Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Aug;174(16):5196–5203. doi: 10.1128/jb.174.16.5196-5203.1992

Reevaluation, using intact cells, of the exclusion limit and role of porin OprF in Pseudomonas aeruginosa outer membrane permeability.

F Bellido 1, N L Martin 1, R J Siehnel 1, R E Hancock 1
PMCID: PMC206352  PMID: 1322882

Abstract

Earlier studies that used model membrane reconstitution methods have come to different conclusions regarding the exclusion limit of the outer membrane of Pseudomonas aeruginosa and whether OprF is the major channel-forming protein in the outer membrane. In this study, a 6.2-kbp SalI fragment, encoding only two cytoplasmic enzymes, alpha-galactosidase and sucrose hydrolase, and the inner membrane raffinose permease, was cloned behind the m-toluate-inducible tol promoter of vector pNM185 to create plasmid pFB71. P. aeruginosa strains harboring pFB71, when grown with inducer, produced both enzymes encoded by the insert and had acquired the ability to grow on the disaccharide melibiose and the trisaccharide raffinose. The rate of growth was dependent on the concentration and size of the saccharide and was decreased three- to fivefold by the absence of OprF, as examined by measuring the growth on melibiose and raffinose of an isogenic OprF-deficient omega insertion derivative, H636(pFB71). At high concentrations, di-, tri-, and tetrasaccharides could pass across the outer membrane to plasmolyze P. aeruginosa, as measured by light scattering and confirmed by electron microscopy. The initial rate kinetics of light-scattering changes were dependent on the size of the saccharide being used. Furthermore, the rates of change in light scattering due to raffinose and stachyose uptake across the outer membrane for strain H636 were fivefold or more lower than for its OprF-sufficient parent H103. These data are consistent with model membrane studies showing that OprF is the most predominant porin for compounds larger than disaccharides in P. aeruginosa and suggest that the exclusion limit for this porin and the outer membrane is greater than the size of a tetrasaccharide. In addition, these data confirmed the existence of other porins with a predominant function in monosaccharide uptake and a more minor function in the uptake of larger saccharides.

Full text

PDF
5196

Images in this article

5201Image
on p.5201

Selected References

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

  1. Alemohammad M. M., Knowles C. J. Osmotically induced volume and turbidity changes of Escherichia coli due to salts, sucrose and glycerol, with particular reference to the rapid permeation of glycerol into the cell. J Gen Microbiol. 1974 May;82(1):125–142. doi: 10.1099/00221287-82-1-125. [DOI] [PubMed] [Google Scholar]
  2. Angus B. L., Carey A. M., Caron D. A., Kropinski A. M., Hancock R. E. Outer membrane permeability in Pseudomonas aeruginosa: comparison of a wild-type with an antibiotic-supersusceptible mutant. Antimicrob Agents Chemother. 1982 Feb;21(2):299–309. doi: 10.1128/aac.21.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aslanidis C., Schmid K., Schmitt R. Nucleotide sequences and operon structure of plasmid-borne genes mediating uptake and utilization of raffinose in Escherichia coli. J Bacteriol. 1989 Dec;171(12):6753–6763. doi: 10.1128/jb.171.12.6753-6763.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benz R., Hancock R. E. Properties of the large ion-permeable pores formed from protein F of Pseudomonas aeruginosa in lipid bilayer membranes. Biochim Biophys Acta. 1981 Aug 20;646(2):298–308. doi: 10.1016/0005-2736(81)90336-9. [DOI] [PubMed] [Google Scholar]
  5. Deretic V., Chandrasekharappa S., Gill J. F., Chatterjee D. K., Chakrabarty A. M. A set of cassettes and improved vectors for genetic and biochemical characterization of Pseudomonas genes. Gene. 1987;57(1):61–72. doi: 10.1016/0378-1119(87)90177-6. [DOI] [PubMed] [Google Scholar]
  6. Gotoh N., Wakebe H., Yoshihara E., Nakae T., Nishino T. Role of protein F in maintaining structural integrity of the Pseudomonas aeruginosa outer membrane. J Bacteriol. 1989 Feb;171(2):983–990. doi: 10.1128/jb.171.2.983-990.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hancock R. E., Bell A. Antibiotic uptake into gram-negative bacteria. Eur J Clin Microbiol Infect Dis. 1988 Dec;7(6):713–720. doi: 10.1007/BF01975036. [DOI] [PubMed] [Google Scholar]
  8. Hancock R. E., Decad G. M., Nikaido H. Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01. Biochim Biophys Acta. 1979 Jul 5;554(2):323–331. doi: 10.1016/0005-2736(79)90373-0. [DOI] [PubMed] [Google Scholar]
  9. Hancock R. E. Role of porins in outer membrane permeability. J Bacteriol. 1987 Mar;169(3):929–933. doi: 10.1128/jb.169.3.929-933.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jap B. K., Walian P. J. Biophysics of the structure and function of porins. Q Rev Biophys. 1990 Nov;23(4):367–403. doi: 10.1017/s003358350000559x. [DOI] [PubMed] [Google Scholar]
  11. KOCH A. L. Some calculations on the turbidity of mitochondria and bacteria. Biochim Biophys Acta. 1961 Aug 19;51:429–441. doi: 10.1016/0006-3002(61)90599-6. [DOI] [PubMed] [Google Scholar]
  12. Mermod N., Ramos J. L., Lehrbach P. R., Timmis K. N. Vector for regulated expression of cloned genes in a wide range of gram-negative bacteria. J Bacteriol. 1986 Aug;167(2):447–454. doi: 10.1128/jb.167.2.447-454.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nakae T. Outer membrane of Salmonella. Isolation of protein complex that produces transmembrane channels. J Biol Chem. 1976 Apr 10;251(7):2176–2178. [PubMed] [Google Scholar]
  14. Nicas T. I., Hancock R. E. Pseudomonas aeruginosa outer membrane permeability: isolation of a porin protein F-deficient mutant. J Bacteriol. 1983 Jan;153(1):281–285. doi: 10.1128/jb.153.1.281-285.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Nikaido H., Rosenberg E. Y. Effect on solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coli. J Gen Physiol. 1981 Feb;77(2):121–135. doi: 10.1085/jgp.77.2.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nikaido H., Rosenberg E. Y. Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins. J Bacteriol. 1983 Jan;153(1):241–252. doi: 10.1128/jb.153.1.241-252.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nikaido H., Vaara M. Molecular basis of bacterial outer membrane permeability. Microbiol Rev. 1985 Mar;49(1):1–32. doi: 10.1128/mr.49.1.1-32.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. RENKIN E. M. Filtration, diffusion, and molecular sieving through porous cellulose membranes. J Gen Physiol. 1954 Nov 20;38(2):225–243. [PMC free article] [PubMed] [Google Scholar]
  20. SCHULTZ S. G., SOLOMON A. K. Determination of the effective hydrodynamic radii of small molecules by viscometry. J Gen Physiol. 1961 Jul;44:1189–1199. doi: 10.1085/jgp.44.6.1189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schmid K., Schmitt R. Raffinose metabolism in Escherichia coli K12. Purification and properties of a new alpha-galactosidase specified by a transmissible plasmid. Eur J Biochem. 1976 Aug 1;67(1):95–104. doi: 10.1111/j.1432-1033.1976.tb10637.x. [DOI] [PubMed] [Google Scholar]
  22. Schmid K., Schupfner M., Schmitt R. Plasmid-mediated uptake and metabolism of sucrose by Escherichia coli K-12. J Bacteriol. 1982 Jul;151(1):68–76. doi: 10.1128/jb.151.1.68-76.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stock J. B., Rauch B., Roseman S. Periplasmic space in Salmonella typhimurium and Escherichia coli. J Biol Chem. 1977 Nov 10;252(21):7850–7861. [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Yoneyama H., Akatsuka A., Nakae T. The outer membrane of Pseudomonas aeruginosa is a barrier against the penetration of disaccharides. Biochem Biophys Res Commun. 1986 Jan 14;134(1):106–112. doi: 10.1016/0006-291x(86)90533-4. [DOI] [PubMed] [Google Scholar]
  27. Yoneyama H., Nakae T. A small diffusion pore in the outer membrane of Pseudomonas aeruginosa. Eur J Biochem. 1986 May 15;157(1):33–38. doi: 10.1111/j.1432-1033.1986.tb09634.x. [DOI] [PubMed] [Google Scholar]
  28. Yoshihara E., Gotoh N., Nakae T. In vitro demonstration by the rate assay of the presence of small pore in the outer membrane of Pseudomonas aeruginosa. Biochem Biophys Res Commun. 1988 Oct 14;156(1):470–476. doi: 10.1016/s0006-291x(88)80865-9. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Yoshimura F., Nikaido H. Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic solutes. J Bacteriol. 1982 Nov;152(2):636–642. doi: 10.1128/jb.152.2.636-642.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yoshimura F., Zalman L. S., Nikaido H. Purification and properties of Pseudomonas aeruginosa porin. J Biol Chem. 1983 Feb 25;258(4):2308–2314. [PubMed] [Google Scholar]

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

RESOURCES