Most Gram-negative bacteria synthesize a plethora of cell surface polysaccharides that play key roles in immune evasion, cell envelope structural integrity and host-pathogen interactions. In the predominant polysaccharide Wzx/Wzy-dependent 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, undecaprenyl pyrophosphate, within the cytoplasmic face of the membrane. This lipid-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 O-antigen 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 that only complete substrates are translocated to the periplasmic face. Here, we labelled serovar Typhimurium group B1 with [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 membrane disruption which opens the lipid-linked oligosaccharide at the cytosolic face to periplasmic ligation to support accumulation occurs at the cytosolic face of the membrane.