, a + 1 or -1 frameshift mutation was observed resulting in a 'gene off' configuration. If the full-length rggC ORF was an ancestral allele, it is possible that alleles showing this frameshift mutation may have been selected for a growth advantage associated with the loss of rggC function. The menadione sensitivity phenotype observed for the rggC8T mutant (whose rggC phase is restored) argued in the favour of that hypothesis.

The results of this study indicate the involvement of the rggC locus in S. thermophilus CNRZ368 oxidative stress response. Accordingly, the Rgg transcriptional regulatory protein from S. pyogenes that coordinates virulence factor synthesis and catabolic activity [START_REF] Chaussee | Rgg coordinates virulence factor synthesis and metabolism in Streptococcus pyogenes[END_REF] was also demonstrated to be involved in thermal and in oxidative stress response [START_REF] Chaussee | Rgg regulates growth phase-dependent expression of proteins associated with secondary metabolism and stress in Streptococcus pyogenes[END_REF]). The S. pyogenes rgg mutant strain was more susceptible than wild-type to paraquat (a hydroxyl ion radicals generating compound) and was characterised by an elevated level of the oxidoreductases AhpC and Nox1 [START_REF] Chaussee | Rgg regulates growth phase-dependent expression of proteins associated with secondary metabolism and stress in Streptococcus pyogenes[END_REF]. All these data suggest a role for the Rgg family members in oxidative stress response, and more generally that these proteins may be important in the streptococcus adaptation to environmental changes.

Fig. 1

 1 Fig. 1 Organisation of the rggC locus of S. thermophilus CNRZ368. (A) The solid line represents the S. thermophilus genomic DNA. ORFs are indicated as open arrows. The broken arrow and the hairpin loop indicate the putative promoter and rho-independant terminator respectively. Putative RBS localisation is indicated by asterisks. Primers used for RT-PCR experiments are represented by small arrows under ORFs. The expected size of the P1-P2 and P3-P4 PCR fragments is 310 bp and 265 bp, respectively. ARN probe localisation is indicated by the black box. (B) Northern blot analysis of rggC2 transcripts. Left: gel electrophoresis staining wih ethidium bromide: lane 1 is RNA size marker and lane 2 is RNA (30g) total extracted from S. thermophilus CNRZ368. 16S and 23S indicate the migrating positions of 16S and 23S rRNA, respectively. Right: result of the hybridisation with the rggC2 RNA probe. (C) Gel electrophoresis showing the RT-PCR products obtained using the primers P1-P2 and P3-P4. PstIdigested  DNA used as a DNA fragment size marker is indicated by  PstI.

Fig. 2

 2 Fig. 2 Survival of rggC1 and rggC2 mutants and the reference strain with increasing

Fig. 3

 3 Fig. 3 Survival of rggC8T mutant and the reference strain with increasing menadione concentrations. The graphs point the average values obtained from experiments performed at least in triplicate. The error bars represent standard deviations

Table 1 . Strains and plasmids

 1

	426
	Strains and	Relevant feature(s)	Source and/or citation
	plasmids
	S. thermophilus
	CNRZ007	Wild-type strain	CNRZ, INRA strain collection
	CNRZ302	Wild-type strain	CNRZ, INRA strain collection
	CNRZ307	Wild-type strain	CNRZ, INRA strain collection
	CNRZ308	Wild-type strain	CNRZ, INRA strain collection
	CNRZ368	Wild-type strain	CNRZ, INRA strain collection
	CNRZ385	Wild-type strain	CNRZ, INRA strain collection
	CNRZ388	Wild-type strain	CNRZ, INRA strain collection
	CNRZ391	Wild-type strain	CNRZ, INRA strain collection
	CNRZ445	Wild-type strain	CNRZ, INRA strain collection
	CNRZ455	Wild-type strain	CNRZ, INRA strain collection
	CNRZ702	Wild-type strain	CNRZ, INRA strain collection
	CNRZ1066	Wild-type strain	CNRZ, INRA strain collection
	CNRZ1068	Wild-type strain	CNRZ, INRA strain collection
	CNRZ1402	Wild-type strain	CNRZ, INRA strain collection
	ATCC19258 Wild-type strain	American Type Culture Collection
	ATCC19987 Wild-type strain	American Type Culture Collection
	LMG18311 Wild-type strain	BCCM/LMG strain collection
	A054	Wild-type strain	Lab collection
	IP6756	Wild-type strain	Institut Pasteur strain collection
	IP6757	Wild-type strain	Institut Pasteur strain collection
	osrC	Em R S. thermophilus CNRZ368 containing	((Fernandez et al. 2004)
	rggC1 rggC2	integrated pGh9::ISS1 into rggC2 CNRZ368 strain carrying a deletion from nt 50 to 127 of rggC1 to 619 of rggC2 CNRZ368 strain carrying a deletion from nt 318	This study This study
	orf2	CNRZ368 strain carrying 2 stop codons in the	This study
		first codon of orf2
	rggC8T	CNRZ368 strain presenting an in-frame rggC	This study
		gene
	E. coli
	EC101	TG1 derived strain [supE hsd-5 thi (lac-proAB)	(Leenhouts 1995)
		F' (traD6proAB + lacI q lacZM15)] containing a
		chromosomal copy of the pWV01 repA gene
	Plasmid
	pBKS	Amp R	Stratagene
	pGhost9	Em R , thermosensitive replication origin from	(Maguin et al. 1996)
		pVE6002
	position 50 to 127 pGh9rggC1 pGh9 plasmid carrying rggC1 deleted from	This study
	position 318 to 619 pGh9rggC2 pGh9 plasmid carrying rggC2 deleted from	This study
	pGh9orf2Z1,2 pGh9 plasmid carrying orf2 with 2 stop codons	This study
		inserted in the first and the second codon
	pGh9rggC8T pGh9 plasmid carrying an in-frame rggC gene	This study

Table 2 . Analysis of the rggC locus in 21 strains of S. thermophilus

 2

	Sequence	Strain name	Mutation
		A054, ATCC19987, CNRZ007,	+1 FS
		CNRZ302, CNRZ307, CNRZ368,
	AGC AAA TTT TTT TTT AGC
		CNRZ385, CNRZ388, CNRZ391,
		CNRZ702, LMG18311, IP6756
	AGC AAA _TT TTT TTT AGC	ATCC19258	/
		CNRZ308, CNRZ445, CNRZ1066,	-1 FS
	AGC AA_ _TT TTT TTT AGC
		CNRZ1068, IP6757
	AGC AA_ __T TTT TTT AGC	CNRZ455, CNRZ1402, LMD9 a	+1 FS

a according to sequence available htpp://www.jgi.doe.gov.

ACKNOWLEDGEMENTS

A.F. was supported by grants from the Ministère de la recherche. F.B. was supported by a grant from the Institut National de la Recherche Agronomique. We are grateful to Arran Johnson for his advice regarding the English formulation of the manuscript.