Dataset: Flow cytometric analysis of interaction between Alteromonas macleodii exudates and Prochlorococcus MIT9312 cells

Strains and culture conditions:
All strains used in this study were taken from those used for a Long-Term Phytoplankton Evolution (LTPE) experiment (1). Prochlorococcus strains were streptomycin-resistant derivates of the high light-adapted strain MIT9312 obtained as described previously (2, 3), either before (Ancestor) or after 500 generations of evolution at either 400 ppm or 800 ppm pCO₂ conditions (i.e., modern day or projected year 2100 conditions (4)). Alteromonas strains were derivatives of strain EZ55, originally isolated from a Prochlorococcus MIT9215 culture (3). As with our Prochlorococcus strains, we used both ancestral and evolved varieties of EZ55 co-evolved with Prochlorococcus at the two pCO₂ treatments and subsequently isolated. Prochlorococcus cultures were revived from cultures cryopreserved with 7.5% DMSO in liquid nitrogen vapor, and Alteromonas cultures were revived from cultures preserved with 20% glycerol stored at -80^o C. Prior to use in experiments, all Prochlorococcus cultures were grown in co-culture with Alteromonas EZ55 helpers (3) and were acclimated to culture conditions for at least 4 generations prior to data collection.

Alteromonas cultures were grown in YTSS medium (5) and Prochlorococcus cultures were grown in Pro99 medium (6) or PEv medium (1), both made in an artificial seawater base (ASW) (1). Prior to addition to co-cultures Alteromonas strains were pelleted at 2000 g for 2 minutes and washed twice in sterile ASW, then added to cultures at approximately 10⁶ cells ml^-1. Alteromonas was grown at 30^o C with 120 rpm shaking. Unless otherwise noted, Prochlorococcus and co-cultures were grown in static 13 mL conical bottom acid-washed glass tubes under approximately 75 mmol photons m^-2 s^-1 cool white light in a Percival incubator set to 23^o C. When medium additions were employed, all solutions were filter sterilized with a 0.2 mm filter. Cell densities of Prochlorococcus cultures to standardize inoculations between experiments were determined using a Guava HT1 flow cytometer (Luminex Corporation, Austin, TX) by the distinctive signature of these cells on plots of forward light scatter vs. red fluorescence (Fig. S1A). Day-to-day culture growth was tracked using the in vivo chlorophyll a module for the Trilogy fluorometer (Turner Designs, San Jose, CA) with a custom 3D-printed adapter designed for conical bottom tubes. Fluorometer measurements and cell counts were linearly related across the range of cells examined in this study (Pearson correlation coefficient 0.835, p = 1.38 x 10^-6, Fig. S1B).

Concentration of Alteromonas exudates:
EZ55 was grown in Pro99 media supplemented with 0.1% glucose to sustain growth in the absence of Prochlorococcus exudates. We scaled cultures up progressively from 12 mL to 2 L. The 2L culture was grown in a vented bottle with an outlet connected to a filter with 0.22 μm pore size. After removing most of the cells by centrifugation, we produced size-fractionated, concentrated exudates using tangential flow filtration using Sartorius Vivaflow 200 cassettes. The 2L culture supernatant was passed first through a 0.22 μm cassette using a Masterflex L/S peristaltic pump (Cole-Parmer) to remove bacterial cells, then through a 50 kDa module and a 5 kDa module in succession to produce >50 kDa and <50 kDa fractions that were each concentrated approximately 100-fold. A portion of the >50 kDa fraction was placed in boiling water for 5 minutes to denature proteins. When these concentrated extracellular products were added to culture media for growth experiments they were diluted 100-fold, returning them to approximately their original concentration prior to filtration.

Exudate interaction with Prochlorococcus: In order to visualize interactions between exudates and Prochlorococcus cells, we covalently labelled the >50 kDa concentrates with an amine-reactive Alexa Fluor 488 5-SDP ester dye (Molecular Probes/ThermoScientific) (7). First, 100 μL of each sample was concentrated with 100 kDa centrifugal ultrafilters (Pall) at 14,000 rpm for 15 minutes and then resuspended with 0.1 M sodium bicarbonate buffer (pH 8.3). Then, 2.5 μL of Alexa Fluor 488 5-SDP ester dye was added and incubated for 1 hour in the dark at room temperature with gentle mixing. The mixture was washed by centrifugation at 14,000 rpm for 15 minutes, removal of the supernatant, and resuspending with 1x PBS three times, with a final resuspension in 100 μL ASW. The labeled and washed exudate was added to 100 μL axenic Prochlorococcus culture and incubated for 2h at 28 ℃ with 120 rpm orbital shaking. Control samples were prepared with Prochlorococcus cells only or with Prochlorococcus stained with Alexa Fluor 488 5-SDP ester dye but without added exudate concentrate. Cells were analyzed with a Guava HT1 flow cytometer tracking cells by size (forward scatter), chlorophyll (red) fluorescence, and dye (green) fluorescence. Flow cytometry data was visualized using the online Floreada platform ( see https://floreada.io). Cells were also examined by using a Nikon 80i epifluorescence compound microscope using the GFP and Texas Red filter cubes. Images were collected and processed using Nikon NIS-Elements imaging software.