Characterisation of Streptococcus thermophilus CNRZ 368 oxidative stress-resistant mutants : involvement of a potential Rgg-like transcriptional regulator

Eight mutants of Streptococcus thermophilus CNRZ368 presenting a menadioneresistant phenotype were selected and the locus mutated in each mutant was identified. Among these clones, 5 were disrupted in a gene of unknown function, 2 were impaired in genes involved in cellular metabolism and the last one (the mutant 15H7) was disrupted in rgg encoding a putative transcriptional regulator. To determine if rgg could be a key regulator of the superoxide defence of S. thermophilus, the 15H7 mutant was further characterised. The results from our work indicate that the rgg gene, mutated in the 15H7 genome, is a pseudogene composed of 2 ORFs (rggA and rggB) that are potentially translated in two different frames. Moreover, transcription analysis demonstrated the existence of a transcript containing rggA, rggB and orf2. Construction of ∆rggA, ∆rggB and orf2 mutants and their phenotypic analysis confirmed the involvement of rggA and rggB in the oxidative stress response. The question of the role of the pseudogene rgg is still open. Streptococcus thermophilus / oxidative stress / Rgg-like transcriptional regulator Résumé – Caractérisation de mutants résistants au stress oxydant chez Streptococcus thermophilus CNZR368 : implication d’un régulateur transcriptionnel putatif de type Rgg. Huit mutants de Streptococcus thermophilus CNRZ368, présentant un phénotype de résistance à la ménadione ont été sélectionnés et le locus muté a été identifié pour chacun d’eux. Parmi ces clones, 5 sont interrompus dans un gène de fonction inconnue, 2 sont mutés dans des gènes impliqués dans le métabolisme cellulaire et un autre (15H7) est interrompu dans le gène rgg codant potentiellement un régulateur transcriptionnel. Afin de déterminer si rgg est un régulateur majeur de la défense contre le stress superoxyde le mutant 15H7 a été plus amplement étudié. Les résultats de notre travail indiquent que le gène rgg, muté dans le génome de 15H7, est un pseudogène composé de deux ORF (rggA and rggB) potentiellement traduites dans deux phases de lecture différentes. De plus, l’analyse transcriptionelle démontre l’existence d’un transcrit englobant rggA, rggB et orf2. La construction des mutants ∆rggA, ∆rggB et orf2 ainsi que l’analyse de leur phénotype confirme l’implication de rggA et rggB dans la réponse contre le stress oxydant. La question concernant le rôle du pseudogène rgg est toujours ouverte. Streptococcus thermophilus / stress oxydant / régulateur transcriptionnel de type Rgg * Corresponding author: bourget@nancy.inra.fr 78 A. Fernandez et al.


INTRODUCTION
S. thermophilus is a Gram-positive bacterium with low G+C content that belongs to the group of lactic acid bacteria (LAB).Due to their ability to metabolise sugar in lactic acid, these microorganisms are commonly used in dairy factories as starters of fermentation of yoghurt and are involved in the process of ripening of cheeses.
S. thermophilus CNRZ368 is considered to be an anaerobic aerotolerant microorganism.Although this bacterium grows more slowly in the presence of oxygen (growth rate = 40 min) than in its absence (growth rate = 27 min), it can survive in the presence of oxygen [20].The ability of S. thermophilus to survive in the presence of oxygen presumably results from the existence of a defence mechanism that helps cells to eliminate reactive oxygen species (ROS) and to repair damage [21].Data concerning the ability of lactic acid bacteria to survive in the presence of oxidative stress are scarce.In the literature, the existence of antioxidant enzymes is reported in Streptococcus genera [1,7,9,14,25].Moreover, the existence of PerR, an oxidative stress-responsive repressor is reported in Streptococcus pyogenes [9,15]; nevertheless, the global network of oxidative stress regulation in streptococci remains to be elucidated.
On the contrary, the oxidative stress response of aerobic organisms such as E. coli is well documented.This response necessitates antioxidant enzymes involved in the detoxification of cells and repair of enzymes necessary to eliminate damage.In E. coli, two networks of proteins can be induced depending on the reactive oxygen species: superoxide radicals induce the SoxR/S regulon, whereas the OxyR/S regulon is induced in the presence of hydrogen peroxide (for a review see [17]).These two regulons are partially overlapping.Although the presence of genes homologous to oxyR and soxR is a characteristic shared by many bacteria, other regulatory proteins have also emerged through evolution.Among them are PerR [15], sigma(B) and OhrR [8] and σ R /RsrA that are involved in the response against H 2 O 2 and superoxide radicals, respectively [12,13].
To identify the genes involved in the oxidative stress response of S. thermophilus CNRZ368, a collection of mutants was generated and screened for the menadioneresistant phenotype.This work allowed the identification of eight loci potentially involved in the mechanism of superoxide tolerance: among them is the locus rgg, described in the literature as a transcriptional regulator [2,3,18,19].

Bacterial strains and growth conditions
S. thermophilus CNRZ368 and E. coli TG1 were purchased from the INRA-CNRZ strain collection (Jouy-en-Josas, France) and from Stratagene (Amsterdam, The Netherlands), respectively.The thermosensitive plasmids pGhost9 and pGhost9:ISS1 were used [11].They carry an erythromycin resistance gene and a thermosensitive origin of replication.Depending on the experiment, S. thermophilus CNRZ368 was cultivated in TPPY or M17 media.For selection of menadione-resistant clones, plates containing TPPY medium supplemented with menadione (concentration going from 1 to 120 µg•mL -1 ) were used.E. coli TG1 cultures were grown on Luria-Bertani medium.

Cloning of the fragments flanking the chromosomal insertion site
Cloning of the fragments flanking the pGhost9:ISS1 insertion locus was done as previously described by Thibessard et al. [23] and allowed the identification of the mutagenised site of each mutant.

General DNA techniques, DNA sequencing and analysis
Isolation of genomic or plasmidic DNA, agarose gel electrophoresis, restriction enzyme digests, Southern hybridisation, PCR, DNA ligation and E. coli transformation were carried out as described in [16].Sequencing was done as previously described in [22].

Construction of S. thermophilus mutants
S. thermophilus mutants were constructed via allelic replacement.The ∆rggA and ∆rggB mutants presented a chromosomal deletion of the residues 14 to 41 of RggA and of the residues 106 to 207 of RggB, respectively.The mutant orf2 presented 2 stop mutations affecting the first two residues of the protein.

RNA preparation, RT-PCR and Northern analysis
Total RNA was extracted from S. thermophilus with a single guanidinium thiocyanate step and phenol-chloroform extraction by using Tri-Reagent (Sigma, Saint-Quentin-Fallavier, France).In the RNA preparation, possible contaminating DNA was eliminated by the action of DNaseI.cDNA was generated from 2.5 µg of RNA reverse transcribed with MMLVreverse transcriptase and hexamer oligonucleotide random primers (Amersham Pharmacia Biotech, Freiburg, Germany).For Northern hybridisation analysis, template RNA samples (30 µg) were electrophoresed into a denaturing (i.e.formaldehyde) agarose gel and transferred to a HybondN + (Amersham Pharmacia Biotech) according to the manufacturer's instructions.Digoxigenin-labelled transcripts complementary to rggB were used as a probe.These rggB transcripts were synthesised in vitro using the T3 promoter RNA polymerase system carried by the pBKS-rggB vector.The latter was constructed by cloning a 533 bp internal fragment of rggB in the pBKS vector.Hybridisation and detection procedures were done according to the manufacturer's instructions (Roche Molecular Biochemicals, Meylan, France).

Sensitivity to oxidising agent
The sensitivity of CNRZ368 derivatives to oxidative stress was checked on exponentially growing cells (OD 600 = 0.6) by the addition of menadione (1 to 20 mg•mL -l ).The cells were left in contact with menadione for 3 h.Aliquots of the culture were diluted in TPPY, and appropriate dilutions plated on TPPY agar plates to determine cell viability.The plates were stocked in jars containing a GENbox anaer system (bioMérieux, Marcy l'Étoile, France) providing hypoxic conditions, and colonies were counted after 20 h of incubation.The percentage of surviving CFU was determined by comparing viability with or without treatment.All experiments were performed in triplicate.

Identification of genes disrupted in S. thermophilus menadioneresistant mutants
To identify the genes implicated in the oxidative stress response of S. thermophilus, a collection of mutants, constructed by insertional mutagenesis using pGhost9:ISS1, was screened for the tolerance of the clones to menadione (a superoxide radical generating molecule).Among the 2112 clones, eight were selected for their increased resistance to menadione and were further characterised.For each mutant, the disrupted locus was cloned and sequenced.Sequence analysis allowed the grouping of these 8 mutants into 3 classes (Fig. 1).The first class assembled the mutants 7H3, 13B12, 14H11, 15H8 and 11C1, that were disrupted in genes of unknown function.For the last clone 11C1, the disrupted gene was specific to the genome of S. thermophilus.The second class brought together the mutants 6H7 and 16C10, that were mutagenised in genes involved in cellular metabolism.The mutant 6H7 was impaired in a gene encoding DeoB, a protein involved in purine metabolism.The mutant 16C10 was affected in gst, that encodes a putative proton-glutamate symport protein.
The third class included the mutant 15H7, that was disrupted in a gene, homologous to a putative transcriptional regulator of Streptococcus mutans, encoding a protein belonging to the Rgg-like family.To determine the role of the rgg locus in menadione tolerance and to understand the mechanism of regulation of S. thermophilus oxidative stress response, we decided to focus our research on the mutant 15H7.

Characterisation of the locus
disrupted in the mutant 15H7

Identification of the rgg locus
Figure 2A shows a scheme of the three ORFs, named rggA, rggB and orf2, identified within the nucleotide sequence flank-ing the pGhost9:ISS1 insertion locus of the genome of 15H7.The rggA ORF potentially encoded a protein of 65 residues that showed 77% identity with the putative transcriptional regulator Rgg of S. mutans.The 232 amino acid residues potentially encoded by rggB presented 56% identity with the same putative transcriptional regulator Rgg of S. mutans.Thus, the homology of the Rgg protein from S. mutans began with the protein RggA and continued with the protein RggB of S. thermophilus in the frame +1.These data indicate that the rgg gene disrupted in the mutant 15H7 is a pseudogene.Finally, orf2 potentially encoded a protein of 35 residues presenting no homology with sequences present in the databases.
The search for transcriptional signals allowed the identification of a potential promoter composed of an extended -10 sequence located 61 pb upstream of the start codon of rggA.Moreover, a putative rho-independent terminator consisting of a 10 pb stem and a 3 pb loop was seen downstream of the stop codon of orf2.Located 8 pb upstream of rggA was a potential Shine-Dalgarno sequence, suggesting that this gene is likely to be translated.On the contrary, no Shine-Dalgarno sequence preceded rggB, thus rendering improbable the synthesis of RggB alone.

Transcriptional organisation of the rgg locus
Northern blot analysis performed with a RNA probe complementary to rggB revealed a strong band of approximately 1200 nucleotides (data not shown).To determine which ORFs were included in this transcript, RT-PCR analyses were carried out.cDNA generated from S. thermophilus CNRZ368 RNA were used in PCR experiments (Fig. 2B).Primers P1 and P2 were designed to verify the presence of a transcript going from rggA to rggB and P3 and P4 to determine the existence of a transcript going from rggB to orf2.In both cases, a PCR product of the expected size (310 pb and 265 pb, respectively) was amplified.These data suggested the existence of one transcript including rggA, rggB and orf2.Thus, the rgg locus showed an operonic structure.

Involvement of the rgg locus in S. thermophilus oxidative stress response
The results from our analysis indicated that impairment of a pseudogene, composed of rggA and rggB, resulted in an increased tolerance of S. thermophilus to menadione.To gain insight into the involve-ment of the rgg locus in the oxidative stress defence, a series of mutants (∆rggA, ∆rggB and orf2) was constructed via allelic replacement.The ∆rggA and ∆rggB mutants presented a deletion of 43% and of 44% of the total protein, respectively.The mutant orf2 carried 2 stop mutations.
The ability of these 3 mutants to survive in the presence of increasing concentrations Figure 3. Survival of the ∆rggA, ∆rggB and orf2 mutants in the presence of increasing concentrations of menadione.Cells grown overnight in milk medium were diluted a hundred-fold into fresh TPPY medium.At OD 600 = 0.6, samples of the culture were treated with menadione for 3 h.Cells were diluted and plated on TPPY solid medium and then incubated for 20 h in jars.Colony forming units (CFUs) were then counted and the percentages of surviving CFUs were estimated relative to non-treated samples.All these experiments were done in triplicate.Results of typical experiments are presented here. of menadione was tested and compared with that of the wild-type strain.As shown in Figure 3, the ∆rggA mutant showed an increased ability to survive in the presence of menadione compared with the wild-type whereas the ability of the ∆rggB clone to tolerate menadione was reduced.Moreover, no difference in behaviour was seen for the mutant orf2 and the wild-type strain.These data suggest that rggA and rggB are both involved in S. thermophilus response to oxidative stress.However, their exact role in this mechanism remains to be solved.In contrast, orf2 does not seem to play a role in the survival of S. thermophilus submitted to oxidative stress.

DISCUSSION
The dairy organism, S. thermophilus, is continuously exposed to stress conditions generated during industrial processes.To identify the mechanisms that confer tolerance to ROS, we isolated menadione-resistant strains by insertional mutagenesis.This study reports the characterisation of 8 genes involved in S. thermophilus CNRZ368 oxidative stress defence.According to their putative function, these genes were classified into 3 groups.The first class, which contains 5/8 genes, brings together genes of unknown function.Thus, the strategy used in this work allowed the identification of new genes involved in oxidative stress: a phenotype of superoxide resistance can now be assigned to their disruption.The second class groups genes implicated in cellular metabolism: deoB encodes a phosphopentomutase that is involved in the purine salvage pathway, whereas gst is a potential glutamate-proton transporter.Both pathways are known to be connected with stress responses.In Lactococcus lactis, the deoB mutation confers multiple stress resistance on the cell [5,6].In S. mutans, genes encoding enzymes responsible for glutamate synthesis are repressed in acid and thermal stress [4].All these results reveal that cellular metabolic pathways are intimately related to stress response and that the flux of particular metabolites are likely to be implicated in stress response in LAB.The last class contains a transcriptional regulator that belongs to the family of Rgg-like proteins.These are DNA-binding proteins including a helix-turn-helix (HTH) motif at their N-terminal end.Rgg-like proteins are only found in Gram-positive bacteria belonging to the Streptococcus, Lactococcus, Lactobacillus and Listeria genera.These regulators are reported to stimulate positively or negatively the transcription of numerous genes of different functions [3].
Our work describes for the first time the involvement of a Rgg-like protein in oxidative stress response.Moreover, it asks the question of how the pseudogene rgg, disrupted in the mutant 15H7, can influence the tolerance of ROS.To address the question of whether RggA or RggB or both were involved in stress response: both ∆rggA and ∆rggB mutants were constructed and their ability to respond to menadione was checked.The results of these analyses indicated that both RggA and RggB are implicated in oxidative stress defence.Taking into account the lack of visible RBS sites downstream of rggB, the independent synthesis of RggB seems improbable.An alternative hypothesis concerns the synthesis of a RggAB protein that would be generated by frameshifting.Sequence analysis of the region overlapping the rggA and rggB ORFs revealed the presence of a frameshift mutation within the sequence TTT TTT TTT.One T-deletion would abolish the frameshift mutation and restore the proper rgg ORF.In the literature, frameshift mutations (and particularly transcriptional frameshifting) are known to arise in sequences composed of multiple thymine or adenine [10,24].Such events can result either from DNA polymerase slippage during replication, or may require RNA polymerase slippage (transcriptional frameshift) or ribosome slippage (translational frameshift).It is now important to know if such a frameshifting event could appear in S. thermophilus, and if so, if it could be stimulated by environmental conditions (for instance, by the presence of oxidative molecules).To fully understand the role of Rgg in the oxidative stress response, the characterisation of the genes regulated by this protein and implicated in stress defence would be essential.

Figure 1 .
Figure 1.Classification of the menadione-resistant mutants according to their putative function.The best homologous gene for ORF disrupted in CNRZ368 mutants is indicated for each clone after the arrows.

Figure 2 .
Figure 2. Organisation of the rgg locus of S. thermophilus CNRZ368.(A) The solid line symbolises the S. thermophilus genomic DNA.ORFs are indicated as open arrows.The black arrowhead indicates the insertion site of pGh9:ISS1 within the genome of the 15H7 mutant.The open flag indicates the putative promoter and the putative rho-independent terminator is represented as a hairpin loop.Grey boxes symbolise the homologous region between rgg from S. thermophilus and from S. mutans: the percentage of amino acid identity is indicated below the double arrows.Primers used for RT-PCR experiments are indicated.The expected size of the P1-P2 and P3-P4 PCR fragments is 310 pb and 265 pb, respectively.(B) Gel electrophoresis showing the RT-PCR products obtained using the primers P1-P2 and P3-P4.