3, 0.8, 1.5, 1.9 and 2.3 (time points A, B, C, D and T, respectively). Aliquots of 20 μg of RNA were treated twice with 2 Units of DNase I with the TURBO DNA-free reagent (Ambion) for 30 min at 37°C. Reverse transcription and quantitative real-time PCR were performed as previously described [25]. PCRs involved a hybridization step of 55°C, except for ramR, SLI0755 and cchB where a temperature of 58°C was used. Each assay was performed in triplicate and repeated with at least two independent RNA samples. The critical threshold cycle (C T ) was defined for each sample. The relative amounts

of cDNA for the tested genes were normalized to that of the hrdB gene transcript which did not vary under our experimental VX-765 research buy conditions (and thus served as an internal standard). The change (n-fold) in a transcript level was calculated using the following equations: ΔC T  = C T(test DNA) - C T(reference cDNA), selleck products ΔΔC T  = ΔC T(target gene) - ΔC T(hrdB), and ratio =  [38]. Student’s t test was CB-839 used to evaluate the significance of differences between the expression level of tested genes and that of a reference gene. A P-value < 0.05 was considered significant. In silico analysis and electrophoretic mobility shift assays (EMSA) Several AdpA-binding site sequences, identified in S. griseus by DNase I footprinting experiments [10, 13, 18, 23], were used with the PREDetector software (version 1.2.3.0)

[39] to generate a S. griseus matrix [25]. This matrix was used with the S. coelicolor genome sequence (the S. lividans genome sequence was not available during the course of this study and is still not available on PREDetector software) to identify putative AdpA-binding sites upstream from S. lividans AdpA-dependent genes (scores > 3). The StrepDB database [7] and Blast were used to identify S. lividans, S.

coelicolor and S. griseus ortholog gene names. Radioactively labelled DNA fragments (180 bp to 496 bp) corresponding to promoter regions of putative S. lividans AdpA-regulated genes were obtained by PCR. Primers (named GSgene in Additional file 1: Table S1) were used to amplify the promoter regions of cchA (opposite orientation to cchB), SLI0755, SLI6586 (opposite Cyclin-dependent kinase 3 orientation to SLI6587), ramR and hyaS as described elsewhere [25]. Purified radiolabelled fragments (10,000 cpm) were then used with purified AdpA histidine-tagged protein (AdpA-His6) in EMSA as previously described [25, 40]. Results Deletion of adpA affects the expression of hundreds of genes during early stationary phase We had previously inactivated adpA in S. lividans and found that this adpA mutant failed to produce aerial mycelium on rich media and that its growth was comparable to that of the parental strain 1326 in liquid YEME medium at 30°C [25]. Expression studies with this S. lividans adpA mutant cultivated in liquid medium identified two differentiation-regulating factors (STI1 and the ClpP1ClpP2 peptidases) whose ORFs were under the direct control of AdpA [25].