Biology subjects

Romby, P.

Publications and source records attributed to Romby, P..

9 recordsLinked to original sources

bioRxiv2025

Complete post-transcriptional modification profiles in individual Staphylococcus aureus tRNA species

Transfer RNAs play a critical role in protein synthesis by matching mRNA codons to their corresponding amino acids. Their post-transcriptional modifications, shaping structure, stability, and codon decoding, are now recognized as key regulators of translation and cellular adaptation, including in bacterial pathogens. Here, we provide a comprehensive analysis of tRNA modifications in Staphylococcus aureus using extensive oligonucleotide mass spectrometry and deep-sequencing methods, generating a high-confidence modification map for each individual tRNA species, including the non-proteogenic tRNAGly. While the overall tRNA modification landscape is conserved among Gram-positive bacteria, our data uncovered unexpected S. aureus-specific features. These include the absence of [m2A]37 in tRNAs despite the presence of the methyltransferase RlmN, a single multi-site DusB2 enzyme catalyzing all tRNA dihydrouridylation, and a dedicated pseudouridine synthase responsible for [{Psi}]32 formation. Besides, heterogenous modification patterns were observed in tRNALeu(UAA) and tRNALys(CAA), highlighting a complex interplay in anticodon hypermodification. Integration of ribosome profiling and Nanopore tRNA sequencing offered a global view of the decoding properties of the reduced S. aureus tRNA set, revealing efficient four-way wobble recognition, slower translation of rare codons by low abundant tRNAs, and distinctive decoding dynamics of Gly codons potentially influenced by the unusual modification status of tRNAGly(UCC). This work establishes a framework for future research aimed at dissecting the role of specific tRNA modifications in S. aureus physiology and pathogenesis.

molecular biology↗

bioRxiv2025

A Zur-dependent regulatory RNA involved in maintaining zinc homeostasis in Staphylococcus aureus

Small regulatory RNAs (sRNAs) are key drivers of bacterial adaptation to environmental fluctuations, including iron and manganese restriction imposed by the host. This study explored the repertoire of sRNAs produced by the human pathogen Staphylococus aureus in response to metal limitation. Two sRNAs, S1077 and ZinS (RsaX20), regulated by zinc (Zn) availability, were identified. Further investigations revealed that, similar to the cnt operon from which it derives, S1077 synthesis is controlled by the transcription factors Zur and Fur. In contrast, zinS transcription is solely repressed by Zur. Amongst the ZinS targets are several Zn-dependent enzymes, such as the alcohol dehydrogenase Adh, whose synthesis is negatively regulated by ZinS. Loss of ZinS does not alter staphylococcal metal accumulation, suggesting a role in a Zn-sparing response. Remarkably, zinS also encodes a small peptide, ZinP. Genomic analysis suggests that the regulatory portion of ZinS emerged from the 3 untranslated region of zinP in S. aureus and closely related species after horizontal gene transfer from phylogenetically distant organisms. All our findings demonstrate that sRNAs also facilitate bacterial adaptation to Zn limitation, and that genetic exchange and subsequent neofunctionalization have enabled S. aureus to adapt to metal-restricted environments.

microbiology↗

bioRxiv2025

Quorum sensing dependent repression of the Staphylococcus aureus transcription termination factor Rho is necessary for pathogenicity

In Staphylococcus aureus, the transcription termination Rho factor has been identified as an attenuator of virulence, highlighting this universal protein as a new player in S. aureus pathophysiology. Moreover, Rho is required for the repression of "pervasive" transcription, which results from transcription at cryptic promoters or from transcriptional readthrough. In our study, we demonstrated that the rho transcript is specifically repressed by the quorum sensing-dependent RNAIII during the stationary phase of growth, allowing exotoxins to be activated at this stage. Rho-dependent RNA targets were identified using RIP-seq and differential transcriptomic analyses, revealing that several of these targets are involved in adhesion, dissemination, and the host responses. Our data provide a molecular explanation for the functions of this well-conserved termination factor, which have evolved in this human pathogen.

microbiology↗

bioRxiv2025

Extended Shine-Dalgarno motifs govern translation initiation in Staphylococcus aureus

Regulation of translation initiation is central to bacterial adaptation, but species-specific mechanisms remain poorly understood. We present high-resolution mapping of translation start sites in S. aureus, revealing distinct features of initiation alongside numerous unannotated small ORFs. Our analysis, combined with cryo-EM of a native mRNA-ribosome complex, shows that S. aureus relies on extended, start codon proximal Shine-Dalgarno (SD) interactions, creating specificity against phylogenetically distant bacteria. Several natural S. aureus initiation sites are not correctly decoded by E. coli ribosomes. We identify new and conserved non-canonical start codons, whose regulatory initiation sites contain these characteristic extended SD sequence motifs. Finally, we characterize a striking example of uORF-mediated translational control in S. aureus, demonstrating that translation of a novel small leader peptide modulates expression of a key biofilm regulator. The described mechanism involves codon rarity, ribosome pausing and arginine availability, linking nutrient sensing to biofilm formation in this major human pathogen. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/660503v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1ea5094org.highwire.dtl.DTLVardef@505be5org.highwire.dtl.DTLVardef@1c14ad6org.highwire.dtl.DTLVardef@118549c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

molecular biology↗

bioRxiv2024

An unprecedented small RNA-riboswitch interaction controls expression of a bifunctional pump that is essential for Staphylococcus aureus infection

Maintaining manganese and iron homeostasis is critical for the human pathogen Staphylococcus aureus. To counteract metal-based host defense strategies (e.g., nutritional immunity, metal poisoning), S. aureus uses a combination of metal-sensing transcription factors and regulatory RNAs to maintain metal homeostasis. In this study, we uncovered an unprecedented interaction between a cis- and a trans-acting regulatory RNA controlling a conditionally essential gene, mntY, encoding a Mn efflux pump. This broadly conserved RNA-RNA interaction between a Fe-responsive sRNA and a Mn-sensing riboswitch allows the integration of Feand Mn-related stresses, notably encountered at the infection site, to fine-tune mntY expression. Remarkably, deletion of the mntY gene is strongly pleomorphic, causing growth defects, altering virulence factor expression, immune evasion, and survival during infection. We demonstrated that MntY is critical for the adaptation of S. aureus to both low and high Mn environments, due to its dual role in metalation of Mn-dependent exoenzymes and Mn detoxification. These findings point to MntY as a promising new therapeutic target to combat multidrug-resistant staphylococcal infections.

microbiology↗

bioRxiv2023

RlmQ: A Newly Discovered rRNA Modification Enzyme Bridging RNA Modification and Virulence Traits in Staphylococcus aureus

rRNA modifications play crucial roles in fine-tuning the delicate balance between translation speed and accuracy, yet the underlying mechanisms remain elusive. Comparative analysis of the ribosomal RNA modifications in taxonomically distant bacteria could help define their general as well as species-specific roles. In this study, we identified a new methyltransferase, RlmQ, in Staphylococcus aureus responsible for the Gram-positive specific m7G2601, which is not modified in E. coli (G2574). We also demonstrate the absence of methylation on C1989, equivalent to E. coli C1962, which is methylated at position 5 by the Gram-negative specific RlmI methyltransferase, a paralogue of RlmQ. Both modifications (S. aureus m7G2601 and E. coli m5C1962) are situated within the same tRNA accommodation corridor, hinting at a potential shared function in translation. Inactivation of S. aureus rlmQ causes the loss of methylation at G2601 and significantly impacts growth, cytotoxicity, and biofilm formation. These findings unravel the intricate connections between rRNA modifications, translation, and virulence in pathogenic Gram-positive bacteria.

molecular biology↗

bioRxiv2022

Complex regulation of gamma-hemolysin expression impacts S. aureus virulence

Staphylococcus aureus gamma-hemolysin CB (HlgCB) is a core-genome encoded pore-forming toxin that targets the C5a receptor, similarly as the phage-encoded Panton-Valentine Leucocidin. Absolute quantification by mass spectrometry of HlgCB in 39 community-acquired pneumonia (CAP) isolates showed considerable variations in HlgC and HlgB yields between isolates. Interestingly, when testing the hypothesis that HlgCB might be associated with severe S. aureus CAP, we found that a high level of HlgCB synthesis was associated with mortality in a rabbit model of pneumonia. To decipher the molecular basis for the variation in hlgCB and hlgB expression and protein production among strains, different regulation levels were analyzed in representative clinical isolates and reference strains. Although HlgC and HlgB are encoded on a single operon, their levels were dissociated in 10% of the clinical strains studied. HlgCB amount and HlgC/HlgB ratio were found to both depend on promotor activity, mRNA stability and translatability, and on the presence of an individual hlgB mRNA processed from the hlgCB transcript. Strikingly, toe-printing and in vitro translation assays revealed that a single SNP in the 5-UTR of hlgCB mRNA strongly impaired hlgC translation in the USA300 strain, leading to a strong decrease in HlgC but not in HlgB; the level of HlgB is likely to have been maintained by the presence of the processed hlgB mRNA. This work illustrates the complexity of virulence factor expression in clinical strains and demonstrates a butterfly effect, where subtle genomic variations have a major impact on phenotype and virulence. Author SummaryThe Gram-positive bacterium Staphylococcus aureus can provoke a wide range of infections due to its ability to produce a large diversity of virulence factors, including immune evasion molecules, adhesins, and toxins. Some of these toxin-encoding genes are localized in mobile genetic elements, and are thus not present in all strains, whilst others are encoded in the core-genome and present in all strains. Gamma-hemolysin CB is a core-genome encoded toxin but its amount varies between community-acquired pneumonia isolates. The regulation mechanisms underlying this variation however, are not well characterized. Here, we show that gamma-hemolysin expression levels vary largely among clinical strains and that, when highly produced, it induces high mortality in a rabbit model of pneumonia. The molecular basis for the variation in gamma-hemolysin expression depends on multiple mechanisms including promoter strength, transcript stability and processing, and translatability (i.e. the amount of protein that is synthetized by the ribosome for a given transcript). Incredibly, all these factors rely on a subtle genetic modification. This work emphasizes the importance of the disparity in virulence factor expression among clinical isolates and points the extreme complexity of the molecular mechanisms underlying their regulation, rendering the prediction of virulence for a clinical isolate difficult.

microbiology↗

bioRxiv2021

Escherichia coli ribosomal protein S1 enhances the kinetics of ribosome biogenesis and RNA decay

Escherichia coli ribosomal protein S1 is essential for translation initiation of mRNAs and for cellular viability. Two oligonucleotide binding (OB)-fold domains located in the C-terminus of S1 are dispensable for growth, but their deletion causes a cold-shock phenotype, loss of motility and deregulation of RNA mediated stress responses. Surprisingly, the expression of the small regulatory RNA RyhB and one of its repressed target mRNA, sodB, are enhanced in the mutant strain lacking the two OB domains. Using in vivo and in vitro approaches, we show that RyhB retains its capacity to repress translation of target mRNAs in the mutant strain but becomes deficient in triggering rapid turnover of those transcripts. In addition, the mutant is defective in of the final step of the RNase E-dependent maturation of the 16S rRNA. This work unveils an unexpected function of S1 in facilitating ribosome biogenesis and RyhB-dependent mRNA decay mediated by the RNA degradosome. Through its RNA chaperone activity, S1 participates to the coupling between ribosome biogenesis, translation, and RNA decay.

microbiology↗

bioRxiv2018

A dual sRNA in Staphylococcus aureus induces a metabolic switch responding to glucose consumption

Pathogenic bacteria must rapidly adapt to ever-changing environmental signals or nutrient availability resulting in metabolism remodeling. The carbon catabolite repression represents a global regulatory system, allowing the bacteria to express genes involved in carbon utilization and metabolization of the preferred carbon source. In Staphylococcus aureus, regulation of catabolite repressing genes is mediated by the carbon catabolite protein A (CcpA). Here, we have identified a CcpA-dependent small non-coding RNA, RsaI that is inhibited by high glucose concentrations. RsaI represses the translation of mRNAs encoding a major permease of glucose uptake, the FN3K enzyme that protects proteins against damages caused by high glucose concentrations, and IcaR, the transcriptional repressor of exopolysaccharide production. Besides, RsaI regulates the activities of other sRNAs responding to the uptake of glucose-6 phosphate or NO. Finally, RsaI inhibits the expression of several enzymes involved in carbon catabolism pathway, and activates genes involved in energy production, fermentation and NO detoxification when the glucose concentration decreases. This multifunctional RNA provides a signature for a metabolic switch when glucose is scarce and growth is arrested.

microbiology↗