Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Aug;171(8):4130–4137. doi: 10.1128/jb.171.8.4130-4137.1989

Pseudomonas aeruginosa outer membrane lipoprotein I gene: molecular cloning, sequence, and expression in Escherichia coli.

M Duchêne 1, C Barron 1, A Schweizer 1, B U von Specht 1, H Domdey 1
PMCID: PMC210182  PMID: 2502533

Abstract

Lipoprotein I (OprI) is one of the major proteins of the outer membrane of Pseudomonas aeruginosa. Like porin protein F (OprF), it is a vaccine candidate because it antigenically cross-reacts with all serotype strains of the International Antigenic Typing Scheme. Since lipoprotein I was expressed in Escherichia coli under the control of its own promoter, we were able to isolate the gene by screening a lambda EMBL3 phage library with a mouse monoclonal antibody directed against lipoprotein I. The monocistronic OprI mRNA encodes a precursor protein of 83 amino acid residues including a signal peptide of 19 residues. The mature protein has a molecular weight of 6,950, not including bound glycerol and lipid. Although the amino acid sequences of protein I of P. aeruginosa and Braun's lipoprotein of E. coli differ considerably (only 30.1% identical amino acid residues), peptidoglycan in E. coli, are identical. Using lipoprotein I expressed in E. coli, it can now be tested whether this protein alone, without P. aeruginosa lipopolysaccharide contaminations, has a protective effect against P. aeruginosa infections.

Full text

PDF
4130

Images in this article

4133Image
on p.4133
4134Image
on p.4134

Selected References

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

  1. Aiba H., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [PubMed] [Google Scholar]
  2. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Brendel V., Trifonov E. N. A computer algorithm for testing potential prokaryotic terminators. Nucleic Acids Res. 1984 May 25;12(10):4411–4427. doi: 10.1093/nar/12.10.4411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  6. Chen R., Henning U. Nucleotide sequence of the gene for the peptidoglycan-associated lipoprotein of Escherichia coli K12. Eur J Biochem. 1987 Feb 16;163(1):73–77. doi: 10.1111/j.1432-1033.1987.tb10738.x. [DOI] [PubMed] [Google Scholar]
  7. Ching G., Inouye M. Expression of the Proteus mirabilis lipoprotein gene in Escherichia coli. Existence of tandem promoters. J Biol Chem. 1986 Apr 5;261(10):4600–4606. [PubMed] [Google Scholar]
  8. Choi D. S., Yamada H., Mizuno T., Mizushima S. Molecular assembly of the lipoprotein trimer on the peptidoglycan layer of Escherichia coli. J Biochem. 1987 Nov;102(5):975–983. doi: 10.1093/oxfordjournals.jbchem.a122174. [DOI] [PubMed] [Google Scholar]
  9. Cryz S. J., Jr, Fürer E., Cross A. S., Wegmann A., Germanier R., Sadoff J. C. Safety and immunogenicity of a Pseudomonas aeruginosa O-polysaccharide toxin A conjugate vaccine in humans. J Clin Invest. 1987 Jul;80(1):51–56. doi: 10.1172/JCI113062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deich R. A., Metcalf B. J., Finn C. W., Farley J. E., Green B. A. Cloning of genes encoding a 15,000-dalton peptidoglycan-associated outer membrane lipoprotein and an antigenically related 15,000-dalton protein from Haemophilus influenzae. J Bacteriol. 1988 Feb;170(2):489–498. doi: 10.1128/jb.170.2.489-498.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Domdey H., Apostol B., Lin R. J., Newman A., Brody E., Abelson J. Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell. 1984 Dec;39(3 Pt 2):611–621. doi: 10.1016/0092-8674(84)90468-9. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Fling S. P., Gregerson D. S. Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea. Anal Biochem. 1986 May 15;155(1):83–88. doi: 10.1016/0003-2697(86)90228-9. [DOI] [PubMed] [Google Scholar]
  14. Frischauf A. M., Lehrach H., Poustka A., Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. doi: 10.1016/s0022-2836(83)80190-9. [DOI] [PubMed] [Google Scholar]
  15. Fung J., MacAlister T. J., Rothfield L. I. Role of murein lipoprotein in morphogenesis of the bacterial division septum: phenotypic similarity of lkyD and lpo mutants. J Bacteriol. 1978 Mar;133(3):1467–1471. doi: 10.1128/jb.133.3.1467-1471.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. George D. G., Barker W. C., Hunt L. T. The protein identification resource (PIR). Nucleic Acids Res. 1986 Jan 10;14(1):11–15. doi: 10.1093/nar/14.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gilleland H. E., Jr, Parker M. G., Matthews J. M., Berg R. D. Use of a purified outer membrane protein F (porin) preparation of Pseudomonas aeruginosa as a protective vaccine in mice. Infect Immun. 1984 Apr;44(1):49–54. doi: 10.1128/iai.44.1.49-54.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Halegoua S., Hirashima A., Inouye M. Existence of a free form of a specific membrane lipoprotein in gram-negative bacteria. J Bacteriol. 1974 Dec;120(3):1204–1208. doi: 10.1128/jb.120.3.1204-1208.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hancock R. E. Intrinsic antibiotic resistance of Pseudomonas aeruginosa. J Antimicrob Chemother. 1986 Dec;18(6):653–656. doi: 10.1093/jac/18.6.653. [DOI] [PubMed] [Google Scholar]
  20. Hantke K., Braun V. Covalent binding of lipid to protein. Diglyceride and amide-linked fatty acid at the N-terminal end of the murein-lipoprotein of the Escherichia coli outer membrane. Eur J Biochem. 1973 Apr;34(2):284–296. doi: 10.1111/j.1432-1033.1973.tb02757.x. [DOI] [PubMed] [Google Scholar]
  21. Huang Y. X., Ching G., Inouye M. Comparison of the lipoprotein gene among the enterobacteriaceae. DNA sequence of Morganella morganii lipoprotein gene and its expression in Escherichia coli. J Biol Chem. 1983 Jul 10;258(13):8139–8145. [PubMed] [Google Scholar]
  22. Hussain M., Ichihara S., Mizushima S. Accumulation of glyceride-containing precursor of the outer membrane lipoprotein in the cytoplasmic membrane of Escherichia coli treated with globomycin. J Biol Chem. 1980 Apr 25;255(8):3707–3712. [PubMed] [Google Scholar]
  23. Inouye S., Wang S., Sekizawa J., Halegoua S., Inouye M. Amino acid sequence for the peptide extension on the prolipoprotein of the Escherichia coli outer membrane. Proc Natl Acad Sci U S A. 1977 Mar;74(3):1004–1008. doi: 10.1073/pnas.74.3.1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Konigsberg W., Godson G. N. Evidence for use of rare codons in the dnaG gene and other regulatory genes of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Feb;80(3):687–691. doi: 10.1073/pnas.80.3.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  26. Marget M., Eckhardt A., Ehret W., von Specht B. U., Duchêne M., Domdey H. Cloning and characterization of cDNAs coding for the heavy and light chains of a monoclonal antibody specific for Pseudomonas aeruginosa outer membrane protein I. Gene. 1988 Dec 30;74(2):335–345. doi: 10.1016/0378-1119(88)90167-9. [DOI] [PubMed] [Google Scholar]
  27. Matthews-Greer J. M., Gilleland H. E., Jr Outer membrane protein F (porin) preparation of Pseudomonas aeruginosa as a protective vaccine against heterologous immunotype strains in a burned mouse model. J Infect Dis. 1987 Jun;155(6):1282–1291. doi: 10.1093/infdis/155.6.1282. [DOI] [PubMed] [Google Scholar]
  28. McClure W. R. Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem. 1985;54:171–204. doi: 10.1146/annurev.bi.54.070185.001131. [DOI] [PubMed] [Google Scholar]
  29. Michel H., Weyer K. A., Gruenberg H., Lottspeich F. The ;heavy' subunit of the photosynthetic reaction centre from Rhodopseudomonas viridis: isolation of the gene, nucleotide and amino acid sequence. EMBO J. 1985 Jul;4(7):1667–1672. doi: 10.1002/j.1460-2075.1985.tb03835.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mizuno T., Kageyama M. Isolation and characterization of major outer membrane proteins of Pseudomonas aeruginosa strain PAO with special reference to peptidoglycan-associated protein. J Biochem. 1979 Oct;86(4):979–989. doi: 10.1093/oxfordjournals.jbchem.a132630. [DOI] [PubMed] [Google Scholar]
  31. Mizuno T., Kageyama M. Isolation of characterization of a major outer membrane protein of Pseudomonas aeruginosa. Evidence for the occurrence of a lipoprotein. J Biochem. 1979 Jan;85(1):115–122. doi: 10.1093/oxfordjournals.jbchem.a132300. [DOI] [PubMed] [Google Scholar]
  32. Movva N. R., Nakamura K., Inouye M. Regulatory region of the gene for the ompA protein, a major outer membrane protein of Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3845–3849. doi: 10.1073/pnas.77.7.3845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mutharia L. M., Nicas T. I., Hancock R. E. Outer membrane proteins of Pseudomonas aeruginosa serotype strains. J Infect Dis. 1982 Dec;146(6):770–779. doi: 10.1093/infdis/146.6.770. [DOI] [PubMed] [Google Scholar]
  34. Nakamura K., Inouye M. DNA sequence of the Serratia marcescens lipoprotein gene. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1369–1373. doi: 10.1073/pnas.77.3.1369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nakamura K., Inouye M. DNA sequence of the gene for the outer membrane lipoprotein of E. coli: an extremely AT-rich promoter. Cell. 1979 Dec;18(4):1109–1117. doi: 10.1016/0092-8674(79)90224-1. [DOI] [PubMed] [Google Scholar]
  36. Perlman D., Halvorson H. O. A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J Mol Biol. 1983 Jun 25;167(2):391–409. doi: 10.1016/s0022-2836(83)80341-6. [DOI] [PubMed] [Google Scholar]
  37. Pier G. B. Safety and immunogenicity of high molecular weight polysaccharide vaccine from immunotype 1 Pseudomonas aeruginosa. J Clin Invest. 1982 Feb;69(2):303–308. doi: 10.1172/JCI110453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Platt T., Yanofsky C. An intercistronic region and ribosome-binding site in bacterial messenger RNA. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2399–2403. doi: 10.1073/pnas.72.6.2399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Weaver R. F., Weissmann C. Mapping of RNA by a modification of the Berk-Sharp procedure: the 5' termini of 15 S beta-globin mRNA precursor and mature 10 s beta-globin mRNA have identical map coordinates. Nucleic Acids Res. 1979 Nov 10;7(5):1175–1193. doi: 10.1093/nar/7.5.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. West S. E., Iglewski B. H. Codon usage in Pseudomonas aeruginosa. Nucleic Acids Res. 1988 Oct 11;16(19):9323–9335. doi: 10.1093/nar/16.19.9323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Woods D. E., Bryan L. E. Studies on the ability of alginate to act as a protective immunogen against infection with Pseudomonas aeruginosa in animals. J Infect Dis. 1985 Apr;151(4):581–588. doi: 10.1093/infdis/151.4.581. [DOI] [PubMed] [Google Scholar]
  45. Yamagata H., Nakamura K., Inouye M. Comparison of the lipoprotein gene among the Enterobacteriaceae. DNA sequence of Erwinia amylovora lipoprotein gene. J Biol Chem. 1981 Mar 10;256(5):2194–2198. [PubMed] [Google Scholar]
  46. Yamaguchi K., Inouye M. Lipoprotein 28, an inner membrane protein of Escherichia coli encoded by nlpA, is not essential for growth. J Bacteriol. 1988 Aug;170(8):3747–3749. doi: 10.1128/jb.170.8.3747-3749.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  48. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. von Specht B. U., Strigl G., Ehret W., Brendel W. Protective effect of an outer membrane vaccine against Pseudomonas aeruginosa infection. Infection. 1987;15(5):408–412. doi: 10.1007/BF01647755. [DOI] [PubMed] [Google Scholar]

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

RESOURCES