Evaluation of Kdo-8-N 3 incorporation into lipopolysaccharides of various Escherichia coli strains.

8-Azido-3,8-dideoxy-α/β-d- manno -oct-2-ulosonic acid (Kdo-8-N 3 ) is a Kdo derivative used in metabolic labeling of lipopolysaccharide (LPS) structures found on the cell membrane of Gram-negative bacteria. Several studies have reported successful labeling of LPS using Kdo-8-N 3 and visualization of LPS by a fluorescent reagent through click chemistry on a selection of Gram-negative bacteria such as Escherichia coli strains, Salmonella typhimurium , and Myxococcus xanthus . Motivated by the promise of Kdo-8-N 3 to be useful in the investigation of LPS biosynthesis and cell surface labeling across different strains, we set out to explore the variability in nature and efficiency of LPS labeling using Kdo-8-N 3 in a variety of E. coli strains and serotypes. We optimized the chemical synthesis of Kdo-8-N 3 and subsequently used Kdo-8-N 3 to metabolically label pathogenic E. coli strains from commercial and clinical origin. Interestingly, different extents of labeling were observed in different E. coli strains, which seemed to be dependent also on growth media, and the majority of labeled LPS appears to be of the 'rough' LPS variant, as visualized using SDS-PAGE and fluorescence microscopy. This knowledge is important for future application of Kdo-8-N 3 in the study of LPS biosynthesis and dynamics, especially when working with clinical isolates.