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Volume 147, Issue 4

Topological investigations of the FomA porin from and identification of the constriction loop L6

The GenBank accession number for the sequence reported in this paper is X72583.

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Abstract

Porin FomA in the outer membrane of is a trimeric protein, which exhibits permeability properties similar to that of the well-known enterobacterial diffusion porins. The proposed topology model of the FomA monomer depicts the β-barrel motif typical of diffusion porins, consisting of 16 antiparallel β-strands. To investigate the accuracy of the FomA model and assess the topological relationship with other porins, individual deletions of variable size in seven of the eight surface-exposed regions of the porin were genetically engineered. Deletions in the predicted loops L1 to L7 were tolerated by the FomA porins, as judged by a normal assembly in the outer membrane of and a sustained pore-forming ability. Deletions in the largest proposed external region, loop L6, made the FomA porins considerably more permeable to antibiotics, indicating larger pore channels. The distinctly increased uptake rates and size exclusion limits displayed by the L6 deletion mutant porins, suggest that loop L6 folds back into the β-barrel thereby constricting the native FomA channel. Thus, the position of the channel constriction loop appears to be shifted towards the C terminus in the FomA porin, as compared to the crystal structures of five non-specific diffusion porins.

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Keyword(s): deletion mutagenesis , eyelet loop , membrane topology , outer membrane protein and β-lactam uptake
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2001-04-01
2024-04-30
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References

  1. Agterberg M., Adriaanse H., Tijhaar E., Resink A., Tommassen J. 1989; Role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein. Eur J Biochem 185:365–370 [CrossRef]
     [Google Scholar]
  2. Bakken V., Jensen H. B. 1986; Outer membrane proteins of Fusobacterium nucleatum Fev1. J Gen Microbiol 132:1069–1078
     [Google Scholar]
  3. Benson S. A., Decloux A. 1985; Isolation and characterization of outer membrane permeability mutants in Escherichia coli K-12. J Bacteriol 161:361–367
     [Google Scholar]
  4. Benson S. A., Occi J. L., Sampson B. A. 1988; Mutations that alter the pore function of the OmpF porin of Escherichia coli K12. J Mol Biol 203:961–970 [CrossRef]
     [Google Scholar]
  5. Bolstad A. I., Tommassen J., Jensen H. B. 1994; Sequence variability of the 40-kDa outer membrane proteins of Fusobacterium nucleatum strains and a model for the topology of the proteins. Mol Gen Genet 244:104–110 [CrossRef]
     [Google Scholar]
  6. Bolstad A. I., Høgh B. T., Jensen H. B. 1995; Molecular characterization of a 40-kDa outer membrane protein, FomA, of Fusobacterium periodonticum and comparison with Fusobacterium nucleatum . Oral Microbiol Immunol 10:257–264 [CrossRef]
     [Google Scholar]
  7. Bosch D., Leunissen J., Verbakel J., Tommassen J, de Jong M., van Erp H. 1986; Periplasmic accumulation of truncated forms of outer-membrane PhoE protein of Escherichia coli K-12. J Mol Biol 189:449–455 [CrossRef]
     [Google Scholar]
  8. Bosch D., Voorhout W., Tommassen J. 1988; Export and localization of N-terminally truncated derivatives of Escherichia coli K-12 outer membrane protein PhoE. J Biol Chem 263:9952–9957
     [Google Scholar]
  9. Bosch D., Scholten M., Verhagen C., Tommassen J. 1989; The role of the carboxy-terminal membrane-spanning fragment in the biogenesis of Escherichia coli K12 outer membrane protein PhoE. Mol Gen Genet 216:144–148 [CrossRef]
     [Google Scholar]
  10. Cowan S. W. 1993; Bacterial porins: lessons from three high-resolution structures. Curr Opin Struct Biol 3:501–507 [CrossRef]
     [Google Scholar]
  11. Cowan S. W., Schirmer T., Rummel G., Steiert M., Ghosh R., Pauptit R. A., Jansonius J. N., Rosenbusch J. P. 1992; Crystal structures explain functional properties of two E. coli porins. Nature 358:727–733 [CrossRef]
     [Google Scholar]
  12. Egli C., Leung W. K., Muller K. H., Hancock R. E., McBride B. C. 1993; Pore-forming properties of the major 53-kilodalton surface antigen from the outer sheath of Treponema denticola . Infect Immun 61:1694–1699
     [Google Scholar]
  13. Hirsch A., Breed J., Saxena K., Richter O. M., Ludwig B., Diederichs K., Welte W. 1997; The structure of porin from Paracoccus denitrificans at 3·1 Å resolution. FEBS Lett 404:208–210 [CrossRef]
     [Google Scholar]
  14. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. 1989; Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59 [CrossRef]
     [Google Scholar]
  15. Huang H., Jeanteur D., Pattus F., Hancock R. E. 1995; Membrane topology and site-specific mutagenesis of Pseudomonas aeruginosa porin OprD. Mol Microbiol 16:931–941 [CrossRef]
     [Google Scholar]
  16. Jeanteur D., Lakey J. H., Pattus F. 1991; The bacterial porin superfamily: sequence alignment and structure prediction. Mol Microbiol 5:2153–2164 [CrossRef]
     [Google Scholar]
  17. Jensen H. B., Skeidsvoll J., Fjellbirkeland A., Høgh B., Puntervoll P., Kleivdal H., Tommassen J. 1996; Cloning of the fomA gene, encoding the major outer membrane porin of Fusobacterium nucleatum ATCC10953. Microb Pathog 21:331–342 [CrossRef]
     [Google Scholar]
  18. Karshikoff A., Spassov V., Cowan S. W., Ladenstein R., Schirmer T. 1994; Electrostatic properties of two porin channels from Escherichia coli . J Mol Biol 240:372–384 [CrossRef]
     [Google Scholar]
  19. Kaufman J., DiRienzo J. M. 1989; Isolation of a corncob (coaggregation) receptor polypeptide from Fusobacterium nucleatum . Infect Immun 57:331–337
     [Google Scholar]
  20. Kinder S. A., Holt S. C. 1993; Localization of the Fusobacterium nucleatum T18 adhesin activity mediating coaggregation with Porphyromonas gingivalis T22. J Bacteriol 175:840–850
     [Google Scholar]
  21. Klebba P. E., Hofnung M., Charbit A. 1994; A model of maltodextrin transport through the sugar-specific porin, LamB, based on deletion analysis. EMBO J 13:4670–4675
     [Google Scholar]
  22. Kleivdal H., Benz R., Jensen H. B. 1995; The Fusobacterium nucleatum major outer-membrane protein (FomA) forms trimeric, water-filled channels in lipid bilayer membranes. Eur J Biochem 233:310–316 [CrossRef]
     [Google Scholar]
  23. Kleivdal H., Benz R., Tommassen J., Jensen H. B. 1999; Identification of positively charged residues of FomA porin of Fusobacterium nucleatum which are important for pore function. Eur J Biochem 260:818–824 [CrossRef]
     [Google Scholar]
  24. Kolenbrander P. E., London J. 1993; Adhere today, here tomorrow: oral bacterial adherence. J Bacteriol 175:3247–3252
     [Google Scholar]
  25. Kreusch A., Neubuser A., Schiltz E., Weckesser J., Schulz G. E. 1994; Structure of the membrane channel porin from Rhodopseudomonas blastica at 2·0 Å resolution. Protein Sci 3:58–63
     [Google Scholar]
  26. Levinthal C., Signer E. R., Fetherolf K. 1962; Reactivation and hybridization of reduced alkaline phosphatase. Proc Natl Acad Sci USA 48:1230–1237 [CrossRef]
     [Google Scholar]
  27. Lou K. L., Saint N., Prilipov A., Rummel G., Benson S. A., Rosenbusch J. P., Schirmer T. 1996; Structural and functional characterization of OmpF porin mutants selected for larger pore size. I. Crystallographic analysis. J Biol Chem 271:20669–20675 [CrossRef]
     [Google Scholar]
  28. Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L. 1975; Electrophoretic resolution of the ‘major outer membrane protein’ of Escherichia coli K12 into four bands. FEBS Lett 58:254–258 [CrossRef]
     [Google Scholar]
  29. Merck K. B., Verheij H. M., Tommassen J, de Cock H. 1997; Topology of the outer membrane phospholipase A of Salmonella typhimurium . J Bacteriol 179:3443–3450
     [Google Scholar]
  30. Nikaido H. 1994; Porins and specific diffusion channels in bacterial outer membranes. J Biol Chem 269:3905–3908
     [Google Scholar]
  31. Nikaido H., Vaara M. 1985; Molecular basis of bacterial outer membrane permeability. Microbiol Rev 49:1–32
     [Google Scholar]
  32. Olsen G. J., Woese C. R., Overbeek R. 1994; The winds of (evolutionary) change: breathing new life into microbiology. J Bacteriol 176:1–6
     [Google Scholar]
  33. Puntervoll P., Kleivdal H., Dahl K. O., Bitter W., Tommassen J., Jensen H. B. 2000; The Fusobacterium nucleatum porin FomA possesses the general topology of the non-specific porins. Microbiology 146:1437–1445
     [Google Scholar]
  34. Rocque W. J., McGroarty E. J. 1990; Structure and function of an OmpC deletion mutant porin from Escherichia coli K-12. Biochemistry 29:5344–5351 [CrossRef]
     [Google Scholar]
  35. Saint N., Lou K. L., Widmer C., Luckey M., Schirmer T., Rosenbusch J. P. 1996; Structural and functional characterization of OmpF porin mutants selected for larger pore size. II. Functional characterization. J Biol Chem 271:20676–20680 [CrossRef]
     [Google Scholar]
  36. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  37. Schmid B., Maveyraud L., Kromer M., Schulz G. E. 1998; Porin mutants with new channel properties. Protein Sci 7:1603–1611 [CrossRef]
     [Google Scholar]
  38. Srikumar R., Dahan D., Arhin F. F., Tawa P., Diederichs K., Coulton J. W. 1997; Porins of Haemophilus influenzae type b mutated in loop 3 and in loop 4. J Biol Chem 272:13614–13621 [CrossRef]
     [Google Scholar]
  39. Tommassen J., Lugtenberg B, van Tol H. 1983; The ultimate localization of an outer membrane protein of Escherichia coli K-12 is not determined by the signal sequence. EMBO J 2:1275–1279
     [Google Scholar]
  40. Van Gelder P., Saint N., Rosenbusch J. P., Tommassen J, van Boxtel R. 1997; Pore functioning of outer membrane protein PhoE of Escherichia coli : mutagenesis of the constriction loop L3. Protein Eng 10:699–706 [CrossRef]
     [Google Scholar]
  41. Weiss M. S., Schulz G. E. 1992; Structure of porin refined at 1·8 Å resolution. J Mol Biol 227:493–509 [CrossRef]
     [Google Scholar]
  42. Zimmermann W., Rosselet A. 1977; Function of the outer membrane of Escherichia coli as a permeability barrier to beta-lactam antibiotics. Antimicrob Agents Chemother 12:368–372 [CrossRef]
     [Google Scholar]
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Topological investigations of the FomA porin from Fusobacterium nucleatum and identification of the constriction loop L6The GenBank accession number for the sequence reported in this paper is X72583.
Microbiology 147, 1059 (2001); https://doi.org/10.1099/00221287-147-4-1059
/content/journal/micro/10.1099/00221287-147-4-1059
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