Department of Molecular Biology, University of Bergen, HiB, Thormøhlensgate 55, N-5020 Bergen, Norway1
Department of Molecular Microbiology and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands2
Author for correspondence: Pl Puntervoll. Tel: +47 55584500. Fax: +47 55589683. e-mail: pal.puntervoll{at}mbi.uib.no
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ABSTRACT |
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Keywords: outer-membrane proteins, porins, Fusobacterium nucleatum, topology, epitope insertion mutagenesis
Abbreviations: wtFomA, wild-type FomA; mFomA, mutant FomA; OMP, outer-membrane protein; SFV, Semliki Forest Virus
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INTRODUCTION |
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Fusobacterium nucleatum is an anaerobic, Gram-negative bacterium and one of the predominant species among the periodontal bacteria (Bolstad et al., 1996 ). It plays a major role in the colonization of the teeth by acting as a bridge between early and late colonizers of the tooth surface (Kolenbrander & London, 1993
). The fusobacterial major outer-membrane protein (OMP), FomA, has been suggested to participate in this coaggregation process by directly binding to Streptococcus sanguis (Kaufman & DiRienzo, 1989
). It has been shown that FomA forms trimeric, water-filled channels in lipid bilayer membranes, acting as non-specific pores (Kleivdal et al., 1995
). The deduced amino acid sequences of the FomA proteins from several strains (Bolstad et al., 1995
, 1994
) revealed no sequence similarity to other known porins. However, a topology model was proposed on the basis of an alignment between the FomA sequences from three F. nucleatum strains and of the structural principles derived from OMPs of Escherichia coli (Bolstad et al., 1994
). The fomA gene of F. nucleatum ATCC 10953 has been successfully cloned and expressed in E. coli (Jensen et al., 1996
) and this has rendered possible the use of mutagenesis as a strategy for probing the proposed topology model.
Epitope insertion mutagenesis has proved to be a valuable experimental approach to obtain structural information on OMPs in the absence of a crystal structure. Since the approach was introduced in the study of LamB (Charbit et al., 1986 ), it has been successfully applied to several bacterial OMPs (Agterberg et al., 1987
; Merck et al., 1997
; Moeck et al., 1994
; Sukhan & Hancock, 1995
; Taylor et al., 1990
). In a recent study on the topology of LamB, results obtained with the epitope insertion method coincided well with the X-ray model (Newton et al., 1996
). In the present study, this approach was applied to the FomA protein.
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METHODS |
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Mutagenesis.
Plasmid pHB14 (Jensen et al., 1996 ) is a pACYC184 derivative and contains the part of the fomA gene encoding the mature part of the FomA protein, immediately downstream of the phoE promoter and signal sequence-encoding part of phoE. The pHB14 plasmid was the parent to all constructs made in this study.
Novel BamHI sites were introduced after the codons for aa 19 and 287 by PCR (Ho et al., 1989 ), resulting in plasmids pSB19 and pSB287, and after the codons for aa 105, 143 and 191 by the double-primer method (Ohmori, 1994
), resulting in plasmids pHK105, pHK143 and pHK191.
For BamHI-linker insertion mutagenesis, pHB14 was randomly linearized by digesting with the frequently cutting restriction endonuclease MseI in the presence of ethidium bromide (1050 ng µl-1). The MseI ends of the linearized plasmid were made blunt by using the Klenow fragment of E. coli DNA polymerase I. Linear blunt-ended pHB14 (10100 ng) was ligated to 1 µg of a dephosphorylated self-annealing 12-mer BamHI oligonucleotide (5'-CGCGGATCCGCG-3'). The great excess of BamHI linker resulted in linear pHB14 with one BamHI oligonucleotide ligated to each end. The ligation mixture was subjected to agarose gel electrophoresis to remove excess BamHI linker, the band corresponding to the linear plasmid was excised from the gel and the DNA was extracted with the JETSORB kit (Genomed). The isolated DNA was heated to 65 °C to remove the non-covalently bound linker strands, followed by slow cooling to room temperature, resulting in circularization of the plasmids due to annealing of the complementary BamHI oligonucleotide strands. The circularized plasmids were used to transform E. coli strain DH5. Transformants were screened by restriction enzyme analysis. Novel BamHI sites were introduced by linker insertions in MseI sites after the codons for aa 97, 116, 130, 138, 167, 211, 215, 236, 245 and 333, resulting in plasmids designated pMB followed by the respective codon numbers.
The constructs containing unique BamHI sites were used to insert a double-stranded oligonucleotide encoding a Semliki Forest Virus (SFV) B-cell epitope (Merck et al., 1997 ; Snijders et al., 1991
), consisting of aa 240255 of the E2 protein of SFV (Table 1
). This was achieved by linearizing the various plasmids with the restriction endonuclease BamHI, followed by ligating 1 µg of the SFV linker to 10100 ng linearized plasmid. The ligation mixture was used to transform E. coli strain DH5
. Linkers encoding the SFV epitope were successfully introduced into 12 of the novel BamHI sites (Table 2
). Insertion of the BamHI linker into the MseI sites (i.e. the pMB plasmids) resulted in a change of reading frame. To accommodate in-frame mutations when inserted into the pMB mutants, linkers C' and E' were used (Table 1
). They are the same as C and E, respectively, but contain one additional base. The plasmids carrying SFV epitope insertions were named after their respective parental plasmids followed by SFV (e.g. pMB236SFV) and the mutant proteins encoded by these plasmids were named SFV followed by the number of the last unchanged amino acid (e.g. SFV-236) (Table 2
).
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All mutations were confirmed by using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit and the ABI PRISM 377 DNA Sequencer (Applied Biosystems).
SDS-PAGE and Western blotting.
SDS-PAGE was performed as described by Lugtenberg et al. (1975) . Prior to electrophoresis, the samples were incubated for 5 min in sample buffer either at room temperature or at 95 °C. Proteins were visualized by staining with the fluorescent dye SYPRO Orange (Molecular Probes) and protein quantification was performed on a Fluorescent Image Analyser FLA-2000 (Fujifilm). Western blotting was performed with the Bio-Rad Mini Trans-Blot System by the procedure described by Towbin et al. (1979)
. The anti-FomA antiserum
239 (Bakken et al., 1989a
) and horseradish-peroxidase-coupled goat anti-rabbit antibody (Bio-Rad) were used as primary and secondary antibody respectively. Immunoblots were developed with 4-chloro-1-naphtol (Bio-Rad).
Preparation of crude cell envelopes.
Crude cell envelopes were prepared by pelleting cells from 10 ml of an overnight culture and resuspending the pellet in 1 ml 50 mM Tris/HCl, 2 mM EDTA (pH 8·5). The cells were disrupted by ultrasonication at 0 °C. Intact cells were removed by centrifugation (1000 g, 20 min) and the crude cell envelopes were pelleted by centrifugation at 15000 g for 45 min. The pellet was resuspended in 60 µl 0·2% Triton X-100 in 2 mM Tris/HCl (pH 8·0).
Trypsin accessibility experiments.
Cells were incubated with trypsin as described by Merck et al. (1997) . Cells from an overnight culture were pelleted and resuspended in a buffer containing 0·1 M Tris/HCl (pH 8·0), 0·25 M sucrose and 10 mM MgCl2. The samples were incubated for 60 min at 37 °C with 0·1 mg trypsin ml-1. The treated cells were washed twice in 1 ml digestion buffer, containing 500 µg trypsin inhibitor ml-1 to stop the reactions and the protein patterns were analysed immediately by SDS-PAGE and Western blotting.
Immunofluorescence microscopy.
Immunofluorescence microscopy was performed as described by Merck et al. (1997) . Mouse mAbs directed either against the SFV B-cell epitope (1:500) (generously provided by C. Kraaijeveld, Utrecht University, The Netherlands) or against the periplasmic C-terminal tail of OmpA (1:100) (generously provided by M. Kleerebezem, University Hospital, Utrecht, The Netherlands) were used as primary antibodies. Texas-red-conjugated goat anti-mouse antibody (1:100) (Southern Biotechnology Associates) was used as secondary antibody. Labelled cells were visualized in a Leica DM IBRE fluorescence microscope by both fluorescence and phase-contrast microscopy at 100x magnification.
In vivo porin activity assay.
Porin activity was assessed by the ß-lactamase assay, originally described by Zimmermann & Rosselet (1977) . In this assay, the diffusion of externally added ß-lactam antibiotics through the outer membrane is the rate-limiting step in their degradation by periplasmic ß-lactamase. The various mutant proteins were expressed in E. coli strain CE1224 carrying plasmid pBR322 to ensure a high expression of ß-lactamase. The bacteria were grown overnight under phosphate-limiting conditions to induce expression of FomA from the phoE promoter. The bacterial cells were washed once with buffer A (10 mM HEPES, 5 mM MgCl2, 0·9% NaCl, pH 7) and subsequently resuspended in buffer A to OD660=1·0. The bacterial suspension was diluted 90 times in buffer A and aliquoted into five tubes, 0·9 ml in each. Starch/iodine reagent was prepared by mixing 10 ml 2 M acetic acid, 4 ml 1 M NaWO4 and 2 ml 2% (w/v) starch solution (in 1 M acetic acid) with 200 µl 8 mM I2, 320 mM KI, and adjusting the volume to 20 ml with water. The assay was performed in a 22 °C water bath and the bacterial suspensions and starch/iodine reagent were preincubated for at least 10 min. The reaction was initiated by adding 100 µl 8 mM cephaloridine to the bacterial suspensions and was stopped after 0, 5, 10, 15 and 20 min by adding 1 ml starch/iodine reagent. A623 was measured exactly 20 min after the reaction was stopped and the rate of uptake was calculated (
A623 min-1). Supernatants of the cell suspensions were examined for ß-lactamase activity, revealing that in all cases studied, leakage of ß-lactamase was negligible.
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RESULTS |
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Surface exposure of the inserted SFV epitope
All mFomAs containing the inserted SFV epitope were recognized by an anti-SFV epitope mAb on a Western blot (not shown). To assess potential exposure of the epitope on the cell surface, indirect immunofluorescence labelling was performed. CE1224 cells expressing wtFomA were used as a negative control. In addition, an mAb directed against the periplasmic C-terminal part of OmpA was used as a control for outer-membrane integrity. None of the cells harbouring mutated fomA genes were labelled with the OmpA mAb (not shown), verifying that the cells assayed were intact. Four of the 11 SFV epitope mutant proteins were clearly recognized by the anti-SFV epitope mAb, namely SFV-105, SFV-116, SFV-191 and SFV-287 (Fig. 3), demonstrating that the insertion sites 105, 116, 191 and 287 are in or near surface-exposed loops. All four proteins had rates of uptake of cephaloridine comparable to wtFomA, consistent with the notion that the proposed loops L3, L5 and L7 are indeed surface-exposed.
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DISCUSSION |
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According to the topology model, insertion sites 97, 105 and 116 are all postulated to be situated in or near loop L3. In the bacterial porins of which the structure has been resolved, including the PhoE protein of E. coli, loop L3 is not exposed to the cell surface, but forms a constriction within the channel at half the height of the membrane (Cowan, 1993 ; Montal, 1996
; Weiss et al., 1991
). Insertion mutants carrying multiple copies of a 9 aa epitope inserted into the third loop of PhoE displayed results that were consistent with loop L3 being located within the pore channel, since at least three copies of the epitope were needed to expose one copy of the epitope to the surface (Struyvé et al., 1993
). The fact that both SFV-105 and SFV-116 were recognized by the anti-SFV mAb on intact cells, together with the trypsin accessibility of SFV-97, strongly indicate that loop L3 of the FomA protein is surface-exposed. Although the permeability rate for cephaloridine seemed to be somewhat affected by the insertion made after aa 97, one would expect the permeability rates to be more strongly affected by such a large insertion if the third loop existed as a constriction loop (Struyvé et al., 1993
). Thus, it seems highly unlikely that the third loop of FomA folds into the barrel in the same manner as the third loop of PhoE (Cowan et al., 1992
).
Whereas the results suggest that site 116 is cell-surface-exposed, this site was postulated to be within a membrane-spanning segment according to the topology model. The SFV epitope appears to be located entirely in the N-terminal part of the insert (Fernandez et al., 1998 ) and the C-terminal part could thus function as a spacer that pushes the epitope to the surface. In addition, the 4 aa C-terminal to the epitope appear to be able to sustain the amphipathic properties of the putative transmembrane segment. Hence, it is entirely possible that insertion site 116 is actually located in a transmembrane segment, whereas the inserted epitope is accessible at the cell surface. This phenomenon has been observed in an epitope insertion mutagenesis study on LamB of E. coli, where some sites predicted to be external were in fact situated in transmembrane segments, as revealed by the X-ray structure (Newton et al., 1996
). The fact that the cephaloridine uptake rate of SFV-116 was at wild-type level strongly suggests that the pore activity remains unaffected by this insertion. This may be in line with the interpretation mentioned above, but could also imply that the model is inaccurate in this area.
It has been suggested that instead of the third loop, the longest loop according to the topology model, loop L6, might form the constriction in the FomA channel (Bolstad et al., 1994 ). The results (Fig. 4
), however, suggest that loop L6 is at least partly exposed to the surface. The fact that the SFV epitope insertions at these sites seemed to be less well tolerated than the 6 aa insertions (Fig. 2
, Table 2
), however, suggests an important structural or functional role. A significant amount of SFV-236 is probably incorrectly assembled in the membrane (Fig. 2
) and hence non-functional. It is thus not safe to conclude that the observed reduction in the uptake rate of cephaloridine is due to a narrowing in the pore.
The surface exposure of L1 could not be demonstrated by the linker insertion approach, but the fact that SFV-19 showed wild-type pore activity may indicate surface exposure, since the only other SFV epitope insertion mutants that displayed wild-type pore activity, namely SFV-105, SFV-116, SFV-191 and SFV-287 all had the SFV epitope exposed to the surface. The surface exposure of L1 is supported by the fact that E29 becomes the new N-terminal amino acid upon proteolytic digestion of intact cells of F. nucleatum (Bakken et al., 1989b ), showing that at least part of the proposed loop L1 is indeed surface-exposed.
The SFV epitope insertion after aa 143 was not detectable on intact cells. However, the results obtained with SFV-138 demonstrate the surface exposure of the region in the proximity of aa 138. The reduced uptake rate of cephaloridine for both mutants carrying insertions in the proposed loop L4 may indicate that L4 is situated more closely to the pore channel than, for instance, loops L5 and L7, and is thus in line with the fact that loop L4 is poorly surface-exposed.
The work presented here demonstrates the surface exposure of five of the postulated loops, namely loops L3 to L7. Although exposure of loop L1 could not be confirmed by SFV-19, Bakken et al. (1989b) have provided chemical evidence that this loop indeed is surface-exposed in F. nucleatum. Thus, loops L2 and L8 are the only loops for which there is no experimental evidence. Kleivdal et al. (1999)
recently identified positively charged amino acids in the FomA protein that were found to be important to pore function, which supported the topology model, where these amino acids are predicted to be located in transmembrane segments 5 and 6 and contribute to a hydrophilic pore lumen. In addition, when the FomA protein sequences were subjected to the recently published neural network topology prediction method (Diederichs et al., 1998
), a virtually identical topology model was predicted (Fig. 1b
), in spite of the fact that the FomA proteins have no sequence similarity to the proteins used to train the neural network. In conclusion, the topology model seems to a great extent to be supported by the presented results, and unlike the other non-specific porins, the third loop of FomA does not seem to be internal.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bakken, V., Aaro, S., Hofstad, T. & Vasstrand, E. N. (1989a). Outer membrane proteins as major antigens of Fusobacterium nucleatum.FEMS Microbiol Immunol 1, 473-483.[Medline]
Bakken, V., Aaro, S. & Jensen, H. B. (1989b). Purification and partial characterization of a major outer-membrane protein of Fusobacterium nucleatum.J Gen Microbiol 135, 3253-3262.[Medline]
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.[Medline]
Bolstad, A. I., Hogh, 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.[Medline]
Bolstad, A. I., Jensen, H. B. & Bakken, V. (1996). Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum.Clin Microbiol Rev 9, 55-71.[Abstract]
Charbit, A., Boulain, J. C., Ryter, A. & Hofnung, M. (1986). Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope; expression at the cell surface.EMBO J 5, 3029-3037.[Abstract]
Cowan, S. W. (1993). Bacterial porins: lessons from three high-resolution structures.Curr Opin Struct Biol 3, 501-507.
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.[Medline]
Dekker, N., Merck, K., Tommassen, J. & Verheij, H. M. (1995). In vitro folding of Escherichia coli outer-membrane phospholipase A.Eur J Biochem 232, 214-219.[Abstract]
Diederichs, K., Freigang, J., Umhau, S., Zeth, K. & Breed, J. (1998). Prediction by a neural network of outer membrane ß-strand protein topology.Protein Sci 7, 2413-2420.
Fernandez, I. M., Harmsen, M., Benaissa-Trouw, B. J., Stuij, I., Puyk, W., Meloen, R. H., Snippe, H. & Kraaijeveld, C. A. (1998). Epitope polarity and adjuvants influence the fine specificity of the humoral response against Semliki Forest virus specific peptide vaccines.Vaccine 16, 1531-1536.[Medline]
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids.J Mol Biol 166, 557-580.[Medline]
Heller, K. B. (1978). Apparent molecular weights of a heat-modifiable protein from the outer membrane of Escherichia coli in gels with different acrylamide concentrations. J Bacteriol 134, 1181-1183.[Medline]
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.[Medline]
Jensen, H. B., Skeidsvoll, J., Fjellbirkeland, A., Hogh, 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.[Medline]
Kaufman, J. & DiRienzo, J. M. (1989). Isolation of a corncob (coaggregation) receptor polypeptide from Fusobacterium nucleatum.Infect Immun 57, 331-337.[Medline]
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.[Abstract]
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.
Kolenbrander, P. E. & London, J. (1993). Adhere today, here tomorrow: oral bacterial adherence.J Bacteriol 175, 3247-3252.[Medline]
Levinthal, C., Signer, E. R. & Fetherolf, K. (1962). Reactivation and hybridization of reduced alkaline phosphatase.Proc Natl Acad Sci USA 48, 1230-1237.[Medline]
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.[Medline]
Merck, K. B., de Cock, H., Verheij, H. M. & Tommassen, J. (1997). Topology of the outer membrane phospholipase A of Salmonella typhimurium.J Bacteriol 179, 3443-3450.[Abstract]
Moeck, G. S., Bazzaz, B. S., Gras, M. F., Ravi, T. S., Ratcliffe, M. J. & Coulton, J. W. (1994). Genetic insertion and exposure of a reporter epitope in the ferrichrome-iron receptor of Escherichia coli K-12.J Bacteriol 176, 4250-4259.[Abstract]
Montal, M. (1996). Protein folds in channel structure. Curr Opin Struct Biol 6, 499-510.[Medline]
Nakae, T. (1976). Identification of the outer membrane protein of E. coli that produces transmembrane channels in reconstituted vesicle membranes.Biochem Biophys Res Commun 71, 877-884.[Medline]
Newton, S. M., Klebba, P. E., Michel, V., Hofnung, M. & Charbit, A. (1996). Topology of the membrane protein LamB by epitope tagging and a comparison with the X-ray model.J Bacteriol 178, 3447-3456.[Abstract]
Ohmori, H. (1994). A new method for strand discrimination in sequence-directed mutagenesis.Nucleic Acids Res 22, 884-885.[Medline]
Snijders, A., Benaissa Trouw, B. J., Oosterlaken, T. A. & 7 other authors (1991). Identification of linear epitopes on Semliki Forest virus E2 membrane protein and their effectiveness as a synthetic peptide vaccine. J Gen Virol 72, 557565.[Abstract]
Struyvé, M., Visser, J., Adriaanse, H., Benz, R. & Tommassen, J. (1993). Topology of PhoE porin: the eyelet region.Mol Microbiol 7, 131-140.[Medline]
Sukhan, A. & Hancock, R. E. (1995). Insertion mutagenesis of the Pseudomonas aeruginosa phosphate-specific porin OprP.J Bacteriol 177, 4914-4920.[Abstract]
Taylor, I. M., Harrison, J. L., Timmis, K. N. & OConnor, C. D. (1990). The TraT lipoprotein as a vehicle for the transport of foreign antigenic determinants to the cell surface of Escherichia coli K12: structure-function relationships in the TraT protein.Mol Microbiol 4, 1259-1268.[Medline]
Tommassen, J. & Lugtenberg, B. (1984). Amino terminus of outer membrane PhoE protein: localization by use of a bla-phoE hybrid gene.J Bacteriol 157, 327-329.[Medline]
Tommassen, J., van Tol, H. & Lugtenberg, B. (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.[Medline]
Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci USA 76, 4350-4354.[Abstract]
Van Gelder, P., Saint, N., Phale, P., Eppens, E. F., Prilipov, A., van Boxtel, R., Rosenbusch, J. P. & Tommassen, J. (1997). Voltage sensing in the PhoE and OmpF outer membrane porins of Escherichia coli: role of charged residues.J Mol Biol 269, 468-472.[Medline]
Weiss, M. S., Abele, U., Weckesser, J., Welte, W., Schiltz, E. & Schulz, G. E. (1991). Molecular architecture and electrostatic properties of a bacterial porin.Science 254, 1627-1630.[Medline]
Welte, W., Nestel, U., Wacker, T. & Diederichs, K. (1995). Structure and function of the porin channel.Kidney Int 48, 930-940.[Medline]
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.[Medline]
Received 17 November 1999;
revised 1 February 2000;
accepted 27 February 2000.