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• W2030897997 abstract "The core lipopolysaccharide (LPS) of Klebsiella pneumoniae is characterized by the presence of disaccharide αGlcN-(1,4)-αGalA attached by an α1,3 linkage to l-glycero-d-manno-heptopyranose II (ld-HeppII). Previously it has been shown that the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS. The presence of GlcNAc instead of GlcN and the lack of UDP-GlcN in bacteria indicate that an additional enzymatic step is required. In this work we identified a new gene (wabN) in the K. pneumoniae core LPS biosynthetic cluster. Chemical and structural analysis of K. pneumoniae non-polar wabN mutants showed truncated core LPS with GlcNAc instead of GlcN. In vitro assays using LPS truncated at the level of d-galacturonic acid (GalA) and cell-free extract containing WabH and WabN together led to the incorporation of GlcN, whereas none of them alone were able to do it. This result suggests that the later enzyme (WabN) catalyzes the deacetylation of the core LPS containing the GlcNAc residue. Thus, the incorporation of the GlcN residue to core LPS in K. pneumoniae requires two distinct enzymatic steps. WabN homologues are found in Serratia marcescens and some Proteus strains that show the same disaccharide αGlcN-(1,4)-αGalA attached by an α1,3 linkage to ld-HeppII. The core lipopolysaccharide (LPS) of Klebsiella pneumoniae is characterized by the presence of disaccharide αGlcN-(1,4)-αGalA attached by an α1,3 linkage to l-glycero-d-manno-heptopyranose II (ld-HeppII). Previously it has been shown that the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS. The presence of GlcNAc instead of GlcN and the lack of UDP-GlcN in bacteria indicate that an additional enzymatic step is required. In this work we identified a new gene (wabN) in the K. pneumoniae core LPS biosynthetic cluster. Chemical and structural analysis of K. pneumoniae non-polar wabN mutants showed truncated core LPS with GlcNAc instead of GlcN. In vitro assays using LPS truncated at the level of d-galacturonic acid (GalA) and cell-free extract containing WabH and WabN together led to the incorporation of GlcN, whereas none of them alone were able to do it. This result suggests that the later enzyme (WabN) catalyzes the deacetylation of the core LPS containing the GlcNAc residue. Thus, the incorporation of the GlcN residue to core LPS in K. pneumoniae requires two distinct enzymatic steps. WabN homologues are found in Serratia marcescens and some Proteus strains that show the same disaccharide αGlcN-(1,4)-αGalA attached by an α1,3 linkage to ld-HeppII. Klebsiella pneumoniae is an important nosocomial pathogen (1Emori T.G. Gaynes R.P. Clin. Microbiol. Rev. 1993; 6: 428-442Crossref PubMed Scopus (1020) Google Scholar) causing infections that may occur at almost all body sites with highest incidence in the urinary and the respiratory tracts. The main populations at risk are neonates, immunocompromised hosts, and patients predisposed by surgery, diabetes, malignancy, etc. (1Emori T.G. Gaynes R.P. Clin. Microbiol. Rev. 1993; 6: 428-442Crossref PubMed Scopus (1020) Google Scholar, 2Hansen D.S. Gottschau A. Holmes K.J. J. Hosp. Infect. 1997; 37: 119-132Google Scholar, 3Hervás J.A. Alomar A. Salvá F. Reina J. Benedí V.J. Clin. Infect. Dis. 1993; 16: 719-724Crossref PubMed Scopus (51) Google Scholar, 4Hansen D.S. Mestre F. Albertí S. Hernández-Alles S. Alvarez D. Domenech-Sánchez A. Gil J. Merino S. Tomás J.M. Benedí V.J. J. Clin. Microbiol. 1999; 37: 56-62Crossref PubMed Google Scholar). K. pneumoniae typically express both smooth lipopolysaccharide (LPS) 3The abbreviations used are:LPSlipopolysaccharideGalAd-galacturonic acidHepheptoseld-Hepl-glycero-d-manno-heptoseld-Heppl-glycero-d-manno-heptopyranoseKdo3-deoxy-d-manno-oct-2-ulosonic acidMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightMSmass spectrometryORFopen reading frameOSoligosaccharideTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 3The abbreviations used are:LPSlipopolysaccharideGalAd-galacturonic acidHepheptoseld-Hepl-glycero-d-manno-heptoseld-Heppl-glycero-d-manno-heptopyranoseKdo3-deoxy-d-manno-oct-2-ulosonic acidMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightMSmass spectrometryORFopen reading frameOSoligosaccharideTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. and capsule polysaccharide (K-antigen) on its surface, and both antigens (LPS and capsule) contribute to the pathogenesis of this species.As in other Enterobacteriaceae in the K. pneumoniae LPS three domains are recognized: the highly conserved and hydrophobic lipid A, the hydrophilic and highly variable O-antigen polysaccharide, and the core oligosaccharide (OS) connecting lipid A and O-antigen. The core domain is usually divided into inner and outer core on the basis of sugar composition.The K. pneumoniae core LPS structure has been determined for several O-serotypes. Although the inner core is highly conserved within the Enterobacteriaceae the K. pneumoniae inner core differs from those of Escherichia coli and Salmonella by the lack of l-glycero-d-manno-heptopyranose I and II (ld-HeppI and -II) phosphoryl modifications and by the presence of a d-glucose (Glc) residue linked by a β1,4 bond to ld-HeppI (5Vinogradov E. Perry M.B. Carbohydr. Res. 2001; 335: 291-296Crossref PubMed Scopus (52) Google Scholar, 6Vinogradov E. Cedzynski M. Ziolkowski A. Swierzko A. Eur. J. Biochem. 2001; 268: 1722-1729Crossref PubMed Scopus (29) Google Scholar, 7Regué M. Izquierdo L. Fresno S. Piqué N. Corsaro M. Naldi T. De Castro C. Waidelich D. Merino S. Tomás J.M. J. Bacteriol. 2005; 187: 4198-4206Crossref PubMed Scopus (44) Google Scholar). Two outer core types (1 and 2) were described in K. pneumoniae, both containing the disaccharide αGlcN-(1,4)-αGalA attached by an α1,3 linkage to ld-HeppII (5Vinogradov E. Perry M.B. Carbohydr. Res. 2001; 335: 291-296Crossref PubMed Scopus (52) Google Scholar, 6Vinogradov E. Cedzynski M. Ziolkowski A. Swierzko A. Eur. J. Biochem. 2001; 268: 1722-1729Crossref PubMed Scopus (29) Google Scholar, 7Regué M. Izquierdo L. Fresno S. Piqué N. Corsaro M. Naldi T. De Castro C. Waidelich D. Merino S. Tomás J.M. J. Bacteriol. 2005; 187: 4198-4206Crossref PubMed Scopus (44) Google Scholar). Type 1 core contains a 3-deoxyd-manno-oct-2-ulosonic acid (Kdo) residue linked by an α2,6 bond to the GlcN residue (5Vinogradov E. Perry M.B. Carbohydr. Res. 2001; 335: 291-296Crossref PubMed Scopus (52) Google Scholar, 6Vinogradov E. Cedzynski M. Ziolkowski A. Swierzko A. Eur. J. Biochem. 2001; 268: 1722-1729Crossref PubMed Scopus (29) Google Scholar), whereas type 2 core contains the disaccharide βGlc-(1,6)-αGlc linked by a α1,4 bond to the GlcN residue (7Regué M. Izquierdo L. Fresno S. Piqué N. Corsaro M. Naldi T. De Castro C. Waidelich D. Merino S. Tomás J.M. J. Bacteriol. 2005; 187: 4198-4206Crossref PubMed Scopus (44) Google Scholar) (Fig. 1).The genes involved in the K. pneumoniae core LPS biosynthesis are clustered in a region (wa) of the K. pneumoniae chromosome, and two different clusters have been identified to be responsible for type 1 and type 2 core biosynthesis (7Regué M. Izquierdo L. Fresno S. Piqué N. Corsaro M. Naldi T. De Castro C. Waidelich D. Merino S. Tomás J.M. J. Bacteriol. 2005; 187: 4198-4206Crossref PubMed Scopus (44) Google Scholar, 8Regué 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) (Fig. 1). Furthermore the functions in both inner and outer core biosynthesis of most of the transferases encoded by the wa gene products have been elucidated (7Regué M. Izquierdo L. Fresno S. Piqué N. Corsaro M. Naldi T. De Castro C. Waidelich D. Merino S. Tomás J.M. J. Bacteriol. 2005; 187: 4198-4206Crossref PubMed Scopus (44) Google Scholar, 8Regué 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, 9Izquierdo L. Merino S. Coderch N. Regué M. Tomás J.M. FEMS Microbiol. Lett. 2002; 216: 211-216PubMed Google Scholar, 10Izquierdo L. Abitiu N. Coderch N. Hita B. Merino S. Gavín R. Tomás J.M. Regué M. Microbiology. 2002; 148: 3485-3496Crossref PubMed Scopus (36) Google Scholar, 11Izquierdo L. Coderch N. Piqué N. Bedini E. Corsaro M. Merino S. Fresno S. Tomas J.M. Regué M. J. Bacteriol. 2003; 185: 7213-7221Crossref PubMed Scopus (68) Google Scholar, 12Frirdich E. Vinogradov E. Whitfield C. J. Biol. Chem. 2004; 279: 27928-27940Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) (Fig. 1). Nevertheless the mechanism leading to the incorporation of the outer core GlcN residue remains unknown. In searching for a candidate glucosaminyltransferase the wabH gene product was characterized (12Frirdich E. Vinogradov E. Whitfield C. J. Biol. Chem. 2004; 279: 27928-27940Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). The WabH protein catalyzes the incorporation of GlcNAc from UDP-GlcNAc into outer core LPS. This transferase activity is dependent on the presence of the outer core d-galacturonic acid (GalA) residue in the acceptor core LPS (12Frirdich E. Vinogradov E. Whitfield C. J. Biol. Chem. 2004; 279: 27928-27940Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). But because GlcN instead of GlcNAc is found in the K. pneumoniae outer core either an unknown glucosaminyltransferase remains to be identified or a mechanism to deacetylate the WabH-transferred GlcNAc residue should be involved. In this work we report the identification and characterization of such a core LPS-GlcNAc deacetylase activity in K. pneumoniae and Serratia marcescens sharing the outer core disaccharide αGlcN-(1,4)-αGalA.MATERIALS AND METHODSBacterial Strains, Plasmids, and Growth Conditions—Bacterial strains and plasmids used in this study are shown in TABLE ONE. Bacterial strains were grown in LB broth and LB agar (13Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972: 433Google Scholar). LB medium was supplemented with kanamycin (50 μg/ml), ampicillin (100 μg/ml), chloramphenicol (20 μg/ml), and tetracycline (25 μg/ml) when needed.TABLE ONEBacterial strains and plasmids usedStrain or plasmidRelevant characteristicsRef. or sourceK. pneumoniae 52145Serovar O1:K2 (core type 2)33Nassif X. Fournier J.M. Arondel J. Sansonetti P.J. Infect. Immun. 1989; 57: 546-552Crossref PubMed Google Scholar 52145Δ waaLNon-polar waaL mutant11Izquierdo L. Coderch N. Piqué N. Bedini E. Corsaro M. Merino S. Fresno S. Tomas J.M. Regué M. J. Bacteriol. 2003; 185: 7213-7221Crossref PubMed Scopus (68) Google Scholar 52145ΔwabNNon-polar wabN mutantThis study 52145ΔwabHNon-polar wabH mutantThis study 52145ΔwaaL wabNDouble non-polar waaL wabN mutantThis study 52145ΔwaaL wabHDouble non-polar waaL wabH mutantThis study C3Serovar O8:K66 (core type 1)34Orskov I. Orskov F. Methods Microbiol. 1984; 14: 143-164Crossref Scopus (99) Google Scholar C3ΔwaaLNon-polar waaL mutant8Regué 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 C3ΔwabNNon-polar wabN mutantThis study C3ΔwabHNon-polar wabH mutantThis study C3ΔwaaL wabNDouble non-polar waaL wabN mutantThis study C3ΔwaaL wabHDouble non-polar waaL wabN mutantThis studyE. coli DH5αF-endA hsdR17 (rk- mk-) supE44 thi-1 recA1 gyr-A96 ϕ80lacZ35Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8126) Google ScholarPlasmid pKO3Cmr temperature-sensitive replication sacB-containing suicide plasmid15Link A.J. Phillips D. Church G.M. J. Bacteriol. 1997; 179: 6228-6237Crossref PubMed Scopus (744) Google Scholar pKO3ΔwabN52145Contains an in-frame internal wabN deletion from strain 52145This study pKO3ΔwabNC3Contains an in-frame internal wabN deletion from strain C3This study pKO3ΔwabH52145Contains an in-frame internal wabH deletion from strain 52145This study pKO3ΔwabHC3Contains an in-frame internal wabH deletion from strain C3This study pGEM-T EasyPCR-generated DNA fragment cloning vector AmprPromega pGEM-T-waaN52145pGEM-T containing the PCR-amplified waaN52145 geneThis study pGEM-T-waaNC3pGEM-T containing the PCR-amplified waaC3geneThis study pGEM-T-waaNSmpGEM-T containing the PCR-amplified waaNSm geneThis study pBAD18-CmArabinose-inducible expression vector16Guzman L.-M. Belin D. Carson M.J. Becwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3906) Google Scholar pBAD18-Cm-wabN52145pBAD18-Cm derivative expressing WabN from strain 52145This study pBAD18-Cm-wabNC3pBAD18-Cm derivative expressing WabN from strain C3This study pBAD18-Cm-wabH52145pBAD18-Cm derivative expressing WabH from strain 52145This study pBAD18-Cm-wabHC3pBAD18-Cm derivative expressing WabH from strain C3This study Open table in a new tab General DNA Methods—Standard DNA manipulations were done essentially as described previously (14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). DNA restriction endonucleases, T4 DNA ligase, E. coli DNA polymerase (Klenow fragment), and alkaline phosphatase were used as recommended by the suppliers.Mutant Construction—K. pneumoniae 52145 and C3 individual genes were mutated by creating in vitro in-frame deletions of each gene (15Link A.J. Phillips D. Church G.M. J. Bacteriol. 1997; 179: 6228-6237Crossref PubMed Scopus (744) Google Scholar). Each mutated gene was transferred to the chromosome by homologous recombination using the temperature-sensitive suicide plasmid pKO3 containing the counterselectable marker sacB (15Link A.J. Phillips D. Church G.M. J. Bacteriol. 1997; 179: 6228-6237Crossref PubMed Scopus (744) Google Scholar). Mutations were made in wabH and wabN. The plasmids containing the engineered in-frame deletions (pKO3ΔwabHC3, pKO3ΔwabH52145, pKO3ΔwaNHC3, and pKO3ΔwabN52145) were transformed into K. pneumoniae 52145, C3, 52145ΔwaaL, and C3ΔwaaL by electroporation. Mutants were selected based on growth in LB agar containing 10% sucrose and loss of the chloramphenicol resistance marker of vector pKO3. The mutations were confirmed by sequencing of the whole constructs in amplified PCR products.The 52145ΔwabN mutant was constructed by asymmetric PCR amplifications using 52145 chromosomal DNA and primers NA52145 (5′-GAAGATCTCACGCCAGCATTTAAAGAAC-3′), NB52145 (5′-CCCATCCACTAAACTTAAACAAACGTCCTCAGGCCATAC-3′), NC52145 (5′-TGTTTAAGTTTAGTGGATGGGTACCAGCGGAAATGCTAAC-3′), and ND52145 (5′-GAAGATCTTCACTATTTTGCCCAGCAC-3′). The primers include BglII sites (underlined). DNA fragments of 743 (NA52145-NB52145) and 628 (NC52145-ND52145) bp were obtained, respectively. DNA fragment NA52145-NB52145 contains from nucleotide 7457, corresponding to the third base of the 9th codon of wabN, to nucleotide 8199 inside waaQ. DNA fragment NC52145-ND52145 contains from nucleotide 5887, inside waaM, to nucleotide 6514 including the last wabN five codons. DNA fragments NA52145-NB52145 and NC52145-ND52145 were annealed at their overlapping region (double underlined letters in primers NB52145 and NC52145) and amplified by PCR as a single fragment using primers NA52145 and ND52145. The fusion product was purified, BglII-digested, ligated into BamHI-digested and phosphatase-treated pKO3 vector, electroporated into E. coli DH5α, and plated on chloramphenicol LB agar plates at 30 °C to obtain plasmid pKO3ΔwabN52145.The C3ΔwabN mutant was constructed by asymmetric PCR amplifications using C3 chromosomal DNA and primers NAC3 (5′-GAAGATCTCGCGCCAGCATTTAAAGAAC-3′), NB52145 (5′-CCCATCCACTAAACTTAAACAAACGTCCTCAGGCCATAC-3′), NCC3 (5′-TGTTTAAGTTTAGTGGATGGGTACCAGCGGAAAATTACAC-3′), and NDC3 (5′-GAAGATCTCCACTTTCTCCCGCTCAT-3′). The primers include BglII sites (underlined). DNA fragments of 745 (NAC3-NB52145) and 623 (NCC3-NDC3) bp were obtained, respectively. DNA fragment NAC3-NBC3 contains from nucleotide 7432, corresponding to the third base of the fourth codon of wabN, to nucleotide 8176 inside waaQ. DNA fragment NCC3-NDC3 contains from nucleotide 5870, inside waaJ, to nucleotide 6492 including the last wabN eight codons. DNA fragments NAC3-NB52145 and NCC3-NDC3 were annealed at their overlapping region (double underlined letters in primers NBC3 and NCC3) and amplified by PCR as a single fragment using primers NAC3 and NDC3. The fusion product was purified, BglII-digested, ligated into BamHI-digested and phosphatase-treated pKO3 vector, electroporated into E. coli DH5α, and plated on chloramphenicol LB agar plates at 30 °C to obtain plasmid pKO3ΔwabNC3.The 52145ΔwabH mutant was constructed using 52145 chromosomal DNA and primers HA52145 (5′-CGCGGATCCAGCGCGAGATTATCGAAG-3′), HB52145 (5′-CCCATCCACTAAACTTAAACAGGGTAAACCGTCAATCAC-3′), HC52145 (5′-TGTTTAAGTTAGTGGATGGGTGTCAGCAGTACCGTCAG-3′), and HD52145 (5′-CGCGGATCCTATGCGACACCGACTCAG-3′). The primers include BamHI sites (underlined). DNA fragments of 688 (HA52145-HB52145) and 715 (HC52145-HD52145) bp were obtained, respectively. DNA fragment HA52145-HB52145 contains from nucleotide 9107, inside wabG to the third base of the 10th codon of wabH, to nucleotide 9794. DNA fragment HC52145-HD52145 contains from nucleotide 11558, inside orf10, to nucleotide 10844 including the last wabH 10 codons. DNA fragments HA52145-HB52145 and HC52145-HD52145 were annealed at their overlapping region (double underlined letters in primers HB52145 and HC52145) and amplified by PCR as a single fragment using primers HA52145 and HD52145. The fusion product was purified, BamHI-digested, ligated into BamHI-digested and phosphatase-treated pKO3 vector, electroporated into E. coli DH5α, and plated on chloramphenicol LB agar plates at 30 °C to obtain plasmid pKO3ΔwabH52145. Because of the extensive identity among wabG, wabH, and orf10 between strains 52145 and C3, the above four primers and 52145 chromosomal DNA were used to construct pKO3ΔwabHC3.Plasmid Constructions for Gene Overexpression and Mutant Complementation Studies—For gene overexpression studies the wabN genes from K. pneumoniae strains 52145 and C3 and S. marcescens N28b were PCR-amplified by using specific primers pairs and transferred into plasmid pABD18-Cm. Each gene was expressed from the arabinose-inducible and glucose-repressible pABD18-Cm promoter (16Guzman L.-M. Belin D. Carson M.J. Becwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3906) 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 chloramphenicol and 0.4% glucose. These cultures were diluted 1:100 in fresh medium (without glucose) and grown until they reached an A600 nm of about 0.2. l-A-rabinose was then added, and the cultures were grown for another 2 h. Repressed controls were maintained in glucose-containing medium.The wabH gene was PCR-amplified from K. pneumoniae C3 chromosomal DNA with primers KPwaa43 (5′-CCTGCACCAGCTAGCGACTCTCA-3′) and KPwaa37 (5′-GAGGGCAGGGGTACCAGTGGGAA-3′) as a 1,613-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 pBAD18-Cm-wabHC3 as reported previously (12Frirdich E. Vinogradov E. Whitfield C. J. Biol. Chem. 2004; 279: 27928-27940Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar).The strain 52145 wabH homologue was PCR-amplified from K. pneumoniae 51245 chromosomal DNA with primers KPwaa43 (5′-CCTGCACCAGCTAGCGACTCTCA-3′) (12Frirdich E. Vinogradov E. Whitfield C. J. Biol. Chem. 2004; 279: 27928-27940Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and NRev (5′-GAGGGCAGAGGATCCAGTGGGAA-3′) as a 1,613-bp fragment. The PCR product was digested with NheI and BamHI (sites underlined) and ligated to the same sites in pBAD18-Cm to make plasmid pBAD18-Cm-wabH52145.For pBAD-18-wabN52145 construction a 2,313-bp DNA fragment containing the wabN gene was PCR-amplified from K. pneumoniae 52145 chromosomal DNA with primers NA52145 and ND52145. This fragment was used as template to amplify an internal 1,649 bp with primers NFw52145 (5′-TTCGATTCTAGAGGGATCAG-3′) and ND52145. This fragment was digested with XbaI and HindIII to obtain a 1,204-bp fragment that was ligated to the same sites in pBAD18-Cm.Plasmid pBAD18-Cm-wabNC3 was constructed by PCR amplification of a 2,308-bp DNA fragment from strain C3 chromosomal DNA using primers and NAC3 and NDC3. This fragment was used as template to amplify an internal 1,644 bp with primers FN52145 and NDC3. This fragment was digested with BglII, blunt-ended, and then digested with XbaI to obtain a 1,638-bp fragment. This fragment was ligated to pBAD18-Cm, HindIII-digested, blunt-ended, and XbaI-digested.Plasmid pBAD18-Cm-wabNSm was constructed by PCR amplification of a 1,076-bp DNA fragment from plasmid pCosFGR16 (17Coderch N. Piqué N. Lindner B. Abitiu N. Merino S. Izquierdo L. Jimenez N. Tomás J.M. Holst O. Regué M. J. Bacteriol. 2004; 186: 978-988Crossref PubMed Scopus (22) Google Scholar) using primers NFwSm (5′-CAACTTAAGCTTCCATTTCTTC-3′) and NRevSm (5′-CAAACCAGGCTAGCAAAGTC-3′). This fragment was digested with NheI and HindIII and ligated into the same sites in pBAD18-Cm. For complementation studies each amplified wabN homologue was ligated to pGEM-T Easy (Promega) and transformed into E. coli DH5α.LPS Isolation and Electrophoresis—LPS was extracted from dry cells of K. pneumoniae grown in LB. The phenol/chloroform/light petroleum ether method (18Galanos C. Lüderitz O. Westphal O. Eur. J. Biochem. 1969; 9: 245-249Crossref PubMed Scopus (1355) Google Scholar) was used for strains producing rough LPS, whereas the phenol/water procedure (19Westphal O. Jann K. Methods Carbohydr. Chem. 1965; 5: 83-91Google Scholar) was used for the strains producing the O-antigen domain (smooth LPS). For screening purposes LPS was obtained after proteinase K digestion of whole cells (20Hitchcock P.J. Brown T.M. J. Bacteriol. 1983; 154: 269-277Crossref PubMed Google Scholar). LPS samples were separated by SDS-PAGE or Tricine-SDS-PAGE and visualized by silver staining as described previously (21Pradel E. Schnaitman C.A. J. Bacteriol. 1991; 173: 6428-6431Crossref PubMed Google Scholar, 22Tsai C.M. Frasch C.E. Anal. Biochem. 1982; 119: 115-119Crossref PubMed Scopus (2294) Google Scholar).Preparation of Oligosaccharides—The LPS (20 mg) was hydrolyzed in 1% acetic acid (100 °C for 120 min), and the precipitate was removed by centrifugation (8,000 × g for 30 min) and lyophilized to give lipid A (10 mg, 50% of LPS). The supernatant was evaporated to dryness, dissolved in water, and lyophilized (6 mg, 30% of LPS).Gas Chromatography-Mass Spectrometry (MS) Analysis—Partially methylated alditol acetates and methyl glycoside acetates were analyzed on an Agilent Technologies 5973N MS instrument equipped with a 6850A gas chromatograph and an RTX-5 capillary column (Restek, 30 m × 0.25-mm inner diameter; flow rate, 1 ml/min; carrier gas, helium). Acetylated methyl glycoside analysis was performed with the following temperature program: 150 °C for 5 min, 150 °C → 250 °C at 3 °C/min, 250 °C for 10 min. For partially methylated alditol acetates the temperature program was: 90 °C for 1 min, 90 °C → 140 °C at 25 °C/min, 140 °C → 200 °C at 5 °C/min, 200 °C → 280 °C at 10 °C/min, 280 °C for 10 min.Glycosyl and Lipid Analysis—LPS (1 mg) was dried over P2O5 overnight and treated with 1 m HCl/CH3OH (1 ml) at 80°C for 20 h to analyze both glycosyl and fatty acid composition. The crude reaction was extracted twice with hexane; the two extracts were pooled, dried under a stream of air, and treated with acetic anhydride (100 μl) at 100 °C for 15 min. The methanol layer was neutralized with Ag2CO3, dried, and acetylated. Both samples were injected into the gas chromatography-MS system, and acetylated fatty acid methyl esters were recovered in the hexane phase, whereas the methylglycoside derivatives were in the methanolic phase.NMR Spectroscopy—1H NMR spectra were recorded on a solution (0.5 ml) of K. pneumoniae 52145ΔwaaL (8 mg) and 52145ΔwaaL wabN (2 mg) core oligosaccharides, respectively, in D2O at 400 MHz with a Bruker DRX 400 Avance spectrometer equipped with a reverse probe in the Fourier transform mode at 303 K.Mass Spectrometry Studies—Positive and negative ions reflectron time-of-flight mass spectra (MALDI-TOF) were acquired on a Voyager DE-PRO instrument (Applied Biosystems) equipped with a delayed extraction ion source. Ion acceleration voltage was 25 kV, grid voltage was 17 kV, mirror voltage ratio was 1.12, and delay time was 150 ns. Samples were irradiated at a frequency of 5 Hz by 337 nm photons from a pulsed nitrogen laser. Postsource decay was performed using an acceleration voltage of 20 kV. The reflectron voltage was decreased in 10 successive 25% steps. Mass calibration was obtained with a maltooligosaccharide mixture from corn syrup (Sigma). A solution of 2,5-dihydroxybenoic acid in 20% CH3CN in water at a concentration of 25 mg/ml was used as the MALDI matrix. One microliter of matrix solution was deposited on the target followed by loading of 1 μl of the sample. The droplets were allowed to dry at room temperature. Spectra were calibrated and processed under computer control using the Applied Biosystems Data Explorer software.Methylation Analysis—A core oligosaccharide sample (1 mg) obtained from 1% AcOH hydrolysis was first reduced with NaBH4 and then methylated (23Ciucanu I. Kerek F. Carbohydr. Res. 1984; 131: 209-217Crossref Scopus (3171) Google Scholar). Linkage analysis was performed as follows. The methylated sample was carboxymethyl-reduced with lithium triethylborohydride (Super-Hydride, Aldrich), hydrolyzed by mild acid to cleave ketosidic linkage, reduced by means of NaBD4, then totally hydrolyzed, reduced with NaBD4, and finally acetylated as described previously (24Forsberg L.S. Ramadas Bhat U. Carlson R.W. J. Biol. Chem. 2000; 275: 18851-18863Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar).Preparation of Cell-free Extracts Containing Core LPS Biosynthetic Enzymes—The K. pneumoniae strains 521453ΔwabH (pBAD18-Cm-wabH52145) and 52145ΔwabN (pBAB18Cm-wabN52145) were used to overexpress the WabH and WabN proteins, respectively. Cultures of these strains and controls (52145ΔwabH and 52145ΔwabN harboring pBAD18-Cm) were grown and arabinose-induced as described above. The cells were harvested, washed once with 50 mm Tris-HEPES (pH 7.5), and then frozen until needed. To prepare lysates cell pellets were 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. Protein expression was monitored by SDS-PAGE, and protein contents of cell-free extracts were determined using the Bio-Rad Bradford assay as directed by the manufacturer.GlcNAc-Core LPS Deacetylase Activity of WabN—The ability of WabN to catalyze the deacetylation of GlcNAc-containing LPS was assayed by using LPS from mutant 52145ΔwabH in a reaction containing both 52145ΔwabH (pBAD18-Cm-wabH52145) and 52145ΔwabN (pBAD18-Cm-wabN52145) cell-free extracts. The assay system was based on the GlcNAc transferase assay described for WabH (12Frirdich E. Vinogradov E. Whitfield C. J. Biol. Chem. 2004; 279: 27928-27940Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar).Assay reactions using UDP-GlcNAc as the substrate were carried out in 0.02 ml at a final concentration of 50 mm Tris-HCl (pH 8.0) containing 10 mm MgCl2, 1mm dithiothreitol, 1 mm UDP-GlcNAc, and 0.003 mg of 52145ΔwabH mutant LPS as the acceptor. The reaction was started by addition of 0.04 mg each of the cell-free extracts from 52145ΔwabH (pBAD18Cm-wabH52145) and 52145ΔwabN (pBAD18-Cm-wabN52145). As controls reactions containing cell-free extracts (0.04 mg) of 52145ΔwabH (pBAD18-Cm-wabH52145), 52145ΔwabH (pBAD18-Cm) and/or 52145ΔwabN (pBAD18-Cm-wabN52145) were used. The mixtures were incubated at 37 °C for 2 h, and the reactions were stopped by adding 0.08 ml of SDS-PAGE sample buffer and boiling for 10 min. Proteinase K diluted in SDS-PAGE sample buffer was then added to a final concentration of 0.8 mg/ml and incubated for 18 h at 55 °C. The reaction products were visualized by SDS-PAGE. When UDP-[14C]GlcNAc was used as the substrate, assay reactions were carried out in a total of 0.1 ml at a final concentration of 50 mm Tris-HCl (pH 8.0) containing 10 mm MgCl2, 1 mm dithiothreitol, 0.3 mg of 52145ΔwabH mutant LPS, and 0.2 mg of the cell-free extracts. The reactions were started by addition of 0.25 μCi of UDP-[14C]GlcNAc (specific activity of 10.2 mCi/mmol; ICN Biomedicals Inc.). Assays were performed at 37 °C for 2 h and were stopped by adding 2 volumes of 0.375 m MgCl2 in 95% ethanol and cooling at –20 °C for 2 h. The LPS was recovered by centrifugation at 12,000 × g for 15 min and resuspended in 100 μl of water. The LPS was precipitated with 2 volumes of 0.375 m MgCl2 in 95% ethanol; this step was repeated three times to eliminate the unincorporated UDP-[14C]GlcNAc. The LPS was hydrolyzed by resuspension in 100 μlof0.1 m HCl and heating to 100 °C for 48 h. The labeled residues from the hydrolyzed LPS samples were separated by TLC (Kieselgel 60, Merck) with n-butanol, methanol, 25% ammonia solution, water (5:4:2:1, v/v/v/v). The labeled residues were detected by autoradiography using as standards [14C]GlcNAc and [14C]GlcN.RESULTSIdentification of a New Core LPS Gene—In" @default.
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• W2030897997 date "2005-11-01" @default.
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• W2030897997 title "The Incorporation of Glucosamine into Enterobacterial Core Lipopolysaccharide" @default.
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