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

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.

[1]  Daniel Kahne,et al.  Identification of a Multicomponent Complex Required for Outer Membrane Biogenesis in Escherichia coli , 2005, Cell.

[2]  T. Silhavy,et al.  Chemical Conditionality A GeneticStrategy to Probe Organelle Assembly , 2005, Cell.

[3]  J. Tommassen,et al.  Identification of an outer membrane protein required for the transport of lipopolysaccharide to the bacterial cell surface. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[4]  H. Nikaido.  Molecular Basis of Bacterial Outer Membrane Permeability Revisited , 2003, Microbiology and Molecular Biology Reviews.

[5]  J. Cronan.  Bacterial membrane lipids: where do we stand? , 2003, Annual review of microbiology.

[6]  William C. Wimley,et al.  The versatile β-barrel membrane protein , 2003 .

[7]  Geoffrey Chang,et al.  Structure of MsbA from Vibrio cholera: a multidrug resistance ABC transporter homolog in a closed conformation. , 2003, Journal of molecular biology.

[8]  P. Roholl,et al.  The Omp85 protein of Neisseria meningitidis is required for lipid export to the outer membrane , 2003, The EMBO journal.

[9]  Yong Chen,et al.  Ring-like pore structures of SecA: Implication for bacterial protein-conducting channels , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[10]  O. Holst,et al.  Folding and Insertion of the Outer Membrane Protein OmpA Is Assisted by the Chaperone Skp and by Lipopolysaccharide* , 2003, The Journal of Biological Chemistry.

[11]  J. Sacchettini,et al.  Crystal structure of Mycobacterium tuberculosis SecA, a preprotein translocating ATPase , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[12]  J. Tommassen,et al.  Role of a Highly Conserved Bacterial Protein in Outer Membrane Protein Assembly , 2003, Science.

[13]  S. Walker,et al.  Substrate analogues to study cell-wall biosynthesis and its inhibition. , 2002, Current opinion in chemical biology.

[14]  C. Raetz,et al.  ATPase Activity of the MsbA Lipid Flippase of Escherichia coli * , 2002, The Journal of Biological Chemistry.

[15]  J. Deisenhofer,et al.  Nucleotide Control of Interdomain Interactions in the Conformational Reaction Cycle of SecA , 2002, Science.

[16]  T. Silhavy,et al.  Imp/OstA is required for cell envelope biogenesis in Escherichia coli , 2002, Molecular microbiology.

[17]  S. Matsuyama,et al.  Elucidation of the function of lipoprotein-sorting signals that determine membrane localization , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[18]  R. Woodbury,et al.  Complex behavior in solution of homodimeric SecA , 2002, Protein science : a publication of the Protein Society.

[19]  Koreaki Ito,et al.  The Sec protein-translocation pathway. , 2001, Trends in microbiology.

[20]  C. Elkins,et al.  Cloning, Overexpression, Purification, and Immunobiology of an 85-Kilodalton Outer Membrane Protein fromHaemophilus ducreyi , 2001, Infection and Immunity.

[21]  W. Wickner,et al.  Functional reconstitution of bacterial Tat translocation in vitro , 2001, The EMBO journal.

[22]  A. Bolhuis,et al.  Protein targeting by the twin-arginine translocation pathway , 2001, Nature Reviews Molecular Cell Biology.

[23]  M. Reedy,et al.  An Escherichia coli Mutant Defective in Lipid Export* , 2001, The Journal of Biological Chemistry.

[24]  Samuel I. Miller,et al.  A PhoP/PhoQ-induced Lipase (PagL) That Catalyzes 3-O-Deacylation of Lipid A Precursors in Membranes ofSalmonella typhimurium * , 2001, The Journal of Biological Chemistry.

[25]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[26]  Samuel I. Miller,et al.  Transfer of palmitate from phospholipids to lipid A in outer membranes of Gram‐negative bacteria , 2000, The EMBO journal.

[27]  C. Andersen,et al.  Chunnel vision , 2000, EMBO reports.

[28]  Colin Hughes,et al.  Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export , 2000, Nature.

[29]  D. Court,et al.  An efficient recombination system for chromosome engineering in Escherichia coli. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[30]  T. Yakushi,et al.  A new ABC transporter mediating the detachment of lipid-modified proteins from membranes , 2000, Nature Cell Biology.

[31]  M. Bogdanov,et al.  Lipid-assisted Protein Folding* , 1999, The Journal of Biological Chemistry.

[32]  A. Oudenaarden,et al.  Brownian ratchets: molecular separations in lipid bilayers supported on patterned arrays. , 1999, Science.

[33]  K. Keegstra,et al.  The evolutionary origin of the protein-translocating channel of chloroplastic envelope membranes: identification of a cyanobacterial homolog. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[34]  M. Fountoulakis,et al.  Use of Genomics To Identify Bacterial Undecaprenyl Pyrophosphate Synthetase: Cloning, Expression, and Characterization of the Essential uppS Gene , 1999, Journal of bacteriology.

[35]  K. Diederichs,et al.  Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide. , 1998, Science.

[36]  T. Silhavy,et al.  Targeting and assembly of periplasmic and outer-membrane proteins in Escherichia coli. , 1998, Annual review of genetics.

[37]  W. Wickner,et al.  The SecA Subunit of Escherichia coliPreprotein Translocase Is Exposed to the Periplasm , 1998, Journal of bacteriology.

[38]  C. Georgopoulos,et al.  Function of Escherichia coli MsbA, an Essential ABC Family Transporter, in Lipid A and Phospholipid Biosynthesis* , 1998, The Journal of Biological Chemistry.

[39]  B. Zomer,et al.  Meningitis bacterium is viable without endotoxin , 1998, Nature.

[40]  P. Tai,et al.  Differential translocation of protein precursors across SecY-deficient membranes of Escherichia coli: SecY is not obligatorily required for translocation of certain secretory proteins in vitro , 1997, Journal of bacteriology.

[41]  Jon Beckwith,et al.  Protein Translocation in the Three Domains of Life: Variations on a Theme , 1997, Cell.

[42]  W. Wickner,et al.  Biogenesis of the Gram-Negative Bacterial Envelope , 1997, Cell.

[43]  V. Ramamurthy,et al.  Topology of the Integral Membrane Form of Escherichia coli SecA Protein Reveals Multiple Periplasmically Exposed Regions and Modulation by ATP Binding* , 1997, The Journal of Biological Chemistry.

[44]  N. W. Davis,et al.  The complete genome sequence of Escherichia coli K-12. , 1997, Science.

[45]  O. Holst,et al.  Deletion of the heptosyltransferase genes rfaC and rfaF in Escherichia coli K-12 results in an Re-type lipopolysaccharide with a high degree of 2-aminoethanol phosphate substitution. , 1997, European journal of biochemistry.

[46]  P. Tai,et al.  SecE-depleted Membranes of Escherichia coli Are Active , 1997, The Journal of Biological Chemistry.

[47]  C. Gross,et al.  SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, participates in the assembly of outer membrane porins. , 1996, Genes & development.

[48]  W. Wickner,et al.  Escherichia coli Preprotein Translocase* , 1996, The Journal of Biological Chemistry.

[49]  J. Lutkenhaus,et al.  Regulation of UDP-3-O-[R-3-hydroxymyristoyl]-N-acetylglucosamine Deacetylase in Escherichia coli , 1996, The Journal of Biological Chemistry.

[50]  H. Nakajima,et al.  Cloning of organic solvent tolerance gene ostA that determines n-hexane tolerance level in Escherichia coli , 1994, Applied and environmental microbiology.

[51]  R. Misra.  OmpF assembly mutants of Escherichia coli K-12: isolation, characterization, and suppressor analysis , 1993, Journal of bacteriology.

[52]  R. F. Wang,et al.  Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. , 1991, Gene.

[53]  C. Raetz,et al.  Biosynthesis and function of phospholipids in Escherichia coli. , 1990, The Journal of biological chemistry.

[54]  B. Washburn,et al.  New method for generating deletions and gene replacements in Escherichia coli , 1989, Journal of bacteriology.

[55]  K. Ito,et al.  Temperature-sensitive sec mutants of Escherichia coli: inhibition of protein export at the permissive temperature , 1989, Journal of bacteriology.

[56]  T. Icho,et al.  Molecular cloning and sequencing of the gene for CDP-diglyceride synthetase of Escherichia coli. , 1985, The Journal of biological chemistry.

[57]  L. Enquist,et al.  Experiments With Gene Fusions , 1984 .

[58]  T. Takagi,et al.  Estimation of molecular weights of membrane proteins in the presence of SDS by low-angle laser light scattering combined with high-performance porous silica gel chromatography. Confirmation of the trimer structure of porin of the E. coli outer membrane. , 1982, Biochimica et biophysica acta.

[59]  J. Beckwith,et al.  Identification of a new gene (secA) and gene product involved in the secretion of envelope proteins in Escherichia coli , 1982, Journal of bacteriology.

[60]  J. Beckwith,et al.  E. coli mutant pleiotropically defective in the export of secreted proteins , 1981, Cell.

[61]  G. Ames,et al.  Protein Composition of the Outer Membrane of Salmonella typhimurium: Effect of Lipopolysaccharide Mutations , 1974, Journal of bacteriology.

[62]  A. Driessen,et al.  The enzymology of protein translocation across the Escherichia coli plasma membrane. , 1991, Annual review of biochemistry.

[63]  H. Nojima,et al.  High efficiency transformation of Escherichia coli with plasmids. , 1990, Gene.

[64]  K. Fridborg,et al.  Structure and function of carbonic anhydrases , 1977 .

[65]  R. Munson,et al.  [67] Separation of the inner (cytoplasmic) and outer membranes of gram-negative bacteria , 1974 .

[66]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .