3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase (WaaA) and Kdo kinase (KdkA) of Haemophilus influenzae are both required to complement a waaA knockout mutation of Escherichia coli

The lipopolysaccharide (LPS) of the deep rough mutant Haemophilus influenza e I69 consists of lipid A and a single 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue substituted with one phosphate at position 4 or 5 (Helander, I. M,, Lindner, B,, Brade, H., Altmann, K,, Lindberg, A. A., Rietschel, E, T,, and Zahringer, U, (1988) fur. J, Biochem, 177, 483-492), The waaA gene encoding the essential LPS-specific Kdo transferase was cloned from this st rain, and its nucleotide sequence was identical to H, influenzae DSM11121, The gene was expressed in the Gram-positive host Corynebacterium glutamicum : and characterized in vitro to encode a monofunctional Kdo transferase. wa aA of H, influenzae could not complement a knockout mutation in the corresp onding gene of an Re-type Escherichia coil strain. However, complementation was possible by coexpressing the recombinant waaA together with the LPS-sp ecific Kdo kinase gene (kdkA) of H, influenzae DSM11121 or I69, respectivel y. The sequences of both kdkA genes were determined and differed in 25 nucl eotides, giving rise to six amino acid exchanges between the deduced protei ns. Both E, coil strains which expressed waaA and kdkA from H, influenzae s ynthesized an LPS containing a single Kdo residue that was exclusively phos phorylated: at position 4, The structure was determined by nuclear magnetic resonance spectroscopy of deacylated LPS, Therefore, the reaction products of both cloned Kdo kinases represent only one of the two chemical structur es synthesized by H, influenzae I69.