Pneumococcal RNase R globally impacts protein synthesis by regulating the amount of actively translating ribosomes

ABSTRACT

Ribosomes are macromolecular machines that carry out protein synthesis. After each round of translation, ribosome recycling is essential for reinitiating protein synthesis. Ribosome recycling factor (RRF), together with elongation factor G (EF-G), catalyse the transient split of the 70S ribosome into subunits. This splitting is then stabilized by initiation factor 3 (IF3), which functions as an anti-association factor. The correct amount of these factors ensures the precise level of 70S ribosomes in the cell. RNase R is a highly conserved exoribonuclease involved in the 3ʹ to 5ʹ degradation of RNAs. In this work we show that pneumococcal RNase R directly controls the expression levels of frr, fusA and infC mRNAs, the corresponding transcripts of RRF, EF-G and IF3, respectively. We present evidences showing that accumulation of these factors leads to a decreased amount of 70S active particles, as demonstrated by the altered sucrose gradient ribosomal pattern in the RNase R mutant strain. Furthermore, the single deletion of RNase R is shown to have a global impact on protein synthesis and cell viability, leading to a ~50% reduction in bacterial CFU/ml. We believe that the fine-tuned regulation of these transcripts by RNase R is essential for maintaining the precise amount of active ribosomal complexes required for proper mRNA translation and thus we propose RNase R as a new auxiliary factor in ribosome reassociation. Considering the overall impact of RNase R on protein synthesis, one of the main targets of antibiotics, this enzyme may be a promising target for antimicrobial treatment.

Introduction

The discovery of bacteria in 1673 by Antony van Leeuwenhoek triggered the beginning of a new era and further investigation on this topic led later to the correlation between bacteria and infections.

Streptococcus pneumoniae is a pathogenic Gram-positive bacterium, being the leading cause of bacterial pneumonia and meningitis. It especially affects young children, the elderly and immunosuppressed individuals. Diseases caused by pneumococcus constitute a major global public health problem. According to the lastest data from the World Health Organization, about 14.5 million episodes of serious pneumococcal disease occurred. Bacterial resistance to antibiotics is increasing and it is imperative to find new strategies to control bacterial infections.

Ribonucleases (RNases) are the enzymes responsible for processing and degrading RNA and are emerging as key effectors in bacterial virulence and survival [1,2]. Ribonuclease R (RNase R) is a member of the RNB family of enzymes, which also includes RNase II and the eukaryotic Rrp44/Dis3, Dis3L1 and Dis3L2 proteins [3]. Members of this family processively hydrolyse RNA in the 3ʹ-5ʹ direction. Although RNase II and RNase R co-exist in many bacteria, in S. pneumoniae only RNase R is present [3]. In Escherichia coli this enzyme has been widely studied and it was reported to be associated with ribosomes [4,5]. Ribosomes are macromolecular machines that translate mRNA into functional proteins and have been used as targets for antibiotic treatment [6]. In bacteria the active form of ribosome (70S subunit) is composed of two subunits, a small 30S subunit and a large 50S subunit. Each subunit is comprised of ribosomal RNA and ribosomal proteins, assembled to form the active 70S ribosome. Association of the small and large subunits to form the active 70S ribosome takes place at the beginning of protein synthesis and is assisted by the translation initiation factors [7,8]. After each round of translation, splitting of ribosomes is a necessary step before the translation reinitiates, and recycling of ribosomes is also dependent on several translation factors [9–12].

RNases are essential for the correct processing of rRNA, converting rRNA precursors into mature rRNA molecules [3]. In E. coli, RNase III is responsible for the cleavage of the initial transcript into 25S, 17S and 9S precursors that, after being processed at their 5ʹ- and 3ʹ-ends, originate mature 23S, 16S and 5S rRNAs, respectively [13,14]. Further maturation of rRNAs 3ʹ- and 5ʹ-ends is carried out by several RNases and is a crucial step for the precise assembly of ribosomal subunits to form a fully functional ribosome [15]. Despite their important roles, studies on pneumococcal RNases are scarce.

This work focuses on the investigation of the role of pneumococcal RNase R on translation. We show that in S. pneumoniae translation is dramatically affected by the lack of its encoding gene. RNase R is shown to affect the amount of 70S ribosomes required for proper mRNA translation. In this paper we demonstrate, for the first time, that RNase R regulates the expression level of the translation factors involved in the dissociation of the ribosome active complex, globally impacting translation and cellular viability.

Results

RNase R association with ribosomes and impact in ribosome profiling

We have previously shown that E. coli RNase R interacts with ribosomes, mostly with the 30S subunit [4]. To date, no studies have been performed regarding this topic in Gram positive bacteria. We have previously biochemically characterised S. pneumoniae RNase R [16] and wanted to investigate whether this enzyme is also associated with ribosomes in this bacterium. For this purpose, we started by carrying out sucrose gradient polysome fractionation. This fractionation method allows the separation along the gradient according to protein size. While soluble proteins remain at the top, ribosomes migrate deeper in the gradient. The graph in Figure 1(a) shows the peaks corresponding to subunits 50S and 70S as well as translating ribosomes (polysomes). Although the complete separation of the 30S subunit from the pool of soluble proteins is not observed, the fractions corresponding to the 30S subunit could be identified by mass spectrometry analysis (Table S1, Supplementary material). As expected, the proportion of ribosomal proteins increases along the gradient (Figure 1(a)) A higher percentage of 30S ribosomal proteins is observed in fraction 4, indicating the 30S subunit corresponding peak. Conversely a higher amount of 50S ribosomal proteins is present in Fraction 6, while fraction 5 seems to contain a mix of both 30S and 50S ribosomal proteins, corresponding to the transition between the two peaks (30S and 50S). Although still containing a higher amount of 50S ribosomal proteins, we believe that fraction 7 corresponds to the transition between the 50S and 70S subunits peak. By mass spectrometry the presence of RNase R was only detected in fractions 5, 6 and 7, suggesting its association mostly with the 50S subunit. Indeed, the trigger factor, known to be associated with the 50S subunit [17], was mostly found in the same fractions. In confirmation of this result, RNase R was mostly detected in fractions 5 and 6, after Western blot with specific antibodies. Even though RNase R could be detected at the top of the gradient, as expected for soluble proteins, it reached maximal intensity in the positions corresponding to the 50S subunit (Figure 1(a)). These results are supportive of the association of pneumococcal RNase R with the ribosomes. However, contrary to the observations in E. coli, RNase R association mostly occurs with the 50S ribosomal subunit in S. pneumoniae.

Figure 1.

RNase R interaction with ribosome and its effect in the ribosomal profile of S. pneumoniae TIGR4. Cellular extracts were separated on 10–30% sucrose gradient and the position of ribosomal subunits, ribosomes and polysomes along the gradient were monitored by UV absorbance at 254 nm on AKTA (GE Healthcare). Peaks corresponding to ribosomal subunits 30S, 50S, 70S and Polisomes are indicated. (a) Ribosomal profile of the wild type (WT) strain. Samples were collected along the gradient (numbered) and analysed by mass spectrometry. The graph shows the relative amount of 30S and 50S ribosomal proteins in each sample. The amount of RNase R in each fraction of the gradient was monitored by Western blot using antibodies against RNase R. Protein samples were separated on a 7% tricine-SDS polyacrylamide gel and blotted to a nitrocellulose membrane. (b) Ribosomal profile of the RNase R mutant (Δrnr) and Δrnr expressing RNase R in trans (Δrnr+R).

Previous studies in E. coli showed that deletion of RNase R does not impact ribosome association and the ribosome profile of the mutant was not changed when compared to the wild type strain [4]. Since RNase R is the only member of the RNB family of enzymes in S. pneumoniae, and considering the observed differences in pneumococcal RNase R association with the ribosomal subunits, we wanted to investigate if deletion of the rnr gene would affect the ribosome profile in pneumococcus. To this end, we have compared the polysome profile from sucrose gradients between the wild-type, the RNase R deletion strain (∆rnr) and the ∆rnr strain expressing RNase R in trans (∆rnr+R). The fractionation profile from the RNase R mutant is shown in Figure 1(b). Comparison to the wild type profile (in Figure 1(a)) reveals an altered proportion of the amount of subunits, with an accentuated decrease of the 70S active particles concomitantly with an increasing level of free 30S and 50S subunits in the mutant strain. The amount of the ribosome subunits observed in the ∆rnr+R profile show a partial reversion to the wild-type migration pattern (Figure 1(b)). These alterations in the ribosome profile caused by the lack of RNase R suggest that this enzyme is probably involved in the assembly of the ribosomal subunits, thus affecting the final level of each subunit in the cell. Furthermore, in the mutant strain almost no polysomes were detected which, together with the decrease in the amount of 70S ribosomes, is strongly indicative that translation might be compromised.

Role of RNase R in 70S subunit dissociation

The observed decrease in the amount of mature 70S ribosome particles could suggest a role for RNase R in ribosome biogenesis. RNases are long known to be involved in the maturation of ribosomal RNAs (rRNAs), whose precise processing is in turn essential for correct ribosome assembly (reviewed in [21,22]). In E. coli, RNase R was indicated as one of the enzymes involved in the 16S precursor (17S rRNA) maturation and the 30S particles containing unprocessed 16S rRNA form 70S ribosomes very poorly [15]). Thus, we started by comparing the levels of 16S rRNA between the strain lacking RNase R and the wild type strain. For that purpose we have performed Northern blot and synergel/agarose gel electrophoresis [23]. However, the amount of 16S in the RNase R deleted strain was roughly the same as in the parental strain and accumulation of 17S was not detected (Figure S1, Supplementary material). Hence, RNase R does not appear to be essential for 16S maturation in S. pneumoniae, and improper maturation of 16S rRNA is probably not the cause for the decreased level of 70S particles observed in the mutant.

During translation, after the ribosome encounters a stop codon, the active 70S particle must dissociate to allow protein synthesis to restart. Three players, RRF (Ribosome Recycling factor), EF-G (Elongation Factor G) and IF3 (Initiation Factor 3) have an important role in the ribosome recycling step. RRF and EF-G bind to the 70S subunit promoting dissociation of the 30S and 50S subunits, after which IF3 binding to the 30S subunit inhibits reassociation of the ribosome [9]. We hypothesized that the decreased levels in the 70S particles observed in the RNase R mutant strain could be due to abnormal regulation of the dissociation factors. If RNase R would be regulating the mRNA levels of the factors involved in 70S ribosome dissociation, one would expect to observe accumulation of these transcripts in the absence of this RNase. To experimentally validate this hypothesis, we compared the mRNA levels of frr, fusA and infC (the respective corresponding transcripts of RRF, EF-G and IF3) in the wild type, the ∆rnr and the ∆rnr+R strains. As shown in Figure 2(a), increased mRNA levels of the three factors were observed in the rnr mutant strain. Addition of RNase R in trans partially restored the wild-type amounts of these transcripts, demonstrating that the absence of RNase R leads to accumulation of frr, fusA and infC messages in the cell. Western blot of the corresponding protein samples with antibodies specific for EF-G also revealed a similar variation in the protein levels of this translation factor in the three strains (Figure 2(a)). In order to test if RNase R is directly involved in the regulation of these transcripts we performed mRNA stability assays in the presence and absence of RNase R (Figure 2(b)). Quantitation of mRNA decay was done by Northern Blot after transcriptional arrest by rifampicin. Samples were taken at the time-points indicated above the images and the amount of each transcript measured. The graphs in Figure 2(c) show a significant stabilization of the three mRNAs in the absence of RNase R. fusA, frr and infC mRNAs were stabilized by about 2-, 4.5- and 4-fold respectively, clearly indicating that RNase R has an important role in the degradation of these transcripts. This might lead to an excess of the corresponding dissociation factors in the RNase R mutant strain, which is most probably the reason for the low level of 70S ribosomes observed in this strain.

Figure 2.

Loss of RNase R leads to accumulation of ribosomal complex dissociation factors. (a) Northern blot and Western blot analysis of RNA and protein samples extracted from the wild type (WT), RNase R mutant (Δrnr) and Δrnr expressing RNase R in trans (Δrnr+R). For Northern blot 10 µg (to detect fusA mRNA – transcript which encodes EF-G) or 15 µg (for detection of frr and infC mRNAs – transcripts which encode RRF and IF3, respectively) of total RNA were separated on respectively 1.2% or 1.5% agarose gel. Separated RNA molecules were transferred to Hybond-N+ membranes and hybridised with specific probes. The membranes were stripped and probed for 23S rRNA as loading control. For Western blot 20 µg of total protein were separated in a 10% tricine-SDS-polyacrylamide gel and blotted to a nitrocellulose membrane. EF-G was detected using specific antibodies. (b) Northern blot analysis of the stability of the same transcripts in the wild type and RNase R mutant strain. Transcription was blocked by addition of rifampicin (0 min) and aliquots harvested at the indicated time-points for RNA extraction. 25 µg of total RNA were separated on 1.2% (for detection of fusA and infC mRNAs) or 1.8% (for detection of frr mRNA) agarose gel. Gels were then blotted to Hybond-N+ membranes and hybridized with specific riboprobes. The membranes of the wild type were exposed longer than those of the mutant strains, due to the different steady state expression levels. (c) Quantification of the fusA, frr and infC transcripts was done by scanning densitometry and values of the amount of RNA at zero minutes considered as 100%. The percentage of transcript remaining after rifampicin addition was plotted as a function of time. Decay rates were evaluated by linear regression analysis.

RNase R has a global effect on translation

Our results are supportive of a role for RNase R in controlling the expression levels of the dissociation factors, ultimately leading to a decreased amount of 70S active ribosomal particles. The low level of 70S subunits, together with the reduced level of polysomes, observed in Δrnr strain, prompted us to investigate if RNase R deletion could have a global effect on translation. This was done by comparing the expression level of a reporter gene on the wild-type and Δrnr strain. A plasmid with a reporter gene encoding the Citrine protein, pBCSJC001 [24], was introduced in both strains and in the Δrnr strain expressing RNase R in trans, and the fluorescence level of the reporter was analysed by spectrofluorimetry. In confirmation of our hypothesis the Δrnr cells presented reduced fluorescence compared to the wild type pneumococcal cells and this phenotype was reverted by RNase R addition in trans (Figure 3). Indeed, quantification of the fluorescent signals emitted by Citrine in live bacteria revealed a more than 50% decrease in the strain without RNase R. As expected, the Citrine protein level also significantly decreased in the RNase R mutant (as detected by western blot), while the corresponding RNA levels remained roughly the same. This fact ruled out any effect at the RNA level and confirmed that protein expression is clearly affected in the rnr deleted strain.

Figure 3.

Influence of RNase R on the global protein expression level. (a) Fluorescent signals emitted by Citrine were detected in live bacteria. Samples of the wild type (WT), RNase R mutant (Δrnr) and Δrnr expressing RNase R in trans (Δrnr+R) were prepared for fluorescence microscopy and visualized using phase contrast (PC) and Citrine (YFP) detection filters. (b) Graphical representation of fluorescence intensity of the same strains as indicated in (a). At least 100 cells of each strain were quantified. (c) Western blot and Northern blot analysis of Citrine and the corresponding yfp mRNA expression. Protein and RNA samples were extracted from the same strains as indicated in (a). Citrine protein (27 kDa) levels were analysed by western blot with GFP antibodies. 40 µg of total protein were separated in a 10% SDS polyacrylamide gel and blotted into a nitrocellulose membrane. The corresponding mRNA (yfp) level was analysed by Northern blot. 10 µg of total RNA were separated on a 1.5% agarose gel, transferred to a Hybond-N+ membrane and hybridised with a specific probe for yfp mRNA. The membrane was stripped and probed for 23S rRNA as loading control.

The reduced level of Citrine expression, together with the lower 70S ribosomal particles and polysomes formation found in the strain lacking RNase R, suggests a global effect of RNase R on protein synthesis, and reinforces the impact of this enzyme on translation.

Deletion of rnr affects cellular viability

By affecting translation RNase R deletion should compromise the expression of many important cellular proteins and this would presumably lead to growth defects. It was of interest to find out whether cellular viability was affected. To address this question, serial dilutions of exponentially growing cultures of the wild type, ∆rnr and ∆rnr+R strains were plated in agar medium. After 24h of incubation the number of colony forming units (CFU) was determined for each strain. As shown in the example of Figure 4, the mutant strain presented a reduced number of colonies, revealing a ~50% decrease in viable bacterial number in comparison with the wild type. Addition of RNase R in trans partially restored the colony growth to the wild type levels (Figure 4), clearly demonstrating the impact of RNase R deletion in cell viability. Taking into account the decreased protein expression levels in the absence of RNase R, we believe that the reduced cellular viability might be related with defects in translation. This is consistent with the low level of translating polysomes observed in the mutant strain.

Figure 4.

Effect of RNase R on cellular viability. Bacterial cells were grown in THY medium until OD₆₀₀ ≈ 0.3. Equal aliquots of the different strains were plated in THY agar medium supplemented with 5% blood and plates were incubated 24h at 37°C in a 5% CO₂ atmosphere. Bacterial growth was compared in the three strains (WT – wild type strain; ΔR – Δrnr strain; Δrnr+R – Δrnr strain expressing RNase R in trans).

Materials and methods

Bacterial strains, plasmids and growth conditions

S. pneumoniae strains were grown in Todd Hewitt medium, supplemented with 0.5% yeast extract (THY) at 37°C without aeration, or in THY agar medium supplemented with 5% sheep blood (Thermo Scientific) at 37°C in a 5% CO₂ atmosphere. When required growth medium was supplemented with 3 μg/ml chloramphenicol (Cm), 1 μg/ml erythromycin (Ery) or 1 μg/ml tetracycline (Tet) as specified.

Bacterial strains and plasmids used in this study are listed in Table 1. All S. pneumoniae strains are isogenic derivatives of the JNR7/87 capsulated strain – TIGR4. Strain CMA611 was transformed with pIL253 and transformants were selected with 1 μg/ml Ery. For the fluorescence experiments the strains CMA607, CMA604 and CMA012 were transformed with an additional plasmid, pBCSJC001, that expresses a YFP variant named Citrine [24,25]. Transformants carrying both plasmids were selected with 1 μg/ml Ery and 1 μg/ml Tet.

Table 1.

List of strains and plasmids used in this work.

Strains	Relevant Markers/Genotype	Source/Reference
JNR7/87 (TIGR4)		[18]
CMA607	TIGR4 carrying pIL253 (EryR)	[19]
CMA611	TIGR4 rnr− (∆rnr-CmR)	[30,19]
CMA604	CMA611 carrying pIL253 (EryR) expressing RNase R	[30]
CMA612	CMA611 carrying pIL253 (EryR)	This work
CMA613	TIGR4 carrying pIL253 (EryR) and pBCSJC001 (TetR)	This work
CMA614	CMA611 carrying pIL253 (EryR) and pBCSJC001 (TetR)	This work
CMA615	CMA611 carrying pIL253 (EryR) expressing RNase R and pBCSJC001 (TetR)	This work
Plasmids	Comment	Source/Reference
pIL253	pAMβ1 derivative (EryR)	[19,20]
pIL253-RNaseR	pIL253 carrying pneumococcal RNase R (EryR)	[30]
pBCSJC001	pBCSMH004 derivative, allowing expression of Citrine (TetR)	[24]

Competent cells/transformation

Pneumococcal transformation was performed essentially as described [26]. Briefly pre-competent pneumococcal cells were grown for 10 minutes at 37°C in a 10-fold volume of CTM-pH 7.8 supplemented with 125 ng/ml CSP-2 (the synthetic competence-stimulating peptide 2). About 200 ng of plasmid DNA were added and cultures were further incubated at 30°C for 30 minutes and then shifted to 37°C. Ninety minutes after the temperature shift, transformants were selected on solid medium supplemented with the appropriate antibiotic.

DNA manipulation

PCR fragments were amplified with DreamTaq (Thermo Fisher). Primers used in this study are listed in Table S2 (Supplementary material) and were synthetized by Stab Vida, Portugal. PCR products and plasmid DNA were purified using the kits ‘PCR Clean-up System’ (Macherey-Nagel) and ‘Wizard H Plus SV Minipreps’ (Promega), respectively.

Riboprobe synthesis and oligoprobe labelling (Table S2, Supplementary material) were performed as previously described [27]. PCR products used as template for the riboprobe synthesis were obtained using the following primer pairs: cbr021/cbr022 for frr transcript, cbr023/cbr024 for infC transcript, smd067/smd097 for fusA transcript and cbr017/cbr018 for yfp mRNA. DNA probes for 16S rRNA and 23S rRNA were generated using oligonucleotides cbr012 and cbr014, respectively, labelled at their 5ʹ-ends with [³²P]-γ-ATP using T4 Polynucleotide kinase (Thermo Fisher).

Viability assays

S. pneumoniae strains were grown in THY, with the respective antibiotics, at 37°C without aeration until OD₆₀₀ ≈ 0.3. The same volume was collected to ensure that the same number of cells were plated. Cells were then harvested by centrifugation and suspended in PBS. Tenfold serial dilutions were performed and plated in THY agar medium supplemented with 5% blood. Plates were incubated at 37°C in a 5% CO₂ atmosphere and after 24h of incubation bacterial cell growth was compared. The final results were obtained from more than 3 independent experiments.

RNA extraction and Northern blotting

Overnight cultures of S. pneumoniae TIGR4 wild type and derivatives were diluted in pre-warmed THY to a final OD₆₀₀ of 0.1 and incubated at 37°C until OD₆₀₀ ≈ 0.3. 20 ml of culture was collected, mixed with 1 volume of stop solution (10 mM Tris pH 7.2, 25 mM NaNO₃, 5 mM MgCl2, 500 µg/ml Cm) and harvested by centrifugation (10 min, 6000 x g, 4°C). For stability experiments, transcription was stopped by addition of rifampicin (0.5 mg/ml) and nalidixic acid (20 µg/ml) to growing cells (OD₆₀₀ ≈ 0.3). At the indicated times, 20 ml of culture was collected, mixed with 1 volume of stop solution and harvested by centrifugation in the same conditions as samples for RNA extraction. In both cases, total RNA was extracted using Trizol reagent (Ambion) essentially as described by the manufacturer, but with some modifications. Pneumococcal cells were lysed by incubation in 650 µl lysis buffer (sodium citrate 150 mM, saccharose 25%, sodium deoxycholate 0.1% and SDS 0.01%) for 15 min at 37°C followed by the addition of 0.1% SDS. After lysis, samples were treated with 10 U Turbo DNase (Ambion) for 1 h at 37°C. The RNA integrity was evaluated by gel electrophoresis and its concentration estimated using a Nanodrop 1000 machine (Nanodrop Technologies).

For Northern blot analysis, total RNA samples were separated under denaturating conditions either by agarose MOPS/formaldehyde gel (1.2% or 1.5%) or in a 6% polyacrylamide/urea 8.3 M gel in TBE. For agarose gels RNA was transferred to Hybond-N+ membranes (GE Healthcare) by capillarity using 20× SSC as transfer buffer, while in the case of polyacrylamide gels, RNA transfer onto Hybond-N+ membranes was performed by electro-blotting (2 hours, 24 V, 4°C) in TAE 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 [27]. Signals were visualized by Phosphorimager (TLA-5100 Series, Fuji) and analysed using the ImageQuant software (Molecular Dynamics).

Sucrose gradient separation

Sucrose gradients were prepared as described [28]. Overnight cultures of S. pneumoniae TIGR4 wild type and derivatives were diluted in pre-warmed THY to a final OD₆₀₀ of 0.1 and incubated at 37°C until OD₆₀₀ ≈ 0.3. Cm was added to the culture (final concentration 0.1 mg/ml) which remained for 3 minutes under the same conditions. Cultures were centrifuged at 4420 x g, for 10 min at 4°C. Pellets were resuspended with 0.6 ml of cold Buffer A (100 mM NH₄Cl, 10 mM MgCl₂, 20 mM Tris-HCl pH 7.5), transferred to an Eppendorf tube, after which lysozyme solution was added to a final concentration of 0.1 µg/µl. Cells were frozen in liquid nitrogen for 5 minutes and then thawed in an ice water bath (this step was repeated twice). Subsequently, 30 µl of 10% Deoxycholate was added to complete the cell lysis and the sample was centrifuged at 17,000 x g for 10 min at 4°C. Supernatant was carefully transferred to a new tube and stored at −80°C. The RNA concentration was determined using a Nanodrop 1000 machine (Nanodrop Technologies) and approximately 600 µg was added to the top of the 10–30% sucrose gradient. Samples were centrifuged at 35,000 rpm for 3h at 4°C, using an SW41ti rotor. Gradients were separated using AKTA equipment (GE Healthcare) and UV spectra were monitored. Proteins from the gradient fractions were collected and precipitated with TCA. Total proteins were separated in a 7% tricine–SDS-polyacrylamide gel, following the modifications described [29].

Total protein extraction

S. pneumoniae cell cultures 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 (defined above) and harvested by centrifugation (10 min, 6000 x g, 4°C). The cell pellet was resuspended in 100 µl of TE buffer supplemented with 1 mM PMSF, 0.15% sodium deoxycholate 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). Total protein samples were separated in a 10% SDS – polyacrylamide gel or in a modified 10% tricine-SDS-polyacrylamide gel as described [29].

Western blotting

After electrophoresis, proteins were transferred into a nitrocellulose membrane (Hybond ECL, GE Healthcare) by electroblotting using the Trans-Blot SD semidry electrophoretic system (Bio-Rad). The membrane with proteins obtained from sucrose gradient separation was probed with 1:500 dilution of anti-RNase R antibodies [30]. For Citrine detection the membrane was probed with 1:100 dilution of anti-GFP antibodies (Invitrogen) and for EF-G detection, the membrane was probed with 1:4000 dilution of anti-EF-G antibodies (Agrisera). In all cases, ECL anti-rabbit IgG peroxidase conjugated (Sigma) was used as the secondary antibody in a 1:10,000 dilution. Immunodetection was conducted via a chemiluminescence reaction using Western Lightning Plus-ECL Reagents (PerkinElmer).

Microscopy and fluorescence quantification

Overnight cultures of S. pneumoniae TIGR4 wild type and derivatives were diluted in pre-warmed THY to a final OD₆₀₀ of 0.1, and incubated at 37°C. At OD₆₀₀ ≈ 0.3 cell cultures were observed by fluorescence microscopy in a thin layer of 1% agarose in PreC medium [31]. Images were obtained using Leica DM6000B microscope equipped with a phase-contrast (PC) Uplan F1 100× objective and an additional 1.6× optavar, a CCD Ixon^EM camera (Andor Technologies), and phase-contrast optics and standard filter for visualization of Citrine (yellow fluorescence protein filter). After acquisition, images were analysed using Metamorph software and Image J software. Fluorescence quantification was performed using the Metamorph software by measuring the integrated fluorescence intensity in a defined region of 2 by 2 pixels and subtracting the minimum background fluorescence obtained from every value. We quantified at least 100 cells of each strain in triplicate, and the obtained values were then normalized to the higher value.

Mass spectrometry analysis and protein identification

Samples were subjected to trypsin in-gel digestion coupled with mass spectrometric analysis (GeLC-MS/MS). Briefly, protein sample was reduced with 10 mM DTT (Sigma) for 40 min at 56 °C followed by alkylation with iodoacetamide (Sigma) 55 mM for 30 min in the dark. Excessive iodoacetamide was quenched by further incubation with DTT (10 mM for 10 min in the dark). The resulting sample was digested overnight with trypsin (Proteomics grade from Promega) at 37 °C (1:50 protein/trypsin ratio), dried and resuspended in 8 µL LCMS water 0.1% formic acid (Fisher Chemicals).

Nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis was performed on an ekspert™ NanoLC 425 cHiPLC® system coupled with a TripleTOF® 6600 with a NanoSpray® III source (Sciex). Peptides were separated through reversed-phase chromatography (RP-LC) in a trap-and-elute mode. Trapping was performed at 2 µl/min with 100% A (0.1% formic acid in water, Fisher Chemicals, Geel, Belgium), for 10 min, on a Nano cHiPLC Trap column (Sciex 200 µm x 0.5 mm, ChromXP C18-CL, 3 µm, 120 Å). Separation was performed at 300 nl/min, on a Nano cHiPLC column (Sciex 75 µm x 15 cm, ChromXP C18-CL, 3 µm, 120 Å). The gradient was as follows: 0–1 min, 5% B (0.1% formic acid in acetonitrile, Fisher Chemicals); 1–91 min, 5–30% B; 91–93 min, 30–80% B; 93–108 min, 80% B; 108–110 min, 80–5% B; 110–127 min, 5% B.

Peptides were sprayed into the MS through an uncoated fused-silica PicoTip™ emitter (360 µm O.D., 20 µm I.D., 10 ± 1.0 µm tip I.D., New Objective). The source parameters were set as follows: 15 GS1, 0 GS2, 30 CUR, 2.5 keV ISVF and 100 °C IHT. An information dependent acquisition (IDA) method was set with a TOF-MS survey scan of 400–2000 m/z for 250 msec. The 50 most intense precursors were selected for subsequent fragmentation and the MS/MS were acquired in high sensitivity mode (150–1800 m/z for 40 msec each). The selection criteria for parent ions included a charge state between +2 and +5 and counts above a minimum threshold of 125 counts per second. Ions were excluded from further MSMS analysis for 12 s. Fragmentation was performed using rolling collision energy with a collision energy spread of 5.

The obtained spectra were processed and analyzed using ProteinPilot™ software, with the Paragon search engine (version 5.0, Sciex). The following search parameters were set: search against Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334/TIGR4) reviewed database from Uniprot/SwissProt (the protein of interest Ribonuclease R was added to the existent reviewed database as tr|A0A0H2UPW1|A0A0H2UPW1_STRPN); Iodoacetamide, as Cys alkylation; Tryspsin, as digestion; TripleTOF 6600, as the Instrument; ID focus as biological modifications and Amino acid substitutions; search effort as thorough; and a FDR analysis. Only the proteins with Unused Protein Score above 1.3 and 95% confidence were considered.

Discussion

We have investigated the association of RNase R with ribosomes in S. pneumoniae and the overall impact of this ribonuclease on translation. We present evidences showing the control of translation factors by RNase R and discuss how this may lead to a reduced amount of active translating ribosomes, globally impacting protein synthesis and bacterial viability. We believe that RNase R, by maintaining the right amount of each dissociation factor, is a new auxiliary factor affecting the reassociation of ribosome subunits.

We show that pneumococcal RNase R is associated with ribosomes. This conclusion is supported by analysis of sucrose polysome gradients combined with antibodies specific for pneumococcal RNase R. S. pneumoniae RNase R clearly migrates along the gradient together with ribosomes, mostly with the 50S subunit. E. coli RNase R has been reported to be associated with the ribosome through the 30S subunit and ribosomal protein S12 [5]. The reason for this difference is not clear. Association of E. coli RNase R with ribosomes has been suggested to be related to ribosomal quality control, and the enzyme was hypothesized to specifically target deficient ribosomal 30S subunits [4]. The interplay of RNase R with the trans-translation effectors tmRNA and SmpB is well known and might also account for the RNase R interaction with ribosomes. Indeed, RNase R is recruited to stalled ribosomes by the trans-translation system, and the enzyme’s stability is known to be regulated by tmRNA-SmpB binding ([32,33], reviewed in [34]). Likewise, we have previously demonstrated that RNase R levels are modulated by SmpB in S. pneumoniae [30]. We have further shown that RNase R also regulates the amount of SmpB and the respective genes are co-transcribed [30]. However, the reason for the preferred interaction with the large ribosomal subunit in S. pneumoniae remains to be elucidated.

Another striking difference is the major impact in ribosome formation caused by the absence of RNase R in S. pneumoniae. In contrast to the previous observations in E. coli, our experimental data shows that the single deletion of rnr gene causes accentuated alterations in the ribosome sedimentation pattern. Lack of RNase R leads to a decrease in the amount of 70S active ribosome particles, concomitantly with the increase of free subunits. Although this could be due to defects in the assembly of 70S subunits, the same amount of mature 16S rRNA was detected in the rnr mutant and in the wild type strain. Thus, misprocessing of the 16S does not appear to account for the low level of 70S ribosomes in the mutant. RNase R has been described as being involved in the processing of this rRNA in E. coli [15], but our results are not indicative of the same role in S. pneumoniae. If, however, pneumococcal RNase R has some function in the maturation of this rRNA, it should be redundant and could be executed by other enzymes. After each round of translation, ribosome recycling is essential for translation to restart. Dissociation of the 70S ribosome into subunits is a critical step for protein synthesis, catalysed by the ribosome-recycling factor (RRF) together with the elongation factor G (EF-G) [9,35]. This transient split of 70S active particles into subunits is then stabilized by IF3, which functions as an anti-association factor. Fluctuations in the amount of these factors were shown to alter the level of the ribosomal subunits [9]. We show that RNase R has a direct influence on the expression level of the genes involved in ribosome recycling. The significant stabilization of frr, fusA and infC transcripts observed in the absence of RNase R indicates that this enzyme is likely involved in the degradation of the mRNAs of the three translation factors. Accumulation of these transcripts in the cell most probably leads to an increase of the corresponding proteins level, as judged by our result with EF-G. Since the fusA mRNA was the less stabilised of the three transcripts we might expect at least a similar increase of RRF and IF3 translation factors. Taking into consideration that protein synthesis is decreased in the rnr mutant strain, the increased EF-G protein levels might even be underestimated relative to the lower level of the total protein observed in this strain (as demonstrated with the Citrine reporter expression). Increased levels of RRF and EF-G would favour the transient dissociation of post-termination ribosomal complex and the higher amount of IF3 would then impede reassociation of the ribosomal subunits. Together these evidences are supportive of the altered sucrose gradient ribosomal pattern observed in the RNase R mutant strain. We believe that the fine-tuned regulation of these transcripts exerted by RNase R will maintain the precise amount of active ribosomal complexes required for proper mRNA translation. This conclusion is further supported by evidences from fluorescent measurement, which demonstrate a decreased expression of Citrine in the absence of RNase R. We show that, despite maintenance of roughly the same amount of the yfp corresponding transcript, a reduced protein and fluorescence level was detected in the mutant strain in comparison to the wild type, demonstrating the overall impact of RNase R deletion in translation. As expected, such an impact on translation has effects on the cellular viability of the mutant strain. The great importance of RNase R is reinforced by our results showing that deletion of its single cognate gene leads to a ~50% reduction in bacterial CFU/ml.

Bacterial infections have become more difficult to overcome due to the appearance of highly drug resistant strains. Moreover, the introduction of new antimicrobials in the market have been decreasing along years, which is leading us towards a serious health problem. Among antibiotic treatment methods, protein synthesis is one of the main targets. This investigation highlights the importance of RNase R by expanding its cellular function to the control of a central cellular mechanism and brings a new potential target to impair bacteria survival. By having a significant effect on translation, RNase R could be a promising candidate to use as target for antimicrobial treatment.

Funding Statement

This work was supported by the European Regional Development Fund (FEDER) through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) under Grant [LISBOA-01-0145-FEDER-007660] (Microbiologia Molecular, Estrutural e Celular); and by FCT — Fundação para a Ciência e a Tecnologia under Grant [PTDC/BIA-MIC/1399/2014]. Cátia Bárria was supported by Grant SFRH/BD/99477/2014 from FCT; and Susana Domingues under Grant 025/BPD/2015 from European Union’s Horizon 2020 research and innovation programme [Ref 635536].

Acknowledgments

We are thankful to Joana Figueiredo from Bacterial cell surfaces and pathogenesis laboratory for kindly providing us with the pBCSJC001 plasmid. We thank Teresa Baptista da Silva for technical assistance. We are grateful to Sandra C. Viegas and Margarida Saramago for critical reading of the manuscript.

Mass spectrometry data provided/obtained by the UniMS – Mass Spectrometry Unit, ITQB/IBET, Oeiras, Portugal.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed here.