Collaborative Research: RUI: Life without lipopolysaccharide- Synthesis of ceramide phosphoglycerate

About This Grant

Gram-negative bacteria are enclosed by two membranes with different compositions. The outer membrane normally has LPS, a lipid with sugars attached to it. We recently discovered that a model bacterium can survive without LPS if it produces CPG2, a type of ceramide lipid. This shows the importance of ceramides in bacterial biology and provides an opportunity to study alternative modes of outer membrane construction in bacteria. The proposed research is anticipated to discover how CPG2 is made and explain how the enzymes that make it work. This knowledge will be essential to discover the numerous roles of bacterial lipids in the various environments in which bacteria live, including the human body. Because many types of bacteria produce ceramides, this advance will contribute to answering important basic and translational research questions related to the production and function of lipids in all bacteria, including those causing disease. More broadly, this project will train students at the high school, undergraduate, and graduate levels. Specifically, these students will be trained in a multidisciplinary setting to develop skills in genetics, biochemistry, and structural biology. These skills are essential to develop the new generation of researchers, and a strong biotech workforce. Gram-negative bacteria are characterized by an envelope consisting of two membranes. The inner membrane is largely composed of phospholipids and functions similarly to the eukaryotic plasma membrane. By contrast, the bacterial outer membrane is unique in that it is asymmetric with an outer leaflet comprised mainly of the glycolipid lipopolysaccharide (LPS) which creates a permeability barrier against hydrophobic molecules including many antibiotics. Given its critical role in Gram-negative bacterial physiology, the genes involved in LPS synthesis are generally essential. Recently, our lab has characterized a mutant strain of Caulobacter crescentus that survives without LPS by synthesizing a novel anionic sphingolipid with a diphosphoglycerate headgroup (CPG2). Genetic analyses identified at least four genes required for CPG2 synthesis, but their specific enzymatic functions remain unclear. We have previously demonstrated that the first enzyme in this pathway is a ceramide kinase (CERK), distinct from the eukaryotic CERK. The remaining enzymes in this pathway have little homology to known lipid-modifying enzymes and provide a platform for uncovering new enzymatic activities and mechanisms. Our central hypotheses are that 1) proteins CCNA_01225 and CCNA_01219 are responsible for adding the first glycerate to ceramide 1-phosphate (C1P) to form ceramide-phosphoglycerate (CPG) and 2) proteins CCNA_01210 and CCNA_01217 add the second phosphoglycerate to form ceramide diphosphoglycerate (CPG2). The broader impacts of this project relate to the training of a new generation of STEM researchers for the biotech workforce, at the high school, undergraduate, and graduate levels. Our multidisciplinary team will provide training in bacterial genetics, biochemistry, and structural biology. 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.