A gene knockout mutant of ferric uptake regulator (Fur) reveals biofilm formation, energy metabolism and transcript regulation are three toxicity regulation strategies of Vibrio harveyi isolated from American eels (Anguilla rostrota)

Abstract Vibrio harveyi is commonly found in salt and brackish water, and is recognized as a serious bacterial pathogen in aquaculture worldwide. In this study, we cloned the ferric uptake regulator ( Fur ) gene from V. harveyi wild-type strain HA_1, which was isolated from diseased American eels ( Anguilla rostrata ) and has a length of 450 bp, encoding 149 amino acids. Then, a mutant strain, HA_1-Δ Fur , was constructed through homologous recombination of a suicide plasmid (pCVD442). The HA_1-Δ Fur mutant exhibited attenuated biofilm formation, intensified swarming motility, and 18-fold decrease (5.5%) in virulence to the American eels, but it showing no difference in growth and hemolysis with the wild-type strain. Transcriptome analysis revealed that 875 genes were differentially expressed in the Δ Fur mutant, with 385 up-regulated and 490 down-regulated DEGs. GO and KEGG enrichment analysis revealed that, compared to the wild-type strain, the type II secretion systems (T2SS), type VI secretion systems (T6SS), amino acid synthesis and transport, and energy metabolism pathways were significantly down-regulated, but the ABC transporters and biosynthesis of siderophore group non-ribosomal peptides pathways were up-regulated in the Δ Fur strain. The qRT-PCR results further confirmed that DEGs responsible for amino acid transport and energy metabolism were positively regulated, but DEGs involved in iron acquisition were negatively regulated in the Δ Fur strain. These findings suggest that the gene Fur contribute to the virulence of V. harveyi through biofilm formation, energy metabolism, and transcript regulation.