| Abstract |
Colanic acid and enterobacterial common antigen (ECA) are cell-surface polysaccharides that are produced by many Escherichia coli isolates. Colanic acid is induced under acidic, low temperature, and high-salt conditions and is important for E. coli resistance to these stresses; however, the role of ECA in these stresses is less clear. Here, we observed that knockout of flippase wzxE, which translocates lipid-linked ECA repeat units from the cytoplasmic side of the inner membrane to the periplasmic side, resulted in the sensitivity of E. coli BW25113 to acidic conditions. The wzxE-knockout mutant showed reduced growth potential and viable counts in vegetable extracts with acidic environments, including cherry tomatoes, carrots, celery, lettuce, and spinach. A double-knockout strain of wzxE and wecF (glycosyltransferase that adds the third-and-final sugar of the lipid-linked ECA repeat unit) was not sensitive to acidic conditions, with similar results obtained for a double-knockout strain of wzxE and wcaJ (glycosyltransferase that initiates colanic acid lipid-linked repeat-unit biosynthesis). The wzxE-knockout mutant was sensitive to low temperatures or high-salt conditions, which induced colanic acid synthesis, and these sensitivities were abolished by the additional knockout of wcaJ. These results suggest that lipid-linked ECA repeat units confer E. coli susceptibility to acidic, low temperatures, and high-salt conditions in a colanic acid-dependent manner and that wzxE suppresses this negative effect.IMPORTANCEThe role of the common enterobacterial antigen, a polysaccharide that is conserved throughout enterobacteria, in stress resistance is unclear. Our results suggest that lipid-linked enterobacterial common antigen repeat units (which are typically translocated across the inner membrane by the flippase WzxE) cause sensitivity of Escherichia coli to acidic, low-temperature, and high-salt conditions in a manner dependent on colanic acid. The wzxE-knockout mutant was sensitive to crude vegetable extracts, suggesting that the development of WzxE inhibitors could lead to novel food poisoning prevention agents. Considering previous findings that lipid-linked ECA repeat units are flipped by both WzxE and the flippase for colanic acid lipid-linked repeat-unit, the colanic acid-dependence of the wzxE-knockout phenotype proposes a model in which a large amount of colanic acid under stress conditions occupies the flippase for colanic acid lipid-linked repeat unit, leading to accumulation of lipid-linked ECA repeat units on the inner membrane.
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