TN916-GENERATED, LIPOOLIGOSACCHARIDE MUTANTS OF NEISSERIA-MENINGITIDIS AND NEISSERIA-GONORRHOEAE

A library of Tn916-generated, tetracycline-resistant (Tc-r) mutants of the group B Neisseria meningitidis strain NMB was screened by using m onoclonal antibodies (MAbs) that recognize structural differences in n eisserial lipooligosaccharide (LOS). The LOS of parental strain NMB ha d a relative molecular mass of 4.5 kDa, reacted with MAbs 3F11 and 6B4 but not with MAb 4C4 or 6E4, and contained a lacto-N-neotetrose unit. Two phenotypically stable mutants, SS3 and R6, altered in LOS, were i dentified by colony immunoblots, electrophoresis, and Western immunobl ots. The LOS of mutant SS3 was 3.4 kDa and reacted with MAbs 4C4 and 6 E4 but not MAb 3F11 or 6B4. The LOS of mutant R6 was 3.1 to 3.2 kDa an d reacted with MAb 6E4 but not MAb 3F11, 6B4, or 4C4. Thus, the LOSS o f the R6 and SS3 mutants were predicted to contain different truncatio ns of the core oligosaccharide. The LOS phenotype of each mutant was l inked to Tc-r, as determined by transformation of the parent strain wi th DNA from the mutant. Southern hybridizations and single-specific-pr imer PCR revealed in each mutant a single truncated Tn916 insertion wh ich had lost genes required for mobilization. Tn916 mutagenesis was us ed to identify two distinct genetic sites in the meningococcal chromos ome involved in biosynthesis of the oligosaccharide chain of LOS and t o create genetically defined LOS mutants Of N. meningitidis and Neisse ria gonorrhoeae.