2, 25 mM NaNO3, 5 mM MgCl2, 500 μg/ml chloramphenicol) and harvested by centrifugation (10 min, 2800 xg, 4°C). Total RNA was extracted using Trizol reagent (Ambion) essentially as described by the manufacturer, with some modifications. Pneumococcal cells were lysed by incubation in 650 μl lysis buffer (sodium citrate 150 mM, saccharose
25 %, sodium deoxicolate 0.1 %, SDS 0.01 %) for 15 min at 37°C, followed by addition of 0.1 % SDS. After lysis, samples were Selleckchem Dorsomorphin treated with 10 U Turbo DNase (Ambion) for 1 h at 37°C. After extraction, the RNA integrity was evaluated by gel electrophoresis and its concentration determined using a Nanodrop 1000 machine (Nanodrop Technologies). For Northern blot analysis, total RNA samples were separated under denaturating conditions either by a 6 % polyacrylamide/urea 8.3 M gel in TBE buffer or by agarose MOPS/formaldehyde gel (1.3 or 1.5 %). For polyacrylamide gels, transfer of RNA onto Hybond-N+ membranes (GE Healthcare)
was performed by electroblotting (2 hours, 24 V, 4°C) in TAE buffer. For agarose gels RNA was transferred to Hybond-N+ membranes by capillarity using 20×SSC as transfer buffer. In both cases, RNA was UV cross-linked to the membrane immediately after transfer. Membranes were then hybridized in PerfectHyb Buffer LXH254 price (Sigma) for 16 h at 68°C for riboprobes and 43°C in the case of oligoprobes. After hybridization, membranes were washed as described [60]. Signals were visualized by PhosphorImaging (Storm Gel and Blot Imaging System, Amersham Bioscience) and analysed using the ImageQuant software (Molecular Dynamics). Hybridization probes Riboprobe synthesis and oligoprobe labelling was performed as previously described [60]. PCR products used as template in the riboprobe synthesis were obtained using the following primer pairs: rnm007/seqT4-3 for rnr, T7tmRNA/P2tmRNA for tmRNA and smd041T7/smd040
for smpB. The DNA probe for 16S rRNA was generated using the primer 16sR labeled at 5’ end with [γ-32P]ATP using T4 Polynucleotide kinase (Fermentas). Reverse transcription-PCR (RT-PCR) RT-PCR reactions were carried out using total RNA, with the OneStep RT-PCR kit (Qiagen), according to the supplier’s instructions. The primer pairs seqT4-2/seqT4-3 and rnm010/smd041 were used to analyse rnr Aurora Kinase and smpB expression, respectively. Amplification of secG+rnr and rnr+smpB fragments was performed with the primer pairs smd038/smd050 and smd064/smd041, respectively. The AICAR chemical structure position of these primers in S. pneumoniae genome is indicated in Figure 2a. As an independent control, 16S rRNA was amplified with specific primers 16sF/16sR. Prior to RT-PCR, all RNA samples were treated with Turbo DNA free Kit (Ambion). Control experiments, run in the absence of reverse transcriptase, yielded no product. Rapid amplification of cDNA ends (RACE) experiments 5’ RACE assays were performed according to Argaman et al.[61] with modifications.