Abstract

The fish pathogen Yersinia ruckeri forms biofilms on abiotic surfaces, contributing to recurrent infections in aquaculture. Increasing evidence suggests that the RNA chaperone Hfq and small non-coding RNAs (sRNAs) are key regulators of bacterial biofilm formation. However, the regulatory mechanisms mediated by these factors remain largely unexplored in Y. ruckeri. In this study, we investigated the roles of Hfq and the Hfq-dependent sRNAs RprA, ArcZ, and RybB in the biofilm formation of Y. ruckeri. We first characterized the sRNAome of biofilm-forming cells, identifying the conserved RprA, ArcZ, and RybB, among the upregulated sRNAs. We then evaluated motility, biofilm formation, and architecture in strains lacking either hfq (?hfq) or these sRNAs (?sRNA). Our results reveal that both ?hfq and ?sRNA strains exhibit significant alterations in biofilm and motility phenotypes, including changes in bacterial morphology and extracellular matrix. Furthermore, expression analyses indicate that these sRNAs modulate the transcription of key regulatory factors, flagellar and phosphodiesterase genes, ultimately influencing intracellular cyclic di-GMP levels, a key second messenger in biofilm formation. Together, our findings demonstrate that Hfq and its associated sRNAs play critical regulatory roles in Y. ruckeri biofilm formation by controlling the expression of genes involved in motility, bacterial envelope proteins, and c-di-GMP metabolism. © 2025 by the authors.