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

Inga Nilsson,Kerri Grove,Dustin Dovala,Tsuyoshi Uehara,Guillaume Lapointe,David A Six

doi:10.1074/jbc.m117.814962

Abstract

3-Deoxy-d-manno-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 Escherichia coli with the chemical reporter 8-azido-3,8-dideoxy-d-manno-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 E. coli 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 E. coli 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 vivo 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 IVA and (Kdo-N3)2-lipid IVA 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 E. coli 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.

Highlights

3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) is an essential component of LPS in the outer leaflet of the Gram-negative bacterial outer membrane
In most Gram-negative bacteria, the first core sugar attached to lipid A is 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) [7]
The green fluorescence observed after labeling E. coli should be localized to the outer membrane (OM) if Kdo-N3-lipid IVA (Kdo)-N3–lipid IVA and (Kdo-N3) is incorporated into the LPS

Summary

Results

Fluorescent labeling of E. coli cells with Kdo-N3 and a fluorescent copper-free click reagent. The fluorescent gel image showed a green fluorescent band in the sample from cells grown with Kdo-N3 and treated with Alexa488-DIBO (Fig. 3A, lanes 5 and 6). The gel image revealed a fluorescently labeled band only for ⌬waaC E. coli grown with Kdo-N3 and reacted with the Alexa488DIBO (Fig. 3C, lanes 5 and 6). After SDS-PAGE and Pro-Q Emerald 300 LPS staining, a band equivalent to rough LPS (core-lipid IVA) was clearly visible from ClearColi K-12 grown with Kdo (Fig. 4B, lane 4). Upon imaging the SDS-polyacrylamide gel for fluorescence (Fig. 4B), the ClearColi sample grown with Kdo-N3 and treated with Alexa488-DIBO revealed two green fluorescent bands (Fig. 4B, lane 6). Observed lipid IVA species in total lipid extracts of ClearColi K-12 evaluated by MALDI-TOF-MS The data are representative of two independent experiments

LPS species

Discussion

When comparing the results for the addition of exogenous

Bacterial strains and growth conditions

Fluorescence microscopy imaging

Pyrophosphate release assay