Transcriptional Analysis of cmr Regulators in C. difficile

For analysis of cmrR and cmrT expression in C. difficile R20291 (WT), cmr-Δ3 ON, and cmr-Δ3 OFF, these strains were grown overnight (16 h) in TY medium, and 5 μL was spotted on BHIS-agar. After 24 h, growth was collected, suspended in 1:1 ethanol:acetone, and stored at −80°C for subsequent RNA isolation. For analysis of transcript abundance in R20291 strains carrying pCmrR, pCmrT, or vector, these strains were grown overnight in TY-Tm medium. Cultures were diluted 1:30 in BHIS-Tm broth. After 2 h of growth, ATc was added to induce gene expression (WT with vector or pCmrR, 10 ng/mL; WT with pCmrT, 2 ng/mL). Samples were collected at the mid-exponential phase and saved in 1:1 ethanol:acetone at −80°C.
RNA was extracted as described previously (46 (link)) and treated with a TURBO DNA-free kit (Life Technologies) to remove contaminating genomic DNA. cDNA was synthesized using the high-capacity cDNA reverse transcription kit (Applied Biosystems) using the manufacturer’s protocols (24 (link), 40 (link)). Real-time PCR was performed using the SensiFAST SYBR and fluorescein kit (Bioline) as previously described (21 (link), 34 (link)). Data were analyzed using rpoC as the reference gene. Primers are as follows: rpoC, R850/R851; cmrR, R2298/R2299; cmrT, R2537/R2538; cmrS, R2539/R2540; TSS1, R2745/R2746; TSS2/3, R2751/R2804; TSS4, R2803/R2716.