title: 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.
abstract: 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 studies, we identified a kinase unique to H. influenzae extracts that phosphorylates Kdo-lipid IV(A), a key precursor of lipopolysaccharide in this organism. We have now identified the gene encoding the Kdo kinase by using an expression 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 achieved ( approximately 8000-fold higher than in H. influenzae membranes). The catalytic properties and the product generated by the overexpressed kinase, assayed with Kdo-lipid IV(A) as the substrate, were the same as observed with H. influenzae membranes. Unexpectedly, the kinase gene was identical to a previously characterized open reading frame (orfZ), which had been shown to be important for establishing bacteremia in an infant rat model (Hood, D. W., Deadman, M. E., Allen, T., Masoud, H., Martin, A., Brisson, J. R., Fleischmann, R., Venter, J. C., Richards, J. C., and Moxon, E. R. (1996) Mol. Microbiol. 22, 951-965). However, based solely on the genome sequence of H. influenzae 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 appear to be required for growth.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: K A
authors.initials: KA
authors.lastname: White
authors.forename: S
authors.initials: S
authors.lastname: Lin
authors.forename: R J
authors.initials: RJ
authors.lastname: Cotter
authors.forename: C R
authors.initials: CR
authors.lastname: Raetz
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003360:Cosmids; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D015698:Genomic Library; D006193:Haemophilus influenzae / Q000235:genetics* / Q000472:pathogenicity; D008050:Lipid A / Q000031:analogs & derivatives* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000096:biosynthesis; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism
chemical_list: D008050:Lipid A; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D009711:Nucleotides; D011994:Recombinant Proteins; C088602:3-deoxy-2-octulosonic acid(2)-lipid IV(A); D014166:Transferases; C019917:3-deoxy-D-manno-octulosonate transferase; C400526:3-deoxy-D-manno-octulosonic acid kinase; D017853:Phosphotransferases (Alcohol Group Acceptor)