Strains and culture conditions
All strains used in this study were taken from those used for a Long-Term Phytoplankton Evolution (LTPE) experiment (Lu et al., 2025). Prochlorococcus strains were streptomycin-resistant derivatives of the high light-adapted strain MIT9312 obtained as described previously (Morris et al., 2011; Morris et al., 2008), 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 (Solomon et al., 2007)). Alteromonas strains were derivatives of strain EZ55, originally isolated from a Prochlorococcus MIT9215 culture (Morris et al., 2008). 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 -80o C. Prior to use in experiments, all Prochlorococcus cultures were grown in co-culture with Alteromonas EZ55 helpers (Morris et al., 2008) and were acclimated to culture conditions for at least 4 generations prior to data collection.
Alteromonas cultures were grown in YTSS medium (Sobecky et al., 1997) and Prochlorococcus cultures were grown in Pro99 medium (Andersen, 2005) or PEv medium (Lu et al., 2025), both made in an artificial seawater base (ASW) (Lu et al., 2025). 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 30o 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 μmol photons m-2 s-1 cool white light in a Percival incubator set to 23o C. When medium additions were employed, all solutions were filter sterilized with a 0.2 μm 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. 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).
Growth tests in conditioned media
We conducted tests using three types of conditioned media: Prochlorococcus (Pro CM), Alteromonas (EZ55 CM), and Prochlorococcus subsequently treated with Alteromonas (Pro CM + EZ55). For Pro CM, we produced axenic Prochlorococcus by adding streptomycin to a final concentration of 100 μg/mL to low-density (~106 cells mL-1) Prochlorococcus cultures. After 48 h exposure to the antibiotic, we confirmed that no Alteromonas EZ55 cells survived by transferring 1 mL into sterile YTSS medium and checking for growth after 24 hours. A 0.5 mL aliquot of this axenic Prochlorococcus culture was transferred to 12 ml fresh Pro99 media and cultivated for 11 days, after which the cells were removed by filtering the medium using a sterile 0.2 μm PVDF syringe filter (Millipore Sigma, Burlington, MA, USA). EZ55 CM and Pro CM + EZ55 were produced by inoculating washed Alteromonas EZ55 cells from YTSS medium at approximately 106 CFU mL-1 to sterile Pro99 (EZ55 CM) or to a sub-sample of the Pro CM described above (Pro CM + EZ55). As with Pro CM, these cultures were cultivated for 11 days and were then filtered to remove the cells; previous work showed that Alteromonas cell densities are stable in ASW without added C over this time frame (Hennon et al., 2017). To initiate experiments, freshly axenic Prochlorococcus (produced as described above) was transferred to replicate 12 mL tubes of each of the 3 conditioned media, and growth was measured by chl-a fluorescence every other day.
Cryo-electron microscopy (cryo-EM)
Samples of the concentrated and desalted >50kDa fractions were kept in a cool box during transport to the UAB Cryo-EM facility where they were immediately cryogenically frozen in preparation for microscopy as previously described (Hennon et al., 2017). Briefly, 3 μL of each sample were applied to glow-discharged Quantifoil R2/2 200 mesh nickel grids (Electron Microscopy Sciences, Hatfield, PA, USA) and vitrified in liquid ethane using an FEI Vitrobot Mark IV (Thermo Fisher Scientific). The grids were observed on an FEI Tecnai F20 electron microscope (Thermo Fisher Scientific) operated at 200 kV with a typical magnification of ~34,500 × and 1-3 μm defocus. 111 images were collected under low-dose conditions in SerialEM using a Gatan K3 direct detector. For single particle reconstruction, micrographs were imported to RELION-3 (Zivanov et al., 2018), and contrast transfer functions were estimated with Gctf (Zhang, 2016). Particles were picked with Topaz (Bepler et al., 2019) using a default model (resnet8_u64). A subset of highly symmetric particles were isolated via iterative 2D classification, then separated for 3D reconstruction. Matches to modeled structures were searched in the Protein Data Bank and these structures were then fit to the reconstruction using ChimeraX (Meng et al., 2023). The diameter of vesicles from CryoEM micrographs was measured manually using the Straight Line Tool in ImageJ (v1.54p). The scale was set to 1.474 pixels Å -1 , which reflects the pixel size images were measured at. A Gaussian Blur with a sigma of 1 was applied to reduce noise. For some dark and noisy images (<10 images), the contrast was also enhanced by saturating pixels by 0.3%. Diameter in Å was then converted to nm.
Sample Metrics
The following table summarizes vesicle size metrics derived from manual measurements of Cryo-EM micrographs for each sample type.
| Sample ID |
Sample Description |
Average length (nm) |
Median length (nm) |
SD (nm) |
Max length (nm) |
Min length (nm) |
n |
| 26 |
ancestor |
20.623 |
19.476 |
5.922 |
68.846 |
11.261 |
896 |
| 397 |
400 ppm |
20.903 |
19.689 |
7.295 |
125.348 |
11.363 |
413 |
| 403 |
800 ppm |
20.146 |
18.775 |
6.164 |
88.396 |
11.837 |
766 |
