Department of Molecular Biology, University of Bergen, HiB, Thormøhlensgate 55, N-5020 Bergen, Norway1
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: Fusobacterium nucleatum, porins, outer-membrane proteins, circular dichroism
Abbreviations: CD, circular dichroism; FomA, fusobacterial outer-membrane protein A; OBG, octyl ß-glucopyranoside; OM, outer membrane; OMP, outer-membrane protein
a Present address: Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Germany.
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INTRODUCTION |
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The Gram-negative anaerobe Fusobacterium nucleatum is an oral bacterium that exists as a part of the normal oral microflora (Bolstad et al., 1996 ). It has a pathogenic potential and is implicated in periodontal diseases as well as being the most common periodontal bacterium in clinical infections of other body sites (Moore & Moore, 1994
). The major OMP of F. nucleatum, FomA, has been shown to function as a non-specific porin in lipid bilayer membranes (Kleivdal et al., 1995
), and to function as a porin in vivo when expressed recombinantly in Escherichia coli (Kleivdal et al., 1999
). A topology model, suggesting that the FomA protein possesses the general topology of the non-specific porins, has been proposed on the basis of structural principles derived for OMPs of E. coli (Bolstad et al., 1994
). This model was largely confirmed by the demonstration of the surface exposure of loops L3 to L7 by epitope insertion mutagenesis (Puntervoll et al., 2000
), together with the fact that deletions in loops L1 to L7 did not impede the pore function (Kleivdal et al., 2001
). In addition, deletions made in loop L6 led to a marked increase in the uptake of antibiotics through the FomA porin (Kleivdal et al., 2001
), suggesting that this loop may function analogously to the pore constriction loop L3 of the structured non-specific porins of E. coli and Rhodobacter capsulatus (Cowan, 1993
). Furthermore, a cluster of arginines that contributes to the constriction of the pore has been identified, similar to the cluster observed in the matrix porin OmpF of E. coli, but positioned closer to the C terminus (Kleivdal et al., 1999
). We recently proposed that these structural deviations from the well-known porins may imply that FomA belongs to a separate subgroup of non-specific porins, reflecting the evolutionary distance between the fusobacteria and the proteobacteria (Kleivdal et al., 2001
), but further structural information is clearly needed to establish whether this actually is the case. In the present study, we present the first physicochemical evidence that FomA is a ß-barrel, and examine the role of the proposed loop L1. The results presented here indicate that the proposed loop L1 is located in the periplasm, and that the FomA protein is a 14-stranded ß-barrel.
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METHODS |
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Escherichia coli strain DH5 (Hanahan, 1983
) was used for cloning purposes. The porin-deficient E. coli K-12 strain CE1224 (Tommassen et al., 1983
) was used to express recombinant FomA protein as previously described (Puntervoll et al., 2000
). The E. coli strains AK101 and DH5
mutS were used with the double-primer method for site-directed mutagenesis (Ohmori, 1994
). E. coli was grown aerobically at 37 °C in LB medium, or, to impose phosphate limitation, in a low-phosphate medium described by Levinthal et al. (1962)
. For large-scale growth under low-phosphate conditions, E. coli was grown in 200 ml LB medium overnight; the cells were harvested and washed twice in a low-phosphate medium and used to inoculate a 1 l low-phosphate medium culture, which was incubated overnight. The antibiotics chloramphenicol (35 µg ml-1), ampicillin (50 µg ml-1) and tetracycline (10 µg ml-1) were added for selective purposes when necessary.
Plasmids and constructs.
The plasmid pHB14 (Jensen et al., 1996 ) contains the part of the fomA gene encoding the mature part of the protein, immediately downstream of the phoE promoter and signal sequence encoding part of phoE, and was used to express the FomA protein recombinantly in E. coli in a functional manner (Jensen et al., 1996
). Plasmid pHB14E2C was constructed by site-directed mutagenesis using the double-primer method (Ohmori, 1994
). The pTZ19Urrh-derived phagemid pHKUrrh containing the chimeric phoE/fomA gene from pHB14 and a defective ori region (Kleivdal et al., 1999
) was used to produce single-stranded DNA (Sambrook et al., 1989
). The E2C (5'-GGTGCAGGCATAACACATGCAGCCTGTACAGATGC-3') and ori repair (5'-GGGAAGCGTGGCGCTTTCTCATAG-3') primers were used, and the site-directed mutagenesis was performed as described by Kleivdal et al. (1999)
. The pHB14E2C plasmid sequence was confirmed by using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit and the ABI PRISM 377 DNA Sequencer (Applied Biosystems).
Isolation of FomA from the outer membrane by detergent extraction.
The procedure described below was used to isolate FomA protein from the outer membrane of both F. nucleatum and E. coli. Bacterial cells from 1 l of the desired culture were harvested in the late exponential phase of growth by centrifugation, washed twice with 100 ml 50 mM Tris/HCl (pH 7·7), resuspended in 25 ml of the same buffer, and disrupted by passage through a French pressure cell three times at 900 p.s.i. (6·2 MPa). The samples were collected in pre-cooled tubes, and intact cells were removed by a low-speed centrifugation at 1000 g for 20 min at 4 °C. All succeeding centrifugations were performed at 20000 g for 45 min. The cell envelopes were isolated by centrifugation at 4 °C. The cell envelopes of F. nucleatum were resuspended in 20 ml 2% Triton X-100, 10 mM MgCl2, 10 mM HEPES (pH 7·4), sonicated for 30 min in a water bath sonicator, incubated for 45 min at room temperature, and subjected to centrifugation at 4 °C. The cell envelopes of E. coli were resuspended in 20 ml 0·05% CHAPS, 50 mM Tris/HCl, 20 mM EDTA (pH 8·0), incubated at room temperature for 10 min on a rotating wheel, and subjected to centrifugation at 14 °C. The supernatant was discarded, and the pellet was resuspended in 10 ml 1% octyl ß-glucopyranoside (OBG), 50 mM Tris/HCl, 20 mM EDTA (pH 8·0), and incubated with vigorous shaking (250 r.p.m.) at 37 °C for 30 min. The residual cell envelope material was pelleted by centrifugation at 24 °C, and the supernatant containing the extracted proteins was collected and stored at 4 °C.
Purification of FomA by chromatography.
Anion-exchange chromatography was used as the first step in the purification of FomA. A 20 ml Q-Sepharose column (1·5 cm diameter) was equilibrated with 20 mM Tris/HCl buffer (pH 8·0), and the flow-through of protein was monitored by measuring A280. When the applied sample contained a detergent other than OBG the column was washed with 20 ml 1 % OBG, 10 mM Tris/HCl (pH 8·0) to exchange the detergent. The protein was eluted from the Q-Sepharose column by using a LiCl gradient. A total volume of 50 ml was used, and the gradient ranged from 0 to 300 mM LiCl with fixed concentrations of 1% OBG and 10 mM Tris/HCl (pH 8·0). Fractions of 1·5 ml were collected, A280 was measured, and fractions of interest were analysed by SDS-PAGE.
Further purification of the FomA protein was achieved by applying size-exclusion chromatography. Fractions enriched in FomA were pooled, and the volume was reduced to less than 2 ml by using a concentration cell (Amicon) with a cut-off filter of 30 kDa. This sample was applied to a Sephacryl 200 column pre-equilibrated with 1% OBG, 10 mM Tris/HCl (pH 8·0), 200 mM LiCl, and size-exclusion chromatography was conducted in the same buffer at a constant flow of 0·5 ml min-1. Fractions of 1·0 ml were collected, A280 was measured, and fractions of interest were analysed by SDS-PAGE.
SDS-PAGE and Western blotting.
SDS-PAGE was performed as described by Lugtenberg et al. (1975) , and the samples were incubated in sample buffer at room temperature or 95 °C for 5 min prior to electrophoresis. The SDS-PA gels were either stained with Coomassie brilliant blue R250 (Sigma) or subjected to Western blot analysis as described by Towbin et al. (1979)
. Anti-FomA antiserum
239 (Bakken et al., 1989b
) and horseradish-peroxidase-coupled goat anti-rabbit antibody (Bio-Rad) were used as primary and secondary antibodies, respectively, and the immunoblots were developed with 4-chloro-1-naphthol (Bio-Rad).
Analysis of secondary structure content by circular dichroism.
Secondary structure content of the FomA protein was evaluated by circular dichroism (CD) spectroscopy using a Jasco J-810 spectropolarimeter equipped with a Jasco PTC-423S Peltier element for temperature control. Prior to analysis, the samples were dialysed against a 1% OBG, 20 mM Tris/HCl (pH 8·0) buffer to reduce the LiCl concentration to less than 5 mM. The CD spectra were recorded at 25 °C with samples containing 0·10·3 mg protein ml-1 by four consecutive scans at 0·5 nm wavelength intervals, and the mean spectra were stored. The temperature-dependent stability of secondary structure was monitored by heating the samples at a programmed rate of 1 °C min-1.
Trypsin treatment of intact cells and cell envelopes containing FomA, and of purified FomA.
Trypsin treatment of intact cells and cell envelopes was in essence performed as described by Merck et al. (1997) . In short, 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, and the samples were incubated on ice for 60 min with 0·1 mg trypsin ml-1. The trypsin reaction was stopped by adding soybean trypsin inhibitor (Sigma) to a final concentration of 500 µg ml-1, and the samples were subsequently washed twice with the above-described buffer containing trypsin inhibitor. Cell envelopes to be trypsin treated were isolated by passage through a French pressure cell and subsequent centrifugation as described by Jensen et al. (1996)
, and subjected to trypsin digestion as described for intact cells. Purified FomA protein was subjected to trypsin digestion by adding trypsin to final concentrations of 10, 1 or 0·1 µg ml-1 and incubating on ice for 60 min; the reaction was stopped as described above. All trypsin-treated samples were immediately analysed by SDS-PAGE and Western blotting to avoid unwanted potential activity of (residual) trypsin.
N-terminal amino acid sequence determination.
Samples containing the FomA protein were subjected to SDS-PAGE, and the Coomassie-stained FomA bands were excised. The protein was extracted from the gel by incubation in 70% formic acid at room temperature overnight (Tsugita et al., 1987 ), and submitted to the protein sequencing facilities at the University of Oslo for N-terminal sequencing.
Single-channel conductance experiments of FomA reconstituted in lipid bilayer membranes.
FomA protein was prepared for single-channel conductance experiments by extracting the protein from OMs with 1% OBG, followed by SDS-PAGE. Samples were incubated with sample buffer at room temperature prior to SDS-PAGE, and run in parallel in all lanes of a 15-lane SDS-PA gel. The outer lanes were stained with Coomassie blue to guide the excision of non-stained native FomA proteins from the gel, and the protein was eluted with 0·5% OBG in 20 mM Tris/HCl (pH 8·0). The reconstitution of pore-forming activity in artificial membranes was performed by using black lipid membranes as described previously (Kleivdal et al., 1995 ).
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RESULTS |
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In order to pursue the hypothesis that the N-terminus is not part of the ß-barrel, the 37 kDa truncated FomA10953TRU was incorporated into artificial bilayer membranes, as described in Methods. Fig. 4 shows that FomA10953TRU indeed functions as a pore, and a mean single-channel conductance of 1·22 nS was found, similar to that observed with full-length FomA10953 (1·17 nS) (Kleivdal et al., 1995
).
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DISCUSSION |
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The trypsin analysis of the FomA protein present in the OM and in solution also provided new structural information. In contrast to earlier published work (Bakken et al., 1989a ), no R or K residues seem to be accessible to trypsin on the surface of intact cells. Instead, the results presented here suggest that the trypsin site reported to be exposed on the cell surface is in fact exposed to the periplasm, since it is fully accessible to trypsin only when cell envelopes are treated with trypsin. Thus residues 128 do not seem to be part of the ß-barrel, since the N-terminal fragment containing these residues apparently dissociates from the rest of the protein, as evident from the increased mobility in SDS-PAGE and the fact that none of these residues can be found by N-terminal sequencing. Furthermore, the first putative membrane-spanning segment of the topology model of the FomA protein contains several alternating proline residues, and hence this segment cannot exist as a classical ß-strand (Bolstad et al., 1994
). The 28 aa N-terminal fragment also seems to dissociate from the FomA protein when subjected to trypsin treatment in solution, since there is a shift in the mobility of both the folded trimers of FomAFev1 and the folded monomers of rFomA10953 in SDS-PAGE (Fig. 3b
, compare lanes 2 and 4). Finally, the fact that several additional trypsin sites were cleaved in solubilized FomA protein (Fig. 3b
, lanes 6 and 7), with no apparent effect on the migration of the folded barrel (Fig. 3b
, lanes 3 and 4), together with the fact that FomA10953TRU functions as a pore (Fig. 4
) with properties similar to those of the FomA10953 protein, strongly supports the hypothesis that the N-terminus is not part of the ß-barrel.
Earlier work has demonstrated that it is possible to replace residues 8 to 23 with the residues RS without the loss of pore function (Kleivdal et al., 2001 ), suggesting that this segment is not critical for maintaining the ß-barrel structure of the FomA protein. In support of this is also the fact that the 37 kDa trypsin fragment of the FomA (FomA10953TRU) protein functioned as an intact pore in artificial bilayer membranes (Fig. 4
), with a mean single-channel conductance similar to that of the full-length FomA10953 protein. On the other hand, the results obtained with the E2C FomA mutant protein suggest that the N-terminus plays a role in stabilizing the quaternary structure, since the rFomA10953E2C protein displays more stable trimers than the wild-type FomA10953 protein under non-reducing conditions, due to a disulphide bond between two monomers within the trimer.
In the light of the results presented here, we propose an alternative topology model where the C-terminal part of the protein forms a 14-stranded ß-barrel (Fig. 6). Although the results do not directly implicate the second putative ß-strand of the original model, the constriction rules for transmembrane ß-barrels formulated by Schultz (2000) imply an even number of ß-strands, thus leading us to propose that the first two strands and loop L1 of the original model are located in the periplasm. The 14-stranded ß-barrel topology model not only conforms to the results presented here, but is also in accordance with previously published results for the FomA porin (Kleivdal et al., 1999
, 2001
; Puntervoll et al., 2000
). The insertion of a foreign epitope after E19 did not result in the surface exposure of this epitope, in contrast to similar insertions in loops L2 to L6 (named according to the current topology model Fig. 6
) (Puntervoll et al., 2000
), in line with a periplasmic location of this part of the protein. Furthermore, neither the insertion of a foreign epitope after E19 (Puntervoll et al., 2000
), nor the deletion of residues 823 (Kleivdal et al., 2001
), severely affected the function of the FomA porin, in line with the N-terminal part not being an integral part of the ß-barrel. Recently, the projection structure of the E. coli OmpG porin was published at 6
(0·6 nm) resolution (Behlau et al., 2001
), and it was suggested that this porin is a 14-stranded ß-barrel. Compared to the classical non-specific proteobacterial porins, the OmpG porin is also atypical in that it does not seem to form trimers, in contrast to the FomA porin (Kleivdal et al., 1995
). In conclusion, the FomA porin, along with the OmpG porin from E. coli, seems to deviate from the common understanding that non-specific porins are 16-stranded ß-barrels. In order to assess the accuracy of this novel 14-stranded topology model, obtaining the crystal structure of FomA would be of utmost value, and this is our current goal. The purification procedures presented here form a solid base for obtaining large amounts of pure protein for crystallization.
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ACKNOWLEDGEMENTS |
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Received 16 July 2002;
accepted 18 July 2002.