PagP Activation in the Outer Membrane Triggers R3 Core Oligosaccharide Truncation in the Cytoplasm of Escherichia coli O157:H7
2007; Elsevier BV; Volume: 283; Issue: 7 Linguagem: Inglês
10.1074/jbc.m708163200
ISSN1083-351X
AutoresAbigail E. Smith, Sang‐Hyun Kim, Feng Liu, Wenyi Jia, Evgeny Vinogradov, Carlton Gyles, Russell E. Bishop,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP is normally a latent enzyme, but it can be directly activated in outer membranes by lipid redistribution associated with a breach in the permeability barrier. We now demonstrate that a lipid A myristate deficiency in an E. coli O157:H7 msbB mutant constitutively activates PagP in outer membranes. The lipid A myristate deficiency is associated with hydrophobic antibiotic sensitivity and, unexpectedly, with serum sensitivity, which resulted from O-antigen polysaccharide absence due to a cytoplasmically determined truncation at the first outer core glucose unit of the R3 core oligosaccharide. Mutational inactivation of pagP in the myristate-deficient lipid A background aggravated the hydrophobic antibiotic sensitivity as a result of losing a partially compensatory increase in lipid A palmitoylation while simultaneously restoring serum resistance and O-antigen attachment to intact lipopolysaccharide. Complementation with either wild-type pagP or catalytically inactive pagPSer77Ala alleles restored the R3 core truncation. However, the intact lipopolysaccharide was preserved after complementation with an internal deletion pagPΔ5-14 allele, which mostly eliminates a periplasmic amphipathic α-helical domain but fully supports cell surface lipid A palmitoylation. Our findings indicate that activation of PagP not only triggers lipid A palmitoylation in the outer membrane but also separately truncates the R3 core oligosaccharide in the cytoplasm. We discuss the implication that PagP might function as an apical sensory transducer, which can be activated by a breach in the outer membrane permeability barrier. The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP is normally a latent enzyme, but it can be directly activated in outer membranes by lipid redistribution associated with a breach in the permeability barrier. We now demonstrate that a lipid A myristate deficiency in an E. coli O157:H7 msbB mutant constitutively activates PagP in outer membranes. The lipid A myristate deficiency is associated with hydrophobic antibiotic sensitivity and, unexpectedly, with serum sensitivity, which resulted from O-antigen polysaccharide absence due to a cytoplasmically determined truncation at the first outer core glucose unit of the R3 core oligosaccharide. Mutational inactivation of pagP in the myristate-deficient lipid A background aggravated the hydrophobic antibiotic sensitivity as a result of losing a partially compensatory increase in lipid A palmitoylation while simultaneously restoring serum resistance and O-antigen attachment to intact lipopolysaccharide. Complementation with either wild-type pagP or catalytically inactive pagPSer77Ala alleles restored the R3 core truncation. However, the intact lipopolysaccharide was preserved after complementation with an internal deletion pagPΔ5-14 allele, which mostly eliminates a periplasmic amphipathic α-helical domain but fully supports cell surface lipid A palmitoylation. Our findings indicate that activation of PagP not only triggers lipid A palmitoylation in the outer membrane but also separately truncates the R3 core oligosaccharide in the cytoplasm. We discuss the implication that PagP might function as an apical sensory transducer, which can be activated by a breach in the outer membrane permeability barrier. Like most enteric Gram-negative bacteria, Escherichia coli surrounds its cytoplasmic membrane with a reticulated peptidoglycan exoskeleton (murein) and an outer membrane (OM), 5The abbreviations used are: OMouter membranel-Ara4N4-amino-4-deoxy-l-arabinoseApampicillinGmgentamycinHepL-glycero-d-manno-heptoseKdo3-deoxy-d-manno-oct-2-ulosonic acidLDAOlauroyldimethylamine-N-oxideLPSlipopolysaccharidePEtNphosphoethanolamineStrstreptomycinTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.5The abbreviations used are: OMouter membranel-Ara4N4-amino-4-deoxy-l-arabinoseApampicillinGmgentamycinHepL-glycero-d-manno-heptoseKdo3-deoxy-d-manno-oct-2-ulosonic acidLDAOlauroyldimethylamine-N-oxideLPSlipopolysaccharidePEtNphosphoethanolamineStrstreptomycinTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. which demarcates the so-called periplasmic space. The enter-obacterial OM is an asymmetric lipid bilayer in which lipopolysaccharide (LPS) exclusively lines the external leaflet, whereas phospholipids line the inner leaflet (1Kamio Y. Nikaido H. Biochemistry. 1976; 15: 2561-2570Crossref PubMed Scopus (248) Google Scholar, 2Nikaido H. Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2850) Google Scholar). The asymmetric lipid organization provides a permeability barrier to hydrophobic antibiotics and detergents encountered in the natural and host environments. Although hydrophobic antibiotics can freely permeate through phospholipid bilayers, negative charges in LPS are bridged by Mg2+ ions to create tight lateral packing interactions, which largely prevent permeation (3Schindler M. Osborn M.J. Biochemistry. 1979; 18: 4425-4430Crossref PubMed Scopus (240) Google Scholar, 4Leive L. Ann. N. Y. Acad. Sci. 1974; 235: 109-129Crossref PubMed Scopus (349) Google Scholar). According to current models, perturbations of OM lipid asymmetry can result from the migration of phospholipids into the external leaflet to create localized rafts of phospholipid bilayers, which render bacteria susceptible to hydrophobic antibiotics (5Nikaido H. Nakae T. Adv. Microb. Physiol. 1979; 20: 163-250Crossref PubMed Scopus (298) Google Scholar, 6Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Crossref PubMed Google Scholar). outer membrane 4-amino-4-deoxy-l-arabinose ampicillin gentamycin L-glycero-d-manno-heptose 3-deoxy-d-manno-oct-2-ulosonic acid lauroyldimethylamine-N-oxide lipopolysaccharide phosphoethanolamine streptomycin N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. outer membrane 4-amino-4-deoxy-l-arabinose ampicillin gentamycin L-glycero-d-manno-heptose 3-deoxy-d-manno-oct-2-ulosonic acid lauroyldimethylamine-N-oxide lipopolysaccharide phosphoethanolamine streptomycin N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. The LPS is a tripartite molecule consisting of the hydrophobic anchor lipid A (endotoxin), the core oligosaccharide, which is divided into the inner and outer core regions, and the O-antigen polysaccharide (7Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3372) Google Scholar). The so-called rough LPS includes only the lipid A-core and is usually distinguished from the smooth LPS that also includes O-antigen. The O-antigen can provide bacterial resistance to serum by preventing deposition of the complement cascade's membrane attack complex (8Joiner K.A. Curr. Top. Microbiol. Immunol. 1985; 121: 99-133PubMed Google Scholar). The entire LPS structure is assembled within three distinct subcellular compartments, namely the cytoplasmic membrane, the periplasmic space, and the OM (9Bishop R.E. Contrib. Microbiol. 2005; 12: 1-27Crossref PubMed Google Scholar). The Raetz pathway for lipid A biosynthesis includes nine Lpx enzymes, which convert UDP-GlcNAc into a β-1′, 6-linked disaccharide of GlcN (7Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3372) Google Scholar). The lipid A molecule is phosphorylated at positions 1 and 4′ and is acylated with R-3-hydroxymyristate in ester linkage at positions 3 and 3′ and in amide linkage at positions 2 and 2′ (Fig. 1). Attachment at position 6′ of two 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) sugars, which belong to the inner-most region of the inner core, is followed by secondary acylation reactions to create the so-called acyloxyacyl linkages. Attachment of laurate at position 2′ is usually followed by attachment of myristate at position 3′, which is catalyzed by the myristoyltransferase MsbB (LpxM) (10Clementz T. Zhou Z. Raetz C.R. J. Biol. Chem. 1997; 272: 10353-10360Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Each of these enzymatic reactions takes advantage of cytoplasmic energy-rich biosynthetic precursors (7Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3372) Google Scholar). The waa (rfa) operons encode cytoplasmic enzymes needed for the stepwise assembly of any one of the five known core oligosaccharides (K-12 and R1-R4) that can exist in E. coli. Although ∼170 distinct O-antigens have been identified in E. coli alone, they are all assembled on the lipid carrier analog of dolichol phosphate known as bactoprenol phosphate. Translocation of the lipid A-core and bactoprenol diphosphate-O-antigen to the periplasmic surface of the inner membrane can be followed by polymerization of the O-antigen polysaccharide with its subsequent en bloc ligation to the outer core. At this periplasmic stage, several regulated partial modifications can occur on both lipid A and the inner core and include the addition of phosphoethanolamine (PEtN), derived from phosphatidylethanolamine, and 4-amino-4-deoxy-l-arabinose (l-Ara4N), derived from bactoprenol phosphate-l-Ara4N (11Raetz C.R. Reynolds C.M. Trent M.S. Bishop R.E. Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (928) Google Scholar). These modifications provide resistance to polymyxin B and can be induced by mildly acidic growth conditions, antimicrobial peptides, or Mg2+ limitation, which together activate the PhoP/PhoQ and PmrA/PmrB two-component signal transduction pathways (12Groisman E.A. J. Bacteriol. 2001; 183: 1835-1842Crossref PubMed Scopus (656) Google Scholar, 13Bader M.W. Sanowar S. Daley M.E. Schneider A.R. Cho U. Xu W. Klevit R.E. Le Moual H. Miller S.I. Cell. 2005; 122: 461-472Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar). E. coli K-12 strains generally synthesize rough LPS lacking O-antigen polysaccharide unless certain genetic factors are exogenously provided (14Heinrichs D.E. Yethon J.A. Whitfield C. Mol. Microbiol. 1998; 30: 221-232Crossref PubMed Scopus (284) Google Scholar). In contrast, smooth LPS with attached O157 polysaccharide is synthesized in enterohemorrhagic E. coli O157:H7. This most common serotype of Shiga toxin-producing E. coli is associated with hemorrhagic colitis and hemolytic-uremic syndrome in humans (15Tarr P.I. Gordon C.A. Chandler W.L. Lancet. 2005; 365: 1073-1086Abstract Full Text Full Text PDF PubMed Scopus (1387) Google Scholar). The core oligosaccharide of E. coli O157:H7 is of the R3 type, which is distinctly different from K-12 in the outer core regions (16Kaniuk N.A. Vinogradov E. Li J. Monteiro M.A. Whitfield C. J. Biol. Chem. 2004; 279: 31237-31250Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The lipid A and inner core structures of the R3 and K-12 LPS are largely identical, with the exception of a few important differences that can be attributed to enzymes encoded in the plasmid pO157 shf locus. The conserved inner core includes, in addition to the two Kdo units, three units of l-glycero-d-manno-heptose (Hep), which can be modified with phosphate and/or PEtN moieties at key positions (Fig. 1). A defining feature of the R3 inner core is the partial modification of HepIII by an α-1,7-linked GlcNAc unit, which is controlled by the shf locus-encoded glycosyltransferase WabB (16Kaniuk N.A. Vinogradov E. Li J. Monteiro M.A. Whitfield C. J. Biol. Chem. 2004; 279: 31237-31250Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Additionally, under normal laboratory growth conditions, where the corresponding enzymes of E. coli K-12 are found to be latent (17Gibbons H.S. Kalb S.R. Cotter R.J. Raetz C.R. Mol. Microbiol. 2005; 55: 425-440Crossref PubMed Scopus (98) Google Scholar), E. coli O157:H7 introduces significant amounts of PEtN into the lipid A phosphate groups (18Kim S.H. Jia W. Parreira V.R. Bishop R.E. Gyles C.L. Microbiology. 2006; 152: 657-666Crossref PubMed Scopus (35) Google Scholar). Finally, E. coli O157:H7 possesses two homologues of the msbB gene: msbB1 encoded on the chromosome, which is equivalent to the single msbB gene of E. coli K-12, and msbB2 encoded on the shf locus. Both msbB orthologues must be inactivated to create a myristate deficiency in the lipid A of E. coli O157:H7, and this is associated with reduced virulence (19Kim S.H. Jia W. Bishop R.E. Gyles C. Infect. Immun. 2004; 72: 1174-1180Crossref PubMed Scopus (28) Google Scholar), as similarly occurs in msbB-deficient Shigella flexneri (20D'Hauteville H. Khan S. Maskell D.J. Kussak A. Weintraub A. Mathison J. Ulevitch R.J. Wuscher N. Parsot C. Sansonetti P.J. J. Immunol. 2002; 168: 5240-5251Crossref PubMed Scopus (117) Google Scholar). The only ascribed function for the single msbB2 gene is in the persistence of E. coli O157:H7 in its agricultural and bovine reservoirs (21Yoon J.W. Lim J.Y. Park Y.H. Hovde C.J. Infect. Immun. 2005; 73: 2367-2378Crossref PubMed Scopus (29) Google Scholar). The entire LPS structure is transported across the periplasmic space and delivered to the OM external leaflet, where lipid A can be further modified (11Raetz C.R. Reynolds C.M. Trent M.S. Bishop R.E. Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (928) Google Scholar). PagP is the only known OM enzyme of LPS biosynthesis in E. coli, and it is controlled by PhoP/PhoQ (22Bishop R.E. Mol. Microbiol. 2005; 57: 900-912Crossref PubMed Scopus (130) Google Scholar). PagP acylates the lipid A position 2 R-3-hydroxymyristate chain with a phospholipid-derived palmitoyl group (23Bishop R.E. Gibbons H.S. Guina T. Trent M.S. Miller S.I. Raetz C.R. EMBO J. 2000; 19: 5071-5080Crossref PubMed Scopus (280) Google Scholar, 24Khan M.A. Neale C. Michaux C. Pomes R. Prive G.G. Woody R.W. Bishop R.E. Biochemistry. 2007; 46: 4565-4579Crossref PubMed Scopus (48) Google Scholar), which provides resistance to cationic antimicrobial peptides (25Guo L. Lim K.B. Poduje C.M. Daniel M. Gunn J.S. Hackett M. Miller S.I. Cell. 1998; 95: 189-198Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar, 26Robey M. O'Connell W. Cianciotto N.P. Infect. Immun. 2001; 69: 4276-4286Crossref PubMed Scopus (101) Google Scholar, 27Pilione M.R. Pishko E.J. Preston A. Maskell D.J. Harvill E.T. Infect. Immun. 2004; 72: 2837-2842Crossref PubMed Scopus (42) Google Scholar) and attenuates the ability of LPS to trigger host defenses through the TLR4 pathway (28Kawasaki K. Ernst R.K. Miller S.I. J. Biol. Chem. 2004; 279: 20044-20048Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 29Miller S.I. Ernst R.K. Bader M.W. Nat. Rev. Microbiol. 2005; 3: 36-46Crossref PubMed Scopus (768) Google Scholar). PagP structure and dynamics demonstrate that the palmitate recognition pocket, known as the hydrocarbon ruler, is only accessible from the OM external leaflet and, thus, requires aberrant translocation of phospholipids into the external leaflet (30Ahn V.E. Lo E.I. Engel C.K. Chen L. Hwang P.M. Kay L.E. Bishop R.E. Prive G.G. EMBO J. 2004; 23: 2931-2941Crossref PubMed Scopus (112) Google Scholar, 31Hwang P.M. Choy W.Y. Lo E.I. Chen L. Forman-Kay J.D. Raetz C.R. Prive G.G. Bishop R.E. Kay L.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13560-13565Crossref PubMed Scopus (278) Google Scholar, 32Hwang P.M. Bishop R.E. Kay L.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9618-9623Crossref PubMed Scopus (92) Google Scholar). Indeed, PagP remains dormant in the OM until perturbations to lipid asymmetry, which compromise the OM permeability barrier, directly trigger PagP activity (33Jia W. Zoeiby A.E. Petruzziello T.N. Jayabalasingham B. Seyedirashti S. Bishop R.E. J. Biol. Chem. 2004; 279: 44966-44975Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). PagP has been proposed to function as a sentinel that can be activated by a breach in the OM permeability barrier (34Bishop R.E. Biochim. Biophys. Acta. 2007; 10.1016/j.bbamem.2007.07.021Google Scholar). We now demonstrate that a lipid A myristoylation mutant of enterohemorrhagic E. coli O157: H7, but not a similar mutant from E. coli K-12, necessarily triggers PagP activity in the OM to exert control on cytoplasmic enzymes that determine its characteristic R3 core oligosaccharide structure. PagP sensory transduction is not controlled by its cell surface catalytic machinery but depends instead on its periplasmic amphipathic α-helix. Materials–32Pi was purchased from PerkinElmer Life Sciences. Antibiotics and Gal were obtained from Sigma. Pyridine, methanol, and 88% formic acid were obtained from Mallinckrodt. Chloroform was purchased from EM Science. Glass-backed Silica Gel 60 TLC plates were from Merck. The QIA-prep spin miniprep, Qiaquick PCR purification, and QIAEX II gel extraction kits were obtained from Qiagen. High fidelity PCR was performed with a proofreading DNA polymerase (Advantage-HF2 PCR kit; BD Biosciences Clontech). Restriction endonucleases, T4 DNA ligase, and dNTP were obtained from Fermentas. Bacto MacConkey agar was obtained from Difco. All other materials were obtained from commercial sources. Bacterial Strains, Plasmids, and Growth Conditions–The bacterial strains and plasmids used in this study are described in Table 1. Cells were generally grown at 37 °C in Luria-Bertani (LB) broth. Antibiotics were added when necessary at final concentrations of 12 μg/ml for tetracycline, 20 μg/ml for chloramphenicol and gentamycin (Gm), 100 μg/ml for ampicillin (Ap), and streptomycin (Str), and 40 μg/ml for kanamycin. Antibiotic concentrations were reduced by a factor of 10 during selection of the hypersensitive E. coli O157:H7 strain 4303-TM. Single colonies were inoculated from plates into 5 ml of liquid medium and grown at 37 °C overnight to stationary phase. A 1% inoculum was then subcultured into the same medium and allowed to resume growth at 37 °C. Cultures were adjusted with EDTA using a stock solution of 250 mm EDTA, pH 8.0, which had been sterilized by using a 0.2-μm filter.TABLE 1Bacterial strains and plasmids used in this studyStrains/plasmidsDescriptionaAbbreviated descriptions used in some figures are included in square brackets.SourceE. coli O157:H74304Wild type (phage-type 14), (Strr)Ref. 19Kim S.H. Jia W. Bishop R.E. Gyles C. Infect. Immun. 2004; 72: 1174-1180Crossref PubMed Scopus (28) Google Scholar4304-DM4304 ΔmsbB1, ΔmsbB2 (Strr) [msbB1/2]Ref. 19Kim S.H. Jia W. Bishop R.E. Gyles C. Infect. Immun. 2004; 72: 1174-1180Crossref PubMed Scopus (28) Google Scholar4304-PM4304 pagP::aacC1 (Strr, Gmr) [pagP]This study4304-TM4304 ΔmsbB1, ΔmsbB2, pagP::aacC1 (Strr, Gmr) [msbB1/2/pagP]This studyE. coli K-12MC1061F-, λ-, araD139, Δ(ara-leu)7697, Δ (lac)X74, galU, galK, hsdR2 (rK-mK+), mcrB1, rpsLRef. 33Jia W. Zoeiby A.E. Petruzziello T.N. Jayabalasingham B. Seyedirashti S. Bishop R.E. J. Biol. Chem. 2004; 279: 44966-44975Abstract Full Text Full Text PDF PubMed Scopus (84) Google ScholarWJ0124MC1061 pagP::amp [pagP]Ref. 33Jia W. Zoeiby A.E. Petruzziello T.N. Jayabalasingham B. Seyedirashti S. Bishop R.E. J. Biol. Chem. 2004; 279: 44966-44975Abstract Full Text Full Text PDF PubMed Scopus (84) Google ScholarSK1061MC1061 msbB::Tn5, pagP::amp [msbB/pagP]This studyMC-msbBMC1061 msbB::Tn5 [msbB]Ref. 18Kim S.H. Jia W. Parreira V.R. Bishop R.E. Gyles C.L. Microbiology. 2006; 152: 657-666Crossref PubMed Scopus (35) Google ScholarBMS67C12msbB::Tn5 mutant of JM83 (Kmr)Ref. 36Somerville Jr., J.E. Cassiano L. Darveau R.P. Infect Immun. 1999; 67: 6583-6590Crossref PubMed Google ScholarSM10thr, leu, tonA, lacY, supE, recA::RP4-2-Tc::Mu, Kmr, λpirRef. 61Edwards R.A. Keller L.H. Schifferli D.M. Gene (Amst.). 1998; 207: 149-157Crossref PubMed Scopus (444) Google ScholarPlasmidspGEM-TPCR product cloning vectorPromegapUCGMGmr cassette (aacC1)-containing plasmidRef. 62Schweizer H.D. BioTechniques. 1993; 15: 831-834PubMed Google ScholarpR7Crc-GmpRE107 carrying pagP::aacC1This studypRE107A suicide vector (oriR6K, RP4 mob, sacB, Apr)Ref. 61Edwards R.A. Keller L.H. Schifferli D.M. Gene (Amst.). 1998; 207: 149-157Crossref PubMed Scopus (444) Google ScholarpCrcATpagPO157 in pGEM-T vectorThis studypWG24waaGO157 cloned into pBAD24 (AmpR)This studypGU184galUO157 cloned into pACYC184 (CmR) [pGalU]This studypAA101E. coli galETK cloned in pBR313Ref. 41Edwards-Jones B. Langford P.R. Kroll J.S. Yu J. Microbiology. 2004; 150: 1079-1084Crossref PubMed Scopus (9) Google ScholarpACPagPE. coli pagP cloned in pACYC184Ref. 33Jia W. Zoeiby A.E. Petruzziello T.N. Jayabalasingham B. Seyedirashti S. Bishop R.E. J. Biol. Chem. 2004; 279: 44966-44975Abstract Full Text Full Text PDF PubMed Scopus (84) Google ScholarpACPagPSer77AlaDerivative of pACPagPThis studypACPagPΔ5-14Previously pACPagPΔ30-39 (precursor numbering)Ref. 33Jia W. Zoeiby A.E. Petruzziello T.N. Jayabalasingham B. Seyedirashti S. Bishop R.E. J. Biol. Chem. 2004; 279: 44966-44975Abstract Full Text Full Text PDF PubMed Scopus (84) Google ScholarpWQ3K. pneumoniae rfbkp01 cloned in pRK404Ref. 48Clarke B.R. Whitfield C. J. Bacteriol. 1992; 174: 4614-4621Crossref PubMed Google ScholarpBAD-B2E. coli msbB2 cloned in pBAD24 [pMsbB2]Ref. 19Kim S.H. Jia W. Bishop R.E. Gyles C. Infect. Immun. 2004; 72: 1174-1180Crossref PubMed Scopus (28) Google ScholarpEP24pagPO157 in pBAD24This studya Abbreviated descriptions used in some figures are included in square brackets. Open table in a new tab DNA Manipulations–Restriction enzyme digestions, ligations, transformations, and DNA electrophoresis were performed as described (35Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The oligonucleotide primers used for DNA sequencing and PCR gene amplification were manufactured by Invitrogen. Purification of plasmids, PCR products, and restriction fragments was performed with the QIAprep, QIAquick, and QIAEX II kits, respectively, according to the manufacturer's instructions (Qiagen). Genomic DNA was purified using the Easy-DNA kit (Invitrogen). DNA sequencing was performed at the ACGT Corp. sequencing facility (Toronto, Canada). Mutant Constructions–An allelic exchange method was employed for creation of a pagP::aacC1 mutation in the double msbB mutant (4304-DM) of E. coli O157:H7 strain 4304 (Table 1). In brief, the pagP gene was amplified from wild type strain 4304 by PCR with primers CrcA (forward, ATGAGCTCAGGTTGACGATA) and CrcR (reverse, TTGAATTCTTGCTGACGTATC) to yield a 1.3-kb product. The amplicon was cloned into the pGEM-T vector, and the recombinant plasmid (pCrcAT) was digested with KpnI, which cuts a single site near the middle of the pagP gene. For insertion of the nonpolar Gm-cassette, the KpnI fragment containing the aacC1 gene was purified from pUCGM carrying the aacC1 gene in the multiple cloning site. The plasmid resulting from ligation of the aacC1 gene into pCrcAT was used as template DNA for a high fidelity PCR with proofreading DNA polymerase and the primer pair CrcA and CrcR. The 2.1-kb amplicon was purified for blunt end ligation with the pRE107 vector digested with SmaI. The resulting suicide vector construct was named pR7Crc-Gm. The SM10 donor E. coli was transformed with the pR7Crc-Gm plasmid harboring the mutated (pagP::aacC1) allele. Strain SM10 (pR7Crc-Gm) was mated with 4304-DM and incubated at 37 °C overnight on blood agar plates. This mating procedure was subsequently repeated using the wild-type strain 4304 to generate a single pagP:aacC1 mutant. The exconjugants were selected on LB agar plates containing appropriate antibiotics (StrR + GmR). The resulting exconjugants were spread on LB agar plates containing 7% sucrose and Gm and incubated at 30 °C in order to select isolates that had undergone a double crossover. A few selected colonies were purified, and the potential pagP::aacC1 mutants (Gmr and Aps) were tested by PCR for confirmation of the mutated allele. The primer pair of CrcA and CrcR was used for amplification of the pagP::aacC1 allele from the mutants. The expected size (2.1 kb) of the amplicon in the mutants was compared with that of the wild type pagP gene (1.3 kb) by 1% agarose-gel electrophoresis. The resulting pagP::aacC1 mutant of E. coli 4304-DM was named 4304-TM, whereas that of E. coli 4304 was named 4304-PM (Table 1). The msbB::Tn5 allele in the E. coli K-12 donor strain BMS67C12 was transferred by P1 transduction to the E. coli K-12 strain WJ0124 to create strain SK1061 (Table 1). A temperature-sensitive P1 cmr-100 lysate of BMS67C12 (36Somerville Jr., J.E. Cassiano L. Darveau R.P. Infect Immun. 1999; 67: 6583-6590Crossref PubMed Google Scholar) was prepared as described elsewhere (18Kim S.H. Jia W. Parreira V.R. Bishop R.E. Gyles C.L. Microbiology. 2006; 152: 657-666Crossref PubMed Scopus (35) Google Scholar) and then mixed with E. coli WJ0124. The resulting msbB::Tn5 mutant of WJ0124 was verified by PCR. Analysis of Lipid A by TLC–Analysis of lipid A compositional profiles was done by TLC separation of 32P-labeled lipid A species released from a mild acid hydrolysis procedure, applied to bacteria cultured with or without EDTA treatment (33Jia W. Zoeiby A.E. Petruzziello T.N. Jayabalasingham B. Seyedirashti S. Bishop R.E. J. Biol. Chem. 2004; 279: 44966-44975Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 37Zhou Z. Lin S. Cotter R.J. Raetz C.R. J. Biol. Chem. 1999; 274: 18503-18514Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). LPS Preparation and SDS-PAGE Analysis–LPS was prepared on a small scale from SDS-proteinase K-treated whole cell lysates (38Hitchcock P.J. Brown T.M. J. Bacteriol. 1983; 154: 269-277Crossref PubMed Google Scholar). Large scale LPS preparations were made by the phenol/chloroform/petroleum ether extraction procedure as described elsewhere (39Galanos C. Luderitz O. Westphal O. Eur. J. Biochem. 1969; 9: 245-249Crossref PubMed Scopus (1366) Google Scholar). The LPS was then separated on a 16% Tricine SDS-polyacrylamide gel (Novex, San Diego, CA) and was visualized by silver staining (40Tsai C.M. Frasch C.E. Anal. Biochem. 1982; 119: 115-119Crossref PubMed Scopus (2311) Google Scholar). PAGE conditions were adjusted as recommended by the manufacturer. NMR Spectroscopy–NMR spectra were recorded at 25 °C in D2O on a Varian UNITY INOVA 500 instrument, using acetone as a reference for proton (2.225 ppm) and carbon (31.5 ppm) spectra. Varian standard programs for COSY, NOESY (mixing time of 400 ms), TOCSY (spin lock time, 120 ms), HSQC, and gHMBC (long range transfer delay, 100 ms) were used. Isolation of the Core Oligosaccharide–LPS (30 mg) was hydrolyzed with 2% acetic acid (3 h, 100 °C). Lipid was removed by centrifugation, and soluble products were separated by gel chromatography on Sephadex G-50 to yield core oligosaccharide (15 mg) and a low molecular mass fraction. The core was additionally purified by anion exchange chromatography on a Hitrap Q column (Amersham Biosciences) using a gradient of NaCl from 0 to 1 m over 1 h, and the major acidic core fraction was desalted by gel chromatography. Hydrazine O-Deacylation of the LPS–LPS (20 mg) was dissolved in anhydrous hydrazine (1 ml) and kept at 60 °C for 1 h, cooled, and poured into acetone (50 ml). Precipitate was collected, washed with acetone, dissolved in water, and freeze-dried to give O-deacylated LPS (12 mg). Monosaccharide Analysis–Hydrolysis was performed with 4 m trifluoroacetic acid (110 °C, 3h), and monosaccharides were conventionally converted into alditol acetates and analyzed by gas chromatography on an Agilent 6850 chromatograph equipped with a DB-17 (30 × 0.25 mm) fused silica column using a temperature gradient from 180 °C (2 min) to 240 °C at 2 °C/min. Mass Spectrometry–Electrospray ionization/mass spectrometry spectra were obtained using a Micromass Quattro spectrometer in 50% acetonitrile with 0.2% formic acid at a flow rate of 15 μl/min with direct injection. A 5-kV electrospray ionization voltage was used. Immunoblotting–The LPS samples of 4304, DM (msbB1/msbB2), and DM (pBAD-B2) were resolved by 16% Tricine SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in standard Tris-buffered saline-Tween 20 buffer. Anti-O157 rabbit serum (Difco) and anti-rabbit IgG coupled with horseradish peroxidase (Sigma) were used as primary and secondary antibodies, respectively. For chemiluminescent detection, the ECL detection kit was used according to the manufacturer's instructions (Amersham Biosciences). Construction of Plasmids for Phenotypic Complementation–The waaG gene of E. coli O157:H7 was amplified by PCR using 4304-WT genomic DNA and the two primers called WG24-Kpn (forward, GACAGGTACGTCGTTATGGTACCTGCTTTTTG) (where underlined italics identify restriction sites) and WG24-H3 (reverse, CTTTACCGCGCCAAAGCTTGGCAAACGGCTC) and then cloned into an arabinose-inducible pBAD24 expression vector digested with KpnI and HindIII. The insertion was verified by agarose gel electrophoresis, and the construct was named pWG24. Also, the galU gene of E. coli O157:H7 was amplified by PCR using 4304-WT genomic DNA and the two primers called GalU-H3 (forward, TGCATTACAAGCTTATGTCGGCTGG) and GalU-Sal (reverse, GTCGATTGGTCGACGCCGTTTCGTG). The 1.1-kb amplicon, including the endogenous promoter region, was digested with HindIII and SalI and inserted into pACYC184 digested with the same restriction enzymes, and the resulting plasmid was named pGU184. The pagP gene of E. coli O157:H7 was amplified by a high fidelity PCR using genomic DNA and the two primers called Pag24-Kpn (forward, TGGTCACQAAATGGTACCGAGTAAATATGTCG) and Pag24-H3 (reverse, GAAGTTACTAAAGCTTCATTTGTCTCAA). The ∼600-bp amplicon was digested with KpnI and HindIII and cloned into an arabinose-inducible pBAD24 expression vector digested with KpnI and HindIII. The insertion was verified by agarose gel electropho
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