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• W1971369615 abstract "The core oligosaccharide region of Klebsiella pneumoniae lipopolysaccharide contains some novel features that distinguish it from the corresponding lipopolysaccharide region in other members of the Enterobacteriaceae family, such as Escherichia coli and Salmonella. The conserved Klebsiella outer core contains the unusual trisaccharide 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-(2,6)-GlcN-(1,4)-GalUA. In general, Kdo residues are normally found in the inner core, but in K. pneumoniae, this Kdo residue provides the ligation site for O polysaccharide. The outer core Kdo residue can also be non-stoichiometrically substituted with an l-glycero-d-manno-heptopyranose (Hep) residue, another component more frequently found in the inner core. To understand the genetics and biosynthesis of core oligosaccharide synthesis in Klebsiella, the gene products involved in the addition of the outer core GlcN (WabH), Kdo (WabI), and Hep (WabJ) residues as well as the inner core HepIII residue (WaaQ) were identified. Non-polar mutations were created in each of the genes, and the resulting mutant lipopolysaccharide was analyzed by mass spectrometry. The in vitro glycosyltransferase activity of WabI and WabH was verified. WabI transferred a Kdo residue from CMP-Kdo onto the acceptor lipopolysaccharide. The activated precursor required for GlcN addition has not been identified. However, lysates overexpressing WabH were able to transfer a GlcNAc residue from UDP-GlcNAc onto the acceptor GalUA residue in the outer core. The core oligosaccharide region of Klebsiella pneumoniae lipopolysaccharide contains some novel features that distinguish it from the corresponding lipopolysaccharide region in other members of the Enterobacteriaceae family, such as Escherichia coli and Salmonella. The conserved Klebsiella outer core contains the unusual trisaccharide 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-(2,6)-GlcN-(1,4)-GalUA. In general, Kdo residues are normally found in the inner core, but in K. pneumoniae, this Kdo residue provides the ligation site for O polysaccharide. The outer core Kdo residue can also be non-stoichiometrically substituted with an l-glycero-d-manno-heptopyranose (Hep) residue, another component more frequently found in the inner core. To understand the genetics and biosynthesis of core oligosaccharide synthesis in Klebsiella, the gene products involved in the addition of the outer core GlcN (WabH), Kdo (WabI), and Hep (WabJ) residues as well as the inner core HepIII residue (WaaQ) were identified. Non-polar mutations were created in each of the genes, and the resulting mutant lipopolysaccharide was analyzed by mass spectrometry. The in vitro glycosyltransferase activity of WabI and WabH was verified. WabI transferred a Kdo residue from CMP-Kdo onto the acceptor lipopolysaccharide. The activated precursor required for GlcN addition has not been identified. However, lysates overexpressing WabH were able to transfer a GlcNAc residue from UDP-GlcNAc onto the acceptor GalUA residue in the outer core. Klebsiella pneumoniae is an important nosocomial pathogen and is implicated in diseases including urinary tract infections, pneumonia, and bacteremia (reviewed in Ref. 1Podschun R. Ullmann U. Clin. Microbiol. Rev. 1998; 11: 589-603Crossref PubMed Google Scholar). It is second only to Escherichia coli as the most common cause of Gram-negative sepsis. The lipopolysaccharide (LPS) 1The abbreviations used are: LPS, lipopolysaccharide(s); OS, oligosaccharide(s); O-PS, O polysaccharide; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acid; Hep, l-glycero-d-manno-heptopyranose; ORF, open reading frame; MS, mass spectrometry; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; ESI, electrospray ionization; d,d-Hep, d-glycero-d-manno-heptopyranose. 1The abbreviations used are: LPS, lipopolysaccharide(s); OS, oligosaccharide(s); O-PS, O polysaccharide; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acid; Hep, l-glycero-d-manno-heptopyranose; ORF, open reading frame; MS, mass spectrometry; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; ESI, electrospray ionization; d,d-Hep, d-glycero-d-manno-heptopyranose. of K. pneumoniae serves as an important virulence factor. It has been shown that isolates of serotypes O1 and O2 during growth release an extracellular toxic complex composed of LPS, capsule, and a small amount of protein. The extracellular toxic complex is associated with the extensive lung damage and high lethality typically seen in mice with pneumonia derived from Klebsiella (2Straus D.C. Atkisson D.L. Garner C.W. Infect. Immun. 1985; 50: 787-795Crossref PubMed Google Scholar, 3Straus D.C. Infect. Immun. 1987; 55: 44-48Crossref PubMed Google Scholar). The degree of virulence and pathology can be directly correlated with the amount of LPS in the complexes, whereas an increase in the amount of extracellular capsule has no effect (4Domenico P. Diedrich D.L. Straus D.C. Can. J. Microbiol. 1985; 31: 472-478Crossref PubMed Scopus (36) Google Scholar). As might be expected for a member of the Enterobacteriaceae, K. pneumoniae LPS shares significant similarity with the well characterized LPS structures of E. coli and Salmonella (5Holst O. Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999: 115-154Google Scholar, 6Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3221) Google Scholar). In all three species, LPS is subdivided into three regions: 1) lipid A, the hydrophobic membrane anchor; 2) a core oligosaccharide (OS); and 3) a polymer of glycosyl (repeat) units known as O polysaccharide (O-PS). The core OS region can be further subdivided on the basis of sugar composition into two regions, the inner and outer core (see Fig. 1B) (5Holst O. Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999: 115-154Google Scholar). The inner core region, which is generally well conserved in the Enterobacteriaceae, contains 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and l-glycero-d-manno-heptopyranose (Hep). In contrast, the outer core region shows more diversity and is usually composed of hexose and acetamidohexose sugars in E. coli and Salmonella. The structures of the core OS of prototype isolates representing the different serotypes of K. pneumoniae have been determined (7Vinogradov E. Perry M.B. Carbohydr. Res. 2001; 335: 291-296Crossref PubMed Scopus (52) Google Scholar). The basic core structure is identical in all the serotypes examined; they differ only in the amounts and positions of non-stoichiometric sugar substitutions (7Vinogradov E. Perry M.B. Carbohydr. Res. 2001; 335: 291-296Crossref PubMed Scopus (52) Google Scholar). The core OS of K. pneumoniae does have some features that distinguish it from the E. coli and Salmonella paradigms (see Fig. 1B). The inner core of K. pneumoniae LPS is made up of the Hep-Kdo backbone seen in other members of the Enterobacteriaceae. In E. coli and Salmonella, the Hep residues are further substituted with phosphate and phosphorylethanolamine (5Holst O. Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999: 115-154Google Scholar). The negative charge provided by these residues plays an important role in maintenance of the barrier function of the outer membrane by providing sites for cross-linking of adjacent LPS molecules with divalent cations (reviewed in Ref. 6Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3221) Google Scholar). K. pneumoniae LPS lacks these phosphate residues, a feature also seen in the LPS of Rhizobium etli and Rhizobium leguminosarum (8Forsberg L.S. Carlson R.W. J. Biol. Chem. 1998; 273: 2747-2757Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and Plesiomonas shigelloides O54 (9Niedziela T. Lukasiewicz J. Jachymek W. Dzieciatkowska M. Lugowski C. Kenne L. J. Biol. Chem. 2002; 277: 11653-11663Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). It is thought that the carboxyl groups of the GalUA sugars in the core may provide the negative charge needed for outer membrane stability. The first residue of the outer core of K. pneumoniae is GalUA. The gene required for the addition of this GalUA residue (wabG) has been identified, and mutants lacking wabG show enhanced sensitivity to hydrophobic compounds (10Izquierdo L. Coderch N. Piqué N. Bedini E. Corsaro M.M. Merino S. Fresno S. Tomás J.M. Regué M. J. Bacteriol. 2003; 185: 7213-7221Crossref PubMed Scopus (66) Google Scholar). The outer core backbone contains another unusual structural motif, the trisaccharide Kdo-(2,6)-GlcN-(1,4)-GalUA. Kdo is generally confined to the inner core, but in K. pneumoniae, it provides the ligation site for O-PS and can also be further substituted with a non-stoichiometric Hep residue (11Vinogradov E. Frirdich E. MacLean L.L. Perry M.B. Petersen B.O. Duus J.O. Whitfield C. J. Biol. Chem. 2002; 277: 25070-25081Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Hep residues are typically confined to the inner core. As a result, the enzymology of K. pneumoniae core OS biosynthesis is considerably different from that of the well characterized E. coli and Salmonella systems. These novel characteristics may be important for the development of novel therapeutic strategies or vaccines against this prevalent nosocomial pathogen. Furthermore, elucidation of the biosynthesis pathway may provide insight into the LPS assembly of other bacteria with outer cores containing Kdo. The genes responsible for core OS biosynthesis in K. pneumoniae are encoded by the waa gene cluster, whose sequence has been reported (12Regué M. Climent N. Abitiu N. Coderch N. Merino S. Izquierdo L. Altarriba M. Tomás J.M. J. Bacteriol. 2001; 183: 3564-3573Crossref PubMed Scopus (53) Google Scholar). The transferases of known function are shown in Fig. 1. Genes responsible for inner core synthesis are readily identified by conserved sequences shared with E. coli and Salmonella, and the enzymology of the biosynthesis of the K. pneumoniae Hep-Kdo inner core backbone has been verified (13Noah C. Brabetz W. Gronow S. Brade H. J. Endotoxin Res. 2001; 7: 25-33Crossref PubMed Google Scholar). Preliminary structural analysis of a non-polar waaE mutant indicates that WaaE is involved in the addition of the inner core β-Glc residue (14Izquierdo L. Merino S. Coderch N. Regué M. Tomás J.M. FEMS Microbiol. Lett. 2002; 216: 211-216PubMed Google Scholar) and that WabG is required for the addition of the initial GalUA residue of the outer core (10Izquierdo L. Coderch N. Piqué N. Bedini E. Corsaro M.M. Merino S. Fresno S. Tomás J.M. Regué M. J. Bacteriol. 2003; 185: 7213-7221Crossref PubMed Scopus (66) Google Scholar). The functions of the remaining transferases have not been assigned. The objective of this study was to resolve the biosynthesis of the outer core OS and to specifically identify the transferases involved in the addition of the novel GlcN, Kdo, and Hep residues. Bacterial Strains, Plasmids, and Growth Conditions—The bacterial strains and plasmids used in this study are summarized in Table I. Bacteria were grown in LB broth at 37 °C (15Miller J.H. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory Press, Plainview, NY1992Google Scholar). The growth media were supplemented with ampicillin (100 μg/ml), chloramphenicol (30 μg/ml), gentamycin (30 μg/ml), kanamycin (50 μg/ml), or tetracycline (15 μg/ml) as necessary. YEG-Cl (0.5% yeast extract, 1% NaCl, 0.4% glucose, 0.2% p-chloro-phenylalanine) plates were used as a counterselection method when using the suicide vector pWQ173 to construct allelic exchange mutants (16Kast P. Gene (Amst.). 1994; 138: 109-114Crossref PubMed Scopus (82) Google Scholar, 17Rahn A. Whitfield C. Mol. Microbiol. 2003; 47: 1045-1060Crossref PubMed Scopus (71) Google Scholar).Table IBacterial strains and plasmidsStrain or plasmidGenotype, serotype, or descriptionRef. or sourceE. coli strainsBL21(λDE3)F-ompT hsdSB(rB-mB-) gal dcm(λDE3)NovagenDH5αK12 Φ80d deoR lacZΔM15 endA1 recA1 hsdR17(rK-mK+) supE44 thi-1 gyrA96 relA1 Δ(lacZYA-argF) U169 F-Ref. 52Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarHB101hsdS20(rB-mB+) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1 supE44 λ- F-Ref. 52Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarLE392hsdR514(rK-mK+) supE44 supF58 lacY1 galK2 galT22 metB1 trpR55 λ- F-Ref. 52Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarK. pneumoniae strainsCWK2O1:K-, derivative of CWK1 (O1:K20); Strr AprRef. 53Whitfield C. Richards J.C. Perry M.B. Clarke B.R. MacLean L.L. J. Bacteriol. 1991; 173: 1420-1431Crossref PubMed Google ScholarCWG399waaL::aacC-1 derivative of CWK2; GmrRef. 11Vinogradov E. Frirdich E. MacLean L.L. Perry M.B. Petersen B.O. Duus J.O. Whitfield C. J. Biol. Chem. 2002; 277: 25070-25081Abstract Full Text Full Text PDF PubMed Scopus (125) Google ScholarCWG600wabI::aphA-3 derivative of CWK2; KmrThis studyCWG601wabJ::aphA-3 derivative of CWK2; KmrThis studyCWG602wabJ::aphA-3 derivative of CWG399; Gmr, KmrThis studyCWG603wabH::aphA-3 derivative of CWG399; Gmr, KmrThis studyCWG628waaQ::aphA-3 derivative of CWK2; KmrThis studyCWG629waaQ::aphA-3 derivative of CWG399; Gmr, KmrThis studyPlasmidspBAD18-CmArabinose-inducible expression vector; CmrRef. 22Guzman L.-M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3876) Google ScholarpBCSK(+)Cloning vector, ColE1 origin; CmrStratagenepET28a(+)IPTG-inducible expression vector; KmrNovagenpGEM5-Zf(+)Cloning vector, fl phage origin; AprPromegapJKB72Expression plasmid for C-terminal His6-KdsB in pCB20 shuttle vector; KmrRef. 23Gronow S. Brabetz W. Brade H. Eur. J. Biochem. 2000; 267: 6602-6611Crossref PubMed Scopus (61) Google ScholarpYA3265Source of non-polar aphA-3 cassette conferring kanamycin resistance; KmrRef. 21Menard R. Sansonetti P.J. Parsot C. J. Bacteriol. 1993; 175: 5899-5906Crossref PubMed Scopus (612) Google ScholarpWQ173Counterselectable (pheS), temperature-sensitive suicide vector based on pKO3; CmrRef. 17Rahn A. Whitfield C. Mol. Microbiol. 2003; 47: 1045-1060Crossref PubMed Scopus (71) Google ScholarpWQ33pWQ173 derivative containing wabI gene interrupted by aphA-3 cassette used to construct CWG600; Cmr, KmrThis studypWQ36pWQ173 derivative containing wabJ gene interrupted by aphA-3 cassette used to construct CWG601 and CWG602; Cmr, KmrThis studypWQ39pWQ173 derivative containing wabH gene interrupted by aphA-3 cassette used to construct CWG603; Cmr, KmrThis studypWQ40pET28a(+) derivative expressing WabI with N-terminal His6 tag; KmrThis studypWQ42pBAD18-Cm derivative expressing His6-WabI; CmrThis studypWQ43pBAD18-Cm derivative expressing WabJ; CmrThis studypWQ44pBAD18-Cm derivative expressing WabH; CmrThis studypWQ45pGEM5 derivative expressing WaaQ; CmrThis studypWQ47pWQ173 derivative containing waaQ gene interrupted by aphA-3 cassette used to construct CWG628 and CWG629; Cmr, KmrThis studypWQ48pBAD18-Cm derivative expressing WaaQ; CmrThis studypWQ161pRK404 derivative carrying waaL coding region of CWK2; TetrRef. 11Vinogradov E. Frirdich E. MacLean L.L. Perry M.B. Petersen B.O. Duus J.O. Whitfield C. J. Biol. Chem. 2002; 277: 25070-25081Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar Open table in a new tab DNA Methods—Plasmid DNA was isolated using the Sigma GenElute plasmid miniprep kit, and chromosomal DNA was prepared by the method of Hull et al. (18Hull R.A. Gill R.E. Hsu P. Minshew B.H. Falkow S. Infect. Immun. 1981; 33: 933-938Crossref PubMed Google Scholar) or by using DNAzol reagent (Invitrogen) in a modified protocol for bacteria. Briefly, the cells from 1.5 ml of an overnight culture were harvested by centrifugation and resuspended in ∼0.05 ml of culture supernatant. 1 ml of DNAzol was added to the cell suspension, and the mixture was incubated for 1 h at room temperature. Cell debris was removed by two 10-min centrifugation steps at 16,100 × g, and then 0.5 ml of cold 100% ethanol was added to the lysate and mixed by inversion. After a 0.5–1 h incubation at room temperature, the precipitated DNA was collected by centrifugation for 30 min at 16 100 × g. The DNA pellet was washed two times with cold 70% ethanol, air-dried, and dissolved overnight in ∼0.03 ml of distilled H2O. PCR amplification was performed in 0.05-ml volumes with either PwoI DNA polymerase (Roche Applied Science) or Platinum Taq DNA polymerase (Invitrogen) under conditions optimized for the primer pair. All PCR products were sequenced to verify that they were error-free. Restriction endonuclease digestions and ligation reactions were performed using standard methods as directed by the manufacturer. Plasmids were maintained in E. coli DH5α, except for pWQ173 derivatives, which were transformed into E. coli HB101. For E. coli strains, transformation was carried out by electroporation following methods described elsewhere (19Binotto J. MacLachlan P.R. Sanderson P.R. Can. J. Microbiol. 1991; 37: 474-477Crossref PubMed Scopus (48) Google Scholar). For some K. pneumoniae strains, a modification of this method was required (20Enderle P.J. Farwell M.A. BioTechniques. 1998; 25: 954-958Crossref PubMed Scopus (73) Google Scholar). Briefly, colonies were scraped off an LB plate (after overnight growth) and resuspended in 0.04 ml of cold distilled H2O. Plasmid DNA was added to the cells, and the DNA/cell suspension was then transferred to an electroporation cuvette for electroporation as described above. In Vitro Mutagenesis and Allelic Exchange—Individual genes were mutated by insertion of a non-polar antibiotic resistance cassette into the target open reading frame (ORF). The same strategy was used for each mutation. The non-polar cassette used was the aphA-3 (kanamycin) resistance cassette from plasmid pYA3265 (21Menard R. Sansonetti P.J. Parsot C. J. Bacteriol. 1993; 175: 5899-5906Crossref PubMed Scopus (612) Google Scholar), and it was cloned in the same orientation for transcription as the target gene. The mutated gene was transferred to the chromosome by homologous recombination using the temperature-sensitive suicide delivery vector pWQ173 containing the counterselectable marker pheS as described previously (17Rahn A. Whitfield C. Mol. Microbiol. 2003; 47: 1045-1060Crossref PubMed Scopus (71) Google Scholar). Mutations were made in waaQ, wabH, wabI, and wabJ. The plasmids containing waaQ::aphA-3 (pWQ47), wabH::aphA-3 (pWQ39), wabI::aphA-3 (pWQ33), and wabJ::aphA-3 (pWQ36) were transformed into K. pneumoniae CWK2 or CWG399 (waaL) by electroporation, as appropriate, for allelic exchange. Mutants were selected based on kanamycin resistance and chloramphenicol sensitivity, and the chromosomal mutations were confirmed by sequencing of the insertion junctions in an amplified PCR product. One representative mutant for each gene was selected for further analysis (Table I). The waaQ::aphA-3 mutant was constructed by first PCR-amplifying waaQ from K. pneumoniae CWK2 chromosomal DNA using KPwaa9 (5′-CGCCCTCGGTACCAATCGTGAT-3′) and KPwaa10 (5′-CCGCGCAGGTACCAGTCTTCAT-3′). The primers include KpnI sites (underlined). The 1673-bp PCR product was ligated using blunt ends to HincII-digested pGEM-5Zf(+). A recombinant plasmid was selected with the waaQ gene inserted in the same orientation as the T7 promoter of pGEM-5Zf(+) to form pWQ45. A HincII fragment carrying the aphA-3 cassette was inserted into HincII- and SmaI-digested pWQ45. The waaQ::aphA-3 gene was then removed as a 1834-bp KpnI fragment (using the sites introduced by the PCR primers) and cloned into pWQ173 to form pWQ47. To construct the wabH::aphA-3 mutant, the wabH coding region was PCR-amplified as a 1614-bp fragment from K. pneumoniae CWK2 chromosomal DNA with primers KPwaa35 (5′-GCCTGCACCAGGGATCCACTCTCAA-3′) and KPwaa37 (5′-GAGGGCAGGGGTACCAGTGGGAA-3′). The PCR product was digested with BamHI and KpnI at sites designed into the primers (underlined) and ligated to the similarly digested pBCSK(+) vector. This plasmid was digested with BglII at a single site within the wabH ORF and ligated to the aphA-3 cassette digested from pYA3265 with BamHI. The 2473-bp wabH::aphA-3 gene was then removed as a BamHI-KpnI fragment and ligated to the suicide delivery vector pWQ173 using the same sites to construct pWQ39. To construct the wabI::aphA-3 mutant, the coding region was first PCR-amplified from K. pneumoniae CWK2 chromosomal DNA using primers KPwaaC1 (5′-GCTGGCAGGAGCTTCTAGATCTGCTG-3′) and KPwaa2 (5′-AGGCGAAGCAGGTACCCTGTGAAGA-3′). The 2588-bp PCR product was digested with XbaI and KpnI at sites introduced by the primers (underlined) and ligated to the similarly digested pBCSK(+) vector. The resulting plasmid was digested with EcoRV at a single site within the wabI ORF and ligated to the HincII fragment containing the aphA-3 cassette from pYA3265. The wabI::aphA-3 gene was removed from pBCSK(+) as a 3425-bp XbaI-KpnI fragment and ligated to the equivalent sites in the suicide delivery vector pWQ173, generating pWQ33. For the wabJ::aphA-3 mutant, wabJ was PCR-amplified from K. pneumoniae CWK2 chromosomal DNA using KPwaa3 (5′-GCGCCTGAGCATCTGGATCCATAC-3′) and KPwaa4 (5′-GCGGCAATGTGGTACCGATGAA-3′). The 1485-bp PCR product was digested with BamHI and KpnI at sites introduced by the primers (underlined) and ligated to pBCSK(+) digested with the same enzymes. The plasmid was digested with HincII and ligated to the HincII fragment carrying the aphA-3 cassette. The wabJ::aphA-3 gene was removed as a 2048-bp BamHI-KpnI fragment and cloned into the suicide delivery vector pWQ173 using the same sites, generating pWQ36. Plasmid Constructs for Mutant Complementation Studies—For complementation, each gene was expressed using a pBAD vector derivative in the relevant mutant strain. Plasmid pBAD18-Cm belongs to a family of expression vectors that uses the arabinose-inducible and glucose-repressible promoter (22Guzman L.-M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3876) Google Scholar). Repression from the araC promoter was achieved by growth in medium containing 0.4% (w/v) glucose, and induction was obtained by adding l-arabinose to a final concentration of 0.02% (w/v). Briefly, a culture was grown for 18 h at 37 °C in LB medium supplemented with ampicillin and 0.4% glucose. This culture was diluted 1:100 in fresh medium (without glucose) and grown until the culture reached A600 nm = 0.2. l-Arabinose was then added, and the culture was grown for another 2 h. Repressed controls were maintained in glucose-containing medium. For expression of the waaQ coding region, the waaQ ORF was removed from pWQ45 as a KpnI fragment (KpnI sites were introduced into the primers used to PCR-amplify waaQ) and ligated to pBAD18-Cm to form pWQ48. The wabJ gene was PCR-amplified from K. pneumoniae CWK2 chromosomal DNA with primers KPwaa30 (5′-GGAAACCTGGGTGCTGTTCT-3′) and KPwaa42 (5′-AGCAGGGCTAGCACCGCCTG-3′). The 1150-bp PCR product was digested with NheI (site underlined) and ligated to pBAD18-Cm digested with NheI and SmaI to generate plasmid pWQ43. The wabI gene was amplified from K. pneumoniae CWK2 chromosomal DNA using primers KPwaa38 (5′-GCTGCTCATATGGGATCCCTGTTTAAGAGGG-3′) and KPwaa39 (5′-CAATCGAGTAAGCTTGTCTGGCGAA-3′). The 994-bp PCR product was digested with NdeI and HindIII (sites underlined) and ligated to the same sites in pET28a(+) to make plasmid pWQ40. This plasmid expresses WabI with an in-frame His6 tag fused to the N terminus. For complementation studies and to verify whether the plasmid His6-tagged WabI was still functional, pWQ42 was constructed with the insert from pWQ40 cloned into pBAD18-Cm to facilitate expression in K. pneumoniae. The wabH gene was PCR-amplified from K. pneumoniae CWK2 chromosomal DNA with primers KPwaa43 (5′-GCCTGCACCAGCTAGCGACTCTCA-3′) and KPwaa37 (5′-GAGGGCAGGGGTACCAGTGGGAA-3′) as a 1613-bp fragment. The PCR product was digested with NheI and KpnI (sites underlined) and ligated to the same sites in pBAD18-Cm to make plasmid pWQ44. Preparation of Cell-free Extracts Containing WabI—The His6-WabI protein was overexpressed from pWQ40 in E. coli BL21(λDE3). This strain was grown for 18 h at 37 °C in LB medium supplemented with kanamycin. The culture was then diluted 1:100 in fresh medium, and incubation was continued until the culture reached A600 nm = 0.6. Expression of His6-WabI was induced by adding isopropyl-1-thio-β-d-galactopyranoside to the culture at a final concentration of 1 mm and continuing incubation for another 3 h. The cells were harvested, washed once with 50 mm HEPES (pH 7.5), and then frozen until needed. The cell pellet was resuspended in 50 mm HEPES (pH 7.5) and sonicated on ice (for a total of 2 min using 10-s bursts followed by 10-s cooling periods). Unbroken cells, cell debris, and the membrane fraction were removed by ultracentrifugation at 100,000 × g for 60 min. For comparison, a soluble extract was prepared using the same protocol from E. coli BL21(λDE3) containing the pET28(+) vector. Protein expression was monitored by SDS-PAGE, and protein contents of the pET28(+) and pWQ40(His6-WabI+) lysates were determined using the Bio-Rad Bradford assay as directed by the manufacturer. Purification of His6-WabI—The His6-WabI protein was overexpressed from pWQ40 in E. coli BL21(λDE3), and the soluble extract was prepared as described above, except that the pellet was washed and resuspended in 20 mm sodium phosphate buffer (pH 7.4) containing 500 mm NaCl (buffer A). His6-WabI was purified on an ÄKTA Explorer 100 system (Amersham Biosciences) using a 1-ml HiTrap chelating HP column (Amersham Biosciences) previously loaded with nickel sulfate and equilibrated with buffer A as recommended by the manufacturer. The column was washed with 5 mm imidazole in buffer A for 10 column volumes, and a step gradient of 50–500 mm imidazole in buffer A was then applied. His6-WabI eluted from the column at an imidazole concentration of 300 mm. The buffer was exchanged into 50 mm HEPES (pH 7.5) with 50 mm NaCl using a HiPrep 26/10 desalting column (Amersham Biosciences) according to the manufacturer's instructions. The protein was concentrated using a Centriplus 10-ml YM-30 centrifugal filter device (Amicon Bioseparations), and typical protein preparations contained yields of ∼0.07 mg/ml as determined by the Bio-Rad Bradford assay. Preparation of Cell-free Extracts Containing WabH—The K. pneumoniae strain CWG603 (waaL wabH) was used to overexpress the WabH protein from pWQ44. Cultures of CWG603(pWQ44) and the CWG603(pBAD18-Cm) control were grown for 18 h at 37 °C in LB medium supplemented with chloramphenicol and kanamycin. The culture was then diluted 1:100 in fresh medium, and incubation was continued until the culture reached A600 nm = 0.2. Expression from the pBAD promoter was induced by adding l-arabinose (0.02% final concentration), and incubation was continued for another 3–4 h. The cells were harvested, washed once with 50 mm Tris-HCl (pH 8.0), and then frozen until needed. Lysates were prepared as described for WabI. Purification of CMP-Kdo Synthetase (KdsB)—Plasmid pJKB72 (23Gronow S. Brabetz W. Brade H. Eur. J. Biochem. 2000; 267: 6602-6611Crossref PubMed Scopus (61) Google Scholar) was transformed into E. coli LE392 for overexpression of KdsB. The purification procedure was a modification of that described elsewhere (23Gronow S. Brabetz W. Brade H. Eur. J. Biochem. 2000; 267: 6602-6611Crossref PubMed Scopus (61) Google Scholar). The strain was grown for 18 h at 37 °C in LB medium supplemented with kanamycin. The culture was then diluted 1:100 in fresh medium and grown until it reached A600 nm = 0.6. Expression of KdsB was induced by adding isopropyl-1-thio-β-d-galactopyranoside to the culture at a final concentration of 0.2 mm, and incubation was continued for another 3.5 h. The cells were harvested and washed once with 50 mm NaH2PO4 (pH 8.0) containing 300 mm NaCl (buffer B), and then the pellet was frozen until required. The frozen pellet was thawed on ice and resuspended in lysis buffer (buffer B containing 20 mm imidazole). RNase and DNase were added to final concentrations of 0.1 mg/ml. The cells were lysed by passage through a French pressure cell four times. MgCl2 was then added to the lysate at a final concentration of 1 mm. Unbroken cells and large cell debris were removed from the lysate by high speed centrifugation for 10 min at 20,000 × g. Cell membranes were removed by ultracentrifugation for 90 min at 100,000 × g. Protease inhibitor mixture tablets (Complete Mini, EDTA-free, Roche Applied Science) were added to the supernatant and dissolved. Next, 1 ml of 50% nickel-nitrilotriacetic acid suspension (QIAGEN Inc.) was added, and the mixture was incubated for 90 min at 4 °C on a rotary shaker. The lysate/nickel-nitrilotriacetic acid mixture was then loaded onto a disposable plastic column (5 ml) with elution by gravity flow. The column was washed twice with 10 column volumes of buffer B containing 40 mm imidazole. The protein was eluted with buffer B containing 250 mm imidazole, and ∼2 ml of eluate was collected in 0.25-ml fractions. The fractions were e" @default.
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• W1971369615 title "Biosynthesis of a Novel 3-Deoxy-D-manno-oct-2-ulosonic Acid-containing Outer Core Oligosaccharide in the Lipopolysaccharide of Klebsiella pneumoniae" @default.
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• W1971369615 doi "https://doi.org/10.1074/jbc.m402549200" @default.
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