Strain CNRZ368 ICESt3cat construction To test the ICESt3 behavior in different S. thermophilus strain background, a filter mating was done as described previously [10] using the donor strain CNRZ385, carrying ICESt3 tagged with the cat gene conferring the chloramphenicol resistance

[10] and the recipient strain CNRZ368ΔICESt1, spontaneous rifampicin and streptomycin-resistant mutant (X. Bellanger unpublished data). Triple-resistant clones were isolated and mapped for cse gene polymorphism [35] to confirm that they are transconjugants harboring CNRZ368 ICESt3cat. Three independent CNRZ368 ICESt3cat clones, which have similar growth parameters, mitomycin C (MMC) minimal inhibitory concentration (MIC) and dnaA/xerS rates (exponential growth phase with and without MMC treatment and stationary phase) than strains CNRZ368 and CNRZ368 cured of ICESt1 were used for each experiments. Growth conditions check details S. thermophilus strains were grown at 42°C in 30 mL of LM17 medium to an optical density at 600 nm of about 0.7. Measures of OD600 nm were performed with the Genesys 20 spectrophotometer (Thermo scientific, Illkirch, France). Cells were diluted

until OD600 nm = 0.05 into 50 mL of preheated medium (42°C) and harvested at early (OD600 nm = 0.2), mid exponential growth phase (OD600 nm = 0.6) or stationary phase (after 1.5 hours at OD600 nm = 1.5) with or without MMC exposure during 2.5 hours at the half of the minimal inhibitory concentration (MIC/2 = 0.1 μg/mL, for all the Protein Tyrosine Kinase inhibitor S. thermophilus strains used in this study) for genomic DNA or RNA extractions. Cultures were centrifuged at 13, 000 g

during 15 min at 42°C and cell pellets were stored at -80°C. DNA manipulation DNA quantity along the MMC exposure was investigated by colorimetric DNA Selleckchem S63845 dosage [36]. Genomic Dipeptidyl peptidase DNA of S. thermophilus was extracted as described previously [37]. Plasmid DNA isolation was performed using Genelute Plasmid Miniprep Kit (Sigma-Aldrich, Lyon, France). DNA fragment recovery was performed using the High Pure PCR Product purification kit (Roche, Neuilly-sur-Seine, France). DNA cloning, ligation and restriction enzyme digestion were all carried out according to standard procedures [33] or according to specific recommendations of the supplier (New England Biolabs, Evry, France). PCR primers were designed with the PrimerQuest software http://​www.​idtdna.​com/​scitools/​applications/​primerquest/​ and synthesized by Eurogentec (Angers, France) at 100 μM. PCR and high fidelity PCR were carried out according to the instructions of the ThermoPol PCR kit (New England Biolabs, Evry, France) and of the Triple Master PCR System (Eppendorf, Le Pecq, France), respectively. Sequencing reactions on RACE PCR amplifications were performed by Cogenics (Beckman Coulter genomics, Villepinte, France).