Production and fate of dimethylsulfoniopropionate (DMSP) in reef-building corals and its integral role in coral health

Bacteria play crucial roles in most biogeochemical cycles in the oceans because of their high abundance and metabolic capabilities. Each square centimetre of coral surface harbours between 10⁶ and 10⁸ bacterial cells, and significantly, bacterial assemblages tend to be highly specific to their coral host. Although the phylogenetic diversity and dynamics of coral-associated bacterial communities have been studied for more than a decade, their ecological and functional roles in the coral holobiont are still poorly understood. The taxonomic composition of these bacterial communities is likely to be greatly influenced by chemicals produced by coral hosts, as well as by their endosymbiotic algae Symbiodinium. Dimethylsulfoniopropionate (DMSP) is a ubiquitous compound found within reefbuilding corals and is a central molecule in the marine sulfur cycle, particularly as a precursor to the climate-regulating gas dimethylsulfide (DMS). Marine bacteria are the primary organisms that degrade DMSP into DMS, and consequently play a critical role in linking the marine environment and the atmosphere in the global sulfur cycle. To date, the role of these organic sulfur compounds in the metabolism of coral-associated bacteria has not been investigated. Consequently, this thesis aims to provide new insights into the roles of DMSP in corals, and more specifically in coral-bacterial associations, with a particular focus on the production and metabolism of this sulfur molecule.\ud \ud To investigate the roles of DMSP in corals, I developed a new direct approach to accurately and rapidly quantify DMSP and one of its breakdown products, acrylate, based on quantitative nuclear magnetic resonance (qNMR) spectroscopy (Chapter 2). This method overcomes inaccuracies associated with indirect methods that convert DMSP to DMS and measure this volatile molecule. The method was tested on a range of coral genera, and enabled simultaneous and direct quantification of multiple molecules from the same extract, as well as rapid processing with high reproducibility. Thus large numbers of samples can be processed in short time periods. The method was successfully applied to environmental samples and provides the first baseline information on diel variation of DMSP and acrylate concentrations in the coral Acropora millepora. The lack of diel variation found raises questions about the role of endosymbiotic dinoflagellates in DMSP biosynthesis in corals.\ud \ud Reef-building corals are among the most prolific DMSP producers in the ocean, but their DMSP production has been attributed entirely to the activities of their algal symbiont, Symbiodinium. Combining chemical, genomic and molecular approaches, I show that coral juveniles from the genus Acropora produce DMSP in the absence of associated microalgae (Chapter 3). DMSP levels increased through time (by up to 54% over 6 days) in coral juveniles raised without access to photosynthetic symbionts. Increased DMSP levels in juvenile and adult corals exposed to experimentally elevated temperature treatments suggest a role for DMSP in thermal stress responses. Discovery of coral orthologs of two algal genes recently identified in DMSP biosynthesis suggests that corals possess the enzymatic machinery necessary for DMSP production. My findings overturn the current paradigm that photosynthetic organisms are the sole biological source of DMSP, and highlight a direct role for corals in climate regulation.\ud \ud In order to investigate the influence of DMSP and DMS on coral-associated bacteria, the bacterial communities of two coral species, Acropora millepora and Montipora aequituberculata, were characterized by both culture-dependent and molecular techniques (Chapter 4). Three genera, Roseobacter, Spongiobacter, and Alteromonas, which were isolated on media with either DMSP or DMS as the sole carbon source, comprised the majority of bacterial communities in these two corals based on both clone library and pyrosequencing approaches. Bacteria capable of degrading DMSP represented 37% of the communities in Montipora and between 67 and 92% in Acropora. These results demonstrate that DMSP and potentially DMS act as nutrient sources for coral-associated bacteria, and that these sulfur compounds are likely to play a role in structuring bacterial communities in corals. Exploration of the publically available metagenome databases revealed that genes implicated in DMSP metabolism are abundant in the viral component of coral-reef-derived metagenomes, indicating that viruses can act as a reservoir for such genes (Chapter 4).\ud \ud The metabolic potential of bacteria in pure culture does not necessarily reflect their metabolic activities within the coral holobiont, therefore I used state-of-the-art imaging techniques (NanoSIMS), coupled with analytical chemistry approaches, to determine linkages between DMSP-synthesising Symbiodinium and DMSP-degrading bacteria (Chapter 5). DMSP-degrading bacteria were coincubated with Symbiodinium cells previously grown in a medium with isotopically labelled sulfate as sole sulfur source. This experiment confirmed that the sulfur used for DMSP biosynthesis comes from sulfate assimilation in Symbiodinium and enabled visualization of sulfur isotope hotspots adjacent to Symbiodinium cells that correlated with the location of bacteria observed with electron microscopy. These results confirm the role of coral-associated bacteria in the sulfur cycle and constitute the first empirical evidence of the bacterial assimilation of Symbiodinium secondary metabolites in vivo.\ud \ud Bacterial communities associated with healthy corals have been suspected to produce antimicrobial compounds that inhibit the colonization and growth of invasive microbes and potential pathogens; however, antimicrobial molecules derived from coral-associated bacteria have not been identified. In chapter 6, I describe the isolation of an antimicrobial compound produced by Pseudovibrio sp., a bacterium commonly associated with reef-building corals and able to degrade dimethylsulfoniopropionate (DMSP). Bioassay-guided fractionation and spectroscopic techniques, including NMR and mass spectrometry (MS), identified the antimicrobial as tropodithietic acid (TDA), a sulfur-containing compound likely derived from DMSP metabolism. TDA was produced in large quantities by Pseudovibrio spp. and prevented the growth of two known coral pathogens, V. coralliilyticus and V. owensii, at very low concentrations (0.5 μg/mL) in agar diffusion assays. Its production was significantly reduced at temperatures causing thermal stress in corals, indicating a role for DMSP-metabolizing bacterial communities in coral disease prevention under ambient temperatures and the potential disruption of this protection during thermal stress events.\ud \ud In summary, this thesis presents novel information on the production and fate of DMSP in reef-building corals. It identifies the coral animal as a DMSP producer, provides corroborative evidence of the important role of DMSP for numerous coral-associated bacteria using both in vitro and in vivo approaches, and isolates an antimicrobial compound likely derived from DMSP metabolism. Together, these results constitute the first comprehensive study of DMSP in reef-building corals and underscore the remarkable contribution of this molecule to coral health