Identification and functional analysis of sRNAs in Pseudomonas aeruginosa quorum sensing and virulence regulation

Small RNAs (sRNAs) act as critical regulators of post-transcriptional activity and diverse cellular processes in bacteria. However, the entire repertoire of sRNAs and their functions has not yet been extensively studied. This study employs a novel combined computational-experimental prediction approach to identify and characterize small RNAs (sRNAs) in Pseudomonas aeruginosa PAO1. We defined the first pan-sRNome, including 1663 sRNAs across 484 P. aeruginosa strains, and classified our 9 newly discovered sRNAs as 3 core and 6 accessory elements within the regulatory network. Functional studies revealed distinct sRNA activities: PA0982.1 was found to be a direct regulator of the rhl quorum sensing system. PA1014.1, PA2734.1, and PA2736.2 enhanced pyocyanin production. PA1014.1 and PA2734.1 facilitated biofilm formation for surface attachment. PA2734.1 and PA2736.2 suppressed motility to regulate dispersal, collectively shaping virulence traits. Additionally, PA1270.1, PA1367.1, and PA3471.1 were associated with reduced interbacterial competitiveness and the downregulation of the Type VI secretion system. Evolutionary analysis across diverse bacterial taxa and archaea demonstrated selective conservation, exposing the evolutionary trajectory of a diverse group of regulatory molecules. Overall, this study not only expands the P. aeruginosa sRNA landscape but also provides a framework for understanding how sRNAs fine-tune virulence and evolutionary adaptation.