Microbial Community Analysis of a Gorgonian Coral, Pseudopterogorgia americana

Christopher Johnston¹, Garriet W. Smith², Cheryl M. Woodley^1,3, and Pamela J. Morris^1,3

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Department of Biology, University of South Carolina Aiken, Aiken, SC

³ Center for Coastal Environmental Health and Biomolecular Research and Hollings Marine Laboratory, Charleston, SC

Coral reefs have long been recognized as the most diverse marine ecosystem; however, relatively little attention has been focused on the prokaryotic diversity associated with coral reefs until recently.  Corals harbor an active microbial community in their surface mucopolysaccharide layer (SML) that may be specific to the host coral over spatial and temporal scales.  We used both culture-dependent and culture-independent approaches to characterize bacteria associated with the SML of a healthy gorgonian coral, Pseudopterogorgia americana, from Rocky Point Reef, San Salvador, Bahamas.  Microorganisms were isolated from the coral SML and microbial community DNA was directly extracted from the SML for 16S rRNA gene analysis.  Of 53 isolates obtained, 30 were distinct ribotypes based on 16S rDNA sequence analysis of the V6-V9 regions.  Seven of the 30 isolates were Gram-positive, and all of the isolates clustered within the Bacillus, γ- and α-Proteobacteria.  We compared four different DNA extraction and purification methods for recovering microbial community DNA directly from SML samples, and determined the efficiency of each method based on total DNA yield and percent recovery from spiked samples.  From the microbial community DNA extracts, we amplified the bacterial 16S rRNA genes and examined the community structure through denaturing gradient gel electrophoresis (DGGE) and 16S rDNA cloning.  DGGE profiles resulted in 15 major bands, 12 of which were excised, sequenced, and phylogenetically characterized.  Of the DGGE sequences obtained, the majority clustered within the β-Proteobacteria.  The 16S rDNA clone library was dominated by the genus Sphingomonas, with almost half of the sequences identified belonging to this genus.  Thus, we observed little congruency between the bacterial 16S rRNA genes identified using culture-dependent and culture-independent approaches.  Additionally, coral SML and SML isolates were screened for antifungal activity in dual culture bioassays and the presence of quorum sensing signaling molecules using two acyl homoserine lactone autoinducer bioassays, thin layer chromatography, and gas chromatography-mass spectrometry.  Of the 30 isolates tested, 2 tested positive for antifungal activity against Aspergillus sydowii and 10 isolates tested positive for signal molecules using the autoinducer bioassays.  Quorum sensing may provide one mechanism by which coral-associated microorganisms control populations within their communities, and prevent invasions by opportunistic pathogens.