Dataset: BB2-AT2 Cell Metal Quota

Location: All physiological work within this dataset was conducted at Woods Hole Oceanographic Institution on a strain of Pseudoalteromonas collected from Scripps Pier, California coast in 1995.

Referenced from Mazzotta, et al. in press:
Marine broth medium was prepared by microwave sterilization49 of 800 mL of 0.2 µm-filtered Vineyard Sound seawater with the addition of 0.2 µm-filter-sterilized solutions of 4 g peptone (Fisher Scientific) and 0.8 g yeast extract (BD Difco). Marine agar plates were prepared with the same composition as that of the marine broth described herein, with the inclusion of 12 g granulated agar (Fisher Scientific) and sterilization achieved by autoclaving for 30 min. Cultures of Pseudoalteromonas sp. BB2-AT2 were revived from a 20% glycerol stock of BB2-AT2 (provided by Kay Bidle, Rutgers University) and used to inoculate approximately 1 mL of marine broth. The inoculated solution was allowed to incubate at 23 °C for 3 hours, then streaked onto a marine agar plate with 50 µL inoculum and incubated at 23 °C overnight. Pellets of BB2-AT2 were grown in volume 500 mL of marine broth medium by inoculation at 23 °C with selection of a single colony from a marine agar plate. Growth curves were obtained with a SpectraMax Me5 unit (Molecular Devices) through absorbance measurements at 600 nm pipetting aliquots into a Corning clear 96 well plate at each time point. Samples were analyzed at 23 °C with a kinetic run over a period of 18 hours with a read interval of 30 minutes, with aliquots added at every timepoint. To validate this approach these plate reader growth rates were successfully intercompared with growth measurements using a Shimadzu UV-1601 spectrophotometer in a quartz cuvette (b = 1 cm) with a 2.5 mL aliquot of inoculated medium (Supplementary Information). Pellets were harvested in mid-exponential phase by centrifugation at 8,000 rpm for 20 minutes using an Eppendorf 5810R centrifuge at 3 °C, the solution was decanted, and the pellet washed with 0.2 µm-filtered seawater (4 mL x 3). Pellets of biomass

The measured quotas reflect contributions of intracellular and extracellular metals. Biomass from triplicate 10 mL cultures were centrifuged at 8,000 rpm for 20 minutes at 3 °C at mid-exponential phase of growth. The cell pellet was washed by resuspension in ~1 mL filtered seawater, transferred to an acid-cleaned microfuge tube and centrifuged again for 15 min at 14,000 RPM at 4 °C. The whole cell pellet was digested in 800 μl of 5% trace metal grade HNO3 (Optima) containing 1 ppb Indium (In) then cells were resuspended multiple times. After digesting for 7 days at room temperature solids were removed by centrifugation, and supernatants were collected for quota analysis. Process blank digestions containing acid but no cells were performed in parallel. Digests were diluted by a factor of 10 with additional 5% nitric acid (also containing 1 ppb In), transferred to acid-washed 96 deepwell plates (Thermo Scientific) before being analyzed on an ICAP-Q inductively coupled plasma-mass spectrometer (Thermo) with an SC-4 DX FAST autosampler (Elemental Scientific, Inc.). Metal concentrations were calibrated to a multi-element standard curve (Spex, Certiprep) over a range of 1 – 50 ppb. Phosphorus concentrations were also measured by ICPMS and calibrated to a separate standard curve ranging from 100–1500 ppb using a stock solution of Na3PO4. Samples were analyzed in KED mode using helium as a collision gas after a 45 s sample uptake window. Mass windows were scanned 3- times during measurements. In was used to correct for variation in sample delivery and plasma suppression between samples. Blanks were subtracted from measured concentrations. Technical replicates (n=3, 3, 2) were averaged for each biological triplicates (Supplemental Information). Averages of biological replicates and their standard deviation were then calculated.

Sample metadata:
All culture samples grown in replete marine broth (see Experimental Procedure for details)
BB2-AT2_1_1 BB2-AT2 biological replicate 1, technical replicate 1
BB2-AT2_1_2 BB2-AT2 biological replicate 1, technical replicate 2
BB2-AT2_1_3 BB2-AT2 biological replicate 1, technical replicate 3
BB2-AT2_2_1 BB2-AT2 biological replicate 2, technical replicate 1
BB2-AT2_2_2 BB2-AT2 biological replicate 2, technical replicate 2
BB2-AT2_2_3 BB2-AT2 biological replicate 2, technical replicate 3
BB2-AT2_3_1 BB2-AT2 biological replicate 3, technical replicate 1
BB2-AT2_3_2 BB2-AT2 biological replicate 3, technical replicate 2

Instruments:

All work performed in N2–filled anaerobic chamber (Coy Laboratory Products Inc.).
Metals analyzed on iCAP-Q inductively coupled plasma-mass spectrometer (Thermo) with an SC-4 DX FAST autosampler (Elemental Scientific, Inc.).