Our goal with this project was to gain a better understanding of how bacteria influence the ecology of the microscopic algae that form the base of the open ocean food web. Our work took place on two fronts. In the first, we looked at organisms taken from an earlier experiment where we evolved bacteria and algae together at either current or projected future CO2 conditions to see how their interactions had changed. What we discovered was that the ancestral bacteria tended to help the algae grow better, but the evolved bacteria had the opposite effect. Moreover, when bacteria that evolved with one kind of algae were mixed with a different kind of algae, this effect was even more pronounced, and in the most extreme cases the bacteria slowed down the growth of the algae relative to how they grew in pure cultures by themselves. We also worked to understand the molecular causes of the changes in the interactions between algae and bacteria from the evolution experiment. When we examined the genomic changes that occurred in the evolving organisms, the biggest changes were found in the bacteria, not the algae. The bacteria we used were originally obtained from a culture of the cyanobacterium Prochlorococcus, and when they were evolved alongside algae that were very different from Prochlorococcus, their genomes changed more, and they became more competitive with Prochlorococcus. We concluded from these experiments that marine bacteria readily adapt to specialize on particular algae when they grow alongside them for long periods of time. Also, while the interactions between algae and bacteria might seem sometimes to be beneficial for both and suggest that they reflect mutualistic cooperation, in reality they are also competing over growth requirements and there can be trade-offs for adapting to one alga that make the bacteria bad partners for other algae. An unexpected discovery from the evolution experiment was that a significant number of interactions between bacteria and algae occurred through extracellular membrane vesicles (EVs) released from the cells, and that the production of EVs appeared to change over the course of evolution. We found that at least some of the growth-promoting aspects for Prochlorococcus of co-culture with bacteria could be achieved by simply purifying EVs from the bacteria and adding those to cultures instead of living bacterial cells. We found that these EVs physically associated with Prochlorococcus cells and carried several measurable enzymatic functions, including the ability to remove free radicals from the environment. We also found that evolved bacteria produced more EVs than their ancestors, and mutations in certain genes were associated with increased EV production, suggesting that the production and packaging of EVs is an intentional activity of bacteria. The other major thrust of the project was to study why Prochlorococcus and Synechococcus, two very similar algae, can co-exist with each other in cultures even though Synechococcus grows much faster and should push Prochlorococcus to extinction according to ecological theory. We found that the two were able to co-exist indefinitely across a wide range of temperatures, light levels, and nutrient concentrations; in fact, we never saw one alga disappear from any culture, and our mathematical models only predicted that one would eventually go extinct at the most extreme temperatures where one of the two were almost incapable of growth in single culture. When we looked at how gene expression changed between single algal cultures and mixed co-cultures, we found evidence of a circular interference network between the two algae and the bacteria that lived in the cultures with them. This interaction is like the game Rock-Paper-Scissors in that each organism beats one other organism and is also beaten by one. Synechococcus beats Prochlorococcus because it grows faster, but it loses to the bacteria, which are better at accessing nutrients when they grow scarce. Prochlorococcus beats the bacteria because they depend on it to produce food from photosynthesis. This result hints at one solution for the famous Paradox of the Plankton which asks why are so many kinds of algae able to live alongside each other even though they are very similar in terms of their nutritional niches. Finally, our project also supported the development and implementation of a unique undergraduate microbiology curriculum using agar art painting with bacteria as a platform for students to brainstorm and execute their own novel microbiology experiments. We were able to produce a free online textbook for our course, which was used by several institutions of higher education, ranging from community colleges to large state schools. We collected classroom data that showed the curriculum increased students self-conception of themselves as scientists, which has been shown to increase the likelihood that students will complete their degree and pursue STEM careers. Last Modified: 09/09/2024 Submitted by: JamesJMorris