Microscale spatial architecture of "pink berry" microbial aggregates and their symbiotic single-cell interactions using BONCAT-CARD-FISH fluorescence imaging

Bacterial symbioses enable microbial metabolisms, such as cryptic sulfur cycling, that escape bulk measurements and can thus only be discerned using microscale methods. While metagenomic information can help us hypothesize on the potential metabolic interactions taking place within a given sample, single-cell level resolution allows us to test these hypotheses and reveal what metabolic interactions are actually taking place therein. "Pink berries", microbial aggregate communities found at the Sippewissett salt marsh in Falmouth, MA, were probed with various electron donors, as well as sulfidic and light/ dark conditions in order to explore the cell-cell interactions between its different microbial species via BONCAT-CARD-FISH. "Pink berries" were used in this study as they are tractable, model microbial communities dominated by two symbiotic bacteria: a purple sulfur bacterium (PSB) and a sulfate reducing bacterium (SRB). These two organisms participate in a cryptic sulfur cycle where the PSB oxidizes sulfide to sulfate, which the SRB reduces back to sulfide. How, then, do various electron donors influence the cryptic cycling of sulfur by the SRB? How does this change as the symbiont is exposed to light and dark conditions? How does this affect other abundant members of the pink berry community? BONCAT (biorthogonal non-canonical amino acid tagging) is a click-chemistry-based assay for new protein synthesis that allows single cell visualization of uncultured microbial activity using fluorescence microscopy, offering a cheaper, viable alternative to nanoSIMS analyses. By pairing this with CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization), one can further identify the phylogenetic identity of these active microbes. Specifically, we were able to observe heterogeneous activity at a microscale throughout the aggregates within six hours of exposure to the non-canonical amino acid. Light/dark conditions, as well as incubations with sulfide and various electron donors resulted in distinct hot spots of microbial activity. This microscale spatial variation reveals differences in the rate and identity of microbial activity, highlighting the need to use microscale methods in order to understand how microbial metabolism drives biogeochemistry in various aquatic environments.