Tolerance Mechanisms in Polysaccharide Biosynthesis: Implications for Undecaprenol Phosphate Recycling in Escherichia coli and Shigella flexneri

Bacterial polysaccharide synthesis is catalysed on the universal lipid carrier, undecaprenol phosphate (UndP). The cellular UndP pool is shared by other polysaccharide synthesis pathways and in peptidoglycan (PG) biogenesis. Disruptions in cytosolic polysaccharide synthesis steps are detrimental to bacterial survival due to affecting UndP recycling. In contrast, bacteria can survive disruptions in the periplasmic steps, suggesting a tolerance mechanism to mitigate UndP sequestration. Here we investigated tolerance mechanisms to disruptions of polymerases that are involved in UndP-releasing steps in two related polysaccharide synthesis pathways: the enterobacterial common antigen (ECA) and the O antigen (OAg), in Escherichia coli and Shigella flexneri. Our study reveals that polysaccharide polymerisation is crucial for efficient UndP recycling. In E. coli K-12, cell survival upon disruptions in OAg polymerase is dependent on a functional ECA synthesis pathway and vice versa. This is because disruptions in OAg synthesis leads to the redirection of the shared lipid-linked sugar substrate UndPP-GlcNAc towards increased ECA production. Conversely, in S. flexneri, the OAg polymerase is essential due to its limited ECA production, which inadequately redirects UndP flow to support cell survival. We propose a model whereby sharing the initial sugar intermediate UndPP-GlcNAc between the ECA and OAg synthesis pathways allows UndP to be redirected towards ECA production, mitigating sequestration issues caused by disruptions in the OAg pathway. These findings suggest an evolutionary buffering mechanism that enhances bacterial survival when UndP sequestration occurs due to stalled polysaccharide biosynthesis, which may allow polysaccharide diversity in the species to increase over time. ImportanceEnzymes involved in bacterial polysaccharide biosynthesis have substrate specificity to ensure the correct polysaccharide is produced at the appropriate place and time. However, this specificity poses a challenge for the diversification of polysaccharide structure and hence function, as the acquisition of a novel oligosaccharide RU would likely disrupt the synthesis pathways, leading to sequestration of the essential universal lipid carrier UndP and ultimately cause cell death. We investigated how cells tolerate disruptions in polysaccharide synthesis pathways and provide evidence that suggests that sharing a common substrate between the synthesis pathways of two common enteric bacterial surface polysaccharides (ECA and OAg), can redirect the flow of UndP. Our study provides insights into the mechanism of how bacteria alleviate sequestration issues, thereby enhancing cell survival which may allow them additional capacity for polysaccharide diversification.