Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1991 Oct;59(10):3740–3749. doi: 10.1128/iai.59.10.3740-3749.1991

Extraction, purification, and characterization of major outer membrane proteins from Wolinella recta ATCC 33238.

W L Kennell 1, S C Holt 1
PMCID: PMC258945  PMID: 1894372

Abstract

The outer membrane of Wolinella recta ATCC 33238 was isolated by French pressure cell disruption and differential centrifugation. Outer membrane proteins (OMPs) were solubilized by Zwittergent 3.14 extraction and separated by DEAE-Sephacel ion-exchange chromatography. The major OMPs that were found in W. recta ATCC 33238 and in several other Wolinella spp. consisted of proteins with apparent molecular masses of 51, 45, and 43 kDa. These three conserved proteins were purified to essential homogeneity by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and characterized chemically. Heating at between 75 and 100 degrees C revealed both the 43- and 51-kDa proteins to be heat modified from apparent molecular masses of 32 and 38 kDa, respectively. The 45-kDa protein was unmodified at all temperatures tested. Two-dimensional isoelectric focusing-SDS-PAGE revealed the 51-kDa protein to be composed of multiple pIs between a pH of 5.0 and greater than 8.0 while the 43- and 45-kDa proteins had a pI of approximately 6.0. N'-terminal amino acid sequence analysis of the first 30 to 40 amino acids and search of the Protein Identification Resource data base for similar proteins only revealed the 43-kDa protein to be similar to the P.69 OMP of Bordetella pertussis; however, the homology was weak (33%). Amino acid analysis revealed the 43-kDa protein to be noncharged and the 45- and 51-kDa proteins to be hydrophilic, containing between 38 to 42% polar residues but no cysteine. This study reports the purification and partial characterization of three conserved proteins in W. recta ATCC 33238.

Full text

PDF
3740

Images in this article

3742Image
on p.3742
3743Image
on p.3743
3743Image
on p.3743
3744Image
on p.3744
3744Image
on p.3744
3744Image
on p.3744
3745Image
on p.3745
3745Image
on p.3745

Selected References

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

  1. Anwar H. Association of a 38 kDa bovine serum protein with the outer membrane of Bordetella pertussis. FEMS Microbiol Lett. 1990 Sep 15;59(3):305–309. doi: 10.1016/0378-1097(90)90238-l. [DOI] [PubMed] [Google Scholar]
  2. Armstrong S. K., Parr T. R., Jr, Parker C. D., Hancock R. E. Bordetella pertussis major outer membrane porin protein forms small, anion-selective channels in lipid bilayer membranes. J Bacteriol. 1986 Apr;166(1):212–216. doi: 10.1128/jb.166.1.212-216.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benz R. Porin from bacterial and mitochondrial outer membranes. CRC Crit Rev Biochem. 1985;19(2):145–190. doi: 10.3109/10409238509082542. [DOI] [PubMed] [Google Scholar]
  4. Blaser M. J., Hopkins J. A., Berka R. M., Vasil M. L., Wang W. L. Identification and characterization of Campylobacter jejuni outer membrane proteins. Infect Immun. 1983 Oct;42(1):276–284. doi: 10.1128/iai.42.1.276-284.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borinski R., Holt S. C. Surface characteristics of Wolinella recta ATCC 33238 and human clinical isolates: correlation of structure with function. Infect Immun. 1990 Sep;58(9):2770–2776. doi: 10.1128/iai.58.9.2770-2776.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buchanan T. M., Hildebrandt J. F. Antigen-specific serotyping of Neisseria gonorrhoeae: characterization based upon principal outer membrane protein. Infect Immun. 1981 Jun;32(3):985–994. doi: 10.1128/iai.32.3.985-994.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Charles I. G., Dougan G., Pickard D., Chatfield S., Smith M., Novotny P., Morrissey P., Fairweather N. F. Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis. Proc Natl Acad Sci U S A. 1989 May;86(10):3554–3558. doi: 10.1073/pnas.86.10.3554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen C. K., Wilson M. E. Outer membrane protein and lipopolysaccharide heterogeneity among Eikenella corrodens isolates. J Infect Dis. 1990 Sep;162(3):664–671. doi: 10.1093/infdis/162.3.664. [DOI] [PubMed] [Google Scholar]
  9. Di Donato A., Draetta G. F., Illiano G., Tufano M. A., Sommese L., Galdiero F. Do porins inhibit the macrophage phagocyting activity by stimulating the adenylate cyclase? J Cyclic Nucleotide Protein Phosphor Res. 1986;11(2):87–97. [PubMed] [Google Scholar]
  10. DiRienzo J. M., Rosan B. Isolation of a major cell envelope protein from Fusobacterium nucleatum. Infect Immun. 1984 May;44(2):386–393. doi: 10.1128/iai.44.2.386-393.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dzink J. L., Tanner A. C., Haffajee A. D., Socransky S. S. Gram negative species associated with active destructive periodontal lesions. J Clin Periodontol. 1985 Sep;12(8):648–659. doi: 10.1111/j.1600-051x.1985.tb00936.x. [DOI] [PubMed] [Google Scholar]
  12. Fauchère J. L., Kervella M., Rosenau A., Mohanna K., Véron M. Adhesion to HeLa cells of Campylobacter jejuni and C. coli outer membrane components. Res Microbiol. 1989 Jul-Aug;140(6):379–392. doi: 10.1016/0923-2508(89)90014-4. [DOI] [PubMed] [Google Scholar]
  13. Galdiero F., Tufano M. A., Galdiero M., Masiello S., Di Rosa M. Inflammatory effects of Salmonella typhimurium porins. Infect Immun. 1990 Oct;58(10):3183–3186. doi: 10.1128/iai.58.10.3183-3186.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gillespie J., Holt S. C. Growth studies of Wolinella recta, a gram-negative periodontopathogen. Oral Microbiol Immunol. 1987 Sep;2(3):105–111. doi: 10.1111/j.1399-302x.1987.tb00271.x. [DOI] [PubMed] [Google Scholar]
  15. Haapasalo M. Bacteroides buccae and related taxa in necrotic root canal infections. J Clin Microbiol. 1986 Dec;24(6):940–944. doi: 10.1128/jcm.24.6.940-944.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Han Y. H., Smibert R. M., Krieg N. R. Wolinella recta, Wolinella curva, Bacteroides ureolyticus, and Bacteroides gracilis are microaerophiles, not anaerobes. Int J Syst Bacteriol. 1991 Apr;41(2):218–222. doi: 10.1099/00207713-41-2-218. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Holt S. C., Bramanti T. E. Factors in virulence expression and their role in periodontal disease pathogenesis. Crit Rev Oral Biol Med. 1991;2(2):177–281. doi: 10.1177/10454411910020020301. [DOI] [PubMed] [Google Scholar]
  19. Kennell W., Holt S. C. Comparative studies of the outer membranes of Bacteroides gingivalis, strains ATCC 33277, W50, W83, 381. Oral Microbiol Immunol. 1990 Jun;5(3):121–130. doi: 10.1111/j.1399-302x.1990.tb00409.x. [DOI] [PubMed] [Google Scholar]
  20. Listgarten M. A. Structure of the microbial flora associated with periodontal health and disease in man. A light and electron microscopic study. J Periodontol. 1976 Jan;47(1):1–18. doi: 10.1902/jop.1976.47.1.1. [DOI] [PubMed] [Google Scholar]
  21. Loeb M. R., Smith D. H. Outer membrane protein composition in disease isolates of Haemophilus influenzae: pathogenic and epidemiological implications. Infect Immun. 1980 Dec;30(3):709–717. doi: 10.1128/iai.30.3.709-717.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Logan S. M., Trust T. J. Outer membrane characteristics of Campylobacter jejuni. Infect Immun. 1982 Dec;38(3):898–906. doi: 10.1128/iai.38.3.898-906.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Malouin F., Campbell G. D., Halpenny M., Becker G. W., Parr T. R., Jr Outer membrane and porin characteristics of Serratia marcescens grown in vitro and in rat intraperitoneal diffusion chambers. Infect Immun. 1990 May;58(5):1247–1253. doi: 10.1128/iai.58.5.1247-1253.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Matsushita K., Adachi O., Shinagawa E., Ameyama M. Isolation and characterization of outer and inner membranes from Pseudomonas aeruginosa and effect of EDTA on the membranes. J Biochem. 1978 Jan;83(1):171–181. doi: 10.1093/oxfordjournals.jbchem.a131888. [DOI] [PubMed] [Google Scholar]
  25. Mizuno T., Chou M. Y., Inouye M. A comparative study on the genes for three porins of the Escherichia coli outer membrane. DNA sequence of the osmoregulated ompC gene. J Biol Chem. 1983 Jun 10;258(11):6932–6940. [PubMed] [Google Scholar]
  26. Munford R. S., Hall C. L., Rick P. D. Size heterogeneity of Salmonella typhimurium lipopolysaccharides in outer membranes and culture supernatant membrane fragments. J Bacteriol. 1980 Nov;144(2):630–640. doi: 10.1128/jb.144.2.630-640.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Munson R., Jr, Grass S. Purification, cloning, and sequence of outer membrane protein P1 of Haemophilus influenzae type b. Infect Immun. 1988 Sep;56(9):2235–2242. doi: 10.1128/iai.56.9.2235-2242.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Naito Y., Ito N., Okuda K., Takazoe I. The heterogeneity of the outer membrane of selected oral black-pigmented Bacteroides species. Bull Tokyo Dent Coll. 1988 Aug;29(3):111–118. [PubMed] [Google Scholar]
  29. Newell D. G., McBride H., Pearson A. D. The identification of outer membrane proteins and flagella of Campylobacter jejuni. J Gen Microbiol. 1984 May;130(5):1201–1208. doi: 10.1099/00221287-130-5-1201. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  32. Progulske A., Holt S. C. Isolation and characterization of the outer membrane and lipopolysaccharide from Eikenella corrodens. Infect Immun. 1984 Jan;43(1):166–177. doi: 10.1128/iai.43.1.166-177.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sleytr U. B., Messner P. Crystalline surface layers on bacteria. Annu Rev Microbiol. 1983;37:311–339. doi: 10.1146/annurev.mi.37.100183.001523. [DOI] [PubMed] [Google Scholar]
  34. Takada H., Ogawa T., Yoshimura F., Otsuka K., Kokeguchi S., Kato K., Umemoto T., Kotani S. Immunobiological activities of a porin fraction isolated from Fusobacterium nucleatum ATCC 10953. Infect Immun. 1988 Apr;56(4):855–863. doi: 10.1128/iai.56.4.855-863.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tanner A. C., Dzink J. L., Ebersole J. L., Socransky S. S. Wolinella recta, campylobacter concisus, bacteroides gracilis, and Eikenella corrodens from periodontal lesions. J Periodontal Res. 1987 Jul;22(4):327–330. doi: 10.1111/j.1600-0765.1987.tb01593.x. [DOI] [PubMed] [Google Scholar]
  36. Tanner A. C., Haffer C., Bratthall G. T., Visconti R. A., Socransky S. S. A study of the bacteria associated with advancing periodontitis in man. J Clin Periodontol. 1979 Oct;6(5):278–307. doi: 10.1111/j.1600-051x.1979.tb01931.x. [DOI] [PubMed] [Google Scholar]
  37. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tufano M. A., Berlingieri M. T., Sommese L., Galdiero F. Immune response in mice and effects on cells by outer membrane porins from Salmonella typhimurium. Microbiologica. 1984 Oct;7(4):353–366. [PubMed] [Google Scholar]
  39. Tufano M. A., Sommese L., Capasso C., Folgore A., Scafa F., Galdiero E. Comparative study of some biological activities of porins extracted from various microorganisms. Microbiologica. 1986 Oct;9(4):431–442. [PubMed] [Google Scholar]
  40. Van Dyke T. E., Dowell V. R., Jr, Offenbacher S., Snyder W., Hersh T. Potential role of microorganisms isolated from periodontal lesions in the pathogenesis of inflammatory bowel disease. Infect Immun. 1986 Sep;53(3):671–677. doi: 10.1128/iai.53.3.671-677.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Williams G. D., Holt S. C. Characteristics of the outer membrane of selected oral Bacteroides species. Can J Microbiol. 1985 Mar;31(3):238–250. doi: 10.1139/m85-046. [DOI] [PubMed] [Google Scholar]
  42. de Melo M. A., Pechère J. C. Identification of Campylobacter jejuni surface proteins that bind to Eucaryotic cells in vitro. Infect Immun. 1990 Jun;58(6):1749–1756. doi: 10.1128/iai.58.6.1749-1756.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES