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J. Biol. Chem., Vol. 275, Issue 45, 34954-34962, November 10, 2000

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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^*

Werner Brabetz, Sven Müller-Loennies, and Helmut Brade

From the Division of Medical and Biochemical Microbiology, Research Center Borstel, Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany

The lipopolysaccharide (LPS) of the deep rough mutant Haemophilus influenzae 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 Zähringer, U. (1988) Eur. J. Biochem. 177, 483-492). The waaA gene encoding the essential LPS-specific Kdo transferase was cloned from this strain, 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. waaA of H. influenzae could not complement a knockout mutation in the corresponding gene of an Re-type Escherichia coli strain. However, complementation was possible by coexpressing the recombinant waaA together with the LPS-specific Kdo kinase gene (kdkA) of H. influenzae DSM11121 or I69, respectively. The sequences of both kdkA genes were determined and differed in 25 nucleotides, giving rise to six amino acid exchanges between the deduced proteins. Both E. coli strains which expressed waaA and kdkA from H. influenzae synthesized an LPS containing a single Kdo residue that was exclusively phosphorylated 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 structures synthesized by H. influenzae I69.

The nucleotide sequences reported in this paper have been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession numbers AJ277814, AJ277816, AJ277815, and AJ277817.

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