Erythromycin in a final concentration of 300 μg/ml for E. coli and 5 μg/ml find more for GAS was used for selection and maintenance of the mutants. Standard DNA techniques Genomic DNA from GAS strains was isolated by DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s
recommendations. Plasmid DNA manipulations, transformation of E. coli and GAS were performed as described previously . S. pyogenes competent cells were prepared in the presence of glycin, mutanolysin and hyaluronidase, as follows: S. pyogenes was grown overnight in 10 ml THY broth supplemented with 20 mM glycin, then 5 ml of the pre-culture was added to 45 ml of THY supplemented with glycine (20 mM) and mutanolysin (10 U/ml) for overnight incubation. Cells were harvested by centrifugation at 3000 rpm, 4°C
for 5 min and washed once with sterile PBS. Pelleted cells were suspended in 1 ml PBS containing 500 U hyaluronidase and incubated for 1 hour at 37°C. The pellet was washed 2 times with ice cooled PBS and 2 times with ice cooled sterile sucrose (0.625 M). Subsequently, the pellet was resuspended in 1.5 ml sucrose (0.625 M) and 100 μl were aliquoted in 1.5 ml Eppendorf tubes. The competent cells were stored at -80°C. RNA isolation Total RNA from GAS strains was isolated from 25 ml of culture at the mid-exponential phase of growth by the usage of FastRNA Pro Kit (MP Biomedicals). In brief, the bacterial pellet was resuspended in 1 ml RNApro solution, transferred to a lysing matrix mTOR inhibitor tube and processed through a Hybaid RiboLyser instrument for 40 seconds at setting of 6.0. After centrifugation, the lysate was subjected to chloroform extraction. The upper phase was mixed with absolute ethanol and incubated at -20°C for 2 hours. After washing with 70% ethanol,
the RNA pellet was dried and resuspended in DEPC-H2O. First-strand cDNA synthesis and reverse transcription PCR pentoxifylline (RT-PCR) DNAse digestion of the obtained RNA was carried out with RNeasy-Free DNase Set (Qiagen). After DNase treatment, 5 μg of total RNA was used for first-strand cDNA synthesis with SuperScript™ II reverse transcriptase using random primers (Invitrogen) according to the manufacturer’s instructions. For the RT-PCR analysis two pairs of primers were used binding to covR and covS, correspondently. A fragment with size of 625 bp appears when using primers binding to covR, CovR_for (5′-CTCTTGAGCTGCAACATGAGG-3′) and CovR_rev (5′-CACGAATAACGTATCCCATGC-3′). A PCR employing primers binding to covS, CovS_for (5-ATCATCTCCTGGCTTGCATGG-3′) and CovS_rev2 (5′-CCAGTCACTGAAAGGTTAATCGC-3′), results in a product with a size of 846 bp. As controls genomic DNA and total RNA were used as template for the PCR analysis with both primer pairs. Construction of recombinant vectors and GAS mutants Using S.