Award: OCE-2046056

The goal of this project was to understand which fungi, bacteria and archaea are active in marine deep sea sediments under close to in situ conditions. During expedition 385 of the International Ocean Discovery Program sediment samples to a depth of down to 154 meters below the seafloor were obtained and incubated on-ship in the presence of the synthetic amino acid L-homoprogargylglycine. Any cells that were active during the several day long incubation took up this amino acid and replaced some of the methionine in proteins that were newly synthesized during time of incubation. At the end of the incubation, samples were preserved for later analyses in the lab. After protocols to efficiently remove cells from sediment particles had been developed, cells were extracted from all samples and newly made proteins in all cells stained with a fluorescence stain using azide-alkyne click chemistry. Cells that were dye-stained (and thus had undergone protein synthesis) were then separated from unstained cells (cells that had not taken up the amino acid or which non active) via fluorescence-activated cells sorting. Sorted cells were lysed and their taxonomic marker genes amplified via polymerase chain reaction. Specifically, a small region of the 16S ribosomal RNA genes of bacteria and archaea and the Intergenic Spacer Region of fungi was amplified. Amplicons from all samples were then sequenced to determine the identity of cells that were metabolically active at the time of incubation. Out results demonstrate that a diverse community of marine fungi, bacteria, and archaea thrive in the deep marine subsurface down to a depth of at least 154 meters. We for the first time applied bioorthogonal labeling and click chemistry to marine subsurface samples and established new cell extraction and labeling protocols that can be widely applied to many marine samples. This will enable other researchers to apply this cost-effective and generalizable technique to answer their specific research questions. Our project provided a unique training opportunity and research experience for a postdoctoral scholar, a graduate student, and a veteran and first generation undergraduate college student. Last Modified: 05/06/2024 Submitted by: RolandHatzenpichler