Skip to main content
SpringerLink
Log in

Characterization of Escherichia coli expressing an Lpp’OmpA(46-159)-PhoA fusion protein localized in the outer membrane

  • Original Paper
  • Applied Genetics and Regulations
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The Lpp′OmpA(46-159) hybrid protein can serve as an efficient targeting vehicle for localizing a variety of procaryotic and eucaryotic soluble proteins onto the E. coli surface, thus providing a system for several possible biotechnology applications. Here we show that fusions between Lpp′OmpA(46-159) and bacterial alkaline phosphatase (PhoA), a normally periplasmic dimeric enzyme, are also targeted to the outer membrane. However, protease accessibility experiments and immunoelectron microscopy revealed that, unlike other periplasmic proteins, the PhoA domain of these fusions is not exposed on the cell surface in cells having an intact outer membrane. Conditions that affect the formation of disulfide bonds and the folding of the PhoA domain in the periplasm not only did not facilitate targeting to the cell surface but led to lethality when the fusion was expressed from a high-copy-number plasmid. Furthermore, E. coli expressing the Lpp′OmpA(46-159)-PhoA fusion exhibited strain- and temperature-dependent alterations in outer-membrane permeability. Our results are consistent with previous studies with other vehicles indicating that PhoA is not displayed on the surface when fused to cell-surface expression vectors. Presumably, the enzyme rapidly assumes a tightly folded dimeric conformation that cannot be transported across the outer membrane. The large size and quaternary structure of PhoA may define a limitation of the Lpp′OmpA(46-159) fusion system for the display of periplasmic proteins on the cell surface. Alkaline phosphatase is a unique protein among a group of five periplasmic proteins (β-lactamase, alkaline phosphatase, Cex cellulase, Cex cellulose-binding domain, and a single-chain Fv antibody fragment), which have been tested as passengers for the Lpp′OmpA(46-159) expression system to date, since it was the only protein not displayed on the surface.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Baneyx F, Georgiou G (1990) In vivo degradation of secreted fusion proteins by the Escherichia coli outer membrane protease OmpT. J Bacteriol 172: 491–494

    CAS  Google Scholar 

  • Bardwell JCA, McGovern K, Beckwith J (1991) Identification of a protein required for disulfide bond formation in vivo. Cell 67: 581–589

    Article  CAS  Google Scholar 

  • Boyd D, Manoil C, Beckwith J (1987) Determinants of membrane protein topology. Proc Natl Acad Sci USA 84: 8525–8529

    Article  CAS  Google Scholar 

  • Brent R, Ptashne M (1981) Mechanism of action of the lex A gene product. Proc Natl Acad Sci USA 78: 4204–4208

    Article  CAS  Google Scholar 

  • Charbit A, Molla A, Saurin W, Hofnung M (1988) Versatility of a vector for expressing foreign polypeptides at the surface of gram-negative bacteria. Gene 70: 181–189

    Article  CAS  Google Scholar 

  • Coulton JW, Reid GK, Campana A. (1988) Export of hybrid proteins FhuA-LacZ and FhuA-PhoA to the cell envelope of Escherichia coli K-12. J. Bacteriol. 170: 2267–2275

    CAS  Google Scholar 

  • Ehrmann M, Boyd D, Beckwith J (1990) Genetic analysis of membrane protein topology by a sandwich gene fusion approach. Proc Natl Acad Sci USA 87: 7574–7578

    Article  CAS  Google Scholar 

  • Enfert Cd’, Pugsley AP (1987) A gene fusion approach to the study of pullulanase export and secretion in Escherichia coli. Mol Microbiol 1: 159–168

    Article  Google Scholar 

  • Filip C, Fletcher G, Wulff JL, Earhart CF (1973) Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosine. J Bacteriol 115: 717–722

    CAS  Google Scholar 

  • Francisco JA, Earhart CF, Georgiou G (1992) Transport and anchoring of β-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci USA 89: 2713–2717

    Article  CAS  Google Scholar 

  • Francisco JA, Campbell R, Iverson BL, Georgiou G (1993a) Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. Proc Natl Acad Sci USA 90: 10444–10448

    Article  CAS  Google Scholar 

  • Francisco JA, Stathopoulos C, Warren RAJ, Kilburn DG, Georgiou G (1993b) Specific adhesion and hydrolysis of cellulose by intact Escherichia coli expressing surface anchored cellulase or cellulose binding domains. Biotechnology 11: 491–495

    Article  CAS  Google Scholar 

  • Freudl R, Braun G, Hindennach I, Henning U (1985) Lethal mutations in the structural gene of an outer membrane protein (OmpA) of Escherichia coli K-12. Mol Gen Genet 201: 76–81

    Article  CAS  Google Scholar 

  • Freudl R, MacIntyre S, Degen M, Henning U (1986) Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12. J Mol Biol 188: 491–494

    Article  CAS  Google Scholar 

  • Gennity JM, Kim H, Inouye M (1992) Structural determinants in addition to the amino-terminal sorting sequence influence membrane localization of Escherichia coli lipoproteins. J Bacteriol 174: 2095–2101

    CAS  Google Scholar 

  • Georgiou G, Shuler ML, Wilson DB (1988) Release of periplasmic enzymes and other physiological effects of β-lactamase overproduction in Escherichia coli. Biotechnol Bioengineer 32: 741–748

    Article  CAS  Google Scholar 

  • Georgiou G, Poetschke HL, Stathopoulos C, Francisco JA (1993) Practical applications of engineering Gram-negative bacterial cell surfaces. Trends Biotechnol 11: 6–10

    Article  CAS  Google Scholar 

  • Georgiou G, Stephens DL, Stathopoulos C, Poetschke HL, Men-denhall J, Earhart CF (1996) Display of β-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp′-OmpA′-β-lactamase fusions. (in press)

  • Ghrayeb J, Inouye M (1984) Nine amino acid residues at the NH2terminal of lipoprotein are sufficient for its modification, processing, and localization in the outer membrane of Escherichia coli. J Biol Chem 259: 463–467

    CAS  Google Scholar 

  • Gunter K, Braun V (1988) Probing FhuA-PhoA fusion proteins for the study of FhuA export into the cell envelope of Escherichia coli K-12. Mol Gen Genet 215: 69–75

    Article  CAS  Google Scholar 

  • Herzberg O (1991) Refined crystal structure of β-lactamase from Staphylococcus aureus PC1 at 2.0 Å resolution. J Mol Biol 217: 701–719

    Article  CAS  Google Scholar 

  • Hofnung M (1991) Expression of foreign polypeptides at the Escherichia coli cell surface. Methods Cell Biol 34: 77–105

    Article  CAS  Google Scholar 

  • Kamitani S, Akiyama Y, Ito K (1992) Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase, a periplasmic enzyme. EMBO J 11: 57–62

    CAS  Google Scholar 

  • Kim EE, Wyckoff HW (1989) Structure and function of alkaline phosphatases. Structure of alkaline phosphatase. Clin Chim Acta 186: 175 188

    Google Scholar 

  • Klauser T, Pohlner J, Meyer TF (1992) Selective extracellular release of cholera toxin B subunit by Escherichia coli: dissection of Neisseria Igaβ-mediated outer membrane transport. EMBO J 11: 2327–2335

    CAS  Google Scholar 

  • Klauser T, Pohlner J, Meyer TF (1993) The secretion pathway of IgA protease-type proteins in gram-negative bacteria. Bioessays 15: 799–805

    Article  CAS  Google Scholar 

  • Kornacker MG, Pugsley AP (1990) The normally periplasmic enzyme β-lactamase is specifically and efficiently translocated through the Escherichia coli outer membrane when it is fused to the cell-surface enzyme pullulanase. Mol Microbiol 4: 1101–1109

    Article  CAS  Google Scholar 

  • Lopes J, Gottfried S, Rothfield L (1972) Leakage of periplasmic enzymes by mutants of Escherichia coli and Salmonella typhimurium: isolation of “periplasmic leaky” mutants. J Bacteriol 109: 520–525

    CAS  Google Scholar 

  • Lugtenberg EJJ, Peters R (1976) Distribution of lipids in cytoplasmic and outer membrane of Escherichia coli K12. Biochim Biophys Acta 441: 38–47

    CAS  Google Scholar 

  • Maniatis T Fritsch EF, Sambrook J (1989) Molecular cloning, a laboratory manual, 2n edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Murphy CK, Klebba PE (1989) Export of FepA:: PhoA fusion proteins to the outer membrane of Escherichia coli K-12. J Bacteriol 171: 5894–5900

    CAS  Google Scholar 

  • Nakamura K, Masui Y, Inouye M (1982) Use of a lac promoteroperator fragment as a transcriptional control switch for expression of the constitutive lpp gene in Escherichia coli. J Mol Appl Genet 1: 289–299

    CAS  Google Scholar 

  • Pogliano KJ, Beckwith J (1993) The Cs sec mutants of Escherichia coli reflect the cold sensitivity of protein export itself. Genetics 133: 763–773

    CAS  Google Scholar 

  • Pohlner J, Klauser T, Kuttler E, Halter R (1992) Sequence-specific cleavage of protein fusions using a recombinant Neisseria type 2 IgA protease. Biotechnology 10: 799–804

    Article  CAS  Google Scholar 

  • Ruppert A, Arnold N, Hobom G (1994) OmpA-FMDV VP1 fusion proteins: production, cell surface exposure and immune responses to the major antigenic domain of foot- and -mouth disease virus. Vaccine 12: 492–498

    Article  CAS  Google Scholar 

  • Silhavy TJ, Shuman HA, Beckwith J, Schwartz M (1977) Use of gene fusions to study outer membrane protein localization in Escherichia coli. Proc Natl Acad Sci USA 74: 5411–5415

    Article  CAS  Google Scholar 

  • Stephens DL, Choe MD, Earhart CF (1995) Escherichia coli periplasmic protein FepB binds ferrienterobactin. Microbiology 141: 1647–1654

    Article  CAS  Google Scholar 

  • Sukupolvi S, Vaara M (1989) Salmonella typhimurium and Escherichia coli mutants with increased outer membrane permeability to hydrophobic compounds. Biochim Biophys Acta 988: 377–387

    CAS  Google Scholar 

  • Vogel H, Jahnig F (1986) Models for the structure of outer-membrane proteins of Escherichia coli derived from Raman spectroscopy and prediction methods. J Mol Biol 190: 191–199

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Microbiology, University of Texas, 78712, Austin, TX, USA

    C. Stathopoulos & C. F. Earhart

  2. Division of Biological Sciences, University of Texas, 78712, Austin, TX, USA

    C. Stathopoulos

  3. Department of Chemical Engineering, University of Texas, 78712, Austin, TX, USA

    G. Georgiou

Authors
  1. C. Stathopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. G. Georgiou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C. F. Earhart
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Stathopoulos, C., Georgiou, G. & Earhart, C.F. Characterization of Escherichia coli expressing an Lpp’OmpA(46-159)-PhoA fusion protein localized in the outer membrane. Appl Microbiol Biotechnol 45, 112–119 (1996). https://doi.org/10.1007/s002530050657

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s002530050657

Keywords

Search

Navigation