The Identification And Functional Study Of Spermidine Binding Protein Gene In Vibrio Anguillarum

The widespread polyamine transport system PotABCD transporter in vivo iscomposed by the four functional proteins: PotA, ATPase, provides energy for thetransport system; PotB and PotC can form transport channels in the double membrane;PotD,which is a cytoplasmic combination protein with a polyamine recognitiondomain, has a higher affinity for spermidine and lower affinity for putrescine.Three presumptive potD genes that encode spermidine binding protein PotD inVibrio anguillarum M3: potD1, potD2and potD3. In order to study the role of thesethree genes in Vibrio anguillarum M3spermidine transporter, as well as the impact onthe virulence of Vibrio anguillarum M3. We used the in-frame deletion technology tobuild the potD1, potD2, and potD3single gene deletion mutants and multi-genedeletion mutants, and in the same time we took advantage of bacteria joints transfertechnology, using low copy number cloning plasmid Pmmb207carrier built acomplementary mutant strains.Ignored the influence of synthesis procedure on intracellular polyamine, wemeasured the polyamine content in chemically defined Minimal Medium M9, andfound in the independent and simultaneous missing of gene potD1or potD3deletionmutants the polyamine content was decreased significantly compared to wild-type Vibrio anguillarum M3, and their growth rate was decreased significantly too. Thisindicated that potD1and potD3genes were spermidine binding protein encoding genesand could participate in the process of polyamine transport in Vibrio anguillarum M3.The gene potD1or potD3indirectly affects the growth capacity of the Vibrioanguillarum M3and pathogenicity to fish by means of the impact on the polyaminetransport process. The potD2gene restrains potD1, and potD3genes, separated potD2gene deletion did not affect the intracellular polyamine content of Vibrio anguillarum.Although it does not participate in the spermidine transporte, potD2can inhibit theexpression of potD1and potD3to a certain extent. Real-time quantitative PCR methodfurther verified the expression of these genes in the mutants, and defined the traits ofVibrio anguillarum changes were caused by the gene expression changes.