Bacteria are often thought to be the very simplest organisms, but this view is being quickly replaced by an appreciation for the very complex behavior and social lives of bacteria. We now know that most bacteria in nature exist in massive, three-dimensional communities called biofilms, which are often likened to 'microbial cities'. Others even suggest that biofilms are most similar to multicellular tissues, like those that make up plants and animals. In either case, we now know that bacteria communicate through the use of chemical signals, migrate in concert to build multicellular structures, and pay a metabolic 'cost' to produce an array of shared, beneficial molecules. This last form of cooperation seems especially challenging to explain: why would evolution favor a cooperative type, if a selfish type can get all of the same benefits without paying a cost? There could be three possible answers to this question: 1) perhaps a cell that produces a beneficial molecule can itself benefit more than a 'free rider' neighbor who does not, 2) because bacterial reproduction is clonal, 'neighbors' in biofilms tend to be clone-mates, which means cooperators will tend to be around cooperators while free riders are near free riders, or 3) entire biofilms with more cooperators will contribute more offspring to the global population than biofilms with more free riders. The proposed experiments will precisely measure competition between cooperators and free riders in the human pathogen Pseudomonas aeruginosa using three-dimensional fluorescent microscopy to map the spread of each type in a growing biofilm. These experiments will strengthen an established, interdisciplinary collaboration between William Driscoll and the geneticists Leland and Elizabeth Pierson and greatly enhance the training of William Driscoll and an undergraduate assistant in methods in microbiology and confocal microscopy. Finally, the results of these important experiments will be presented at the international scientific conference Evolution in 2011 and published in a peer-reviewed scientific journal.
(Note: the aims of this project were revised after late-breaking results from a colleague cast serious doubts on the originally proposed research on bacterial biofilms.) Toxic algae are an increasing problem in the USA, especially as new invasive species spread into nutrient-fertilized areas and form highly damaging toxic blooms. Particularly in the Southwest, Prymnesium parvum, also known as ‘golden algae,’ are a major and growing problem. This species takes over entire bodies of water during the winter months by killing competing algae, zooplankton that normally control algae by grazing, and even massive numbers of fish. Even worse, P. parvum is able to consume other algae and zooplankton, so rather than stop P. parvum growth, these organisms end up providing even more nutrients to fuel P. parvum growth. Despite the major importance of P. parvum due to its destructive capacity, we know almost nothing about its genetics, or the natural variability in toxicity, growth rate, or other ecological aspects of this species. This information is especially important, considering that P. parvum has very short generation times (about one day per generation) and can thus evolve very quickly. Will P. parvum continue to become more destructive as it invades new habitats, or might evolution lead to less destructive varieties? Is there any way to selectively favor less toxic strains in habitats that are already dominated by P. parvum, potentially allowing native algae and zooplankton to coexist more successfully with this toxic species? The theory of evolution by natural selection suggests that there may be weaknesses to the P. parvum strategy. While the entire population of P. parvum can gain major benefits through ‘working together’ to produce toxins that eliminate competition and predators, individual cells may nonetheless gain a ‘selfish’ benefit by avoiding the costs of producing toxins. These ‘cheaters’ can instead use the energy that normally goes into toxin production for reproduction, and should be able to grow more rapidly than the toxic ‘cooperators’, and perhaps even replace them. In this way, evolution may lead to less toxic populations of P. parvum, but only under certain conditions. A major part of this project was to assess the different ‘strategies’ that we see in a bloom population. Are all individuals identical in these populations, or are they genetically diverse? If all individuals are from the same clone, we expect to see cooperation; if they are genetically distinct, we might see evolutionary competition between the lineages, which may lead some to act selfishly and reduce the population’s toxicity. Indeed, we found that there was considerable genetic diversity in these populations (which provides the raw materials for evolution), and some strains of P. parvum appeared more toxic than others. Interestingly, we found one strain in particular that was only very weakly toxic, but grew to much higher numbers than its toxic neighbors. This strain was susceptible to competition from other species of algae, suggesting that there may be a tradeoff between growth and toxicity in P. parvum. Furthermore, in mixed populations, the less toxic strain partially replaced the toxic strain over three weeks—suggesting that the less toxic strain may truly be a cheater, and gain an advantage over the more toxic strain when they coexist. Finally, we have sequenced the expressed genes in both toxic and nontoxic strains of P. parvum. This database allows us to see the actual RNA sequences of the genes (which can be compared to RNA sequences in better-known organisms, like yeast and plants, to better understand the function of the gene in P. parvum), and how strongly the gene is expressed. The more copies of any one gene we find, the higher the level of expression. We found several important genes were expressed at much higher levels in the toxic strain, including genes that appear likely to be directly involved in toxicity. Currently there is very little information on the genetics of toxin production in this species, and as we finish our analysis, we hope that we will add substantially to this. This knowledge will help us develop laboratory and field tools to better understand natural toxic variation among strains of P. parvum, and also the conditions that stimulate or repress toxicity in this species. This project has helped to identify the ecological roles and impacts of toxicity and the natural variation in this important aspect of P. parvum. Furthermore, we are beginning to unravel the genetics and physiology of toxicity, which will help us better understand the mode of action of toxins, and how this damaging behavior is controlled in response to the environment. This project also established the basis for a next phase of our research, to begin next year, which will further explore the cell’s behavior as they kill prey, in the hopes of better understanding this successful ecological strategy, and ultimately identifying potential weak points.
