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Applied and Environmental Microbiology, October 2002, p. 4722-4730, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.4722-4730.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Functional Expression of Enterobacterial O-Polysaccharide Biosynthesis Enzymes in Bacillus subtilis

Christina Schäffer,1,2* Thomas Wugeditsch,1,2 Paul Messner,1 and Chris Whitfield2

Zentrum für Ultrastrukturforschung and Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur Wien, A-1180 Vienna, Austria,1 Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada2

Received 13 May 2002/ Accepted 16 July 2002

The expression of heterologous bacterial glycosyltransferases is of interest for potential application in the emerging field of carbohydrate engineering in gram-positive organisms. To assess the feasibility of using enzymes from gram-negative bacteria, the functional expression of the genes wbaP (formerly rfbP), wecA (formerly rfe), and wbbO (formerly rfbF) from enterobacterial lipopolysaccharide O-polysaccharide biosynthesis pathways was examined in Bacillus subtilis. WbaP and WecA are initiation enzymes for O-polysaccharide formation, catalyzing the transfer of galactosyl 1-phosphate from UDP-galactose and N-acetylglucosaminyl 1-phosphate from UDP-N-acetylglucosamine, respectively, to undecaprenylphosphate. The WecA product (undecaprenylpyrophosphoryl GlcNAc) is used as an acceptor to which the bifunctional wbbO gene product sequentially adds a galactopyranose and a galactofuranose residue from the corresponding UDP sugars to form a lipid-linked trisaccharide. Genes were cloned into the shuttle vectors pRB374 and pAW10. In B. subtilis hosts, the genes were effectively transcribed under the vegII promoter control of pRB374, but the plasmids were susceptible to rearrangements and deletion. In contrast, pAW10-based constructs, in which genes were cloned downstream of the tet resistance cassette, were stable but yielded lower levels of enzyme activity. In vitro glycosyltransferase assays were performed in Escherichia coli and B. subtilis, using membrane preparations as sources of enzymes and endogenous undecaprenylphosphate as an acceptor. Incorporation of radioactivity from UDP-{alpha}-D-14C-sugar into reaction products verified the functionality of WbaP, WecA, and WbbO in either host. Enzyme activities in B. subtilis varied between 20 and 75% of those measured in E. coli.

* Corresponding author. Mailing address: Zentrum für Ultrastrukturforschung, Universität für Bodenkultur Wien, Gregor-Mendel-Straße 33, A-1180 Vienna, Austria. Phone: 43-1-47654, ext. 2203. Fax: 43-1-4789112. E-mail: CRS{at}edv1.boku.ac.at.

Applied and Environmental Microbiology, October 2002, p. 4722-4730, Vol. 68, No. 10
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.10.4722-4730.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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