The structures of lipopolysaccharides (LPSs) of lic1 and lic1/lic2 mutants from Haemophilus influenzae type b strain Eagan (RM153) were investigated using methylation analysis, electrospray ionization – mass spectrometry, and nuclear magnetic resonance spectroscopy on O-deacylated, O- and N-deacylated core oligosaccharide (OS); and deacylated, dephosphorylated, and terminally reduced samples. The backbone OS derived from the major LPS glycoforms were determined to consist of the inner-core triheptosyl unit, l-α-d-Hepp-(1-2)-l-α-d-Hepp-(1-3)-l-α-d-Hepp-(1-, common to all H. influenzae strains investigated to date that is linked to the lipid A region of the molecule via a Kdo residue to which β-d-Glcp and β-d-Galp residues are attached in 1,4 and 1,2 linkages to the proximal (HepI) and distal (HepIII) heptose residues, respectively. It was found that the lic1 mutant predominately elaborates the Hex4 LPS glycoforms previously identified in the parent strain where a β-d-Glcp-(1-4)-α-d-Glcp unit is linked in a 1,3 linkage to the central heptose (HepII) of the triheptosyl moiety. The lic1 locus consists of 4 genes (lic1A to lic1D) in a single transcriptional unit that directs phase variable expression of phosphocholine. The lic1A gene is phased off in the RM153 isolate of strain Eagan. LPS from the double mutant, lic1/lic2 had a similar structure to that of lic1 mutant except that there was no chain extension from the central heptose in the inner core (HepII). The lic2 locus consists of 4 genes (lic2A to lic2D). Our structural data were consistent with the proposed function of lic2C, providing the first definitive evidence for its role as the glycosyltransferase required for chain initiation from HepII. The presence of an O-acetyl group at O-3 of the distal heptose (HepIII) was elucidated by 1H NMR on the mild acid liberated core OS samples.
