ABSTRACT: Pseudomonas aeruginosa, X-ray irradiation, inner membrane blebbing, membrane vesicles Almost all bacteria produce membrane vesicles (MVs) that have been found to be generally composed of lipopolysaccharides (LPSs), phospholipids (PLs), outer membrane proteins, periplasmic proteins and peptidoglycan (PG), cytoplasmic proteins, as well as nucleic acids, metabolites and signaling molecules. We have recently demonstrated that X-ray irradiation strongly induced the MV production in P. aeruginosa, but the detailed biogenesis mechanisms and protein components of MVs were largely unclear. In this study, we performed a quantitative proteomics analysis of the MVs; a total of 2320 proteins were identified, among which 72.33% were cytoplasmic proteins, followed by periplasmic proteins (12.59%), IMPs (8.45%), OMPs (5.26%) and others (1.38%). Gene ontology (GO) enrichment analysis of biological processes showed that nucleoside phosphate metabolic process, ribose phosphate metabolic process, nucleoside metabolic process, and purine-containing compound biosynthetic process were significantly enriched. As expected, cellular component analysis confirmed the enrichment of cytoplasm. Notably, molecular functional enrichment analysis revealed that a large proportion of proteins that were related to RNA binding, tRNA binding, RNA methyltransferase activity, aminoacyl-tRNA ligase activity, and ligase activity were highly enriched. KEGG enrichment analysis showed that pathways involved in ribosome, purine metabolism, tRNA biogenesis and RNA degradation were enriched. These results suggest that under X-ray irradiation stress, P. aeruginosa PAO1 cells could transport the damaged nucleic acids by producing MVs, and the RNA binding and degradation proteins contained in MVs may help P. aeruginosa to identify and degrade damaged RNA.