The Design of a PCR-Based Assay to Detect and Isolate the Serine Palmitolytransferase Gene From Environmental Bacteria

Sphingolipids are a natural class of lipids that function as structural elements of cell membranes and signaling molecules for important cellular activities such as cell growth, differentiation, apoptosis, recognition, and adhesion. These lipids can be found universally in eukaryotic cells as well as some species of bacteria, such as those found in the human gut microbiome and in the environment in soils. Though sphingolipid production is rare in bacteria, both eukaryotic and prokaryotic sphingolipid biosynthesis begin with the condensation of serine and palmitoyl CoA into 3-ketodihydrosphingosine catalyzed by the enzyme serine palmitoyltransferase (SPT). In recent years, several studies have shown the connection between sphingolipid homeostasis and various diseases such as Alzheimer’s disease and hereditary sensory neuropathy type I disease. Furthermore, these lipids play a significant role in the innate immune system as mediators of the inflammatory response and the CD1d pathway. These various biological functions and ready access to the compounds from bacterial producers have presented sphingolipids as possible targets for therapeutic development. However, our knowledge of bacterial sphingolipids and their therapeutic potential is limited. The purpose of this research is to develop an effective means of identifying environmental sphingolipid producers for further analysis of the structure of their sphingolipids and characterization of their bioactivity. Because the SPT gene is a commonality between all sphingolipid producers, our research has centered around creating a set of polymerase chain reaction (PCR) primers that can accurately test for the presence of this gene amongst bacterial isolates from soil. Using previous literature and our own sequencing data, we have developed a set of primers that can detect the presence of the SPT gene in species of Sphingomonas bacteria and some species of Novosphingobium, both known sphingolipid producers. Additionally, our team has been working on using various methods of selective culturing to add to the efficiency of the screening process and optimize the procedure of finding environmental sphingolipid producers. Future work on this project will continue to analyze genetic sequences to create a complete set of primers that can isolate naturally occurring sphingolipid producers across several different species. Overall, we aim to create a screening technique that is efficient and useful in discovering more sphingolipid-producing bacteria from the environment with the hopes of isolating novel sphingolipids for development as medicines