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

Abstract

3-Deoxy-d-<i>manno</i>-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 <i>Escherichia coli</i> with the chemical reporter 8-azido-3,8-dideoxy-d-<i>manno</i>-oct-2-ulosonic acid (Kdo-N₃) has been reported, its incorporation into LPS has not been directly shown. We have now verified Kdo-N₃ incorporation into <i>E. coli</i> LPS at the molecular level. Using microscopy and PAGE analysis, we show that Kdo-N₃ is localized to the outer membrane and specifically incorporates into rough and deep-rough LPS. In an <i>E. coli</i> 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 <i>in vivo</i> in the presence of sufficient exogenous Kdo. In contrast, exogenous Kdo-N₃ only restored a small fraction of core LPS with the majority incorporated into truncated LPS. The truncated LPS were identified as Kdo-N₃-lipid IV_A and (Kdo-N₃)₂-lipid IV_A by MS analysis. The low level of Kdo-N₃ incorporation could be partly explained by a 6-fold reduction in the specificity constant of the CMP-Kdo synthetase KdsB with Kdo-N₃ compared with Kdo. These results indicate that the azido moiety in Kdo-N₃ interferes with its utilization and may limit its utility as a tracer of LPS biosynthesis and transport in <i>E. coli</i> 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.