Details of experimental procedures are described in the ‘Methods’

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 Akt inhibitor specific antibodies. An RNase R- mutant strain was used as a negative control. A non-specific band (Control) detected with the same see more antibodies was used as loading control. A representative membrane of several independent Western blots is shown. (Lower panel) Analysis SN-38 solubility dmso 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. It has been recently shown that the interaction of SmpB and tmRNA with E. coli RNase R destabilizes the ribonuclease [28]. To see if the levels of pneumococcal RNase R were affected by SmpB, comparative

Western blot analysis was performed in the presence or absence of SmpB. For this purpose we have constructed an isogenic mutant lacking smpB (SmpB-) and followed the expression of RNase R at 15°C and 37°C in the wild type, the SmpB- strain, and the SmpB- strain complemented with a plasmid encoding SmpB. As shown in Figure 1, at 15°C the levels of RNase R were roughly the same as in the wild type, but at 37°C there was an increase of the RNase R levels in the SmpB- strain (~2 fold higher than the wild type). The fact that RNase R levels were restored after SmpB expression in trans, confirms that SmpB is implicated in the regulation of RNase R. Progesterone This regulation is probably post-translational, since the rnr mRNA levels are roughly the same in the absence of smpB.

Interestingly, the effect of SmpB on RNase R is only observed at 37°C. This suggests that the modulation of RNase R by SmpB is probably growth stage-dependent, as it was shown in E. coli[29]. Altogether these results indicate that in S. pneumoniae SmpB may be one important factor in controlling the levels of RNase R. Nonetheless, the significant increase of the rnr mRNA levels under cold-shock may certainly account for the final levels of RNase R in the cell. The RNase R transcriptional unit: rnr and smpB are co-transcribed The cooperation of RNase R and SmpB in important cellular functions, together with the proximal location of their respective coding sequences in the genome of S. pneumoniae, led us to further characterize the expression of these two genes.

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