Annabelle Thibessard

Frédéric Borges

Annabelle Fernandez

Brigitte Gintz

Bernard Decaris
email: decaris@nancy.inra.fr

Nathalie N Leblond-Bourget

Thibessard Annabelle

Borges Frédéric

Fernandez Annabelle

Gintz Brigitte

Decaris Bernard

Pr Bernard Decaris

Identification of Streptococcus thermophilus CNRZ368 genes involved in

Keywords: Streptococcus thermophilus, superoxide stress, insertional mutagenesis, ironsulfur cluster, RNA maturation, cell wall Streptococcus thermophilus, superoxide stress, insertional mutagenesis, ironsulfur cluster, RNA maturation, cell wall

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INTRODUCTION

Oxygen is an essential element for the viability of most living organisms through its involvement in oxidative phosphorylation that takes place during the aerobic respiration process. However, oxygen can also be harmful, due to the production, especially during respiration, of toxic derivatives namely ROS (reactive oxygen species) such as hydrogen peroxide, hydroxyl and superoxide radicals (for a review, (25)). Accumulation of these molecules, triggers lipid, protein and nucleic acid oxidation, causing lethal cellular damage (for reviews (7,25,53)). In response to such attack, aerobic micro-organisms have evolved anti-oxidant mechanisms (eg catalase, peroxidase and superoxide dismutase activities) to protect and adapt cells against ROS excess (6,18,24,65,68).

Although they do not achieve aerobic respiration, anaerobic micro-organisms can be subject to ROS. The strict anaerobes Bacteroides fragilis and Clostridium perfringens are able to survive in the presence of a low amount of oxygen, due to the induction of an adaptative oxidative stress response that is activated under aerobic conditions (13,(46)(47)(48)(49). The peroxide response of B. fragilis shares some characteristics with that of E. coli such as the involvement of the redoxsensitive transcriptional activator OxyR (46) and some antioxidant activities under its control (47,49).

The lactic acid bacterium (LAB) Streptococcus thermophilus is a Gram-positive bacterium widely used in dairy factories, mainly as a starter of fermentation in yogurt and cheese production. During fermentation processes, S. thermophilus is exposed to oxygen and to its deleterious effects that can lead to a reduction of cell viability and consequently may have repercussions on texture, flavor and safety of the final product.

S. thermophilus is an anaerobic aerotolerant organism that can grow in presence of oxygen and survive to low concentrations of superoxide and hydroxyl radicals (59). This suggests that it displays a defense system that provides protection against the effects of ROS. In the literature, data about its oxidative stress response are scarce. The existence of at least one 6 incubated at 42°C for 3h. The cultures were then transferred on TPPY agar plates containing erythromycin (2 µg/ml) using a hedgehog. The ability of the mutants to survive to the menadione stress was assessed, after 20 h incubation at 42°C, by observation of the presence or the absence of colonies.

For each experiment, clones were considered as sensitive when they did not grow on a plate where more than 90% of the clones were growing. Both procedures were applied twice and the results had been compiled to reveal the mutants raising the more severe phenotype.

DNA manipulations. Preparation of chromosomal, plasmid and recombinant DNA and Southern analysis were performed according to standard protocols (50). All cloning junctions and cloned PCR products were checked by sequencing using Dye-Terminator chemistry on an ABI Prism 377 Genetic Analyser (PE Biosystems). Sequence data were analysed using Genbank, BLAST (2,3), TMHMM (51) and HMMTOP (61) software. For all analysed sequences, The DNA sequences reported in this paper have been deposited in GenBank under accession numbers AY386239, AY386240, AY386241, AY386242, AY386243, AY386244, AY386245.

Construction of the ∆sufD and iscU 97 mutants. Oligonucleotides used to construct the ∆sufD and iscU 97 mutants were purchased from MWG Biotech AG (Ebersberg, Germany). To construct non-polar truncated ∆sufD mutant, the 5' and 3' ends of the sufD gene were independently amplified by PCR. The 5' end was amplified by using the primers GAGCGTGTTGAATTCCACCG containing an EcoRI site, symbolized in bold characters and AACTTAACTGCAGCACCGTCT containing a PstI site. The 3' end was amplified by using the primers GGTGTTCCAGAATTCAAATAACC containing an EcoRI site and TGGTCTGTCATCAGGTCG. This last primer does not contain any PstI site, but the PCR product generated does contain a PstI restriction site. These two PCR products were digested by PstI and ligated with each other. The resulting fragment was amplified by PCR, then digested by EcoRI and cloned into pGh9. The final construct (pGh9∆sufD) was used to transform S. thermophilus CNRZ368. For IscU protein inactivation, the strategy consisted in replacing the TGT codon encoding the conserved Cys 97 by a TAT codon encoding a Tyr. This was achieved by replacing, in oligonucleotides, the 3 underlined nucleotides of the sequence (AGG TGT ACA) generating an EcoRV restriction site (AGA TAT CCA). Thus, the three Fitzgerald (41). The following gene replacements were done as previously described (8) but in TPPY medium and at 30°C/42°C as permissive and restrictive temperature, respectively.

Physiological characterization of S. thermophilus mutant strains.

Exposure to methyl viologen. For "short" time exposure, cultures of CNRZ368 and its derivative strains were grown in TPPY medium. At an OD 600 of 0.6, cultures were divided into two subcultures (in a ratio of liquid to air space of approximately 1/1). Methyl viologen (paraquat; Sigma-Aldrich) was added to one of them with a final concentration of 50 mM. After 3 h incubation at 42°C, cultures were diluted to appropriate concentrations and spread onto 8 TPPY agar plates. For "long" time methyl viologen exposure, strains were grown in TPPY medium until OD 600 of 0.6. Cultures were then diluted to a final theorical OD 600 of 0.001 in TPPY medium (in a ratio of liquid to air space of approximately 5/6) supplemented with 1.7 mM methyl viologen, or not for control experiment. After 18h incubation at 42°C, cultures were diluted in TPPY to appropriate concentrations and spread onto TPPY agar plates. In both procedures, methyl viologen sensitivity was estimated by comparing CFU/ml obtained with treated cultures, with respect to non treated cultures.

Heat shock. The ability of S. thermophilus and its derivatives to respond to a heat stress was assessed as follow. Each strain is grown at 42°C in TPPY medium until early log phase (OD 600 = 0.3). Then the culture was divided into two. One part was kept at 42°C (control experiment); the other one was immediately transferred to 60°C for 15 min. Appropriate dilutions were spread on TPPY agar plates. The fraction of surviving cells was calculated by dividing the surviving CFU/ml by the count for the control experiment.

Streptonigrin sensitivity. Streptonigrin sensitivity was monitored on cells grown overnight

in TPPYC medium at 42°C. Cells were harvested by centrifugation, washed twice in TPPYC medium, resuspended in TPPYC, diluted to OD 600 = 0.01 and cultivated in TPPYC for 9 h at 42°C. Then, the cultures were diluted again to OD 600 = 0.005 and divided into 4 parts which were all incubated for 21 h at 42°C. One part was supplemented with 0.4% (v/v) dimethyl formamide (control experiment), the 3 other parts were supplemented with different concentrations of streptonigrin (1 µg/ml, 2 µg/ml and 4 µg/ml dissolved in dimethyl formamide). As a control experiment, cultures were also carried out in TPPYC supplemented with 1 mM of 2'2-dipyridyl (an Fe chelator). In each experiment, OD 600 value was used to estimate the cellular density after 21h incubation. Relative optic density was determined by comparing cellular density obtained with and without streptonigrin. All experiments were performed in triplicate.

RESULTS

1. Selection of S. thermophilus CNRZ368 mutants with altered oxidative stress response.

A collection of 2112 mutants of S. thermophilus CNRZ368 was generated by insertional mutagenesis using the plasmid pGh9:ISS1 (35,56). This plasmid carries an erythromycin resistance gene for its selection and the insertional element ISS1 that allows random integration of the plasmid into S. thermophilus genome. The 2112 mutants obtained were screened to select clones sensitive to menadione (a superoxide-generating compound). Two distinct selection procedures were used to identify mutants susceptible to menadione (see materials and methods). The characterization of 10 mutants, disrupted in loci originally named ossA to ossJ (for oxidative stress sensitive locus) is described in this paper.

Sensitivity of S. thermophilus CNRZ368 mutants to methyl viologen. Previously, we

demonstrated that ossB and ossC mutants (previously named 6G4 and 18C3, respectively) are compromised in their ability to survive superoxide stress (57). To confirm that the other 8 mutants selected were sensitive to superoxide stress, their sensitivity to methyl viologen was examined (Fig. 1). In this experiment, we used the non-polar mutants ∆sufD and iscU 97 constructed in this work (see further) rather than the potential polar clones ossI and ossJ.

Short-term methyl viologen exposure revealed the extreme sensitivity of ossG, as well as the 4 to 5-fold increased sensitivity of ossD, ossH and iscU 97 mutants (Fig. 1A). Although less severe the ∆sufD mutant exhibited about 2-fold less survival following such stress. Under these conditions, the other 3 mutants (ossA, ossE and ossF) did not exhibit any significant sensitivity, however long term methyl viologen exposure confirmed their sensitivity. Indeed, long term methyl viologen exposure (Fig. 1B) showed that the ossA mutant strain displayed a relative survival more than 10-fold lower than that of the wild type strain. The ossE and ossF mutants also showed a 2-fold lower growth compared to the wild type strain.

3. Survival of the mutants exposed to heat stress. To assess the specificity of these 10 loci to oxidative stress defense, the mutants ability to survive to heat shock was estimated. Five out of the 10 mutants (ossA, ossB, ossC, ossD, and ossJ) isolated as oxidative stress sensitive clones also showed a reduced survival to heat shock compared with WT (Fig. 2). These data suggest that the genes impaired in these mutants are not specific to oxidative stress defense process but are probably involved in a more global response to stresses.

In contrast, the ossE, ossF and ossG mutants did not differ significantly from the wild type, indicating that the genes disrupted in these strains did not influence cells ability to respond to heat shock. Interestingly, the ossG mutant, that was highly sensitive to methyl viologen, showed no significant decrease of survival to heat shock compared with WT, suggesting that ossG gene, essential for oxidative stress defense, could be specific to such a stress.

4.

Identification of genes implicated in oxidative stress tolerance. The disrupted locus was identified for each of the 10 mutants by cloning and sequencing the junctions as previously described (56). All of the insertions were mapped within coding sequences. Sequence similarity searches and analysis revealed similar sequences in other bacteria (see Table 1)

with an E-value lower than 10 -10 for BLASTX searches. In 9/10 cases, data about the function of these genes were available. To facilitate their description, these genes were grouped into 5 classes (from I to V) according to the putative function and renamed when relevant (Table 1).

Class I: Genes potentially involved in cell wall structure and cell shape determination.

The ossA, ossB and ossC putative gene products shared sequence identity with MreD, RodA and Pbp2b, respectively, which putatively direct the elongation of the lateral cell wall in E. coli, conferring the cell its characteristic rod shape (5,28,29). The ossA mutant was defective in a gene encoding a protein, with 30% identity with MreD of Streptococcus mutans. Sequence analysis of the surrounding region indicated that S. thermophilus mreD gene (ossA) was clustered with a gene encoding a putative protein similar to MreC. Moreover, hydrophopathy profiles of MreD (OssA) determined by using TMHMM and HMMTOP showed 5 regions of sufficient length and hydrophobicity to be membrane-spanning segments (data not shown), suggesting that this protein is the counterpart of MreD. The relatively low identity (30%) observed between S. thermophilus and S. mutans MreD was in agreement with the general idea that the structure of this protein is more highly conserved than its primary sequence (17). To study the effect of mreD disruption in S. thermophilus, TPPY cultures of the wild type and mreD mutant strains were observed under light microscopy. In contrast to wild type cells, mreD mutant cells formed very long chains, up to 180 cells per chain (Fig. 3).

Moreover, a reduction of about 25% of the cell size was observed in the mutant. Growth of the mreD mutant and of the wild type in TPPY medium were monitored by measuring the OD 600 . Both strains displayed a similar doubling time of 30 min, but the maximal OD 600 of the mreD mutant (OD max = 1.7 ± 0.1) was lower than that of the wild type (OD max = 2.4 ± 0.2)

showing that the mreD mutant entered stationary phase earlier than wild type cells. The characterization of rodA and pbp2b mutants of S. thermophilus CNRZ368 is described in a previous paper (57).

Class II: Gene potentially involved in exopolysaccharide translocation. The sequence disrupted in the mutant ossD was partially determined. Its analysis indicated that the C-terminal portion of the predicted encoded protein showed 98% identity to CpsX, a putative membrane protein from S. thermophilus MR-1C. The role of CpsX is still unknown but the conserved location of cpsX near eps genes of S. thermophilus (14) and the presence of a putative gene related to an ABC transport system in its vicinity argue in favor of CpsX being involved in exopolysaccharide translocation (15). Moreover, the TMHMM and HMMTOP software predictions indicated that CpsX (OssD) from S. thermophilus CNRZ368 would contain highly hydrophobic domains compatible with a location in the cytoplasmic membrane. Although the cpsX function is not known yet, our work indicate that its disruption is associated with sensitivity not only to superoxide stress but also to heat shock.

Class III: Genes involved in RNA maturation. In the ossE and ossF mutants, the plasmid inserted into genes whose predicted products share sequence identity with enzymes involved in RNA maturation. The ossE gene encodes a putative tRNA guanine transglycosylase (TGT) which shares 92% identity with the putative Streptococcus pneumoniae TGT. TGT enzyme takes part in the process which removes the guanine residue at position 34 (wobble position of the anticodon loop) of tRNA His , tRNA Asp , tRNA Asn and tRNA Tyr and replaces it by the modified residue queuosine (39,40).

ossF potentially encodes a protein that shows 77 % identity with a putative RNA methyltransferase of Streptococcus agalactiae (Gbs0448) belonging to the TrmA family.

Enzymes of the TrmA family catalyze the formation of the 5-methyluridine in position 54

(localized in the TΨC loop) of tRNA (9). The presence of a TRAM-like domain (RNA binding domain) in the N-terminal part of OssF supports the idea of an interaction between this protein and RNA.

Class IV: Gene of unknown function. The ossG product shows 67% identity with the product of a gene of unknown function (smu.1428c) of S. mutans (Table 1). Although ossG function is not known yet, the mutation of this gene is now associated with a sensitivity phenotype to superoxide stress. The TMHMM and HMMTOP software predictions suggest the existence of two transmembrane domains in the N-terminal region of OssG, making of OssG a potential membrane protein.

Class V: Genes involved in iron metabolism.

(i) Ferric iron transport. The putative product of ossH shows 55% identity with FatD from S. pneumoniae, a potential membrane spanning permease of an Fe 3+ ABC-transporter. Analysis of the adjacent sequence reveals the presence of a gene encoding a putative FatC similar protein downstream of fatD (ossH), in the same orientation. The fatC gene is also clustered with fatD in S. pneumoniae genome. Hydropathy profile of FatD (OssH) from S. thermophilus, assesses with TMHMM and HMMTOP, reveals that this protein display ten putative membrane spanning segments compatible with its expected cytoplasmic membrane location.

(ii) [Fe-S] cluster biosynthesis. The sequence affected in the ossI mutant encodes a product that shows 75% identity with Sag0142, a putative protein deduced from the complete genome sequence of S. agalactiae and similar to SufD-like proteins. Analysis of totally sequenced microbial genomes reveals that sufD-like genes are conserved in bacteria and also in some archaea such as Halobacterium sp. NRC-1, Thermoplasma acidophilum and Aeropyrum pernix (23). In E. coli and Erwinia chrysanthemi, the sufD gene belongs to the sufABCDSE cluster identified as being involved in iron metabolism and more precisely in [Fe-S] cluster biogenesis and/or repair (37,42). The ossJ gene product shows sequence identity to the gbs0140 product from S. agalactiae (Table 1), probably encoding a IscU protein, a NifU family protein. NifU proteins have been proposed to serve as scaffold proteins in [Fe-S] cluster assembly in Synechocystis PCC6803 and would function as a mediator for iron-sulfur cluster delivery (38).

In some genomes including that of S. pyogenes, nifU-like genes are clustered with the suf genes (23). To determine if sufD (ossI) and iscU (ossJ) from S. thermophilus were genetically linked together, the chromosomal DNA regions flanking the pGhost9:ISS1 insertion site of both mutants were sequenced until overlapping. Examination of the genes in the neighborhood of sufD and iscU revealed that they both belong to a cluster of 5 genes all orientated in the same direction. This cluster, named sufCDS iscU sufB cluster, is shown in Fig. 4. The highest sequence identities of these gene products are found with putative proteins from S. agalactiae (Table 2). They also share sequence similarity with proteins encoded by E. coli and Er. chrysanthemi suf gene clusters. The putative SufC, SufD, SufS and SufB proteins encoded by the S. thermophilus cluster present respectively 72%, 48%, 65%, and 57% similarity to their E. coli counterparts. Moreover, IscU presents 47% similarity with IscU from E. coli.

Sequences corresponding to promoter regions were searched by inspection of the total sequence of the sufCDS iscU sufB cluster. A unique extended -10 sequence

(5'-TTTGTTATAAT-3') is found upstream sufC, the first ORF of the cluster. A potential rho-independent transcriptional terminator is identified 63 bp downstream sufB, the last ORF of the cluster (∆G = -10 kcal/mol). These features, together with the short or missing intergenic regions and the same orientation of the genes, suggest that these 5 ORFs form an operon, as it is the case for the Er. chrysanthemi suf locus.

Molecular characterization of the sufCDS iscU sufB cluster.

5.1. Construction of ∆sufD and iscU 97 mutants. Since S. thermophilus suf genes are potentially clustered in an operon, non polar ∆sufD and iscU 97 mutants were constructed and analysed. The sufD mutation consists of an internally in frame deletion leading to the loss of 43% of the whole protein (see materials and methods and Fig. 4). Sequence comparison of NifU family proteins from various organisms indicates that S. thermophilus IscU protein contains the three highly conserved cystein residues that have been shown to be the assembly site of a "transitory" [2Fe-2S] center within NifU (70). Thus, the construction of a null iscU mutant was achieved by replacing one of these conserved residues, the Cys 97 , by a Tyr (see materials and methods and Fig. 4). Sequencing of these two constructs confirmed that they did not contain any secondary mutation.

Role of sufD and iscU in iron metabolism.

To investigate the possibility that ∆sufD and iscU 97 mutations impair growth due to the lack of functional [Fe-S] cluster biosynthesis, the mutants were grown in TPPY medium and compared to wild type. Growth of the ∆sufD and iscU 97 mutants showed no significant difference compared to the WT strain. Assuming that the high iron concentration of the TPPY medium could allow self assembling of the [Fe-S] clusters (71), and consequently could be responsible for the absence of difference in growth between strains, the same experiment was conducted in TPPYC, a divalent cation depleted medium. Under these conditions, the mutants displayed a growth rate similar of that of the WT strain but their maximal OD 600 was lower than that of the wild type, showing their premature entry in stationary phase (Table 3).

Moreover, this difference disappeared when the cultures were grown on TPPYC supplemented by 100 µM of FeSO 4 . Taken together, these results suggest that (i) iscU and sufD genes of S. thermophilus are involved in iron metabolism and (ii) their presence would be of particular importance under iron-limiting conditions.

To confirm the involvement of sufD and iscU in iron metabolism, the iron intracellular concentration of ∆sufD and iscU 97 was checked by measuring the sensitivity of these strains to streptonigrin (an iron-activated antibiotic). As shown in Fig. 5 A, the iscU mutant was more sensitive to streptonigrin than the wild type strain whereas the ∆sufD mutant was more resistant. Moreover, addition of 2,2'-dipyridyl (a ferrous iron chelator) abolished the difference of growth between the strains (Fig. 5 B) indicating that iron was responsible for the streptonigrin sensitivity observed without 2,2'-dipyridyl. Taken altogether these results

suggest that both iscU and sufD of S. thermophilus play a role in iron metabolism.

Discussion

To investigate the oxidative stress defense system of S. thermophilus, a collection of random insertion mutants was screened to select clones with high sensitivity to menadione. Using this approach, we aimed to identify genes involved in superoxide stress defense without presuming their function. It can be noted that no gene encoding a protein considered, in other organisms, as major antioxidant enzyme were found using this approach. This can be due to the fact that (i) the collection of mutants was non-exhaustive (ii) the corresponding mutant could have a weak phenotype (iii) such mutations could be lethal (iv) except from SOD, most anti-oxidant enzymes are protective against H 2 O 2 and not O2 .-. The genes identified in the present work were classified into 5 groups according to their function: genes potentially involved in cell wall metabolism, exopolysaccharide translocation, tRNA modification, and iron metabolism and gene of unknown function.

Perhaps the most striking observation of this study is that 5 out of the 10 sensitive mutants selected are disrupted in a gene encoding an membrane protein (OssH, MreD, RodA, PBP2b and CpsX). This could reflect that the cell envelope is the main target of the stress applied.

Among these mutants, three (mreD, rodA and pbp2b) are impaired in genes potentially encoding proteins described in E. coli as playing a role in peptidoglycan biosynthesis during cell elongation (28,52,63). Phenotypic analysis of these three S. thermophilus mutants showed that they all present a reduced cell size associated with a modification of the cell shape, as observed in rodA, pbp2 and mre mutants of E. coli or Salmonella typhimurium (5,20,28,29,63,64). Moreover, the 3 S. thermophilus mutants also presented an increased sensitivity to heat shock compared to the wild type strain suggesting that the protective role of MreD, RodA and PBP2b is not specific to oxidative stress but could be enlarged to a more global stress response. In support of this hypothesis is the existence in Lactococcus lactis of mutations in pbp genes that negatively affect acid tolerance of a guaA mutant (16).

The major function assigned to cell wall is the maintenance of cell integrity by protecting cells not only against osmotic stress but also against other stress conditions. For instance, in LAB, it has already been shown that compromised biosynthesis of peptidoglycan results in an increased sensitivity to either UV (22) or acid (11) stress. Additionally, disruption of mreD, rodA or pbp2b may cause a modification of the peptidoglycan architecture in S. thermophilus, reducing the cell protection against aggressions such as oxidative and heat stress.

Transfer RNA modification is a universal feature of all living organisms, although some described modifications have no assigned function yet. Modifying enzymes affect the tRNA precursor either by chemical modification of an existing residue (in the base or in the sugar)

or by nucleotide substitution. These modifications, in particular those present in the anticodon region can affect translation efficiency and fidelity (for a review (10)). In this study, two clones disrupted in the loci tgt and ossF, encoding proteins showing high identity with tRNA modifying enzymes were selected for their high sensitivity to oxidative stress. This sensitivity could suggest that the expression of genes involved in oxidative stress defense may require tRNA maturation or, that such modifications could help tRNA stabilization under stress conditions. In agreement with the former hypothesis Shigella flexneri pathogenicity is regulated by virF mRNA translation efficiency under TGT activity control (21). Additionally, several environmental conditions are suggested to influence tRNA modification: in the case of queuosine (whose synthesis and substitution is carried out by several enzymes among which is TGT), synthesis is sensitive to the presence of iron (31) and TGT requires oxygen for its full activity (54). Therefore, further investigations on this topic should provide an interesting new regulatory model and could contribute towards the emerging observation that translational regulation plays an important role in oxidative stress response, a role mainly ascribed to transcriptional regulation thus far.

Iron is essential for cellular metabolism, due to its requirement as a cofactor for a large number of enzymes. However, an excess of iron is toxic because of its involvement in the Fenton reaction that enhances the formation of ROS. In this work, a gene homologous to fatD, encoding a putative Fe 3+ ABC-transporter was found to be involved in S. thermophilus oxidative stress response. To test whether or not S. thermophilus FatD is indeed an iron transporter, the sensitivity of the fatD mutant to streptonigrin (an iron activated-antibiotic) was assessed. No significant difference between the mutant and the wild type strains was detected (data not shown) suggesting that disruption of fatD would have no effect on intracellular iron concentration. Thus, either FatD is not a major iron transporter under the experimental procedure we used or S. thermophilus fatD encodes an ABC transporter for another metallic cation. A possible cation could be Mn 2+ known to have a protective function against oxidative damage either via direct dismutase activity, such as reported in other organisms (4,26,60), or via its role as a cofactor of the only SOD reported into S. thermophilus (19). Thus, inactivation of a Mn 2+ ABC transporter could trigger sensitivity to oxidative stress.

[Fe-S] clusters are oxygen sensitive prosthetic groups, incorporated into proteins via covalent bonds to cysteines. Iron-sulfur proteins, which are found in more than 120 classes of enzymes and proteins (30), are known to play important physiological roles in electron transfer, metabolic reactions and in transcriptional regulation (30,32). Indeed, because of their intrinsic redox properties, they are likely to sense oxygen or its derivatives as it is the case in SoxR and FNR transcriptional regulators, responding to oxidative stress and oxygen availability, (for a review (32)). IscR is also a iron-sulfur transcriptional regulator that is suggested to respond to Fe-S cluster limiting conditions and potentially to hydrogen peroxide in E. coli (32). Although it has been shown that [Fe-S] cluster assembly can occur spontaneously in vitro under anaerobic conditions (36), this process can also be catalyzed enzymatically in vivo. Genes of the suf operon and of the isc cluster (iron sulfur cluster) are conserved and encode [Fe-S] cluster biosynthesis and/or repair pathways (23). More precisely, it has been recently proposed that Isc proteins are necessary for biosynthesis, while Suf proteins are essentially involved in repair of oxidatively damaged [Fe-S] clusters (37). Additionally, S. thermophilus sufD and iscU mutants displayed an increased sensitivity to superoxide radicals compared to wild type. Thus, SufD and IscU are likely to play a major role in oxidative stress defense, probably resulting from their predicted role in [Fe-S] cluster assembly and/or repair. The involvement of the suf operon in oxidative stress resistance during the early steps of infection by Er. chrysanthemi has also been reported (37), as well as 20 the induction of the suf gene cluster of E. coli by the hydrogen peroxide response regulator OxyR (72). In spite of a possible spontaneous formation, our data suggest that under oxidative stress, [Fe-S] cluster assembly and/or repair becomes a limiting step in the formation of ironsulfur proteins, and that the cell requires an additional, dedicated system, encoded by the suf gene cluster, to optimize [Fe-S] cluster formation or repair.

Conclusion:

The strategy used in this work allowed the identification of genes involved in S. thermophilus oxidative stress defense system. Data concerning the function of some loci is already available in the literature, whereas the role of others is not known. In both cases, the exact roles of these genes in the defense of S. thermophilus against oxidative stress remains to be elucidated.

LEGENDS FIGURES

INTRODUCTION

Oxygen is an essential element for the viability of most living organisms through its involvement in oxidative phosphorylation that takes place during the aerobic respiration process. However, oxygen can also be harmful, due to the production, especially during respiration, of toxic derivatives namely ROS (reactive oxygen species) such as hydrogen peroxide, hydroxyl and superoxide radicals (for a review, ( 25)). Accumulation of these molecules, triggers lipid, protein and nucleic acid oxidation, causing lethal cellular damage (for reviews (7,25,53)). In response to such attack, aerobic micro-organisms have evolved anti-oxidant mechanisms (eg catalase, peroxidase and superoxide dismutase activities) to protect and adapt cells against ROS excess (6,18,24,65,68).

Although they do not achieve aerobic respiration, anaerobic micro-organisms can be subject to ROS. The strict anaerobes Bacteroides fragilis and Clostridium perfringens are able to survive in the presence of a low amount of oxygen, due to the induction of an adaptative oxidative stress response that is activated under aerobic conditions (13,(46)(47)(48)(49). The peroxide response of B. fragilis shares some characteristics with that of E. coli such as the involvement of the redoxsensitive transcriptional activator OxyR (46) and some antioxidant activities under its control (47,49).

The lactic acid bacterium (LAB) Streptococcus thermophilus is a Gram-positive bacterium widely used in dairy factories, mainly as a starter of fermentation in yogurt and cheese production. During fermentation processes, S. thermophilus is exposed to oxygen and to its deleterious effects that can lead to a reduction of cell viability and consequently may have repercussions on texture, flavor and safety of the final product.

S. thermophilus is an anaerobic aerotolerant organism that can grow in presence of oxygen and survive to low concentrations of superoxide and hydroxyl radicals (59). This suggests that it displays a defense system that provides protection against the effects of ROS. In the literature, data about its oxidative stress response are scarce. The existence of at least one ROS defense system and its inducibility by hydrogen peroxide are established in S. thermophilus CNRZ368 (58). This organism possesses a single manganese-containing superoxide dismutase (19) and a glutathione reductase (43). However, no catalase activity was found. In other streptococci, antioxidant enzymes were reported to play a role in the defense against oxidative stress (27,33,44,66) and in pathogenesis (45,67,69).

The aim of this work was to identify genes involved in the oxidative stress defense of S. thermophilus CNRZ368 without presuming their function. For this purpose a collection of mutants was created by random insertional mutagenesis and the mutants were screened for their sensitivity to menadione (a superoxide generating agent). The locus disrupted in 10 S. thermophilus menadione sensitive clones was identified and the potential physiological role of the mutated genes in oxidative stress defense is discussed. The implication of these 10 loci during heat shock was also evaluated to assess their specificity to oxidative stress defense.

Moreover, the characterization of two of these mutants, disrupted in sufD and iscU genes which may participate in Fe-S clusters assembly or repair, is more detailed. The results presented in this paper indicate that the inactivation of sufD and iscU genes caused a higher sensitivity to superoxide stress and suggest that Fe-S cluster repair or assembly could play an important role in the oxidative stress defense of anaerobic bacteria.

MATERIALS AND METHODS

Bacterial strains, growth conditions. S. thermophilus CNRZ368 was purchased from the INRA-CNRZ strain collection (Jouy en Josas, France). Depending on the experiments, S. thermophilus CNRZ368 and its derivatives were cultivated at 30°C or 42°C in milk medium (for pre-cultures), TPPY (12) or M17 (55) media, without shaking. Erythromycin was added at 2 µg.ml -1 , when required. Streptonigrin sensitivity was monitored using a TPPYC medium which contained a reduced concentration of metallic dications (including Fe 2+ ).

TPPYC was performed according to the protocol described by Visca et al. (62) and modified as follow: Chelex beads (20 g/l, sigma) were added to TPPY medium and stirred at 4°C for 48 h and the medium was then supplemented with 1 mM MgCl 2 and 1 mM CaCl 2 .

Recombinant plasmids were transformed into E. coli EC101, a TG1 strain [supE hsd-5 thi ∆(lac-proAB) F' (traD6 proAB + lacI q lacZ∆M15)] containing a chromosomal copy of the pWV01 repA gene ( 34) and selected at 37°C on Luria-Bertani (LB) (50) agar plates containing 150 µg/ml of erythromycin.

Insertional mutagenesis. The collection of mutants was made by insertional mutagenesis using the plasmid pGh9:ISS1 that carries Em R gene (35). The mutagenesis procedure and the cloning of the fragments flanking the pGh9:ISS1 insertion locus were carried out on S. thermophilus CNRZ368 genome, as previously described (56).

Selection of mutants impaired in their ability to tolerate menadione. For selection of menadione sensitive clones, two different procedures, differing mainly by the length of the menadione-exposure, were assayed. Both assays were preceded by a pre-culture in milk medium containing 2 µg/ml erythromycin: pre-cultures of the 2112 mutants were performed in 96-well microtitre plates.

For "long" time menadione exposure, each pre-culture was transferred, using a hedgehog, onto M17 agar plates containing 2 µg/ml erythromycin and increasing concentrations of menadione (from 0 µg/ml to 120 µg/ml). After 20h incubation at 42°C, the presence (or the absence) of colonies was observed.

For "short" time menadione exposure, each pre-culture was diluted (with a 1/100 ratio) in 5 different 96-well microtitre plates containing liquid TPPY medium supplemented with 2 µg/ml erythromycin (in final volume of 100µl, resulting in an approximately 1/1 liquid to air space ratio). After a 3h30 growth period at 42°C, 5 different concentrations of menadione (from 0 mg/ml to 15 mg/ml) were added to each of the 5 microtitre plates. Microtitre plates were incubated at 42°C for 3h. The cultures were then transferred on TPPY agar plates containing erythromycin (2 µg/ml) using a hedgehog. The ability of the mutants to survive to the menadione stress was assessed, after 20 h incubation at 42°C, by observation of the presence or the absence of colonies.

For each experiment, clones were considered as sensitive when they did not grow on a plate where more than 90% of the clones were growing. Both procedures were applied twice and the results had been compiled to reveal the mutants raising the more severe phenotype.

DNA manipulations. Preparation of chromosomal, plasmid and recombinant DNA and

Southern analysis were performed according to standard protocols (50). All cloning junctions and cloned PCR products were checked by sequencing using Dye-Terminator chemistry on an ABI Prism 377 Genetic Analyser (PE Biosystems). Sequence data were analysed using Genbank, BLAST (2, 3), TMHMM (51) and HMMTOP ( 61

S. thermophilus competent cells was performed as previously described by O'sullivan and

Fitzgerald (41). The following gene replacements were done as previously described ( 8) but in TPPY medium and at 30°C/42°C as permissive and restrictive temperature, respectively.

Physiological characterization of S. thermophilus mutant strains.

Exposure to methyl viologen. For "short" time exposure, cultures of CNRZ368 and its derivative strains were grown in TPPY medium. At an OD 600 of 0.6, cultures were divided into two subcultures (in a ratio of liquid to air space of approximately 1/1). Methyl viologen (paraquat; Sigma-Aldrich) was added to one of them with a final concentration of 50 mM. After 3 h incubation at 42°C, cultures were diluted to appropriate concentrations and spread onto 8 TPPY agar plates. For "long" time methyl viologen exposure, strains were grown in TPPY medium until OD 600 of 0.6. Cultures were then diluted to a final theorical OD 600 of 0.001 in TPPY medium (in a ratio of liquid to air space of approximately 5/6) supplemented with 1.7 mM methyl viologen, or not for control experiment. After 18h incubation at 42°C, cultures were diluted in TPPY to appropriate concentrations and spread onto TPPY agar plates. In both procedures, methyl viologen sensitivity was estimated by comparing CFU/ml obtained with treated cultures, with respect to non treated cultures.

Heat shock. The ability of S. thermophilus and its derivatives to respond to a heat stress was assessed as follow. Each strain is grown at 42°C in TPPY medium until early log phase (OD 600 = 0.3). Then the culture was divided into two. One part was kept at 42°C (control experiment); the other one was immediately transferred to 60°C for 15 min. Appropriate dilutions were spread on TPPY agar plates. The fraction of surviving cells was calculated by dividing the surviving CFU/ml by the count for the control experiment.

Streptonigrin sensitivity. Streptonigrin sensitivity was monitored on cells grown overnight

in TPPYC medium at 42°C. Cells were harvested by centrifugation, washed twice in TPPYC medium, resuspended in TPPYC, diluted to OD 600 = 0.01 and cultivated in TPPYC for 9 h at 42°C. Then, the cultures were diluted again to OD 600 = 0.005 and divided into 4 parts which were all incubated for 21 h at 42°C. One part was supplemented with 0.4% (v/v) dimethyl formamide (control experiment), the 3 other parts were supplemented with different concentrations of streptonigrin (1 µg/ml, 2 µg/ml and 4 µg/ml dissolved in dimethyl formamide). As a control experiment, cultures were also carried out in TPPYC supplemented with 1 mM of 2'2-dipyridyl (an Fe chelator). In each experiment, OD 600 value was used to estimate the cellular density after 21h incubation. Relative optic density was determined by comparing cellular density obtained with and without streptonigrin. All experiments were performed in triplicate.

RESULTS

1. Selection of S. thermophilus CNRZ368 mutants with altered oxidative stress response.

A collection of 2112 mutants of S. thermophilus CNRZ368 was generated by insertional mutagenesis using the plasmid pGh9:ISS1 (35,56). This plasmid carries an erythromycin resistance gene for its selection and the insertional element ISS1 that allows random integration of the plasmid into S. thermophilus genome. The 2112 mutants obtained were screened to select clones sensitive to menadione (a superoxide-generating compound). Two distinct selection procedures were used to identify mutants susceptible to menadione (see materials and methods). The characterization of 10 mutants, disrupted in loci originally named ossA to ossJ (for oxidative stress sensitive locus) is described in this paper.

Sensitivity of S. thermophilus CNRZ368 mutants to methyl viologen. Previously, we

demonstrated that ossB and ossC mutants (previously named 6G4 and 18C3, respectively) are compromised in their ability to survive superoxide stress (57). To confirm that the other 8 mutants selected were sensitive to superoxide stress, their sensitivity to methyl viologen was examined (Fig. 1). In this experiment, we used the non-polar mutants ∆sufD and iscU 97 constructed in this work (see further) rather than the potential polar clones ossI and ossJ.

Short-term methyl viologen exposure revealed the extreme sensitivity of ossG, as well as the 4 to 5-fold increased sensitivity of ossD, ossH and iscU 97 mutants (Fig. 1A). Although less severe the ∆sufD mutant exhibited about 2-fold less survival following such stress. Under these conditions, the other 3 mutants (ossA, ossE and ossF) did not exhibit any significant sensitivity, however long term methyl viologen exposure confirmed their sensitivity. Indeed, long term methyl viologen exposure (Fig. 1B) showed that the ossA mutant strain displayed a relative survival more than 10-fold lower than that of the wild type strain. The ossE and ossF mutants also showed a 2-fold lower growth compared to the wild type strain.

3. Survival of the mutants exposed to heat stress. To assess the specificity of these 10 loci to oxidative stress defense, the mutants ability to survive to heat shock was estimated. Five out of the 10 mutants (ossA, ossB, ossC, ossD, and ossJ) isolated as oxidative stress sensitive clones also showed a reduced survival to heat shock compared with WT (Fig. 2). These data suggest that the genes impaired in these mutants are not specific to oxidative stress defense process but are probably involved in a more global response to stresses.

In contrast, the ossE, ossF and ossG mutants did not differ significantly from the wild type, indicating that the genes disrupted in these strains did not influence cells ability to respond to heat shock. Interestingly, the ossG mutant, that was highly sensitive to methyl viologen, showed no significant decrease of survival to heat shock compared with WT, suggesting that ossG gene, essential for oxidative stress defense, could be specific to such a stress.

Identification of genes implicated in oxidative stress tolerance.

The disrupted locus was identified for each of the 10 mutants by cloning and sequencing the junctions as previously described (56). All of the insertions were mapped within coding sequences. Sequence similarity searches and analysis revealed similar sequences in other bacteria (see Table 1)

with an E-value lower than 10 -10 for BLASTX searches. In 9/10 cases, data about the function of these genes were available. To facilitate their description, these genes were grouped into 5 classes (from I to V) according to the putative function and renamed when relevant (Table 1).

Class I: Genes potentially involved in cell wall structure and cell shape determination.

The ossA, ossB and ossC putative gene products shared sequence identity with MreD, RodA and Pbp2b, respectively, which putatively direct the elongation of the lateral cell wall in E. coli, conferring the cell its characteristic rod shape (5,28,29). The ossA mutant was defective in a gene encoding a protein, with 30% identity with MreD of Streptococcus mutans. Sequence analysis of the surrounding region indicated that S. thermophilus mreD gene (ossA) was clustered with a gene encoding a putative protein similar to MreC. Moreover, hydrophopathy profiles of MreD (OssA) determined by using TMHMM and HMMTOP showed 5 regions of sufficient length and hydrophobicity to be membrane-spanning segments (data not shown), suggesting that this protein is the counterpart of MreD. The relatively low identity (30%) observed between S. thermophilus and S. mutans MreD was in agreement with the general idea that the structure of this protein is more highly conserved than its primary sequence (17). To study the effect of mreD disruption in S. thermophilus, TPPY cultures of the wild type and mreD mutant strains were observed under light microscopy. In contrast to wild type cells, mreD mutant cells formed very long chains, up to 180 cells per chain (Fig. 3).

Moreover, a reduction of about 25% of the cell size was observed in the mutant. Growth of the mreD mutant and of the wild type in TPPY medium were monitored by measuring the OD 600 . Both strains displayed a similar doubling time of 30 min, but the maximal OD 600 of the mreD mutant (OD max = 1.7 ± 0.1) was lower than that of the wild type (OD max = 2.4 ± 0.2)

showing that the mreD mutant entered stationary phase earlier than wild type cells. The characterization of rodA and pbp2b mutants of S. thermophilus CNRZ368 is described in a previous paper (57).

Class II: Gene potentially involved in exopolysaccharide translocation. The sequence disrupted in the mutant ossD was partially determined. Its analysis indicated that the C-terminal portion of the predicted encoded protein showed 98% identity to CpsX, a putative membrane protein from S. thermophilus MR-1C. The role of CpsX is still unknown but the conserved location of cpsX near eps genes of S. thermophilus (14) and the presence of a putative gene related to an ABC transport system in its vicinity argue in favor of CpsX being involved in exopolysaccharide translocation (15). Moreover, the TMHMM and HMMTOP software predictions indicated that CpsX (OssD) from S. thermophilus CNRZ368 would contain highly hydrophobic domains compatible with a location in the cytoplasmic membrane. Although the cpsX function is not known yet, our work indicate that its disruption is associated with sensitivity not only to superoxide stress but also to heat shock.

Class III: Genes involved in RNA maturation. In the ossE and ossF mutants, the plasmid inserted into genes whose predicted products share sequence identity with enzymes involved in RNA maturation. The ossE gene encodes a putative tRNA guanine transglycosylase (TGT) which shares 92% identity with the putative Streptococcus pneumoniae TGT. TGT enzyme takes part in the process which removes the guanine residue at position 34 (wobble position of the anticodon loop) of tRNA His , tRNA Asp , tRNA Asn and tRNA Tyr and replaces it by the modified residue queuosine (39,40).

ossF potentially encodes a protein that shows 77 % identity with a putative RNA methyltransferase of Streptococcus agalactiae (Gbs0448) belonging to the TrmA family.

Enzymes of the TrmA family catalyze the formation of the 5-methyluridine in position 54

(localized in the TΨC loop) of tRNA (9). The presence of a TRAM-like domain (RNA binding domain) in the N-terminal part of OssF supports the idea of an interaction between this protein and RNA.

Class IV: Gene of unknown function. The ossG product shows 67% identity with the product of a gene of unknown function (smu.1428c) of S. mutans (Table 1). Although ossG function is not known yet, the mutation of this gene is now associated with a sensitivity phenotype to superoxide stress. The TMHMM and HMMTOP software predictions suggest the existence of two transmembrane domains in the N-terminal region of OssG, making of OssG a potential membrane protein. (ii) [Fe-S] cluster biosynthesis. The sequence affected in the ossI mutant encodes a product that shows 75% identity with Sag0142, a putative protein deduced from the complete genome sequence of S. agalactiae and similar to SufD-like proteins. Analysis of totally sequenced microbial genomes reveals that sufD-like genes are conserved in bacteria and also in some archaea such as Halobacterium sp. NRC-1, Thermoplasma acidophilum and Aeropyrum pernix (23). In E. coli and Erwinia chrysanthemi, the sufD gene belongs to the sufABCDSE cluster identified as being involved in iron metabolism and more precisely in [Fe-S] cluster biogenesis and/or repair (37,42). The ossJ gene product shows sequence identity to the gbs0140 product from S. agalactiae (Table 1), probably encoding a IscU protein, a NifU family protein. NifU proteins have been proposed to serve as scaffold proteins in [Fe-S] cluster assembly in Synechocystis PCC6803 and would function as a mediator for iron-sulfur cluster delivery (38).

In some genomes including that of S. pyogenes, nifU-like genes are clustered with the suf genes (23). To determine if sufD (ossI) and iscU (ossJ) from S. thermophilus were genetically linked together, the chromosomal DNA regions flanking the pGhost9:ISS1 insertion site of both mutants were sequenced until overlapping. Examination of the genes in the neighborhood of sufD and iscU revealed that they both belong to a cluster of 5 genes all orientated in the same direction. This cluster, named sufCDS iscU sufB cluster, is shown in Fig. 4. The highest sequence identities of these gene products are found with putative proteins from S. agalactiae (Table 2). They also share sequence similarity with proteins encoded by E. coli and Er. chrysanthemi suf gene clusters. The putative SufC, SufD, SufS and SufB proteins encoded by the S. thermophilus cluster present respectively 72%, 48%, 65%, and 57% similarity to their E. coli counterparts. Moreover, IscU presents 47% similarity with IscU from E. coli.

Sequences corresponding to promoter regions were searched by inspection of the total sequence of the sufCDS iscU sufB cluster. A unique extended -10 sequence

(5'-TTTGTTATAAT-3') is found upstream sufC, the first ORF of the cluster. A potential rho-independent transcriptional terminator is identified 63 bp downstream sufB, the last ORF of the cluster (∆G = -10 kcal/mol). These features, together with the short or missing intergenic regions and the same orientation of the genes, suggest that these 5 ORFs form an operon, as it is the case for the Er. chrysanthemi suf locus.

Molecular characterization of the sufCDS iscU sufB cluster.

5.1. Construction of ∆sufD and iscU 97 mutants. Since S. thermophilus suf genes are potentially clustered in an operon, non polar ∆sufD and iscU 97 mutants were constructed and analysed. The sufD mutation consists of an internally in frame deletion leading to the loss of 43% of the whole protein (see materials and methods and Fig. 4). Sequence comparison of NifU family proteins from various organisms indicates that S. thermophilus IscU protein contains the three highly conserved cystein residues that have been shown to be the assembly site of a "transitory" [2Fe-2S] center within NifU (70). Thus, the construction of a null iscU mutant was achieved by replacing one of these conserved residues, the Cys 97 , by a Tyr (see materials and methods and Fig. 4). Sequencing of these two constructs confirmed that they did not contain any secondary mutation.

Role of sufD and iscU in iron metabolism.

To investigate the possibility that ∆sufD and iscU 97 mutations impair growth due to the lack of functional [Fe-S] cluster biosynthesis, the mutants were grown in TPPY medium and compared to wild type. Growth of the ∆sufD and iscU 97 mutants showed no significant difference compared to the WT strain. Assuming that the high iron concentration of the TPPY medium could allow self assembling of the [Fe-S] clusters (71), and consequently could be responsible for the absence of difference in growth between strains, the same experiment was conducted in TPPYC, a divalent cation depleted medium. Under these conditions, the mutants displayed a growth rate similar of that of the WT strain but their maximal OD 600 was lower than that of the wild type, showing their premature entry in stationary phase (Table 3).

Moreover, this difference disappeared when the cultures were grown on TPPYC supplemented by 100 µM of FeSO 4 . Taken together, these results suggest that (i) iscU and sufD genes of S. thermophilus are involved in iron metabolism and (ii) their presence would be of particular importance under iron-limiting conditions.

To confirm the involvement of sufD and iscU in iron metabolism, the iron intracellular concentration of ∆sufD and iscU 97 was checked by measuring the sensitivity of these strains to streptonigrin (an iron-activated antibiotic). As shown in Fig. 5 A, the iscU mutant was more sensitive to streptonigrin than the wild type strain whereas the ∆sufD mutant was more resistant. Moreover, addition of 2,2'-dipyridyl (a ferrous iron chelator) abolished the difference of growth between the strains (Fig. 5 B) indicating that iron was responsible for the streptonigrin sensitivity observed without 2,2'-dipyridyl. Taken altogether these results suggest that both iscU and sufD of S. thermophilus play a role in iron metabolism.

The major function assigned to cell wall is the maintenance of cell integrity by protecting cells not only against osmotic stress but also against other stress conditions. For instance, in LAB, it has already been shown that compromised biosynthesis of peptidoglycan results in an increased sensitivity to either UV (22) or acid (11) stress. Additionally, disruption of mreD, rodA or pbp2b may cause a modification of the peptidoglycan architecture in S. thermophilus, reducing the cell protection against aggressions such as oxidative and heat stress.

Transfer RNA modification is a universal feature of all living organisms, although some described modifications have no assigned function yet. Modifying enzymes affect the tRNA precursor either by chemical modification of an existing residue (in the base or in the sugar)

or by nucleotide substitution. These modifications, in particular those present in the anticodon region can affect translation efficiency and fidelity (for a review (10)). In this study, two clones disrupted in the loci tgt and ossF, encoding proteins showing high identity with tRNA modifying enzymes were selected for their high sensitivity to oxidative stress. This sensitivity could suggest that the expression of genes involved in oxidative stress defense may require tRNA maturation or, that such modifications could help tRNA stabilization under stress conditions. In agreement with the former hypothesis Shigella flexneri pathogenicity is regulated by virF mRNA translation efficiency under TGT activity control (21). Additionally, several environmental conditions are suggested to influence tRNA modification: in the case of queuosine (whose synthesis and substitution is carried out by several enzymes among which is TGT), synthesis is sensitive to the presence of iron (31) and TGT requires oxygen for its full activity (54). Therefore, further investigations on this topic should provide an interesting new regulatory model and could contribute towards the emerging observation that translational regulation plays an important role in oxidative stress response, a role mainly ascribed to transcriptional regulation thus far.

Iron is essential for cellular metabolism, due to its requirement as a cofactor for a large number of enzymes. However, an excess of iron is toxic because of its involvement in the Fenton reaction that enhances the formation of ROS. In this work, a gene homologous to fatD, encoding a putative Fe 3+ ABC-transporter was found to be involved in S. thermophilus oxidative stress response. To test whether or not S. thermophilus FatD is indeed an iron transporter, the sensitivity of the fatD mutant to streptonigrin (an iron activated-antibiotic) was assessed. No significant difference between the mutant and the wild type strains was detected (data not shown) suggesting that disruption of fatD would have no effect on intracellular iron concentration. Thus, either FatD is not a major iron transporter under the experimental procedure we used or S. thermophilus fatD encodes an ABC transporter for another metallic cation. A possible cation could be Mn 2+ known to have a protective function against oxidative damage either via direct dismutase activity, such as reported in other organisms (4,26,60), or via its role as a cofactor of the only SOD reported into S. thermophilus (19). Thus, inactivation of a Mn 2+ ABC transporter could trigger sensitivity to oxidative stress.

[Fe-S] clusters are oxygen sensitive prosthetic groups, incorporated into proteins via covalent bonds to cysteines. Iron-sulfur proteins, which are found in more than 120 classes of enzymes and proteins (30), are known to play important physiological roles in electron transfer, metabolic reactions and in transcriptional regulation (30,32). Indeed, because of their intrinsic redox properties, they are likely to sense oxygen or its derivatives as it is the case in SoxR and FNR transcriptional regulators, responding to oxidative stress and oxygen availability, (for a review (32)). IscR is also a iron-sulfur transcriptional regulator that is suggested to respond to Fe-S cluster limiting conditions and potentially to hydrogen peroxide in E. coli (32). Although it has been shown that [Fe-S] cluster assembly can occur spontaneously in vitro under anaerobic conditions (36), this process can also be catalyzed enzymatically in vivo. Genes of the suf operon and of the isc cluster (iron sulfur cluster) are the induction of the suf gene cluster of E. coli by the hydrogen peroxide response regulator OxyR (72). In spite of a possible spontaneous formation, our data suggest that under oxidative stress, [Fe-S] cluster assembly and/or repair becomes a limiting step in the formation of ironsulfur proteins, and that the cell requires an additional, dedicated system, encoded by the suf gene cluster, to optimize [Fe-S] cluster formation or repair.

Conclusion:

The strategy used in this work allowed the identification of genes involved in S. thermophilus oxidative stress defense system. Data concerning the function of some loci is already available in the literature, whereas the role of others is not known. In both cases, the exact roles of these genes in the defense of S. thermophilus against oxidative stress remains to be elucidated. (TPPYC medium supplemented with MgCl 2 (1 mM) and CaCl 2 (1 mM)). After one night growth, cells were washed twice and resuspend either in FeSO 4 -deficient medium or supplemented with 100 µM FeSO 4 . The growth of the cells was then followed by measuring the OD 600 . The OD 600 were measured after 24h incubation at 42°C. This experiment was done in triplicate.

LEGENDS FIGURES

  amino-acids Gly 96 Cys 97 Tyr 98 were replaced by Arg 96 Tyr 97 Thr 98 in the mutated protein. used with two additional external primers (AAGCGGAATTCAAGCATCCACTCAGGC containing an EcoRI site and TGTGACAAGCTTGTAGAGGCAATTCTT containing an HindIII site), respectively, to amplify 5' and 3' end of iscU. The two PCR products of 515 bp and 262 bp were digested by EcoRI and EcoRV and by HindIII and EcoRV respectively before being ligated to each other. The resulting fragment was cloned into pGh9 digested by EcoRI and HindIII. The final construct, (pGh9iscU 97 ) was used to transform S. thermophilus CNRZ368. Preparation of S. thermophilus competent cells was performed as previously described by O'sullivan and

  Study of ∆sufD and iscU 97 non-polar mutants indicates that SufD and IscU proteins are required for growth in an iron-deficient medium. SufD and IscU inactivation conferred an increased resistance and sensitivity respectively to streptonigrin. These results indicate that S. thermophilus SufD and IscU are involved in intracellular iron balance. Additionally, SufD and IscU of S. thermophilus, similar to SufD and NifU family proteins from other microorganisms, are encoded by genes clustered with sufB, sufC and sufS homologues, also potentially involved in [Fe-S] cluster repair. Hence the participation of S. thermophilus SufD and IscU proteins in iron intracellular balance is probably the result of their involvement in [Fe-S] cluster repair, as in Er. chrysanthemi(37). The NifU family protein function is to provide a scaffold for assembly of clusters mediated by NifS activity, and such clusters can then be transferred into apo forms of [Fe-S] cluster containing proteins(1, 70). The role of the SufD in [Fe-S] cluster biosynthesis is not well established. However, in S. thermophilus, the increased resistance to streptonigrin of the ∆sufD mutant suggests that its intracellular iron concentration is lower than in the wild type. At least two alternative hypotheses can be drawn: (i) SufD could be directly involved in the uptake or assimilation of extracellular iron, (ii) SufD could inhibit the synthesis and/or repair of the [Fe-S] clusters. In this latter case, SufD inactivation would result in an increase synthesis of [Fe-S] clusters, and consequently in a reduced intracellular free iron concentration.

FIG. 1 .FIG. 2 .FIG. 3 .FIG. 4 .FIG. 5 .

 12345 FIG. 1. Relative methyl viologen sensitivity. (A). Results of short term methyl viologen

  ) software. For all analysed sequences, The DNA sequences reported in this paper have been deposited in GenBank under accession numbers AY386239, AY386240, AY386241, AY386242, AY386243, AY386244, AY386245.Construction of the ∆sufD and iscU 97 mutants. Oligonucleotides used to construct the ∆sufD and iscU 97 mutants were purchased from MWG Biotech AG (Ebersberg, Germany). To construct non-polar truncated ∆sufD mutant, the 5' and 3' ends of the sufD gene were independently amplified by PCR. The 5' end was amplified by using the primers GAGCGTGTTGAATTCCACCG containing an EcoRI site, symbolized in bold characters and AACTTAACTGCAGCACCGTCT containing a PstI site. The 3' end was amplified by using the primers GGTGTTCCAGAATTCAAATAACC containing an EcoRI site and TGGTCTGTCATCAGGTCG. This last primer does not contain any PstI site, but the PCR product generated does contain a PstI restriction site. These two PCR products were digested by PstI and ligated with each other. The resulting fragment was amplified by PCR, then digested by EcoRI and cloned into pGh9. The final construct (pGh9∆sufD) was used to transform S. thermophilus CNRZ368. For IscU protein inactivation, the strategy consisted in replacing the TGT codon encoding the conserved Cys 97 by a TAT codon encoding a Tyr. This was achieved by replacing, in oligonucleotides, the 3 underlined nucleotides of the sequence (AGG TGT ACA) generating an EcoRV restriction site (AGA TAT CCA). Thus, the three amino-acids Gly 96 Cys 97 Tyr 98 were replaced by Arg 96 Tyr 97 Thr 98 in the mutated protein. used with two additional external primers (AAGCGGAATTCAAGCATCCACTCAGGC containing an EcoRI site and TGTGACAAGCTTGTAGAGGCAATTCTT containing an HindIII site), respectively, to amplify 5' and 3' end of iscU. The two PCR products of 515 bp and 262 bp were digested by EcoRI and EcoRV and by HindIII and EcoRV respectively before being ligated to each other. The resulting fragment was cloned into pGh9 digested by EcoRI and HindIII. The final construct, (pGh9iscU 97 ) was used to transform S. thermophilus CNRZ368. Preparation of

Class V :

 : Genes involved in iron metabolism.(i) Ferric iron transport. The putative product of ossH shows 55% identity with FatD from S. pneumoniae, a potential membrane spanning permease of an Fe 3+ ABC-transporter. Analysis of the adjacent sequence reveals the presence of a gene encoding a putative FatC similar protein downstream of fatD (ossH), in the same orientation. The fatC gene is also clustered with fatD in S. pneumoniae genome. Hydropathy profile of FatD (OssH) from S. thermophilus, assesses with TMHMM and HMMTOP, reveals that this protein display ten putative membrane spanning segments compatible with its expected cytoplasmic membrane location.

. 1 .

 1 FIG. 1. Relative methyl viologen sensitivity. (A). Results of short term methyl viologen

FIG. 2 .FIG. 3 .FIG. 4 .FIG. 5 .

 2345 FIG. 2. Survival to heat shock. The heat shock consisted of 15 min treatment at 60°C. The

Va.

  Initial gene name with, in brackets and when applicable, the definitive gene name. b. Disrupted ORF of S. thermophilus CNRZ368 that have been partially sequenced. c. Organism, gene name and, in brackets, length of the protein sequence giving the best alignment score. d. Percentage of sequence identity with the protein showing the best alignment score to each S. thermophilus Oss protein. In brackets is indicated the portion of the protein sharing this identity (numbers correspond to the amino acid positions).e. The numbers correspond to the deduced amino acid position of the pGh9:ISS1-generated disruption in the full length protein showing the best alignment score to the Oss proteins.

TABLE 1 .

 1 Products of oss genes: database similarities and putative fonctions. Oss protein. In brackets is indicated the portion of the protein sharing this identity (numbers correspond to the amino acid positions).e. The numbers correspond to the deduced amino acid position of the pGh9:ISS1-generated disruption in the full length protein showing the best alignment score to the Oss proteins.

	Gene name a	Accession number	Gene encoding the most similar product c	amino acid identity d	Insertion site e	Putative function Class
	ossA (mreD)	AY386242 S. mutans mreD (168)	30% (1-168)	127	Cell shape maintenance
	ossB (rodA)	AF399832 S. mutans rodA (408)	65% (1-391)	304	Peptidoglycan elongation	I
	ossC (pbp2b) b AF399833	S. thermophilus Sfi6 pbp2b (704)	98% (641-704)	640	Peptidoglycan elongation
	ossD (cpsX) b AY386241 S. thermophilus cpsX (378)	98% (212-378)	264	Membrane exopolysaccharide translocation of	II
	ossE (tgt) b AY386243 S. pneumoniae tgt (380) ossF b AY386244 S. agalactiae gbs0448 (451)	92% (1-333) 77% (1-204)	99 51	tRNA-guanine transglycosylase methyltransferase RNA	III
	ossG	AY386239 S. mutans smu.1428c (284) 67% (1-280)	170	Unknown conserved function IV
	ossH (fatD)	AY386240 S. pneumoniae fatD (324) 55% (22-323) 119	Iron ABC transporter
	ossI (sufD)	AY386245	S. agalactiae sag0142 (420)	76% (1-420)	144	Assembly of Fe/S clusters	V
	ossJ (iscU)	AY386245	S. agalactiae gbs0140 (147)	78% (1-141)	57	Assembly of Fe/S clusters

a. Initial gene name with, in brackets and when applicable, the definitive gene name. b. Disrupted ORF of S. thermophilus CNRZ368 that have been partially sequenced. c. Organism, gene name and, in brackets, length of the protein sequence giving the best alignment score. d. Percentage of sequence identity with the protein showing the best alignment score to each S. thermophilus

TABLE 3 .

 3 Growth of wild type and mutant strains in TPPYC and in TPPYC supplemented with Cells were grown in milk medium for 15°h at 42°C and diluted in FeSO 4 -deficient medium (TPPYC medium supplemented with MgCl 2 (1 mM) and CaCl 2 (1 mM)). After one night growth, cells were washed twice and resuspend either in FeSO 4 -deficient medium or supplemented with 100 µM FeSO 4 . The growth of the cells was then followed by measuring the OD 600 . The OD 600 were measured after 24h incubation at 42°C. This experiment was done in triplicate.

	iron.
		Final OD 600 resulting from growth a in:
	Strain	TPPYC	TPPYC + FeSO 4
	WT	0.96	1.32
	∆sufD	0.58	1.48
	iscU 97	0.58	1.15

a

TABLE 1 .

 1 Products of oss genes: database similarities and putative fonctions.

	Gene name a	Accession number	Gene encoding the most similar product c	amino acid identity d	Insertion site e	Putative function Class
	ossA (mreD)	AY386242 S. mutans mreD (168)	30% (1-168)	127	Cell shape maintenance
	ossB (rodA)	AF399832 S. mutans rodA (408)	65% (1-391)	304	Peptidoglycan elongation	I
	ossC (pbp2b) b AF399833	S. thermophilus Sfi6 pbp2b (704)	98% (641-704)	640	Peptidoglycan elongation
	ossD (cpsX) b AY386241 S. thermophilus cpsX (378)	98% (212-378)	264	Membrane exopolysaccharide translocation of	II
	ossE (tgt) b AY386243 S. pneumoniae tgt (380) ossF b AY386244 S. agalactiae gbs0448 (451)	92% (1-333) 77% (1-204)	99 51	tRNA-guanine transglycosylase methyltransferase RNA	III
	ossG	AY386239 S. mutans smu.1428c (284) 67% (1-280)	170	Unknown conserved function IV
	ossH (fatD)	AY386240 S. pneumoniae fatD (324) 55% (22-323) 119	Iron ABC transporter
	ossI (sufD)	AY386245	S. agalactiae sag0142 (420)	76% (1-420)	144	Assembly of Fe/S clusters
	ossJ (iscU)	AY386245	S. agalactiae gbs0140 (147)	78% (1-141)	57	Assembly of Fe/S clusters

TABLE 3 .

 3 Growth of wild type and mutant strains in TPPYC and in TPPYC supplemented with iron.Final OD 600 resulting from growth a in: Cells were grown in milk medium for 15°h at 42°C and diluted in FeSO 4 -deficient medium

	Strain	TPPYC	TPPYC + FeSO 4
	WT	0.96	1.32
	∆sufD	0.58	1.48
	iscU 97	0.58	1.15

a

ACKNOWLEDGEMENTS

A. T and A. F. were 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 Paul Hoskinson for his advice regarding the English formulation of the manuscript.

ACKNOWLEDGEMENTS

A. T and A. F. were 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 Paul Hoskinson for his advice regarding the English formulation of the manuscript.

ABSTRACT

To better understand the defense mechanism of Streptococcus thermophilus against superoxide stress, molecular analysis of 10 menadione-sensitive mutants, obtained by insertional mutagenesis, was undertaken. This analysis allowed the identification of 10 genes that, with respect to their putative function, were classified in 5 categories (i) those involved in cell wall metabolism, (ii) those involved in exopolysaccharide translocation, (iii) those involved in RNA modification (iv) those involved in iron homeostasis and (v) those whose function is still unknown.

The behavior of the 10 menadione sensitive mutants exposed to heat shock was investigated.

Data from these experiments allowed us to distinguish genes whose action might be specific to oxidative stress defense (tgt, ossF, ossG) from those whose action may be generalized to other stressful conditions (mreD, rodA, pbp2b, cpsX, iscU).

Among the mutants, two harboured an independently inserted copy of pGh9:ISS1 in two loci close to each other. More precisely, these two loci are homologous to sufD and iscU genes, involved in the biosynthesis of iron-sulfur clusters. This region, called the suf region, was further characterized in S. thermophilus CNRZ368 by sequencing and by construction of ∆sufD and iscU 97 non polar mutants. Streptonigrin sensitivity level of both mutants suggested that these two genes were involved in iron metabolism.

Discussion

To investigate the oxidative stress defense system of S. thermophilus, a collection of random insertion mutants was screened to select clones with high sensitivity to menadione. Using this approach, we aimed to identify genes involved in superoxide stress defense without presuming their function. It can be noted that no gene encoding a protein considered, in other organisms, as major antioxidant enzyme were found using this approach. This can be due to the fact that (i) the collection of mutants was non-exhaustive (ii) the corresponding mutant could have a weak phenotype (iii) such mutations could be lethal (iv) except from SOD, most anti-oxidant enzymes are protective against H 2 O 2 and not O2 .-. The genes identified in the present work were classified into 5 groups according to their function: genes potentially involved in cell wall metabolism, exopolysaccharide translocation, tRNA modification, and iron metabolism and gene of unknown function.

Perhaps the most striking observation of this study is that 5 out of the 10 sensitive mutants selected are disrupted in a gene encoding an membrane protein (OssH, MreD, RodA, PBP2b and CpsX). This could reflect that the cell envelope is the main target of the stress applied.

Among these mutants, three (mreD, rodA and pbp2b) are impaired in genes potentially encoding proteins described in E. coli as playing a role in peptidoglycan biosynthesis during cell elongation (28,52,63). Phenotypic analysis of these three S. thermophilus mutants showed that they all present a reduced cell size associated with a modification of the cell shape, as observed in rodA, pbp2 and mre mutants of E. coli or Salmonella typhimurium (5,20,28,29,63,64). Moreover, the 3 S. thermophilus mutants also presented an increased sensitivity to heat shock compared to the wild type strain suggesting that the protective role of MreD, RodA and PBP2b is not specific to oxidative stress but could be enlarged to a more global stress response. In support of this hypothesis is the existence in Lactococcus lactis of mutations in pbp genes that negatively affect acid tolerance of a guaA mutant (16).

conserved and encode [Fe-S] cluster biosynthesis and/or repair pathways (23). More precisely, it has been recently proposed that Isc proteins are necessary for biosynthesis, while Suf proteins are essentially involved in repair of oxidatively damaged [Fe-S] clusters (37). Additionally, S. thermophilus sufD and iscU mutants displayed an increased sensitivity to superoxide radicals compared to wild type. Thus, SufD and IscU are likely to play a major role in oxidative stress defense, probably resulting from their predicted role in [Fe-S] cluster assembly and/or repair. The involvement of the suf operon in oxidative stress resistance during the early steps of infection by Er. chrysanthemi has also been reported (37), as well as

Study of ∆sufD and iscU


Annabelle Thibessard

Annabelle Fernandez

Brigitte Gintz

Nathalie N Leblond-Bourget

Bernard Decaris

Hydrogen peroxide effects on Streptococcus thermophilus CNRZ368 cell viability

come   L'archive ouverte pluridisciplinaire

Introduction

The oxidative stress response has been well characterized for some microorganisms and especially for the facultative aerobic Gram-negative bacteria Escherichia coli (for a review, [START_REF] Storz | Bacterial stress responses[END_REF]). In this organism, two distinct regulons, OxyR [START_REF] Christman | OxyR, a positive regulator of hydrogen peroxide-inducible genes in Escherichia coli and Salmonella typhimurium, is homologous to a family of bacterial regulatory proteins[END_REF] and SoxR/S [START_REF] Greenberg | Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli[END_REF][START_REF] Tsaneva | soxR, a locus governing a superoxide response regulon in Escherichia coli K-12[END_REF], have been identified and are known to be induced by two distinct oxidative molecules, H 2 O 2 and O -

2

• respectively. The case of streptococci is less well documented since only the existence of some anti-oxidant enzymes have been reported [3, 8, 10, 13 -15, 19]. Moreover, these bacteria lack catalase [START_REF] Sneath | Bergey's manual of systematic bacteriology[END_REF].

Our research focuses on the facultative anaerobic Gram-positive bacterium Streptococcus thermophilus, widely used in dairy product fermentation. Its ability to grow under aerobic conditions raises the question of the existence of a defence system against oxidative stress. Indeed, such a system might be necessary to rescue cells from damage caused by the inevitable reactive oxygen species produced under aerobic growth conditions.

The aim of our work was to test the effect of oxidative stress on S. thermophilus survival. For this purpose, H 2 O 2 was used as the oxidative agent: hydrogen peroxide is notably known to react with reduced iron or copper ions to generate hydroxyl radicals (OH•) in the Fenton reaction [START_REF] Imlay | Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro[END_REF][START_REF] Imlay | DNA damage and oxygen radical toxicity[END_REF].

Materials and methods

Strains and culture conditions

S. thermophilus CNRZ368 was purchased from the Centre National de la Recherche Zootechnique belonging to the INRA strains collection (France). D 1 was isolated in the progeny of strain CNRZ368 after serial subclonings. All experiments were performed on this clone. First, D 1 was grown overnight at 42 • C in milk medium. Overnight culture was then diluted 100-fold in fresh TPPY [START_REF] Bracquart | An agar medium for the differential enumeration of Streptococcus thermophilus and Lactobacillus bulgaricus in yoghurt[END_REF] (0.7% tryptose, 0.7% proteose-peptone, 0.2% bacto yeast extract, 2% lactose) and grown at 42 • C.

Sensitivity to oxidizing agents

D 1 was grown overnight in milk medium, then inoculated into fresh TPPY medium. During exponential growth (OD 600 = 0.6), culture was exposed to oxidative stress by the addition of hydrogen peroxide (0.25 to 30 mM). Cells were left in contact with hydrogen peroxide for 15 min. Aliquots of the culture were diluted in TPPY, and appropriate dilutions plated on TPPY agar plates to determine cell viability.

Plates were stocked in jars provided with the GENbox Anaer system (bioMérieux). Colonies were counted after 20-h incubation under anaerobic conditions. Percentage survival was determined by comparing viability with or without treatment. All experiments were performed at least in triplicate.

Results and discussion

Effect of increasing concentrations of H 2 O 2 on survival

The ability of D 1 (derived from CNRZ368) to respond to 15-min exposure to a range of hydrogen peroxide concentrations (0.25-30 mM) was investigated and led to a depletion of cell viability (figure 1A). When D 1 was treated with low doses, the H 2 O 2 dose response curve was found to be biphasic: between 0 and 0.5 mM H 2 O 2 viability decreased, but then rose again above 0.5 to 1.0 mM (figure 1B). Further increases in the level of H 2 O 2 above 1 mM resulted in a further loss of viability. The kill curves seen in figure 1 are very similar to those seen in E. coli, although S. thermophilus appears to be inherently more sensitive to H 2 O 2 .

Two hypotheses can be proposed to explain this biphasic shape: the curve may reflect two different modes of killing by H 2 O 2 . Such an explanation has been proposed to account for two kinetically distinguishable modes of cell killing by H 2 O 2 in E. coli. In the first mode, cells are killed primarily by DNA damage caused by OH• radicals produced during the Fenton reaction. However, higher levels of H 2 O 2 are thought to scavenge OH•, thus suppressing mode one killing. As the concentration of H 2 O 2 rises still further, viability is decreased by a second mode of killing, although the cellular targets in this instance are not known [START_REF] Imlay | Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro[END_REF][START_REF] Imlay | DNA damage and oxygen radical toxicity[END_REF]. Alternatively, the curve shape could account for the induction of a protective response above a concentration of 0.5 mM H 2 O 2 . Above the concentration of 1 mM, viability again decreased, because of the saturation of the protective system.

The influence of preexposure to H 2 O 2 on resistance to oxidative stress

The ability of E. coli to resist stress has been shown to increase following prior exposure to low doses of the same stress [START_REF] Crawford | Adaptative response and oxidative stress[END_REF]. Therefore, the viability of D 1 was tested with or without pretreatment with a sublethal level of H 2 O 2 . As shown in figure 2, a 0.25-mM pretreatment led to a 15-fold increase in survival when challenged with 5 mM H 2 O 2 , compared to strains that had not been pretreated. Moreover, when challenged with 10 mM H 2 O 2 , no more than 80% of the 0.25 mM H 2 O 2 pretreated population was killed, compared to 99% loss of viability for the non-pretreated control. A 1-mM H 2 O 2 preexposure led to even higher levels of survival to subsequent exposure: no more than 30% of the population was killed when subsequently challenged with 10 mM H 2 O 2 . Thus, D 1 , when exposed to sublethal levels of H 2 O 2 , was better able to cope with subsequent toxic doses. These results suggest that S. thermophilus cells respond to H 2 O 2 , probably by activating groups of genes encoding antioxidant and repair enzymes.

The effect of growth phase on H 2 O 2 tolerance

In E. coli and S. typhimurium, stationary-phase cells are more resistant to environmental stress than exponentially growing cells [START_REF] Demple | Regulation of bacterial oxidative stress genes[END_REF]. To test whether this is the case for S. thermophilus, the effect of growth phase on H 2 O 2 tolerance was investigated. A culture of D 1 was split into five subcultures, and H 2 O 2 was added at four different time points during the ensuing growth period. One culture remained untreated and served to determine the growth rate, which proceeded in TPPY with a doubling time of 30 min -1 (figure 3A). Figure 3B shows H 2 O 2 tolerance as a function of the growth phase. H 2 O 2 had little effect on the viability of stationary phase cells (OD 600 = 2.1), even up to 5 mM. In contrast, in early exponential phase (OD 600 = 0.15) were extremely sensitive to H 2 O 2 , and resistance increased throughout the growth period (OD 600 = 0.6; OD 600 = 0.85). Thus, nongrowing S. thermophilus cells appeared to be more tolerant to H 2 O 2 than their actively growing counterparts as found in E. coli. Moreover, since the resistance is settled down progressively through the growth curve, the oxidative stress response in S. thermophilus would have at least two modes of induction, one by H 2 O 2 and the other by starvation, as seen in Bacillus subtilis [START_REF] Dows | Relationship among oxidative stress, growth cycle, and sporulation in Bacillus subtilis[END_REF].

Conclusion

Our results suggest the existence of an inducible defence system against H 2 O 2 in S. thermophilus CNRZ368, possibly of the type OxyR-like regulon. Although the search for the structural homologue of OxyR (the regulator protein of the OxyR regulon) in Streptococcus mutans has failed [START_REF] Yamamoto | Cloning, nucleotide sequence, and disruption of Streptococcus mutans glutathione reductase gene (gor )[END_REF], the hypothesis of a functional substitution for the OxyR protein can be drawn. Such functional substitution has already been identified in another Gram-positive bacteria as PerR in Bacillus subtilis [START_REF] Bsat | Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors[END_REF].

Figure 1 .

 1 Figure 1. Sensitivity of D 1 isolate to killing by H 2 O 2 . Survival was determined by plating appropriate dilutions of samples after a 15min exposure to H 2 O 2 of exponentially growing cells (OD 600 nm = 0.6). Surviving fractions were calculated relative to colony counts without treatment. These experiments were performed in triplicate and the results of typical experiments are displayed in the figure. A. High concentrations of H 2 O 2 . B. Low concentrations of H 2 O 2 .

Figure 2 .

 2 Figure 2. Effect of H 2 O 2 pretreatment on cells viability. Cells were preincubated for 15 min at 42 • C in TPPY medium in the absence ( ) or in the presence of 0.25 mM H 2 O 2 ( ) or 1 mM H 2 O 2 ( ). Cells were then treated again with hydrogen peroxide at the indicated concentration and plated. Percentage of surviving cells is indicated on the graph.

Figure 3 .

 3 Figure 3. A. Growth curve of S. thermophilus D1 strain. B. Change in H 2 O 2 tolerance as a function of physiological state. Each point represents the average of a triplicate experiment. Cells were grown in TPPY medium. H 2 O 2 was added at different times during the ensuing growth period as a function of the OD 600 .Cells were treated at OD 600nm = 0.15 (#), OD 600nm = 0.60 ( ), OD 600nm = 0.85 ( ) and OD 600nm = 2.10 ( ).

Acknowledgements

We are grateful to Mark Paget for his critical reading and his help in the English formulation of the manuscript.

This work was supported by Le Ministère de l'Education Nationale, de la Recherche et de la Technologie.