Analysis of the icsBA locus required for biosynthesis of the inner core region from Neisseria meningitidis lipopolysaccharide.
By deletion mutagenesis in the entire meningococcal chromosome, we have previously identified the icsA gene, which encodes the glycosyltransferase required for adding GlcNAc to Hep-II in the inner core of meningococcal LPS. This gene has homology to several LPS glycosyltransferases, notably to rfaK from Salmonella typhimurium and bplH from Bordetella pertussis, both of which encode GlcNAc transferases. Directly upstream of icsA is an ORF showing significant homology to the hypothetical protein HI0653 from the Haemophilus influenzae genome sequence, and to a lesser degree to putative glycosyltransferases from Streptococcus thermophilus and Yersinia enterocolitica. Insertional inactivation of this ORF resulted in a meningococcal strain with truncated LPS. We have named this new LPS-involved gene icsB. Differences in binding of monoclonal antibodies and in mobility on Tricine-SDS-PAGE showed that LPS from icsA and icsB mutants is similar but not identical. On the basis of these results, we postulated that the new gene encodes the glycosyltransferase required for adding Glc to Hep-I. Structural analysis of purified mutant LPS by electrospray mass spectrometry was used to verify this hypothesis. The composition determined for icsA and icsB is lipidA-(KDO)2-(Hep)2.PEA and lipidA-(KDO)2-(Hep)2.PEA-GlcNAc, respectively. The icsA and icsB genes thus form an operon encoding the glycosyltransferases required for chain elongation from the lipidA-(KDO)2-(Hep)2 basal structure, with IcsA first adding GlcNAc to Hep-II and IcsB subsequently adding Glc to Hep-I. Only then is completion of the lacto-N-neotetraose structure possible through the action of the IgtA-E genes.
van der Ley
P
P
Laboratory of Vaccine Development and Immune Mechanisms, National Institute of Public Health and the Environment, Bilthoven, The Netherlands. peterl@rivm.nl
Kramer
M
M
Martin
A
A
Richards
J C
JC
Poolman
J T
JT
D000117:Acetylglucosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015139:Blotting, Southern; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D006539:Heptoses / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D009345:Neisseria meningitidis / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D012703:Serotyping
D001426:Bacterial Proteins; D006539:Heptoses; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D016695:Glycosyltransferases; D000117:Acetylglucosamine