IDENTIFICATION OF GENES RELEVANT TO BACTERIAL BIOSYNTHESIS OF GLYCOSPHINGOLIPIDS

Sphingolipids and glycosphingolipids are amphiphilic molecules present in the plasma membrane of all eukaryotic cells where they play an important role in membrane structure and permeability, cell-cell recognition and interaction, and in modulating the immune response. Sphingolipids and glycosphingolipids were also found in several groups of bacteria including human pathogens, commensals and soil bacteria with a large range of metabolic capabilities. Bacterial sphingolipids were mainly characterized in two groups of Gram-negative bacteria, the genus of mutualistic bacteria - Bacteroides and in a family of soil bacteria called Sphingomonadaceae. However, very little is known about the biosynthetic pathway and the role those molecules play in bacterial physiology. Since bacterial glycosphingolipids are located on the outer leaflet of the cell membrane, they most likely take part in several important processes such as shaping the interaction of bacteria with their environment, mediate their ability to occupy unique ecological niches, and facilitate their ability to utilize recalcitrant carbon sources. To explore the connotation between sphingolipids and these processes, we sought to identify and characterize genes associated with bacterial glycosphingolipids biosynthesis. We have developed a comprehensive genetic screen based on transposon mutagenesis and detection of surface carbohydrates using various methods. The screen is targeted to identify genes relevant to the synthesis, processing and transport of bacterial glycosphingolipids. We use Sphingomonadaceae as our model system since in this family lipopolysaccharides are completely replaced by glycosphingolipids. The screen enable us to isolate mutants hampered in their ability to present sugars on the outer membrane, easily identify the associated genes and characterizing their role in glycosphingolipids biosynthesis.