Investigation of the roles of T6SS genes in motility, biofilm formation, and extracellular protease Asp production in Vibrio alginolyticus with modified Gateway-compatible plasmids

Aims: The aims of this study were to create and evaluate the Gateway‐compatible plasmids for investigating the function of genes in Vibrio alginolyticus and other Gram‐negative bacteria. Methods and Results: In this study, Gateway‐compatible plasmids were successfully constructed for rapid and comprehensive function analysis of genes. Taking advantage of these plasmids, the in‐frame deletion mutant strains and their complemented strains of five T6SS genes, including dotU1, VEPGS_0008, VEPGS_0011, hcp2 and ppkA2, were obtained. The results illustrated that all the mutant strains showed no significant effects on extracellular protease production, expression of Hcp1, and biofilm formation when compared to the wild‐type strain, but in‐frame deletion of VEPGS_0008 resulted in obvious biofilm reduction and the complemented strain restored to the level of the wild‐type strain. Besides, in‐frame deletion of dotU1, VEPGS_0008 and ppkA2 abolished the swarming ability. Conclusions: A set of Gateway‐compatible vectors for internal insertion, in‐frame deletion and complementation of the target genes is constructed to facilitate the general and rapid function analysis of genes involved in T6SS in Vibrio alginolyticus. Significance and Impact of the Study: The modified Gateway‐compatible plasmids greatly facilitate the high‐throughput and convenient function analysis of the unidentified genes.