RUI: Mechanisms and physiological functions of bacterial sphingolipids

All cells are surrounded by a membrane which serves as a barrier between the contents of the cell and the outside world. In addition to their barrier function, membranes contribute to the mechanical stability of the cell. A major component of these membranes are lipids; these lipids have water-soluble heads that are oriented towards the outside of the membrane and oil-soluble tails facing the inside. There are numerous types of lipids that vary in their headgroups and tails, and the focus of this research is on sphingolipids. Sphingolipids are found throughout plants, fungi, and animals and were recently shown to be widespread in bacteria as well. Since the pathway for producing sphingolipids in bacteria was only recently discovered, there are many questions remaining about their synthesis and function. The goals of this project are to 1) determine the localization of the sphingolipid synthetic enzymes within bacteria, 2) identify the genes required for transporting the sphingolipids within the cell, and 3) use computational and genetic tools to understand how sphingolipids impact the structure and function of the bacterial outer membrane. The Broader Impacts of this project are to provide students with computational and quantitative skills necessary for modern biological research as well as stimulate excitement in pursuing STEM careers. Students at the LEAP Academy High School in Camden, NJ will have summer research internships in which they will learn bacterial genetics. Undergraduates from underrepresented minority groups will be recruited to the biology honor’s thesis program and graduate students in Computational and Integrative Biology will receive cross-disciplinary training in experimental and computational approaches for studying bacterial membranes.Sphingolipid are found ubiquitously in eukaryotes from fungi, to plants, to animals. By contrast, these lipids have been reported in only a small number of bacterial taxa. Despite the limited number of sphingolipid-producing bacteria, there is a wide range of physiological roles for sphingolipids including modulation of host-microbe interactions, protection from bacteriophage, bacterial life cycle and sporulation, and microbial predation. The recent identification of the complete pathway required for bacterial sphingolipid biosynthesis showed that sphingolipid synthesis is, in fact, far more widespread among bacteria than previously thought. The objectives of this project are to characterize the spatial organization of sphingolipid production and the mechanism of their transport to the outer membrane and computationally investigate how ceramides affect outer membrane functionality and biophysical properties. Specifically, a series of biochemical and genetic analyses will determine whether these enzymes are soluble or membrane-anchored, as well as whether their activity is in the cytoplasm versus the periplasm. The role of LptF/G and LptC homologues will be investigated for their role in sphingolipid transport. Lastly, Molecular Dynamics simulations (MD) will be used to assess lipid orientation, lipid clustering, and interactions between sphingolipids and membrane proteins and antibiotics. The predicted interactions identified by MD will be experimentally validated in C. crescentus. The expected findings will provide a vertical advance in the field of bacterial cell biology by contributing to critical basic research questions related to bacterial membrane synthesis and function.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.