Synergies between RNA degradation and trans-translation in Streptococcus pneumoniae: cross regulation and co-transcription of RNase R and SmpB
- Ricardo N Moreira†1,
- Susana Domingues†1,
- Sandra C Viegas1,
- Mónica Amblar2 and
- Cecília M Arraiano1Email author
© Moreira et al.; licensee BioMed Central Ltd. 2012
Received: 19 July 2012
Accepted: 31 October 2012
Published: 20 November 2012
Ribonuclease R (RNase 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 (tmRNA/SsrA 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 (SmpB and tmRNA/SsrA). 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.
KeywordsRNA turnover Post-transcriptional control Quality control Transcriptional unit Non-stop RNA decay
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 [2, 3]. 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 (RNase R) is a processive 3’-5’ exoribonuclease that belongs to the RNase II family of enzymes [4–7]. 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 [9–15]. 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 [11, 12, 17]. 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[18–21]. This enzyme has also been involved in the quality control of defective tRNA and rRNA molecules [13, 22]. Furthermore, E. coli RNase R was shown to participate in the maturation of the transfer-messenger RNA (tmRNA, also called SsrA) , 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 [23, 24]. 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 (for a review see references [25, 26]). 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 (reviewed by [25, 26]). During trans-translation RNase R is the key exoribonuclease involved in the degradation of the faulty mRNAs after the release of the halted ribosomes [2, 27]. 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 [28, 29].
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 (rnr). 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 [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 RNase R expression and study its interplay with the trans-translation machinery of S. pneumoniae.
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 (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. 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 smpBare co-transcribed
The fact that the same pattern was obtained from wild type and RNase R- samples (Figure 4b) 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
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 TIGR4 SmpB showed a significant increase in the protein levels in the absence of RNase R (~13-fold at 15°C and ~7-fold at 37ºC) (Figure 5b). 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.
RNase R levels and activity are known to increase in stationary phase and under certain stress situations, namely cold-shock and starvation [11, 12, 17]. 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 [12, 18, 24, 33, 34]. Mutants lacking any of the trans-translation components (tmRNA and SmpB) also have a variety of stress phenotypes. These range from attenuated antibiotic resistance to problems in adaptation to oxidative stress, cold- and heat-shock [35, 36]. 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 (a protein from the same family of RNase R) 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 [28, 29]. 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[23, 24]. Additional evidences included a direct role in the selective degradation of non-stop mRNAs [2, 27] 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.
Organization of the RNase R genomic region in some Gram+ and Gram- bacteria
secG -yvaK- rnr-smpB -ssrA
secG -LMHCC_0148- rnr-smpB
secG -SAB0735- rnr-smpB
secG - rnr -surE- smpB
secG - rnr - smpB
secG -EF2619-EF2618- rnr - smpB
nsrR- rnr -rlmB-yjfI a
yjeT-purA-yjeB- rnr -yjfH-yjfI
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 (downstream), and secG (upstream). 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.
Bacterial growth conditions
E. coli was cultivated in Luria-Bertani broth (LB) at 37°C with agitation, unless differently specified. Growth medium was supplemented with 100 μg/ml ampicillin (Amp) when required.
S. pneumoniae strains were grown in Todd Hewitt medium, supplemented with 0.5 % yeast extract (THY) at 37°C without shaking, except when differently described. When required growth medium was supplemented with 3 μg/ml chloramphenicol (Cm), 1 or 5 μg/ml Erythromycin (Ery) or 250 μg/ml kanamycin (Km) as specified bellow.
Oligonucleotides, bacterial strains and plasmids
Unless differently specified all DNA sequencing and oligonucleotide synthesis (Additional file 2: Table S1) were performed by STAB Vida. All PCR reactions to perform the constructions below were carried out with Phusion DNA polymerase (Finnzymes).
List of strains used in this work
F' fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17a
E. coli DH5α carrying pSDA-02
F–ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])
E. coli BL21(DE3) overexpressing His-tagged RNase R from S. pneumoniae TIGR4
E. coli BL21(DE3) carrying pSDA-02
TIGR4 RNase R-
TIGR4 rnr - (Δrnr-CmR)
C. Arraiano and P. Lopez Labsa
TIGR4 rnr - (Δrnr-CmR) carrying pIL253 (EryR) expressing RNase R
TIGR4 smpB - (ΔsmpB-KanR)
TIGR4 smpB - (ΔsmpB-KanR) carrying pLS1GFP (EryR) expressing SmpB
The S. pneumoniae RNase R- derivative is an in frame deletion of rnr that preserves the transcriptional and translational relationships between smpB and the upstream ORF. A chloramphenicol-resistance cassette replaces nucleotides +1 to +2288 of the rnr gene (Mohedano, Domingues et al., manuscript in preparation)
The S. pneumoniae smpB - deficient mutant was created through allelic replacement mutagenesis  using a DNA fragment containing the smpB flanking regions, in which smpB is replaced by a kanamycin resistance cassette. km marker was amplified from pR410  with primers smd019 and smd020. The upstream and downstream smpB flanking regions were amplified by PCR using respectively the primer pairs smd053/smd054 and smd055/smd056. Both smd054 and smd055 primers contained 3’ extensions complementary to the 5’- and 3’- ends of the km marker, respectively. The combination of these three PCR products was used as template in another PCR reaction performed with primers smd053 and smd056. The resulting PCR product corresponded to a ~3.9 kb fragment containing the smpB flanking genes (~1.5 kb each side) and a km marker replacing nucleotides +38 to +467 of the smpB gene. This fragment was used to transform TIGR4 competent cells of S. pneumoniae. Competent cultures of S. pneumoniae TIGR4 were prepared in Todd-Hewitt medium (TH) plus 0.5 % glycine and 0.5 % yeast extract by several cycles of dilutions and growing at 37°C up to an OD at 650 nm of 0.3. Competent cells in a concentration 1.5 x 107 CFU/ml were then grown in a casein hydrolase-based medium (AGCH) with 0.2 % sucrose (Suc) and 0.001 % CaCl2, and treated with 100 ng/ml of CSP-2 for 14 min at 30°C. Then 590 ng of DNA were added, and the culture was incubated at 30 °C for 40 min. The culture was then transferred to 37°C and incubated for 120 min before plating on media plates (AGCH medium with 1 % agar plus 0.3 % Suc and 0.2 % yeast extract) containing 250 μg/ml Km. Transformants were grown at 37°C in a 5 % CO2 atmosphere. A KmR transformant was selected, and the insertion/deletion mutation was confirmed by DNA sequencing at the Genomic Service of Instituto de Salud Carlos III.
In order to express SmpB in trans, the TIGR4 SmpB coding sequence was obtained by PCR amplification with primers smd003 and smd004 and was inserted into the unique XbaI site of pLS1GFP ). This construction, expressing SmpB from the pneumococcal PM promoter of this plasmid , was transformed into the TIGR4 SmpB- strain. Transformants were selected with 1 μg/ml Ery.
The lactococcal plasmid vector pIL253  was used to express TIGR4 RNase R. We have recently shown that this plasmid replicates in S. pneumoniae and is suitable for the expression of cloned genes in this bacterium (C. Arraiano, manuscript in preparation). The rnr coding sequence was amplified using primers smd093 and smd094 and was inserted into the unique SmaI/PstI sites of pIL253. pIL253 carrying TIGR4 rnr was transformed into S. pneumoniae TIGR4 RNase R- and transformants were selected with 5 μg/ml Ery.
E. coli SmpB overexpressed in the absence of tmRNA is insoluble . Hence, in order to overexpress and purify pneumococcal SmpB, its coding region was cloned in fusion with pneumococcal ssrA (the gene encoding tmRNA) to allow co-expression of both. smpB was amplified by PCR with primers rnm010 and rnm011, which contains a 3’ extension complementary to the 5’-end of ssrA. ssrA was amplified using the primer pair smd057/smd058. The two PCR fragments were then mixed and used as template in a PCR with primers rnm010 and smd058. The resulting PCR product was digested with NdeI and BamHI (Fermentas), and cloned into the pET-15b vector (Novagen) previously cleaved with the same restriction enzymes. This construction, named pSDA-02, was first obtained in E. coli DH5α and then transferred to E. coli BL21(DE3) to allow the expression of His-SmpB. This construct was confirmed by DNA sequencing.
Overexpression and purification of proteins
RNase R from S. pneumoniae was purified as previously described . For purification of SmpB, BL21(DE3) cells containing pSDA-02 plasmid were grown at 37°C in 250 ml of LB medium supplemented with 100 μg/ml Amp to an OD600 of 0.5. Overexpression of SmpB was then induced by addition of 1 mM IPTG; induction proceeded for 3 hours at 37°C. Cells were harvested by centrifugation and stored at −80°C. Purification was performed by histidine affinity chromatography using HisTrap Chelating HP columns (GE Healthcare) and AKTA HPLC system (GE Healthcare) as follows. Frozen cells were thawed and resuspended in lysis buffer (50 mM HEPES pH 7.5, 1 M NH4Cl, 5 mM MgCl2, 2 mM β-mercaptoethanol, 10 mM imidazole). Cell suspensions were lysed using a French Press at 9000 psi in the presence of 1 mM PMSF. The crude extracts were treated with Benzonase (Sigma) to degrade the nucleic acids and clarified by a 30 min centrifugation at 10000 xg. The clarified extracts were then loaded onto a HisTrap Chelating Sepharose 1 ml column equilibrated with buffer A (20 mM sodium phosphate pH 7.4, 0,5 M NaCl, 20 mM imidazole). Protein elution was achieved by a continuous imidazole gradient (from 20 mM to 500 mM) in buffer A. The fractions containing the purified protein were pooled together and concentrated by centrifugation at 4°C in an Amicon Ultra Centrifugal Filter Device with a molecular mass cutoff of 10 kDa (Millipore). Protein concentration was determined using the Bradford method .
SmpB and RNase R purified proteins were loaded in a SDS-PAGE gel and Coomassie blue stained for band excision (data not shown). Bands corresponding to a total of 500 μg of each protein were used to raise antibodies against the respective pneumococcal proteins (Eurogentec).
RNA extraction and northern blotting
Overnight cultures of S. pneumoniae TIGR4 wild type and mutant derivatives were diluted in pre-warmed THY to a final OD600 of 0.1, and incubated at 37°C until OD600 ~ 0.3. At this point, cultures were split in two aliquots and each aliquot was further incubated at 15°C or 37°C for 2 h. 20 ml culture samples were collected, mixed with 1 volume of stop solution (10 mM Tris pH 7.2, 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 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 (Sigma) for 16 h at 68°C for riboprobes and 43°C in the case of oligoprobes. After hybridization, membranes were washed as described . Signals were visualized by PhosphorImaging (Storm Gel and Blot Imaging System, Amersham Bioscience) and analysed using the ImageQuant software (Molecular Dynamics).
Riboprobe synthesis and oligoprobe labelling was performed as previously described . 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 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 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. with modifications. 5’ triphosphates were converted to monophosphates by treatment of 6 μg of total RNA with 10 units of tobacco acid pyrophosphatase (TAP) (Epicentre Technologies) at 37°C for 30 min in a total reaction volume of 50 μl. The same amount of RNA was used in a parallel reaction where TAP was not added to the sample. To both tubes, 500 pmol of RNA linker and 100 μl of H2O were added. Enzyme and buffer were removed by phenol/chloroform/isoamyl alcohol extraction followed by ethanol precipitation. Samples were resuspended in 28 μl of H2O and heated-denatured 5 min at 90°C. The adapter was ligated at 4°C for 12h with 40 units of T4 RNA ligase (Fermentas). Enzyme and buffer were removed as described above. Phenol chloroform-extracted, ethanol-precipitated RNA was then reverse-transcribed with gene-specific primers (2 pmol each: smd039 for secG; smd050 for rnr; rnm011 for smpB) using Transcriptor Reverse Transcriptase (Roche) according to the manufacturer’s instructions. Reverse transcription was performed in three subsequent 20 min steps at 55°C, 60°C and 65°C, followed by RNase H treatment. The products of reverse transcription were amplified using 2 μl aliquot of the RT reaction, 25 pmol of each gene specific primer (smd039 for secG; smd051 for rnr; smd041 for smpB) and adapter-specific primer (asp001), 250 μM of each dNTP, 1,25 unit of DreamTaq (Fermentas) and 1x DreamTaq buffer. Cycling conditions were as follows: 95°C/10 min; 35 cycles of 95°C/40 s, 58°C/40 s, 72°C/40 s; 72°C/7 min. Products were separated on 1.5% agarose gels, and bands of interest were excised, gel-eluted (Nucleospin extract: Macherey-Nagel) and cloned into pGEM-T Easy vector (Promega). Bacterial colonies obtained after transformation were screened for the presence of inserts of appropriate size by colony PCR. The plasmids with inserts of interest were purified (ZR plasmid miniprep–classic: Zymo Research) and sequenced.
Primer extension analysis
Total RNA was extracted as described above. Primers rnm016, rnm014 and rnm002, respectively complementary to the 5’-end of rnr, secG and smpB, were 5’-end-labeled with [γ-32P]ATP using T4 polynucleotide kinase (Fermentas). Unincorporated nucleotides were removed using a MicroSpinTM G-25 Column (GE Healthcare). 2 pmol of the labeled primer were annealed to 5 μg of RNA, and cDNA was synthesized using 10U of Transcriptor Reverse Transcriptase (Roche). In parallel, an M13 sequencing reaction was performed with Sequenase Version 2.0 sequencing kit (USB) using a sequence specific primer, according to the supplier instructions. The primer extension products were run together with the M13 sequencing reaction on a 5 % polyacrylamide / urea 8 M sequencing gel. The gel was exposed, and signals were visualized in a PhosphorImager (Storm Gel and Blot Imaging System, Amersham Bioscience). The size of the extended products was determined by comparison with the M13 generated ladder enabling the 5’-end mapping of the respective transcripts.
Total protein extraction and western blotting
Cell cultures used to prepare protein extracts were grown in the same conditions as described above for RNA extraction. 20 ml culture samples were collected, mixed with 1 volume of stop solution [10 mM Tris (pH 7.2), 25 mM NaN3, 5 mM MgCl2, 500 μg/ml chloramphenicol] and harvested by centrifugation (10 min, 2800 xg, 4°C). The cell pellet was resuspended in 100 μl TE buffer supplemented with 1 mM PMSF, 0.15 % sodium deoxicolate and 0.01 % SDS. After 15 min incubation at 37°C, SDS was added to a final concentration of 1 %. Protein concentration was determined using a Nanodrop 1000 machine (NanoDrop Technologies). 20 μg of total protein were separated in a 7 % (for RNase R detection) or 10 % (for SmpB detection) tricine-SDS-PAGE gel, following the modifications described by . After electrophoresis, proteins were transferred to a nitrocellulose membrane (Hybond ECL, GE Healthcare) by electroblotting using the Trans-Blot SD semidry electrophoretic system (Bio-Rad). Membranes were then probed with a 1:1000 or 1:500 dilution of anti-SmpB or anti-RNase R antibodies, respectively. ECL anti-rabbit IgG peroxidase conjugated (Sigma) was used as the secondary antibody in a 1:10000 dilution. Immunodetection was conducted via a chemiluminescence reaction using Western Lightning Plus-ECL Reagents (PerkinElmer).
In silico predictions of putative promoters were performed using the BPROM SoftBerry software (http://linux1.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb) and Neural Network Promoter Prediction (http://www.fruitfly.org/seq_tools/promoter.html)  bioinformatics tools.
We thank Andreia Aires for technical assistance. R. Moreira (Doctoral fellow), S. Domingues (Postdoctoral fellow) and S. Viegas (Postdoctoral fellow) received fellowships from FCT-Fundação para a Ciência e Tecnologia, Portugal. This work was supported by several grants from FCT, including grant PEst-OE/EQB/LA0004/2011 and the work at Instituto de Salud Carlos III was supported by Fondo de Investigación Sanitaria (FIS) (PI08/0442 and PI11/00656), CIBER Enfermedades Respiratorias (initiative of the Instituto de Salud Carlos III) in Spain, and by the Bilateral Collaboration program between Conselho Reitores Universidades Portuguesas (CRUP) from Portugal and Ministerio de Ciencia e Innovación (MICINN) (HP2008-0041) Acciones Integradas of Spain.
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