Metabolic Incorporation of Azido-Sugars into LPS to Enable Live-Cell Fluorescence Imaging

Metabolic labeling of lipopolysaccharides (LPS) with the exogenous azido analog of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) or Kdo-N₃ allows for both live-cell and molecular analysis of the outer membrane composition and biosynthesis in different Gram-negative bacteria. Here, we describe Kdo-N₃ incorporation into bacterial cells, followed by click labeling with a fluorescent dye. The fluorescently labeled LPS can be analyzed from lysed cells by SDS-PAGE and from intact cells by microscopy and flow cytometry. These methods have been applied to the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae, which possess the sialic acid transporter NanT that is also capable of transporting exogenous Kdo and Kdo analogs into the cytoplasm for incorporation into nascent LPS.