Critical functions and key interactions mediated by the RNase E scaffolding domain in Pseudomonas aeruginosa
Sandra Amandine Marie Geslain, Stéphane Hausmann, Johan Geiser, George Edward Allen, Diego Gonzalez, Martina Valentini
Abstract
The RNA degradosome is a bacterial multi-protein complex mediating mRNA processing and degradation. In Pseudomonadota, this complex assembles on the C-terminal domain (CTD) of RNase E through short linear motifs (SLiMs) that determine its composition and functionality. In the human pathogen Pseudomonas aeruginosa, the RNase E CTD exhibits limited similarity to that of model organisms, impeding our understanding of RNA metabolic processes in this bacterium.
Introduction
In bacteria, the processing and degradation of mRNAs is primarily orchestrated by the RNA degradosome, a multi-enzyme complex that includes ribonucleases, RNA helicases, and various other proteins [1–4]. The RNA degradosome plays a crucial role in enabling bacteria to rapidly adapt to changing environmental conditions by swiftly modifying mRNA levels and broadly regulating gene expression [5,6].
Materials and method
Bacterial strains and growing conditions
A list of bacterial strains used can be found in S1 Table. Unless otherwise specified, P. aeruginosa PAO1 was grown at 37°C in NYB (25 g of Difco nutrient broth, 5g of Difco yeast agar per liter) with shaking at 180 rpm or on Nutrient Agar (NA) (40 g Oxoid blood agar base, 5 g Difco yeast agar per liter). E. coli strains were similarly cultivated in LB broth or on LB agar instead. When required, antibiotics were added to these media at the following concentrations: 100 µg/ml ampicillin, 50 μg/mL kanamycin, 20 μg/mL tetracycline and 10 µg/ml gentamicin for E. coli; and 50 μg/mL or 100 μg/mL tetracycline and 50 µg/ml gentamicin for P. aeruginosa.
Results
The P. aeruginosa RNase E harbours several uncharacterised SLiMs conserved within the Pseudomonadales
P. aeruginosa RNase E (Pa RNase E) consists of an NTD (amino acids 1-529) that shares high sequence identity (70%) and similarity (90%) with the NTD of E. coli RNase E (Ec RNase E), and a CTD (amino acids 530-1057) that shows only 25% identity and 37% similarity with the CTD of Ec RNase E (S1 Fig). Previous work by Aït-Bara et al. (2015) identified seven different putative SLiMs on the Pa RNase E CTD (MTS, AR4, REE, M29, AR1, M20 and NDPR) [29], which are shown in comparison to the Ec RNase E in S2A Fig. Of note, the MTS is the only SLiM of Pa RNase E CTD with a propensity to form a secondary structure while in Ec RNase E, the enolase and PNPase binding sites also exhibit this feature (Fig 1 and [11]).
Discussion
The composition and the functions of the RNA degradosome vary among bacterial species, likely reflecting diverse lifestyles and niche adaptations [29]. Studying the RNA degradosome in various bacterial species is therefore crucial for elucidating how bacteria respond and adapt to different environmental conditions and sustain various types of stress. In this study, we characterized the RNA degradosome of P. aeruginosa, a versatile opportunistic pathogen and widely used model organism for bacterial pathogenesis and environmental adaptation.
Acknowledgments
We thank Sylvain Guex-Crosier for his assistance with RNA extraction and Nathanaël Guggenheim for assistance with protein purification and cloning experiments. We also acknowledge the Proteomics Core Facility and the Bioinformatics Support Platform from the University of Geneva for assistance with the realisation of LC-MS-MS and bioinformatics analyses, respectively.
Citation: Geslain SAM, Hausmann S, Geiser J, Allen GE, Gonzalez D, Valentini M (2025) Critical functions and key interactions mediated by the RNase E scaffolding domain in Pseudomonas aeruginosa . PLoS Genet 21(3): e1011618. https://doi.org/10.1371/journal.pgen.1011618
Editor: Ankur B. Dalia, Indiana University Bloomington, UNITED STATES OF AMERICA
Received: December 6, 2024; Accepted: February 12, 2025; Published: March 17, 2025
Copyright: © 2025 Geslain et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Raw counts of RNA seq are accessible in the NCBI GEO database with the accession number GSE287326. Total peptide counts from pull-down experiments are provided in supplementary S5 Table.
Funding: Work in M.V. laboratory is funded by Swiss National Science Foundation (Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, PCEFP3_203343 to MV) and Fondation Pierre Mercier pour la Science (to MV). This work is additionally funded by the iGE3 PhD Salary awards 2024 (to SG), by the Swiss National Science Foundation (Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, PZ00P3_180142 to DG) and by the Velux Foundation (grant 1814 to PJ and DG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
