Quantification of bacterial gene expression by qRT-PCR

Bacteria were grown overnight at 20°C in KB broth containing 50 μg/mL rifampicin to an OD₆₀₀ = ~1.0. Cells were centrifuged for 10 min at 21,000 x g and the resulting bacterial pellets were frozen in liquid nitrogen and stored at -80°C prior to use. RNA was extracted from the bacterial pellets and 500 ng converted into cDNA in 25 μL reactions as described previously [9]. Quantitative RT-PCR (qRT-PCR) reactions were performed in 10 μL volumes using 5 μL of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad), 4 μL of a mix containing 0.5 μM of each transcript-specific primer (S2 Table), and 1 μL of cDNA. SYBR Green fluorescence from each qRT-PCR reaction was monitored using the C1000 Thermal Cycler with CFX96 Real-Time System (Bio-Rad). Relative abundance of transcripts was calculated relative to gyrA using the formula Transcript Abundance = PCR efficiency^{-(Ct[gene]-Ct[gyrA])} [48]. PCR efficiency values were calculated for each qRT-PCR reaction using LinRegPCR [49].