Mee-Ngan Frances Yap

Ribosomes are essential macromolecules that convert mRNA to polypeptides. Ribosomes can be regulated by multiple mechanisms, including limiting the biogenesis of ribosomal subunits, sequestering 70S ribosomes, dimerizing 70S complexes into inactive 100S ribosomes, and “silencing” the 50S subunit to prevent the assembly of active 70S ribosomes. In this review, we focus on the protein that carries out ribosomal silencing, called ribosomal silencing factor S (RsfS), that has been shown in multiple species to bind to the uL14 ribosomal protein in the 50S subunit and prevent 50S-to-30S subunit joining. RsfS is a highly conserved small protein found across most bacterial species and in eukaryotes. The direct target of RsfS, uL14 ( rplN ), is one of the most conserved ribosomal proteins. RsfS has been primarily studied for binding interactions with uL14 and the downstream effects of preventing 70S ribosome assembly. rsfS null mutant strains exhibit defects in several bacterial species in vitro (stationary phase, biofilm, growth, nutrient limitation, and oxidative stress) and in vivo (macrophage and murine models). However, other topics remain underexplored, highlighting areas for future study, such as the regulation of RsfS and uL14 expression, additional roles in antibiotic tolerance, whether RsfS has physiological roles beyond ribosomal silencing, and if other factors dictate ribosomal interactions. Overall, while RsfS is clearly important in many bacteria, there remain many areas for future studies.

SUMMARY: Protein synthesis by ribosomes proceeds through the sequential stages of initiation, elongation, termination, and ribosome recycling. In most bacteria, coupled transcription-translation enhances the efficiency of transcription but limits opportunities to repair or eliminate faulty mRNAs, thereby increasing the likelihood that ribosomes encounter incomplete or damaged transcripts. Furthermore, mRNA integrity can be compromised by transcriptional errors, abortive transcription, nucleolytic cleavage, or chemical damage. Translation of such defective transcripts, as well as other hard-to-translate mRNAs, can impede elongation or termination, resulting in stalled ribosome complexes that sequester ribosomes and tRNAs, thereby reducing their availability for subsequent rounds of translation. To maintain translation capacity, bacteria employ dedicated ribosome rescue and quality control pathways that resolve stalled complexes and promote the turnover of aberrant mRNAs and nascent polypeptides. Here, we summarize the molecular mechanisms underlying bacterial ribosome stalling, describe the pathways that rescue stalled ribosomes, and discuss current technologies for detecting ribosome stalling. We also highlight the physiological roles of programmed ribosome stalling and explore the potential of ribosome rescue pathways as druggable sites.

Bacterial ribonucleases regulate gene expression in response to environmental stress and host interactions. In Staphylococcus aureus , the hibernation-promoting factor (Hpf) induces the formation of RNase R-resistant 100S ribosomes. We previously showed that the 3’-5’ exoribonuclease YhaM cleaves the hpf transcript, reducing Hpf synthesis and leading to ribosome degradation. No structure of any YhaM homolog bound to RNA is available, and biological investigations of YhaM remain limited. Here, we find that deletion of yhaM attenuates S. aureus virulence in a Galleria mellonella infection model. We further determined electron cryomicroscopy structures of YhaM–RNA complexes. YhaM adopts a hexameric complex arranged in a ring, with its N-terminal oligonucleotide/oligosaccharide-binding (OB) domains positioned on both sides of the ring while the catalytic histidine/aspartate-rich (HD) domain active sites are buried within the interior. The OB-1’’ domains recognize the hpf hairpin by the formation of complementary minor groove interactions. RNA binding by two YhaM OB domains is mediated through engagement of both the backbones and nucleobases of the RNA substrate, where stacking of aromatic residues and nucleobases likely contributes to substrate recognition. Structures of YhaM bound to a single-stranded RNA reveal how the 3’ ends of two RNAs are positioned within the HD domain poised for catalysis. Although six YhaM active sites are present, only two engage in RNA cleavage and further point to the importance of the remaining YhaM monomers as structural scaffolds for guiding RNA to the active site. In summary, these findings provide insights into the unique assembly of an understudied bacterial RNase.

Macrolide, lincosamide, and streptogramin (MLS) antibiotics are structurally distinct molecules that inhibit protein synthesis by binding overlapping sites within the 23S rRNA of bacterial ribosomes. The clinical utility of MLS antibiotics has diminished due to the dissemination of e rythromycin r esistance rRNA m ethyltransferase (Erm) genes. Staphylococcus aureus ErmB methylates the universally conserved A2058 nucleotide of the 23S rRNA (m ⁶ A2058), resulting in cross-resistance to all MLS antibiotics by reducing drug-binding affinity. The operonic upstream ribosome stalling peptide ErmBL was thought to be the sole regulatory element controlling ErmB synthesis and the extent of MLS resistance. Unexpectedly, our laboratory evolution experiments revealed that numerous loss-of-function mutations outside the bicistronic ermBL-ermB operon amplify ErmB-mediated MLS resistance. Among these are mutations in genes critical for purine de novo biosynthesis and the salvage pathway. Specifically, inactivation of the gene encoding the purine biosynthesis transcriptional repressor (PurR) converts an otherwise moderately resistant ermBL-ermB (ermB ⁺ ) strain into one exhibiting pronounced hyper-resistance to MLS antibiotics. This cooperative resistance phenotype is not attributable to increased ermB expression or elevated m ⁶ A2058 modification. Instead, purR inactivation leads to derepression of purine and pyrimidine biosynthesis, accompanied by increased expression of ribosome components. We find that the elevated ribosome abundance and translational capacity of the ermB ⁺ ∆ purR are directly proportional to its accelerated growth rate, thereby priming S. aureus for survival under high MLS concentrations. These findings support a model in which expanded nucleotide metabolites and a surplus of antibiotic-free ribosomes drive global translation to buffer the inhibitory effects of MLS antibiotics. IMPORTANCE: The Erm rRNA methyltransferase superfamily represents the most prevalent determinant of MLS resistance in nosocomial Gram-negative and Gram-positive bacteria. Previous studies have established that erm expression is primarily governed by upstream ribosome stalling peptide and the 5′ untranslated regions. Using the widespread S. aureus ermBL-ermB ( ermB ⁺ ) operon as a model system, we unexpectedly identified second-site mutations in purR that synergistically enhance MLS resistance in an ermB ⁺ background. Loss of purR function derepresses nucleotide biosynthesis and ribosome production, thereby promoting bacterial growth under antibiotic stress. While numerous purR single-nucleotide polymorphisms across multiple species have been associated with antibiotic resistance, no study has directly linked these sequence polymorphisms to their regulatory function. Our results highlight the critical role of ribosome abundance and nucleotide metabolism in shaping antibiotic efficacy.

Glutamate dehydrogenase (GDH) resides at the crossroads of nitrogen and carbon metabolism, catalyzing the reversible conversion of L-glutamate to α-ketoglutarate and ammonium. GDH paralogs are ubiquitous across most species, presumably enabling functional specialization and genetic compensation in response to diverse conditions. Staphylococcus aureus harbors a single housekeeping GDH (GudB), whereas Bacillus subtilis encodes both a major and a minor GDH, GudB and RocG, respectively. In an unsuccessful attempt to identify an alternative GDH in S. aureus, we serendipitously discovered previously unrecognized GDH activity in two metabolic enzymes of B. subtilis. The hexameric Val/Leu/Ile dehydrogenase Bcd (formerly YqiT) catabolizes branched-chain amino acids and to a lesser extent glutamate using NAD⁺ as a cofactor. Removal of gudB and rocG unmasks the dual NAD(P)⁺-dependent GDH activity of RocA, which otherwise functions as a 3-hydroxy-1-pyrroline-5-carboxylate dehydrogenase. Bcd homologs are prevalent in free-living and obligate bacteria but are absent in most, if not all, staphylococci. Despite low sequence homology, Bcd structurally resembles the GudB/RocG family and can functionally compensate for the loss of GudB in S. aureus. Bcd is essential for the full maturation of biofilms. B. subtilis lacking GDHs exhibits severe impairments in rugose architecture and colony expansion of biofilms. This study underscores the importance of metabolic redundancy and highlights the critical role of substrate promiscuity in GDHs during biofilm development.

Antimicrobial resistance is a major global health challenge, diminishing the efficacy of many antibiotics, including macrolides. InStaphylococcus aureus, an opportunistic pathogen, macrolide resistance is primarily mediated by Erm-family methyltransferases, which mono- or dimethylate A2058 in the 23S ribosomal RNA, reducing drug binding. Although macrolide–ribosome interactions have been characterized in nonpathogenic species, their structural basis in clinically relevant pathogens remains limited. In this study, we investigate the impact ofermB-mediated resistance on drug binding by analyzing ribosomes fromS. aureusstrains with varying levels ofermBexpression and activity. Using cryo-electron microscopy, we determined the high-resolution structures of solithromycin-bound ribosomes, including those with dimethylated A2058. Our structural analysis reveals the specific interactions that enable solithromycin binding despite double methylation and resistance, as corroborated by microbiological and biochemical data, suggesting that further optimization of ketolide–ribosome interactions could enhance macrolide efficacy against resistantS. aureusstrains.

The erythromycin resistance RNA methyltransferase ( erm ) confers cross-resistance to all therapeutically important macrolides, lincosamides, and streptogramins (MLS phenotype). The expression of erm is often induced by the macrolide-mediated ribosome stalling in the upstream co-transcribed leader sequence, thereby triggering a conformational switch of the intergenic RNA hairpins to allow the translational initiation of erm . We investigated the evolutionary emergence of the upstream erm regulatory elements and the impact of allelic variation on erm expression and the MLS phenotype. Through systematic profiling of the upstream regulatory sequences across all known erm operons, we observed that specific erm subfamilies, such as ermB and ermC , have independently evolved distinct configurations of small upstream ORFs and palindromic repeats. A population-wide genomic analysis of the upstream ermB regions revealed substantial non-random allelic variation at numerous positions. Utilizing machine learning-based classification coupled with RNA structure modeling, we found that many alleles cooperatively influence the stability of alternative RNA hairpin structures formed by the palindromic repeats, which, in turn, affects the inducibility of ermB expression and MLS phenotypes. Subsequent experimental validation of 11 randomly selected variants demonstrated an impressive 91% accuracy in predicting MLS phenotypes. Furthermore, we uncovered a mixed distribution of MLS-sensitive and MLS-resistant ermB loci within the evolutionary tree, indicating repeated and independent evolution of MLS resistance. Taken together, this study not only elucidates the evolutionary processes driving the emergence and development of MLS resistance but also highlights the potential of using non-coding genomic allele data to predict antibiotic resistance phenotypes. IMPORTANCE: Antibiotic resistance (AR) poses a global health threat as the efficacy of available antibiotics has rapidly eroded due to the widespread transmission of AR genes. Using Erm-dependent MLS resistance as a model, this study highlights the significance of non-coding genomic allelic variations. Through a comprehensive analysis of upstream regulatory elements within the erm family, we elucidated the evolutionary emergence and development of AR mechanisms. Leveraging population-wide machine learning (ML)-based genomic analysis, we transformed substantial non-random allelic variations into discernible clusters of elements, enabling precise prediction of MLS phenotypes from non-coding regions. These findings offer deeper insight into AR evolution and demonstrate the potential of harnessing non-coding genomic allele data for accurately predicting AR phenotypes.

The hibernation-promoting factor (Hpf) in Staphylococcus aureus binds to 70S ribosomes and induces the formation of the 100S complex (70S dimer), leading to translational avoidance and occlusion of ribosomes from RNase R-mediated degradation. Here, we show that the 3′-5′ exoribonuclease YhaM plays a previously unrecognized role in modulating ribosome stability. Unlike RNase R, which directly degrades the 16S rRNA of ribosomes in S. aureus cells lacking Hpf, YhaM destabilizes ribosomes by indirectly degrading the 3′-hpf mRNA that carries an intrinsic terminator. YhaM adopts an active hexameric assembly and robustly cleaves ssRNA in a manganese-dependent manner. In vivo, YhaM appears to be a low-processive enzyme, trimming the hpf mRNA by only 1 nucleotide. Deletion of yhaM delays cell growth. These findings substantiate the physiological significance of this cryptic enzyme and the protective role of Hpf in ribosome integrity, providing a mechanistic understanding of bacterial ribosome turnover.

Bacterial hibernating 100S ribosomes (the 70S dimers) are excluded from translation and are protected from ribonucleolytic degradation, thereby promoting long-term viability and increased regrowth. No extraribosomal target of any hibernation factor has been reported. Here, we discovered a previously unrecognized binding partner (YwlG) of hibernation-promoting factor (HPF) in the human pathogenStaphylococcus aureus. YwlG is an uncharacterized virulence factor inS. aureus. We show that the HPF–YwlG interaction is direct, independent of ribosome binding, and functionally linked to cold adaptation and glucose metabolism. Consistent with the distant resemblance of YwlG to the hexameric structures of nicotinamide adenine dinucleotide (NAD)–specific glutamate dehydrogenases (GDHs), YwlG overexpression can compensate for a loss of cellular GDH activity. The reduced abundance of 100S complexes and the suppression of YwlG-dependent GDH activity provide evidence for a two-way sequestration between YwlG and HPF. These findings reveal an unexpected layer of regulation linking the biogenesis of 100S ribosomes to glutamate metabolism.

All newly synthesized proteins must pass through a channel in the ribosome named the exit tunnel before emerging into the cytoplasm, membrane, and other compartments. The structural characteristics of the tunnel could govern protein folding and gene expression in a species-specific manner but how the identity of tunnel elements influences gene expression is less well-understood.