Fig 1.
Schematic representation of metabolic lipopolysaccharide labeling using Kdo-N3.
Culture of E. coli in the presence of Kdo-N3 results in incorporation of the bioorthogonal azido function with the bacterial LPS. This incorporation can be further visualized by Copper-catalyzed Azide Alkyne Cycloaddition (CuAAC), in the presence of Cu(I) and a terminal alkyne. Alternatively, Strain-Promoted Alkyne Azide Cycloaddition (SPAAC) with a cyclooctyne-type reagent results in copper-free ligation.
Fig 2.
Kdo-N3 metabolically labels E. coli LPS.
(A) Metabolically incorporated Kdo-N3 in E. coli was revealed by copper-free click chemistry (sulfo-DBCO-biotin followed by an anti-biotin A488 antibody). (B) Frequency distribution of the bacterial fluorescence values in the presence (green bars) or absence of Kdo-N3 (black bars). Scale bar = 1 μm. More than ten independent experiments have been performed and a representative experiment is depicted.
Fig 3.
Kdo-N3 specifically labels culturable E. coli.
Detection of culturable E. coli (arrows) mixed with m-cherry dead E. coli (asterisk) (ratio = 106:106) by copper-free click chemistry (sulfo-DBCO-biotin followed by an anti-biotin A488 antibody) in the absence (A) or presence (B) of metabolically incorporated Kdo-N3. Scale bar = 1 μm. Frequency distribution of each bacterial fluorescence values in the absence (A) or presence of Kdo-N3 (B). Four independent experiments have been performed and a representative experiment is depicted.
Fig 4.
Kdo-N3 specifically labels E. coli culturable Gram negative bacterial membrane.
Detection of culturable E. coli (arrows) mixed with culturable GFP B. subtilis (asterisk) (ratio = 106:3x106) by copper-free click chemistry (sulfo-DBCO-biotin followed by an anti-biotin A594 antibody) in the absence (A) or presence (B) of metabolically incorporated Kdo-N3. Scale bar = 1 μm. Frequency distribution of each bacterial fluorescence values in the absence (A) or presence of Kdo-N3 (B). Five independent experiments have been performed and a representative experiment is depicted.
Fig 5.
Specific isolation of culturable E. coli among a bacterial mix with magnetic beads.
Determination of culturable E. coli recovery in the supernatant (grey bars) and magnetic streptavidin bead fraction (white bars) with or without incorporation of Kdo-N3 followed by copper-free click chemistry (sulfo-DBCO-biotin). Different amount of culturable E. coli alone or mixed with 106 B. subtilis were tested: 106 (A), 103 (B), 102 (C), 101 (D). Pictures of culturable E. coli (arrows) recovered or not in the magnetic streptavidin bead fraction either in the presence or absence of Kdo-N3, respectively (E). Bacteria were stained with DAPI and Kdo-N3 incorporation within LPS was revealed with an anti-biotin A488 antibody. Scale bar = 1 μm. Data are means ± SD of four independent experiments.
Table 1.
Percentage of culturable E. coli recovery mixed or not with culturable B. subtilis in the supernatant fraction and magnetic streptavidin beads fraction with or without incorporation of Kdo-N3.
Table 2.
Percentage of culturable bacteria recovery in the supernatant fraction and magnetic streptavidin beads fraction with or without incorporation of Kdo-N3.
Fig 6.
Isolation of all culturable E. coli cells incubated with 25 mM of Kdo-N3 within 2 h.
Determination of culturable E. coli recovery in the supernatant (grey bars) and magnetic streptavidin bead fraction (white bars) with or without incorporation of Kdo-N3 5mM or 25 mM for 2 and 4 h followed by copper-free click chemistry (sulfo-DBCO-biotin). Data are means ± SD of four independent experiments.