Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • BCO-DMO Processing Description
• Parameters
• Instruments
• Project Information
• Funding

This lab experiment was conducted at Wellesley College, Welleslsey MA in August 2024. Alteromonas macleodii MIT1002 were grown at 24 °C in a natural seawater-based media containing 0.2 µM filtered, autoclaved seawater supplemented with 3 mM NH₄Cl, 50 µM NaH₂PO₄, Pro99 trace metals, and 0.01% (w/v) glucose. Overnight cultures of Alteromonas were washed twice in media lacking added glucose (-C). Cultures were diluted to 5e6 cells/mL and supplemented with with either 1x PBS (negative control), Prochlorococcus extracellular vesicles, Alteromonas extracellular vesicles, or glucose as a carbon source, each in triplicate. Cultures were incubated at 24°C with shaking at 120 rpm. Samples were collected at two timepoints (representing early and late exponential growth phase) by pelleting at 10,000 xg for 15 minutes, flash freezing on dry ice, and storing at -80 °C. Cells were resuspended and lysed in 600 µL Zymo TriReagent and RNA was extracted using the Zymo Trizol RNA Extraction RNA spin column kit following the manufacturer’s protocol. The columns were washed and treated with DNAse per manufacturers recommendation. RNA was eluted in 50 µL of RNase-free water and quantified using the Qubit RNA High Sensitivity kit (Invitrogen). Ribosomal RNA was depleted using the Qiagen QIAseq FastSelect kit, then the Kapa RNA Hyperprep kit. Paired-end 150nt sequencing data was generated on an Illumina NovaSeq X. RNA library preparation and sequencing was carried out by the  Bauer Core Facility (Harvard University).

- Loaded "Alteromonas_GEO_accessions.tsv" (TSV format) using filename as resource name, with "nd" and empty strings treated as missing values
- Deleted field "GEO_link"
- Reordered fields to: Biosample_ID, SRA_Experiment_ID, GEO_Sample_ID, Bacterium, Carbon_Substrate, Timepoint, Replicate
- Renamed resource to "997637_v1_alteromonas_geo_accessions"
- Output saved as 997637_v1_alteromonas_geo_accessions.csv

NSF Award Abstract:
Microbial production and consumption of organic carbon play critical roles in the marine food web and global carbon cycling. Bacteria release organic matter in a variety of chemical forms and in diverse contexts, ranging from individual molecules to small aggregates and larger biological particles. In recent years we have come to understand that most, if not all, marine microbes release nanoscale structures called extracellular vesicles from their surfaces. These discrete particles, which are abundant in the oceans, are capable of transporting multiple classes of organic molecules between organisms and can serve as a potential nutrient source for other microbes. Extracellular vesicles thus represent a potentially important component of marine microbial food webs, but the magnitude and dynamics of this contribution are unknown. Further, the packaging of material within vesicles may influence the accessibility of this organic material as compared with truly ‘dissolved’ substances to different groups of marine organisms, potentially biasing nutrient exchanges. Broader impacts of this work is providing hands-on research experiences for female undergraduate students - including those from groups historically underrepresented in STEM fields - and training in data analysis tools.

The goal of this project is to advance the understanding of the role that extracellular vesicles play in marine dissolved organic carbon pools and microbial food webs. To determine the contribution of vesicles to organic matter release by marine microbes, the investigators are quantifying the fraction of excreted carbon and nitrogen associated with vesicles released by multiple marine cyanobacteria and heterotrophs. The project is examining how vesicles are ‘consumed’ by heterotrophs to calculate a mass balance of vesicle utilization and produce detailed gene expression data to explore how cells respond to the presence of vesicles. Finally, experiments with coastal and oligotrophic marine communities are providing insights into which organisms utilize vesicles in the field, and whether they are broadly accessible to all microbes or are instead preferentially consumed by a subset of microbes. Collectively, these experiments are opening up a new area of research into the mechanisms underlying the microbial loop and provide foundational insights into the roles of extracellular vesicles in ocean ecosystems.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.