Dataset: Ruegeria pomeroyi DOP hydrolysis rates

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

DOP hydrolysis rates were determined by following the production of Pi, as described previously (Diaz et al., 2018, 2019). R. pomeroyi culture samples were collected at the same time from both +Pi and -P media, according to the time period of midlog and stationary phases for +Pi cultures. Two types of samples were prepared from each culture. First, whole culture samples were diluted 1:10 or 1:20 with sterile-filtered (0.2 μm) natural seawater (Diaz et al., 2019). Second, cultures were filtered (0.22 μm) in order to generate the cell-free filtrates. Diluted whole culture and filtrate samples were then added to clear 96-well plates and amended with the DOP sources ATP, AMP, 3polyP, 45polyP, or MUF-P (18 μM, final molecular concentration) or Pi (18 μM, final). Briefly, Pi was quantified as soluble reactive phosphorus (SRP) following the method of Hansen and Koroleff (1999) using a multimode plate reader (Molecular Devices) with a detection limit of 800 nmol L-1 P (Diaz et al., 2018). Samples were reacted at 4 to 6 specific timepoints up to 24 hr. Each Pi measurement that was derived from a diluted whole culture sample was corrected for cellular Pi uptake as described previously (Diaz et al., 2019), and hydrolysis rates were calculated as the slope of Pi production over time using a simple linear regression (typically R2 > 0.95). Hydrolysis rates in diluted whole culture samples were corrected for dilution after regression analysis. To assess abiotic DOP hydrolysis, culture samples were filtered (0.2 μm) and boiled (99°C, 15 min). DOP hydrolysis in these controls was negligible.

APA competition plates

APA was assessed at midlog and stationary phases in undiluted whole culture samples and cell-free filtrates in the presence and absence of DOP sources by following hydrolysis of the fluorogenic substrate MUF-P (Millipore Sigma). MUF-P hydrolysis was tracked using a multimode plate reader (Molecular Devices), using 359 nm and 449 nm as the excitation and emission wavelengths, respectively. Final molecular concentrations of DOP substrates were 0-100 μM (Pi as a control, ATP, AMP, 3polyP, and 45polyP). The final concentration of MUF-P was 10 μM. Experiments were run in kinetic mode for 15 minutes collecting data every 30 seconds. Enzyme activity was calculated as a percentage of the control hydrolysis rate (no DOP added) to illustrate inhibition of MUF-P by the unlabeled DOP substrates. IC50 values for each inhibiting DOP substrate were calculated from the linear regression of log normalized substrate concentrations versus the percent inhibition of MUF-P by each substrate (R2 typically >0.90). IC50 values were calculated according to the formula: IC50 = (0.5-b)/a, where b is the y-intercept and a is the slope of the linear regression.

Flow Cytometry (see Related dataset " Ruegeria pomeroyi OD and FCM"  https://www.bco-dmo.org/dataset/897371).

Taxonomic Identifiers (Species, LSID):
Ruegeria pomeroyi, urn:lsid:marinespecies.org:taxname:567965