Identification of the Functional Roles of Six Key Proteins in the Biosynthesis of Enterobacteriaceae Colanic Acid.

When subjected to harsh conditions such as low pH, pathogenic Escherichia coli can secrete colanic acid to establish a protective barrier between the organism and the acidic environment. The colanic acid consists of a six-sugar repeating unit polymer comprised of glucose, fucose, galactose, and glucuronic acid. The region of the E. coli genome that encodes colanic acid biosynthesis has been reported, and the first enzyme in the biosynthesis pathway has been biochemically characterized. However, the specific roles of the remaining genes required for colanic acid biosynthesis have not been identified. Here we report the in vitro reconstitution of the next six steps in the assembly of the colanic acid repeating unit. To do this, we have cloned and overexpressed each gene within the colanic acid biosynthesis operon. We then tested the activity of the protein product of these genes using high-performance liquid chromatography analysis and a fluorescent analogue of the isoprenoid anchor bactoprenyl diphospho-glucose as a starting substrate. To ensure that retention time changes were associated with varying sugar additions or modifications, we developed a liquid chromatography-mass spectrometry method for analysis of the products produced by each enzyme. We have identified the function of all but one encoded glycosyltransferase and have identified the function of two acetyltransferases. This work demonstrates the centrality of acetylation in the biosynthesis of colanic acid and provides insight into the activity of key proteins involved in the production of an important and highly conserved bacterial glycopolymer.