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 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.
Drazek
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Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100, USA.
Stein
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Deal
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D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000096:biosynthesis*; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D003429:Cross Reactions; D016368:Frameshift Mutation; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009344:Neisseria gonorrhoeae / Q000201:enzymology / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides; C037213:ADP-L-glycero-D-mannoheptose-6-epimerase; D002238:Carbohydrate Epimerases