Accumulation of a Lipid A Precursor Lacking the 4′-Phosphate following Inactivation of the Escherichia coli lpxKGene
1998; Elsevier BV; Volume: 273; Issue: 20 Linguagem: Inglês
10.1074/jbc.273.20.12457
ISSN1083-351X
AutoresTeresa A. Garrett, Nanette L. S. Que, Christian R.H. Raetz,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoThe lpxK gene has been proposed to encode the lipid A 4′-kinase in Escherichia coli (Garrett, T. A., Kadrmas, J. L., and Raetz, C. R. H. (1997)J. Biol. Chem. 272, 21855–21864). In cell extracts, the kinase phosphorylates the 4′-position of a tetraacyldisaccharide 1-phosphate precursor (DS-1-P) of lipid A, but the enzyme has not yet been purified because of instability. lpxK is co-transcribed with an essential upstream gene, msbA, with strong homology to mammalian Mdr proteins and ABC transporters.msbA may be involved in the transport of newly made lipid A from the inner surface of the inner membrane to the outer membrane. Insertion of an Ω-chloramphenicol cassette into msbA also halts transcription of lpxK. We have now constructed a strain in which only the lpxK gene is inactivated by inserting a kanamycin cassette into the chromosomal copy oflpxK. This mutation is complemented at 30 °C by a hybrid plasmid with a temperature-sensitive origin of replication carryinglpxK +. When this strain (designated TG1/pTAG1) is grown at 44 °C, the plasmid bearing thelpxK + is lost, and the phenotype of anlpxK knock-out mutation is unmasked. The growth of TG1/pTAG1 was inhibited after several hours at 44 °C, consistent with lpxK being an essential gene. Furthermore, 4′-kinase activity in extracts made from these cells was barely detectable. In accordance with the proposed biosynthetic pathway for lipid A, DS-1-P (the 4′-kinase substrate) accumulated in TG1/pTAG1 cells grown at 44 °C. The DS-1-P from TG1/pTAG1 was isolated, and its structure was verified by 1H NMR spectroscopy. DS-1-P had not been isolated previously from bacterial cells. Its accumulation in TG1/pTAG1 provides additional support for the pathway of lipid A biosynthesis inE. coli. Homologs of lpxK are present in the genomes of other Gram-negative bacteria. The lpxK gene has been proposed to encode the lipid A 4′-kinase in Escherichia coli (Garrett, T. A., Kadrmas, J. L., and Raetz, C. R. H. (1997)J. Biol. Chem. 272, 21855–21864). In cell extracts, the kinase phosphorylates the 4′-position of a tetraacyldisaccharide 1-phosphate precursor (DS-1-P) of lipid A, but the enzyme has not yet been purified because of instability. lpxK is co-transcribed with an essential upstream gene, msbA, with strong homology to mammalian Mdr proteins and ABC transporters.msbA may be involved in the transport of newly made lipid A from the inner surface of the inner membrane to the outer membrane. Insertion of an Ω-chloramphenicol cassette into msbA also halts transcription of lpxK. We have now constructed a strain in which only the lpxK gene is inactivated by inserting a kanamycin cassette into the chromosomal copy oflpxK. This mutation is complemented at 30 °C by a hybrid plasmid with a temperature-sensitive origin of replication carryinglpxK +. When this strain (designated TG1/pTAG1) is grown at 44 °C, the plasmid bearing thelpxK + is lost, and the phenotype of anlpxK knock-out mutation is unmasked. The growth of TG1/pTAG1 was inhibited after several hours at 44 °C, consistent with lpxK being an essential gene. Furthermore, 4′-kinase activity in extracts made from these cells was barely detectable. In accordance with the proposed biosynthetic pathway for lipid A, DS-1-P (the 4′-kinase substrate) accumulated in TG1/pTAG1 cells grown at 44 °C. The DS-1-P from TG1/pTAG1 was isolated, and its structure was verified by 1H NMR spectroscopy. DS-1-P had not been isolated previously from bacterial cells. Its accumulation in TG1/pTAG1 provides additional support for the pathway of lipid A biosynthesis inE. coli. Homologs of lpxK are present in the genomes of other Gram-negative bacteria. Lipopolysaccharide (LPS) 1The abbreviations used are: LPS, lipopolysaccharide; DS-1-P, tetraacyldisaccharide 1-phosphate; HPTLC, high performance thin layer chromatography; kb, kilobase pair(s); PBS, phosphate-buffered saline; COSY, two-dimensional 1H correlation spectroscopy; Kdo, 3-deoxy-d-manno-octulosonic acid; UDP-DAG, UDP-2,3-diacylglucosamine. is an essential glycolipid of Gram-negative bacteria (1Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1041) Google Scholar, 2Raetz C.R.H. J. Bacteriol. 1993; 175: 5745-5753Crossref PubMed Scopus (236) Google Scholar, 3Raetz C.R.H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 4Rietschel E.T. Brade H. Sci. Am. 1992; 267: 54-61Crossref PubMed Scopus (456) Google Scholar, 5Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar). It is a complex molecule that forms the outer leaflet of the outer membrane and is important in forming an effective permeability barrier (3Raetz C.R.H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 6Nikaido H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 29-47Google Scholar, 7Vaara M. Antimicrob. Agents Chemother. 1993; 37: 2255-2260Crossref PubMed Scopus (141) Google Scholar). The lipid A portion of LPS is required for bacterial viability and is a potent immunostimulant (1Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1041) Google Scholar, 2Raetz C.R.H. J. Bacteriol. 1993; 175: 5745-5753Crossref PubMed Scopus (236) Google Scholar, 3Raetz C.R.H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 4Rietschel E.T. Brade H. Sci. Am. 1992; 267: 54-61Crossref PubMed Scopus (456) Google Scholar, 5Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar). Indeed, Gram-negative sepsis is thought to be mediated by over-stimulation of the immune system by bacterially derived lipid A (1Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1041) Google Scholar, 2Raetz C.R.H. J. Bacteriol. 1993; 175: 5745-5753Crossref PubMed Scopus (236) Google Scholar, 3Raetz C.R.H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 4Rietschel E.T. Brade H. Sci. Am. 1992; 267: 54-61Crossref PubMed Scopus (456) Google Scholar, 5Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar). In Escherichia coli K12, lipid A is a disaccharide of glucosamine that is phosphorylated at the 1- and 4′-positions and acylated at the 2-, 3-, 2′-, and 3′-positions with (R)-3-hydroxymyristate (Fig.1) (1Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1041) Google Scholar, 2Raetz C.R.H. J. Bacteriol. 1993; 175: 5745-5753Crossref PubMed Scopus (236) Google Scholar, 3Raetz C.R.H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 4Rietschel E.T. Brade H. Sci. Am. 1992; 267: 54-61Crossref PubMed Scopus (456) Google Scholar, 5Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar). Two additional fatty acyl chains are esterified to the 2′- and 3′-hydroxymyristoyl chains to form acyloxyacyl moieties characteristic of lipid A (1Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1041) Google Scholar, 2Raetz C.R.H. J. Bacteriol. 1993; 175: 5745-5753Crossref PubMed Scopus (236) Google Scholar, 3Raetz C.R.H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar, 4Rietschel E.T. Brade H. Sci. Am. 1992; 267: 54-61Crossref PubMed Scopus (456) Google Scholar, 5Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar). The biosynthetic pathway for making lipid A in E. coli is well understood (1Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1041) Google Scholar, 2Raetz C.R.H. J. Bacteriol. 1993; 175: 5745-5753Crossref PubMed Scopus (236) Google Scholar, 3Raetz C.R.H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar). Nine enzymes are required to synthesize Kdo2-lipid A (Fig. 1) (1Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1041) Google Scholar, 2Raetz C.R.H. J. Bacteriol. 1993; 175: 5745-5753Crossref PubMed Scopus (236) Google Scholar, 3Raetz C.R.H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar). With the recent identification of the gene encoding the lipid A 4′-kinase (8Garrett T.A. Kadrmas J.L. Raetz C.R.H. J. Biol. Chem. 1997; 272: 21855-21864Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), the genes encoding 8 of the 9 enzymes required for the biosynthesis of Kdo2-lipid A have been identified (3Raetz C.R.H. Neidhardt F.C. 2nd Ed. Escherichia coli and Salmonella: Cellular and Molecular Biology. 1. American Society for Microbiology, Washington, D. C.1996: 1035-1063Google Scholar). The lipid A 4′-kinase catalyzes the transfer of the γ-phosphate from ATP to the 4′-position of tetraacyldisaccharide 1-phosphate (DS-1-P) to form tetraacyldisaccharide 1,4′-bis-phosphate (lipid IVA) (Fig. 1) (9Ray B.L. Raetz C.R.H. J. Biol. Chem. 1987; 262: 1122-1128Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of the 4′-OH group is necessary for the action of distal biosynthetic enzymes, such as the Kdo transferase (10Brozek K.A. Hosaka K. Robertson A.D. Raetz C.R.H. J. Biol. Chem. 1989; 264: 6956-6966Abstract Full Text PDF PubMed Google Scholar, 11Belunis C.J. Raetz C.R.H. J. Biol. Chem. 1992; 267: 9988-9997Abstract Full Text PDF PubMed Google Scholar), and for recognition of lipid A by mammalian cells during endotoxin stimulation (5Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zähringer U. Seydel U. Di Padova F. Schreier M. Brade H. FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar). The lipid A 4′-kinase gene was recently identified as orfE(a previously reported open reading frame of unknown function) (12Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (125) Google Scholar), and it is now referred to as lpxK (8Garrett T.A. Kadrmas J.L. Raetz C.R.H. J. Biol. Chem. 1997; 272: 21855-21864Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). lpxK forms an operon with an essential upstream gene, called msbA, which has homology to ABC transporters and mammalian Mdr proteins (12Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (125) Google Scholar,13Polissi A. Georgopoulos C. Mol. Microbiol. 1996; 20: 1221-1233Crossref PubMed Scopus (110) Google Scholar). msbA has been implicated in the transport of lipid A from its site of biosynthesis on the inner surface of the inner membrane to the outer membrane (12Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (125) Google Scholar, 13Polissi A. Georgopoulos C. Mol. Microbiol. 1996; 20: 1221-1233Crossref PubMed Scopus (110) Google Scholar, 43Zhou Z. White K.A. Polissi A. Georgopoulos C. Raetz C.R.H. J. Biol. Chem. 1998; 273: 12466-12475Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Georgopoulos and co-workers (12Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (125) Google Scholar, 13Polissi A. Georgopoulos C. Mol. Microbiol. 1996; 20: 1221-1233Crossref PubMed Scopus (110) Google Scholar) constructed a strain with an Ω-cam cassette inserted in the msbA gene. Because lpxK is co-transcribed with msbA (12Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (125) Google Scholar, 13Polissi A. Georgopoulos C. Mol. Microbiol. 1996; 20: 1221-1233Crossref PubMed Scopus (110) Google Scholar), this insertion stops expression of both msbA and lpxK. Complementation analysis showed that both msbA and lpxK were required for growth (12Karow M. Georgopoulos C. Mol. Microbiol. 1993; 7: 69-79Crossref PubMed Scopus (125) Google Scholar). It has also been found that glucosamine-labeled LPS precursors accumulate in the inner membrane ofmsbA/lpxK knock-outs (13Polissi A. Georgopoulos C. Mol. Microbiol. 1996; 20: 1221-1233Crossref PubMed Scopus (110) Google Scholar). This phenomenon was attributed to the loss of the putative transport protein, MsbA. However, given the fact that lpxK plays an integral role in the biosynthesis of lipid A (8Garrett T.A. Kadrmas J.L. Raetz C.R.H. J. Biol. Chem. 1997; 272: 21855-21864Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), the apparent accumulation of LPS in the inner membrane might be due to the build up of lipid A precursor(s), which could accumulate when the 4′-kinase is inactivated. These precursors may not be efficiently transported to the outer membrane by the putative lipid A transport machinery and may even inhibit transport of lipid A. Direct evidence for the function of MsbA as a lipid A transporter in strains bearing extra copies oflpxK is presented in the accompanying manuscript (43Zhou Z. White K.A. Polissi A. Georgopoulos C. Raetz C.R.H. J. Biol. Chem. 1998; 273: 12466-12475Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). In the present work, we have constructed a strain with an insertion mutation in only the lpxK gene. In strain TG1/pTAG1 the chromosomal copy of lpxK is inactivated by insertion of a kanamycin resistance cassette into the center of the lpxKgene. This insertion mutation is complemented by a plasmid carryinglpxK + and a temperature-sensitive origin of replication. Thus, the lpxK knock-out genotype can be induced by growth at 44 °C. At this temperature the hybrid plasmid carrying lpxK + is no longer maintained in the cells, and both the lpxK gene and its product are gradually lost. Under these conditions, the phenotype of cells with no lipid A 4′-kinase can be examined. We now show that the 4′-kinase (LpxK) is indeed required for growth, and upon its depletion, the expected precursor, DS-1-P, accumulates in cells. 32Pi was obtained from NEN Life Science Products; 0.25-mm glass-backed Silica Gel 60 thin layer chromatography plates and high performance thin layer chromatography (HPTLC) plates were from E. Merck; yeast extract and tryptone were from Difco; restriction enzymes and Klenow DNA polymerase (large fragment) were from New England Biolabs; T4 DNA ligase was from Life Technologies, Inc.; shrimp alkaline phosphatase was from U. S. Biochemical Corp.; DEAE-cellulose (DE52) was from Whatman; and octadecylsilane (C18) silica was from Baker. All solvents were reagent grade from Malinckrodt. CDCl3 and CD3OD were purchased from Aldrich. Table I lists the E. coli K-12 strains used in this study. Cells were cultured at 30, 37, or 44 °C in Luria Broth (LB) consisting of 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per liter (14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor, Cold Spring Harbor, NY1989: 1.74-1.84Google Scholar). Antibiotics were added as necessary at 50 μg/ml for ampicillin, 12 μg/ml for tetracycline, 30 μg/ml for chloramphenicol, and 30 μg/ml for kanamycin. Mini preparations of plasmid DNA were made using the Qiaprep Spin Miniprep Kit (Qiagen). Large scale preparations of plasmid DNA were made using the Bigger Prep kit from 5 Prime → 3 Prime, Inc., Boulder, CO. DNA fragments were isolated from agarose gels using the Qiagen Qiaex II gel extraction kit. Restriction enzymes, T4 DNA ligase, and Klenow DNA polymerase were used according to the manufacturers' instructions. Transformation of E. coli with plasmid DNA was done using salt-competent cells (14Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor, Cold Spring Harbor, NY1989: 1.74-1.84Google Scholar).Table IPlasmids and E. coli strains used in this studyPlasmid or strainRelevant genotypeSourcepJK2lpxK in pET3a8pMAK705cmr, temperature-sensitive replicon15pKEM 14–5valAB construct in pTZ18U, ampr17pUC4KkanrAmersham Pharmacia BiotechpNGH1Low copy, lac promoter, cmr16pACYC177Low copy, amprNew England BiolabspNGH1-amppNGH1 promoter region with pACYC origin and ampr cassetteThis workpTAG1pMAK705 withXbaI/BamHI fragment from pJK2, cmrThis workpTAG2pTAG1 with PstI excised kan cassette from pUC4K in NsiI site, cmr, kanrThis workpTAG6pNGH1-amp withXbaI/BamHI fragment from pJK2, amprThis workpTAG8valB only,NdeI/HindIII fragment from pKEM14–5 in pACYC177, amprThis workMC1061Wild type, rec A +18XLI-BluerecA1,endA1, lacStratageneBLR(DE3)pLysSΔ(srl-recA) 306::Tn10NovagenTG1/pTAG1lpxK::kan,recA::Tn10, pTAG1 (cmr)This workTG1/pTAG6lpxK::kan,recA::Tn10, pTAG6 (ampr)This workTG1/pTAG8lpxK::kan,recA::Tn10, pTAG8 (ampr)This work Open table in a new tab Table I lists all of the plasmids used in this study. pTAG1 contains the lpxK gene cloned into pMAK705, a vector with a temperature-sensitive origin of replication (15Hamilton C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar). pJK2 (8Garrett T.A. Kadrmas J.L. Raetz C.R.H. J. Biol. Chem. 1997; 272: 21855-21864Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) and pMAK705 were digested with XbaI andBamHI. The 1-kb lpxK gene from pJK2 and the 6-kb linearized pMAK705 were gel-purified from a 1% agarose gel. ThelpxK gene was ligated into pMAK705. A portion of the ligation mixture was transformed into competent E. coliXL1-Blue (Stratagene), and colonies resistant to chloramphenicol were selected. Plasmid DNA was isolated from chloramphenicol-resistant clones and digested with XbaI and BamHI to identify those constructs with the desired insert. This plasmid is called pTAG1. This plasmid was tested for its ability to promote LpxK expression. A plasmid analogous to pTAG1 was constructed with a kanamycin cassette inserted into the NsiI site of lpxK gene. pJK2 was digested with NsiI, and pUC-4K (Amersham Pharmacia Biotech) was digested with PstI. The 5.5-kb linearized pJK2 and the 1.2-kb kanamycin cassette from pUC-4K were gel-purified and ligated together. A portion of the ligation was transformed intoE. coli XL1-Blue, and colonies resistant to ampicillin were selected. Plasmids were isolated from ampicillin-resistant colonies and digested with NdeI and BamHI to verify the presence of the correct 2.2-kb insert. ThelpxK::kan construct described above was digested with XbaI and BamHI and cloned into pMAK705 exactly as for pTAG1, yielding pTAG2. pNGH1-amp was constructed from pNGH1 (16Odegaard T.J. Kaltashov I.A. Cotter R.J. Steeghs L. van der Ley P. Khan S. Maskell D.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 19688-19696Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). pNGH1 was digested withBamHI and SalI yielding 3.9- and 1.6-kb fragments. pACYC177 was digested with BamHI andXhoI yielding 2.5- and 1.4-kb fragments. The 2.5-kb pACYC177 fragment which contains the β-lactamase gene and 1.6-kb pNGH1 fragment which contains the trc promoter were ligated together to form pNGH1-amp. To construct pTAG6, pJK2 was digested with NdeI, and the 5′-overhang was filled in with Klenow DNA polymerase according to manufacturer's directions (New England Biolabs). The lpxKgene was excised by further digestion with BamHI, yielding a 985-base pair fragment with one blunt end and one BamHI sticky end. pNGH1-amp was digested with SmaI andBamHI. The lpxK fragment was ligated into the digested pNGH1-amp. A portion of the ligation was transformed into XL1-Blue competent cells, and colonies resistant to ampicillin were selected. Plasmid DNA was isolated from ampicillin-resistant clones and was digested with BamHI and SacI to verify the presence of the correct insert. One such plasmid was called pTAG6. pTAG8 is the Francisella novicida 4′-kinase gene,valB, cloned into pACYC177. pKEM14-5 (17Mdluli K.E. Anthony L.S. Baron G.S. McDonald M.K. Myltseva S.V. Nano F.E. Microbiology. 1994; 140: 3309-3318Crossref PubMed Scopus (31) Google Scholar) was digested withNdeI, and the 5′-overhang was filled in with Klenow DNA Polymerase (New England Biolabs). A fragment containing valBand a portion of the downstream polA gene was excised by further digestion with HindIII, yielding a ∼1800-base pair fragment with one blunt end and one HindIII sticky end. pACYC177 was digested with SmaI and HindIII. ThevalB fragment was ligated into the digested pACYC177. A portion of the ligation was transformed into XL1-Blue competent cells, and colonies resistant to ampicillin were selected. Plasmid DNA was isolated from ampicillin-resistant clones and was digested withEcoRV and NdeI to verify the presence of the correct insert. One such plasmid was called pTAG8. TG1/pTAG1 was constructed following the method of Hamilton et al. (15Hamilton C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar) (Fig.2). Competent MC1061 cells (18Crowell D.N. Anderson M.S. Raetz C.R.H. J. Bacteriol. 1986; 168: 152-159Crossref PubMed Google Scholar) were transformed with pTAG2 and grown at 30 °C to anA 600 of 0.6. Next, 1 × 105cells were plated on prewarmed LB plates containing 30 μg/ml chloramphenicol and incubated at 44 °C. This selects for cells in which pTAG2 has integrated into the genome. A single colony was used to inoculate 1 ml of LB containing chloramphenicol and grown at 30 °C to stationary phase. A portion of the culture was diluted 1:1000 into fresh LB containing chloramphenicol and again grown at 30 °C to stationary phase. The above outgrowth was repeated once more. During this outgrowth, the integrated plasmid will occasionally excise carrying either the wild type lpxK or thelpxK::kan allele (Fig. 2) (15Hamilton C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar). The cells were plated on LB containing chloramphenicol at 30 °C. Cells in which the plasmid had excised were identified by their inability to grow at 44 °C in the presence of chloramphenicol. Plasmids were then isolated from 14 such temperature-sensitive strains and digested withXbaI and BamHI. Of the 14 colonies, 11 contained the pTAG2 insert. Three, however, had the pTAG1 insert, indicating that the lpxK::kan allele had replaced the wild typelpxK gene on the chromosome (15Hamilton C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar). One of these strains was made recA − by P1 transduction using BLR(DE3)pLysS (Novagen) as the donor. The presence of the recA− phenotype was verified by the strain's sensitivity to UV light (19Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1989: 1.4.7Google Scholar). This strain is designated TG1/pTAG1. Strain TG1/pTAG6 was made by transforming TG1/pTAG1 with pTAG6 and selecting for transformants at 30 °C on LB plates containing ampicillin. Single colonies were then streaked to LB plates containing ampicillin to 44 °C. pTAG1 is then lost, and pTAG6 (which is not temperature-sensitive for replication) provides wild type lpxK. TG1/pTAG8 was constructed in a similar manner. Cell-free extracts for determination of 4′-kinase activity in cells grown at 44 °C were prepared as follows. Single colonies of TG1/pTAG1 and TG1/pTAG6 were inoculated into 5 ml of LB medium containing the appropriate antibiotics and grown at 30 °C overnight. Portions of the overnight cultures were diluted into 200 ml of LB broth (containing no antibiotics) to A 600of 0.01, and the growth temperature was shifted to 44 °C. When the cultures reached A 600 of 0.2–0.3, portions were diluted 10-fold into fresh pre-warmed medium, and the growth cycles were continued. The cells from the remaining cultures (about 180 ml) were collected by centrifugation at 10,000 × g for 15 min at 4 °C. The cell pellets were washed with 200 ml of 50 mm HEPES, pH 7.4; the centrifugation was repeated, and the final cell pellets were resuspended in 1 ml of the same buffer. Meanwhile, the growth cycles of the diluted cultures were continued as above, diluting further as necessary to maintain logarithmic growth. The volumes of the cultures were adjusted to provide 180–200 ml of cell culture at A 600 of about 0.3 for preparation of cell-free extracts at the indicated times after the shift to 44 °C. To prepare cell-free extracts, cells were broken in a French pressure cell at 20,000 p.s.i., and unbroken cells were removed by centrifugation at 3500 × g. The protein concentrations were determined using the Bio-Rad protein assay kit with bovine serum albumin as a standard. 4′-Kinase activity was assayed as described previously (8Garrett T.A. Kadrmas J.L. Raetz C.R.H. J. Biol. Chem. 1997; 272: 21855-21864Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Typically, 100 μm DS-1-32P (1000 cpm/nmol), 1 mg/ml cardiolipin, 50 mm Tris chloride, pH 8.5, 5 mm ATP, 1% Nonidet P-40, and 5 mmMgCl2 were mixed with 2 to 0.05 mg/ml protein fractions and incubated at 30 °C for various times. Reactions were stopped by spotting a portion onto a Silica Gel 60 thin layer chromatography plate. Plates were developed in chloroform/methanol/water/acetic acid (25:15:4:2, v:v), dried, and exposed to a Molecular Dynamics PhosphorImager screen. Conversion of DS-1-32P to 1-32P-lipid IVA was quantified using ImageQuant software (Molecular Dynamics). DS-1-32P and carrier DS-1-P were prepared as described previously (20Radika K. Raetz C.R.H. J. Biol. Chem. 1988; 263: 14859-14867Abstract Full Text PDF PubMed Google Scholar). Single colonies of TG1/pTAG1 and TG1/pTAG6 were inoculated into separate 3-ml cultures of LB medium containing the appropriate antibiotics and grown overnight at 30 °C. Each overnight culture was then diluted into two 25-ml portions of fresh LB medium (containing no antibiotics) to anA 600 of 0.01. One was grown at 30 °C and the other at 44 °C. The cells were labeled with32Pi for about two doubling times.32Pi (5 μCi/ml) was added to the cultures grown at 30 °C when the A 600 reached 0.15 and grown to an A 600 of 0.5. The cultures grown at 44 °C were diluted 10-fold into fresh prewarmed medium whenever theA 600 reached ∼0.2. When the cumulative growth yield was 13.4 for TG1/pTAG1 (the point at which the growth of TG1/pTAG1 begins to slow down) and 27.5 for TG1/pTAG6, both cultures were labeled with 5 μCi/ml 32Pi. TG1/pTAG1 was labeled for 3 h, and TG1/pTAG6 was labeled for 1.5 h. Each culture was split into three tubes (∼8 ml per tube). The cells were collected by centrifugation at 3000 × g, and the pellets were frozen at −20 °C for further analysis. Using one tube of each labeled culture, the lipid A to glycerophospholipid ratio was then determined as described previously (16Odegaard T.J. Kaltashov I.A. Cotter R.J. Steeghs L. van der Ley P. Khan S. Maskell D.J. Raetz C.R.H. J. Biol. Chem. 1997; 272: 19688-19696Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 21Galloway S.M. Raetz C.R.H. J. Biol. Chem. 1990; 265: 6394-6402Abstract Full Text PDF PubMed Google Scholar) with the following modification. Samples were analyzed by thin layer chromatography in a system containing chloroform/methanol/water/ammonia (40:25:4:2, v/v). HPTLC plates were used for rapid chromatography in this solvent system because the formation of an ammonia-catalyzed deacylation product was minimized. A large batch of TG1/pTAG1 that had been shifted to 44 °C was prepared as follows. Overnight cultures of TG1/pTAG1 and TG1/pTAG6 grown at 30 °C were used to inoculate LB medium to an A 600 of 0.01. Cultures were then grown at 44 °C and were diluted 10-fold as necessary to keep the optical density below 0.3 for 10.5 h. TG1/pTAG1 cultures were diluted into successively larger volumes to a final volume of 3 liters. A TG1/pTAG6 culture was maintained at 50 ml. Cells were harvested by centrifugation at 10,000 × g for 15 min at 4 °C, washed once with PBS (1 liter for TG1/pTAG1 and 10 ml for TG1/pTAG6), and resuspended in PBS (30 ml for TG1/pTAG1 and 2 ml for TG/pTAG6). Lipid A precursor accumulation was examined in the non-labeled cells of TG1/pTAG1 and TG1/pTAG6 shifted to 44 °C for 10.5 h. TG1/pTAG1 cells (200 μl of the above 30-ml suspension) were brought to a volume of 2 ml by the addition of PBS. These TG1/pTAG1 cells and the entire 2-ml suspension of TG1/pTAG6 (as prepared above) were then extracted with a neutral single phase Bligh Dyer system (chloroform/methanol/PBS, 1:2:0.8) (22Bligh E.G. Dyer J.J. Can. J. Biochem. Physiol. 1959; 37: 911-918
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