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• W2085239309 abstract "In most members of the Enterobacteriaceae, including Escherichia coli and Salmonella, the lipopolysaccharide core oligosaccharide backbone is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. Mutants lacking the core heptose region and the phosphate residues display pleiotrophic defects collectively known as the deep-rough phenotype, characterized by changes in outer membrane structure and function. Klebsiella pneumoniae lacks phosphoryl residues in its core, but instead contains galacturonic acid. The goal of this study was to determine the contribution of galacturonic acid as a critical source of negative charge. A mutant was created lacking all galacturonic acid by targeting UDP-galacturonic acid precursor synthesis through a mutation in glaKP. GlaKP is a K. pneumoniae UDP-galacturonic acid C4 epimerase providing UDP-galacturonic acid for core synthesis. The glaKP gene was inactivated and the structure of the mutant lipopolysaccharide was determined by mass spectrometry. The mutant displayed characteristics of a deep-rough phenotype, exhibiting a hypersensitivity to hydrophobic compounds and polymyxin B, an altered outer membrane profile, and the release of the periplasmic enzyme β-lactamase. These results indicate that the negative charge provided by the carboxyl groups of galacturonic acid do play an equivalent role to the core oligosaccharide phosphate residues in establishing outer membrane integrity in E. coli and Salmonella. In most members of the Enterobacteriaceae, including Escherichia coli and Salmonella, the lipopolysaccharide core oligosaccharide backbone is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. Mutants lacking the core heptose region and the phosphate residues display pleiotrophic defects collectively known as the deep-rough phenotype, characterized by changes in outer membrane structure and function. Klebsiella pneumoniae lacks phosphoryl residues in its core, but instead contains galacturonic acid. The goal of this study was to determine the contribution of galacturonic acid as a critical source of negative charge. A mutant was created lacking all galacturonic acid by targeting UDP-galacturonic acid precursor synthesis through a mutation in glaKP. GlaKP is a K. pneumoniae UDP-galacturonic acid C4 epimerase providing UDP-galacturonic acid for core synthesis. The glaKP gene was inactivated and the structure of the mutant lipopolysaccharide was determined by mass spectrometry. The mutant displayed characteristics of a deep-rough phenotype, exhibiting a hypersensitivity to hydrophobic compounds and polymyxin B, an altered outer membrane profile, and the release of the periplasmic enzyme β-lactamase. These results indicate that the negative charge provided by the carboxyl groups of galacturonic acid do play an equivalent role to the core oligosaccharide phosphate residues in establishing outer membrane integrity in E. coli and Salmonella. Lipopolysaccharide (LPS) 1The abbreviations used are: LPS, lipopolysaccharide; core OS, core oligosaccharide; O-PS, O-polysaccharide; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acid; Hep, l-glycero-d-manno-heptopyranose; OM, outer membrane; GalUA, galacturonic acid; OMP, outer membrane protein; GlcUA, glucuronic acid; His6, hexahistidine; MALDI-TOF, matrix-assisted laser desorption-ionization time-of-flight; PMB, polymyxin B; MIC, minimum inhibitory concentration; Ara4N, 4-amino-4-deoxyarabinose; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ESI, electrospray ionization. 1The abbreviations used are: LPS, lipopolysaccharide; core OS, core oligosaccharide; O-PS, O-polysaccharide; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acid; Hep, l-glycero-d-manno-heptopyranose; OM, outer membrane; GalUA, galacturonic acid; OMP, outer membrane protein; GlcUA, glucuronic acid; His6, hexahistidine; MALDI-TOF, matrix-assisted laser desorption-ionization time-of-flight; PMB, polymyxin B; MIC, minimum inhibitory concentration; Ara4N, 4-amino-4-deoxyarabinose; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ESI, electrospray ionization. is a major virulence determinant in Gram-negative bacteria (1Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3356) Google Scholar). The Klebsiella pneumoniae LPS molecule shares significant similarity with the well characterized LPS structures from other members of the Enterobacteriaceae, like Escherichia coli and Salmonella (1Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3356) Google Scholar, 2Holst 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), whereas the outer region shows more diversity. Conservation in the inner core may reflect its crucial role in the essential barrier function of the outer membrane (OM). There is a single core OS structure in K. pneumoniae and small variations between isolates in the addition of non-stoichiometric substituents (3Vinogradov E. Perry M.B. Carbohydr. Res. 2001; 335: 291-296Crossref PubMed Scopus (54) Google Scholar). The genes responsible for core OS biosynthesis in K. pneumoniae are encoded by the waa gene cluster, whose sequence has been reported (4Regué M. Climent N. Abitiu N. Coderch N. Merino S. Izquierdo L. Altarriba M. Tomás J.M. J. Bacteriol. 2001; 183: 3564-3573Crossref PubMed Scopus (54) Google Scholar). The biosynthesis of the K. pneumoniae core OS region has now been well characterized, with functions having been assigned to all but one of the genes encoded in the gene cluster; the exception is orf10 (4Regué M. Climent N. Abitiu N. Coderch N. Merino S. Izquierdo L. Altarriba M. Tomás J.M. J. Bacteriol. 2001; 183: 3564-3573Crossref PubMed Scopus (54) Google Scholar, 5Vinogradov 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 (128) Google Scholar, 6Frirdich E. Vinogradov E. Whitfield C. J. Biol. Chem. 2004; 279: 27928-27940Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 7Izquierdo L. Coderch N. Pique N. Bedini E. Corsaro M.M. Merino S. Fresno S. Tomas J.M. Regue M. J. Bacteriol. 2003; 185: 7213-7221Crossref PubMed Scopus (69) Google Scholar, 8Izquierdo L. Abitiu N. Coderch N. Hita B. Merino S. Gavin R. Tomas J.M. Regue M. Microbiology. 2002; 148: 3485-3496Crossref PubMed Scopus (36) Google Scholar, 9Izquierdo L. Merino S. Coderch N. Regue M. Tomas J.M. FEMS Microbiol. Lett. 2002; 216: 211-216PubMed Google Scholar). The only unassigned function is the addition of the non-stoichiometric β-galacturonic acid (GalUA) residues in the inner core (see Fig. 1), which potentially involves orf10 activity. One major feature distinguishing the K. pneumoniae core OS from those of E. coli and Salmonella is the absence of phosphoryl substitutions (Fig. 1). The negative charges provided by these phosphate residues in E. coli and Salmonella play a critical role in maintaining the barrier function of the OM by providing sites for cross-linking of adjacent LPS molecules with divalent cations or polyamines (reviewed in Refs. 1Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3356) Google Scholar, 10Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Crossref PubMed Google Scholar, and 11Nikaido H. Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2838) Google Scholar). In addition, the negative charges are important in mediating interactions between LPS and the positive charges on certain OM proteins (OMPs) (12Ferguson A.D. Welte W. Hofmann E. Lindner B. Holst O. Coulton J.W. Diederichs K. Structure Fold. Des. 2000; 8: 585-592Abstract Full Text Full Text PDF Scopus (182) Google Scholar, 13Vandeputte-Rutten L. Kramer R.A. Kroon J. Dekker N. Egmond M.R. Gros P. EMBO J. 2001; 20: 5033-5039Crossref PubMed Scopus (212) Google Scholar). Mutants with highly truncated core OS structures lacking the inner core Hep region display a pleiotrophic phenotype known as the deep-rough phenotype, characterized by changes in structure and composition of the OM (reviewed in Refs. 10Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Crossref PubMed Google Scholar, 11Nikaido H. Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2838) Google Scholar, 14Schnaitman C.A. Klena J.D. Microbiol. Rev. 1993; 57: 655-682Crossref PubMed Google Scholar, and 15Heinrichs D.E. Yethon J.A. Whitfield C. Mol. Microbiol. 1998; 30: 221-232Crossref PubMed Scopus (284) Google Scholar). In E. coli and Salmonella, these mutants show a decrease in the amount of OMPs and a corresponding increase in phospholipids. The loss of OMPs is likely because of improper folding and assembly of OMPs in the absence of LPS negative charge (12Ferguson A.D. Welte W. Hofmann E. Lindner B. Holst O. Coulton J.W. Diederichs K. Structure Fold. Des. 2000; 8: 585-592Abstract Full Text Full Text PDF Scopus (182) Google Scholar, 16Sen K. Nikaido H. J. Bacteriol. 1991; 173: 926-928Crossref PubMed Google Scholar, 17de Cock H. Tommassen J. EMBO J. 1996; 15: 5567-5573Crossref PubMed Scopus (65) Google Scholar, 18Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (661) Google Scholar, 19de Cock H. Brandenburg K. Wiese A. Holst O. Seydel U. J. Biol. Chem. 1999; 274: 5114-5119Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 20Kramer R.A. Zandwijken D. Egmond M.R. Dekker N. Eur. J. Biochem. 2000; 267: 885-893Crossref PubMed Scopus (92) Google Scholar, 21Hagge S.O. de Cock H. Gutsmann T. Beckers F. Seydel U. Wiese A. J. Biol. Chem. 2002; 277: 34247-34253Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 22Bulieris P.V. Behrens S. Holst O. Kleinschmidt J.H. J. Biol. Chem. 2003; 278: 9092-9099Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). These mutants are also hypersensitive to hydrophobic compounds, because of the appearance of phospholipids in the outer leaflet of the OM that may facilitate rapid penetration of these compounds through the phospholipid bilayer regions of the membrane. Other characteristics of deep-rough mutants seen in E. coli include the release of periplasmic enzymes, the loss of cell surface organelles (e.g. pili and flagella), secretion of an inactive form of hemolysin, and increased susceptibility to polymorphonuclear leukocyte lysosomal fractions and phagocytosis by macrophages (reviewed in Refs. 1Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3356) Google Scholar, 10Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Crossref PubMed Google Scholar, 11Nikaido H. Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2838) Google Scholar, 14Schnaitman C.A. Klena J.D. Microbiol. Rev. 1993; 57: 655-682Crossref PubMed Google Scholar, and 15Heinrichs D.E. Yethon J.A. Whitfield C. Mol. Microbiol. 1998; 30: 221-232Crossref PubMed Scopus (284) Google Scholar). In addition, there is an up-regulation of colanic acid production because of induction of the RcsC/YojN/RcsB phosphorelay system (14Schnaitman C.A. Klena J.D. Microbiol. Rev. 1993; 57: 655-682Crossref PubMed Google Scholar, 23Parker C.T. Kloser A.W. Schnaitman C.A. Stein M.A. Gottesman S. Gibson B.W. J. Bacteriol. 1992; 174: 2525-2538Crossref PubMed Google Scholar). The RcsC/YojN/RcsB activating signal is unknown, but it is responsive to OM perturbations (24Gottesman S. Hoch J.A. Silhavy T.J. Two-component Signal Transduction. ASM Press, Washington, D. C.1995: 253-262Google Scholar, 25Conter A. Sturny R. Gutierrez C. Cam K. J. Bacteriol. 2002; 184: 2850-2853Crossref PubMed Scopus (42) Google Scholar, 26Mouslim C. Groisman E.A. Mol. Microbiol. 2003; 47: 335-344Crossref PubMed Scopus (94) Google Scholar, 27Mouslim C. Latifi T. Groisman E.A. J. Biol. Chem. 2003; 278: 50588-50595Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). One difficulty in relating some of the elements of the deep-rough phenotype to the LPS structure is the fact that the original findings were based on severely truncated LPS molecules with unknown genetic defects, rather than precise mutations. Precise mutations that eliminate core phosphorylation in E. coli and Salmonella enterica sv. Typhimurium yield strains that exhibit the major characteristics of the deep-rough phenotype with an increase in susceptibility to hydrophobic compounds, but there is no alteration in the profile of the major OMPs (28Yethon J.A. Heinrichs D.E. Monteiro M.A. Perry M.B. Whitfield C. J. Biol. Chem. 1998; 273: 26310-26316Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 29Yethon J.A. Gunn J.S. Ernst R.K. Miller S.I. Laroche L. Malo D. Whitfield C. Infect. Immun. 2000; 68: 4485-4491Crossref PubMed Scopus (77) Google Scholar). The S. enterica sv. Typhimurium mutant also caused a complete attenuation of virulence in a mouse model (29Yethon J.A. Gunn J.S. Ernst R.K. Miller S.I. Laroche L. Malo D. Whitfield C. Infect. Immun. 2000; 68: 4485-4491Crossref PubMed Scopus (77) Google Scholar). The only source of negative charge outside the lipid A domain of K. pneumoniae is contributed by the GalUA residues in the core OS region. This is also seen in the core OS regions of other environmental isolates, Rhizobium etli (30Forsberg L.S. Carlson R.W. J. Biol. Chem. 1998; 273: 2747-2757Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), Rhizobium leguminosarum (30Forsberg 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 (31Niedziela 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 has been proposed that having GalUA residues instead of phosphoryl substitutions may give these organisms an ecological advantage in habitats that are low in phosphate and low in the divalent cations involved in cross-linking adjacent LPS molecules, because carboxyl groups become more easily protonated, decreasing the repulsion between LPS molecules (11Nikaido H. Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2838) Google Scholar). To determine the role that the carboxyl groups on GalUA residues play in OM stability and whether they fulfill the same functions as the negative charges provided by the phosphate residues in E. coli and Salmonella, a mutant was created that lacked all GalUA residues from the core OS (the GalUA in the inner core as well as the non-stoichiometric substitutions in the inner core). The transferase (WabG) involved in adding the GalUA onto HepII of the inner core has been identified (7Izquierdo L. Coderch N. Pique N. Bedini E. Corsaro M.M. Merino S. Fresno S. Tomas J.M. Regue M. J. Bacteriol. 2003; 185: 7213-7221Crossref PubMed Scopus (69) Google Scholar). A wabG mutant showed enhanced sensitivity to hydrophobic compounds. However, this mutant may still possess some GalUA in its inner core, because the transferase(s) responsible for the addition of the non-stoichiometric substitutions onto the HepIII and β-Glc of the inner core is presently unknown. Therefore, to entirely eliminate GalUA from the K. pneumoniae core OS, UDP-GalUA precursor synthesis was targeted. UDP-GalUA is formed from UDP-GlcUA by the UDP-GalUA C4-epimerase (formerly uge but henceforth referred to as Gla) (32Regué M. Hita B. Pique N. Izquierdo L. Merino S. Fresno S. Benedi V.J. Tomas J.M. Infect. Immun. 2004; 72: 54-61Crossref PubMed Scopus (74) Google Scholar, 33Frirdich E. Whitfield C. J. Bacteriol. 2005; 187: 4104-4115Crossref PubMed Scopus (20) Google Scholar). A mutant in glaKP was constructed to ascertain the effect of this mutation on the OM stability of K. pneumoniae. Bacterial Strains, Plasmids, and Growth Conditions—The bacterial strains and plasmids used in this study are summarized in Table I. Bacteria were grown at 37 °C in Luria-Bertani broth (34Miller J.H. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 439Google Scholar). Growth media were supplemented with chloramphenicol (15 or 7.5 μg/ml), gentamicin (30 μg/ml), kanamycin (25 μg/ml), or streptomycin (200 μg/ml), as necessary. For mutant complementation, wild-type copies of glaKP were expressed using a pBAD-vector derivative, which is a family of expression vectors that use the arabinose-inducible and glucose-repressible araC promoter (35Guzman L.-M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3940) Google Scholar). For induction, a culture was grown at 37 °C for 18 h in LB supplemented with the appropriate antibiotics and 0.4% (w/v) glucose. This culture was diluted 1:100 into fresh medium without glucose and grown until the culture reached an A600 of 0.2. l-Arabinose, 0.02% (w/v), was then added and the culture was grown for another 2 h. Repressed controls were diluted 1:100 into fresh medium with 0.4% (w/v) glucose.Table IBacterial strains and plasmidsStrain or plasmidGenotype, serotype, or descriptionRef. or sourceE. coliDH5αK12 ϕ80d deoR lacZΔM15 endA1 recA1 hsdR17(rK-mK+) supE44 thi-1 gyrA96 relA1 Δ(lacZYA-argF) U169 F-79Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarDH5α [λ pir]K12 ϕ80d deoR lacZΔM15 endA1 recA1 hsdR17(rK-mK+) supE44 thi-1 gyrA96 relA1 Δ(lacZYA-argF) U169 F- [λ pir]80Edwards R.A. Keller L.H. Schifferli D.M. Gene (Amst.). 1998; 207: 149-157Crossref PubMed Scopus (444) Google ScholarSM10 [λ pir]K-12 thi-1 thr-1 leuB6 tonA21 lacY1 supE44 glnV44 recA::RP4-2-Tc::Mu [λ pir]40Miller V.L. Mekalanos J.J. J. Bacteriol. 1988; 170: 2575-2583Crossref PubMed Scopus (1709) Google ScholarK. pneumoniaeCWK2O1:K-; derivative of CWK1 (O1:K20); Strr81Whitfield C. Richards J.C. Perry M.B. Clarke B.R. MacLean L.L. J. Bacteriol. 1991; 173: 1420-1431Crossref PubMed Google ScholarCWG399waaL::aacC1 derivative of CWK2; Gmr5Vinogradov 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 (128) Google ScholarCWG603wabH::aphA-3 derivative of CWG399; Gmr Kmr6Frirdich E. Vinogradov E. Whitfield C. J. Biol. Chem. 2004; 279: 27928-27940Abstract Full Text Full Text PDF PubMed Scopus (26) Google ScholarCWG630glaKP::pRE112 derivative of CWK2; CmrThis studyPlasmidspBAD18-CmArabinose-inducible expression vector; Cmr35Guzman L.-M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3940) Google ScholarpRE112Mobilizable suicide vector used for chromosomal insertions requiring λ pir for replication; Cmr80Edwards R.A. Keller L.H. Schifferli D.M. Gene (Amst.). 1998; 207: 149-157Crossref PubMed Scopus (444) Google ScholarpWQ67pET28a(+)-derivative expressing GlaKP with an N-terminal His6 tag; Kmr33Frirdich E. Whitfield C. J. Bacteriol. 2005; 187: 4104-4115Crossref PubMed Scopus (20) Google ScholarpWQ68pRE112-derivative containing an internal fragment of glaKP, used to construct CWG630; CmrThis studypWQ69pBAD18-Km-derivative expressing His6-GlaKP; Kmr33Frirdich E. Whitfield C. J. Bacteriol. 2005; 187: 4104-4115Crossref PubMed Scopus (20) Google Scholar Open table in a new tab DNA Methods—Plasmid DNA was isolated using the Sigma GenElute Plasmid Miniprep Kit (Sigma) and chromosomal DNA was prepared by the method of Hull et al. (36Hull 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 (6Frirdich E. Vinogradov E. Whitfield C. J. Biol. Chem. 2004; 279: 27928-27940Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). PCR were performed in 0.05-ml volumes with either PwoI DNA polymerase (Roche) or Platinum Taq DNA polymerase (Invitrogen), using conditions optimized for the primer pair. Oligonucleotide primer synthesis and automated DNA sequencing were performed at the Guelph Molecular Supercenter (University of Guelph, Ontario, Canada). All PCR products were sequenced to verify that they were error-free. Plasmids were maintained in E. coli DH5α, except for pRE112 derivatives, which were maintained in DH5α [λ pir]. For E. coli strains, transformation was carried out by electroporation and methods described elsewhere (37Binotto J. MacLachlan P.R. Sanderson P.R. Can. J. Microbiol. 1991; 37: 474-477Crossref PubMed Scopus (49) Google Scholar). For some K. pneumoniae strains, a modification of this method was required (6Frirdich E. Vinogradov E. Whitfield C. J. Biol. Chem. 2004; 279: 27928-27940Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 38Enderle P.J. Farwell M.A. BioTechniques. 1998; 25 (958): 954-956Crossref PubMed Scopus (74) Google Scholar). Insertion Mutagenesis—The mutation glaKP (CWG630) was constructed by insertion of the pRE112 plasmid into the glaKP gene on the K. pneumoniae chromosome, using methods described previously (39Nesper J. Kraiss A. Schild S. Blass J. Klose K.E. Bockemuhl J. Reidl J. Infect. Immun. 2002; 70: 2419-2433Crossref PubMed Scopus (41) Google Scholar). Briefly, a pRE112-derivative containing a glaKP internal fragment was transformed into E. coli SM10 [λ pir] and then transferred by conjugation to the recipient strain, K. pneumoniae CWK2. The plasmid pRE112 requires the pir gene product to replicate (40Miller V.L. Mekalanos J.J. J. Bacteriol. 1988; 170: 2575-2583Crossref PubMed Scopus (1709) Google Scholar), so for the plasmid to be maintained in CWK2, it must be integrated into the chromosome by homologous recombination within glaKP. CWK2 mutant derivatives in which pRE112 has been inserted into glaKP were selected by resistance to streptomycin (resistance carried by CWK2) and chloramphenicol (antibiotic marker on the plasmid). An internal fragment of glaKP was amplified from K. pneumoniae CWK2 chromosomal DNA using primers KPwbnF8 (5′-AACTTGAACAGCGCCATATC-3′) and KPwbnF9 (5′-GGCATAATGTTGTCGGAATC-3′) and the blunt ends of the 510-bp product were ligated to pRE112 digested with SmaI, giving pWQ68. The resulting glaKP mutant was designated K. pneumoniae CWG630. The colony morphology of the mutant was indistinguishable from the parent. Polyacrylamide Gel Electrophoresis Analysis—For PAGE analysis, LPS was isolated on a small scale from proteinase K-digested whole cell lysates as described by Hitchcock and Brown (41Hitchcock P.J. Brown T.M. J. Bacteriol. 1983; 154: 269-277Crossref PubMed Google Scholar). The LPS was then separated on 4–12% BisTris NuPAGE gels from Invitrogen, and visualized by silver staining (42Tsai G.M. Frasch C.E. Anal. Biochem. 1982; 119: 115-119Crossref PubMed Scopus (2309) Google Scholar). For SDS-PAGE of proteins, the protein samples were solubilized in SDS-containing sample buffer (43Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207002) Google Scholar) by boiling at 100 °C for 15 min and then separated on 12% SDS-PAGE gels. Proteins were visualized by Coomassie Brilliant Blue staining. Determination of the Structure of Deacylated LPS—K. pneumoniae CWG630 (glaKP) and the complemented strain CWG630 (pWQ69; His6-Gla+KP) were grown in a fermenter (2× 10 liters) in LB for 21 h, harvested, and then lyophilized. For the CWG630 (glaKP) rough strain lacking O-PS (rough LPS), the LPS was isolated from the dried cells by the phenol/chloroform/light petroleum method (44Galanos C. Luderitz O. Westphal O. Eur. J. Biochem. 1969; 9: 245-249Crossref PubMed Scopus (1366) Google Scholar). For the complemented strain producing smooth LPS, CWG630 (pWQ69; Gla+KP), the hot water phenol method of LPS extraction was used (45Westphal O. Jann K. Methods Carbohydr. Chem. 1965; 5: 83-91Google Scholar). LPS was deacylated by procedures described elsewhere (46Holst O. Thomas-Oates J.E. Brade H. Eur. J. Biochem. 1994; 222: 183-194Crossref PubMed Scopus (80) Google Scholar, 47Holst O. Holst O. Methods in Molecular Biology, Bacterial Toxins: Methods and Protocols. 145. Humana Press Inc., Totowa, NJ2000: 345-353Google Scholar). Briefly for O-deacylation, the LPS (100 mg) was dissolved in anhydrous hydrazine (3 ml), incubated for 1 h at 40 °C, and then poured into cold acetone. The precipitated O-deacylated LPS was collected by centrifugation, washed with acetone, and then lyophilized. Electrospray ionization (ESI) mass spectra were obtained using a Micromass Quattro spectrometer (GV Instruments Inc., Hudson, NH) in 50% MeCN with 0.2% HCOOH, at a flow rate of 15 μl/min with direct injection. OM Protein Preparations—For OM preparations, bacteria were grown overnight at 37 °C. The culture was then diluted 1:100 in 100 ml of fresh medium and incubation was continued at 37 °C for 18 h. The cells were harvested and frozen until required. The cell pellet was thawed, resuspended in 10 ml of 10 mm Tris, pH 8.0, and sonicated on ice (for a total of 2 min using 10-s bursts, followed by 10-s cooling periods). Unbroken cells and cell debris was removed by centrifugation (10 min at 20,000 × g). The membrane fraction was isolated by ultracentrifugation (60 min at 100,000 × g). The membrane fraction was resuspended in 1 ml of 10 mm Tris, pH 8.0, containing 2% Sarkosyl (sodium lauryl sarcosine) and incubated on a rotary shaker at room temperature for 30 min. The OM is resistant to Sarkosyl solubilization, whereas the inner membrane is not (48Filip C. Fletcher G. Wulff J.L. Earhart C.F. J. Bacteriol. 1973; 115: 717-722Crossref PubMed Google Scholar). The Sarkosyl-insoluble OM was separated from the soluble inner membrane by centrifugation at 100,000 × g for 15 min. The OM fraction was washed again with 2% Sarkosyl, washed twice with 10 mm Tris, pH 8.0, and then resuspended in 0.2 ml of 10 mm Tris, pH 8.0. The protein content of each sample was determined using the Bio-Rad DC protein assay, according to the manufacturer's instructions. Unlike E. coli, the inner and outer membranes of these K. pneumoniae strains could not be reliably separated by isopycnic sucrose gradient centrifugation. The molecular basis for this is unclear, but it is not a unique situation (49Larsen B.S. Biedermann K. Anal. Biochem. 1993; 214: 212-221Crossref PubMed Scopus (6) Google Scholar). Identification of individual OM proteins was performed at the Biological Mass Spectrometry Facility, University of Guelph, by peptide mass fingerprinting and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) analysis. Data base searching was done using the ProFound search engine (prowl.rockefeller.edu). Sensitivity to Hydrophobic Compounds and Polymyxin B (PMB)— For SDS sensitivity, serial 2-fold dilutions of SDS (from 6.25 to 0.098 mg/ml) were made in 5 ml of LB. An 18-h culture of each strain was used to inoculate each 5-ml tube at a ratio of 1:100. After incubation for 8 h at 37 °C, a tube was rated positive for growth if the optical density at 600 nm was greater than 0.2. For sensitivity to novobiocin, vancomycin, and PMB, a plate assay was performed. An 18-h culture grown at 37 °C was added to an LB plate supplemented with antibiotics, where appropriate, and the excess was poured off. The plates were dried and 0.01 ml of serial 2-fold diluted novobiocin (from 51.2 to 0.2 mg/ml), vancomycin (from 40 to 0.0098 mg/ml), or PMB (from 780 to 0.095 μg/ml) were spotted onto the plates. After a 24-h incubation at 37 °C, the minimum inhibitory concentration (MIC) was determined as the highest dilution resulting in clearing. All assays were performed in triplicate. Spectrophotometric Assay for Detection of β-Lactamase Activity—To measure endogenous β-lactamase activity, bacteria were grown at 37 °C for 18 h in LB supplemented with the appropriate antibiotics. The culture was then diluted 1:50 in 200 ml of fresh medium and incubation was continued at 37 °C for 4 h. 1 ml of cells were pelleted and the culture supernatant was retained and kept on ice. The remaining cells were harvested and the cell pellet was resuspended in 5 ml of cold LB. The cells were lysed by sonication and the unbroken cells and cell debris were removed by low speed centrifugation (15 min at 5000 × g). The cell lysate was diluted to 10 times the original culture volume, because of high levels of β-lactamase activity. 160 μl of supernatant and diluted cell lysate from each strain was aliquoted in triplicate into 96-well polystyrene microplates (Evergreen Scientific Inc., Los Angeles, CA). Reactions were started by the addition of 40 μl of a 0.5 mg/ml nitrocephin stock (0.1 mg/ml final concentration; Oxoid, Basingstoke, UK) and the absorbance at 560 nm was measured every minute for 30 min using a FLUOstar OPTIMA microplate reader (BMG Labtechnologies Inc., Offenburg, Germany). A sample of 160 μl of LB growth medium with 40 μl of nitrocephin was used to measure background absorbance. Each strain was assayed in triplicate in multiple experiments. GlaKP Is Required for GalUA Addition to the Core—To examine the role of glaKP in core OS biosynthesis, a gene disruption mutant K. pneumoniae (CWG630) was constructed. The lipid A-core fraction of the LPS of the glaKP mutant migrated significantly faster" @default.
• W2085239309 created "2016-06-24" @default.
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• W2085239309 date "2005-07-01" @default.
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• W2085239309 title "The Role of Galacturonic Acid in Outer Membrane Stability in Klebsiella pneumoniae" @default.
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• W2085239309 doi "https://doi.org/10.1074/jbc.m504987200" @default.
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