Abstract
Vibrio parahaemolyticus (V. parahaemolyticus) is a globally prevalent seafood-borne pathogen and a leading cause of gastroenteritis. It readily forms biofilms on shrimp, shellfish, and food packaging surfaces, enhancing its environmental tolerance, survival, and risk of cross-contamination during processing and storage. In this study, we constructed a transposon mutant library to systematically identify genes involved in biofilm regulation. Quantitative screening of 4000 mutants revealed 103 candidate genes, with 28 mutants showing reduced biofilm formation and 75 showing enhanced formation. Enrichment analysis indicated that these genes are primarily associated with the two-component systems (TCS), pyrimidine metabolism, and amino acid biosynthesis pathways. Two previously uncharacterized genes were further analyzed. Results showed that vp2252, encoding a component of a TCS, and vp2888, encoding a diguanylate cyclase with cyclic di-GMP (c-di-GMP) synthetase activity, broadly regulate biofilm formation, motility, and virulence. Transcriptomic data suggest that vp2252 mediates the transition from free-swimming to surface-associated swarming lifestyles. These findings provide new insights into the genetic regulation of V. parahaemolyticus biofilm development, highlight potential molecular targets for biofilm control, and lay the groundwork for future studies on its regulatory networks.