In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz

Abstract

Polysaccharides constitute a major component of bacterial cell surfaces and play critical roles in bacteria–host interactions. The biosynthesis of such molecules, however, has mainly been characterized through in vivo genetic studies, thus precluding discernment of the details of this pathway. Accordingly, we present a chemical approach that enabled reconstitution of the E. coli O-polysaccharide biosynthetic pathway in vitro. Starting with chemically prepared undecaprenyl-diphospho-N-acetyl-D-galactosamine, the E. coli O86 oligosaccharide repeating unit was assembled by means of sequential enzymatic glycosylation. Successful expression of the putative polymerase Wzy using a chaperone coexpression system then allowed demonstration of polymerization in vitro using this substrate. Analysis of more substrates revealed a defined mode of recognition for Wzy toward the lipid moiety. Specific polysaccharide chain length modality was furthermore demonstrated to result from the action of Wzz. Collectively, polysaccharide biosynthesis was chemically reconstituted in vitro, providing a well defined system for further underpinning molecular details of this biosynthetic pathway.

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Figure 1: wzy-dependent pathway of O-polysaccharide biosynthesis (E. coli O86:B7 O-polysaccharide as an example).
Figure 2: In vitro reconstitution of E. coli O86 polysaccharide repeating unit biosynthesis and associated product characterization.
Figure 3: Analysis of the Wzy polymerization reaction with the Und-based donor by SDS-PAGE and visualization with autoradiography.

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Acknowledgements

We are grateful to J. Liu (University of North Carolina) for providing the GroES/EL expression vector and helpful discussions of expression conditions. R.W. acknowledges the NIH Predoctoral Trainee Program (T32 GM008512). L.L. acknowledges support from the China Scholarship Council (2007102057). P.G.W. acknowledges the US National Cancer Institute (R01 CA118208), US National Institutes of Health (R01 GM085267), US National Science Foundation (CHE-0616892) and Bill & Melinda Gates Foundation (51946) for financial support.

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R.W., W.Y. and P.G.W. designed research. R.W., L.L., W.Y., G.Z., H.E., P.R.S., H.G., J.K.S., E.M., L.C., P.K., X.L., W.H., W.Z., Y.D. and M.L. performed research. L.L., W.Y., G.Z., H.E. and P.G.W. analyzed data. R.W., L.L., W.Y., G.Z. and P.G.W. wrote the paper.

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Correspondence to Peng George Wang.

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Woodward, R., Yi, W., Li, L. et al. In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz. Nat Chem Biol 6, 418–423 (2010). https://doi.org/10.1038/nchembio.351

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