INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Bundle-forming pili (BFP) are type IV fimbriae expressed by enteropathogenic Escherichia coli (EPEC), a bacterium that causes infantile diarrhea (25). BFP are a demonstrated EPEC virulence factor (6) and can elicit an immune response in naturally occurring infections (40). The presence of BFP is required for two notable EPEC phenotypes. Localized adherence, the classical phenotype, describes the binding of EPEC to cultured epithelial cells in a characteristic clustered formation (56). A similar pattern has been noted in EPEC infections in vivo (52). Autoaggregation, the second phenotype, describes the formation of dynamic multicellular clusters of EPEC grown in tissue culture medium (6). The relevance of this phenomenon to EPEC infection of the human intestinal tract is unclear.

Few details are known about the molecular mechanisms of type IV fimbrial biogenesis. Our current concept of BFP synthesis is as follows (21). BFP fibers are polymers of a pilin protein known as bundlin. Coincident with or following their synthesis, bundlin precursors are probably anchored in the cytoplasmic membrane (via a stretch of hydrophobic residues located near their N termini), with the majority of the polypeptide being exported to the periplasm. On the cytoplasmic side of the membrane, the N-terminal leader peptide of prebundlin is removed by the BfpP prepilin peptidase (75). In the periplasm, the formation of a disulfide bond required for bundlin stability is catalyzed by the oxidant DsbA (74). Following these events, the modified bundlin monomers are somehow removed from the membrane and polymerized into fimbriae that are extruded through the outer membrane.

BFP biogenesis is dependent on a cluster of 14 bfp genes located on a large plasmid found in many EPEC strains (58, 60). Expression of these 14 genes in a K-12 laboratory strain of E. coli that is normally nonpiliated is sufficient to elicit BFP formation (60). The bfpA gene encodes prebundlin (17, 57), while the bfpP gene encodes the prepilin peptidase (4, 75). A third gene, bfpB, encodes an outer membrane lipoprotein that is a member of the secretin family and is required for BFP biogenesis (4, 49). The bfpF gene encodes a putative cytoplasmic membrane protein that is not essential for BFP biogenesis but is required for the dynamic behavior of EPEC autoaggregates and BFP bundles (3, 6, 33). The precise functions of the 10 remaining bfp gene products are unknown. At least four of them (bfpC, bfpD, bfpG, and bfpL) are required for BFP biogenesis, while bfpH is not (4, 6, 58, 60). The seventh gene in the bfp cluster, bfpE, encodes a putative polytopic cytoplasmic membrane protein, BfpE, which has been detected using inducible T7 RNA polymerase expression systems (58, 60). BfpE is a member of the GspF family of proteins, which includes components of other systems that transport macromolecules or macromolecular assemblies (including type IV pili) across bacterial envelopes (48, 53). In this report, we describe preliminary analyses of the function and structure of BfpE.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains and growth conditions. The E. coli strains used in this study are listed in Table 1. Strains were routinely cultured in Luria-Bertani (LB) broth or on LB agar at 37°C. Antibiotics and/or chromogenic substrates were added as necessary at the following concentrations: ampicillin, 200 µg/ml; chloramphenicol, 20 µg/ml; nalidixic acid, 50 µg/ml; kanamycin, 50 µg/ml; 5-bromo-4-chloro-3-indolylphosphate (BCIP), 40 µg/ml; 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal), 40 µg/ml. Dual-indicator plates were prepared as described elsewhere (1).

Autoaggregation assay. EPEC strains were cultured overnight at 37°C in LB (plus ampicillin as appropriate). The resulting stationary-phase cultures were diluted 1:100 or 1:250 (making adjustments for the optical density at 600 nm [OD₆₀₀]) into 20 ml of Dulbecco's modified Eagle medium plus nutrient mixture F-12 (DMEM/F-12) containing 15 mM HEPES buffer and lacking phenol red (Gibco-BRL Life Technologies no. 11039-021). The DMEM/F-12 cultures were shaken at 250 rpm for 5 h in 50-ml conical polypropylene tubes at 37°C. Autoaggregation was gauged by visually inspecting the cultures for bacterial aggregates and sedimentation, by examining 5-µl aliquots of the culture under phase-contrast microscopy, and by determining the aggregation index (AI) (4). To determine AI, two equivalent 1-ml samples were removed from each culture. The OD₆₀₀ of the first sample was measured immediately using a spectrophotometer. The second sample was agitated for 1 min on a vortex mixer before the OD₆₀₀ was measured. AI was calculated by subtracting the OD₆₀₀ of the first sample from that of the second, dividing the result by the value of the first sample, and multiplying by 100.

Localized adherence assay. Localized adherence to HEp-2 laryngeal carcinoma cells was assayed as described previously (19), using the eight-well chamber slide modification.

Transmission electron microscopy. To prepare samples for electron microscopy, 1-ml aliquots were removed from EPEC cultures grown in DMEM for 6 h and the bacteria were pelleted by brief centrifugation. Most of the medium was decanted, and the bacterial pellet was gently resuspended in the remainder. A 10-µl aliquot was spotted onto electron microscopy grids, which were dried for 10 min and then stained and examined as described previously (3, 60).

Sequence analysis. Eight computer programs available on the Internet were used to examine the BfpE protein sequence for the presence of hydrophobic regions having the potential to be transmembrane (TM) segments. These programs are DAS (13) (http://www.sbc.su.se/~miklos/DAS/), HMMTOP (65) (www.enzim.hu/hmmtop/), PHDhtm/PHDtopology (50, 51) (www.embl-heidelberg.de/predictprotein/), PSORT (43) (http://psort.nibb.ac.jp/), SOSUI (29) (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html), TMHMM (59) (www.cbs.dtu.dk/services/TMHMM-1.0/), TMpred (30) (www.isrec.isb-sib.ch/software/TMPRED_form.html), and TopPred2 (12, 68) (http://www.sbc.su.se /~erikw/toppred2/). Five of these programs (HMMTOP, PHDtopology, TMHMM, TMpred, and TopPred2) were also used to predict the membrane topology of BfpE.

Plasmids, primers, PCR, and sequencing. Source plasmids used for this study are listed in Table 2. The plasmids constructed during the course of this work are described below. Oligonucleotide primers used for PCR and plasmid sequencing are listed in Table 3. The University of Maryland School of Medicine Biopolymer Laboratory performed all primer syntheses as well as plasmid sequencing. Standard techniques were used for DNA manipulation (54). Plasmids were maintained in strain DH5 or CC118 and were prepared using the Wizard Plus SV Minipreps DNA Purification System (Promega). PCR was carried out using Taq (Gibco-BRL) or Pfu (Stratagene) polymerase as specified by the manufacturer.

Construction of strain UMD934 containing a nonpolar bfpE mutation. A previously described allelic replacement strategy (20) was modified to disrupt the bfpE gene in wild-type EPEC strain E2348/69. Plasmid pHZZ4B1 was linearized at a unique AflIII site specified by codons 130 and 131 of bfpE and then treated with Klenow fragment of DNA polymerase I. The blunted ends were ligated to the 850-bp SmaI fragment from pUC18K carrying the nonpolar aphA-3 cassette specifying kanamycin resistance (41), creating pTEB43-K1A. Restriction analysis followed by sequencing using the Donne-200 primer confirmed that the aphA-3 gene was inserted in the same orientation as bfpE and was in the proper reading frame to reinitiate translation from the 3' end of the transcribed cassette. Plasmid pTEB43-K1A and suicide vector pCVD442 were both digested with SalI and then ligated to form pTEB44, in which the bfpE::aphA-3 and sacB genes are transcribed in opposite orientations. Plasmid pTEB44 was next digested with SacI to remove a 5.9-kb fragment containing the pACYC184 origin of replication, chloramphenicol resistance gene, and a portion of the bfp cluster. The remaining 9.3-kb SacI fragment was religated to form the bfpE::aphA-3 suicide plasmid pTEB45, which was introduced into DH5pir by electroporation. HB101 containing helper plasmid pRK2073 was used to mobilize pTEB45 from DH5pir into EPEC strain E2348/69. EPEC transconjugants were selected on LB plates containing nalidixic acid and kanamycin. From among these, an isolate was identified that was resistant to kanamycin and 5% sucrose but sensitive to ampicillin. Replacement of the wild-type bfpE allele by the bfpE::aphA-3 allele in this strain, UMD934, was confirmed by PCR analysis using primers Donne-243 and Donne-256.

Construction of plasmids for membrane topology analysis. A matched set of plasmids for membrane topology analysis was derived from the low-copy expression vector pTrc99A (Amersham Pharmacia Biotech). One plasmid, pTrcphoA, carries the 'phoA gene (lacking codons Met 1 through Met 26 encoding the signal sequence), while the other, pTrclacZ, carries the 'lacZ gene (lacking codons Met 1 through Ser 7). To construct pTrcphoA, overlap extension PCR was first used to eliminate the NcoI site from 'phoA. Specifically, a portion of 'phoA upstream of and overlapping the NcoI site was amplified using primers Donne-222 and Donne-224, with plasmid pCVD433::31TnphoA as the template. A second portion of 'phoA overlapping and downstream of the NcoI site was prepared using primers Donne-211 and Donne-223. The two portions were annealed and amplified with primers Donne-224 and Donne-211, creating a 'phoA fragment carrying a silent mutation of CAT to CAC at histidine codon 272. The ends of the 'phoA fragment were digested with BamHI and KpnI and inserted into the multiple-cloning site of BamHI-KpnI-digested pTrc99A. To construct pTrclacZ, the 'lacZ gene was amplified by PCR using Donne-227 and Donne-228 as primers and pMD103 as template. The ends of the 'lacZ PCR product were digested with BamHI and KpnI and then ligated to BamHI-KpnI-digested pTrc99A. In both pTrclacZ and pTrcphoA, the reporter genes are fused in frame to a start codon directly downstream of the trc promoter. Fusion genes can be created in these plasmids by inserting gene segments between restriction sites present between the start codon and reporter gene.

Construction of random bfpE'::'phoA and bfpE'::'lacZ gene fusions. PCR using primers Donn-243 and Donne-256 and pKDS135 as template was used to construct the bfpE355 allele, which differs slightly from wild-type bfpE at both ends. At the 5' end of the PCR product, the initiator Met codon (Met-1) was replaced with a sequence creating an NcoI site and an extra Ala codon between Met-1 and Lys-2. At the 3' end of PCR product, the TGA termination codon was replaced with a sequence encoding Leu-Glu-STOP and multiple restriction sites. The bfpE355 PCR product was digested with NcoI and XmaI and ligated to the large fragments of pTrcphoA and pTrclacZ resulting from digestion with the same enzymes, creating pTEB41 and pTEB42. In both plasmids, the bfpE355 gene is followed by a TAA termination codon and a 13-bp linker sequence, rendering the 'lacZ or 'phoA genes out of frame. Plasmid pTEB65 carrying bfpE but no reporter gene was constructed by deleting a 1.36-kb NheI-XbaI fragment containing 'phoA from pTEB41.

Construction of specific bfpE'::'phoA and bfpE'::'lacZ fusions. The randomly generated bfpE' alleles described above were transferred as NcoI-AvaI fragments to the vector (pTrcphoA digested with NcoI-AvaI, or pTrclacZ digested with NcoI-XmaI) containing the complementary reporter gene. PCR was used to construct additional plasmids containing bfpE' segments of specified sizes. Each of the PCR amplifications used pTEB65 as template and Donne-243 as the upstream primer. The downstream primers were Donne-305, Donne-316, Donne-317, Donne-318, and Donne-319. PCR products containing bfpE' segments were digested with NcoI and AvaI and introduced into pTrcphoA digested with NcoI-AvaI or into pTrclacZ digested with NcoI-XmaI.

Construction of internal deletions in bfpE. Three plasmids carrying bfpE::'phoA fusions containing internal deletions in bfpE were constructed by introducing downstream segments of bfpE into existing bfpE'::'phoA fusion plasmids. To construct the bfpE292'(210-233)::'phoA plasmid, primers Donne-343 and Donne-344 were used to amplify a fragment from the bfpE292'::'phoA plasmid containing codons 234 through 292 of bfpE plus the proximal end of 'phoA. The resulting 722-bp PCR product was digested with NgoMIV and RsrII and then introduced into the bfpE78'::'phoA plasmid that had been digested with AvaI and RsrII. To construct the bfpE352(116-301)::'phoA plasmid, primers Donne-315 and Donne-344 were used to amplify a fragment from the bfpE352::'phoA plasmid containing codons 302 through 352 of bfpE plus the proximal end of 'phoA. The resulting 697-bp PCR product was digested with NgoMIV and RsrII and then introduced into the bfpE115'::'phoA plasmid that had been digested with AvaI and RsrII. To construct the bfpE352(210-233)::'phoA plasmid, primers Donne-222 and Donne-343 were used to amplify a fragment from the bfpE352::'phoA plasmid containing codons 234 through 352 of bfpE plus the proximal end of 'phoA. The resulting 1,202-bp PCR product was digested with NgoMIV and RsrII and then introduced into the bfpE209'::'phoA plasmid that had been digested with AvaI and RsrII. A linker sequence specifying Ala-Pro-Gly replaced the deleted bfpE codons in each of the new plasmids. A plasmid carrying bfpE with a deletion of sequences specifying HS3 was constructed by replacing an NcoI-EcoO109I fragment of pTEB65 with the corresponding fragment from the bfpE352(210-233)::'phoA plasmid.

Construction of bfpE'::'phoA-lacZ::'bfpE sandwich fusions. Sandwich fusion plasmids were constructed by in-frame insertion of a dual-reporter 'phoA-lacZ cassette from pMA632 into unique restriction sites within the bfpE gene of pTEB65. The cassette was inserted into the BtrI site as a SmaI-EcoRV fragment. The cassette was amplified from pMA632 by PCR using primers Donne-383 and Donne-384, cut with AvaI, and inserted into the BspEI site. Likewise, the cassette was amplified using primers Donne-385 and 386, cut with EcoO109I, and then inserted into the EcoO109I sites of both pTEB65 and its HS3 derivative. A plasmid carrying a tandem repeat of the cassette at the EcoO109I site of the full-length bfpE gene was a by-product of this construction. The cassette was inserted into the filled-in AvaI site as a SmaI-NruI fragment. The NcoI site in the phoA gene of the AvaI insertion plasmid was eliminated by replacing a 331-bp EcoRI fragment with the corresponding fragment from pTrcphoA, generating pTEB120. Digestion of pTEB120 with NcoI and AvaI or XhoI will precisely remove the bfpE gene, allowing any gene fragments to be placed there to construct novel C-terminal dual-reporter fusions.

Construction of bfpE derivatives with a C-terminal epitope tag. The full-length bfpE gene (codons 1 to 352) was amplified by PCR from pTEB65 using primers Donne-243 and Donne-507, digested with NcoI and XbaI, and inserted into the corresponding sites in the multiple-cloning site of pBAD/myc-His B. Partial bfpE genes were amplified using primers Donne-486 and Donne-507 (codons 234 to 352) or primers Donne-486 and Donne-508 (codons 234 to 323), digested with NcoI and XbaI, and inserted into the corresponding sites in the multiple-cloning site of pBAD/gIII A.

Immunoblotting. For the bundlin immunoblot, overnight LB broth cultures of EPEC were diluted 1:100 into 20 ml of DMEM/F-12. After 6 h of growth at 37°C with shaking, the bacteria were pelleted by centrifugation at 2,500 × g for 10 min and then resuspended in 1.2 ml of cell lysis buffer (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100). The remaining procedures for sample preparation, protein concentration measurement, and electrophoresis were as previously described (4). The blots were blocked with phosphate-buffered saline containing 0.1% Tween-20 plus 5% nonfat dry milk and then incubated sequentially with a 1:10,000 dilution of ICA4, a monoclonal immunoglobulin G1 antibody raised against BFP (26), with an anti-mouse horseradish peroxidase (HRP) conjugate at a 1:30,000 dilution, and, finally, with enhanced chemiluminescence (ECL) Western blotting detection reagents (Amersham Pharmacia Biotech).

Enzyme assays. To assay the enzyme activities of BfpE-PhoA fusions, cultures were grown as described above for immunoblotting. Samples were prepared and alkaline phosphatase assays were carried out by a microtiter plate procedure described previously (14), with the following modifications. The volume of diluted, permeabilized cultures added to the wells of the microtiter plate was 220 µl, and the volume of para-nitrophenyl phosphate solution was 20 µl. After a 15-min incubation at room temperature, the reactions were stopped by adding 20 µl of 1 M KH₂PO₄ plus 4 µl of 0.5 M EDTA (pH 8.0) to each well. OD readings were recorded at 620 nm (cell density), 414 nm (yellow color), and 540 nm (light scattering by cell debris). To assay the activities of BfpE-LacZ C-terminal fusions, -galactosidase assays were carried out in a similar fashion, with the following modifications. Bacteria from 1-ml culture samples were pelleted, washed with 1 ml of 1 M Tris-HCl (pH 8.0), and resuspended in 1 ml of Z buffer (42). Aliquots of cultures that had been diluted 1:4 or 1:9 in Z buffer and then permeabilized were placed in the wells of a microtiter plate. The reaction was started by adding 37.5 µl of 10-mg/ml ortho-nitrophenyl--D-galactoside (ONPG) prepared in Z buffer and allowed to continue for 20 min at room temperature. The reactions were stopped by adding 75 µl of 1 M Na₂CO₃. Activity units were calculated by standard methods (37, 42), with appropriate adjustments for the dilution of the original cultures. The -galactosidase activities of the BfpE-dual-reporter sandwich fusions could not be detected using ONPG. They were detectable using the more sensitive fluorescent substrate 4-methylumbelliferyl--D-galactoside (MUG). The MUG assay was carried out as described previously (42), except that a Shimadzu fluorescence spectrophotometer was utilized. A standard curve of 12.5 to 400 pM 4-methylumbelliferone was used as a reference to calculate units of specific -galactosidase activity (42).

Sucrose density flotation gradient fractionation. TOP10 strains containing plasmids with epitope-tagged bfpE derivatives were grown overnight in LB medium, diluted 1:100 into 30 ml of fresh LB medium, and shaken at 37°C for 4 h, after which arabinose was added to 0.2% to induce protein expression and shaking was continued for an additional 3 h. The remainder of the procedure was performed as previously described (4).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Phenotypes of a bfpE mutant. To determine whether the bfpE gene product plays a role in BFP biogenesis and BFP-associated phenotypes, we constructed UMD934, an EPEC strain containing a nonpolar insertion mutation in the bfpE gene. UMD934 was examined for BFP formation directly by transmission electron microscopy and indirectly by assaying for the BFP-dependent phenomena of autoaggregation and localized adherence. The isogenic wild-type EPEC strain E2348/69 and the bfpA mutant strain UMD901 were included in these experiments as positive and negative controls, respectively. BFP were readily detected emanating from cells of strain E2348/69 (Fig. 1A) but were not detected on cells of strain UMD901 or UMD934. When E2348/69 was grown in DMEM/F-12 tissue culture medium for at least 3 h, visible bacterial aggregates formed (Fig. 1D). In contrast, UMD901 and UMD934 cultures did not develop visible or even microscopic aggregates. To quantify the extent of autoaggregation in these strains, an AI was calculated. AI is a measure of the percent increase in OD that occurs after agitation of the culture to disperse bacterial aggregates (3, 4). E2348/69 exhibited a high AI value, while the bfpE mutant UMD934 exhibited a low AI value similar to that of UMD901 (Table 4). When tested for localized adherence to HEp-2 epithelial cells in tissue culture, E2348/69 exhibited the characteristic clustered pattern (Fig. 1G). UMD901 and UMD934 adhered poorly. To determine whether BfpE plays a role in bundlin expression and processing, extracts of EPEC strains were subjected to immunoblotting using an anti-bundlin antibody. UMD934 was found to produce completely processed bundlin at normal levels (Fig. 2).

View larger version (135K): [in this window] [in a new window]   FIG. 1.   Assay of BFP formation, autoaggregation, and localized adherence by EPEC. Shown are the wild-type EPEC strain E2348/69 (A, D, and G) and the isogenic bfpE mutant UMD934 bearing either the vector pTrcphoA (B, E, and H) or plasmid pTEB41 carrying the bfpE gene (C, F, and I). The top row (A to C) displays transmission electron micrographs of EPEC cultured in DMEM for 6 h. Magnifications, ×20,000 (A) and ×12,000 (B and C). Bar, 200 nm (A) or 500 nm (B and C). BFP can be seen in panels A and C. The center row (D to F) displays phase-contrast micrographs (magnifications, approximately ×460 for panels D and F and ×580 for panel E) of EPEC cultured in DMEM for 7 h and then examined in hanging drop slides. EPEC aggregates are seen in panels D and F. The bottom row (G to I) displays phase-contrast micrographs (magnification, ×630) of HEp-2 cells incubated for 3 hours with EPEC, washed, and fixed. Adherent EPEC microcolonies are seen in panels G and I. Arrows point to two of the microcolonies in each panel.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Examination of bundlin expression and processing by EPEC. Whole-cell extracts were prepared from EPEC strains (left to right) E2348/69, UMD932, UMD901, UMD934, E2348/69 (pTrcphoA), UMD934 (pTrcphoA), and UMD934 (pTEB41) after growth in DMEM/F-12 for 6 h. Extracts were separated by SDS-PAGE using a 15% polyacrylamide gel. A monoclonal antibody was used to detect bundlin.

Complementation of the bfpE mutation. The low-copy plasmid pTEB41, which carries the cloned bfpE gene, was introduced into strain UMD934 to complement the bfpE mutation. UMD934 bearing pTEB41 exhibited BFP formation, autoaggregation, and localized adherence (Fig. 1C, G, and I; Table 4), while UMD934 bearing the control vector pTrcphoA did not (Fig. 1B, E, and H; Table 4). These results demonstrated conclusively that bfpE is required for BFP biogenesis. In pTEB41, a pTrc99A derivative, the bfpE gene is expressed under the control of the trc promoter. This promoter is supposed to be efficiently repressed by the product of the lacI^q gene present on the plasmid and can be derepressed by the addition of isopropyl--D-thiogalactopyranoside (IPTG) (2). However, successful complementation of the bfpE mutant did not require the addition of IPTG. The repressed level of bfpE expression from pTrc99A derivatives was sufficient not only for complementation but also for the production of BfpE fusion proteins at a level that can be readily detected by immunoblotting and enzyme assays (see below).

Predictions of BfpE topology and TM segments. Hydropathy plots and computer programs were used to identify potential TM segments in BfpE and to suggest the most likely topology of the protein (see Materials and Methods). Four hydrophobic segments potentially long enough to span the cytoplasmic membrane were identified by all programs. These segments, designated HS1 through HS4, are located at residues 115 to 139, 171 to 191, 212 to 232, and 322 to 342, respectively, of BfpE (modal values from eight programs). In the predominant topology model (predicted by three of five programs), all four of the hydrophobic segments cross the membrane and both termini of the protein are found in the cytoplasm.

Construction of random and specific bfpE'::'phoA and bfpE'::'lacZ fusions. To determine the topology of BfpE experimentally, we turned to the construction and analysis of bfpE'::'lacZ and bfpE'::'phoA fusions. Such fusions are commonly used to study membrane protein topology (reviewed in references 37, 48, 64, and 66). Enzymatically active -galactosidase (LacZ) fusions are expected to identify regions of BfpE that reside in the cytoplasm, while active alkaline phosphatase (PhoA) fusions are expected to identify regions of BfpE that reside in the periplasm. The bfpE gene was introduced into the expression vector pTrc99A upstream from and out of frame with 'lacZ or 'phoA reporter genes. Exonuclease III was used to degrade the bfpE gene from the 3' end and create a nested set of bfpE' C-terminal deletion fusions to 'phoA or 'lacZ. The plasmid pool carrying these gene fusions was introduced into E. coli DH5. Colonies containing enzymatically active BfpE-LacZ and BfpE-PhoA fusions were identified on medium containing the chromogenic substrates X-Gal or BCIP.

View larger version (71K): [in this window] [in a new window]   FIG. 3.   Expression of BfpE-LacZ fusion proteins. Whole-cell extracts were prepared from plasmid-bearing derivatives of E. coli CC118 and separated by SDS-PAGE on a 6% polyacrylamide gel. The fusion proteins were detected with an anti--galactosidase antibody. The positions of molecular mass markers are displayed to the left of the blot. The first three lanes display samples from strains carrying control plasmids pTEB65 (no LacZ), pTrclacZ (no BfpE), and pTEB42 (substrate for exonuclease III digestion). The remaining lanes display samples from strains carrying plasmids with bfpE'::'lacZ fusion genes. The number of the terminal amino acid in the BfpE portion of the fusion protein is noted above each lane. The arrow to the right of the blot indicates a prominent degradation product of many of the fusions that is similar in size to -galactosidase (LacZ).

View larger version (43K): [in this window] [in a new window]   FIG. 4.   Activities of BfpE-LacZ and BfpE-PhoA fusion proteins. In the diagrams to the left, the shaded bars represent the extent of BfpE included in each fusion protein and the black boxes represent potential transmembrane segments. Black lines and triangles indicate deleted portions of BfpE. Alkaline phosphatase or -galactosidase enzyme assays were performed on permeabilized cultures of E. coli CC118 carrying fusion plasmids. The bar graph data represent the mean and standard error values (in units of enzyme activity) for four enzyme activity determinations from a single set of permeabilized cells per sample. Similar results were obtained in repeated experiments. Each datum point corresponds to the fusion depicted to the left of it. Four points are absent from the LacZ data (indicated by asterisks) because a plasmid expressing the fusion was not constructed (residue 149) or a fusion protein was not detected by immunoblotting (residues 157, 249, and 352).

Analyses of fusion protein enzyme activity and expression. To indicate a cytoplasmic or periplasmic location for the reporter enzyme moieties of the BfpE fusion proteins, alkaline phosphatase or -galactosidase activities were determined for permeabilized E. coli CC118 bearing bfpE'::'phoA or bfpE'::'lacZ plasmids (Fig. 4). To determine whether BfpE-LacZ and BfpE-PhoA fusion proteins were expressed, whole-cell extracts were prepared from these strains and subjected to immunoblotting using anti-LacZ and anti-PhoA antiserum (Fig. 3 and 5).

View larger version (73K): [in this window] [in a new window]   FIG. 5.   Expression of BfpE-PhoA fusion proteins. Whole-cell extracts were prepared from plasmid-bearing derivatives of E. coli CC118 and separated by SDS-PAGE on a 6.5% polyacrylamide gel. The fusion proteins were detected with an anti-PhoA antibody. The positions of molecular mass markers are displayed to the left of the blot. The first three lanes display samples from strains carrying control plasmids pTrcphoA (no BfpE), pTEB41, and pTEB65 (no PhoA). The remaining lanes display samples from strains carrying plasmids with bfpE'::'phoA fusion genes. The number of the terminal amino acid in the BfpE portion of the fusion protein is noted above each lane. The arrow to the right of the blot indicates a prominent degradation product of many of the fusions that is similar in size to alkaline phosphatase (PhoA).

Analyses of dual-reporter sandwich fusions. To further analyze the topology of BfpE, we constructed sandwich fusions, where the reporter moiety was placed at sites internal to the complete BfpE protein. This approach can provide a more reliable picture of a protein's topology than the C-terminal deletion fusion approach, especially when regions of the protein located both N- and C-terminal to the reporter enzyme must interact to establish the correct topology (22, 66). In particular, we wished to use such fusions to test the hypothesis, suggested by the BfpE-PhoA fusions, that HS3 of BfpE does not act as a TM domain when HS4 is also present. To make sandwich fusions in bfpE, we utilized a dual-reporter cassette containing the fused phoA gene and lacZ fragment, which allows both PhoA and LacZ enzyme activities to be analyzed in the context of a single protein (1). The cassette was inserted in frame into four restriction sites unique within the bfpE gene of plasmid pTEB65. As indicated in Fig. 6, these insertion sites correspond to regions in BfpE near the N terminus (BtrI and BspEI), between HS3 and HS4 (EcoO109I), and at the extreme C terminus (AvaI). Two variants of the EcoO109I construct were also prepared: one which carried two tandem copies of the dual reporter and one in which the dual reporter was inserted into a bfpE gene that lacked sequences encoding the putative TM domain HS3 (codons 210 to 233). The sandwich fusion plasmids were introduced into E. coli TG1. When plated on Red-Gal/BCIP dual indicator medium (1), TG1 colonies carrying the EcoO109I, EcoO109I×2, and AvaI insertions exhibited a blue color 1 day after plating, suggesting a periplasmic location for the dual reporter. In contrast, colonies carrying the BtrI, BstEI, and EcoO109I(210-233) insertions exhibited a purplish red color that was not detectable the day after plating but was clearly seen after 1 week of storage at 4°C. This result suggests a cytoplasmic location for the dual reporter in these constructs. The sandwich fusion proteins were analyzed for enzyme activity and expression (Fig. 6). Their enzyme activities were extremely low but detectable, allowing certain conclusions. The relatively high alkaline phosphatase and low -galactosidase activities produced by the AvaI insertion indicate that the C-terminal portion of the fusion is periplasmic. The striking reversal in the relative activities produced by the EcoO109I insertions that occurs on deletion of HS3 strongly supports a periplasmic location for the segment between HS3 and HS4 as well. These changes argue that HS3 acts as a TM segment even in the presence of HS4. The other activities were too low or inconsistent to yield firm conclusions.

View larger version (41K): [in this window] [in a new window]   FIG. 6.   Analyses of BfpE-dual-reporter sandwich fusion proteins. The labels indicate restriction sites in the bfpE gene into which a dual-reporter ('phoA-lacZ) cassette was inserted. (A) Activities of sandwich fusion proteins. The BfpE protein is depicted as in Fig. 4, with the arrows indicating the approximate point at which the dual reporter is inserted into the protein. Alkaline phosphatase or -galactosidase enzyme assays were performed on cultures of E. coli TG1 carrying sandwich fusion plasmids. The bar graph data represent the mean and standard error values for three separate experiments, with four (alkaline phosphatase) or three (-galactosidase) enzyme activity determinations per experiment. (B) Expression of sandwich fusion proteins. Whole-cell extracts were prepared from the strains described above and separated by SDS-PAGE on a 6% polyacrylamide gel. The fusion proteins were detected with an anti-PhoA antibody. The first three lanes display samples from strains carrying control plasmids, while the remaining lanes display samples from strains carrying sandwich fusions.

HS4 can act as a TM segment. The substantial alkaline phosphatase activities produced by fusions of PhoA on either side of HS4 called into question the ability of this region to act as a TM segment in BfpE. To explore this issue further, we analyzed three bfpE'::'phoA constructs containing specific deletions within the bfpE gene (Fig. 4). In the first construct, sequences encoding HS3 (codons 210 to 233) were deleted from an otherwise full-length fusion of bfpE to phoA. This construct produced a weakly detectable protein (Fig. 6) that had moderately high activity (Fig. 4). As a control, a partial bfpE gene ending before the sequence encoding HS4 (at codon 292) and lacking sequences encoding HS3 was fused to phoA. The construct expressed a readily detectable protein (Fig. 5), but, in contrast to both the first construct and the C-terminal PhoA fusion at 292, it had no activity (Fig. 4). These data reconfirm the membrane-spanning capabilities of HS3 and, more importantly, suggest that HS4 can also act as a TM segment, exporting PhoA to the periplasm in the absence of HS3. To confirm this notion, a third phoA fusion construct was analyzed which contained a bfpE gene from which codons 116 through 301 had been deleted. The resulting fusion protein lacked hydrophobic segments HS1, HS2, and HS3, leaving HS4 as the only potential TM segment. This fusion construct produced a readily detectable protein with considerable PhoA activity (Fig. 4 and 5), clearly demonstrating that HS4 is capable of spanning the membrane. The limitation of these constructs was that HS4 was in an orientation opposite from the one it would be expected to hold in the native BfpE protein.

View larger version (40K): [in this window] [in a new window]   FIG. 7.   Sucrose density flotation gradient fractionation of three epitope-tagged BfpE derivatives. (A) Representations of epitope-tagged BfpE constructs. Each construct carries a C-terminal myc-His double epitope tag (not shown). For each construct, the number in the left column indicates the range of BfpE amino acids that are present. The middle column shows a linear representation of each protein. The right column displays the expected topology of each protein. Black boxes and cylinders indicate TM segments, while white boxes and cylinders indicate an exogenous N-terminal signal sequence, whose presumed removal is indicated by an arrow. (B) Lysates of TOP10 E. coli carrying plasmids producing the constructs shown in panel A were fractionated on sucrose density flotation gradients by centrifugation. Fractions were collected from the top (least dense portion) of the gradient, separated by SDS-PAGE on a 15% polyacrylamide gel, and subjected to immunoblotting using an antiserum recognizing the epitope tag. Fractions progress from the least dense on the left to the most dense on the right.

Complementation studies indicate the importance of HS3 and HS4 in the activity of BfpE. To determine the extent of the BfpE C terminus that is required for activity, we tested the longest BfpE-PhoA fusions for the ability to restore autoaggregation to the bfpE mutant EPEC strain UMD934. Both the full-length bfpE352::'phoA fusion and the bfpE349'::'phoA fusion enabled UMD934 to form microscopic aggregates, with the aggregates being consistently larger and more numerous in the bfpE352::'phoA sample. In contrast, PhoA C-terminal fusions at BfpE residues 337, 323, 320, or 292 did not promote autoaggregation. These results indicate that an intact HS4 is required for the activity of BfpE. We note that it is unclear whether the intact BfpE-PhoA fusion or the BfpE portion remaining after the release of the PhoA moiety (see above) is the complementing protein in these experiments.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

BfpE is required for BFP biogenesis. The biogenesis of type IV fimbriae requires multiple protein components in addition to pilin, the fimbrial structural element. It is likely that in EPEC, many or all of the products of the bfp gene cluster constitute a molecular machine that assembles BFP fimbriae and extrudes them through the outer bacterial membrane. Other type IV fimbriae are presumably assembled by similar machines. In this study, we have demonstrated the requirement for BfpE, one component of the BFP assembly apparatus. We found that a bfpE mutant strain of EPEC fails to form detectable BFP and fails to carry out autoaggregation and localized adherence. The cloned bfpE gene, transcribed from a heterologous promoter on a low-copy plasmid, complements each of the defects of the mutant. These results indicate that the BfpE protein is essential for type IV fimbrial biogenesis in EPEC. The bfpE mutation does not alter the expression or leader peptide processing of prebundlin. Since catalysis of disulfide bond formation in bundlin by the periplasmic protein DsbA is required for pilin stability (74), BfpE probably participates in an aspect of BFP synthesis that takes place after the insertion of bundlin into the cytoplasmic membrane. For example, it may mediate the extraction of bundlin from the membrane, the polymerization of bundlin into BFP, the extrusion of BFP through the outer membrane, or the anchoring of BFP to the cell envelope. BfpE could associate with bundlin directly or could influence it indirectly through other components of the BFP synthesis machinery. Mutations introduced into many of the bfp genes, including bfpB, bfpC, bfpD, bfpG, and bfpL, elicit phenotypes identical to those found in the bfpE mutant (4, 6, 49, 58, 60). To understand the specific requirement for the products of each of these bfp genes in detail, it will be necessary to develop new means of examining BFP synthesis at the molecular level.

Topology of BfpE. As a further step toward understanding the structure and function of BfpE and other GspF proteins, we have determined the arrangement of BfpE in the cytoplasmic membrane. For this purpose we isolated and constructed fusions of 3'-truncated bfpE derivatives to phoA (alkaline phosphatase) and lacZ (-galactosidase) genes. These had a wide range of BfpE endpoints, covering each of the hydrophilic domains of the protein. We also constructed a small number of sandwich fusions. The ability to identify both active and inactive PhoA and LacZ fusions strongly supports the notion that BfpE is a cytoplasmic membrane protein, as do the results of sucrose density gradient centrifugation with an epitope-tagged version of BfpE. A study of the relative activities and levels of the fusion proteins allowed us to evaluate the locations and orientations of four putative TM segments, HS1 through HS4, identified through sequence analysis. The activities of the BfpE-PhoA fusion proteins, as well as the dual-reporter sandwich fusions, varied in a manner consistent with the topology shown in Fig. 8. This topology meets the criteria of the positive-inside rule (67), with the majority of the arginine and lysine residues being located in the cytoplasm. The region between HS3 and HS4 contains 12 such residues and is periplasmic but can be considered exempt from the rule due to its length of greater than 60 residues (69, 70). Overall, the BfpE protein can be thought of as being divided into thirds. The N-terminal third is located in the cytoplasm. The middle third serves as a membrane anchor, containing three membrane-spanning segments. The C-terminal third is located primarily in the periplasm, with its end anchored in the membrane. As discussed in more detail below, this organization has profound implications for the function of BfpE.

View larger version (49K): [in this window] [in a new window]   FIG. 8.   Proposed topology of the BfpE protein. The amino acids composing BfpE are represented by circles. These are displayed in a manner that specifies their arrangement in the E. coli cytoplasmic membrane. HS1 through HS4 denote TM segments. Positively charged amino acids (arginines and lysines) that may be topology determinants are shaded. C-terminal fusion protein junctions are indicated by a line and the number of the terminal BfpE residue in the fusion. The insertion sites for the dual reporter in sandwich fusions are indicated by a restriction enzyme name.

BfpE has a different topology from another GspF protein. The topology of OutF, a GspF protein from the type II protein secretion system of Erwinia carotovora, was previously determined using -lactamase gene fusions (61). Like BfpE, OutF has a large N-terminal cytoplasmic segment plus TM domains that are analogs of HS1 and HS2. However, the C-terminal portion of the OutF topology is surprisingly divergent from that proposed here for BfpE. A TM domain corresponding to HS3 does not exist in OutF. As a result, most of the C-terminal third of OutF is located in the cytoplasm while the C-terminal third of BfpE is found mostly in the periplasm. The third TM segment in OutF is analogous to HS4 of BfpE in terms of its position in the protein sequence. However, in OutF this segment crosses the membrane from the cytoplasmic to the periplasmic side, while in BfpE it appears to have the opposite orientation. The topologies of the two proteins could be identical if HS3 of BfpE was excluded from crossing the membrane in the presence but not in the absence of HS4. However, this possibility was ruled out by BfpE sandwich fusion data, which indicate that the region downstream of HS3 is located in the periplasm even when HS4 is present. In summary, the presence of HS3 and disposition of HS4 define the differences between BfpE and OutF.