DNA electrophoretic mobility shift assay (EMSA) The DNA binding of the His6-tagged Rgg0182 protein to the shp 0182 and pep 0182 promoter regions was tested by EMSA using the LightShift Chemiluminescent EMSA Kit (Thermo Scientific). The promoter regions of ldh (P ldh , 110pb), shp 0182 (P shp0182 , 126 bp) and pep 0182 (P pep0182 , 165 bp) were amplified by PCR using the TPCA-1 Pldh-5′/Pldh-3′, Pshp-3′/Pshp-5′ and Ppep-3′/Ppep-5′ primers, respectively. These were 3′-end biotin labelled with Biotin 3′ End DNA Labeling Kit (Thermo Scientific) and used in EMSA according to the manufacturer’s instructions. Chemiluminescent detection of biotin DNA on membranes
was realised with the Chemi-Doc apparatus (Bio-Rad). RNA extraction and quantitative RT-PCR (qPCR) experiments RNA extractions www.selleckchem.com/small-molecule-compound-libraries.html were adapted from Kieser et al. (1999) . RNAs were extracted from cultures grown in CDM or LM17 medium in exponential, transition, or stationary PI3K/Akt/mTOR inhibitor phase at 30 or 42°C. RNAs were also extracted from stationary phase cells exposed to a 30 min temperature shift from 30 to 52°C. The
RNAs were treated with amplification grade DNase I (Euromedex). The quantity and quality of the RNA samples were verified by agarose gel electrophoresis and by measuring their absorbance at 260 and 280 nm (NanoDrop-1000). Reverse transcription was performed according to the manufacturer’s instructions (MMLV-reverse transcriptase, Invitrogen). cDNA
was generated from 1.25 μg of DNA-free RNA and used for qPCR analysis of transcription of rgg 0182 gene and its potential target genes transcript levels. Gene transcripts quantification was done using the CFX96 manager software (Bio-Rad) with the following program: 1 cycle at 98°C for 3 min and 40 cycles at 95°C for 10 s and at 58°C for 45 s. The amplification reactions were carried out with GNA12 SYBR Green Supermix (Bio-Rad). Melting curve analysis was performed with 0.5°C increments every 10 s from 55 to 95°C to check that the cDNA amplification did not lead to secondary products. The primers used for qPCR are listed in Table 2. The efficiency of all primers pairs was checked in qPCR using serial dilutions of cDNA, and ranged from 90 to 100%. The level of gene transcript was calculated with ldh gene as the internal control gene for normalization . Physiological characterization of the Δrgg 0182 mutant Stationary phase cells were harvested from cultures grown in CDM at 30°C by centrifugation at 4,500 rpm for 10 min. Cells were washed twice and resuspended in 10 mM sterile phosphate buffer, pH 7.0 with a final OD600nm of 1.0. Then, for heat stress, cells were treated by incubation at 52°C during 15, 30, 45 and 60 min (heat stress condition) or not (control condition). Cultures were then diluted to appropriate concentrations, spread on LM17 agar plates and incubated overnight at 42°C under anaerobic conditions.