Chemical polyglycosylation and nanolitre detection enables single-molecule recapitulation of bacterial sugar export.
The outermost protective layer of both Gram-positive and Gram-negative bacteria is composed of bacterial capsular polysaccharides. Insights into the interactions between the capsular polysaccharide and its transporter and the mechanism of sugar export would not only increase our understanding of this key process, but would also help in the design of novel therapeutics to block capsular polysaccharide export. Here, we report a nanolitre detection system that makes use of the bilayer interface between two droplets, and we use this system to study single-molecule recapitulation of sugar export. A synthetic strategy of polyglycosylation based on tetrasaccharide monomers enables ready synthetic access to extended fragments of K30 oligosaccharides and polysaccharides. Examination of the interactions between the Escherichia coli sugar transporter Wza and very small amounts of fragments of the K30 capsular polysaccharide substrate reveal the translocation of smaller but not larger fragments. We also observe capture events that occur only on the intracellular side of Wza, which would complement coordinated feeding by adjunct biosynthetic machinery.
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK.
Lingbing
L
Kong
School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
Andrew
A
Almond
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK.
Hagan
H
Bayley
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK.
Benjamin G
BG
Davis
D001692:Biological Transport; D002236:Carbohydrate Conformation; D004926:Escherichia coli / Q000378:metabolism; D006031:Glycosylation; D008051:Lipid Bilayers / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D011135:Polysaccharides, Bacterial / Q000737:chemistry* / Q000378:metabolism
D008051:Lipid Bilayers; D011135:Polysaccharides, Bacterial