Experimental Procedures
Culture conditions and growth tracking
R. pomeroyi DSS-3 was cultured in media modified from the recipe of Rivers et al. (2016). Briefly, media (100 mL) were prepared using 0.2 μm-filtered natural seawater collected from the Scripps Institution of Oceanography pier that was autoclaved (121°C, 20 min) in 125 mL acid-washed glass Erlenmeyer flasks. Sterile-filtered (0.2 μm) glucose and nutrient stocks, including P sources, were aseptically added to the sterile seawater base in a laminar flow hood. Phosphate-replete media (+Pi) contained 18 μM P. P depleted media (-P) were prepared by adding phosphate to a final concentration of 1.8 μM P. ATP (Millipore Sigma), AMP (Fisher Scientific), 3polyP (Millipore Sigma), or 45polyP (Millipore Sigma) were added to -P media at a final concentration of 18 μM P. All media were inoculated with 50 μL of R. pomeroyi grown to stationary phase in +Pi media, in order to limit the carryover of P. Cultures were grown in a Thermo shaker/incubator at 30˚C with shaking at 150rpm for 10 days. Samples for optical density (600 nm) and flow cytometry were taken daily. Flow cytometry samples were prepared by sampling 2ml of cultures into cryovials, preserved with a final concentration of 0.5% glutaraldehyde at 4˚C for 10 minutes, and frozen at -80°C until analysis. Growth rates were calculated over the interval of log-linear growth in +Pi cultures. Growth rates in -P cultures were calculated over the same time period as +Pi cultures. All growth experiments were performed in triplicate.
DOP hydrolysis and APA competition plates (see related "Ruegeria pomeroyi DOP hydrolysis rates" dataset https://www.bco-dmo.org/dataset/897359)
Flow Cytometry
For cell counts, culture samples were preserved in filtered (0.22 μm) glutaraldehyde (0.5% final concentration), left to fix at 4˚C for 10 minutes, and frozen at -80°C until analysis. Preserved samples were thawed and counted on a Guava EasyCyte HT flow cytometer (Millipore), and instrument calibration was performed using instrument-specific beads (Luminex). Prior to running on the flow cytometer, samples were prepared in clear, round-bottom 96 well plates (Fisher Scientific) and diluted with filtered (0.22 μm) seawater either 100X (T0, T1) or 1000X (T2 - T8). Triplicate blanks prepared with filtered (0.22 μm) seawater and glutaraldehyde (0.5% final concentration) were run with samples, and the average blank cell count was subtracted from all samples. Blanks and diluted samples were stained with diluted SYBR Green nucleic acid gel stain (diluted in deionized water to 100X; Fisher Scientific) and left in the dark for 30 minutes. After staining, bacterial cell concentrations were analyzed at a low flow rate (0.24 μL s-1) for 3 minutes, and cells were counted based on diagnostic forward scatter versus green fluorescence signals.
Taxonomic Identifiers (Species, LSID):
Ruegeria pomeroyi, urn:lsid:marinespecies.org:taxname:567965
Time ranges: Experiments were performed 3/9/21 - 3/17/21. Fixed flow cytometry samples were run from 3/18/21 - 3/23/21
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