BACKGROUND
the ability of bacteria to sense and adapt to environmental changes is critical to survival.
under stress conditions, prokaryotic cells rapidly adjust their gene expression to deal with a changing environment  <cit> .
rna molecules provide the dynamic link between dna-encoded information and protein synthesis.
a rapid response to a changing environment involves not only transcriptional but also post-transcriptional regulation  <cit> .
mrna decay is of prime importance for controlling gene expression, and the labile nature of the rna molecules is critical as it allows a rapid adjustment of proteins levels.
ribonuclease r  is a processive 3’-5’ exoribonuclease that belongs to the rnase ii family of enzymes  <cit> .
orthologues have been found in most sequenced genomes  <cit>  and have been implicated in the processing and degradation of different types of rna, such as trna, rrna, mrna and the small rna tmrna  <cit> .
rnase r is the only exoribonuclease able to degrade highly structured rna molecules and therefore, it is particularly important in the removal of rna fragments with extensive secondary structures  <cit> .
cold-shock treatment is a condition which thermodynamically favours the formation of highly structured rna molecules, and this fact probably leads to the marked increase of rnase r under this stress situation.
in fact, escherichia coli rnase r is a general stress-induced protein whose levels are highly upregulated under cold-shock  <cit> .
stress resistance and virulence are intimately related since many pathogenic bacteria are challenged with very harsh conditions during the process of infection.
not surprisingly, rnase r has been implicated in the establishment of virulence in a growing number of pathogens.
these include aeromonas hydrophila, shigella flexneri, enteroinvasive e. coli, and helicobacter pylori <cit> .
this enzyme has also been involved in the quality control of defective trna and rrna molecules  <cit> .
furthermore, e. coli rnase r was shown to participate in the maturation of the transfer-messenger rna   <cit> , an important small rna involved in trans-translation.
in pseudomonas syringae and caulobacter crescentus, degradation of tmrna was also shown to be dependent on rnase r  <cit> .
tmrna together with smpb are the main components of the trans-translation system, an elegant surveillance pathway that directs deficient proteins and mrnas for degradation while rescuing stalled ribosomes .
trans-translation allows bacteria to efficiently respond to a variety of stresses and is required for the viability and for the establishment of virulence in many pathogenic bacteria .
during trans-translation rnase r is the key exoribonuclease involved in the degradation of the faulty mrnas after the release of the halted ribosomes  <cit> .
moreover, in e. coli the stability of rnase r was shown to be regulated by interaction with tmrna/smpb, which in turn seems to depend on previous rnase r acetylation  <cit> .
in previous studies we have purified and biochemically characterized rnase r from streptococcus pneumoniae <cit> , an important human pathogen that is one of the leading causes of nosocomial infections.
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  <cit>  and our analysis of the pneumococcal genome revealed that the coding sequence of smpb is located immediately downstream of the gene encoding rnase r .
these observations 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 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 <cit> , 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  <cit> .
it is also known that rnase r is functionally related with the trans-translation system in a wide variety of bacteria  <cit> .
altogether these observations encouraged us to characterize 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 .
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  of the rnr transcript was observed under cold-shock .
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.
it has been recently shown that the interaction of smpb and tmrna with e. coli rnase r destabilizes the ribonuclease  <cit> .
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  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  <dig>  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 .
the fact that rnase r levels were restored after smpb expression in trans, confirms that smpb is implicated in the regulation of rnase r. 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 <cit> .
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.
the fact that rnr is located upstream and partially overlaps with smpb  indicates that these genes may be co-transcribed as part of an operon.
to check this possibility rt-pcr experiments were carried out using primers smd <dig>  and smd <dig>  .
as shown in figure 2b a fragment that results from the amplification of a transcript containing both rnr and smpb could be observed, indicating that smpb is co-transcribed with rnr.
a global overview of the rnr/smpb genomic region revealed the existence of several orfs oriented in the same direction .
the first orf  that is preceded by an orf oriented in opposite direction is located about 5kb upstream of rnr.
these orfs are closely located, some with overlapping regions and, using a specific probe for smpb in northern blot experiments, we detected a high molecular weight transcript   that could arise from co-transcription of these orfs.
we used the same rt-pcr approach for each pair of consecutive orfs  in order to establish which orfs were in the same transcriptional unit.
a fragment corresponding to the amplification of each orf pair could be detected .
the last orf of this transcriptional unit is most probably tehb , since under the same experimental conditions no amplification product containing tehb and the downstream gene could be obtained .
in fact, inspection of the sequence revealed a rho-independent transcription terminator downstream of tehb.
interestingly, all the amplification products were detected in a higher amount at 15°c than at 37°c  and this is also the case of the high molecular weight transcript detected in the northern blot experiments .
these results could be indicative of a cold-shock operon.
taken the above observations a complex regulation of the operon, with multiple promoters and transcripts containing different sets of genes, cannot be ruled out.
since we were particularly interested in rnr and smpb we have searched for promoters in the vicinity that could regulate the expression of this particular set of genes.
even though bioinformatics analysis indicated a putative promoter immediately upstream of rnr, we could not detect any active promoter, either by primer extension analysis or by 5’ race mapping .
upstream of rnr lays a small orf that encodes a protein with homology to secg, an auxiliary protein in the sec-dependent protein export pathway.
a transcript containing secg and rnr was detected and was also mainly expressed under cold shock .
in fact, a putative promoter upstream this orf was identified in silico, which could also drive rnr transcription .
therefore, primer extension and race experiments were conducted to check this possibility.
a single fragment was extended from a primer that hybridizes with the 5’-end of the secg mrna  as shown in figure 3a.
the size of this fragment, as determined by comparison with the m <dig> phage sequence, shows that its 5’-end matches the transcription start site  of the in silico predicted promoter .
to confirm this result the 5’-end of the transcript was mapped by 5’ race following a protocol that makes use of the tobacco acid pyrophosphatase  enzyme  <cit> .
this method allows distinguishing between 5’-ends of primary transcripts from those generated by cleavage/processing.
a 5’ race product that was only obtained from the tap-treated samples  indicates that it carries a 5’-triphosphate group characteristic of primary transcripts.
cloning and sequencing of this race product allowed us to identify the + <dig> site at the same position as that identified by primer extension.
these results clearly show that this promoter is active and drives the expression of secg.
considering the lack of a promoter upstream rnr and since a transcription terminator could neither be identified in this region, we believe that the secg promoter may also contribute to the rnr expression.
since our data indicate that rnr and smpb are co-transcribed, this promoter most likely directs smpb transcription as well.
nonetheless, we searched for alternative promoters of smpb.
we started by analysing the 5’-end of the smpb transcript by primer extension using a primer specific for the smpb 5’-end region .
as shown in figure 4a, two different fragments were extended from this primer .
analysis of the sequence revealed that the 5’-ends of both fragments are located right before the overlapping region between rnr and smpb .
since by visual inspection we could not identify any putative promoter upstream this region, the extended fragments likely correspond to different processing sites.
to clarify the primer extension result and confirm this hypothesis, 5’ race experiments were conducted before and after treatment with tap to discriminate primary transcripts from those originated by processing.
the gel in figure 4b shows several 5’ race products that are most probably derived from processed molecules as inferred by the similar intensity of tap-treated samples.
thereby, under these experimental conditions we did not identify any active promoter upstream smpb.
this result further corroborates the rnr and smpb co-transcription hypothesis.
the fragments that were not detected in the negative control  were cloned, and the sequence of several independent clones allowed us to infer the respective 5’-ends.
as expected by the smeared-appearance of fragment  <dig>  sequence analysis revealed different transcripts with distinct 5’-ends .
all of these fragments mapped in the 3’-end of rnr upstream the overlapping region with smpb , in agreement with the primer extension results.
however, only one exactly matched the nucleotide position of one of the extended fragments .
we do not know the reason for this, but one hypothesis is that these fragments could be the result of trimming by a 5’-3’ exoribonuclease, predicted in this gram-positive bacterium.
interestingly all the sequences mapped before the putative rbs upstream smpb and thus, these processing events may generate a functional independent smpb transcript.
the sequences of the clones corresponding to the other race product  mapped inside smpb after the overlapping region.
while inactivating smpb mrna, this cleavage spares the rnr transcript, which may thereby be independently translated.
the fact that the same pattern was obtained from wild type and rnase r- samples  further confirms that the processing of the rnr/smpb transcript is not affected in the rnase r- strain.
taken together these results indicate that the pneumococcal rnr transcript is expressed as part of an extensive operon.
this large transcript is most probably subject to a complex regulation with several promoters and multiple processing events leading to smaller transcripts.
indeed, a promoter identified upstream secg may be responsible for the independent regulation of the downstream genes, secg, rnr and smpb.
processing of the operon to yield mature gene products is likely to occur.
since we could not identify other active promoters upstream rnr or smpb, we believe that transcription of rnr and smpb does not occur independently and is most probably driven by the promoter identified upstream of secg.
smpb mrna and protein levels are modulated by rnase r
we have just seen that in s. pneumoniae rnr is co-transcribed with smpb.
on the other hand, in e. coli smpb was shown to modulate the stability of rnase r  <cit> .
in e. coli processing of tmrna under cold-shock is dependent on rnase r  <cit> , and this enzyme has also been involved in tmrna degradation in c. crescentus and p. syringae <cit> .
thus, we were interested in clarifying which could be the involvement of rnase r with the main components of the trans-translation system in s. pneumoniae.
for this purpose, we compared both smpb and tmrna expression by northern blot between the wild type, an isogenic mutant lacking rnase r  and the rnase r– strain complemented with a plasmid expressing rnase r constitutively.
the results showed that the accumulation of the tmrna precursor form  at low temperature is similar in the wild-type and the deletion mutant , and an increase in the tmrna levels was neither observed in the absence of rnase r. hence, under our experimental conditions, rnase r from s. pneumoniae does not seem to be involved in the tmrna processing or turnover.
nonetheless, analysis of the smpb mrna levels revealed a strong accumulation of the transcript in the absence of rnase r, especially under cold-shock .
comparison of smpb levels between the wild type and the rnase r- strain revealed an increase of about 25-fold at 15°c, while the variation of smpb levels at 37°c appeared very low.
the lesser accumulation of the smpb transcript at 37°c may suggest that in this condition the role of rnase r in the control of this transcript is probably less important.
this is in agreement with the low levels of rnase r detected at this temperature .
the involvement of rnase r was further substantiated by complementation of the rnase r- strain with rnase r expressed from pil <dig>  at 15°c addition of rnase r partially restored the wild type smpb levels, leading to a ~17-fold decrease relatively to the rnase r- strain .
interestingly, in the rnase r complementation strain the variation of smpb levels between 15°c and 37°c is lower, suggesting that the temperature-dependent regulation of smpb levels is compromised.
this is probably due to the fact that rnase r expression from pil <dig> is constitutive contrary to the temperature-regulated expression pattern observed in the wild type.
together, these results strongly suggest that rnase r has a role in smpb degradation.
to check if the increment observed on the rna levels would influence the final levels of protein in the cell, we analysed the expression of smpb under the same conditions.
smpb expression was compared by western blot in the wild type, the rnase r- mutant derivative and the rnase r- strain complemented with rnase r expressed in trans.
analysis of smpb levels with specific antibodies raised against purified tigr <dig> smpb showed a significant increase in the protein levels in the absence of rnase r  .
this phenotype was partially restored in the strain complemented with rnase r, suggesting that rnase r is determinant for the final levels of smpb in the cell.
discussion
rnase r levels and activity are known to increase in stationary phase and under certain stress situations, namely cold-shock and starvation  <cit> .
rnase r is the unique exoribonuclease able to degrade rna molecules with extensive secondary structures, and the increase of rnase r under multiple stress conditions may indicate a general modification of structured rna in response to environmental changes.
in fact this enzyme was shown to be important for growth and viability of several bacteria especially under cold-shock, a condition where rnase r levels are considerably increased  <cit> .
mutants lacking any of the trans-translation components  also have a variety of stress phenotypes.
these range from attenuated antibiotic resistance to problems in adaptation to oxidative stress, cold- and heat-shock  <cit> .
in this report we have studied the regulation of the rnase r expression and the interplay of this exoribonuclease with the components of the trans-translation system in the human pathogen s. pneumoniae.
our results show that, as occurs in e. coli, pneumococcal rnase r is induced after a downshift from 37°c to 15°c.
according to our data, both rnr mrna and protein levels are elevated after cold-shock treatment, which could suggest that the higher levels of protein would be directly related with the increased amount of mrna molecules in the cell.
however, the expression of rnase r seems to be also modulated by smpb.
in the absence of this protein the levels of rnase r are similar at 15°c and 37°c and the temperature-dependent regulation observed in the wild type seems to be lost.
this result resembles the e. coli situation where rnase r was shown to be destabilized by smpb during exponential phase in a tmrna-dependent manner  <cit> .
interestingly, e. coli rnase ii  was reported to be destabilized by gmr, which is encoded by a gene located immediately downstream  <cit> .
our data suggests that pneumococcal smpb, which is encoded downstream of rnr, may also have a role in the control of rnase r stability.
in e. coli destabilization of rnase r by smpb was shown to be dependent on previous acetylation of the enzyme.
acetylation only occurs during exponential growth and was proposed to release the c-terminal lysine-rich region of rnase r  <cit> .
this domain of rnase r is directly bound by smpb in a tmrna-dependent manner, and this interaction would ultimately target rnase r for proteolytic degradation  <cit> .
we have analysed the pneumococcal rnase r sequence and also identified a lysine-rich c-terminal domain, which could mediate an association between rnase r and smpb.
it seems reasonable to speculate that in s. pneumoniae, a similar interaction is taking place.
interestingly, the lysine-rich domain of rnase r is essential for the enzyme’s recruitment to ribosomes that are stalled and for its activity on the degradation of defective transcripts  <cit> .
a proper engagement of rnase r is dependent on both functional smpb and tmrna, and seems to be determinant for the enzyme’s role in trans-translation.
all these observations point to an interaction between the pneumococcal rnase r and smpb, which may destabilize the exoribonuclease.
however, we believe that the strong increment of the rnr mrna levels detected at 15°c may also account for the final expression levels of rnase r in the cell.
a higher amount of mrna may compensate the low translation levels under cold-shock.
one of the first indications for the involvement of e. coli rnase r in the quality control of proteins was its association with a ribonucleoprotein complex involved in ribosome rescue  <cit> .
this exonuclease was subsequently shown to be required for the maturation of e. coli tmrna under cold-shock  <cit> , and for its turnover in c. crescentus and p. syringae <cit> .
additional evidences included a direct role in the selective degradation of non-stop mrnas  <cit>  and destabilization of rnase r by smpb  <cit> .
in this work we strengthen the functional relationship between rnase r and the trans-translation machinery by demonstrating that rnase r is also implicated in the modulation of smpb levels.
a marked accumulation of both smpb mrna and smpb protein was observed in a strain lacking rnase r. the increment in mrna levels is particularly high at 15°c, the same condition where rnase r expression is higher.
this fact suggests that the enzyme is implicated in the control of smpb mrna levels.
the higher smpb mrna levels detected at 15°c could also suggest a temperature-dependent regulation of this message.
however, the steady state levels of smpb protein in the rnase r- strain were practically the same under cold-shock or at 37°c.
translational arrest caused by the temperature downshift may be responsible for the difference between the protein and rna levels.
alternatively, we may speculate that the interaction between rnase r and smpb could also mediate smpb destabilization.
this hypothesis would imply that rnase r/smpb protein-protein association would direct both proteins for degradation.
further work is necessary to investigate this attractive possibility.
analysis of the s. pneumoniae rnase r genomic region revealed the presence of several orfs that may be part of a large transcript shown to be mainly expressed under cold-shock.
some of them are essential for growth, as it is the case of the gtp-binding protein era and of the dephospho-coa kinase.
others are important in the resistance to some drugs or mutagens, as for instance formamidopyrimidine-dna glycosylase, the multi-drug resistance efflux pump pmra and the tellurite resistance protein tehb.
the first gene of this large operon – ybey, a putative metalloprotease - appears to be essential for translation under high temperature growth conditions.
however, besides rnase r and smpb none of these genes have known links to cold-stress.
smpb is located downstream of rnr and we show that both genes are co-transcribed.
although we were not able to identify an active promoter immediately upstream of rnr or smpb that could drive the transcription of these genes independently, a promoter upstream of secg was identified.
secg is a small orf located immediately upstream of rnr and transcription from its promoter is likely to drive expression of the downstream genes.
indeed, we have demonstrated that this promoter is active and most probably drives the coupled transcription of secg, rnr and smpb.
identification of processing sites in the overlapping region between rnr and smpb indicates that this message is processed, yielding either rnr or smpb.
the fact that the coding regions of these genes overlap makes it impossible to have simultaneously both mature mrnas.
thus, processing of the original transcript always results in disruption of one of the mrnas.
this is in agreement with our results and substantiates the hypothesis of the mutual dependency observed between smpb and rnase r. in terms of cell physiology it is very interesting to note that when the cell is in need of rnase r and raises its production, the higher amount of enzyme lowers the levels of smpb mrna.
since smpb destabilizes rnase r, by lowering the amount of smpb, the cell guarantees that rnase r will not be degraded.
the fact that smpb mrna is disrupted when rnr mrna is matured adds another level of regulation to this complex system.
on the other hand when smpb is required, not only rnase r is destabilized, but its mrna is also disrupted.
comparison of the rnr genomic region of different gram-negative and gram-positive bacteria revealed that this genomic organization  seems to be a common feature among gram-positive bacteria .
the rnr gene is clustered with secg and smpb in numerous bacteria.
does this close localization have a biological meaning?
it is known that bacterial genes involved in the same pathway are frequently co-localized  <cit> .
what could then be the physiological significance of the secg association with two proteins involved in the trans-translation system?
secg is an integral membrane protein that is part of the secyeg complex involved in the recognition and translocation of appropriate polypeptides through the membrane .
recent data has suggested that trans-translation might be linked with other crucial co-translational processes, such as protein folding and secretion  <cit> .
indeed, problems with folding of nascent polypeptides were recently shown to promote trans-translation  <cit> .
this new hypothesis may provide a plausible explanation for the wide array of phenotypes associated with inactivation of tmrna or smpb  <cit> .
most bacterial proteins are secreted through the secyeg translocator, either during or after translation.
when a translocator is blocked in a nascent polypeptide, secy is degraded, which can be lethal or severely impair cell growth because this protein is required to assemble new translocators  <cit> .
an attractive model for a role of tmrna in releasing blocked sec translocators postulates that trans-translation activity over a ribosome stalled on a non-stop mrna during co-translational translocation would allow a tagged protein to be translocated  <cit> .
the subcellular localization of tmrna and smpb is also consistent with a link between trans-translation and protein secretion.
tmrna and smpb are concentrated in a helix-like structure similar to that observed for secy, sece, and secg  <cit> .
the close genomic location of secg, smpb and rnr uncovered in this work also points to a functional relationship.
this interesting possibility certainly deserves further investigation.
CONCLUSIONS
in s. pneumoniae the rnase r coding region is shown to be part of a large transcript that is mainly expressed under cold-shock.
we demonstrate that rnr is co-transcribed with the flanking genes- smpb , and secg .
a promoter identified upstream of secg is likely to control the expression of the downstream genes.
several processing sites in the overlapping region between rnr and smpb were mapped, indicating that the polycistronic message is processed to yield mature independent mrnas.
the gene cluster “secg rnr smpb” appears ubiquitous among gram-positive bacteria.
this finding supports the recently proposed link between trans-translation and other crucial co-translational processes, such as protein folding and secretion  <cit> .
this work shows that the expression of the pneumococcal rnase r is modulated by temperature and higher mrna and protein levels were observed under cold-shock.
additionally it is demonstrated that the trans-translation mediator, smpb, is involved in the regulation of the enzyme expression, leading to increased rnase r levels at 37°c when it is absent.
we postulate that in s. pneumoniae smpb may destabilize rnase r at 37°c through a direct protein-protein interaction, as it was shown for e. coli <cit> .
conversely, a strong accumulation of both smpb mrna and smpb protein was observed in the absence rnase r. this was mainly observed under cold-shock, the main condition where the rnase r levels are higher.
this fact strengthens the role of rnase r in smpb degradation at 15°c.
the implication of rnase r in the control of smpb levels reinforces the functional relationship between rnase r and the trans-translation machinery, and illustrates the mutual dependency and cross-regulation of these two proteins.

 the ability of bacteria to sense and adapt to environmental changes is critical to survival. under stress conditions, prokaryotic cells rapidly adjust their gene expression to deal with a changing environment. rna molecules provide the dynamic link between dna-encoded information and protein synthesis. a rapid response to a changing environment involves not only transcriptional but also post-transcriptional regulation. mrna decay is of prime importance for controlling gene expression, and the labile nature of the rna molecules is critical as it allows a rapid adjustment of proteins levels. ribonuclease r is a processive 3’-5’ exoribonuclease that belongs to the rnase ii family of enzymes. orthologues have been found in most sequenced genomes and have been implicated in the processing and degradation of different types of rna, such as trna, rrna, mrna and the small rna tmrna. rnase r is the only exoribonuclease able to degrade highly structured rna molecules and therefore, it is particularly important in the removal of rna fragments with extensive secondary structures. cold-shock treatment is a condition which thermodynamically favours the formation of highly structured rna molecules, and this fact probably leads to the marked increase of rnase r under this stress situation. in fact, escherichia coli rnase r is a general stress-induced protein whose levels are highly upregulated under cold-shock. stress resistance and virulence are intimately related since many pathogenic bacteria are challenged with very harsh conditions during the process of infection. not surprisingly, rnase r has been implicated in the establishment of virulence in a growing number of pathogens. these include aeromonas hydrophila, shigella flexneri, enteroinvasive e. coli, and helicobacter pylori. this enzyme has also been involved in the quality control of defective trna and rrna molecules. furthermore, e. coli rnase r was shown to participate in the maturation of the transfer-messenger rna, an important small rna involved in trans-translation. in pseudomonas syringae and caulobacter crescentus, degradation of tmrna was also shown to be dependent on rnase r. tmrna together with smpb are the main components of the trans-translation system, an elegant surveillance pathway that directs deficient proteins and mrnas for degradation while rescuing stalled ribosomes. trans-translation allows bacteria to efficiently respond to a variety of stresses and is required for the viability and for the establishment of virulence in many pathogenic bacteria. during trans-translation rnase r is the key exoribonuclease involved in the degradation of the faulty mrnas after the release of the halted ribosomes. moreover, in e. coli the stability of rnase r was shown to be regulated by interaction with tmrna/smpb, which in turn seems to depend on previous rnase r acetylation. in previous studies we have purified and biochemically characterized rnase r from streptococcus pneumoniae, an important human pathogen that is one of the leading causes of nosocomial infections. 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 and our analysis of the pneumococcal genome revealed that the coding sequence of smpb is located immediately downstream of the gene encoding rnase r. these observations 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 under the control of rnase r. this finding uncovers an unsuspected additional connection of rnase r with the trans-translation machinery.  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, 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. it is also known that rnase r is functionally related with the trans-translation system in a wide variety of bacteria. altogether these observations encouraged us to characterize 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. 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 of the rnr transcript was observed under cold-shock. 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. it has been recently shown that the interaction of smpb and tmrna with e. coli rnase r destabilizes the ribonuclease. 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 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  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. the fact that rnase r levels were restored after smpb expression in trans, confirms that smpb is implicated in the regulation of rnase r. 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. 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. the fact that rnr is located upstream and partially overlaps with smpb indicates that these genes may be co-transcribed as part of an operon. to check this possibility rt-pcr experiments were carried out using primers smd and smd. as shown in figure 2b a fragment that results from the amplification of a transcript containing both rnr and smpb could be observed, indicating that smpb is co-transcribed with rnr. a global overview of the rnr/smpb genomic region revealed the existence of several orfs oriented in the same direction. the first orf that is preceded by an orf oriented in opposite direction is located about 5kb upstream of rnr. these orfs are closely located, some with overlapping regions and, using a specific probe for smpb in northern blot experiments, we detected a high molecular weight transcript that could arise from co-transcription of these orfs. we used the same rt-pcr approach for each pair of consecutive orfs in order to establish which orfs were in the same transcriptional unit. a fragment corresponding to the amplification of each orf pair could be detected. the last orf of this transcriptional unit is most probably tehb, since under the same experimental conditions no amplification product containing tehb and the downstream gene could be obtained. in fact, inspection of the sequence revealed a rho-independent transcription terminator downstream of tehb. interestingly, all the amplification products were detected in a higher amount at 15°c than at 37°c and this is also the case of the high molecular weight transcript detected in the northern blot experiments. these results could be indicative of a cold-shock operon. taken the above observations a complex regulation of the operon, with multiple promoters and transcripts containing different sets of genes, cannot be ruled out. since we were particularly interested in rnr and smpb we have searched for promoters in the vicinity that could regulate the expression of this particular set of genes. even though bioinformatics analysis indicated a putative promoter immediately upstream of rnr, we could not detect any active promoter, either by primer extension analysis or by 5’ race mapping. upstream of rnr lays a small orf that encodes a protein with homology to secg, an auxiliary protein in the sec-dependent protein export pathway. a transcript containing secg and rnr was detected and was also mainly expressed under cold shock. in fact, a putative promoter upstream this orf was identified in silico, which could also drive rnr transcription. therefore, primer extension and race experiments were conducted to check this possibility. a single fragment was extended from a primer that hybridizes with the 5’-end of the secg mrna as shown in figure 3a. the size of this fragment, as determined by comparison with the m phage sequence, shows that its 5’-end matches the transcription start site of the in silico predicted promoter. to confirm this result the 5’-end of the transcript was mapped by 5’ race following a protocol that makes use of the tobacco acid pyrophosphatase enzyme. this method allows distinguishing between 5’-ends of primary transcripts from those generated by cleavage/processing. a 5’ race product that was only obtained from the tap-treated samples indicates that it carries a 5’-triphosphate group characteristic of primary transcripts. cloning and sequencing of this race product allowed us to identify the + site at the same position as that identified by primer extension. these results clearly show that this promoter is active and drives the expression of secg. considering the lack of a promoter upstream rnr and since a transcription terminator could neither be identified in this region, we believe that the secg promoter may also contribute to the rnr expression. since our data indicate that rnr and smpb are co-transcribed, this promoter most likely directs smpb transcription as well. nonetheless, we searched for alternative promoters of smpb. we started by analysing the 5’-end of the smpb transcript by primer extension using a primer specific for the smpb 5’-end region. as shown in figure 4a, two different fragments were extended from this primer. analysis of the sequence revealed that the 5’-ends of both fragments are located right before the overlapping region between rnr and smpb. since by visual inspection we could not identify any putative promoter upstream this region, the extended fragments likely correspond to different processing sites. to clarify the primer extension result and confirm this hypothesis, 5’ race experiments were conducted before and after treatment with tap to discriminate primary transcripts from those originated by processing. the gel in figure 4b shows several 5’ race products that are most probably derived from processed molecules as inferred by the similar intensity of tap-treated samples. thereby, under these experimental conditions we did not identify any active promoter upstream smpb. this result further corroborates the rnr and smpb co-transcription hypothesis. the fragments that were not detected in the negative control were cloned, and the sequence of several independent clones allowed us to infer the respective 5’-ends. as expected by the smeared-appearance of fragment sequence analysis revealed different transcripts with distinct 5’-ends. all of these fragments mapped in the 3’-end of rnr upstream the overlapping region with smpb, in agreement with the primer extension results. however, only one exactly matched the nucleotide position of one of the extended fragments. we do not know the reason for this, but one hypothesis is that these fragments could be the result of trimming by a 5’-3’ exoribonuclease, predicted in this gram-positive bacterium. interestingly all the sequences mapped before the putative rbs upstream smpb and thus, these processing events may generate a functional independent smpb transcript. the sequences of the clones corresponding to the other race product mapped inside smpb after the overlapping region. while inactivating smpb mrna, this cleavage spares the rnr transcript, which may thereby be independently translated. the fact that the same pattern was obtained from wild type and rnase r- samples further confirms that the processing of the rnr/smpb transcript is not affected in the rnase r- strain. taken together these results indicate that the pneumococcal rnr transcript is expressed as part of an extensive operon. this large transcript is most probably subject to a complex regulation with several promoters and multiple processing events leading to smaller transcripts. indeed, a promoter identified upstream secg may be responsible for the independent regulation of the downstream genes, secg, rnr and smpb. processing of the operon to yield mature gene products is likely to occur. since we could not identify other active promoters upstream rnr or smpb, we believe that transcription of rnr and smpb does not occur independently and is most probably driven by the promoter identified upstream of secg. smpb mrna and protein levels are modulated by rnase r we have just seen that in s. pneumoniae rnr is co-transcribed with smpb. on the other hand, in e. coli smpb was shown to modulate the stability of rnase r. in e. coli processing of tmrna under cold-shock is dependent on rnase r, and this enzyme has also been involved in tmrna degradation in c. crescentus and p. syringae. thus, we were interested in clarifying which could be the involvement of rnase r with the main components of the trans-translation system in s. pneumoniae. for this purpose, we compared both smpb and tmrna expression by northern blot between the wild type, an isogenic mutant lacking rnase r and the rnase r– strain complemented with a plasmid expressing rnase r constitutively. the results showed that the accumulation of the tmrna precursor form at low temperature is similar in the wild-type and the deletion mutant, and an increase in the tmrna levels was neither observed in the absence of rnase r. hence, under our experimental conditions, rnase r from s. pneumoniae does not seem to be involved in the tmrna processing or turnover. nonetheless, analysis of the smpb mrna levels revealed a strong accumulation of the transcript in the absence of rnase r, especially under cold-shock. comparison of smpb levels between the wild type and the rnase r- strain revealed an increase of about 25-fold at 15°c, while the variation of smpb levels at 37°c appeared very low. the lesser accumulation of the smpb transcript at 37°c may suggest that in this condition the role of rnase r in the control of this transcript is probably less important. this is in agreement with the low levels of rnase r detected at this temperature. the involvement of rnase r was further substantiated by complementation of the rnase r- strain with rnase r expressed from pil at 15°c addition of rnase r partially restored the wild type smpb levels, leading to a ~17-fold decrease relatively to the rnase r- strain. interestingly, in the rnase r complementation strain the variation of smpb levels between 15°c and 37°c is lower, suggesting that the temperature-dependent regulation of smpb levels is compromised. this is probably due to the fact that rnase r expression from pil is constitutive contrary to the temperature-regulated expression pattern observed in the wild type. together, these results strongly suggest that rnase r has a role in smpb degradation. to check if the increment observed on the rna levels would influence the final levels of protein in the cell, we analysed the expression of smpb under the same conditions. smpb expression was compared by western blot in the wild type, the rnase r- mutant derivative and the rnase r- strain complemented with rnase r expressed in trans. analysis of smpb levels with specific antibodies raised against purified tigr smpb showed a significant increase in the protein levels in the absence of rnase r. this phenotype was partially restored in the strain complemented with rnase r, suggesting that rnase r is determinant for the final levels of smpb in the cell. discussion rnase r levels and activity are known to increase in stationary phase and under certain stress situations, namely cold-shock and starvation. rnase r is the unique exoribonuclease able to degrade rna molecules with extensive secondary structures, and the increase of rnase r under multiple stress conditions may indicate a general modification of structured rna in response to environmental changes. in fact this enzyme was shown to be important for growth and viability of several bacteria especially under cold-shock, a condition where rnase r levels are considerably increased. mutants lacking any of the trans-translation components also have a variety of stress phenotypes. these range from attenuated antibiotic resistance to problems in adaptation to oxidative stress, cold- and heat-shock. in this report we have studied the regulation of the rnase r expression and the interplay of this exoribonuclease with the components of the trans-translation system in the human pathogen s. pneumoniae. our results show that, as occurs in e. coli, pneumococcal rnase r is induced after a downshift from 37°c to 15°c. according to our data, both rnr mrna and protein levels are elevated after cold-shock treatment, which could suggest that the higher levels of protein would be directly related with the increased amount of mrna molecules in the cell. however, the expression of rnase r seems to be also modulated by smpb. in the absence of this protein the levels of rnase r are similar at 15°c and 37°c and the temperature-dependent regulation observed in the wild type seems to be lost. this result resembles the e. coli situation where rnase r was shown to be destabilized by smpb during exponential phase in a tmrna-dependent manner. interestingly, e. coli rnase ii was reported to be destabilized by gmr, which is encoded by a gene located immediately downstream. our data suggests that pneumococcal smpb, which is encoded downstream of rnr, may also have a role in the control of rnase r stability. in e. coli destabilization of rnase r by smpb was shown to be dependent on previous acetylation of the enzyme. acetylation only occurs during exponential growth and was proposed to release the c-terminal lysine-rich region of rnase r. this domain of rnase r is directly bound by smpb in a tmrna-dependent manner, and this interaction would ultimately target rnase r for proteolytic degradation. we have analysed the pneumococcal rnase r sequence and also identified a lysine-rich c-terminal domain, which could mediate an association between rnase r and smpb. it seems reasonable to speculate that in s. pneumoniae, a similar interaction is taking place. interestingly, the lysine-rich domain of rnase r is essential for the enzyme’s recruitment to ribosomes that are stalled and for its activity on the degradation of defective transcripts. a proper engagement of rnase r is dependent on both functional smpb and tmrna, and seems to be determinant for the enzyme’s role in trans-translation. all these observations point to an interaction between the pneumococcal rnase r and smpb, which may destabilize the exoribonuclease. however, we believe that the strong increment of the rnr mrna levels detected at 15°c may also account for the final expression levels of rnase r in the cell. a higher amount of mrna may compensate the low translation levels under cold-shock. one of the first indications for the involvement of e. coli rnase r in the quality control of proteins was its association with a ribonucleoprotein complex involved in ribosome rescue. this exonuclease was subsequently shown to be required for the maturation of e. coli tmrna under cold-shock, and for its turnover in c. crescentus and p. syringae. additional evidences included a direct role in the selective degradation of non-stop mrnas and destabilization of rnase r by smpb. in this work we strengthen the functional relationship between rnase r and the trans-translation machinery by demonstrating that rnase r is also implicated in the modulation of smpb levels. a marked accumulation of both smpb mrna and smpb protein was observed in a strain lacking rnase r. the increment in mrna levels is particularly high at 15°c, the same condition where rnase r expression is higher. this fact suggests that the enzyme is implicated in the control of smpb mrna levels. the higher smpb mrna levels detected at 15°c could also suggest a temperature-dependent regulation of this message. however, the steady state levels of smpb protein in the rnase r- strain were practically the same under cold-shock or at 37°c. translational arrest caused by the temperature downshift may be responsible for the difference between the protein and rna levels. alternatively, we may speculate that the interaction between rnase r and smpb could also mediate smpb destabilization. this hypothesis would imply that rnase r/smpb protein-protein association would direct both proteins for degradation. further work is necessary to investigate this attractive possibility. analysis of the s. pneumoniae rnase r genomic region revealed the presence of several orfs that may be part of a large transcript shown to be mainly expressed under cold-shock. some of them are essential for growth, as it is the case of the gtp-binding protein era and of the dephospho-coa kinase. others are important in the resistance to some drugs or mutagens, as for instance formamidopyrimidine-dna glycosylase, the multi-drug resistance efflux pump pmra and the tellurite resistance protein tehb. the first gene of this large operon – ybey, a putative metalloprotease - appears to be essential for translation under high temperature growth conditions. however, besides rnase r and smpb none of these genes have known links to cold-stress. smpb is located downstream of rnr and we show that both genes are co-transcribed. although we were not able to identify an active promoter immediately upstream of rnr or smpb that could drive the transcription of these genes independently, a promoter upstream of secg was identified. secg is a small orf located immediately upstream of rnr and transcription from its promoter is likely to drive expression of the downstream genes. indeed, we have demonstrated that this promoter is active and most probably drives the coupled transcription of secg, rnr and smpb. identification of processing sites in the overlapping region between rnr and smpb indicates that this message is processed, yielding either rnr or smpb. the fact that the coding regions of these genes overlap makes it impossible to have simultaneously both mature mrnas. thus, processing of the original transcript always results in disruption of one of the mrnas. this is in agreement with our results and substantiates the hypothesis of the mutual dependency observed between smpb and rnase r. in terms of cell physiology it is very interesting to note that when the cell is in need of rnase r and raises its production, the higher amount of enzyme lowers the levels of smpb mrna. since smpb destabilizes rnase r, by lowering the amount of smpb, the cell guarantees that rnase r will not be degraded. the fact that smpb mrna is disrupted when rnr mrna is matured adds another level of regulation to this complex system. on the other hand when smpb is required, not only rnase r is destabilized, but its mrna is also disrupted. comparison of the rnr genomic region of different gram-negative and gram-positive bacteria revealed that this genomic organization seems to be a common feature among gram-positive bacteria. the rnr gene is clustered with secg and smpb in numerous bacteria. does this close localization have a biological meaning? it is known that bacterial genes involved in the same pathway are frequently co-localized. what could then be the physiological significance of the secg association with two proteins involved in the trans-translation system? secg is an integral membrane protein that is part of the secyeg complex involved in the recognition and translocation of appropriate polypeptides through the membrane. recent data has suggested that trans-translation might be linked with other crucial co-translational processes, such as protein folding and secretion. indeed, problems with folding of nascent polypeptides were recently shown to promote trans-translation. this new hypothesis may provide a plausible explanation for the wide array of phenotypes associated with inactivation of tmrna or smpb. most bacterial proteins are secreted through the secyeg translocator, either during or after translation. when a translocator is blocked in a nascent polypeptide, secy is degraded, which can be lethal or severely impair cell growth because this protein is required to assemble new translocators. an attractive model for a role of tmrna in releasing blocked sec translocators postulates that trans-translation activity over a ribosome stalled on a non-stop mrna during co-translational translocation would allow a tagged protein to be translocated. the subcellular localization of tmrna and smpb is also consistent with a link between trans-translation and protein secretion. tmrna and smpb are concentrated in a helix-like structure similar to that observed for secy, sece, and secg. the close genomic location of secg, smpb and rnr uncovered in this work also points to a functional relationship. this interesting possibility certainly deserves further investigation.  in s. pneumoniae the rnase r coding region is shown to be part of a large transcript that is mainly expressed under cold-shock. we demonstrate that rnr is co-transcribed with the flanking genes- smpb, and secg. a promoter identified upstream of secg is likely to control the expression of the downstream genes. several processing sites in the overlapping region between rnr and smpb were mapped, indicating that the polycistronic message is processed to yield mature independent mrnas. the gene cluster “secg rnr smpb” appears ubiquitous among gram-positive bacteria. this finding supports the recently proposed link between trans-translation and other crucial co-translational processes, such as protein folding and secretion. this work shows that the expression of the pneumococcal rnase r is modulated by temperature and higher mrna and protein levels were observed under cold-shock. additionally it is demonstrated that the trans-translation mediator, smpb, is involved in the regulation of the enzyme expression, leading to increased rnase r levels at 37°c when it is absent. we postulate that in s. pneumoniae smpb may destabilize rnase r at 37°c through a direct protein-protein interaction, as it was shown for e. coli. conversely, a strong accumulation of both smpb mrna and smpb protein was observed in the absence rnase r. this was mainly observed under cold-shock, the main condition where the rnase r levels are higher. this fact strengthens the role of rnase r in smpb degradation at 15°c. the implication of rnase r in the control of smpb levels reinforces the functional relationship between rnase r and the trans-translation machinery, and illustrates the mutual dependency and cross-regulation of these two proteins. 
rnase r has also been implicated in the trans-translation process. in this work we study the expression of rnase r in the human pathogen streptococcus pneumoniae and analyse the interplay of this enzyme with the main components of the trans-translation machinery. we show that rnase r is induced after a 37°c to 15°c temperature downshift and that its levels are dependent on smpb. on the other hand, our results revealed a strong accumulation of the smpb transcript in the absence of rnase r at 15°c. this study unravels an additional contribution of rnase r to the trans-translation system by demonstrating that smpb is regulated by this exoribonuclease. rnase r in turn, is shown to be under the control of smpb. the data presented here shed light on the interactions between rnase r, trans-translation and cold-shock response in an important human pathogen. it is a stress-induced protein shown to be important for the establishment of virulence in several pathogenic bacteria. ribonuclease r is an exoribonuclease that recognizes and degrades a wide range of rna molecules. transcription of these genes is driven from a promoter upstream of secg and the transcript is processed to yield mature independent mrnas.
 ribonuclease r is an exoribonuclease that recognizes and degrades a wide range of rna molecules. it is a stress-induced protein shown to be important for the establishment of virulence in several pathogenic bacteria. rnase r has also been implicated in the trans-translation process. transfer-messenger rna and smpb are the main effectors of trans-translation, an rna and protein quality control system that resolves challenges associated with stalled ribosomes on non-stop mrnas. trans-translation has also been associated with deficiencies in stress-response mechanisms and pathogenicity.  in this work we study the expression of rnase r in the human pathogen streptococcus pneumoniae and analyse the interplay of this enzyme with the main components of the trans-translation machinery. we show that rnase r is induced after a 37°c to 15°c temperature downshift and that its levels are dependent on smpb. on the other hand, our results revealed a strong accumulation of the smpb transcript in the absence of rnase r at 15°c. transcriptional analysis of the s. pneumoniae rnr gene demonstrated that it is co-transcribed with the flanking genes, secg and smpb. transcription of these genes is driven from a promoter upstream of secg and the transcript is processed to yield mature independent mrnas. this genetic organization seems to be a common feature of gram positive bacteria, and the biological significance of this gene cluster is further discussed.  this study unravels an additional contribution of rnase r to the trans-translation system by demonstrating that smpb is regulated by this exoribonuclease. rnase r in turn, is shown to be under the control of smpb. these proteins are therefore mutually dependent and cross-regulated. the data presented here shed light on the interactions between rnase r, trans-translation and cold-shock response in an important human pathogen. rna turnoverpost-transcriptional controlquality controltranscriptional unitnon-stop rna decay 