Characterization of the lipopolysaccharide from Pasteurella multocida Heddleston serovar 9: Identification of a proposed bi-functional dTDP-3-acetamido-3,6-dideoxy-α-d-glucose biosynthesis enzyme

Pasteurella multocida strains are classified into 16 different lipopolysaccharide (LPS) serovars using the Heddleston serotyping scheme. Ongoing studies in our laboratories on the LPS aim to determine the core oligosaccharide ( OS) structures expressed by each of the Heddleston type strains and identify the genes and transferases required for the biosynthesis of the serovar-specific OSs. In this study, we have determined the core OS of the LPS expressed by the Heddleston serovar 9 type strain, P2095. Structural information was established by a combination of monosaccharide and methylation analyses, nuclear magnetic resonance spectroscopy and mass spectrometry revealing the following structure: alpha-Rha-(1-2)-beta-Qui3NAc-(1-2)-beta-Qui3NAc-(1-2)-alpha-Rha-(1-2)-alpha-Rha(1-4)- alpha-Glc P-(PEtn) D,D-alpha-Hep-(1-6)-beta-Glc-(1-4)-L,D-alpha-Hep(3)(6)-1-5-alpha-Kdo(4) L,D-alpha-Hep(2)(1)-3-PEtn L,D-alpha-Hep(1) The serovar 9 OS contains an inner core that is conserved among P. multocida strains with an elaborate outer core extension containing rhamnose (Rha), a D-glycero-D-manno isomer of heptose, and the unusual deoxyamino sugar, 3-acetamido-3,6-dideoxy-alpha-D-glucose (Qui3NAc). Genetic analyses of the LPS outer core biosynthesis locus revealed that in addition to the glycosyltransferases predicted to transfer the sugars to the nascent LPS molecule, the locus also contained the complete set of genes required for the biosynthesis of the nucleotide sugar donors dTDP-Rha and dTDP-Qui3NAc. One of the genes identified as part of the dTDP-Qui3NAc biosynthesis pathway, qdtD, encodes a proposed bi-functional enzyme with N-terminal amino acid identity to dTDP-4-oxo-6-deoxy-D-glucose-3,4oxoisomerase and C-terminal amino acid identity to dTDP3-oxo-6-deoxy-a-D-glucose transacetylase.