RNA isolation

For qRT–PCR 25-ml cultures were inoculated with a preculture to an OD₆₀₀ of 0.05 and grown for 6 h. Cells were immediately harvested and RNA stabilized by addition of 2 × vol. RNAprotect bacteria reagent (Quiagen). After centrifugation the supernatant was removed, and the pellet was quickly frozen with liquid N₂.Then the pellets were resuspended in 1 ml TRIZOL solution (Invitrogen—Life Technologies Corporation, Darmstadt, Germany) and lysed with 0.3 ml zirconia-silica beads (Karl Roth GmbH, Karlsruhe, Germany; 0.1-mm diameter) in a high-speed benchtop homogenizer (FastPrep-24, MP Biomedicals, Germany). The lysed samples were mixed with 200 μl chloroform followed by centrifugation for 15 min at 4 °C and 15 000 r.p.m. The clear layer was transferred to a new tube and mixed with 500 μl isopropanol. After a further centrifugation and two washing steps with ethanol the RNA was carefully resuspended in RNAse-free water. To get rid of DNA contamination 5 μg ml⁻¹ of each sample was mixed with 2 μl DNAse I (DNase Treatment and Removal kit, Ambion, Life Technologies) and 1 μl recombinant RNasin ribonuclease inhibitor (Promega, Madison, WI, USA) and incubated for 30 min at room temperature. Then DNAse was inactivated by adding inactivation reagent (DNase Treatment and Removal kit, Ambion, Life Technologies and RNA concentration was analysed.