RUI: Understanding the Biosynthesis and Function of Headgroup Acylated Glycerophospholipids in Escherichia coli

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Intellectual MeritLipids form the semi-permeable bi-layer membrane that surrounds all cells and organelles, compartmentalizing important cellular reactions and metabolites. There is a large diversity of lipids required to form a functional membrane. Glycerophospholipids (GPLs), which contain a glycerol phosphate backbone with two fatty acyl chains esterified to the glycerol and an alcohol added to the phosphate, compromise a significant portion of the membrane lipids. In addition to the major GPLs, E. coli also contains a variety of low abundance lipids whose function is not well understood. One such class of low abundance lipids contains an additional acyl chain added to the headgroup. Headgroup-acylated lipids have been previously shown to impact essential cell functions such as cell division. This research program will characterize two enzymes that impact headgroup-acylated GPL levels in E. coli in order to further shed light on the biological function of headgroup-acylated GPLs. The E. coli enzyme PldB catalyzes the formation of the headgroup-acylated GPL, acyl phosphatidylglycerol (acyl PG) by transferring an acyl chain from a monoacylated GPL (lyso GPL) to the headgroup of PG. The Arabidopsis thaliana enzyme At1g78690p, which acylates lyso GPLs to form di-acylated GPLs in vitro, interestingly, leads to the accumulation of the headgroup-acylated GPL, acyl PG, when over-expressed in E. coli. This A. thaliana protein is a useful tool for probing the interaction between lyso GPL and headgroup-acylated GPL metabolism. In order to illuminate how lyso GPL and headgroup-acylated GPL metabolism interact the substrate specificity of PldB and At1g78690p will be determined. A variety of acyl donors and acyl acceptors will be tested to determine the substrate preference for each enzyme. The enzymatic properties of PldB and At1g78690p will also be investigated. Amino acids critical for catalysis and substrate preference will be identified by testing the activity of mutated PldB and At1g78690p with different acyl donors and acceptors. The biological role of headgroup-acylated GPLs will also be investigated by genetically manipulating E. coli lipid biochemical pathways. Enzymes that alter the levels of headgroup-acylated GPLs will be introduced into wild-type and mutant E. coli strains. For each strain, alterations to the cellular lipid composition will be assessed using liquid chromatography electrospray ionization mass spectrometry. In addition, the growth rate, antibiotic sensitivity and temperature sensitivity of the strains will be tested to determine the phenotypic impacts of alterations of headgroup-acylated GPL levels on cells in a variety of genetic backgrounds. This work will provide the basis to understand the function of these lipids and help, in future work, explain the complexity of lipids found in all cells.Broader ImpactThis research program will impact the field of lipid biochemistry by helping to explain the interaction between lyso GPL and headgroup-acylated GPL metabolism. In addition, undergraduates at Vassar College will have opportunities to engage in substantial research in lipid biochemistry. Every aspect of this research will directly involve undergraduate student researchers. Students will prepare lipid substrates and protein extracts, perform the in vitro enzyme assays, purify the enzymes to homogeneity, and construct the E. coli. They will present their work at scientific conferences as poster and oral presentations and publish in peer-reviewed journals. Particular attention will be given to involve of students at all levels, including freshman and students from under-represented groups, in research leading to increased retention of these students in STEM fields.This project is jointly supported by the Biomolecular Dynamics, Structure and Function Cluster in the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes program in the Chemistry Division.