Infections like pneumonia, meningitis or sepsis caused by S. pneumoniae place this bacterium among the leading causes of mortality from infectious diseases, affecting

especially young children and the elderly. Expression of tmRNA in S. pneumoniae have been recently demonstrated [31] and our analysis of the pneumococcal see more genome revealed that the coding sequence of SmpB is located immediately downstream of the gene encoding RNase R (rnr). These observations eFT-508 ic50 prompted us to study RNase R expression in this bacterium and to analyse the involvement of this exoribonuclease with the trans-translation machinery of S. pneumoniae. In this report we show that the pneumococcal rnr gene is co-transcribed with the flanking genes secG and smpB from a promoter upstream of secG. This conserved location among Gram-positive bacteria may have a relevant biological meaning. We demonstrate that RNase R expression is induced under cold-stress and that the enzyme levels are modulated by SmpB. Conversely we found that SmpB mRNA and protein levels are INCB28060 concentration under the control of RNase R. This finding uncovers an unsuspected additional connection of RNase R with the trans-translation machinery. Results RNase R levels

are regulated by temperature and modulated by SmpB In a previous work, we have biochemically characterized RNase R, the only hydrolytic exoribonuclease described in S. pneumoniae[30], but nothing is known about its expression and regulation. In E. coli RNase R was previously described to be

modulated in response to different stress situations, namely after cold-shock [11, 12, 17]. It is also known that RNase R is functionally related with the trans-translation system in a wide variety of bacteria [12, 23, 24, 27]. Altogether these observations encouraged us to characterize Celecoxib RNase R expression and study its interplay with the trans-translation machinery of S. pneumoniae. To study the expression of RNase R, total protein extracts obtained under physiological temperature and cold-shock were analysed by Western blot using specific polyclonal antibodies that we raised against the purified pneumococcal RNase R. Two hours after a downshift from 37°C to 15°C the protein levels increased around 3-fold (Figure 1). Thus, the expression of the pneumococcal RNase R is modulated by temperature, increasing under cold-shock. In order to determine whether the induction of RNase R could be related with a higher level of the rnr transcript under the same conditions, the variation of the rnr mRNA levels was evaluated by RT-PCR. A strong increase (~6.5-fold) of the rnr transcript was observed under cold-shock (Figure 1). Therefore, the higher levels of RNase R at 15°C are, at least in part, a consequence of the strong increase of the respective mRNA amount. Figure 1 Pneumococcal RNase R is more abundant under cold-shock and its levels are modulated by SmpB.