A tale of two reefs: Organohalide cycling in marine sponges

Biogeochemical cycles are the pathways by which elements circulate in ecosystems. The most well-known examples of these cycles are the water, carbon, nitrogen, phosphorus, and sulfur cycles; however, there is evidence of another that remains poorly defined - the organohalogen cycle. Organohalogens, or organohalides, are organic molecules with at least one halogen atom, such as chlorine, bromine, or iodine. While often thought of as anthropogenic contaminants, organohalides are not unusual or uncommon molecules in nature. Over 8000 naturally occurring organohalide compounds have been identified, with sources including marine plants, corals, sponges, and fungi. I am particularly interested in marine sponges due to the variety of compounds they produce and their ubiquitous presence in Earth’s oceans. Despite the wealth of information concerning natural production, there is limited information available about the natural dehalogenation of organohalides in marine environments. Reductive dehalogenation by bacterial symbionts of marine sponges has been previously described, but aspects such as host-specificity and diversity of the symbionts are unclear. A detailed understanding of the organohalogen cycle is required to predict outcomes of potential disturbances such as global climate change and influx of anthropogenic halogenated contaminants. Additionally, the way organohalides cycle naturally should inform bioremediation efforts. In this work, I aimed 1) to evaluate the host-specificity of sponge microbiomes with a focus on bacteria of importance to biogeochemical cycling, and 2) to isolate and characterize sponge-associated dehalogenating organisms implicated in the natural organohalogen cycle. I hypothesized that sponge microbiomes are host-specific at the strain level, including dehalogenating bacterial symbionts. I hypothesized that the organohalide metabolites produced by sponges would select for growth of unique dehalogenating bacteria that vary based on host sponge.Sponges were collected from the Great Barrier Reef, Australia and Kāne‘ohe Bay, Hawaii. Enrichment cultures were established from homogenized sponge biomass and amended with 2,6-dibromophenol as the sole terminal electron acceptor. Debromination to phenol was monitored by high-performance liquid chromatography. Debrominating activity was maintained over successive transfers. To evaluate strain-level host-specificity of sponge microbiomes, DNA was extracted from both homogenized sponge biomass and debrominating enrichments. Ribosomal RNA operon amplicons were sequenced using the Oxford Nanopore MinION to characterize bacterial communities. Sponge microbiomes were clearly host-specific at the strain level, but this specificity did not extend to dehalogenating bacteria. Nitrospira and Synechococcus strains displayed clear host-specificity while Desulfoluna strain distribution appeared stochastic, implying different modes of transmission for individual sponge-associated bacteria.To isolate and further characterize sponge-associated debrominating bacteria, later generations of enrichment cultures were inoculated into anaerobic soft-agar tubes in a serial dilution. Colonies were removed and inoculated into liquid medium amended with 2,6-dibromophenol as the electron acceptor. DNA was extracted from debrominating enrichments and isolates for ribosomal RNA operon amplicon sequencing. Whole genomes of isolates were also sequenced. Three strains of a novel species, for which we propose the name Desulfoluna pacifica, were isolated from the sponges Cliona orientalis and Cymbastela coralliophila in the Great Barrier Reef and Suberites aurantiacus in Kāne‘ohe Bay, Hawaii. Phylogenetic analysis of rRNA operons from enrichments and isolates indicates D. pacifica is the key debrominating Desulfoluna species in Pacific Ocean sponges. This is in contrast to Desulfoluna spongiiphila found in Mediterranean sponges. Other putative dehalogenating genera, including Halodesulfovibrio and Desulfuromusa, were detected in some enrichments and represent novel clades of sponge-associated dehalogenating bacteria. One strain of a novel genus, for which we propose the name Dehalogenimusa anaincola, was isolated from a Suberites aurantiacus sponge in Kāne‘ohe Bay, Hawaii. There are eight reductive dehalogenase genes encoded by the D. anaincola genome, which may confer the ability to dehalogenate a wide range of brominated substrates.The symbiotic relationship of marine sponges and dehalogenating bacteria is likely driven by the organobromide metabolites produced by sponges. The results of this work implicate sponges as reservoirs of unique dehalogenating bacteria relevant to natural organohalide cycling. Sponge-associated dehalogenating bacteria are far more diverse than previously thought, and separation of species may be related to host location. The isolates obtained here and additional isolates from diverse locations should be further characterized to elucidate their role in natural organohalide cycling.