Sequestration of dead-end undecaprenyl phosphate-linked oligosaccharide intermediate

In the predominant Wzx/Wzy-dependent bacterial surface polysaccharide biosynthetic pathway, synthesis is divided between the cytoplasmic and periplasmic faces of the membrane. Initially, an oligosaccharide composed of 3-8 sugars is synthesized on a membrane-embedded lipid carrier, Und-P, within the cytoplasmic face of the membrane. This Und-P-linked oligosaccharide is then translocated to the periplasmic face by the Wzx flippase, where it is polymerized into a repeat-unit polysaccharide. Structural alterations to the repeating oligosaccharide significantly reduce polysaccharide yield and lead to cell death or morphological abnormalities. These effects are attributed to the substrate recognition function of the Wzx flippase, which we postulated to act as a gatekeeper to ensure only complete substrates are translocated to the periplasmic face. Here, we labelled Salmonella enterica serovar Typhimurium Group B1 with [ ¹⁴ C] D-galactose. Our results showed that strains unable to synthesize the full O-antigen repeat unit accumulate significantly higher levels of Und-P-linked material (∼10-fold). Importantly, this sequestration is alleviated by mild membrane disruption which opens the cytosolic face Und-PP-linked material to O-antigen ligation that supports the accumulation to occur at the cytosolic face of the membrane.