Defining Early Steps in Biofilm Biosynthesis.

Unlabelled: The extracellular biofilm matrix includes an exopolysaccharide that is critical for the architecture and function of the community. To date, our understanding of the biosynthetic machinery and the molecular composition of the exopolysaccharide of remains unclear and incomplete. This report presents synergistic biochemical and genetic studies built from a foundation of comparative sequence analyses targeted at elucidating the activities of the first two membrane-committed steps in the exopolysaccharide biosynthetic pathway. By taking this approach, we determined the nucleotide sugar donor and lipid-linked acceptor substrates for the first two enzymes in the biofilm exopolysaccharide biosynthetic pathway. EpsL catalyzes the first phosphoglycosyl transferase step using UDP-di- -acetyl bacillosamine as phospho-sugar donor. EpsD is a GT-B fold glycosyl transferase that facilitates the second step in the pathway that utilizes the product of EpsL as an acceptor substrate and UDP- -acetyl glucosamine as the sugar donor. Thus, the study defines the first two monosaccharides at the reducing end of the growing exopolysaccharide unit. In doing so we provide the first evidence of the presence of bacillosamine in an exopolysaccharide synthesized by a Gram-positive bacterium.

Importance: Biofilms are the communal way of life that microbes adopt to increase survival. Key to our ability to systematically promote or ablate biofilm formation is a detailed understanding of the biofilm matrix macromolecules. Here we identify the first two essential steps in the biofilm matrix exopolysaccharide synthesis pathway. Together our studies and approaches provide the foundation for the sequential characterization of the steps in exopolysaccharide biosynthesis, using prior steps to enable chemoenzymatic synthesis of the undecaprenol diphosphate-linked glycan substrates.