Making the Enterobacterial Common Antigen Glycan and Measuring Its Substrate Sequestration.

The enterobacterial common antigen (ECA), a three-sugar repeat unit polysaccharide produced by Enterobacteriaceae family members, impacts bacterial outer membrane permeability, and its biosynthesis affects the glycan landscape of the organism. ECA synthesis impacts the production of other polysaccharides by reducing the availability of shared substrates, the most notable of which is the 55-carbon polyisoprenoid bactoprenyl phosphate (BP), which serves as a carrier for the production of numerous bacterial glycans including ECA, peptidoglycan, O-antigen, and more. Here, using a combination of enzymatic synthesis and liquid chromatography-mass spectrometry (LC-MS) analysis of bacterial lysates, we provide biochemical evidence for the effect on endogenous polyisoprenoid pools from cell culture that arises from glycan pathway disruption.