Structural characterization of hibernating ribosomes in four Gram-negative pathogenic bacteria

Abstract Ribosome hibernation is a conserved bacterial stress-response mechanism that promotes translational shutdown and enhances survival under adverse conditions, contributing to persistence and tolerance to ribosome-targeting antibiotics. Here, we report high-resolution cryo-electron microscopy (cryo-EM) structures (2.5–2.8 Å) of hibernating 70S ribosomes from four clinically important ESKAPE pathogens: Pseudomonas aeruginosa , Enterobacter hormaechei , Klebsiella quasipneumoniae , and Acinetobacter baumannii . Structural analyses identified the bound hibernation factors as HPF in P. aeruginosa and E. hormaechei , and YfiA in K. quasipneumoniae and A. baumannii . Despite sequence divergence, HPF and YfiA adopt a conserved fold and occupy the same ribosomal binding site, interacting primarily with 16S rRNA and ribosomal proteins uS7 and uS9 and occupying the A- and P-sites while extending toward the E-site. Comparative analyses revealed a conserved core interaction network, including contacts with functionally important modified 16S rRNA nucleotides, alongside species-specific adaptations that preserve overall ribosome-binding architecture. Analysis of hibernating ribosome populations uncovered substantial heterogeneity in E-site tRNA occupancy, bS21 association, and mRNA binding among species, suggesting that these features are species-dependent rather than determined solely by the identity of the hibernation factor. Together, these structures provide a comprehensive comparative view of HPF- and YfiA-mediated ribosome hibernation across major Gram-negative pathogens, revealing conserved molecular principles and species-specific adaptations that expand our understanding of bacterial translational dormancy and establish a structural framework for future antibacterial strategies targeting ribosome hibernation.