Mapping Hot Spots of Metabolic Potential in Salt Marsh Sediments with Multiplexed Fluorescence In Situ Hybridization
Authors
Jeffrey J. Marlow* (jjmarlow@bu.edu), Paul Rousteau, Sandra Molnar, Ellie Bosacoma
Institutions
Boston University, Boston, MA
Abstract
Sediment-hosted microbial communities pose several challenges for direct observation and precise localization of individual organisms and their metabolic functions. High-throughput sequencing sacrifices spatial context. Static measurements complicate measures of activity, and background fluorescence can obscure stains and dyes. The team addresses these challenges by developing a novel technique for microscopic mapping of metabolic networks within salt marsh sediments. Salt marshes display high levels of microbial diversity due to elevated organic matter input and active tidal cycling, supporting a wide array of metabolisms that are centered around key genes known as “functional genes.” The project uses functional genes as a proxy for broader pathways that dictate biogeochemical cycling and focuses on carbon and sulfur metabolisms at the “hot spots” of plant root surfaces.
The team deploys fluorescence in situ hybridization (FISH)—a powerful tool that allows direct visual confirmation of a particular target nucleotide sequence at microscale resolution. FISH microscopy has focused on the 16S rRNA gene, providing reliable information on phylogenetic diversity but lacking information on metabolic function. Functional genes are ideal targets to elucidate pathway dynamics and putative interactions. By combining 16S rRNA gene FISH and functional gene FISH with multiplexed probe designs, researchers quantitatively link microbial diversity with putative pathway distributions in salt marsh sediments. This novel approach uses hybridization chain reaction FISH (HCR-FISH), a fluorescent technique that enhances the contrast between labeled microorganisms and the sediment matrix. Furthermore, several genes can be targeted in the same organisms, resulting in a diagnostic color code that allows the team to identify microorganisms and quantify distinct functional guilds at different distances with respect to plant roots and certain minerals. From these observations, researchers evaluate the spatial distribution of plant carbon exudate consumption, sulfur oxidation, sulfate reduction, and methanogenesis; such data allow initiation of a biogeochemical model of salt marsh sediments. More broadly, the team suggests that using multiplexed FISH with functional genes to complement 16S rRNA gene markers is a compelling approach for mapping hot spots in complex microbial communities.