Determining the function of sterol lipids in the bacterial domain

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The overall goal of this project is to determine the functions cholesterol-like lipid molecules have in bacteria. Cholesterol plays many essential roles in the biology of higher organisms, but the importance of cholesterol-like lipids in simpler organisms like bacteria is not well understood. The hypothesis to be tested here is that sterols allow bacteria to sense and react to low levels of oxygen that adversely affect their ability to grow. The work has the potential to expand our understanding of a fundamental cellular pathway and can inform future studies that have broad societal impacts - from human physiology to response to environmental change. This project will also contribute to the goal of broadening participation in STEM fields by providing scientific training for postdoctoral scholars as well as graduate and undergraduate students from diverse backgrounds. Undergraduate underrepresented minority students will participate in this project through two summer programs at Stanford to undertake research projects that are directly relevant to the scientific goals of this proposal. Further, a seminar series that will bring early career underrepresented scientists to Stanford to present their research and interact with students and postdoctoral scholars will be developed and executed by the principle investigators, both of whom are underrepresented minority women scientists themselves.In this project, a variety of molecular studies will be undertaken to investigate the functional role of cholesterol-like lipids, or sterols, in the bacterium Methylococcus capsulatus. M. capsulatus, an environmentally relevant microbe that grows by consuming methane and oxygen, was the first bacterium shown to produce sterols over 40 years ago. However, the function of these very important lipids in bacteria remains a mystery. This project will test the hypothesis that the biosynthesis of certain sterol structures within this microbe signals to the cell that it is experiencing low oxygen stress (termed hypoxia), a condition that adversely affects the bacterium's physiology. To test this hypothesis, the researchers will: (1) determine how varying oxygen levels affect sterol synthesis in M. capsulatus; (2) identify and characterize proteins necessary for transporting specific sterol structures to the outer bacterial membrane under different oxygen conditions; and 3) determine what genes are turned on and off when M. capsulatus sterol-deficient mutant cells are grown under oxygen-replete and oxygen-limited conditions. Together, these studies have the potential to identify unique sterol-binding proteins and transporters, as well as novel sterol regulatory mechanisms in bacteria. This type of a regulatory role for sterols has been shown in eukaryotes but would represent a novel function in the bacterial domain.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.