Molecular analysis of a locus for the biosynthesis and phase-variable expression of the lacto-N-neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis.
Lipopolysaccharide (LPS) is a major determinant of Neisseria, meningitidis virulence. A key feature of meningococcal LPS is the phase-variable expression of terminal structures which are proposed to have disparate roles in pathogenesis. In order to identify the biosynthetic genes for terminal LPS structures and the control mechanisms for their phase-variable expression, the lic2A gene, which is involved in LPS biosynthesis in Haemophilus influenzae, was used as a hybridization probe to identify a homologous gene in N. meningitidis strain MC58. The homologous region of DNA was cloned and nucleotide sequence analysis revealed three open reading frames (ORFs), two of which were homologous to the H. influenzae lic2A gene. All three ORFs were mutagenized by the insertion of antibiotic-resistance cassettes and the LPS from these mutant strains was analysed to determine if the genes had a role in LPS biosynthesis. Immunological and tricine-SDS-PAGE analysis of LPS from the mutant strains indicated that all three genes were probably transferases in the biosynthesis of the terminal lacto-N-neotetraose structure of meningococcal LPS. The first ORF of the locus contains a homopolymeric tract of 14 guanosine residues within the 5'-end of the coding sequence. As the lacto-N-neotetraose structure in meningococcal LPS is subject to phase-variable expression, colonies that no longer expressed the terminal structure, as determined by monoclonal antibody binding, were isolated. Analysis of an 'off' phase variant revealed a change in the number of guanosine residues resulting in a frameshift mutation, indicating that a slipped-strand mispairing mechanism, operating in the first ORF, controls the phase-variable expression of lacto-N-neotetraose.
Jennings
M P
MP
Molecular Infectious Diseases Group, John Radcliffe Hospital, Headington, Oxford, UK. mjennings@molbiol.ox.ac.uk
Hood
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Peak
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Virji
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Moxon
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D016368:Frameshift Mutation; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009345:Neisseria meningitidis / Q000235:genetics* / Q000472:pathogenicity; D009844:Oligosaccharides / Q000235:genetics*; D016366:Open Reading Frames; D010957:Plasmids; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014166:Transferases / Q000235:genetics
D001426:Bacterial Proteins; D008070:Lipopolysaccharides; D009844:Oligosaccharides; C102237:lic2A protein, Haemophilus influenzae; C013084:lacto-N-neotetraose; D014166:Transferases; C042308:phosphatidylglycerol glyceryl transferase