A Comprehensive Analysis of the Microbial Diversity Associated with Eunicea Fusca and Related Plexauridae Octocorals, and the Isolation of New Natural Products from Selected, Associated Bacteria

Marine invertebrates, including octocorals, are a prolific source of marine natural products (MNPs) and are also known to host diverse microbial communities. It is well-established that marine microbes are producers of bioactive secondary metabolites. Accordingly, it is hypothesized that bioactive metabolites extracted from some marine invertebrates may actually be produced by associated microbes rather than the invertebrate host.;This research aimed to characterize the microbial community of E. fusca and related Plexauridae octocorals and to discover novel, bioactive compounds biosynthesized by associated microorganisms. E. fusca was the main focus of this study because its microbial community has not been thoroughly characterized, and it is the sole source of the potent anti-inflammatory diterpenes fuscol, eunicol, and the fuscosides. Investigations of the unexplored E. fusca microbial community could therefore lead to the discovery of novel MNPs from unexplored microbes, as well as provide insights into the biosynthetic source of these diterpenes.;The microbiome of Floridian and Bahamian E. fusca and related Plexauridae octocorals collected in June 2009 were characterized using culture-independent (454-pyrosequencing, denaturing gradient gel electrophoresis, cloning, and species-specific primers) and -dependent (dilution plating) methods. The culture-independent analysis revealed that E. fusca hosted a geographically-heterogeneous bacterial community, where Endozoicomonas relatives dominated the Florida sites, and Mycoplasma relatives dominated at The Bahamas sites. There were, however, a few stable bacterial associates found in all E. fusca at all sites, including relatives of the Endozoicomonas, Mycoplasma , and Oceanospirillales, suggesting that these bacteria may be critical to holobiont functioning, and differences in environmental conditions at the locations may affect the dominant community member. E. fusca's bacterial community also differed from that of closely-related Plexauridae octocorals and the surrounding seawater. All octocorals contained the same clade of dinoflagellate, Symbiodinium B1/B184. However, the fungal and archaeal communities were not consistent between Plexauridae species or location.;In the culture-dependent study, 137 unique bacteria and 11 unique fungi were cultured from the Plexauridae octocorals; 31 of the isolated bacteria were putative novel species based on 16S rDNA analysis (<97% sequence identity of 16S rDNA). Two of the novel species, Endozoicomonas euniceicola sp. nov. and Endozoicomonas gorgoniicola sp. nov., had nearly identical 16S rDNA sequences to the dominant members in the culture-independent bacterial community, providing the extraordinary opportunity to explore the biology and chemistry of these putative octocoral symbionts. These two bacteria were formally characterized, revealing that they were genotypically, phenotypically, and chemotypically different from the three Endozoicomonas spp. cultures previously isolated from other marine invertebrates.;From the culture-dependent library, selected bacteria were fermented and analyzed for MNP production. Under the evaluated fermentation conditions, two bacteria were found to produce novel MNPs. A Labrenzia sp. produced a family of novel fatty acid derivatives, and a Euzebyella sp. produced a novel 2,5-dialkylresorcinol with good gram-positive bioactivity.;In conclusion, Eunicea fusca hosts a geographically heterogeneous and diverse microbial community that differs from that of other closely-related Plexauridae octocorals and the surrounding environment. This microbial community has a varied metabolic repertoire, which can ultimately lead to the discovery of novel, microbial MNPs.