Rotor gene 3000a

MG1655 and MG1655 ΔminCDE cells were grown to an OD₆₀₀ of 0.18. Rifampicin (400 μg/ml) was then added for 15 min. Samples were collected every 3 min after the addition of rifampicin. Total RNA was isolated, and RNA concentrations were determined using a NanoDrop machine (NanoDrop Technologies). DNA was removed by DNase treatment and 1 µg of DNA-free RNA was used for cDNA synthesis. cDNA was quantified by real-time PCR using SYBR-green mix in Rotor Gene 3000A (Corbett) according to the manufacturer’s instructions. Primers were designed for polar and non-polar mRNAs, and their expression was normalized using 16S rRNA levels. The relative amount of cDNA was calculated using the standard curve method obtained from PCR on serially diluted genomic DNA templates. The corrected values were plotted as a percent of the initial value versus time, and smoothed curves were fitted using an exponential function I(t) = I0 exp(−kt), where I0 is the initial mRNA concentration, I(t) is the concentration after t min, and k is the decay coefficient in minutes−1, extracted from the exponential fit to the decay plot. The half-life was calculated from this decay coefficient using the relation t1/2 = ln(2)/k.

Total RNA was isolated from cultured cells using TRI-reagent (T9424; Sigma) according to the manufacturer’s instructions. RNA quality and quantity was measured using a NanoDrop ND-1000 spectrometer (NanoDrop Technologies, Wilmington, DE). Reverse transcription was performed using the M-MLV Reverse Transcriptase Kit (M368B; Promega, Wallisellen, Switzerland) according to the manufacturer’s instructions. Relative quantification of mRNA expression levels was performed by real-time qPCR. Briefly, cDNA (10 ng), gene-specific oligonucleotide primers (Table S1) (200 nmol/L), and KAPA SYBR FAST qPCR reagent (KK4600; Kapa systems, Wilmington, DE) (5 μL), in a final volume of 10 μL, were analyzed by qPCR in a rotor gene 3000A (Corbett Research, Sydney, Australia). Thermal cycler parameters were as follows: denaturation for 15 min at 95°C, followed by amplification of cDNA for 40 cycles with melting for 15 sec at 94°C, annealing for 30 sec at 56°C, and extension for 30 sec at 72°C. Relative gene expression normalized to the internal control gene coding for cyclophilin A (PPIA) was obtained by the 2^−ΔΔCt method 23 (link).

RNA concentrations were determined using a NanoDrop machine (NanoDrop Technologies). DNA was removed by DNase treatment according to the manufacturer's instructions (RQ1 RNase‐free DNase, Promega). 2 μg of DNA‐free total RNA was used for cDNA synthesis using MMLV reverse transcriptase and random primers (Promega). kil cDNA levels were analyzed by real‐time PCR using specific primers (2531–2532) and SYBR green mix (Absolute SYBR GREEN ROX MIX, ABgene) with Rotor‐gene 3000A (Corbett) according to the manufacturer's instructions. The level of 16S rRNA (rrsB; primers 1309–1310) (Park et al, 2007) was used to normalize kil levels. The relative amount of cDNA was calculated using the standard curve method. A standard curve was obtained from PCR on serially diluted genomic DNA as templates and was analyzed using Rotor‐gene analysis software 6.0.