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 (1.4K) on a 4.1 K Escherichia coli lipopolysaccharide (LPS) structure, producing a 5.5K 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 7.2kb PstI-BamHI fragment sequentially modified an E. coli4.1 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 7.2kb PstI-BamHI restriction fragment contained a cluster of at least three genetic loci whose products acted sequentially in LOS biosynthesis.