Abstract
Black band disease (BBD) of corals is a complex polymicrobial disease considered to be a threat to coral reef health, as it can lead to mortality of massive reef-building corals. The BBD community is dominated by gliding, filamentous cyanobacteria with a highly diverse population of heterotrophic bacteria. Microbial interactions such as quorum sensing (QS) and antimicrobial production may be involved in BBD disease pathogenesis. In this study, BBD (whole community) samples, as well as 199 bacterial isolates from BBD, the surface mucopolysaccharide layer (SML) of apparently healthy corals, and SML of apparently healthy areas of BBD-infected corals were screened for the production of acyl homoserine lactones (AHLs) and for autoinducer-2 (AI-2) activity using three bacterial reporter strains. AHLs were detected in all BBD (intact community) samples tested and in cultures of 5.5% of BBD bacterial isolates. Over half of a subset (153) of the isolates were positive for AI-2 activity. AHL-producing isolates were further analyzed using LC-MS/MS to determine AHL chemical structure and the concentration of (S)-4,5-dihydroxy-2,3-pentanedione (DPD), the biosynthetic precursor of AI-2. C6-HSL was the most common AHL variant detected, followed by 3OC4-HSL. In addition to QS assays, 342 growth challenges were conducted among a subset of the isolates, with 27% of isolates eliciting growth inhibition and 2% growth stimulation. 24% of BBD isolates elicited growth inhibition as compared to 26% and 32% of the bacteria from the two SML sources. With one exception, only isolates that exhibited AI-2 activity or produced DPD inhibited growth of test strains. These findings demonstrate for the first time that AHLs are present in an active coral disease. It is possible that AI-2 production among BBD and coral SML bacteria may structure the microbial communities of both a polymicrobial infection and the healthy coral microbiome.
Highlights
Coral diseases are widely believed to play a key role in the deterioration of coral reefs on a global basis [1,2], with black band disease (BBD) identified as one of the major coral diseases contributing to this decline [1,3]
11 (5.5%) of the 199 isolates strains were positive for at least one of the two acyl homoserine lactones (AHLs) reporter strain assays, and two of these isolates (i.e., BBD-FTL-6j and BBD-FTL-8c) tested positive in both assays. These two isolates were both positive in the patch test with the C. violaceum CV026 assay and the culture filtrates (CFs) test with A. tumefaciens NTL4(pZLR4), indicating that they produce short-chain AHLs when grown in benthic form and medium- to long-chain AHLs when grown in planktonic form
The two isolates that tested positive for both assays were each a 99% (16S rRNA gene) sequence match in GenBank to one strain of Vibrio rotiferianus; they were obtained from BBD infections on two separate coral colonies and exhibited different colony morphologies when grown on agar
Summary
Coral diseases are widely believed to play a key role in the deterioration of coral reefs on a global basis [1,2], with black band disease (BBD) identified as one of the major coral diseases contributing to this decline [1,3]. BBD is distinguished in the reef environment (Figure 1), manifesting as a dark-colored band separating healthy coral tissue from recently exposed coral skeleton. A recent microscopic study [19] documented cellular necrosis (i.e., loss of tissue confluence, cellto-cell adhesion, cytoplasmic disintegration, nuclear breakdown, and the presence of autophagous bodies, pyknotic nuclei, and apoptotic bodies) in the coral tissue surrounding cyanobacterial filaments in active BBD infections. These microscopic observations support the previous studies demonstrating the role cyanobacterial toxins in BBD pathogenicity
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