Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Aug;174(15):4977–4985. doi: 10.1128/jb.174.15.4977-4985.1992

Analysis of the Pseudomonas aeruginosa major outer membrane protein OprF by use of truncated OprF derivatives and monoclonal antibodies.

R L Finnen 1, N L Martin 1, R J Siehnel 1, W A Woodruff 1, M Rosok 1, R E Hancock 1
PMCID: PMC206311  PMID: 1378429

Abstract

TnphoA mutagenesis of the cloned oprF gene was utilized to generate 16 classes of fusions encoding differing lengths of the amino terminus of OprF fused to either alkaline phosphatase or to peptide tags of 1 to 20 amino acids, depending on the orientation and reading frame into which TnphoA was inserted. Representatives of each of the 16 classes were sequenced to determine the precise fusion joint. Four of these 16 representatives which produced in-frame fusions to alkaline phosphatase and another 8 with fusion joints in the amino-terminal half of OprF failed to react with a panel of 10 specific monoclonal antibodies. In contrast, OprF derivatives with predicted fusion joints at amino acids 180, 204, 289, and 299 reacted with one to five of the monoclonal antibodies. Four other immunoreactive OprF derivatives were created by subcloning and encoded amino acids 1 to 187, 188 to 326, 1 to 273 and 1 to 170 plus 301 to 326. On the basis of reactivity with the TnphoA-truncated derivatives and subclones of oprF, the epitopes for all 10 monoclonal antibodies were localized, in part, to specific regions of OprF. Nnie of the 10 monoclonal antibodies, 8 of which recognize surface-exposed epitopes, mapped within the carboxy-terminal region of OprF that is homologous to the Escherichia coli outer membrane protein OmpA. Thus, we concluded that parts of the carboxy terminus of OprF are exposed on the external face of the outer membrane. In addition, a clone containing only the first two cysteine residues of OprF demonstrated reactivity with monoclonal antibodies MA4-4 and MA7-8 that was destroyed by 2-mercaptoethanol treatment, as was reactivity with intact OprF. Thus, we conclude that this first pair of cysteines at residues 176 and 185 of mature OprF form a disulfide bond.

Full text

PDF
4977

Images in this article

4980Image
on p.4980
4980Image
on p.4980
4981Image
on p.4981
4982Image
on p.4982

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bremer E., Cole S. T., Hindennach I., Henning U., Beck E., Kurz C., Schaller H. Export of a protein into the outer membrane of Escherichia coli K12. Stable incorporation of the OmpA protein requires less than 193 amino-terminal amino-acid residues. Eur J Biochem. 1982 Feb;122(1):223–231. doi: 10.1111/j.1432-1033.1982.tb05870.x. [DOI] [PubMed] [Google Scholar]
  2. Charbit A., Ronco J., Michel V., Werts C., Hofnung M. Permissive sites and topology of an outer membrane protein with a reporter epitope. J Bacteriol. 1991 Jan;173(1):262–275. doi: 10.1128/jb.173.1.262-275.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen R., Schmidmayr W., Krämer C., Chen-Schmeisser U., Henning U. Primary structure of major outer membrane protein II (ompA protein) of Escherichia coli K-12. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4592–4596. doi: 10.1073/pnas.77.8.4592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Coulton J. W., Reid G. K., Campana A. Export of hybrid proteins FhuA'-'LacZ and FhuA'-'PhoA to the cell envelope of Escherichia coli K-12. J Bacteriol. 1988 May;170(5):2267–2275. doi: 10.1128/jb.170.5.2267-2275.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Counts G. W., Schwartz R. W., Ulness B. K., Hamilton D. J., Rosok M. J., Cunningham M. D., Tam M. R., Darveau R. P. Evaluation of an immunofluorescent-antibody test for rapid identification of Pseudomonas aeruginosa in blood cultures. J Clin Microbiol. 1988 Jun;26(6):1161–1165. doi: 10.1128/jcm.26.6.1161-1165.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Duchêne M., Schweizer A., Lottspeich F., Krauss G., Marget M., Vogel K., von Specht B. U., Domdey H. Sequence and transcriptional start site of the Pseudomonas aeruginosa outer membrane porin protein F gene. J Bacteriol. 1988 Jan;170(1):155–162. doi: 10.1128/jb.170.1.155-162.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Elish M. E., Pierce J. R., Earhart C. F. Biochemical analysis of spontaneous fepA mutants of Escherichia coli. J Gen Microbiol. 1988 May;134(5):1355–1364. doi: 10.1099/00221287-134-5-1355. [DOI] [PubMed] [Google Scholar]
  8. Freudl R., MacIntyre S., Degen M., Henning U. Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12. J Mol Biol. 1986 Apr 5;188(3):491–494. doi: 10.1016/0022-2836(86)90171-3. [DOI] [PubMed] [Google Scholar]
  9. Goff S. A., Casson L. P., Goldberg A. L. Heat shock regulatory gene htpR influences rates of protein degradation and expression of the lon gene in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6647–6651. doi: 10.1073/pnas.81.21.6647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gutierrez C., Barondess J., Manoil C., Beckwith J. The use of transposon TnphoA to detect genes for cell envelope proteins subject to a common regulatory stimulus. Analysis of osmotically regulated genes in Escherichia coli. J Mol Biol. 1987 May 20;195(2):289–297. doi: 10.1016/0022-2836(87)90650-4. [DOI] [PubMed] [Google Scholar]
  11. Günter K., Braun V. Probing FhuA'-'PhoA fusion proteins for the study of FhuA export into the cell envelope of Escherichia coli K12. Mol Gen Genet. 1988 Dec;215(1):69–75. doi: 10.1007/BF00331305. [DOI] [PubMed] [Google Scholar]
  12. Hancock R. E., Carey A. M. Outer membrane of Pseudomonas aeruginosa: heat- 2-mercaptoethanol-modifiable proteins. J Bacteriol. 1979 Dec;140(3):902–910. doi: 10.1128/jb.140.3.902-910.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Labarca C., Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem. 1980 Mar 1;102(2):344–352. doi: 10.1016/0003-2697(80)90165-7. [DOI] [PubMed] [Google Scholar]
  14. Lloyd A. D., Kadner R. J. Topology of the Escherichia coli uhpT sugar-phosphate transporter analyzed by using TnphoA fusions. J Bacteriol. 1990 Apr;172(4):1688–1693. doi: 10.1128/jb.172.4.1688-1693.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Morona R., Klose M., Henning U. Escherichia coli K-12 outer membrane protein (OmpA) as a bacteriophage receptor: analysis of mutant genes expressing altered proteins. J Bacteriol. 1984 Aug;159(2):570–578. doi: 10.1128/jb.159.2.570-578.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Murphy C. K., Kalve V. I., Klebba P. E. Surface topology of the Escherichia coli K-12 ferric enterobactin receptor. J Bacteriol. 1990 May;172(5):2736–2746. doi: 10.1128/jb.172.5.2736-2746.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Murphy C. K., Klebba P. E. Export of FepA::PhoA fusion proteins to the outer membrane of Escherichia coli K-12. J Bacteriol. 1989 Nov;171(11):5894–5900. doi: 10.1128/jb.171.11.5894-5900.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mutharia L. M., Hancock R. E. Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa. Can J Microbiol. 1985 Apr;31(4):381–386. doi: 10.1139/m85-073. [DOI] [PubMed] [Google Scholar]
  20. Nicas T. I., Hancock R. E. Outer membrane protein H1 of Pseudomonas aeruginosa: involvement in adaptive and mutational resistance to ethylenediaminetetraacetate, polymyxin B, and gentamicin. J Bacteriol. 1980 Aug;143(2):872–878. doi: 10.1128/jb.143.2.872-878.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nikaido H., Nikaido K., Harayama S. Identification and characterization of porins in Pseudomonas aeruginosa. J Biol Chem. 1991 Jan 15;266(2):770–779. [PubMed] [Google Scholar]
  22. Paul C., Rosenbusch J. P. Folding patterns of porin and bacteriorhodopsin. EMBO J. 1985 Jun;4(6):1593–1597. doi: 10.1002/j.1460-2075.1985.tb03822.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schnaitman C. A. Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J Bacteriol. 1971 Oct;108(1):545–552. doi: 10.1128/jb.108.1.545-552.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sonntag I., Schwarz H., Hirota Y., Henning U. Cell envelope and shape of Escherichia coli: multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins. J Bacteriol. 1978 Oct;136(1):280–285. doi: 10.1128/jb.136.1.280-285.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sugawara E., Nikaido H. Pore-forming activity of OmpA protein of Escherichia coli. J Biol Chem. 1992 Feb 5;267(4):2507–2511. [PubMed] [Google Scholar]
  26. Ullstrom C. A., Siehnel R., Woodruff W., Steinbach S., Hancock R. E. Conservation of the gene for outer membrane protein OprF in the family Pseudomonadaceae: sequence of the Pseudomonas syringae oprF gene. J Bacteriol. 1991 Jan;173(2):768–775. doi: 10.1128/jb.173.2.768-775.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Vogel H., Jähnig F. Models for the structure of outer-membrane proteins of Escherichia coli derived from raman spectroscopy and prediction methods. J Mol Biol. 1986 Jul 20;190(2):191–199. doi: 10.1016/0022-2836(86)90292-5. [DOI] [PubMed] [Google Scholar]
  28. Winans S. C., Kerstetter R. A., Ward J. E., Nester E. W. A protein required for transcriptional regulation of Agrobacterium virulence genes spans the cytoplasmic membrane. J Bacteriol. 1989 Mar;171(3):1616–1622. doi: 10.1128/jb.171.3.1616-1622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Woodruff W. A., Hancock R. E. Construction and characterization of Pseudomonas aeruginosa protein F-deficient mutants after in vitro and in vivo insertion mutagenesis of the cloned gene. J Bacteriol. 1988 Jun;170(6):2592–2598. doi: 10.1128/jb.170.6.2592-2598.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Woodruff W. A., Hancock R. E. Pseudomonas aeruginosa outer membrane protein F: structural role and relationship to the Escherichia coli OmpA protein. J Bacteriol. 1989 Jun;171(6):3304–3309. doi: 10.1128/jb.171.6.3304-3309.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Woodruff W. A., Parr T. R., Jr, Hancock R. E., Hanne L. F., Nicas T. I., Iglewski B. H. Expression in Escherichia coli and function of Pseudomonas aeruginosa outer membrane porin protein F. J Bacteriol. 1986 Aug;167(2):473–479. doi: 10.1128/jb.167.2.473-479.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yoshihara E., Nakae T. Identification of porins in the outer membrane of Pseudomonas aeruginosa that form small diffusion pores. J Biol Chem. 1989 Apr 15;264(11):6297–6301. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES