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. 1996 Feb;62(2):480–485. doi: 10.1128/aem.62.2.480-485.1996

Outer Membrane Protein Heterogeneity within Pseudomonas fluorescens and P. putida and Use of an OprF Antibody as a Probe for rRNA Homology Group I Pseudomonads

L Kragelund, K Leopold, O Nybroe
PMCID: PMC1388773  PMID: 16535235

Abstract

The electrophoretic patterns of outer membrane proteins of strains representing the biovars of Pseudomonas fluorescens and Pseudomonas putida were analyzed by gel electrophoresis. The outer membrane protein profiles were variable, and they were not useful for assigning strains to a specific biovar. However, three or four predominant outer membrane proteins migrating at 42 to 46 kDa, 33 to 38 kDa, and 20 to 22 kDa were conserved among the strains. They could be tentatively identified as OprE (44 kDa), OprF (38 kDa), OprH (21 kDa), and OprL (20.5 kDa), which are known proteins from Pseudomonas aeruginosa. A 37-kDa OprF-like protein was purified from P. fluorescens DF57 and used to raise a polyclonal antibody. In Western blot (immunoblot) analysis, this antibody reacted with OprF proteins from members of Pseudomonas rRNA homology group I but not with proteins from nonpseudomonads. The heterogeneity in M(infr) of OprF was greater among P. fluorescens strains than among P. putida strains. Immunofluorescence microscopy of intact cells demonstrated that the antibody recognized epitopes that were accessible only after unmasking by EDTA treatment. The antibody was used in a colony blotting assay to determine the percentage of rRNA homology group I pseudomonads among bacteria from the rhizosphere of barley. The bacteria were isolated on 10% tryptic soy agar, King's B agar, and the pseudomonad-specific medium Gould S1 agar. The estimate of OprF-containing CFU in rhizosphere soil obtained by colony blotting on 10% tryptic soy agar was about 2 and 14 times higher than the values obtained from King's agar and Gould S1 agar, respectively, indicating that not all fluorescent pseudomonads are scored on more specific media. The colonies reacting with the OprF antibody were verified as being rRNA homology group I pseudomonads by using the API 20NE system.

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Selected References

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  1. Anderson P. J. The recovery of nitrocellulose-bound protein. Anal Biochem. 1985 Jul;148(1):105–110. doi: 10.1016/0003-2697(85)90634-7. [DOI] [PubMed] [Google Scholar]
  2. Barrett E. L., Solanes R. E., Tang J. S., Palleroni N. J. Pseudomonas fluorescens biovar V: its resolution into distinct component groups and the relationship of these groups to other P. fluorescens biovars, to P. putida, and to psychrotrophic pseudomonads associated with food spoilage. J Gen Microbiol. 1986 Oct;132(10):2709–2721. doi: 10.1099/00221287-132-10-2709. [DOI] [PubMed] [Google Scholar]
  3. Christensen H., Boye M., Poulsen L. K., Rasmussen O. F. Analysis of fluorescent pseudomonads based on 23S ribosomal DNA sequences. Appl Environ Microbiol. 1994 Jun;60(6):2196–2199. doi: 10.1128/aem.60.6.2196-2199.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. De Mot R., Proost P., Van Damme J., Vanderleyden J. Homology of the root adhesin of Pseudomonas fluorescens OE 28.3 with porin F of P. aeruginosa and P. syringae. Mol Gen Genet. 1992 Feb;231(3):489–493. doi: 10.1007/BF00292721. [DOI] [PubMed] [Google Scholar]
  5. De Mot R., Schoofs G., Roelandt A., Declerck P., Proost P., Van Damme J., Vanderleyden J. Molecular characterization of the major outer-membrane protein OprF from plant root-colonizing Pseudomonas fluorescens. Microbiology. 1994 Jun;140(Pt 6):1377–1387. doi: 10.1099/00221287-140-6-1377. [DOI] [PubMed] [Google Scholar]
  6. González I., Martín R., García T., Morales P., Sanz B., Hernández P. E. A sandwich enzyme-linked immunosorbent assay (ELISA) for detection of Pseudomonas fluorescens and related psychrotrophic bacteria in refrigerated milk. J Appl Bacteriol. 1993 Apr;74(4):394–401. doi: 10.1111/j.1365-2672.1993.tb05144.x. [DOI] [PubMed] [Google Scholar]
  7. Gould W. D., Hagedorn C., Bardinelli T. R., Zablotowicz R. M. New selective media for enumeration and recovery of fluorescent pseudomonads from various habitats. Appl Environ Microbiol. 1985 Jan;49(1):28–32. doi: 10.1128/aem.49.1.28-32.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Hancock R. E., Chan L. Outer membranes of environmental isolates of Pseudomonas aeruginosa. J Clin Microbiol. 1988 Nov;26(11):2423–2424. doi: 10.1128/jcm.26.11.2423-2424.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hancock R. E., Siehnel R., Martin N. Outer membrane proteins of Pseudomonas. Mol Microbiol. 1990 Jul;4(7):1069–1075. doi: 10.1111/j.1365-2958.1990.tb00680.x. [DOI] [PubMed] [Google Scholar]
  11. Hoff K. A. Rapid and simple method for double staining of bacteria with 4',6-diamidino-2-phenylindole and fluorescein isothiocyanate-labeled antibodies. Appl Environ Microbiol. 1988 Dec;54(12):2949–2952. doi: 10.1128/aem.54.12.2949-2952.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hughes E. E., Gilleland L. B., Gilleland H. E., Jr Synthetic peptides representing epitopes of outer membrane protein F of Pseudomonas aeruginosa that elicit antibodies reactive with whole cells of heterologous immunotype strains of P. aeruginosa. Infect Immun. 1992 Sep;60(9):3497–3503. doi: 10.1128/iai.60.9.3497-3503.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Judd R. C. Purification of outer membrane proteins of the gram-negative bacterium Neisseria gonorrhoeae. Anal Biochem. 1988 Sep;173(2):307–316. doi: 10.1016/0003-2697(88)90194-7. [DOI] [PubMed] [Google Scholar]
  14. Kragelund L., Nybroe O. Culturability and Expression of Outer Membrane Proteins during Carbon, Nitrogen, or Phosphorus Starvation of Pseudomonas fluorescens DF57 and Pseudomonas putida DF14. Appl Environ Microbiol. 1994 Aug;60(8):2944–2948. doi: 10.1128/aem.60.8.2944-2948.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Martin N. L., Rawling E. G., Wong R. S., Rosok M., Hancock R. E. Conservation of surface epitopes in Pseudomonas aeruginosa outer membrane porin protein OprF. FEMS Microbiol Lett. 1993 Nov 1;113(3):261–266. doi: 10.1111/j.1574-6968.1993.tb06524.x. [DOI] [PubMed] [Google Scholar]
  16. Martínez-Maza O., Fehniger T. E., Ashman R. F. Antibody-secreting cell precursor frequencies among the sheep-erythrocyte-binding cells after immunization. Scand J Immunol. 1983 Apr;17(4):345–354. doi: 10.1111/j.1365-3083.1983.tb00799.x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Mutharia L. M., Hancock R. E. Surface localization of Pseudomonas aeruginosa outer membrane porin protein F by using monoclonal antibodies. Infect Immun. 1983 Dec;42(3):1027–1033. doi: 10.1128/iai.42.3.1027-1033.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Olsen P. E., Rice W. A. Rhizobium strain identification and quantification in commercial inoculants by immunoblot analysis. Appl Environ Microbiol. 1989 Feb;55(2):520–522. doi: 10.1128/aem.55.2.520-522.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Parekh B. S., Mehta H. B., West M. D., Montelaro R. C. Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem. 1985 Jul;148(1):87–92. doi: 10.1016/0003-2697(85)90631-1. [DOI] [PubMed] [Google Scholar]
  22. Rawling E. G., Martin N. L., Hancock R. E. Epitope mapping of the Pseudomonas aeruginosa major outer membrane porin protein OprF. Infect Immun. 1995 Jan;63(1):38–42. doi: 10.1128/iai.63.1.38-42.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Zaat S. A., Slegtenhorst-Eegdeman K., Tommassen J., Geli V., Wijffelman C. A., Lugtenberg B. J. Construction of phoE-caa, a novel PCR- and immunologically detectable marker gene for Pseudomonas putida. Appl Environ Microbiol. 1994 Nov;60(11):3965–3973. doi: 10.1128/aem.60.11.3965-3973.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. de Weger L. A., van Boxtel R., van der Burg B., Gruters R. A., Geels F. P., Schippers B., Lugtenberg B. Siderophores and outer membrane proteins of antagonistic, plant-growth-stimulating, root-colonizing Pseudomonas spp. J Bacteriol. 1986 Feb;165(2):585–594. doi: 10.1128/jb.165.2.585-594.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

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