A MUTATION IN THE NEISSERIA-GONORRHOEAE RFAD HOMOLOG RESULTS IN ALTERED LIPOOLIGOSACCHARIDE EXPRESSION

The gonococcal lsi-6 locus was cloned and shown by DNA sequence analys is to have homology with the E. coli rfaD gene, which encodes ADP-L-gl ycero-D-mannoheptose epimerase. This enzyme is involved in the biosynt hesis of the lipopolysaccharide precursor ADP-L-glycero-D-mannoheptose . A site-directed frameshift mutation in lsi-6 was constructed by PCR amplification acid introduced into the chromosome of Neisseria gonorrh oeae MS11 P+ by transformation. The lipooligosaccharides (LOS) of muta nt and parental strains were characterized by sodium dodecyl sulfate-p olyacrylamide gel electrophoresis (SDS-PAGE), The lsi-6 mutant produce d LOS components with apparent molecular masses of 2.6 and 3.6 kDa as compared with a 3.6-kDa band of the MS11 P+ strain. The parental LOS p henotype was expressed when a revertant was constructed by transformat ion of the cloned wild-type gene into the lsi-6 mutant. The immunoreac tivity of LOS from parental and constructed strains was examined by SD S-PAGE and Western blotting. Only the parental and reconstructed wild- type strains produced a 3.6-kDa LOS component that reacted with monocl onal antibody 2-1-L8. These results suggest that the lsi-6 locus is in volved in gonococcal LOS biosynthesis and that the nonreactive mutant 3.6-kDa LOS component contains a conformational change or altered sacc haride composition that interferes with immunoreactivity.