Bacterial diversity of the octocoral Pseudopterogorgia elisabethae via culture-dependent and culture-independent methods and screening of selected bacteria for antimicrobial activity

摘要

Alcyonacean octocorals (Order Alcyonacea, amended from Order, Gorgoniidae) are both dominant on Caribbean reefs and prolific sources of bioactive natural products. Although extensive literature has been published with respect to stony coral-associated bacteria, very little is known regarding bacterial populations in healthy octocorals. In this thesis, the octocoral Pseudopterogorgia elisabethae was selected as a model organism to investigate owing to its commercial significance and its understudied bacterial community. The overall hypothesis of this thesis is that the bacterial community of P. elisabethae is diverse and can be cultured to produce a valuable and sustainable source of novel natural products. To test this hypothesis, three studies were performed. In the first investigation, pyrosequencing was used to examine the culture independent bacterial community in P. elisabethae. In the second study, bacteria were isolated from P. elisabethae using nine diverse isolation media to maximize the number of isolates. The final approach involved assessing the ability of bacterial isolates to produce antimicrobial compounds via screening fermentation extracts against five clinically relevant pathogens.Pyrosequencing results from 14 coral-associated bacterial sequence libraries suggested P. elisabethae individuals are a rich source of diverse and novel bacteria. Bacterial communities were analyzed at the class and the species level, where a lack of conservation between conspecifics within and between reefs in the Bahamas was observed. From the culturing approach, 852 bacteria were isolated and were de-replicated by MALDI-TOF MS to approximately 78 unique isolates. In addition, sequences generated from de-replicated isolates were compared to the pyrosequencing community at 97% sequence similarity, yet very little overlap (0.44%) was observed. Regardless, de-replicated isolates were screened for antimicrobial activity, where a Streptomyces sp. RKBH-B54 and genetically-related Pseudoalteromonas spp. were reproducibly bioactive against yeast and Gram-positive bacteria respectively. Due to the potent bioactivity of RKBH-B54, a bioassay guided fractionation was performed in an attempt to isolate the antifungal compound produced. Dihydromaltophilin and maltophilin were proposed as the most likely compounds responsible for the observed antifungal activity from RKBH-B54 based on comparison of UPLC/HRMS data to analytical databases; unfortunately, the instability of these compounds prevented complete purification and structural elucidation. In conclusion, this thesis has demonstrated that P. elisabethae is a rich source of diverse bacteria, yet collecting multiple individuals throughout the Bahamas is required to truly evaluate the microbial biodiversity in this octocoral. It is also concluded that the potent and reproducible bioactivity of selected bacterial extracts from P. elisabethae can be used as a resource for developing potentially new therapeutic agents.