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Loss of Outer Membrane Proteins without Inhibition of Lipid Export in an Escherichia coli YaeT Mutant

2005; Elsevier BV; Volume: 280; Issue: 30 Linguagem: Inglês

10.1074/jbc.m504796200

ISSN

1083-351X

Autores

William T. Doerrler, Christian R.H. Raetz,

Tópico(s)

RNA and protein synthesis mechanisms

Resumo

Escherichia coli yaeT encodes an essential, conserved outer membrane (OM) protein that is an ortholog of Neisseria meningitidis Omp85. Conflicting data with N. meningitidis indicate that Omp85 functions either in assembly of OM proteins or in export of OM lipids. The role of YaeT in E. coli was investigated with a new temperature-sensitive mutant harboring nine amino acid substitutions. The mutant stops growing after 60 min at 44 °C. After 30 min at 44 °C, incorporation of [35S]methionine into newly synthesized OM proteins is selectively inhibited. Synthesis and export of OM phospholipids and lipopolysaccharide are not impaired. OM protein levels are low, even at 30 °C, and the buoyant density of the OM is correspondingly lower. By Western blotting, we show that levels of the major OM protein OmpA are lower in the mutant in whole cells, membranes, and the growth medium. SecA functions as a multicopy suppressor of the temperature-sensitive phenotype and partially restores OM proteins. Our data are consistent with a critical role for YaeT in OM protein assembly in E. coli. Escherichia coli yaeT encodes an essential, conserved outer membrane (OM) protein that is an ortholog of Neisseria meningitidis Omp85. Conflicting data with N. meningitidis indicate that Omp85 functions either in assembly of OM proteins or in export of OM lipids. The role of YaeT in E. coli was investigated with a new temperature-sensitive mutant harboring nine amino acid substitutions. The mutant stops growing after 60 min at 44 °C. After 30 min at 44 °C, incorporation of [35S]methionine into newly synthesized OM proteins is selectively inhibited. Synthesis and export of OM phospholipids and lipopolysaccharide are not impaired. OM protein levels are low, even at 30 °C, and the buoyant density of the OM is correspondingly lower. By Western blotting, we show that levels of the major OM protein OmpA are lower in the mutant in whole cells, membranes, and the growth medium. SecA functions as a multicopy suppressor of the temperature-sensitive phenotype and partially restores OM proteins. Our data are consistent with a critical role for YaeT in OM protein assembly in E. coli. The envelope of Gram-negative bacteria is composed of an inner membrane (IM) 1The abbreviations used are: IM inner membrane; OM, outer membrane; ts, temperature-sensitive; LPS, lipopolysaccharide. (1Cronan Jr., J.E. Gennis R.B. Maloy S.R. Neidhardt F.C. Escherichia coli and Salmonella typhimurium. 1. American Society for Microbiology, Washington, D. C.1987: 31-55Google Scholar, 2Cronan J.E. Annu. Rev. Microbiol. 2003; 57: 203-224Crossref PubMed Scopus (265) Google Scholar) and an outer membrane (OM) (3Nikaido H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 29-47Google Scholar, 4Nikaido H. Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2863) Google Scholar), separated by the periplasmic space and peptidoglycan (5Park J.T. Neidhardt F. Escherichia coli and Salmonella typhimurium. 1. American Society for Microbiology, Washington, D. C.1987: 663-671Google Scholar, 6Lazar K. Walker S. Curr. Opin. Chem. Biol. 2002; 6: 786-793Crossref PubMed Scopus (49) Google Scholar). The OM has a lipid and protein profile distinct from that of the IM (4Nikaido H. Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2863) Google Scholar). The OM contains phospholipids on its inner surface and lipopolysaccharide (LPS) on its outside surface (3Nikaido H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 29-47Google Scholar, 4Nikaido H. Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2863) Google Scholar, 7Raetz C.R.H. Whitfield C. Annu. Rev. 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Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 1005-1014Google Scholar, 11Masuda K. Matsuyama S. Tokuda H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7390-7395Crossref PubMed Scopus (86) Google Scholar, 12Yakushi T. Masuda K. Narita S. Matsuyama S. Tokuda H. Nat. Cell Biol. 2000; 2: 212-218Crossref PubMed Scopus (206) Google Scholar). The lipids and proteins of the OM are synthesized on the cytoplasmic surface of the IM, transported across the IM and periplasm, and assembled into the OM (2Cronan J.E. Annu. Rev. Microbiol. 2003; 57: 203-224Crossref PubMed Scopus (265) Google Scholar, 3Nikaido H. Neidhardt F.C. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 29-47Google Scholar, 4Nikaido H. Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2863) Google Scholar, 7Raetz C.R.H. Whitfield C. 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Kleinschmidt J.H. J. Biol. Chem. 2003; 278: 9092-9099Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 28Rouviere P.E. Gross C.A. Genes Dev. 1996; 10: 3170-3182Crossref PubMed Scopus (251) Google Scholar, 29Bogdanov M. Dowhan W. J. Biol. Chem. 1999; 274: 36827-36830Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 30Danese P.N. Silhavy T.J. Annu. Rev. Genet. 1998; 32: 59-94Crossref PubMed Scopus (189) Google Scholar). Subsequent steps are uncertain. Some OM proteins, like FhuA, have high affinity LPS-binding sites (31Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (664) Google Scholar). OM proteins are generally not required for growth (4Nikaido H. Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2863) Google Scholar). However, in Neisseria meningitidis, Omp85 is a minor, essential OM protein (32Voulhoux R. Bos M.P. Geurtsen J. Mols M. Tommassen J. Science. 2003; 299: 262-265Crossref PubMed Scopus (584) Google Scholar, 33Genevrois S. Steeghs L. Roholl P. Letesson J.J. Van Der Ley P. EMBO J. 2003; 22: 1780-1789Crossref PubMed Scopus (96) Google Scholar). Depletion experiments have led to contradictory conclusions regarding Omp85 function. Genevrois et al. (33Genevrois S. Steeghs L. Roholl P. Letesson J.J. Van Der Ley P. EMBO J. 2003; 22: 1780-1789Crossref PubMed Scopus (96) Google Scholar) showed that transport of lipids to the OM is selectively blocked, as in MsbA mutants (22Doerrler W.T. Reedy M.C. Raetz C.R.H. J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). However, Voulhoux et al. (32Voulhoux R. Bos M.P. Geurtsen J. Mols M. Tommassen J. Science. 2003; 299: 262-265Crossref PubMed Scopus (584) Google Scholar) demonstrated that OM protein folding and oligomerization are defective. Both groups employed a construct with a chromosomal deletion of omp85 and an intact copy of the omp85 gene behind an inducible promoter on a plasmid. This strategy required a prolonged (>6 h) incubation to deplete pre-existing Omp85. Recently, Kahne and co-workers (34Wu T. Malinverni J. Ruiz N. Kim S. Silhavy T.J. Kahne D. Cell. 2005; 121: 235-245Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar) discovered that YaeT exists in a heterooligomeric OM complex with three lipoproteins of unknown function: YfgL, NlpB, and YfiO. Their findings confirm that YaeT is essential for viability in Escherichia coli and demonstrate a role for YaeT in OM biogenesis. However, lipid trafficking, global outer membrane protein composition, and buoyant density were not examined. YaeT is the E. coli ortholog of Omp85 (35Blattner F.R. Plunkett G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (6024) Google Scholar). We have now examined the function of YaeT. We confirm that yaeT is essential in E. coli, as in other bacteria (32Voulhoux R. Bos M.P. Geurtsen J. Mols M. Tommassen J. Science. 2003; 299: 262-265Crossref PubMed Scopus (584) Google Scholar, 33Genevrois S. Steeghs L. Roholl P. Letesson J.J. Van Der Ley P. EMBO J. 2003; 22: 1780-1789Crossref PubMed Scopus (96) Google Scholar, 36Reumann S. Davila-Aponte J. Keegstra K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 784-789Crossref PubMed Scopus (160) Google Scholar, 37Thomas K.L. Leduc I. Olsen B. Thomas C.E. Cameron D.W. Elkins C. Infect. Immun. 2001; 69: 4438-4446Crossref PubMed Scopus (35) Google Scholar). The yaeT gene in E. coli is situated upstream of lpxA, lpxB, and lpxD (7Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3380) Google Scholar) and downstream of uppS (38Apfel C.M. Takacs B. Fountoulakis M. Stieger M. Keck W. J. Bacteriol. 1999; 181: 483-492Crossref PubMed Google Scholar), cdsA (39Icho T. Sparrow C.P. Raetz C.R.H. J. Biol. Chem. 1985; 260: 12078-12083Abstract Full Text PDF PubMed Google Scholar), and yaeL (40Alba B.M. Gross C.A. Mol. Microbiol. 2004; 52: 613-619Crossref PubMed Scopus (282) Google Scholar) (Fig. 1A). We have constructed a yaeT deletion mutant covered by a plasmid that expresses a temperature-sensitive (ts) allele of yaeT that stops growing after 60 min at 44 °C. The levels of major OM proteins are greatly reduced, and the OM buoyant density is reduced. The level of new OM proteins is low at the permissive temperature and is almost undetectable after only 30 min at 44 °C, but lipid synthesis and export are relatively unaffected. Moderate overexpression of SecA, a component of the pre-protein translocase (41Oliver D.B. Beckwith J. Cell. 1981; 25: 765-772Abstract Full Text PDF PubMed Scopus (290) Google Scholar, 42Oliver D.B. Beckwith J. J. Bacteriol. 1982; 150: 686-691Crossref PubMed Google Scholar), largely restores OM proteins and growth to normal rates at 44 °C. Our data are consistent with a critical role for YaeT in the assembly of OM proteins in E. coli. Materials—Tryptone and yeast extract were from Difco. Radioisotopes and Enhance were purchased from PerkinElmer Life Sciences. Restriction enzymes were purchased from New England Biolabs. T4 DNA ligase, shrimp alkaline phosphatase, and custom-made primers were from Invitrogen. Pfu DNA polymerase and the GeneMorph PCR mutagenesis kit were obtained from Stratagene. Protein concentrations were determined with the BCA Protein Assay Reagent from Pierce, using bovine serum albumin as the standard (43Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18645) Google Scholar). All other chemicals were purchased from either Sigma or Mallinckrodt. Plasmid Construction—Standard recombinant DNA techniques were utilized in the construction of plasmids (44Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). W3110 chromosomal DNA was obtained using the Easy DNA kit from Invitrogen. Plasmids were isolated using QIAprep spin miniprep kit from Qiagen. The Qiaex II gel extraction kit from Qiagen was used to extract DNA from agarose gels. Restriction enzymes, T4 DNA ligase, and shrimp alkaline phosphatase were used according to the manufacturer's instructions. Competent cells were prepared according to Inoue et al. (45Inoue H. Nojima H. Okayama H. Gene (Amst.). 1990; 96: 23-28Crossref PubMed Scopus (1569) Google Scholar). Plasmids and strains are listed in Table I.Table IStrains and plasmids used in this studyStrainRelevant genotypeSource or Ref.E. coliW3110Wild-type, F-, λ-E. coli Genetic Stock Center, Yale UniversityDY330W3110 Δ lacU169 gal490 λcI857 Δ(cro-bioA)48Yu D. Ellis H.M. Lee E.C. Jenkins N.A. Copeland N.G. Court D.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5978-5983Crossref PubMed Scopus (1369) Google ScholarWD401W3110 ΔyaeT::kanThis workXL1-Blue MRΔmcrABC recA1 endA1 gyrA96 relA1 supE44 thi-1 lacStratagenePlasmidspUC4KkanrAmersham BiosciencespET23aExpression Vector; T7 lac promoter, amprNovagenpMAK705Ts replicon, cmr46Hamilton C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google ScholarpWSK29Low copy vector, lac promoter, ampr47Wang R.F. Kushner S.R. Gene (Amst.). 1991; 100: 195-199Crossref PubMed Scopus (1011) Google ScholarpACYC184Low copy vector, tetr cmrNew England BiolabspWTD25pET23a containing E. coli yaeT (NdeI/BamHI sites)This workpWTD27XbaI/BamHI fragment of pWTD25 cloned into pMAK705This workpWTD30XbaI/BamHI fragment of pWTD25 cloned into pWSK29This workpWTD30-9YaeT9 (ts) cloned into XbaI/BamHI sites of pWSK29This workpWTD31pET23a containing E. coli secA (NdeI/BamHI sites)This workpWTD32XbaI/BamHI fragment of pWTD31 cloned into pACYC184This workpWTD33XbaI/BamHI fragment of pWTD31 cloned into pWSK29This work Open table in a new tab E. coli yaeT was amplified from W3110 genomic DNA by PCR with Pfu turbo, according to the manufacturer's instructions. The forward primer was YaeTNdeI, and the reverse primer was YaeTBamHI (Table II). These primers were used at a final concentration of 2.5 ng/μl in a 100-μl PCR mixture containing 100 ng of genomic DNA and 5 units of Pfu polymerase. The reaction conditions were as follows: 94 °C denaturation for 1 min followed by 25 cycles of 94 °C (denaturation) for 1 min, 55 °C (annealing) for 1 min, and 72 °C (extension) for 2.5 min. This was followed by a 10-min run-off at 72 °C. The gel-purified PCR product was digested with NdeI and BamHI and then ligated into an NdeI/BamHI- and shrimp alkaline phosphatase-treated pET23a vector (Novagen), yielding pWTD25. Plasmid pWTD27 was constructed by cloning the XbaI/BamHI fragment of pWTD25 (containing a 5′ ribosomal binding site) into XbaI/BamHI- and shrimp alkaline phosphatase-treated vector, pMAK705 (46Hamilton C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar). Plasmid pWTD30 was constructed by cloning the XbaI/BamHI fragment of pWTD25 into XbaI/BamHI- and shrimp alkaline phosphatase-treated vector, pWSK29 (47Wang R.F. Kushner S.R. Gene (Amst.). 1991; 100: 195-199Crossref PubMed Scopus (1011) Google Scholar).Table IIOligonucleotide primers used in this workNameSequenceYaeTNdeI5′-GGC CAT ATG GCG ATG AAA AAG TTG CTC-3′YaeTBamHI5′-GGC GGA TCC TTA CCA GGT TTT ACC GAT G-3′SecANdeI5′-GGC ATA TGC TAA TCA AAT TGT TAA CTA AAG-3′SecABamHI5′-GGC GGA TCC TTA TTG CAG GCG GCC ATG G-3′YaeTkoA5′-GAT TTC TCT CGG TTA TGA GAG TTA GTT AGG AAG AAC GCA TAA TAA CGA TGA GCC ATA TTC AAC GGG AAA CG-3′YaeTkoB5′-CCT AAA GTC ATC GCT ACA CTA CCA CTA CAT TCC TTT GTG GAG AAC ACT TAG AAA AAC TCA TCG AGC ATC-3′T7 promoter5′-TAA TAC GAC TCA CTA TAG GG-3′pACYC184/BamHIA5′-CTA TCG ACT ACG CGA TCA TG-3′pACYC184/BamHIB5′-CGG TGA TGT CGG CGA TAT AG-3′ Open table in a new tab pWTD31 was constructed by amplifying E. coli secA from a plasmid that was isolated from an E. coli genomic library, as described below, based on its ability to restore growth to strain WD401/pWTD30-9 at 42 °C. Primers were SecANdeI and SecABamHI (Table II). pWTD32 was constructed by cloning the XbaI/BamHI fragment of pWTD31 (containing a 5′ ribosomal binding site) into XbaI/BamHI- and shrimp alkaline phosphatase-treated pACYC184. pWTD33 was constructed by cloning the XbaI/BamHI fragment of pWTD31 into XbaI/BamHI- and shrimp alkaline phosphatase-treated pWSK29. Plasmids were sequenced at the Duke University sequencing facility. When appropriate, antibiotics were added as follows: chloramphenicol, 30 μg/ml; kanamycin, 25 μg/ml; and ampicillin, 90 μg/ml. In experiments with the ts mutant, the nonpermissive temperature was defined as 42 °C for plate grown bacteria and 44 °C for liquid grown bacteria. Replacement of the yaeT Gene—Replacement of yaeT with the kan gene from pUC4K was done using λ-RED-mediated recombination (48Yu D. Ellis H.M. Lee E.C. Jenkins N.A. Copeland N.G. Court D.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5978-5983Crossref PubMed Scopus (1369) Google Scholar). The kan gene was amplified by PCR from plasmid DNA using primers YaeTkoA and YaeTkoB (Table II). The gel-purified DNA was treated with DpnI to remove contaminating methylated plasmid DNA and was used to transform E. coli strain DY330/pWTD27 by electroporation. Following a 2-h outgrowth in LB broth (49Miller J.R. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar) at 30 °C, the cells were plated on LB agar plates containing kanamycin and incubated overnight at 30 °C. A P1vir lysate made from DY330 (yaeT::kan)/pWTD27 was used to transduce W3110/pWTD27 to KmR (50Silhavy T.J. Berman M.L. Enquist L.W. Experiments with Gene Fusions. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1984Google Scholar). The resulting strain was designated WD401/pWTD27. Isolation of ts yaeT Mutation by Error-prone PCR—PCR mutagenesis was carried out using the GeneMorph kit from Stratagene. The target DNA was pWTD25, which was used at a final concentration of 300 or 30 pg/μl. Primers were YaeTBamHI and T7 promoter (Table II). A 50-μl reaction mixture contained the Mutazyme reaction buffer (1×), 200 μm dNTPs, 125 ng of primers, and 2.5 units of Mutazyme DNA polymerase. The reaction conditions were as follows: a 94 °C denaturation step for 1 min was followed by 30 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 2.5 min. This was followed by a 10-min run-off at 72 °C. The DNAs from both high and low target concentration reactions were pooled and gel-purified. The DNA was then digested with BamHI/XbaI and used in a ligation reaction containing similarly digested vector pWSK29. Following ligation, E. coli W3110 cells were transformed with the plasmid DNA containing mutagenized yaeT. Following an overnight incubation at 37 °C, ∼1000 AmpR colonies were scraped and pooled into 5 ml of LB broth. These cells were diluted into 50 ml of LB broth containing ampicillin, grown overnight, and used as the recipient cells for a P1vir transduction using a lysate prepared from WD401/pWTD27. KmR colonies (which were also AmpR because of the presence of the vector pWSK29) were screened by streaking on LB/ampicillin plates and incubating overnight at 30 and 42 °C. A representative construct with the desired ts phenotype was designated WD401/pWTD30-9. E. coli Genomic DNA Library Construction—Genomic DNA from E. coli W3110 (50 μg) was partially digested with 2 units of Sau3A1 for 15 min at 37 °C in 0.2 ml of Sau3A1 buffer. Then 6 μlof0.5 m EDTA, pH 8.0, was added to stop the reaction, and the mixture was incubated at 65 °C for 20 min to inactivate the enzyme. The DNA was resolved on a 1% agarose gel, and fragments corresponding to 2–6.5 kb were recovered. Plasmid pACYC184 was opened with BamHI, gel-purified, and treated with shrimp alkaline phosphatase. For the ligation reaction, 200 ng of partially digested genomic DNA was incubated at 16 °C overnight with 200 ng of treated plasmid and 2 units of T4 DNA ligase in a total volume of 25 μl of ligase buffer. E. coli XL1-Blue cells were transformed with the ligation mixture, and CmR transformants were selected. Ligation reactions containing both the vector and insert yielded ∼10-fold more colonies than a control ligation reaction containing vector alone. Following overnight incubation at 37 °C, ∼20,000 CmR colonies were recovered. These were pooled with a cell scraper and transferred to 250 ml of SOC medium (44Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar) containing chloramphenicol. Cells were grown to stationary phase. Plasmid DNA was isolated, divided into aliquots, and stored at –80 °C. Multicopy Suppressors of the ts Growth of WD401/pWTD30-9 —Competent WD401/pWTD30-9 cells were transformed with 100 ng of the pACYC184-based E. coli genomic DNA library, described above. Following a 2-h outgrowth at 30 °C in the absence of antibiotic, the cells were plated on LB broth containing chloramphenicol. A few plates were incubated at 30 °C to assess transformation efficiency, and the rest were incubated at 42 °C. Colonies appeared at 42 °C at a frequency of ∼0.1% versus 30 °C. Plasmid DNA was isolated and used to transform E. coli XL1-Blue cells to CmR. Plasmid DNA was then isolated from these recipient cells, and the ends of the inserts were sequenced with primers designed to flank the BamHI site of pACYC184 (pACYC184/BamHIA and pACYC184/BamHIB; Table II). Labeling, Membrane Preparation, and Sucrose Gradients—Cells were grown in 25 ml of LB broth to A600 = 1.0 at 30 °C and then diluted 4-fold into fresh, pre-warmed LB broth at 44 °C. After 30 min of growth at 44 °C, cells were labeled with 4 μCi/ml 32Pi (9000 Ci/mmol) or 10 μCi/ml [35S]methionine/cysteine (1175 Ci/mmol) for 10 min. Cells were cooled rapidly on ice and harvested by centrifugation. Spheroplasts were prepared at 0 °C by lysozyme/EDTA treatment and broken by mild sonic irradiation (22Doerrler W.T. Reedy M.C. Raetz C.R.H. J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 51Osborn M.J. Munson R. Methods Enzymol. 1974; 31: 642-653Crossref PubMed Scopus (227) Google Scholar). The cell lysate was cleared by centrifugation at 4000 × g at 4 °C for 20 min. Membranes were prepared by two sequential ultracentrifugation steps at 100,000 × g for 60 min with a wash of the first membrane pellet in 10 ml of 10 mm Tris acetate, pH 7.8, 25% (w/v) sucrose, as described previously (22Doerrler W.T. Reedy M.C. Raetz C.R.H. J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 51Osborn M.J. Munson R. Methods Enzymol. 1974; 31: 642-653Crossref PubMed Scopus (227) Google Scholar). Membranes were separated at 4 °C on a 30–60% (w/w) isopycnic sucrose gradient prepared in 10 mm Tris acetate, pH 7.8, and 0.5 mm EDTA, which was centrifuged at 155,000 × g for 18 h in a Beckman SW41 rotor (22Doerrler W.T. Reedy M.C. Raetz C.R.H. J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 51Osborn M.J. Munson R. Methods Enzymol. 1974; 31: 642-653Crossref PubMed Scopus (227) Google Scholar). OM phospholipase A, IM NADH oxidase, total protein, and radioactivity were measured for each 0.5-ml fraction (21Zhou 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, 22Doerrler W.T. Reedy M.C. Raetz C.R.H. J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Thin Layer Chromatography of 32Pi-Labeled Lipids Extracted from IMs and OMs—Portions of fractions 3–18 from each gradient were hydrolyzed at 100 °C in 12.5 mm sodium acetate, pH 4.5, 1% SDS to release lipid A from LPS. Lipid A and phospholipids were extracted into organic solvent and spotted onto Silica Gel 60 thin layer plates (Merck). Plates were developed with chloroform, pyridine, 88% formic acid, H2O (50:50:16:5, v/v) and analyzed with a PhosphorImager (21Zhou 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, 22Doerrler W.T. Reedy M.C. Raetz C.R.H. J. Biol. Chem. 2001; 276: 11461-11464Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). SDS-PAGE of 35S-Labeled Proteins from IMs and OMs—Protein samples were denatured at 40 °C for 30 min using sample buffer containing 50 mm Tris-Cl, pH 6.8, 3% SDS, 50 mm dithiothreitol, 12.5% glycerol, and 0.02% bromphenol blue. These conditions do not dissociate porin trimers (52Kameyama K. Nakae T. Takagi T. Biochim. Biophys. Acta. 1982; 706: 19-26Crossref PubMed Scopus (40) Google Scholar). Samples were resolved with 10% SDS-PAGE, followed by fixation in 40% methanol, 10% acetic acid. The gel was soaked in Enhance (PerkinElmer Life Sciences) for 1 h, water for 30 min, dried, and exposed to a PhosphorImager screen. Pre-stained, low range molecular weight standards from Bio-Rad were used as markers. Western Blotting—Western blot analysis was performed according to the method of Sorensen et al. (53Sorensen P.G. Lutkenhaus J. Young K. Eveland S.S. Anderson M.S. Raetz C.R.H. J. Biol. Chem. 1996; 271: 25898-25905Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Anti-pro-OmpA and anti-SecA antibodies were generously provided by Dr. William Wickner (Dartmouth University) and Dr. Timothy Yahr (University of Iowa) and were used at titers of 1:20,000 and 1:10,000, respectively. Horseradish peroxidase-conjugated anti-rabbit IgG from Pierce was used at a titer of 1:200,000. The ECL Plus kit from Amersham Biosciences was used for detection. YaeT Is Essential in E. coli—To study YaeT function in E. coli, we attempted to disrupt the chromosomal copy of yaeT by replacing it with a kanamycin resistance cassette (kan). Experiments without a covering plasmid were unsuccessful. Therefore, we amplified yaeT from E. coli W3110 genomic DNA (Tables I and II) and cloned it into the vector pMAK705, which carries a ts origin of replication (46Hamilton C.M. Aldea M. Washburn B.K. Babitzke P. Kushner S.R. J. Bacteriol. 1989; 171: 4617-4622Crossref PubMed Google Scholar). The resulting hybrid plasmid, pWTD27, was transformed into E. coli DY330 (Table I), and the chromosomal copy of yaeT was then replaced with the kan cassette by homologous recombination at 30 °C with linear DNA (48Yu D. Ellis H.M. Lee E.C. Jenkins N.A. Copeland N.G. Court D.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5978-5983Crossref PubMed

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