Effects of Lipopolysaccharide Core Sugar Deficiency on Colanic Acid Biosynthesis in Escherichia coli

Abstract

When 10 Escherichia coli mutant strains with defects in lipopolysaccharide (LPS) core biosynthesis were grown on agar medium at 30°C, four of them, the ⌬waaF, ⌬waaG, ⌬waaP, and ⌬waaB strains, formed mucoid colonies, while the other six, the ⌬waaU, ⌬waaR, ⌬waaO, ⌬waaC, ⌬waaQ, and ⌬waaY strains, did not. Using light microscopy with tannin mordant staining, the presence of exopolysaccharide around the cells of the mutants that formed mucoid colonies could be discerned. The ⌬waaF mutant produced the largest amounts of exopolysaccharide, regardless of whether it was grown on agar or in liquid medium. The exopolysaccharide was isolated from the liquid growth medium of ⌬waaF cells, hydrolyzed, and analyzed by high-performance liquid chromatography with an ion-exchange column, and the results indicated that the exopolysaccharide was consistent with colanic acid. When the key genes related to the biosynthesis of colanic acid, i.e., wza, wzb, wzc, and wcaA, were deleted in the ⌬waaF background, the exopolysaccharide could not be produced any more, further confirming that it was colanic acid. Colanic acid could not be produced in strains in which rcsA, rcsB, rcsD, or rcsF was deleted in the ⌬waaF background, but a reduced level of colanic acid production was detected when the rcsC gene was deleted, suggesting that a change of lipopolysaccharide structure in ⌬waaF cells might be sensed by the RcsCDB phosphorelay system, leading to the production of colanic acid. The results demonstrate that E. coli cells can activate colanic acid production through the RcsCDB phosphorelay system in response to a structural deficiency of lipopolysaccharide. IMPORTANCELipopolysaccharide and colanic acid are important forms of exopolysaccharide for Escherichia coli cells. Their metabolism and biological significance have been investigated, but their interrelation with the cell stress response process is not understood. This study demonstrates, for the first time, that E. coli cells can activate colanic acid production through the RcsCDB phosphorelay system in response to a structural change of lipopolysaccharide, suggesting that bacterial cells can monitor the outer membrane integrity, which is essential for cell survival and damage repair. Bacteria produce a wide range of exopolysaccharides, which have specific monomer compositions, substituent decorations, and biosynthetic pathways. Genes involved in biosynthesis of these exopolysaccharides are often clustered in specific loci within the genome and encode glycosyltransferases, polymerizing and branching enzymes, and enzymes responsible for addition of substituents (1). Escherichia coli K-12 cells contain several gene clusters related to biosynthesis of polysaccharides (2-7), such as lipopolysaccharide (LPS) and colanic acid (CA) (Fig. 1). Understanding the fundamental processes involved in exopolysaccharide biosynthesis and the regulation of these processes is important.As a major molecule in the outer membrane of E. coli K-12 cells (8, 9), LPS consists of a hydrophobic lipid A domai...

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