BackgroundThe 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.ResultsMutations 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0617-z) contains supplementary material, which is available to authorized users.

ADDITIONAL INDEX WORDS. Vaccinium macrocarpon, gypsy moth, cranberry weevil, red-headed flea beetle ABSTRACT. Several insect herbivores have been anecdotally reported to prefer 'Howes' cranberry leaves (Vaccinium macrocarpon) over those of 'Early Black'. A series of studies were undertaken to determine whether these anecdotal reports are accurate and to compare phenolic profiles in the foliage of 'Early Black' and 'Howes' for compounds that differ in concentration and could be further investigated as possible feeding deterrents. Gypsy moth larvae (Lymantria dispar) demonstrated a significant feeding preference for 'Howes' over 'Early Black'. Red-headed flea beetle adults (Systena frontalis) demonstrated a similar but not statistically significant trend, whereas cranberry weevil (Anthonomus musculus) did not prefer either cultivar. Compounds giving rise to six peaks in the phenolic profile were significantly greater in concentration in 'Early Black' than 'Howes' on at least one of three sampling dates during the growing season. Five of these compounds were isolated from leaves harvested at the June time point coinciding with gypsy moth infestation and identified as: 5-O-caffeoylquinic acid, 3-O-p-coumaroylquinic acid, 5-Op-coumaroylquinic acid, quercetin-3-O-galactoside, and quercetin-3-O-rhamnoside.

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