Pneumococcal RNase R is more abundant under cold-shock and its levels are modulated by SmpB. Western blot and RT-PCR analysis of protein and RNA samples extracted from wild type and mutant strains as indicated on top of each lane. Details of experimental procedures are described in the ‘Methods’ section. (Upper panel) Analysis of RNase R (~92 kDa) expression by Western blot. RNase R levels were compared in the wild type (WT), the SmpB- mutant and the SmpB- strain expressing SmpB from pLS1GFP at different temperatures (15°C and 37°C). 20 μg of each protein sample were separated in a 7 % tricine-SDS-polyacrylamide gel and blotted to a nitrocellulose membrane. RNase R was detected using specific antibodies. An RNase R- mutant strain was used as a negative control. A non-specific band (Control) detected with the same antibodies was used as loading control. A representative membrane of several independent Western blots is shown. (Lower panel) Analysis of rnr mRNA levels by RT-PCR. RT–PCR experiments were carried out with primers specific for rnr using 100 ng of total RNA extracted from the wild type (WT) and derivatives at 15°C or 37°C, as indicated on top of the lanes. The RNase R- mutant derivative was used as a negative control. RT-PCR with primers specific for 16S rRNA shows that there were not significant variations in the amount of RNA used in each sample.