Scientific research. There has been an explosion of research directed at understanding the mechanisms of how bacteria communicate and cooperate to perform a variety of multicellular behaviors. Not until very recently have microbiologists also begun to investigate these behaviors from the perspective of social evolution. Dr. Schuster's long-term goal is to integrate mechanistic and evolutionary approaches to investigate communication, also termed quorum sensing (QS), and cooperation in the model bacterium Pseudomonas aeruginosa. P. aeruginosa communicates via diffusible acyl-homoserine lactone (acyl-HSL) signals to coordinate the expression of hundreds of genes, many of which encode extracellular factors. Acyl-HSL QS is a widespread phenomenon in bacteria and regulates diverse group behaviors such as light production, virulence, and biofilm formation. The specific objective of this project is to conduct a comprehensive characterization of novel forms of social behavior. Initially, the focus will be on the recently discovered signal-blind obligate cheating and re-evolved cooperation phenomenon that the Schuster group identified during an experimental evolution study of P. aeruginosa. The obligate cheaters do not contribute to the production of quorum-controlled public goods because they harbor a loss-of-function mutation in the gene encoding the central QS regulator. Such mutants have been isolated from natural environments suggesting that cheaters arise also in vivo. The re-evolved cooperators emerged from cheaters are predicted to harbor mutations elsewhere in the genome that restore QS-dependent phenotypes. Very little is known about the genetic basis of such transitions from an obligate to an autonomous life style. Candidate mutations in the genome of a re-evolved cooperator point towards a novel regulatory mechanism of QS gene expression. The specific aims of this project are to (1) characterize the mutation(s) and underlying molecular mechanisms responsible for the cheater-to-cooperator conversion, (2) screen environmental P. aeruginosa isolates for the presence of social mutations and determine their frequency within the population, and (3) characterize evolutionary fitness and cooperative behavior of the cheater-turned-cooperator under social and asocial conditions. This research is relevant to the evolution of many bacteria that rely on social traits for evolutionary success, and has important implications for our understanding of the evolution of cooperative behavior across all domains of life.
Educational impact. The described project provides excellent educational opportunities for students. Dr. Schuster has and will continue to train graduate and undergraduate students in the laboratory. Dr. Schuster will also provide educational opportunities for high-school students through an established program at Oregon State University. He has found that the incorporation of evolutionary concepts in the study of microbiological problems greatly enhances student curiosity and interest. Most experiments are conceptually and technically straight-forward and are particularly well suited for the engagement of high-school and undergraduate students in the scientific process.
Our work is at the interface of microbiology and evolutionary biology. The proposed research has afforded the opportunity to understand social evolution of a model bacterium at multiple levels of biological organization. Specifically, we obtained insights into social cheating and maintenance of sociality. Bacteria are ideally suited for these studies. They reproduce quickly and can be genetically manipulated, allowing the rigorous experimental testing of evolutionary concepts in a laboratory setting. Thus far, our careful analysis of the selective pressures that allow mutations in different QS systems to invade wild type populations contributed to our understanding of the evolution of QS and the emergence of QS mutants in synthetic and natural environments. Our results indicate that non-social mutations can have a profound effect on the maintenance of social behavior. Characterization of P. aeruginosa populations from individual cystic fibrosis patients revealed great diversity of behavioral types, suggesting that the selective pressures in the lung are also diverse. Therefore, in vivo, a single selective mechanism may contribute to but fails to solely explain the emergence of behavioral variants. The observed diversity also shows that conclusions about P. aeruginosa populations in the cystic fibrosis lung cannot be drawn from the analysis of one or a few selected isolates. Finally, our investigation of the role of social conflict in the emergence of QS drug resistance provides a completely new dimension to antivirulence drug research.