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Analysis of Haemophilus influenzae type b lipooligosaccharide‐synthesis genes that assemble or expose a 2‐keto‐3‐deoxyoctulosonic acid epitope
Author(s) -
Abu Kwaik Y.,
McLaughlin B. E.,
Apicella M. A.,
Spinola S. M.
Publication year - 1991
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1991.tb02092.x
Subject(s) - biology , haemophilus influenzae , subcloning , epitope , escherichia coli , gene , microbiology and biotechnology , haemophilus , gene cluster , recombinant dna , genetics , bacteria , antigen
Summary 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–Bam HI fragment sequentially modified an E. coli 4.1 K LPS structure, generating novel species of 4.5K, 5.1K and 5.5K. Only the 5.5K 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‐Bam Hl restriction fragment contained a cluster of at least three genetic loci whose products acted sequentially in LOS biosynthesis.