JB Accepts, published online ahead of print on 23 October 2009

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J. Bacteriol. doi:10.1128/JB.00558-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Functional characterization of Lpt3 and Lpt6, the inner core lipooligosaccharide phosphoethanolamine transferases from Neisseria meningitidis

Cory Q. Wenzel^*, Frank St. Michael, Jacek Stupak, Jianjun Li, Andrew D. Cox, and James C. Richards

Institute of Biological Sciences, 100 Sussex Drive, National Research Council, Ottawa, ON, Canada K1A 0R6

^* To whom correspondence should be addressed. Email: Cory.Wenzel{at}nrc-cnrc.gc.ca.

The lipooligosaccharide (LOS) of Neisseria meningitidis contains heptose residues that are modified with phosphoethanolamine (PEtn) at the 3- and/or 6-position. The lpt3 (NMB2010) and lpt6 (NMA0408) genes of N. meningitidis, which are proposed to encode the required HepII 3- and 6-PEtn transferases respectively, were cloned and overexpressed as C-terminally polyhistidine-tagged fusion proteins in Escherichia coli and found to localize to the inner-membrane based on sucrose density gradient centrifugation. Lpt3-His₆ and Lpt6-His₆ were purified from Triton X-100-solubilized membranes by nickel-chelation chromatography, and dot-blot analysis of enzymatic reactions using 3-PEtn- and 6-PEtn-specific monoclonal antibodies demonstrated conclusively that Lpt3 and Lpt6 are phosphatidylethanolamine-dependent LOS HepII 3- and 6-PEtn transferases, respectively, and that both enzymes are capable of transferring PEtn to both fully-acylated and de-O-acylated (de-O-Ac) LOS. Further enzymatic studies using capillary electrophoresis-MS demonstrated that both Lpt3 and Lpt6 are capable of transferring PEtn to de-O-Ac LOS molecules already containing PEtn at the 6- or 3-positions of HepII, respectively, demonstrating that there is no obligate order of PEtn addition in the generation of 3,6-di-PEtn LOS moieties in vitro.