INNER-CORE BIOSYNTHESIS OF LIPOOLIGOSACCHARIDE (LOS) IN NEISSERIA-MENINGITIDIS SEROGROUP-B - IDENTIFICATION AND ROLE IN LOS ASSEMBLY OF THEALPHA-1,2 N-ACETYLGLUCOSAMINE TRANSFERASE (RFAK)

A lipooligosaccharide (LOS) mutant of Neisseria meningitidis serogroup B strain NMB (immunotype L3,7,9) was identified in a Tn916 (tetM) mut ant bank by loss of reactivity with monoclonal antibody 3F11, which re cognizes the terminal Gal beta 1-->4GlcNAc epitope in the lacto-N-neot etraose moiety of the wild-type LOS structure. The mutant, designated 559, was found to express a truncated LOS of 3.0 kDa, Southern and PCR analyses demonstrated that there was a single intact Tn916 insertion (class I) in the mutant 559 chromosome. Linkage of the LOS phenotype a nd the Tn916 insertion was confirmed by transformation of the wild-typ e parent, Nucleotide sequence analysis of the region surrounding the t ransposition site revealed a 1,065-bp open reading frame (ORF), A homo logy search of the GenBank/EMBL database revealed that the amino acid sequence of this ORF had 46.8% similarity and 21.2% identity with the alpha 1,2 N-acetylglucosamine transferase (RfaK) from Salmonella typhi murium. Glycosyl composition and linkage analysis of the LOS produced by mutant 559 revealed that the lacto-N-neotetraose group which is att ached to heptose I (HepI) and the N-acetylglucosamine and glucose resi dues that are attached to HepII in the inner core of the parental LOS were absent, These analyses also showed that the HepII residue in both the parent and the mutant LOS molecules was phosphorylated, presumabl y by a phosphoethanolamine substituent. The insertion of nonpolar and polar antibiotic resistance cartridges into the parental rfaK gene res ulted in the expression of LOS with the same mobility as that produced by mutant 559. This result indicated that the inability to add the la cto-N-neotetraos group to the 559 LOS is not due to a polar effect on a gene(s) downstream of rfaK. Our data indicate that we have identifie d the meningococcal alpha 1,2 N-acetylglucosamine transferase responsi ble for the addition of N-acetylglucosamine to HepII. We propose that the lack of alpha-chain extension from HepI in the LOS of mutant 559 m ay be due to structural constraints imposed by the incomplete biosynth esis of the LOS inner core.