Molecular characterization and verification of azido-3,8-dideoxy-d--oct-2-ulosonic acid incorporation into bacterial lipopolysaccharide.

3-Deoxy-d--oct-2-ulosonic acid (Kdo) is an essential component of LPS in the outer leaflet of the Gram-negative bacterial outer membrane. Although labeling of with the chemical reporter 8-azido-3,8-dideoxy-d--oct-2-ulosonic acid (Kdo-N) has been reported, its incorporation into LPS has not been directly shown. We have now verified Kdo-N incorporation into LPS at the molecular level. Using microscopy and PAGE analysis, we show that Kdo-N is localized to the outer membrane and specifically incorporates into rough and deep-rough LPS. In an strain lacking endogenous Kdo biosynthesis, supplementation with exogenous Kdo restored full-length core-LPS, which suggests that the Kdo biosynthetic pathways might not be essential in the presence of sufficient exogenous Kdo. In contrast, exogenous Kdo-N only restored a small fraction of core LPS with the majority incorporated into truncated LPS. The truncated LPS were identified as Kdo-N-lipid IV and (Kdo-N)-lipid IV by MS analysis. The low level of Kdo-N incorporation could be partly explained by a 6-fold reduction in the specificity constant of the CMP-Kdo synthetase KdsB with Kdo-N compared with Kdo. These results indicate that the azido moiety in Kdo-N interferes with its utilization and may limit its utility as a tracer of LPS biosynthesis and transport in We propose that our findings will be helpful for researchers using Kdo and its chemical derivatives for investigating LPS biosynthesis, transport, and assembly in Gram-negative bacteria.