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• W2008238093 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 IVA, 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 inE. coli behind a T7 promoter, massive overproduction of kinase activity was achieved (∼8000-fold higher than in H. influenzae membranes). The catalytic properties and the product generated by the overexpressed kinase, assayed with Kdo-lipid IVA as the substrate, were the same as observed withH. 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. 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 IVA, 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 inE. coli behind a T7 promoter, massive overproduction of kinase activity was achieved (∼8000-fold higher than in H. influenzae membranes). The catalytic properties and the product generated by the overexpressed kinase, assayed with Kdo-lipid IVA as the substrate, were the same as observed withH. 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. lipopolysaccharide 3-deoxy-d-manno-octulosonic acid polymerase chain reaction kilobase pairs matrix-assisted laser desorption/ionization The Gram-negative pathogen Haemophilus influenza is a common cause of otitis media, upper respiratory infections, and meningitis in children (1Klein J.O. Pediatr. Infect. Dis. J. 1997; 16: 5-8Crossref PubMed Scopus (72) Google Scholar, 2Moxon E.R. Wilson R. Rev. Infect. Dis. 1991; 13 Suppl. 6: 518-527Crossref Scopus (60) Google Scholar, 3Moxon E.R. J. Infect. Dis. 1992; 165 Suppl. 1: 77-81Crossref Scopus (19) Google Scholar, 4Murphy T.F. Yi K. Ann. N. Y. Acad. Sci. 1997; 830: 353-360Crossref PubMed Scopus (14) Google Scholar). Like other Gram-negative bacteria (5Nikaido H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 29-47Google Scholar,6Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar), the outer surface of the H. influenzae outer membrane consists predominantly of lipopolysaccharide (LPS)1 (7Hood D.W. Deadman M.E. Allen T. Masoud H. Martin A. Brisson J.R. Fleischmann R. Venter J.C. Richards J.C. Moxon E.R. Mol. Microbiol. 1996; 22: 951-965Crossref PubMed Scopus (147) Google Scholar). LPS provides the organism with a permeability barrier to certain antibacterial agents (5Nikaido H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 29-47Google Scholar, 6Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar). LPS is anchored into the outer leaflet of the outer membrane by its lipid A moiety (Fig. 1). Lipid A is an acylated disaccharide of glucosamine that is usually phosphorylated at positions 1 and 4′ and triggers many of the inflammatory responses associated with infections (6Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 8Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar, 9Wyckoff T.J.O. Raetz C.R.H. Jackman J.E. Trends Microbiol. 1998; 6: 154-159Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The enzymatic steps of lipid A biosynthesis have been fully delineated in Escherichia coli(6Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 10Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1041) Google Scholar). Most of the relevant structural genes are required for viability (6Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 9Wyckoff T.J.O. Raetz C.R.H. Jackman J.E. Trends Microbiol. 1998; 6: 154-159Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The current surge in bacterial genome sequencing projects has made it possible to identify homologues of the E. coli lipid A genes (6Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 9Wyckoff T.J.O. Raetz C.R.H. Jackman J.E. Trends Microbiol. 1998; 6: 154-159Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) in H. influenzae (11Fleischmann R.D. Adams M.D. White O. Clayton R.A. Kirkness E.F. Kerlavage A.R. Bult C.J. Tomb J.-F. Dougherty B.A. Merrick J.M. McKenney K. Sutton G. FitzHugh W. Fields C. Gocayne J.D. Scott J. Shirley R. Liu L.-I. Glodek A. Kelley J.M. Weidman J.F. Phillips C.A. Spriggs T. Hedblom E. Cotton M.D. Utterback T.R. Hanna M.C. Nguyen D.T. Saudek D.M. Brandon R.C. Fine L.D. Fritchman J.L. Furhmann J.L. Geohagen N.S.M. Gnehm C.L. McDonald L.A. Small K.V. Fraser C.M. Smith H.O. Venter J.C. Science. 1995; 269: 496-512Crossref PubMed Scopus (4702) Google Scholar) and other pathogens (12Tomb J.F. White O. Kerlavage A.R. Clayton R.A. Sutton G.G. Fleischmann R.D. Ketchum K.A. Klenk H.P. Gill S. Dougherty B.A. Nelson K. Quackenbush J. Zhou L. Kirkness E.F. Peterson S. Loftus B. Richardson D. Dodson R. Khalak H.G. Glodek A. McKenney K. Fitzgerald L.M. Lee N. Adams M.D. Venter J.C. et al.Nature. 1997; 388: 539-547Crossref PubMed Scopus (3028) Google Scholar, 13Andersson S.G. Zomorodipour A. Andersson J.O. Sicheritz-Ponten T. Alsmark U.C. Podowski R.M. Naslund A.K. Eriksson A.S. Winkler H.H. Kurland C.G. Nature. 1998; 396: 133-140Crossref PubMed Scopus (1338) Google Scholar, 14Kalman S. Mitchell W. Marathe R. Lammel C. Fan J. Hyman R.W. Olinger L. Grimwood J. Davis R.W. Stephens R.S. Nat. Genet. 1999; 21: 385-389Crossref PubMed Scopus (585) Google Scholar). In E. coli and Salmonella typhimurium, lipid A is glycosylated with a non-repeating oligosaccharide known as the core (6Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar,15Schnaitman C.A. Klena J.D. Microbiol. Rev. 1993; 57: 655-682Crossref PubMed Google Scholar), which begins with the unusual sugar 3-deoxy-d-manno-octulosonic acid (Kdo) (Fig. 1). Many strains are further glycosylated with a distal repeating oligosaccharide, known as the O-antigen (not shown) (6Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 15Schnaitman C.A. Klena J.D. Microbiol. Rev. 1993; 57: 655-682Crossref PubMed Google Scholar, 16Whitfield C. Trends Microbiol. 1995; 3: 178-185Abstract Full Text PDF PubMed Scopus (268) Google Scholar).H. influenzae lacks the O-antigen but contains a more extensively branched core oligosaccharide than E. coli (7Hood D.W. Deadman M.E. Allen T. Masoud H. Martin A. Brisson J.R. Fleischmann R. Venter J.C. Richards J.C. Moxon E.R. Mol. Microbiol. 1996; 22: 951-965Crossref PubMed Scopus (147) Google Scholar,17Masoud H. Moxon E.R. Martin A. Krajcarski D. Richards J.C. Biochemistry. 1997; 36: 2091-2103Crossref PubMed Scopus (123) Google Scholar). Portions of the H. influenzae core are highly variable from strain to strain (17Masoud H. Moxon E.R. Martin A. Krajcarski D. Richards J.C. Biochemistry. 1997; 36: 2091-2103Crossref PubMed Scopus (123) Google Scholar). The phenomenon of phase variation provides a mechanism for core alteration within a given strain (18Weiser J.N. Love J.M. Moxon E.R. Cell. 1989; 59: 657-665Abstract Full Text PDF PubMed Scopus (242) Google Scholar, 19High N.J. Deadman M.E. Moxon E.R. Mol. Microbiol. 1993; 9: 1275-1282Crossref PubMed Scopus (93) Google Scholar, 20Jarosik G.P. Hansen E.J. Infect. Immun. 1994; 62: 4861-4867Crossref PubMed Google Scholar). The variability in core structures may provide a means for the bacteria to evade host immune responses (18Weiser J.N. Love J.M. Moxon E.R. Cell. 1989; 59: 657-665Abstract Full Text PDF PubMed Scopus (242) Google Scholar, 19High N.J. Deadman M.E. Moxon E.R. Mol. Microbiol. 1993; 9: 1275-1282Crossref PubMed Scopus (93) Google Scholar, 20Jarosik G.P. Hansen E.J. Infect. Immun. 1994; 62: 4861-4867Crossref PubMed Google Scholar). Recent studies have established an important role for proper core biosynthesis in the virulence ofH. influenzae. Hood et al. (7Hood D.W. Deadman M.E. Allen T. Masoud H. Martin A. Brisson J.R. Fleischmann R. Venter J.C. Richards J.C. Moxon E.R. Mol. Microbiol. 1996; 22: 951-965Crossref PubMed Scopus (147) Google Scholar) prepared a series of mutants with insertions in the genes postulated to be involved in core biosynthesis. The virulence of the mutant strains was then tested by their ability to cause bacteremia in an infant rat model (7Hood D.W. Deadman M.E. Allen T. Masoud H. Martin A. Brisson J.R. Fleischmann R. Venter J.C. Richards J.C. Moxon E.R. Mol. Microbiol. 1996; 22: 951-965Crossref PubMed Scopus (147) Google Scholar). A correlation was found between the extent of core glycosylation and virulence (7Hood D.W. Deadman M.E. Allen T. Masoud H. Martin A. Brisson J.R. Fleischmann R. Venter J.C. Richards J.C. Moxon E.R. Mol. Microbiol. 1996; 22: 951-965Crossref PubMed Scopus (147) Google Scholar). For instance, mutants lacking heptose, which have the minimal LPS structure capable of supporting growth (Fig. 1), did not cause significant bacteremia (7Hood D.W. Deadman M.E. Allen T. Masoud H. Martin A. Brisson J.R. Fleischmann R. Venter J.C. Richards J.C. Moxon E.R. Mol. Microbiol. 1996; 22: 951-965Crossref PubMed Scopus (147) Google Scholar). The first step of core biosynthesis in all Gram-negative bacteria is the addition of Kdo to the 6-OH of the precursor, lipid IVA(Fig. 1) (6Raetz C.R.H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 10Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1041) Google Scholar). In both E. coli and H. influenzae, the transfer of Kdo to lipid IVA is essential for viability (7Hood D.W. Deadman M.E. Allen T. Masoud H. Martin A. Brisson J.R. Fleischmann R. Venter J.C. Richards J.C. Moxon E.R. Mol. Microbiol. 1996; 22: 951-965Crossref PubMed Scopus (147) Google Scholar, 21Belunis C.J. Clementz T. Carty S.M. Raetz C.R.H. J. Biol. Chem. 1995; 270: 27646-27652Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The E. coli Kdo transferase, encoded by the kdtA gene (22Clementz T. Raetz C.R.H. J. Biol. Chem. 1991; 266: 9687-9696Abstract Full Text PDF PubMed Google Scholar), is an unusual bi-functional enzyme (Fig. 1) that catalyzes the sequential addition of two Kdo residues in distinct glycosidic linkages to lipid IVA (23Belunis C.J. Raetz C.R.H. J. Biol. Chem. 1992; 267: 9988-9997Abstract Full Text PDF PubMed Google Scholar). Most other Gram-negative bacteria similarly contain at least two Kdo residues in their inner core (24Rietschel E.T. Brade L. Lindner B. Zähringer U. Morrison D.C. Ryan J.L. Bacterial Endotoxic Lipopolysaccharides. CRC Press, Inc., Boca Raton, FL1992: 3-41Google Scholar). In H. influenzae, however, only a single Kdo is present (25Zamze S.E. Ferguson M.A.J. Moxon E.R. Dwek R.A. Rademacher T.W. Biochem. J. 1987; 245: 583-587Crossref PubMed Scopus (34) Google Scholar, 26Helander I.M. Lindner R.B. Brade H. Altmann K. Lindberg A.A. Rietschel E.T. Zähringer U. Eur. J. Biochem. 1988; 177: 483-492Crossref PubMed Scopus (171) Google Scholar, 27Phillips N.J. Apicella M.A. Griffiss J.M. Gibson B.W. Biochemistry. 1992; 31: 4515-4526Crossref PubMed Scopus (119) Google Scholar). This Kdo is phosphorylated at its 4-OH position (17Masoud H. Moxon E.R. Martin A. Krajcarski D. Richards J.C. Biochemistry. 1997; 36: 2091-2103Crossref PubMed Scopus (123) Google Scholar), the same site at which the second Kdo residue is attached in E. coli (Fig. 1). By using a non-typeable strain of H. influenzae, we previously demonstrated that the H. influenzae Kdo transferase is mono-functional (28White K.A. Kaltashov I.A. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 16555-16563Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), i.e. capable of adding only a single Kdo residue to lipid IVA. In addition, we provided the first evidence for the presence of a Kdo kinase unique to extracts of H. influenzae (28White K.A. Kaltashov I.A. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 16555-16563Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) (Fig. 1). A homologue encoding a protein with 70% predicted similarity to E. coliKdtA was readily apparent by inspection of the H. influenzaegenome (11Fleischmann R.D. Adams M.D. White O. Clayton R.A. Kirkness E.F. Kerlavage A.R. Bult C.J. Tomb J.-F. Dougherty B.A. Merrick J.M. McKenney K. Sutton G. FitzHugh W. Fields C. Gocayne J.D. Scott J. Shirley R. Liu L.-I. Glodek A. Kelley J.M. Weidman J.F. Phillips C.A. Spriggs T. Hedblom E. Cotton M.D. Utterback T.R. Hanna M.C. Nguyen D.T. Saudek D.M. Brandon R.C. Fine L.D. Fritchman J.L. Furhmann J.L. Geohagen N.S.M. Gnehm C.L. McDonald L.A. Small K.V. Fraser C.M. Smith H.O. Venter J.C. Science. 1995; 269: 496-512Crossref PubMed Scopus (4702) Google Scholar). Analysis of the reaction product generated by the overexpressed recombinant H. influenzae KdtA confirmed this genomic assignment and the mono-functional activity of the protein. 2K. A. White and C. R. H. Raetz, manuscript in preparation. However, since no protein sequence was available for the Kdo kinase, the genome sequence alone was insufficient to permit identification of the kinase gene. We now report the expression cloning and biochemical characterization of the Kdo kinase structural gene of H. influenzae. A cosmid library containing DNA fragments of the H. influenzae strain used for the genome project (Rd) (11Fleischmann R.D. Adams M.D. White O. Clayton R.A. Kirkness E.F. Kerlavage A.R. Bult C.J. Tomb J.-F. Dougherty B.A. Merrick J.M. McKenney K. Sutton G. FitzHugh W. Fields C. Gocayne J.D. Scott J. Shirley R. Liu L.-I. Glodek A. Kelley J.M. Weidman J.F. Phillips C.A. Spriggs T. Hedblom E. Cotton M.D. Utterback T.R. Hanna M.C. Nguyen D.T. Saudek D.M. Brandon R.C. Fine L.D. Fritchman J.L. Furhmann J.L. Geohagen N.S.M. Gnehm C.L. McDonald L.A. Small K.V. Fraser C.M. Smith H.O. Venter J.C. Science. 1995; 269: 496-512Crossref PubMed Scopus (4702) Google Scholar) was constructed in E. coli. Lysates of single colonies harboring individual hybrid cosmids of the library were assayed for the presence of Kdo kinase activity, which is absent in E. coli (28White K.A. Kaltashov I.A. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 16555-16563Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) (Fig. 1). A single cosmid that directs the expression of the kinase was found. Interestingly, the gene encoding the kinase had previously been described as a possible open reading frame of unknown function, termedorfZ (7Hood D.W. Deadman M.E. Allen T. Masoud H. Martin A. Brisson J.R. Fleischmann R. Venter J.C. Richards J.C. Moxon E.R. Mol. Microbiol. 1996; 22: 951-965Crossref PubMed Scopus (147) Google Scholar), in H. influenzae. Although OrfZ had been shown to be essential for virulence, no biochemical function could be assigned to the protein based solely upon its sequence and the apparently normal electrophoretic properties of the LPS isolated fromorfZ mutants (7Hood D.W. Deadman M.E. Allen T. Masoud H. Martin A. Brisson J.R. Fleischmann R. Venter J.C. Richards J.C. Moxon E.R. Mol. Microbiol. 1996; 22: 951-965Crossref PubMed Scopus (147) Google Scholar). In light of our discovery thatorfZ encodes the Kdo kinase, the genetic and pathogenic studies by Hood et al. (7Hood D.W. Deadman M.E. Allen T. Masoud H. Martin A. Brisson J.R. Fleischmann R. Venter J.C. Richards J.C. Moxon E.R. Mol. Microbiol. 1996; 22: 951-965Crossref PubMed Scopus (147) Google Scholar) can now be re-interpreted to suggest that the absence of Kdo phosphorylation in H. influenzae dramatically reduces virulence but does not stop bacterial growth. Given the identification of the biochemical function of orfZ, we suggest that the gene now be designatedkdkA (for “Kdo kinase A”) (Fig. 1). Consistent with the occurrence of phosphorylated Kdo residues in some Gram-negative bacteria (29Chaby R. Szabo R. Eur. J. Biochem. 1975; 59: 277-280Crossref PubMed Scopus (53) Google Scholar, 30Le Dur A. Caroff M. Chaby R. Szabo L. Eur. J. Biochem. 1978; 84: 579-589Crossref PubMed Scopus (37) Google Scholar, 31Brade H. J. Bacteriol. 1985; 161: 795-798Crossref PubMed Google Scholar, 32Bock K. Vinogradov E.V. Holst O. Brade H. Eur. J. Biochem. 1994; 225: 1029-1039Crossref PubMed Scopus (54) Google Scholar), significant homologues of kdkA are present in Bordetella pertussis, Vibrio cholerae,Actinobacillus actinomycetemcomitans, Shewanella putrefaciens, and Pateurella multocida. [γ-32P]ATP was purchased from NEN Life Science Products. Kdo, HEPES, EDTA, EGTA, NAD+, heme, CTP, ATP, and other nucleotides were purchased from Sigma. Triton X-100 was Surfact-Amps grade from Pierce. Yeast extract, tryptone, and brain heart infusion were obtained from Difco. All other chemicals and solvents were reagent grade. DEAE-cellulose (DE52) was purchased from Whatman. The 0.25-mm glass backed Silica Gel 60 thin layer chromatography plates were from Merck. The various strains and plasmids utilized for the experiments described are detailed in Table I. H. influenzae strain Rd (catalog number 51907) was purchased from ATCC. The H. influenzae cells were grown at 37 °C in brain heart infusion medium (37 g/liter) supplemented with heme (10 μg/ml) and NAD+ (10 μg/ml) (33Barcak G.J. Chandler M.S. Redfield R.J. Tomb J.F. Methods Enzymol. 1991; 204: 321-342Crossref PubMed Scopus (150) Google Scholar). E. coli strains were grown at 37 °C on Luria broth, consisting of 10 g of NaCl, 10 g of tryptone, and 5 g of yeast extract per liter (34Miller J.R. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). When applicable, the cultures were supplemented with 50 μg/ml ampicillin and/or 10 μg/ml chloramphenicol.Table IRelevant bacterial strains and plasmidsBacterial strain or plasmidRelevant genotypeSourceStrains H. influenzae, strain 722Non-typeable wild type28White K.A. Kaltashov I.A. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 16555-16563Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar H. influenzae, strain RdWild typeATCC (51907) XL1 Blue-MRΔmcrABC, recA1, lacStratagene Surelac/ qZΔM15, TetrStratagene BLR(DE3)/pLysSΔ(sr1-recA)306::Tn10(DE3), Tetr/CmrStratagene B67EXL1 Blue-MR containing cosmid 7EThis workPlasmids pWE15Cosmid, Ampr pBluescript KS IIlacZ, AmprStratagene 7EpWE15 encoding the Kdo kinase activity (kdkA/orfZ), AmprThis work pE3.24556-bp EcoRI fragment cloned into pBluescript, kdkA +, AmprThis work pB6A1192-bp BamHI deletion from pE3.2,kdkA +, AmprThis work pIB100Intergenic region between orfM andopsX cloned into pBluescript, kdkA +, AmprThis work pET21aVector containing a T7 promoter, AmprNovagen pKdkApET21a containing Kdo kinase (orfZ) coding region, AmprThis work Open table in a new tab Milligram quantities of the precursor lipid IVA were prepared as described previously (35Raetz C.R.H. Purcell S. Meyer M.V. Qureshi N. Takayama K. J. Biol. Chem. 1985; 260: 16080-16088Abstract Full Text PDF PubMed Google Scholar). Prior to use in assays and the synthesis of Kdo-lipid IVA, the lipid was subjected to reverse phase chromatography (36Hampton R.Y. Golenbock D.T. Raetz C.R.H. J. Biol. Chem. 1988; 263: 14802-14807Abstract Full Text PDF PubMed Google Scholar). Unlabeled Kdo-lipid IVA was prepared by a previously described method (28White K.A. Kaltashov I.A. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 16555-16563Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Both unlabeled and Kdo2[4′-32P]lipid IVA were prepared by the published methods (37Brozek K.A. Hosaka K. Robertson A.D. Raetz C.R.H. J. Biol. Chem. 1989; 264: 6956-6966Abstract Full Text PDF PubMed Google Scholar, 38Basu S.S. York J.D. Raetz C.R.H. J. Biol. Chem. 1999; 274: 11139-11149Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The [4′-32P]lipid IVA was prepared by the method of Brozek et al. (39Brozek K.A. Bulawa C.E. Raetz C.R.H. J. Biol. Chem. 1987; 262: 5170-5179Abstract Full Text PDF PubMed Google Scholar), using membranes isolated from theE. coli strain pJK2/BLR(DE3), which overexpresses the 4′-kinase (40Garrett T.A. Kadrmas J.L. Raetz C.R.H. J. Biol. Chem. 1997; 272: 21855-21864Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The Kdo[4′-32P]lipid IVA was synthesized by a slight modification of the published method (28White K.A. Kaltashov I.A. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 16555-16563Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Briefly, [4′-32P]lipid IVA was prepared as usual (38Basu S.S. York J.D. Raetz C.R.H. J. Biol. Chem. 1999; 274: 11139-11149Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), but at the end of the reaction, the volume was adjusted to 180 μl with water. The solution was then converted to a two-phase Bligh/Dyer system, consisting of CHCl3/methanol/H2O (2:2:1.8, v/v) (41Bligh E.G. Dyer J.J. Can. J. Biochem. Physiol. 1959; 37: 911-918Crossref PubMed Scopus (43133) Google Scholar, 42Nishijima M. Raetz C.R.H. J. Biol. Chem. 1979; 254: 7837-7844Abstract Full Text PDF PubMed Google Scholar), by the addition of 200 μl of both CHCl3 and methanol. The tube was mixed thoroughly and centrifuged at 20,800 ×g for 5 min at room temperature to separate the phases. The chloroform-rich lower phase was removed and transferred to a fresh tube. The upper phase was then washed twice with pre-equilibrated acidic lower phase, i.e. a lower phase generated by mixing chloroform/methanol/0.1 m HCl (2:2:1.8, v/v). The resulting lower phases were pooled with the initial lower phase and dried under a stream of nitrogen. The reaction components for the mono-functional Kdo transferase reaction (28White K.A. Kaltashov I.A. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 16555-16563Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) were then added to the tube. Following the Kdo transferase reaction, the Kdo[4′-32P]lipid IVA was isolated by preparative thin layer chromatography as described (28White K.A. Kaltashov I.A. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 16555-16563Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). All lipids were stored as aqueous dispersions at −20 °C and were dispersed again after thawing by sonic irradiation in a bath for 30–60 s prior to use. Recombinant E. coliCMP-Kdo synthetase (43Goldman R.C. Bolling T.J. Kohlbrenner W.E. Kim Y. Fox J.L. J. Biol. Chem. 1986; 261: 15831-15835Abstract Full Text PDF PubMed Google Scholar) was partially purified as described by Brozeket al. (37Brozek K.A. Hosaka K. Robertson A.D. Raetz C.R.H. J. Biol. Chem. 1989; 264: 6956-6966Abstract Full Text PDF PubMed Google Scholar). H. influenzae Rd genomic DNA was prepared as described previously (33Barcak G.J. Chandler M.S. Redfield R.J. Tomb J.F. Methods Enzymol. 1991; 204: 321-342Crossref PubMed Scopus (150) Google Scholar). Plasmid DNA was isolated using the Qiagen Mini-Prep purification system (Qiagen). Restriction endonucleases (New England Biolabs), T4 DNA ligase (Life Technologies, Inc.), and shrimp alkaline phosphatase (U. S. Biochemical Corp.) were used according to the manufacturers' instructions. DNA sequencing was performed at the Duke University Medical Center shared DNA sequencing facility. Kdo transfer from the donor, CMP-Kdo, to the acceptor, [4′-32P]lipid IVA, was assayed as described previously (28White K.A. Kaltashov I.A. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 16555-16563Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The reaction mixtures (typically 10–20 μl) contained 50 mm HEPES, pH 7.5, 2 mm Kdo, 0.1% Triton X-100, 100 μm[4′-32P]lipid IVA (3000–6000 cpm/nmol), 5 mm CTP, 10 mm MgCl2, and 1.8 milliunits of partially purified, recombinant CMP-Kdo synthase. Assays (at 30 °C) were initiated by the addition of enzyme, usuallyH. influenzae membrane preparations, as indicated. The reactions were terminated by spotting 5-μl portions onto a thin layer plate. The plate was dried under a cool air stream and developed in the solvent chloroform/pyridine/88% formic acid/water (30:70:16:10, v/v). The solvent was evaporated with a hot air stream, and the plate was exposed to a PhosphorImager screen for 12–16 h. The conversion of32P-labeled substrate to product was quantified using a Molecular Dynamics PhosphorImager equipped with ImageQuant software. The Kdo kinase was assayed using the acceptor Kdo[4′-32P]lipid IVA, as described previously (28White K.A. Kaltashov I.A. Cotter R.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 16555-16563Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The conditions were very similar to those used for the Kdo transferase. Briefly, reaction mixtures (10–20 μl) contained 50 mm HEPES, pH 7.5, 0.1% Triton X-100, 10 mmMgCl2, 5 mm ATP, and 100 μmKdo[4′-32P]lipid IVA (3000–6000 cpm/nmol). The reactions were initiated with enzyme and incubated for designated times at 30 °C. The assays were terminated, and the substrate and product were resolved by thin layer chromatography, as described above for the Kdo transferase. When membranes from pKdkA/BLR(DE3)/pLysS were used in the assays, 1 mg/ml bovine serum albumin was included in the reaction mixture. Other minor modifications to the standard reaction conditions are noted in the figure legends. A cosmid library of H. influenzae strain Rd (11Fleischmann R.D. Adams M.D. White O. Clayton R.A. Kirkness E.F. Kerlavage A.R. Bult C.J. Tomb J.-F. Dougherty B.A. Merrick J.M. McKenney K. Sutton G. FitzHugh W. Fields C. Gocayne J.D. Scott J. Shirley R. Liu L.-I. Glodek A. Kelley J.M. Weidman J.F. Phillips C.A. Spriggs T. Hedblom E. Cotton M.D. Utterback T.R. Hanna M.C. Nguyen D.T. Saudek D.M. Brandon R.C. Fine L.D. Fritchman J.L. Furhmann J.L. Geohagen N.S.M. Gnehm C.L. McDonald L.A. Small K.V. Fraser C.M. Smith H.O. Venter J.C. Science. 1995; 269: 496-512Crossref PubMed Scopus (4702) Google Scholar) was constructed inE. coli XL1-Blue MR (Strategene), utilizin" @default.
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