Contributions of Tropodithietic Acid and Biofilm Formation to the Probiotic Activity of Phaeobacter Inhibens

Background: The probiotic bacterium Phaeobacter inhibens strain S4Sm, isolated from the inner shell surface of a healthy oyster, secretes the antibiotic tropodithietic acid (TDA), is an excellent biofilm former, and increases oyster larvae survival when challenged with bacterial pathogens. In this study, we investigated the specific roles of TDA secretion and biofilm formation in the probiotic activity of S4Sm. Results: Mutations in clpX (ATP-dependent ATPase) and exoP (an exopolysaccharide biosynthesis gene) were created by insertional mutagenesis using homologous recombination. Mutation of clpX resulted in the loss of TDA production, no decline in biofilm formation, and loss of the ability to inhibit the growth of Vibrio tubiashii and Vibrio anguillarum in co-colonization experiments. Mutation of exoP resulted in a ~60 % decline in biofilm formation, no decline in TDA production, and delayed inhibitory activity towards Vibrio pathogens in co-colonization experiments. Both clpX and exoP mutants exhibited reduced ability to protect oyster larvae from death when challenged by Vibrio tubiashii. Complementation of the clpX and exoP mutations restored the wild type phenotype. We also found that pre-colonization of surfaces by S4Sm was critical for this bacterium to inhibit pathogen colonization and growth. Conclusions: Our observations demonstrate that probiotic activity by P. inhibens S4Sm involves contributions from both biofilm formation and the production of the antibiotic TDA. Further, probiotic activity also requires colonization of surfaces by S4Sm prior to the introduction of the pathogen. Keywords: Phaeobacter inhibens, Tropodithietic acid, Biofilm formation, Probiotic, Marine pathogens, Vibrio tubiashii, Vibrio anguillarum, Oyster disease, ClpX, Exo