A Haemophilus influenzae gene that encodes a membrane bound 3-deoxy-D-manno-octulosonic acid (Kdo) kinase - Possible involvement of Kdo phosphorylation in bacterial virulence

The lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy- D-manno-octulosonic acid (Kdo) residue derivatized with either a phosphate or an ethanolamine pyrophosphate moiety at the 4-OH position. In previous s tudies, we identified a kinase unique to H. influenzae extracts that phosph orylates Kdo-lipid IVA, a key precursor of lipopolysaccharide in this organ ism. We have now identified the gene encoding the Kdo kinase by using an ex pression cloning approach. A cosmid library containing random DNA fragments from H. influenzae strain Rd was constructed in Escherichia coli. Extracts of 472 colonies containing individual hybrid cosmids were assayed for Kdo kinase activity. A single hybrid cosmid directing expression of the kinase was found. The kinase gene was identified by activity assays, sub-cloning, and DNA sequencing. When the putative kinase gene was expressed in E. coli behind a T7 promoter, massive overproduction of kinase activity was achieve d (similar to 8000-fold higher than in H. influenzae membranes). The cataly tic properties and the product generated by the overexpressed kinase, assay ed with Kdo-lipid IV,as the substrate, were the same as observed with H. in fluenzae membranes. Unexpectedly, the kinase gene was identical to a previo usly characterized open reading frame (orfZ), which had been shown to be im portant for establishing bacteremia in an infant rat model (Hood, D. W., De adman, M. E., Alien, T., Masoud, H., Martin, A., Brisson, J. R., Fleischman n, R., Venter, J, C., Richards, J. C., and Moxon, E. R. (1996) Mol. Microbi ol. 22, 951-965). However, based solely on the genome sequence of H. influe nzae Rd, no biochemical function had been assigned to the product of orfZ, which we now designate kdkA ("Kdo kinase A"). Although Kdo phosphorylation may be critical for bacterial virulence of H. influenzae, it does not appea r to be required for growth.