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J. Bacteriol., 05 1995, 2321-2327, Vol 177, No. 9
Copyright © 1995, American Society for Microbiology

A mutation in the Neisseria gonorrhoeae rfaD homolog results in altered lipooligosaccharide expression

ES Drazek, DC Stein and CD Deal
Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100, USA.

The gonococcal lsi-6 locus was cloned and shown by DNA sequence analysis to have homology with the E. coli rfaD gene, which encodes ADP- L-glycero-D-mannoheptose epimerase. This enzyme is involved in the biosynthesis of the lipopolysaccharide precursor ADP-L-glycero-D- mannoheptose. A site-directed frameshift mutation in lsi-6 was constructed by PCR amplification and introduced into the chromosome of Neisseria gonorrhoeae MS11 P+ by transformation. The lipooligosaccharides (LOS) of mutant and parental strains were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lsi-6 mutant produced 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 phenotype was expressed when a revertant was constructed by transformation of the cloned wild-type gene into the lsi-6 mutant. The immunoreactivity of LOS from parental and constructed strains was examined by SDS-PAGE and Western blotting. Only the parental and reconstructed wild-type strains produced a 3.6-kDa LOS component that reacted with monoclonal antibody 2-1-L8. These results suggest that the lsi-6 locus is involved in gonococcal LOS biosynthesis and that the nonreactive mutant 3.6-kDa LOS component contains a conformational change or altered saccharide composition that interferes with immunoreactivity.

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