FEMS MicrobiologyLetters92(1992)47-56 ~ 1992Federation of European MicrobiologicalSocieties0378-1{197/92/$05.1111 Published by Elsevier

FEM {14823

Molecular cloning and expression of a major surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis in Escherichia coli Kan-ichi Watanabe % Toshihide Takasawa ", Fuminobu Yoshimura ~, Masami Ozeki ~, Masamitsu Kawanami ~ and Hiroshi Kato " "Departmentof Periodontdogy,&'hod of Dentist~', HokkaidoUniversiO',5apporo, ~Laborato~'of Chemisto', LiberalArts Dirision, Obihiro Unirersityo]'Agriadtureand Veter#ran"Medicine.Obihiro,and ' Departmentof MicrobiokJgy,Schoolo[ Dentistrt', Aichi-Gakuin University,Nagoya,Japan Received 1 November t991 Accepted 21 January 1992 Key words: Porphyromonas bacteroides; Bacteroides gingivalis; Periodontopathogen; Major surface protein; Gene cloning; Gene expression; Expression vector system

1. SUMMARY A major immunodominant surface protein (the 75-kDa protein)of Porphyromonas (Bacteroides) gingivalis 381 has been purified and its aminoterminal amino acid sequence has been determined. Using oligonucleotide probes corresponding to the sequence, we identified a recombinant plasmid clone carrying a single 4.2-kb BamH1 fragment from pUCI9 libraries of P. gingivalis. The BamHl fragment transferred to the bacteriophage T7 RNA polymerase/promoter expression vector system produced a slightly larger (77-kDa) protein, a precursor form, immunoreactive to the antibody against the 75-kDa protein, suggesting that the cloned DNA fragment probably carried

Correspondenceto: F. Yoshimura, Department of Microbiology,Schoolof Dentist"y,Aichi-GakuinUniversity,Chikusa-ku. Nagoya464, Japan.

an entire gene for the 75-kDa protein. Genomic Southern analysis revealed a single copy of the 75-kDa protein gene per genome among all P. gingiralis strains tested, and that no homologous genes are present in other black-pigmented BacteroMes species. These observations suggest that the 75-kDa protein gene may be useful as a specific DNA probe to classify or to detect this organism.

2. INTRODUCTION Porphyromonas (Bacteroides) gingivalis is a Gram-negative oral anaerobe thought to be one of the most prominent periodontopathogens [13]. This organism has unique surface components such as fimbriae, hemagglutinin and protease [4]. They are referred to as major pathogenic factors in this organism. Cell membrane proteins may also contribute to bacterial pathogenicity, pre-

sumably because they are preferentially recognized by host defense mechanisms. Among them a major surface protein (the 75-kDa protein) of P. gingica.lis has recently been purified and characterized [5]: it is one of the immunodominant materials of this bacterium, and is considered to be present as a large stable complex (about 2000 kDa) in its outermost part. The 75-kDa protein exhibits a strong humeral antibody response in humans (refs. 5, 6; K. Watanabe and F. Yoshimura, unpublished data) and a strong B-cell mitogenic activity in in vitro assays (K. Watanabe and T. Umemoto, unpublished data). In this study, we have sought to isolate a recombinant clone carrying the 75-kDa protein gene from P. gingicalis 381. Our specific aims are to provide basic information on the structure of the 75.kDa protein, which might provide further clues to its function and oligomeric structure and result in the development of a specific hybridization probe that would allow us to study the expression of the gene in P. gingivalis.

isolated before use by plating on Luria agar plates

[8]. 3.2. Purification of the 75-kDa protein, determination of the amino-terminal amino acid, and amino-terminal sequencing Purification of the 75-kDa protein from the bacterial envelope fraction was carried out as previously described [5]. Identification of the amino-terminal amino acid was performed by the dansyl method [9]. An Applied Biosystems 470A sequencer equipped with a 130A separation system as a PTH-amino acid analyzer was us.:d for the automatic amino-terminal amino acid sequencing. About 0.6 nmot of protein was used for each run. Polybrene was used as a non-protein carrier to retain the protein on the filter as described by Tarr et al. [10].

3.3. Isolation of genomic and plasmid DNA DNA was isolated from P. gingicalis 381 by a modification of the method of Marmur [11]. Standard procedures were used for enzymatic reactions and for isolation of plasmid DNA [8].

3. MATERIALS AND METHODS

3.4. Construction and testing of oligonucleotide probes

3.1. Bacterial strains and growth conditions. The P. gingicalis strains used were 381, 1112,

Two 20-residue mixed-base probes were synthesized to correspond to the N-terminal coding sequence of the 75-kDa protein by using phosphoramidite chemistry [12] on an Applied Biosysterns model 381A DNA synthesizer. The probes were 5' end-labeled with 32p using a MEGALABEL kit (Takara, Kyoto) as recommended by the manufacturer. The reaction mixture was incubated at 37°C for 30 min, and the labeled probes were used without further purification. To test 20-mers as probes for detecting the 75-kDa protein gene, P. gingicalis 381 genomic DNA was digested with several restriction enzymes including Pstl and BamHl, electrophoresed on 1% agarose gels, and transferred to nylon membranes [13]. The membrane filters were baked, rinsed at 680C for 1 h in 3 × SSC (1 × SSC contains 0.15 M NaC! and 0.015 M sodium citrate, pH 7.0)-0.1% sodium dodecyi sulfate (SDS) and then prehybridized with 6 × SSC-Denhardt solution [8]-100 p,g of sheared denatured salmon sperm DNA per ml, Hybridization with the probes was performed

14018, ATCC 33277, W83, JKG-10, D40C-28, D67D.9, and Shirai. Other bacterial strains were B. asaccharolyticus ATCC 25260, B. endodontalis HG 370, B. intermedius ATCC 25611 and ATCC 33563, B. corporis VPI 9342, B. melaninogenicus ATCC 25845, B. denticola ATCC 33185, B. loescheii ATCC 15930, B. fragilis lid 1638, B. macacae ATCC 33141, B. levii B-151, Actinobaciilus actinomycetemcomitans Y4, Fusobacterium nucleatum FN-2, Streptococcus mutans ATCC 27351, and S. sanguis ATCC 10557. Bacteroides and other anaerobes were grown anaerobically at 37°C as described previously [7]. Escherichia coil JM83 [ara, A(lac.proAB), rpsL, thi, strA, 4~80dlacZzlM15] was used as the host strain for pUC19 plasmid. E. coil NM522 {supE, thi, hsd 5, A(lac-proAB), IF', proAi3, lac lqZ AM15]} was used as the host strain for the expression vector. Cells were maintained as frozen glycerol stocks, and a single colony of each strain was

in fresh prehybridization mixture containing 1-3 x 10" cpm/ml of probe. Hybridization was continued overnight at 370C, after which the blots were rinsed three times at room temperature, followed by further washings at 37°C for 15 min and at 40°C for 15 rain, with 100 ml of 6 x SSC0.1% SDS. To establish probe specificities and wash stringencies, the filters were washed at various temperatures in the beginning.

lope and soluble fractions were separated as described previously [7]. The proteins in some gels were stained with Coomassie brilliant blue, and those from other gels were electrophoretically transferred to nitrocellulose membranes for immunobiotting as described by Towbin et al. [16].

3.5. Plasmid cloning To clone P. gingit'alis genomic DNA fragments into pUC19, P. gingit'alis genomic DNA was separately digested to completion with Hindlll, Pst l and BamHl restriction endonuclease. The latter

To detect gene product from the cloned DNA fragment, the 4.2-kb BamHl fragment was excised from pUC19Bg75, and recloned into multiple cloning sites downstream from the highly specific bacteriophage T7 promoter on pTZI8R (Pharmacia). The DNA fragment was inserted in the two possible directions to the promoter. The two recombinants obtained were separately introduced into E. coli NM522 carrying pGP1-2 by transformation. The pGP1-2 plasmid provides for expression of T7 RNA polymerase. It consists of the T7 RNA polymerase gene under the control of inducible lambda PL promoter, and the gene for the heat-sensitive lambda repressor, ei857 [17]. Clones with both pTZ18R recombinant and pGP1-2 were grown in an enriched medium (2% tryptone, 1% yeast extract, 0.5% NaCI, 0.2% glycerol, 50 mM potassium phosphate, pH 7.2) in the presence of antibiotics (50 #g/ml ampicillin and kanamycin), followed by heat induction and by the addition of rifampicin (100 v.g/ml). Bacterial cells were then suspended in 5 mM Tris. HCI, pH 7.4, and the cell suspension was sonicated for cell lysis. Whole-cell extracts were obtained as a supernatant after unbroken cells were removed by centrifugation at 1000 × g for 10 min. The cell extracts were separated into soluble (supernatant) and envelope (pellet) fractions by ultracentrifugation at 143 000 × g for 60 rain. Then protein patterns in the whole-cell lysates, and in the soluble and envelope fractions of these cells, were examined by SDS-PAGE and immunoblotting as described above.

two enzymes were specially chosen because they produced 1.7-kb and 4.2-kb genomic DNA fragments, respectively, that carried sequences complementary to the probes. The pUC19 vector was separately linearized with endonucleases. Genomic DNA digested separately with Hindlll, Pstl and BamHl was ligated into the compatible vectors, and the ligation mixtures were used to transform JM83 cells rendered competent by calcium chloride treatment. 5-Bromo-4-chloro-3-indolyl-B-o-galactopyranoside (X-gal) was used to identify recombinants as white colonies.

3.6. Screening of HindlIl, Pstl and BamH! clones Recombinants carrying cloned sequences were identified by replication onto membrane filters, followed by colony lysis and either immunoblotting or probe hybridization. One clear, positive colony carrying a cloned BamHl fragment was found from among approximately 5000 transformants. The purified recombinant plasmid was characterized by restriction endonuclease analysis and Southern blotting. A putative clone carrying the 75-kDa protein gene was designated as pUC19Bg75.

3. Z Analysis of proteins by SDS.polyacrylamide gel electrophoresis (SDS-PAGE) and immunobiotting Whole cell lysates were prepared as previously described [14] from JM83 cells containing selected pUC19 recombinants grown under the various culture conditions. The whole cell lysates (50-100 ~g protein) were electrophoresed on 12% SDS-polyacrylamide gels [15]. Whole enve-

3.8. Detection of gene product expressed by the bacteriophage T7 RNA polymerase /promoter expression t'ector system

3.9. Southern-blot analysis of black-pigmented Bacteroides species with the 75-kDa protein clone The 1.7-kb Pst I fragment carrying the greater part of the 75-kDa protein gene, derived from the

4.2-kb BamHi insert in pUClgBg75, was labeled by the random primed method to a specific activity of > 2 × i0 s cpm//.tg with [alpha-3~'P]dCTP. Genomic DNA was isolated from various strains of black-Pigmented Bacteroides species, essentially as described above for P. gingivalis. After restriction of genomic DNA from each strain with BamHl, the DNA fragments were electrophoresed on a 1% agarose gel, and separated DNA fragments were transferred to nylon membranes as described above. The baked membrane filters were rinsed at 68°C in 3 × SSC-0.1% SDS. The membranes were then prehybridized for 3 h at 68°C in 50 ml of 6 × SSC-5 x Denhardt solution-1% SDS-100 #g/ml denatured salmon sperm DNA per membrane. Hybridization with the probes was performed in fresh prehybridization mixture containing 1-5 × 107 cpm/ml of probe. Hybridization was continued overnight at 68°C, after which the blots were rinsed 2 times at 68°C with 100 ml of 2 × SSC-I% SDS. The blots were next rinsed once at 68°C with 100 ml of l × SSC1% SDS. The dried filters were then exposed to x-ray film with an intensifying screen at -70°C for various times.

4. RESULTS

4.1. Amino-terminal amhlo acid sequence and oligonucleotide probes for detecting the 75-kDa protein gene To construct oligonucleotidc probes for detecting the 75-kDa protein gene, we purified the 75-kDa protein and sequenced N-terminal amino acids with a gas-phase sequencer. The sequence up to 40 residues is shown in Fig. !. As shown in the figure, two 20-mer oligonucleotides, designated as probe-I and probe-2, corresponding to the nucleotide sequences of the two oligopeptide spans between Glu-17 and Lys-23 and between 11e-29 and Gly-35, respectively, were designed and synthesized. This was because the two oligopeptides contained Trp and Met, which are each represented by a single codon, UGG and AUG, respectively. The synthesized probes were radiolabeled and tested by Southern-blot hybridization analysis of P. gingivalis genomie DNA.

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Both probe-I and probe-2 yielded a single band of approx. 1.7 kb and 4.2 kb with P. gingivalis genomic DNA fragments restricted by Pstl and BamHl, respectively (data not shown). This observation confirmed that both probes were useful in screening for recombinants carrying sequences of the 75-kDa protein gent with them. Other restriction enzymes tested such as EcoRI, Hindill, Kpnl, and Sacl did not show any distinct hybridizing band with Southern-blot analysis. We therefore screened P. gingivalis genomic libraries prepared by using Psti and BarnHi.

4.2. Plasmid cloning We initially screened Hindlll and Pstl libraries with the polyclonai antibody against the 75-kDa protein. Despite screening over 4000 clones, no immunoreactive clone was detected. Since Southern-blot data were available and indicated that Pst 1 produced a smaller genomic DNA fragment than BamHl did, the Pstl genomic DNA library of P. gingivalis was screened with the oligonudeotide probes. Despite screening

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Fig. 2. Relevant restriction enzyme map of a recombinant plasmid, pUCBg75, carrying it 4.2-kb Bamlll DNA fragment. The solid bars represent pUCI9 plasmid reciter DNA, (P.. hw promoter: O., ha' operator) and the thin line reprc.,,ents cloned P. gbtgit'ali.r DNA. Abbreviations indicate rcstrictivn enzyme sties for BamH! iBm), P~II (Psi, Sinai (Sin}. Sail (SI), Hincll (He), Bglll (Bg), /:'¢oRV {Ev) and ('lal (('1). I~, N-terminal region complememary It} the oligonudeolide prnbe. The 1.7-kb P~'tl fragment that ,~e initia[l~ soughl lt~ clone is indicated by the arrow. This DNA fragment wa.~ used for the genomic Southern analysis of other strains.

over 2000 clones containing P. gingiralis genomic Pst l fragments by the colony hybridization method, we failed to detect any clone that gave a significant hybridization signal. Therefore, another plasmid library was prepared with BamHI and similarly screened. Of the 311t)11 clones screened, one clone showed a significant positive signal and was found to carry a 4.2-kb BamHi insert, a size consistcnt with the genomie DNA fragment that BamH! showed in Southern analysis. This clone was designated as pUClgBg75.

4.3. Characterization of pUClgBg75 insert DNA The 4.2-kb BamH! insert DNA fragment was characterized by restriction enzyme mapping and Southern analysis to specify the N-terminal region complementary to the oligonucleotide probes {Fig. 2). As shown in the figure, we found that an approx, 0.5-kb DNA region between the Sall(Hincll) and Bglll sites contained sequences complementary to the probes. The cloned BamHl fragment was found to include the 1.7-kb Pstl fragment that hybridized with the probes and that we initially sought to clone from genomic DNA. Thcse results suggest that even if the C-terminal region of the 75-kDa protein gene should be present in the direction of the downstream BamHi site, the BamHl fragment could include most of the gene based on the estimated length of the DNA fragment between Sail and the downstream BamHI site.

4.4. l:~rpre,~'skmoj' gene product To determine whether the antibody against 75-kDa protein reacted with a peptide of the same appropriate size, whole cell lysate of E. coli JM83 carrying pUClgBg75 was analyzed by immunoblotting. Since no immunoreactive bands were detected, we constructed a recombinant plasmid carrying the 4.2-kb BamH! fragment in an orientation opposite to that of the parent clone. This plasmid was designated as pUClgBg 75(- ). Although both clones were grown under various culture conditions with or without an inducer, isopropyl beta-o-thiogalactoside (IPTG), again, none of the proteins reactive to the antibody was detected in the clones. One possible explanation for our failure to detect gene product was that transcription by E. coil RNA polymerase from the htc promoter terminates bcfore the gent and thc polymerase is not efficient in synthesizing RNA from P. gmgiralis DNA. Recloning the fragment to a strong expression vector system could obviate this potential difficulty. For this purpose, we chose the bacteriophage T7 RNA polymerase/promoter vector system. The BamHI fragment excised from pUCI9Bg75 was inserted in the two possible directions into multiple cloning sites downstream from the T7 promoter on pTZISR. The two recombinants obtained wcrc designated as pKWi01 and pKWl02 as shown in Fig. 3A. These rccombinants were introduced into E. coli NM522 carrying pGPI-2 by transformation, and the resulting clones were grown and then induced by heat treatment. In the clone carrying pKWI01, a protein with an apparent molecular weight of 77,000 (77-kDa) that was immunorcactive to the 75-kDa protein antibody (Fig. 3B and C) was induced. The size of the expressed protein was confirmed by several experiments. The amount expressed in E. coil was comparable to that in P. gh~git'alis based on band intensity in Western-blot analysis. The results suggested that the entire 75-kDa protein gene was placed in the BamHl insert and. consequently, that the gene was expressed as the 77kDa protein, a slightly larger precursor, presumably with a leader peptide. This has to be confirmed by determination of the DNA sequence of the gene.

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Fig, 5. Southern-blot analysis of BamHl-digested genomic DNA fragments from various strains of P. gingiculis (A) and from various species other than this organism (B). (A) Lanes: S. 32P-labeled iambda DNA digested with BstP! as standard markers: C, pUCBg75 digested with BamHI; 1,381; 2, l ll2: 3, 14018: 4, ATCC 33277: 5, W83; 6. JKG-10: 7. D40C-28; 8, D67D-9: 9, Shirai. (B) The letter C is the same as that specified for panel A. Lanes: 1, B. asaccharolyticus ATCC 25260; 2. B. endodontalis HG370; 3, B. bltermedius ATCC 25611: 4, B. huermedius ATCC 33563; 5. B, cor~ris VPi 9342: 6: B. melaninogenicus ATCC 25845; 7, B. denticola ATCC 33185; 8, B. loescheii ATCC 15930; 9. B. fragilis l i d 1638; 10, B. ma,'acae ATCC 33141; It. B. lecii B-151; 12. A. actimJmycetemcomitans Y4; 13, F. nucleatum FN-2; 14, S. mutans ATCC 27351; 15, S, sanguis ATCC 10557.

4.5. Localization of gene product ( 77-kDa plvtein) bt the E. coil cell The whole cell extracts of two induced cultures, E. coli NM522 (pGPI-2, pKWI01) and NM522 (pGPI-2, pKWI02), were separated into soluble and envelope fractions by ultracentrifugation, and then the protein patterns of the fractions were examined in SDS-PAGE, after which immunoblot analysis was carried out using the antibody against the 75-kDa protein. As shown in Fig. 4, the induced 77-kDa protein was localized in the envelope (insoluble) fraction, but not in the soluble fraction.

4.6. Southern.blot analysis of black-~,igmented Bacteroides species To test the possibility that homologous genes might exist in other black-pigmented Bacteroides species, we carried out Southern-blot analysis of BamHi-digested genomic DNA isolated from various species, including P. gingit'alis strains, with the 1.7-kb Pstl fragment in pUC19Bg75 as the probe. As shown in Fig. 5A, all P. gingicalis strains tested except for W83 gave a strong, single hybridizing band, suggesting that although some strains showed restriction fragment length polymorphism of DNA, P. gingi~'a!is strains appeared to have a single-copy genc for the 75-kDa protein. None of the other species tested gave a significant hybridization signal (Fig. 5B).

5. DISCUSSION In this study, we present the cloning and initial characterization of the gene for the major surface protein, the 75-kDa protein, of P. gingit,alis, During the course of studies on genes for fimbriae [18,19] and protease [7] of this organism, we could not clone P. gingicalis genes by detection of gene producis either with enzyme activities or antibodies (F. Yoshimura, unpublished results), and consequently suspected that the genes may not be properly expressed in E. coll. Therefore, we intended to use DNA probes in order to screen P. gingicalis gene libraries because they are essential to clone genes when we cannot expect proper expression of foreign genes in E. coli. While we

were first attempting to screen a gene library with the antibody against the 75-kDa protein, aminoterminal amino acid sequencing of the 75-kDa protein was carried out to obtain adequate information on the sequences of the DNA probes. By screening a pUC plasmid library of P. gingicalis genomic DNA with oligonucleotide probes corresponding to N-terminal amino acid sequences, we have identified a clone carrying a sequence homologous to the probes. As we suspected, the cloned gene on pUC plasmid in the proper orientation was not expressed, and the gene product was not detected at all even in immunoblot analysis. However, the gene was indeed expressed as a 77-kDa precursor protein in the bacteriophage T7 RNA polymerase/promoter expression vector system, suggesting that a possible, presumably strong, P. gingi~'alis promoter upstream from the 75-kDa protein gene was not recognized by E. coil RNA polymerase. A recent report on P. gingicalis RNA polymerase supports this idea [20]. RNA polymerase of P. gingil,alis was reported to be unrelated to that of E. coli, and they seemed to be incompatible. On the other hand, phage T7 RNA polymerase is unique and strong among known RNA polymerases, in that all transcription by this enzyme is directed from the unique T7 promoters and transcription by the enzyme from a T7 promoter on a plasmid results in transcripts several times the plasmid length [21]. Since expression of the immunoreactive 77-kDa protein was dependent upon heat induction and proper orientation of the inserted DNA fragment to the T7 promoter on the plasmid (Fig. 3B and C), the expression is considered to be due to the transcription from the T7 promoter. The 77-kDa protein could be expressed more clearly if the upstream region, for instance the region between BamHl and Sail, of the 75-kDa protein gene were deleted. As described in a previous report [5], some P. gingicalis strains such as 14018, W83, JKG-10, D40C-28, and D67D-9 did not contain serologicaUy related 75-kDa proteins. However, all these strains have the 75-kDa protein gene and none of the other species tested had homologous sequences (Fig. 5A and B), suggesting that the 75-kDa protein gene can be species specific and

may be useful as a specific D N A probe to classify or to detect this organism, Complete D N A sequencing of the 75-kDa protein gene is in progress,

ACKNOWLEDGMENTS We thank T. Ikeda for critical review, Y. Takahashi, A. Uchida and K. Sagisaka for carrying out some studies, and Y. Shimizu and S. Ichihara for their valuable suggestions. This work was supported by grants from the Akiyama Foundation, Ciba-Geigy Foundation (Japan) for the Promotion of Science, and the ;qaito Foundation and by Grants-in-Aid for Scientific Research and Co-operative Research from the Ministry of Education, Science and Culture of Japan. We also thank K. Barrymore for helpful advice and discussion on the manuscript, and H. Nikaido for providing us with the strains of the bacteriophage T7 R N A p o l y m e r a s e / p r o m o t e r vector system.

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