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1.
Org Biomol Chem ; 17(24): 5920-5924, 2019 06 18.
Article in English | MEDLINE | ID: mdl-31165848

ABSTRACT

Utilising a fast and sensitive screening method based on imidazolium-tagged probes, we report unprecedented reversible activity of bacterial ß1,4-galactosyltransferases to catalyse the transgalactosylation from lactose to N-acetylglucosamine to form N-acetyllactosamine in the presence of UDP. The process is demonstrated by the preparative scale synthesis of pNP-ß-LacNAc from lactose using ß1,4-galactosyltransferase NmLgtB-B as the only biocatalyst.


Subject(s)
Amino Sugars/biosynthesis , Galactosyltransferases/metabolism , Lactose/metabolism , Amino Sugars/chemistry , Biocatalysis , Galactosyltransferases/chemistry , Lactose/chemistry , Molecular Structure , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
2.
Plant J ; 85(3): 437-47, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26676799

ABSTRACT

In plants, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) is a monosaccharide that is only found in the cell wall pectin, rhamnogalacturonan-II (RG-II). Incubation of 4-day-old light-grown Arabidopsis seedlings or tobacco BY-2 cells with 8-azido 8-deoxy Kdo (Kdo-N3 ) followed by coupling to an alkyne-containing fluorescent probe resulted in the specific in muro labelling of RG-II through a copper-catalysed azide-alkyne cycloaddition reaction. CMP-Kdo synthetase inhibition and competition assays showing that Kdo and D-Ara, a precursor of Kdo, but not L-Ara, inhibit incorporation of Kdo-N3 demonstrated that incorporation of Kdo-N3 occurs in RG-II through the endogenous biosynthetic machinery of the cell. Co-localisation of Kdo-N3 labelling with the cellulose-binding dye calcofluor white demonstrated that RG-II exists throughout the primary cell wall. Additionally, after incubating plants with Kdo-N3 and an alkynated derivative of L-fucose that incorporates into rhamnogalacturonan I, co-localised fluorescence was observed in the cell wall in the elongation zone of the root. Finally, pulse labelling experiments demonstrated that metabolic click-mediated labelling with Kdo-N3 provides an efficient method to study the synthesis and redistribution of RG-II during root growth.


Subject(s)
Arabidopsis/ultrastructure , Cell Wall/ultrastructure , Nucleotidyltransferases/antagonists & inhibitors , Pectins/chemistry , Sugar Acids/chemistry , Azides/chemistry , Cells, Cultured , Plant Roots/ultrastructure , Seedlings/ultrastructure , Staining and Labeling , Nicotiana/ultrastructure
3.
PLoS One ; 10(6): e0127700, 2015.
Article in English | MEDLINE | ID: mdl-26061695

ABSTRACT

Currently, identification of pathogenic bacteria present at very low concentration requires a preliminary culture-based enrichment step. Many research efforts focus on the possibility to shorten this pre-enrichment step which is needed to reach the minimal number of cells that allows efficient identification. Rapid microbiological controls are a real public health issue and are required in food processing, water quality assessment or clinical pathology. Thus, the development of new methods for faster detection and isolation of pathogenic culturable bacteria is necessary. Here we describe a specific enrichment technique for culturable Gram negative bacteria, based on non-lethal click chemistry and the use of magnetic beads that allows fast detection and isolation. The assimilation and incorporation of an analog of Kdo, an essential component of lipopolysaccharides, possessing a bio-orthogonal azido function (Kdo-N3), allow functionalization of almost all Gram negative bacteria at the membrane level. Detection can be realized through strain-promoted azide-cyclooctyne cycloaddition, an example of click chemistry, which interestingly does not affect bacterial growth. Using E. coli as an example of Gram negative bacterium, we demonstrate the excellent specificity of the technique to detect culturable E. coli among bacterial mixtures also containing either dead E. coli, or live B. subtilis (as a model of microorganism not containing Kdo). Finally, in order to specifically isolate and concentrate culturable E. coli cells, we performed separation using magnetic beads in combination with click chemistry. This work highlights the efficiency of our technique to rapidly enrich and concentrate culturable Gram negative bacteria among other microorganisms that do not possess Kdo within their cell envelope.


Subject(s)
Click Chemistry/methods , Gram-Negative Bacteria/isolation & purification , Immunomagnetic Separation/methods , Azides/analysis , Cell Culture Techniques , Sugar Acids/analysis
4.
Angew Chem Int Ed Engl ; 53(5): 1275-8, 2014 Jan 27.
Article in English | MEDLINE | ID: mdl-24446310

ABSTRACT

Legionella pneumophila is a pathogenic bacterium involved in regular outbreaks characterized by a relatively high fatality rate and an important societal impact. Frequent monitoring of the presence of this bacterium in environmental water samples is necessary to prevent these epidemic events, but the traditional culture-based detection and identification method requires up to 10 days. Reported herein is a method allowing identification of Legionella pneumophila by metabolic lipopolysaccharide labeling which targets, for the first time, a precursor to monosaccharides that are specifically present within the O-antigen of the bacterium. This new approach allows easy detection of living Legionella pneumophila, while other Legionella species are not labeled.


Subject(s)
Legionella pneumophila/isolation & purification , Lipopolysaccharides/chemistry , Click Chemistry , Fluorescent Dyes/chemistry , Legionella pneumophila/metabolism , Lipopolysaccharides/metabolism , Microscopy, Confocal , Monosaccharides/chemistry , O Antigens/chemistry , O Antigens/metabolism , Serotyping
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