Analysis of Haemophilus influenzae type b lipooligosaccharide-synthesis genes that assemble or expose a 2-keto-3-deoxyoctulosonic acid epitope.
We recently isolated a recombinant phage from a Haemophilus influenzae type b (Hib) library that assembles an oligosaccharide with an apparent molecular weight of 1400 (1.4 K) on a 4.1 K Escherichia coli lipopolysaccharide (LPS) structure, producing a 5.5 K LPS species that contains a KDO (2-keto-deoxyoctulosonic acid) epitope. Subcloning and deletional analysis of the 14 kb Haemophilus insert showed that three overlapping restriction fragments contained within a 7.2 kb Pstl-BamHl fragment sequentially modified an E. coli 4.1 K LPS structure, generating novel species of 4.5 K, 5.1 K and 5.5 K. Only the 5.5 K species contained the KDO epitope. We confirmed the relationship between the cloned genes and Hib lipooligosaccharide (LOS) biosynthesis by constructing a mutant that expressed an altered LOS. Thus, the Hib 7.2 kb Pstl-BamHl restriction fragment contained a cluster of at least three genetic loci whose products acted sequentially in LOS biosynthesis.
Abu Kwaik
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Department of Medicine, School of Medicine, State University of New York, Buffalo 14215.
McLaughlin
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Apicella
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Spinola
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SM
D015153:Blotting, Western; D002872:Chromosome Deletion; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006193:Haemophilus influenzae / Q000235:genetics* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000096:biosynthesis* / Q000302:isolation & purification; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D015183:Restriction Mapping; D013400:Sugar Acids / Q000032:analysis / Q000276:immunology / Q000378:metabolism*
D004269:DNA, Bacterial; D000939:Epitopes; D008070:Lipopolysaccharides; D013400:Sugar Acids; C023023:lipid-linked oligosaccharides; C002532:2-keto-3-deoxyoctonate