Ribosome hibernation factor promotes Staphylococcal survival and differentially represses translation

Abstract

In opportunistic Gram-positive Staphylococcus aureus, a small protein called hibernation-promoting factor (HPF_Sa) is sufficient to dimerize 2.5-MDa 70S ribosomes into a translationally inactive 100S complex. Although the 100S dimer is observed in only the stationary phase in Gram-negative gammaproteobacteria, it is ubiquitous throughout all growth phases in S. aureus. The biological significance of the 100S ribosome is poorly understood. Here, we reveal an important role of HPF_Sa in preserving ribosome integrity and poising cells for translational restart, a process that has significant clinical implications for relapsed staphylococcal infections. We found that the hpf null strain is severely impaired in long-term viability concomitant with a dramatic loss of intact ribosomes. Genome-wide ribosome profiling shows that eliminating HPF_Sa drastically increased ribosome occupancy at the 5′ end of specific mRNAs under nutrient-limited conditions, suggesting that HPF_Sa may suppress translation initiation. The protective function of HPF_Sa on ribosomes resides at the N-terminal conserved basic residues and the extended C-terminal segment, which are critical for dimerization and ribosome binding, respectively. These data provide significant insight into the functional consequences of 100S ribosome loss for protein synthesis and stress adaptation.

INTRODUCTION

Protein synthesis is the most energy-consuming cellular process (1–3). Thus, suppression of translation during stress conditions is a universal adaptation mechanism used by all living organisms to mitigate wasteful energy consumption. In bacteria, the majority of non-translating ribosomes accumulate as 70S monomers and dimers of 70S (100S complex). Gammaproteobacteria possess three 70S ribosome-silencing factors that bind to the 30S subunit near the interface of the 30S and 50S subunits (4,5). In Escherchia coli, RMF_Ec and HPF_Ec (formerly YhbH) concertedly induce the formation of 100S ribosomes, whereas YfiA_Ec (also known as pY or RaiA) (6) stabilizes the inactive 70S ribosome analogously to PSRP1 in plant plastids (7). Crystal structures of the T. thermophilus ribosome in complex with RMF_Ec, HPF_Ec and YfiA_Ec have suggested that translational inactivation is due to the steric interference of these factors with the binding of tRNA, mRNA and initiation factors to the ribosomal decoding center on the 30S subunit (4). Electron microscopy studies have shown in vitro and in situ that the two 70S monomers in the 100S dimer are joined together via the 30S subunits (8–10). Of note, E. coli hibernating ribosomes are exclusively found during the stationary growth phase when nutrients are limited (11–13). Upon starvation relief, they are split into subunits and presumably are recycled for new rounds of translation by a poorly defined mechanism (11,14–16). Although an E. coli rmf_Ec mutant is reduced in long-term viability and stress tolerance during stationary phase growth (13,17,18), the deletion of hpf_Ec or yfiA_Ec does not appear to elicit strong cellular defects (19).

In stark contrast to E. coli, the clinically important Gram-positive bacterium Staphylococcus aureus does not contain RMF or YfiA homologs; instead, it carries only a long form of HPF (HPF_Sa) that is twice the size of HPF_Ec (Figure 1) (20–23). In fact, the long C-terminal extension, which shares some degrees of homology with RMF_Ec, is a hallmark of the HPF family in plant plastids and all Gram-positive bacteria (10,22,24,25). Curiously, HPF_Sa-mediated 100S ribosomes are present throughout all growth phases even when nutrients are abundant (23,26). The physiological relevance of these translationally incompetent ribosomes during exponential growth remains unknown. Nevertheless, the deletion of hpf in Listeria monocytogenes leads to attenuated virulence in a mouse model of infection (24) and sensitivity to prolonged antibiotic exposure (27). The Lactococcus lactis hpf mutant fails to resuscitate from nutrient-depleted growth (10).

Figure 1.

Similarity of S. aureus HPF_Sa to E. coli ribosome hibernating factors HPF_Ec, RMF_Ec and YfiA_Ec. Black patches indicate the conserved basic residues (K27, R30, K84 and R87, HPF_Sa numbering) on the two predicted α-helices. A question mark denotes the unknown C terminus folding.

In this study, we sought to define the role of HPF_Sa in a methicillin-resistant S. aureus (MRSA) strain by examining the biochemical features of HPF_Sa and the effect of the hpf_Sa null mutation on in vivo global translation. We found that the hpf_Sa mutant has an extremely low survival rate in long-term cultures that coincides with the rapid degradation of ribosomes. We identified HPF_Sa regions that are critical for ribosome dimerization and binding. Using in vivo ribosome profiling and in vitro cell-free translation, we provided experimental evidence that HPF_Sa attenuates translation of a subset of genes.

MATERIALS AND METHODS

Bacterial strains and reagents

Strains JE2 and NE838 are derivatives of the community-associated methicillin-resistant (CA-MRSA) S. aureus isolate USA300 (28). The hpf_Sa mutant strain (NE838) carries a bursa aurealis transposon insertion at 816475 that disrupts the synthesis of hpf_Sa. Complementing plasmid pLI50hpf was constructed by PCR to amplify the promoter and coding region of hpf_Sa with primers P651 (5′-CGG GAT CCA TAC AAC TGG ATT AAC AAT TCA TCG TGC AGG GTG-3′) and P627 (5′-TGA AGC TTT AAA CTT AAT TTA TTG TTC ACT AGT TTG AAT CAA GCC-3′) and the genomic DNA of JE2 as a template. The PCR fragment was then cloned into the BamHI and HindIII sites of pLI50 (29). The overexpression of recombinant His₆-tagged HPF on pET28a (Novagen) in E. coli BL21(DE3) has been previously described (26). To prepare ribosomal protein S7 and L22 antibodies, the rpsG region was PCR amplified using primers P524 (5′-GGC ATA TGC CTC GTAAAG GAT CAG TAC CTA-3′) and P525 (5′-CGC TCG AGT TAC CAA CGG TAG TGA GCA AAT GCT-3′), the rplV region was PCR amplified using P522 (5′- GGC ATA TGC CTC GTA AAG GAT CAG TAC CTA-3′) and P523 (5′- CGC TCG AGT TAC CAA CGG TAG TGA GCA AAT GCT-3′) and the genomic DNA of JE2 as template. The rpsG fragment was cloned into the NdeI and XhoI sites, whereas the rplV fragment was cloned into the NheI and XhoI sites of pET28a and overexpressed in E. coli BL21(DE3). The N-terminally His₆-tagged proteins were purified under native conditions as previously described (26). Polyclonal anti-S7 and anti-L22 were raised in rabbits (Josman, LLC). Polyclonal anti-HPF_Sa was prepared as previously described (26). S. aureus strains were routinely grown in tryptic soy broth (TSB, BD Difco), brain heart infusion (BHI, BD Difco), or chemically defined minimal medium (30) supplemented with chloramphenicol (10 μg/ml) when necessary. E. coli bearing pET28a derivatives was grown in LB supplemented with 50 μg/ml kanamycin. All strains were cultured at 37°C unless otherwise noted.

Stress assays

S. aureus strains were grown in tryptic soy broth (TSB) or minimal medium (MM) at 37°C until the mid-log or stationary phase, followed by exposure to various concentrations of stressors. Cell viability was measured at 1, 2 and 4 hr after stress exposure by enumerating colony-forming units (CFU). Cells treated with antimicrobials were washed twice with 1 ml of sterile 1x phosphate buffered saline (PBS) to eliminate carryover; the cells subsequently underwent serial dilution and were spotted on TSB agar plates. The final concentrations of stressors were as follows: rifampicin (5–50 μg/ml), mitomycin (0.5–2 μg/ml), kanamycin (5–50 μg/ml), streptomycin (5–50 μg/ml), tetracycline (50–200 μg/ml), 5% ethanol and hydrogen peroxide (5–20 mM). Cell survival was also evaluated under conditions of low pH (pH 4.3), osmosis (2.5 M NaCl), heat shock (52°C) and cold shock (10°C).

Purification of recombinant protein HPF_Sa

Recombinant HPF_Sa variants were purified with nickel affinity chromatography (MCLAB) under native conditions according to the manufacturer's manual with the following modifications: sodium phosphate buffer was replaced by 25 mM HEPES (pH 7.5) and NaCl was substituted with 500 mM KCl. Eluted proteins were stabilized in 25% (v/v) glycerol. His₆-tag was removed by the thrombin-CleanCleave Kit (Sigma) in 1× cleavage buffer (20 mM HEPES (pH 7.5)/14 mM MgOAc) and incubated at room temperature overnight. The cleaved products were collected by centrifugation at 500 ×g for 2 min.

Preparation of salt-washed ribosomes

The hpf_Sa mutant was grown in 1 l of BHI at 37°C until OD₆₀₀ = 0.6. The cells were poured over an equal volume of sterile ice cubes and harvested by centrifugation at 11 540 ×g (Beckman JLA 8.1 rotor) for 20 min at 4°C. The cell pellets were washed once with buffer A (20 mM HEPES (pH 7.5)/14 mM MgOAc/100 mM KOAc/6 mM 2-marcaptoethanol/0.5 M PMSF) and then resuspended in buffer B (20 mM HEPES (pH 7.5)/14 mM MgOAc/100 mM KOAc/1 mM DTT/0.5 mM PMSF) to a concentration of 0.5 g wet cells/ml. The cell pellet was flash-frozen in liquid nitrogen and pulverized on a cryomiller (Retch MM400) using eight 3 min cycles at 15 Hz in a 10-ml grinding jar with a 15-mm grinding ball. Grinding jars were re-chilled between each cycle in liquid nitrogen. Milled cells containing liquid nitrogen were placed in a −80°C freezer to allow for evaporation. The resulting milled cells were thawed in a 30°C water bath for 5–8 min and then immediately placed in an ice bath for 10 min. The lysate was centrifuged at 20 000 ×g for 10 min at 4°C. Clarified lysate was recovered and spun at 20,817 ×g at 4°C for 5 min to remove residual debris. To remove factors associated with ribosomes, equal volumes of lysate and 2× high-salt buffer (28 mM MgOAc/1 M KOAc/40 mM HEPES (pH 7.5)/1 mM EDTA) were mixed and incubated at 4°C on a rotator for 1 h. The mixture was layered on 1 M sucrose composed of 1× high-salt buffer and spun in a Beckman TLA-100 rotor at 4°C and 435 000 ×g for 30 min. The ribosome pellets were resuspended in × volume of buffer B. The ribosome concentration was determined with a NanoDrop spectrophotometer (Thermo Scientific) and was calculated using 1 Abs₂₆₀ = 23 pmol/ml 70S ribosome (31).

Crude lysate preparation and sucrose density gradient fractionation

TSB cultures at 37°C were harvested at 2, 28 and 96 h. Crude lysate was prepared by cryomilling as described above. Five Abs₂₆₀ units of lysate were layered on 5–25% sucrose (w/v) gradient prepared with buffer C (20 mM HEPES (pH 7.5)/10 mM MgCl₂/100 mM NH₄Cl) and equilibrated with a BioComp Gradient Master. The gradients were spun on a Beckman Coulter L7–66 ultracentrifuge at 210 000 ×g at 4°C in a SW41 rotor for 2.5 hr. Fractionation was performed using a Brandel fractionation system equipped with a UA-6 detector. A total of 46 fractions were collected, each with a size of 200 μl. Sucrose fractions were precipitated with 10% trichloroacetic acid, and the pellets were washed once with acetone, neutralized with 50 mM Tris base containing Laemmli sample buffer and resolved using 4–20% TGX SDS-PAGE (Bio-Rad). Proteins were transferred onto a nitrocellulose membrane using a Trans-blot Turbo system (Bio-Rad). A dilution of 1/8000 of anti-HPF_Sa and 1/1500 of anti-S7 and anti-L22 were used for Western blots.

In vitro 100S ribosome formation

Different molar ratios of salt-washed hpf mutant ribosomes to purified HPF_Sa were mixed in a 100 μL reaction containing 1× binding buffer (10×: 50 mM HEPES (pH 7.5)/500 mM KOAc/100 mM NH₄Cl/100 mM MgOAc). A final concentration of 0.3 μM ribosomes was routinely used in a typical experiment because no difference was observed with ribosome concentrations ranging from 0.1–0.4 μM. The mixture was incubated at 37°C for 30 min before layering on 5%-25% sucrose gradients made in buffer C. Ultracentrifugation, fractionation and immunoblotting were as described above.

Ribosome profiling

Exponentially grown S. aureus were harvested at OD₆₀₀ = 0.4–0.5 after 2 min pre-treatment with 100 μg/ml of chloramphenicol. Ribosome footprints were prepared exactly as described (26), except that Ribo-Zero rRNA removal kit (Epicentre) was used in place of the MICROBExpress (Ambion). Raw FastQ sequencing data were processed using a locally installed Galaxy platform. The rRNA-less reads were aligned to the USA300_FPR3757 reference genome (GenBank CP000255.1) using Bowtie v.0.12.0. The alignment .map files were used as inputs for the modified Python scripts(32). The normalized ribosome densities, measured as reads per million reads (RPM), were visualized in MochiView (33). The gene expression levels (reads per kilobase per million mapped reads, RPKM) were calculated using Python scripts (32). Translation efficiency (TE) was calculated as the relative number of ribosome footprints to mRNA-seq reads in log₂ ratios (34). Sequencing data were deposited in the NCBI GEO database with accession number GSE74197.

Hybrid in vitro translation

The hybrid in vitro translation system consists of the salt-washed S. aureus Δhpf_Sa ribosome and purified translation factors from PURExpressΔribosome Kit (New England BioLabs). Ribosomes were pre-incubated with or without HPF_Sa in 1x binding buffer at 37°C for 30 min. HPF_Sa targets were translated by programming T7 promoter-containing DNA templates (final 10 ng/μl), 10 μCi Tran³⁵S-label (MP Biomedicals) and 0.8 μM of ribosome/HPF_Sa or ribosome alone premix. After 1 hr incubation at 37°C, protein samples were precipitated by 4× volumes of acetone, resolved on 4–20% TGX SDS-PAGE (BioRad) and autoradiographed.

RESULTS

HPF_Sa is required for long-term viability of S. aureus

The phenotypic manifestations of hpf_Sa remain unexplored, and studies from other bacterial counterparts point to hibernating ribosomes playing a role in the stress response. We subjected wild-type and hpf_Sa mutant strains to a variety of agents that are commonly used to assess virulence. All of the tested stressors, including hydrogen peroxide, high salt, low pH, transcription inhibition (rifampicin), DNA damage induction (mitomycin) and ribosome-targeting antibiotics (kanamycin and tetracycline), sensitized the wild-type and mutant cells equally, regardless of the dosage, bacterial growth phase or medium type. However, we found that the viability of the hpf_Sa mutant declined rapidly in long-term minimal medium (MM) culture and eventually lost >95% of cell viability at day eight, yielding approximately 400-fold fewer CFU than the wild-type strain (Figure 2A). A similar downtrend was observed in rich tryptic soy broth (TSB) medium, although cell death was less pronounced (approximately 10-fold) than in MM (Figure 2B). In both cases, complementation of the hpf_Sa mutant with a plasmid-borne copy of hpf_Sa under control of its native promoter fully rescued the survival defect, confirming that hpf_Sa is essential for S. aureus long-term viability.

Figure 2.

HPF_Sa mediates long-term cell survival. (A) Cell viability in minimal medium (MM) over an 8-day period. (B) Cell viability in tryptic soy broth (TSB) over a 6-day period. Viability was assessed by enumerating colony-forming units (CFU) from wild-type, hpf null and complemented strain carrying pLI50hpf. Error bars indicate the standard deviation of three independent experiments. An arrow denotes the detection limit.

Loss of hpf_Sa sharply reduces the intact ribosome pool during the stationary phase

To investigate the link between 100S ribosome formation and the reduced survival rate in the hpf_Sa mutant, we monitored changes in ribosome species during stationary culturing by sucrose density gradient fractionation. Crude ribosomes were prepared from cells grown at different stages and extracted by cryomilling, a method known to faithfully preserve ribosome complexes and translational states (26,35,36). Consistent with previous studies (23,26), we observed an accumulation of 100S ribosomes in rich medium during early logarithmic growth (OD₆₀₀∼ 0.3–0.4) (Figure 3 (top left), S1A). Western blots showed that the majority of HPF_Sa was associated with the 100S complex and 70S ribosome (Figure 3 (top left)). In the complemented strain in which plasmid-borne HPF_Sa was expressed approximately 10-fold more than wild type, the 100S ribosome peak was substantially elevated and a fraction of HPF_Sa also bound to the 30S subunits (Figure 3 (top right)). Curiously, the increased production of 100S ribosomes did not significantly slow bacterial growth over 24 h in the TSB culture (Supplementary Figure S1A). During the stationary phase, a substantial amount of the ribosome pool diminished in the wild-type and complemented strains (Figure 3 (middle and bottom)), presumably due to massive ribosome degradation to provide cells with energy and nutrients. The disappearance of the 100S complex and the basal 70S ribosome level in these strains suggested that 100S particles may have been redistributed to form translationally competent ribosomes. The exact sequence of 100S to 70S inter-conversion is unclear. In contrast, most of the ribosomes in the hpf_Sa mutant were degraded, which coincided with the onset of cell death in prolonged culture (Figure 2B). Free 30S and 50S subunits are more prone to ribonuclease attack than the 70S ribosome because of their exposed rRNA interface (37). The accumulation of 70S dimers in the wild-type cells may thus serve as a nuclease-resistant ribosome repository.

Figure 3.

Accelerated ribosome degradation in the S. aureus hpf null strain. Sucrose gradient profiles of S. aureus ribosomes harvested from TSB cultures at the logarithmic phase (top panels), the stationary phase (middle panels) and the late stationary phase (bottom panels). The y-axis corresponds to the absorbance at 254 nm of the ribosome species separated on a 5–25% sucrose gradient. Abs₂₅₄ values on the right serve as a ruler to compare relative ribosome peak height. Each panel corresponds to five Abs₂₆₀ units of total RNA input. The distribution of HPF_Sa was detected via immunoblotting. The 30S ribosomal subunit protein S7 is a marker control for fractionation. ‘(+)’ denotes loading control with the purified recombinant HPF_Sa.

HPF_Sa alone is sufficient to induce the formation of 100S ribosomes in vitro

To confirm that HPF_Sa is the sole contributor to the formation of 100S ribosomes in vivo, we recapitulated 70S ribosome dimerization in vitro using purified HPF_Sa and S. aureus ribosomes isolated from the hpf_Sa null strain. The ribosomes were subjected to a high-salt wash to remove any associated factors. Hexahistidine-tagged HPF_Sa was overexpressed in E. coli and purified by nickel affinity chromatography. The affinity tag was removed by thrombin cleavage because 6His-HPF_Sa was prone to aggregation, possibly due to misfolding, and was nonfunctional in 100S complex formation (Figure 4A). Purified HPF_Sa was incubated with high-salt washed ribosomes at different molar ratios, and the ribosome profiles were obtained by sucrose density gradient fractionation. At a 1:1 ribosome to HPF_Sa ratio, HPF_Sa induced the dimerization of 70S ribosomes to a level comparable to that observed in vivo (Figure 3 (top left), 4B (middle)). Increasing the amount of HPF_Sa did not enhance dimerization, although immunoblotting showed that the protein effectively bound to the 70S ribosome. In fact, we found that a modest increase of the ribosome to HPF_Sa ratio to 1:2 completely abrogated dimerization but did not affect ribosome association (Figure 4B, bottom). The observation that a higher concentration of HPF_Sa is counterproductive could be attributable to HPF_Sa self-oligomerization or/and non-specific binding of HPF_Sa to the ribosome when HPF_Sa is in large molar excess. Nevertheless, our data demonstrate that optimal in vitro formation of the 100S ribosome occurs when HPF_Sa and the ribosome are present at a 1:1 molar ratio, which is in agreement with previous in vivo estimation of the E. coli 100S ribosome (23).

Figure 4.

HPF_Sa induces 100S dimer formation in vitro with a 1:1 ribosome to HPF_Sa ratio. (A) SDS-PAGE gel showing the purity of recombinant wild-type HPF_Sa and its mutants. ‘-’ indicates His₆-tag-free proteins that have been subjected to thrombin cleavage. (B) Sucrose density gradient (5-25%) profiles of salt-washed ribosome isolated from S. aureus hpf null strain (top) and mixture of ribosomes and HPF_Sa in a ratio of 1:1 (middle) and 1:2 (bottom). Abs₂₅₄ values on the right serve as a ruler to compare relative ribosome peak height. Association of HPF_Sa with ribosomes was confirmed by immunoblotting using anti-HPF_Sa (1:8000 dilution) and the internal control anti-S7 (1:1500 dilution). ‘(+)’ denotes a loading control with the purified recombinant HPF_Sa.

Ribosome dimerizing activity of HPF_Sa is highly sensitive to perturbation mutagenesis

Our in vitro dimerization results showed that HPF_Sa binding to the ribosome is not sufficient to promote 100S formation. The result prompted us to investigate the domains that are critical for dimerization. HPF_Ec adopts a βαβββα fold, with two basic patches each on the two α-helices (Supplementary Figure S2) (4). These positively charged residues are conserved in all HPF and YfiA homologs (Figure 1) and have been implicated in direct interactions with the 16S rRNA bases at the decoding channel (4,5). In vitro dimerization was repeated using purified HPF_Sa mutant proteins harboring the desired mutations in the basic patches (Figure 4A). Although the double mutation K27A/R30A on the first α-helix did not completely impair dimerization, the double mutation K84A/R87A on the second α-helix did but did not abolish ribosome binding, as a large fraction of mutant HPF_Sa was still associated with the 70S ribosomes (Figure 5A and B). To abrogate HPF_Sa binding to the ribosome, concurrent mutations (K27A/R30A/K84A/R87A) on both helices were required, as judged by the sedimentation of mutant HPF_Sa in the light sucrose fraction. (Figure 5B, bottom). Notably, several mutations outside the α-helices did not perturb 70S dimerization (Supplementary Figure S3). HPF_Sa contains a long C-terminal tail that is absent in HPF_Ec (Figure 1). Deletion of the last 42-amino acid segment reduced ribosome binding, whereas a larger truncation (up to 90 amino acids) exacerbated the binding defect further (Supplementary Figure S4). The data suggest that the basic residues on the second α-helix (K84/R87) are critical for dimerization, whereas both α-helices and the C-terminal tail are important for ribosome binding and/-or the stabilization of binding.

Figure 5.

Mutations that affect HPF_Sa-mediated 100S dimer formation and binding to the ribosome. (A) Sucrose density gradient (5-25%) profiles of salt-washed ribosomes reacted with an equimolar ratio of purified HPF_Sa and its mutant variants. (B) Western blots showing the association of HPF_Sa in each ribosomal fraction. The 30S ribosomal subunit protein S7 serves as a control. A K27A/R30A double mutant has reduced 100S dimer formation. A K84A/R87A double mutant impairs dimerization but not binding. The quadruple mutant abolishes binding.

Loss of hpf_Sa causes unusual ribosome occupancy around start codons under nutrient-limited conditions

The paradoxical observation that 100S ribosomes are prevalent in S. aureus growing logarithmically in rich culture medium with a growth rate that is unaffected by HPF_Sa loss (Supplementary Figure S1A, 3 (top)) is inconsistent with the notion that HPF acts as a global ribosome inhibitor (4,20,22). It is conceivable that ribosomes are in excess in rich medium, and the subpopulation of 100S ribosomes is unable to titrate the bulk of ribosomes from normal translation, resulting in a lack of growth defect. It may be that the 100S ribosome is simply a quiescent product to conserve ribosome and has no direct role in regulating translation. Alternatively, the formation of 100S ribosomes may downregulate the translation of only a fraction of mRNAs that are not essential for exponential growth when nutrients are plentiful. This prediction is supported by the observation that when nutrients were limited, both wild-type and complemented strains were delayed in entering the stationary phase compared with the hpf_Sa mutant (Supplementary Figure S1B). It seems plausible that HPF_sa directly participates in translational repression and leads to a slower growth rate in minimal medium. To test this model, we performed a deep sequencing-based ribosome profiling analysis to compare the translatomes of exponentially growing wild-type and hpf_Sa mutant strains in both minimal and rich media. Ribosome profiling captures a global snapshot of ribosome positioning and density on template mRNAs with single-nucleotide resolution (34,36). A high number of ribosome-protected footprints (RPFs) mapping to the transcriptome (mRNA-seq) at a unique position or being distributed across the transcript is indicative of ribosome stalling or active translation, respectively.

Our initial attempt to construct RPF libraries from stationary phase cells was unsuccessful. We opted for mid-log cells, as the 100S ribosomes are already prevalent at this stage. Exponentially grown S. aureus were collected at OD₆₀₀ 0.4–0.5. Unlike other cell lysate preparations described in this study, the cells subject to ribosome profiling were pretreated with 100 μg/ml chloramphenicol for 2 min to stabilize the polysomes (translating ribosomes) before harvest (Supplementary Figure S5). Consistent with previous observations (26), the addition of chloramphenicol appears to trap HPF_Sa in all ribosomal fractions (Supplementary Figure S5). Cryogenic cell disruption, construction of cDNA libraries and data analyses have been previously described in detail (26,32,36). A total of 16 RPFs and total mRNA-seq libraries were generated from the wild-type and hpf_Sa mutant strains, representing two independent biological replicates from rich and minimal media (Supplementary Table S1).

To obtain an overview of global differences in ribosome density at the beginning and end of open reading frames (ORFs), metagene analysis was performed to analyze the distribution of ribosomes along an average transcript of the most abundantly expressed genes (≥50 reads/transcript). Elevated ribosome density around the start and stop codons corresponds to slower kinetics of initiation and termination than those of the elongation rate (36). Based on the prediction that HPF_Sa inhibits translation, we expected that loss of HPF_Sa would lead either to clustering of ribosomes at the very N-terminus of ORFs, reflecting derepression of translation initiation, or to disappearance of occupancy at unique positions within ORFs, suggesting a relief of elongation arrest, or to an increase in ribosome occupancy along the transcripts, indicating elevated translation efficiency. As predicted, the loss of HPF_Sa disproportionately increased ribosome occupancy around start codons transcriptome-wide in cells grown in minimal medium relative to the wild-type (Figure 6A, Supplementary Figure S6). Unexpectedly, despite a feature that suggests higher translation initiation, downstream translation in the hpf_Sa mutant did not increase to a level that positively correlates with the elevated ribosome density at the initiation site (Figure 6A, top). The unusual accumulation of ribosomes at the 5′-end could be an artifact derived from the chloramphenicol treatment as a recent study showed that the addition of translation inhibitors could distort ribosome coverage profiles in eukaryotes (38). This is highly unlikely in our case because the same elevated ribosome density was not observed in rich medium samples (Figure 6A, bottom) and MM-grown WT samples (Figure 6A, top). In rich TSB medium, a modest increase of ribosome density along the entire transcript was observed in the hpf_Sa mutant, suggesting moderate inhibition of translation by HPF_Sa (Supplementary Figure S6).

Figure 6.

Effects of hpf_Sa null mutation on global translatome in minimal and rich culture conditions. (A) Ribosome density as a function of position. Metagene analysis of read densities in minimal medium (MM, top) and tryptic soy broth (TSB, bottom) samples. The normalized reads per million mapped reads (RPM) correspond to the average ribosome density across the most abundantly translated ORFs (>50 reads), which were aligned relative to the start and stop codons. Ribosome density was drastically elevated around start codons in the hpf_Sa mutant grown in MM, whereas such an increase was not observed in TSB culture. (B) Comparison between translation efficiency (TE) in the wild-type and hpf_Sa mutant in MM (left) and TSB (right). Each dot corresponds to a single gene. Genes with TE improvement upon hpf_Sa loss are shown in red. TE was moderately increased in the hpf_Sa mutant and more pronounced in MM cultures. TE was calculated as the log₂ ratios of the RPFs to the mRNA reads that were measured in RPKM.

hpf_Sa null mutation increases translational efficiency of a subset of mRNAs

To evaluate how efficiently an individual transcript was translated, we calculated the TE in the wild-type and mutant strains by dividing ribosome footprints by mRNA-seq density (Figure 6B). Plotting the TEs from the hpf_Sa mutant against WT provides information about the changes in ribosome occupancy between strains. An improvement of hpf_Sa TE will be indicated by a log₂ TE value of hpf_Sa (x-axis) that is larger than the log₂ TE value of the wild-type (y-axis), whereas high R² correlations suggest that TE is less affected by the loss of hpf_Sa. In general, TE in most genes remained unchanged between the wild-type and mutant. TE improvement in the hpf_Sa mutant was more pronounced in MM than in TSB culture (R² = 0. 67 (MM) and 0.81 (TSB)). Consistent with the metagene analysis, we found that genes displaying high ribosome density at the 5′-end were not always correlated with the overall improvement of TE. As a result, fewer than 10% of the actively transcribed mRNAs exhibited a significant TE increase (2-fold) in the absence of HPF_Sa (Supplementary Tables S2 and S3). Thus, the loss of hpf_Sa appears to have a modest impact on overall protein output during exponential growth.

HPF_Sa-mediated translational inhibition in vitro

To determine the effects of HPF_Sa on the protein yield from transcripts that were identified from ribosome profiling, we used a cell-free hybrid translation system that combines purified E. coli translation factors and Δhpf_Sa ribosomes. Because the sequence context at the 5′-end might influence the translation efficiency of individual genes, we preserved the native Shine-Dalgarno (SD) sequence and start codon in T7 promoter-driven DNA templates. Representative genes whose TEs were elevated by >2-fold were randomly selected based on known function (Figure 7A and B). Translation reactions were programmed with ³⁵S-methionine and various molar ratios of ribosomes to HPF_Sa. The protein production varied because each template carried a different translation initiation signal sequence. The intensities of the anticipated protein products were quantitated by normalization to an ∼17 kDa non-specific band, whose levels remained constant in a minus template control and throughout HPF_Sa titration (Figure 7C). The non-specific band did not appear to be aminoacyl-tRNAs as the labile ester bond would be hydrolyzed on the alkaline SDS-PAGE. In brief, the inhibition of translation by HPF_Sa could be recapitulated in vitro, but the effects were less pronounced than the in vivo ribosome profiling effects, presumably due to subtle incompatibility of the purified E. coli components with the S. aureus ribosome. The inhibition was specific to HPF_Sa targets because the same inhibition was not seen with the non-target prmA or the heterologous E. coli dhfr.

Figure 7.

In vitro translation with S. aureus ribosomes recapitulates HPF_Sa-mediated translational repression. (A) Ribosome density profiles showing the strong increase in ribosome occupancy around start codons of selected genes in the hpf_Sa mutant grown in minimal medium (MM). (B) Ribosome density plot of genes derived from tryptic soy broth (TSB) samples that exhibit an overall increase in ribosome occupancy along the gene body. (C) In vitro translation validating the repression of translation in the genes shown in panels A and B. Translation products were resolved on an alkaline Tris-glycine-based 4–20% TGX SDS-PAGE. An asterisk depicts the non-specific band used as an internal reference for intensity quantitation. A cursor indicates a second non-specific band that was fortuitously intensified in a specific translation reaction. The predicted gene product of SAUSA300_0432 is a 50 kDa sodium transporter; it runs aberrantly faster on SDS-PAGE, which is not uncommon for membrane proteins.

DISCUSSION

The bacterial 100S ribosome was first observed more than five decades ago (40,41). Its function has remained obscured despite its pervasiveness at different bacterial growth phases. Likewise, little is known about the biochemical properties of the factors (e.g. HPF, RMF) that promote 100S formation. Here, we integrate biochemistry and ribosome profiling to characterize the biological and biochemical features of HPF_Sa in the opportunistic pathogen S. aureus. HPF_Sa is a peculiar homolog in that it is unusually long, is constitutively expressed, and is functionally equivalent to RMF_Ec and HPF_Ec combined. Whereas 100S formation in E. coli is stationary phase-specific, it occurs in S. aureus as early as the post-lag phase. We show that the loss of hpf_Sa causes massive ribosome breakdown upon entering the stationary phase that correlates with the onset of cell death. It seems likely that 100S ribosomes provide a ‘readiness plan’ in advance of future growth constraints when they can be redistributed for translation reinitiation (Figure 8). Beyond their role as a ‘reservoir’, the formation of 100S ribosomes in logarithmically growing cells reduces the translation efficiency of a fraction of genes. Furthermore, structure-guided mutagenesis suggests important roles of the C-terminal segment of HPF_Sa in ribosome binding and of the conserved basic residues in dimerization.

Figure 8.

A model depicting the role of HPF_Sa. (A) In minimal medium, HPF_Sa-mediated formation of 100S dimers causes a reduction of translation capacity, and the cells grow slower than the hpf_Sa mutant. Upon entering the stationary phase, 100S dimers in the wild-type are converted to recyclable ribosome subunits to support a viable state, whereas all post-termination ribosomes in the hpf_Sa mutant are degraded, leading to cell death. (B) In rich medium, the sequestration of excess ribosomes in the form of 100S complexes does not affect cell growth, but the translation efficiency of a small set of genes is reduced. hpf_Sa mutation increases the active ribosome pool and increases the ribosome occupancy of specific genes. Hyperactive translation in the hpf_Sa mutant leads to rapid degradation of ribosomes during the stationary phase. A black circle denotes HPF_Sa and a star denotes an unknown factor that splits the 100S dimer.

Although the 100S ribosomes are prevalent in bacteria, translationally silent ribosomes in eukaryotes exist as 80S monomers held by the clamping protein Stm1 (43). The mammalian equivalent of 100S, a dimer of 80S monomers (110 complex), occurs infrequently except under nutrient starvation conditions (42).

The rapid degradation of ribosomes in hpf null mutants has not been reported. The degradation of S. aureus ribosomes likely involves RNase III and RNase J1/J2 (44), as implicated by the accumulation of 100S dimers in Streptomyces rnc and rnj mutants (45). Curiously, decay of the GFP-tagged E. coli ribosome is not aggravated in rmf_Ec and hpf_Ec mutants. Under our experimental conditions, the S. aureus USA300 strain enters the stationary phase much earlier than do other Gram-positive bacteria (10,22,24). The differences are likely due to the choice of medium, culture conditions and strains. The strain-specific abundance of 100S ribosomes has been observed in E. coli (47). In our hands, S. aureus RN4200 and N315 produce 30% more 100S ribosomes than the strain (USA300) we used in this study under the same conditions.

We propose a model for the role of HPF_Sa (Figure 8). In minimal medium, the translation of a fraction of genes is suppressed because a subpopulation of ribosomes is sequestered away from active translation, leading to a slower growth rate. The reason underlying the specific targeting of mRNA has yet to be determined. It is possible that some mRNAs are more sensitive to altered cellular concentrations of active ribosomes; e.g. the translation of genes with highly structured mRNA or weaker SD sequence may require higher concentrations of ribosome. A loss of hpf_Sa liberates the ribosome for translation initiation but only partially increases the overall translational output, presumably due to low cellular concentrations of charged-tRNAs and other translation factors. Both wild-type and hpf_Sa null strains undergo ribosome degradation, but the former retains a fraction of the functional ribosomes that are derived from the 100S pool (Figure 8A). The process by which 100S dimers are disassembled and converted to active 70S ribosome is unclear, although the participation of the initiation factors IF1 and IF3 and ribosome recycling factor RRF have been implicated in E. coli (4,11,14). In rich medium, excess unused ribosomes are preserved as silent 100S dimers. The formation of the 100S complex does not significantly affect wild-type cell growth but is important for viability during the stationary phase, when the ribosome pool is low. The absence of HPF_Sa increases ribosome occupancy but only modestly upregulates translation efficiency (Figure 8B). The reason is unclear, although it is possible that elevated ribosome occupancy causes ribosomal traffic jams, which in turn may perturb polysome organization (48) and limit translation.

Although an increase in ribosome density at the 5′ regions of coding sequences suggests elevated translation initiation, it is possible that secondary effects in the absence of hpf_Sa, such as proteotoxic stress, may lead to ribosome stalling at 5′ proximal regions (49). Why is only a subset of genes derepressed in the hpf_Sa null strain, particularly at the initiation phase? These genes do not belong to a specific functional category, although several important virulence factors are among the list (Supplementary Tables S2 and S3). No correlation with gene length or the mRNA's abundance was observed. Sequence context, mRNA secondary structure and codon usage bias may shape the expression levels of individual genes (50). Moreover, the first 50 codons have been shown to dictate translation efficiency (51). Our attempts to identify unique sequence signatures upstream and downstream of the start codons were unsuccessful, although we noted that the spacer between the SD sequence and start codon in HPF_Sa targets is highly enriched in uracil in contrast to the non-targets’ enrichment in adenine. Furthermore, mRNA secondary structures that occlude the SD sequence and initiation site appear to be overrepresented in the HPF_Sa-affected genes. The significance of these features remains to be determined.

The formation and stability of 100S ribosomes are sensitive to ionic strength and magnesium concentration (21). The binding of RMF_Ec and HPF_Ec induces a structural rearrangement of the 30S subunit head domain that is necessary for dimerization (4). Whereas dimerization is impaired by minor perturbation of the conserved basic patches of HPF_Sa, ribosome binding is abrogated when a large deletion or concurrent mutations on the two predicted α-helices are introduced. These data imply that HPF_Sa interacts with the ribosome at multiple sites and that a single-site defect does not abolish binding. Likewise, dimerization does not occur prior to the stabilization of ribosome binding. Previous work (10) has shown that the L. lactis HPF_Ll C-terminal tail (residues 126–185) is functionally equivalent to that of RMF_Ec. Truncation of this region does not impair ribosome binding, which is completely opposite to HPF_Sa. These differences suggest some idiosyncrasy of long-form HPF homologs in interacting with their cognate ribosomes.

Our data imply that HPF/100S ribosomes serve two purposes in S. aureus. The first is to suppress the translation of specific mRNA through titrating active ribosomes concomitantly with reducing energy consumption. The second is to avoid rapid ribosome turnover by dimerizing 70S monomers so that the dimers can be dissociated and reassembled into active ribosomes during stationary phase. Bacterial persistence has been linked to the suppression of macromolecule biosynthesis and translation (52–54) and S. aureus is particularly adept at establishing long-term survival inn the host, which leads to recalcitrant and relapsing infections (55). Given that the hpf_Sa null strain is non-viable in long-term minimal medium culture, strategies that disrupt the 100S ribosome may vastly synergize the efficacy of conventional antibiotics and may serve as a new anti-staphylococci drug target to treat recalcitrant infections.

ACCESSION NUMBER

NCBI GEO GSE74197.

FUNDING

National Institutes of Health [R00GM094212 to M.N.Y.]; Edward Mallinckrodt Jr. Foundation; Saint Louis University faculty start-up fund. M.N.Y. is a Pew Scholar in the Biomedical Sciences, supported by the Pew Charitable Trusts. Funding for open access charge: The Pew Charitable Trusts [00002920].

Conflict of interest statement. None declared.