A 55-Kilodalton Immunodominant Antigen of Porphyromonas ... · DNA sequence data were analyzed by...

INFECTION AND IMMUNITY,0019-9567/99/$04.0010

Mar. 1999, p. 1157–1171 Vol. 67, No. 3

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

A 55-Kilodalton Immunodominant Antigen of Porphyromonasgingivalis W50 Has Arisen via Horizontal Gene Transfer

SHIRLEY A. HANLEY, JOSEPH ADUSE-OPOKU, AND MICHAEL A. CURTIS*

MRC Molecular Pathogenesis Group, Department of Oral Microbiology, St. Bartholomew’s and The Royal LondonSchool of Medicine and Dentistry, Queen Mary and Westfield College, London E1 2AA, United Kingdom

Received 28 May 1998/Returned for modification 18 August 1998/Accepted 30 October 1998

A 55-kDa outer membrane protein of Porphyromonas gingivalis W50 is a significant target of the serumimmunoglobulin G antibody response of periodontal disease patients and hence may play an important role inhost-bacterium interactions in periodontal disease. The gene encoding the 55-kDa antigen (ragB, for receptorantigen B) was isolated on a 9.5-kb partial Sau3AI fragment of P. gingivalis W50 chromosomal DNA in pUC18by immunoscreening with a monoclonal antibody to this antigen. The 1.6-kb open reading frame (ORF)encoding RagB was located via subcloning and nested-deletion analysis. Sequence analysis demonstrated thepresence of an upstream 3.1-kb ORF (ragA) which is cotranscribed with ragB. A number of genetic charac-teristics suggest that the ragAB locus was acquired by a horizontal gene transfer event. These include asignificantly reduced G1C content relative to that of the P. gingivalis chromosome (42 versus 48%) and thepresence of mobility elements flanking this locus in P. gingivalis W50. Furthermore, Southern blotting and PCRanalyses showed a restricted distribution of this locus in laboratory and clinical isolates of this bacterium. Theassociation of ragAB1 P. gingivalis with clinical status was examined by PCR analysis of subgingival samples.ragAB1 was not detected in P. gingivalis-positive shallow pockets from periodontal disease patients but waspresent in 36% of the P. gingivalis-positive samples from deep pockets. These data suggest that the ragAB locuswas acquired by certain P. gingivalis strains via horizontal gene transfer and that the acquisition of this locusmay facilitate the survival of these strains at sites of periodontal destruction.

Porphyromonas gingivalis is a gram-negative anaerobic bac-terium considered to be a major etiological agent in destructiveperiodontal disease (38). The periodontal diseases are chronicinflammatory conditions of the tooth-supporting tissues whichin severe cases result in the destruction of the periodontium,including alveolar bone, leading to eventual tooth loss. In pa-tients with periodontitis, P. gingivalis is frequently isolated inhigh numbers from heavily inflamed subgingival lesions, whereit may constitute a significant proportion of the oral microflora.P. gingivalis may also be isolated from periodontally healthypopulations but at a much lower percentage of the total mi-croflora (5, 28). It is not known whether strains colonizingperiodontally healthy individuals are genetically distinct fromthose associated with periodontitis, but DNA fingerprintingstudies have identified a large number of distinct P. gingivalisgenotypes (23, 25, 30). The pathogenic potentials of differentP. gingivalis strains have been investigated in animal models ofsoft tissue destruction and do vary according to the strain used,suggesting that strain variation may influence the virulencepotential of this organism (20, 32).

The interplay between the periodontal microflora and thehost in destructive periodontal disease is composed of a com-plex set of interactions, involving extracellular and surface de-terminants of the bacteria and the host’s inflammatory andspecific immune responses. In order to identify the determi-nants of P. gingivalis which participate in this dynamic interac-tion, we have analyzed the specific immunoglobulin G serumantibody responses of periodontal disease patients and

matched healthy controls to preparations of P. gingivalis W50outer membranes and cell sonicates (11). These studies led tothe identification of three immunodominant surface antigens(115, 55, and 47 kDa) of this organism which, by extrapolation,are expressed in vivo and interact with the immune systems ofperiodontal disease patients.

Subsequent molecular genetic and immunochemical inves-tigations have demonstrated that the 47-kDa antigen is a hem-agglutinin/adhesin component of a major arginine-specific pro-tease, RgpA, of this organism and that the coding region forthis antigen is present in multiple genes of P. gingivalis (1, 12).Members of this family of gene products all have putative rolesin proteolytic, adherence, or surface transport processes andare thought to represent key virulence determinants (2, 14, 18).The distribution of these genes has been examined, and theevidence obtained to date suggests that they are present in alllaboratory and clinical isolates, although some between-straingenetic polymorphism at some of these loci is detectable.These data are analogous to the findings for another importantcolonization determinant of P. gingivalis, the surface fimbriae,the gene for which is similarly ubiquitous in different strains(24). To our knowledge, there are no examples to date ofvirulence determinant genes of this bacterium which show arestricted distribution which could account for between-strainvariance in virulence potential.

The properties of the remaining two antigens (115 and 55kDa), identified on the basis of their reactivity with periodontaldisease case sera, have not been described. However, produc-tion of a monoclonal antibody to the 55-kDa antigen by Millaret al. (31) facilitated the analysis of the expression of thisantigen. These studies confirmed that the 55-kDa antigen con-stitutes a major fraction of the outer membrane of P. gingivalisW50 and, furthermore, that it is expressed in only approxi-mately 60% of laboratory strains and clinical isolates (31). Thepurpose of the present study was to identify and characterize at

* Corresponding author. Mailing address: MRC Molecular Patho-genesis Group, Department of Oral Microbiology, St. Bartholomew’sand The Royal School of Medicine and Dentistry, Queen Mary andWestfield College, 32, Newark St., London E1 2AA, United Kingdom.Phone: 44 171 377 0444. Fax: 44 171 247 3428. E-mail: [email protected].

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the molecular level the genetic component encoding the 55-kDa antigen.

MATERIALS AND METHODS

Bacterial strains and culture conditions. P. gingivalis W50, W50Be1, W50Br1,W83, LB13D-3, 381, 11834, WPH-34, and WPH-35 and Porphyromonas asaccha-rolytica ATCC 25260 have been described previously (1, 3). Chromosomal DNAsfrom 38 clinical isolates (3) were used in PCR analysis. Porphyromonas specieswere grown on a blood agar base containing 5% defibrinated horse blood or inbrain heart infusion broth supplemented with hemin (5 mg ml21) in an anaerobicchamber (Don Whitley, Shipley, United Kingdom) with an atmosphere of 85%N2, 10% H2, and 5% CO2 at 37°C. Escherichia coli XL-1 Blue (Stratagene Ltd.,Cambridge, United Kingdom) was used for all cloning experiments and wasgrown aerobically on Luria-Bertani medium (1% tryptone, 0.5% yeast extract,0.5% NaCl) with 20 mg of tetracycline ml21 added for F9 episome selection. Theplasmid pUC18 was used for cloning in E. coli XL-1 Blue and was selected onLuria-Bertani medium supplemented with ampicillin (50 mg ml21). Blue or whitecolony color selection was used to distinguish between nonrecombinant andrecombinant E. coli clones, respectively, by means of X-Gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) and IPTG (isopropyl-b-D-thiogalactopyrano-side). General cloning procedures were as described by Sambrook et al. (35).

Protein analysis. E. coli XL-1 Blue transformants were immobilized at a highdensity on nitrocellulose membranes and permeabilized with chloroform vaporfor 30 minutes followed by lysis with lysozyme in the presence of pancreaticDNase I for 16 h. Membranes were blocked in 5% bovine serum albumin inphosphate-buffered saline and incubated with the primary antibody (DRU55.5,which recognizes the 55-kDa outer membrane antigen, at a 1:500 dilution) in 1%bovine serum albumin in phosphate-buffered saline. The antibody-antigen reac-tion was detected with horseradish peroxidase-conjugated antimouse antiserumat a 1:500 dilution, and the reaction was developed by using H2O2 and diami-nobenzidine (0.05%).

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was per-formed as described by Laemmli (22) on 10% separating gels. Transfer tonitrocellulose membranes for immunoblot analysis was carried out in a bicar-bonate transfer buffer (3 mM Na2CO3, 10 mM NaHCO3 [pH 9.9], 20% metha-nol) at a constant current of 400 mA for 2 h. Immunostaining of the membraneswas performed with horseradish peroxidase as described above.

Outer membranes of P. gingivalis W50 were prepared by Sarkosyl solubiliza-tion, and P. gingivalis whole cells were also prepared as previously described (11).

DNA manipulations and sequencing. Two sets of P. gingivalis libraries wereconstructed by cloning partially Sau3AI-digested P. gingivalis W50 chromosomalDNA in the ranges of 1.8 to 5 kb and 5 to 12 kb into the plasmid pUC18 andtransforming it into competent E. coli XL-1 Blue cells. The E. coli transformantswere immobilized on nitrocellulose membranes and screened by using the mono-clonal antibody DRU55.5, which was raised against the immunodominant 55-kDa antigen. Approximately 10,000 colonies from each library were screened.The library consisting of the smaller insert sizes of 1.8 to 5 kb was nonreactivewith the antibody; however, one immunoreactive clone was identified from thelibrary containing the larger inserts of 5 to 12 kb. The recombinant plasmidpPM1, which expressed the 55-kDa protein, was isolated from the E. coli clonefor retransformation and characterization. Restriction analysis indicated thatpPM1 contained a 9.5-kb P. gingivalis chromosomal insert. Removal of a 2.0-kbSmaI fragment from one end of pPM1 followed by pPM1 religation gave rise toa 7.5-kb insert, pSM, which also expressed the 55-kDa protein. The plasmid pSMwas used as a convenient template for the construction of a set of nested-deletionderivatives. The pSM plasmid was digested with SphI and XbaI, both of whichspecifically restrict the DNA within the multiple cloning site of pUC18, and thenwas subjected to a progressive unidirectional digestion with exonuclease III. Atotal of 24 derivatives were prepared by using the Nested Deletion kit (Amer-sham Pharmacia Biotech, St. Albans, United Kingdom). Plasmid DNA wasisolated from E. coli by the method described by Birnboim and Doly (6).

For DNA sequence analysis, sequencing-grade plasmid DNA was prepared byion-exchange chromatography on columns from Qiagen Ltd. (West Sussex,United Kingdom). The nested-deletion plasmids were subjected to Taq cyclesequencing by using the dideoxy dye termination chemistry from Applied Bio-systems and the universal M13 forward and reverse primers. The products wereanalyzed on an Applied Biosystems model 377 automated DNA sequencer.Complete coverage of both strands of the pSM subclone and linkage of contigswere achieved by primer walking. All oligonucleotide primers were purchasedfrom Amersham Pharmacia.

DNA sequence data were analyzed by using Gene Jockey II (Biosoft, Cam-bridge, United Kingdom), the Wisconsin Genetics Computer Group software(13), and the Staden DNA and protein analysis software (National Institute forMedical Research, London, United Kingdom).

Southern hybridization analysis. Genomic DNA was electrophoresed in aga-rose gels, depurinated in 0.25 M HCl, and then transferred onto Hybond N1membranes (Amersham Pharmacia) by vacuum blotting (Vacu-Aid; Life Sci-ences International, Hampshire, United Kingdom) under alkaline conditions(0.4 M NaOH). After blotting, the membranes were prehybridized for 1 h at 65°Cin Rapid-Hyb buffer (Amersham Pharmacia) and hybridized for 2 h in the samesolution with heat-denatured 32P-labelled probes. The probe DNA for ragA and

ragB was prepared by PCR (see below), and that for orf3 was prepared by EcoRVrestriction digestion of pPM1. Probes were labelled by the random-primermethod with the Ready To Go labelling system (Amersham Pharmacia). Mem-branes were washed twice in final stringent washes of 0.23 standard saline citrate(SSC) (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate) containing 0.1%SDS at 65°C. Autoradiography was performed at 270°C with intensifying screensfor variable exposure times on Kodak-X Omat LS Films (Genetic ResearchInstrumentation, Essex, United Kingdom).

Northern hybridization analysis. RNA was prepared from P. gingivalis W50cells after 6 h of growth in brain heart infusion by using a Total RNA IsolationReagent Kit (Advanced Biotechnologies, Epsom, United Kingdom). RNA spe-cies were resolved in denaturing formaldehyde agarose gel (35) and then trans-ferred to Hybond N1 membranes by vacuum blotting (Vacu-Aid; Life SciencesInternational) essentially as described above for Southern blotting except thatthe transfer was performed in 203 SSC. RNA was fixed to the membrane byusing UV light (l 5 312 nm) for 5 min. The blot was stained with methylene blue(0.04% in 0.5 M Na-acetate, pH 6) (21) to locate 16S RNA, 23S RNA, and RNAmarkers (0.24 to 9.5 kb; Life Sciences International) and then briefly destainedwith water and prehybridized in Rapid-Hyb buffer (Amersham Pharmacia) at65°C. Northern hybridization was as described above for Southern hybridization.

PCR analysis of ragA and ragB gene distributions. Probes for Southern anal-ysis of ragA and ragB distributions were prepared by amplification of the com-plete 1.5-kb ragB gene and an internal 1.6-kb fragment of the ragA gene. ThePCR products were purified by using the Wizard cleanup kit (Promega,Southampton, United Kingdom). Amplification was performed in a thermalcycler (Omnigene; Life Sciences International) with Red Hot DNA polymerase(Advanced Biotechnologies). The reaction mixture consisted of approximately20 ng of pSM plasmid template, 10 ml of 103 PCR amplification buffer (100 mMTris, 500 mM KCl, 1% [vol/vol] Triton X-100), 0.5 mg of forward and reverseragA.1 or ragB.1 primers (Table 1), 250 nM deoxynucleoside triphosphates (Am-ersham Pharmacia), and 2 U of DNA polymerase in a 100-ml volume adjusted bythe addition of distilled water. The program conditions for both primer sets wereas follows: 2 min at 95°C followed by 25 cycles of 95°C for 1 min, 50°C for 1 min,and 72°C for 3 min. After the final cycle, the extension reaction was allowed toproceed at 72°C for 3 min.

For analysis of ragA and ragB distributions in clinical isolates, PCR was usedto amplify smaller amplicons of 435 and 509 bp of ragB and ragA, respectively.Chromosomal DNAs purified from 38 P. gingivalis clinical isolates (3) were usedas templates in the PCR analysis. The reactions were carried out in a 100-mlvolume consisting of 50 ml of Taq Master Mix (Qiagen Ltd.), approximately 200ng of template chromosomal DNA, 0.5 mg of forward or reverse ragA.2 andragB.2 primers (Table 1), and 47 ml of H2O. The program conditions for bothprimer sets were as follows: a hot start of 95°C for 2 min followed by 25 cycles of95°C for 1 min, 60°C for 1 min, and 72°C for 1 min. After the final cycle, theextension reaction was allowed to proceed at 72°C for 3 min.

PCR detection of ragB in clinical samples. Subgingival plaque samples fromadult periodontitis patients were obtained from patients attending the periodon-tal clinic (mean age, 44 years; range, 28 to 60 years; n 5 28) at the School ofMedicine and Dentistry. Control samples were obtained from periodontallyhealthy students and members of the staff (mean age, 32 years; range, 22 to 50years; n 5 33). Clinical samples were collected by the procedures outlined byAshimoto et al. (4). Briefly, sample sites were isolated with cotton rolls and airdried. Paper points were inserted to the depth of the pockets. Samples fromperiodontal disease patients were taken from at least three of the deepestpockets, and a sample was also taken from a site without evidence of severeperiodontal destruction (,4-mm pocket depth). Two samples were taken fromeach member of the control group, from the lower right and lower left first molar.All pockets sampled in the control group were ,4 mm. The paper points wereremoved 5 to 10 s after insertion and placed in a vial containing 1 ml of fastidiousanaerobic broth (Oxoid, Basingstoke, United Kingdom). The samples in fastid-ious anaerobic broth were mixed well on a Vortex mixer, 0.5 ml of the microbialsuspension was removed and centrifuged, and the pellet was washed three timeswith distilled water. The bacterial pellets were resuspended in 0.1 ml of distilledwater, boiled for 10 min, and placed on ice. After centrifugation to remove celldebris, the supernatant was used for PCR analysis.

The reactions were carried out in a 100-ml volume consisting of 50 ml of TaqMaster Mix (Qiagen Ltd.), 10 ml of sample, and 0.5 mg of forward and reverse P.gingivalis 16S RNA or ragB.2 primers (Table 1) made up to 100 ml with distilledH2O. The program conditions for both primer sets were as follows: 2 min at 95°Cfollowed by 36 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 1 min. Afterthe final cycle, the extension reaction was allowed to proceed at 72°C for 3 min.Primers designed for the catalytic domain of the rgpA protease gene (Table 1)were also used for P. gingivalis detection in samples which were negative for P.gingivalis with the 16S RNA primers but positive with the ragB.2 primers. ThePCRs were performed with Taq Master Mix (Qiagen Ltd.) as outlined above, andthe conditions used were as described by Allaker et al. (3). The results werestatistically analyzed by Spearman’s rho correlation analysis with Stata software.

Nucleotide sequence accession number. The nucleotide sequence data re-ported in this study have been assigned GenBank accession no. AJ130872.

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RESULTS

Identification of an E. coli clone, PM1, expressing the 55-kDa antigen. Immunoblot analysis of colony lifts by using amonoclonal antibody (DRU55.5) to the 55-kDa antigen (31)resulted in the identification of one E. coli clone from thelibrary containing plasmids with the larger inserts of 5 to 12 kbonly. The E. coli clone identified, PM1, contains a 9.5-kb frag-ment of P. gingivalis DNA in pUC18 and expresses the 55-kDaprotein (Fig. 1B).

To localize the gene expressing the 55-kDa protein, subcloneanalysis was performed on the 9.5-kb pPM1 insert by restric-tion digestion. SmaI digestion of pPM1 and recircularization

generated a 7.5-kb subclone, pSM (Fig. 2), which maintainedexpression of the 55-kDa protein (Fig. 1B). Restriction analysiswith the enzymes BglII, HindIII, BamHI, and PstI, whose rec-ognition sites lie within pPM1, did not generate subclonesexpressing the 55-kDa protein (Fig. 2).

Localization of the gene encoding the 55-kDa antigen withinpSM. The 7.5-kb P. gingivalis insert in pSM, which expressedthe 55-kDa antigen, was chosen as a template to generate a setof nested-deletion derivatives. pSM was subjected to progres-sive unidirectional digestion by the enzyme exonuclease IIIfollowed by blunting with S1 nuclease. This resulted in theproduction of 24 pSM derivatives. The insert sizes of these

FIG. 1. (A) Protein composition of the outer membrane of P. gingivalis W50 in SDS–10% polyacrylamide gel electrophoresis. Lane 1, low-molecular-weightstandards; lane 2, outer membrane preparation of P. gingivalis W50. Arrows indicate the positions of the immunodominant 115- and 55-kDa outer membrane proteins.(B) Western blot analysis of whole-cell preparations of E. coli XL-Blue containing pUC18 (lane 1), E. coli clone PM1 containing a 9.5-kb fragment of P. gingivalis W50DNA cloned in pUC18 (lane 2), E. coli subclone SM containing a 7.5-kb fragment of P. gingivalis W50 DNA (lane 3), and an outer membrane preparation of P. gingivalisW50 (lane 4). Western analysis was performed with the monoclonal antibody DRU55.5.

TABLE 1. PCR amplification primers

Amplification (reference) Primer pairs (59-39) Direction Base position(amplicon length [bp])

ragA.1 TGT CTG GTG CGA TAG CGA ATA G Forward 3198–4904 (1,600)TAC ATA GGT GAG TTT GAG ATT C Reverse

ragB.1 AAT ACT GAA AAT CCA CGA Forward 5275–6686 (1,500)TAG GGG CTG CGA CAA AAA Reverse

ragA.2 CAA CCA CCA AGC ACT CGT Forward 4033–4542 (509)CGA CTT GAC CAG GAA CAT AC Reverse

ragB.2 GAC TCT TGG CTC GTT TAC TG Forward 6001–6436 (436)ACG CAA ACG ACT ACC CTC A Reverse

P. gingivalis 16S RNA (4) AGG CAG CTT GCC ATA CTG CG Forward 729–1132 (404)ACT GTT AGC AAC TAC CGA TGT Reverse

P. gingivalis rpgA catalytic domain (3) AGT GAG CGA AAC TTC GGA GC Forward 1199–2902 (1,709)CCT GTC CAA TAT GGG TCA CTA TGG Reverse

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derivatives ranged from 0.2 to 7.5 kb. Western blot analysis ofthe E. coli clones with the monoclonal antibody DRU55.5revealed a positive reaction with the nine largest derivatives(5.4 to 7.5 kb), while the smaller derivatives were nonreactivewith the antibody. These results, coupled with the results of theWestern analysis of the subclones of pPM1 generated by re-striction digestion, localized the coding region for the DRU55.5epitope to within the initial 3 to 4 kb of the pSM subclone (Fig.2).

Sequence analysis of the pSM insert and identification ofthe ragAB locus. Nucleotide sequence analysis of pSM andpPM1 indicated that there are two major open reading frames(ORFs), which are flanked by two potentially mobile elements.A 1.6-kb ORF encoding a protein of approximately 55 kDa isfound in the first 3 to 4 kb of pSM. Western blot analysis of thenested-deletion clones had previously shown the epitope rec-ognized by DRU55.5 to lie within this region, indicating thatthe 1.6-kb ORF identified encodes the antigenic 55-kDa outer

FIG. 2. Schematic diagram of restriction digest-generated derivatives of pPM1 and their reaction with the monoclonal antibody DRU55.5. The fragment shown iscloned in the BamHI site of pUC18, with the residual pUC18 multiple cloning sites of EcoRI and HindIII located to the left and right of the insert, respectively.Restriction enzyme sites in pPM1: S, SmaI; Bg, BglII; H, HindIII; B, BamHI; and P, PstI. 1, positive reaction with DRU55.5 in Western blot analysis; 2, nonreactivitywith DRU55.5 antibody. The arrow depicts the direction of exonuclease III digestion in the construction of the nested-deletion derivatives. The dashed line representsthe point at which loss of reactivity with DRU55.5 occurs, between the first 9 nested deletion derivatives and the remaining 15 derivatives.

FIG. 3. Schematic diagram of the genetic organization of P. gingivalis W50 DNA in pPM1 and pSM. The horizontal bar represents the 9.5- and 7.5-kb P. gingivalisDNA fragments cloned in pPM1 and pSM, respectively; the vertical bars indicate positions of restriction enzyme sites. Restriction enzyme sites: S, SmaI; Bg, BglII; H,HindIII; B, BamHI; P, PstI. The arrows indicate the locations and directions of ORFs, with known motifs shaded: ■, TonB box sites; u, signal sequence motif oflipoproteins. The heavier outlined arrows of ragA and ragB represent the difference in the G1C contents of these genes in comparison to the G1C content of P.gingivalis genes.



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membrane protein. A 3.1-kb ORF encoding a protein with acalculated molecular size of 115 kDa is situated 30 bp up-stream of the 1.6-kb gene (Fig. 3). Due to the direction oftranscription of these two ORFs and their close proximity toeach other, the gene for the 115-kDa protein was named ragA(for receptor antigen A), and the gene for the 55-kDa outermembrane protein was named ragB.

An incomplete ORF, orf3, is present at the 39 end of thepSM insert approximately 1 kb downstream of ragB (Fig. 3).Database homology searches of the region between ragB andorf3 revealed sequence similarity at the amino acid level withlocalized regions of a number of potentially mobile elements,including a putative transposase from Vibrio cholerae (acces-sion no. AF004384) and the P. gingivalis insertion sequenceelement PGIS2 (41). In addition, regional similarity was alsofound with the H repeat-associated protein (42) of E. coli(accession no. P28917).

In the 59 region of pSM and upstream of ragA is 300 bpcorresponding to the C-terminal region of the product of the1.3-kb P. gingivalis insertion sequence element IS1126 (27).Subsequent sequence analysis of the 59 end of pPM1 revealedthe additional 1 kb of this insertion sequence ORF, thus iden-tifying a complete copy of IS1126 (Fig. 3). Sequence compar-ison between IS1126W83, sequenced by Maley and Roberts(27) from P. gingivalis W83, and the copy of IS1126W50 presentin pPM1 demonstrated 97% homology at the nucleic acid level.Examination of the restriction enzyme sites of IS1126W50 re-vealed an additional PstI site at position 806 (Fig. 4), indicatingheterogeneity within the transposase between the two P. gin-givalis strains.

Examination of the G1C content revealed a ratio of 47.5%for IS1126, which is comparable to the expected 46 to 48%ratio for P. gingivalis genes. However, analysis of ragA and ragBrevealed a significantly lower G1C ratio of approximately42%. Furthermore, the noncoding region downstream of ragBwith deduced sequence similarity to transposases also dis-played a G1C ratio (39%) different from that of the remainderof the chromosome. The difference in the G1C ratios suggeststhat this entire locus may have originated from a source otherthan P. gingivalis via a horizontal gene transfer event.

Analysis of the nucleic acid sequence upstream of the ragAstart codon revealed putative promoter sequences at positions1791 to 1796 and 1835 to 1840 which share 100 and 85%homology, respectively, with the 235 and 210 E. coli consen-sus promoter sequences. An E. coli consensus ribosome bind-ing site (RBS) (37) could not be located. Examination of thesequence upstream of the ragB start codon could not identify apromoter sequence, suggesting either that ragB does not con-tain a typical E. coli promoter sequence or that transcription ofragB may be linked to that of ragA. Upstream of orf3 aresecond putative promoter sequences at positions 7126 to 7131and 7167 to 7173 which share 100 and 85% identity with the E.coli 235 and 210 promoter sequences, respectively. A putativeRBS with 85% identity to the E. coli RBS is located at posi-tions 7490 to 7495 (Fig. 4) preceding orf3.

Database analysis of the ragA, ragB, and orf3 genes. Nucleicacid and amino acid homology searches of the National Centerfor Biotechnology Information database with the sequenceencoding the 55-kDa protein (RagB) did not yield significantsimilarities with any existing sequences. However, a proteinmotif search with the deduced amino acid sequence revealedthe presence of a signal sequence typical of lipoproteins (19).A cysteine residue at position 20 in the amino acid sequence(Fig. 4) denotes the specific cleavage site for signal peptidaseII, the enzyme responsible for signal sequence cleavage oflipoproteins. Other features typical of a signal sequence in-

clude a net positive charge at the N terminus (the start methi-onine followed by two lysine residues), a hydrophobic core(residues 4 to 16), and, following signal peptidase II cleavage,a net negative charge at the N terminus of the mature proteinstarting at residue 20. Hydropathy plot analysis of the aminoacid sequence of RagB also demonstrated the hydrophobicregion at the N terminus typical of signal sequences (notshown).

Significant homology was found in database searches withthe 3.1-kb ragA gene. RagA was found to have significantsequence similarity to several protein sequences, including theTonB-dependent outer membrane receptor protein SusC (forstarch utilization system gene C) of Bacteroides thetaiotamicron(34), the TonB-dependent colicin I receptor (CirA) of E. coli(16), and the TonB-linked adhesin (Tla) of P. gingivalis (2).The homology between RagA and SusC is significantly greater(probability value of 285) than those between RagA and CirAand between RagA and Tla (probability values of 24) andspans the entire length of the SusC protein sequence. SusC isa TonB-linked receptor which forms part of a complex in-volved in the uptake and utilization of malto-oligosaccharidesand starch (34).

The regions of RagA with greatest homology to SusC arelocated primarily in the first 300 residues of the N terminus andthe last 30 residues at the extreme C terminus of the proteinand suggest a relationship between RagA and TonB-linkedreceptors in other gram-negative bacteria. A multiple align-ment of the putative TonB boxI and TonB boxIII regions ofRagA with the TonB boxes of SusC and other TonB-linkedreceptors and their percent similarity values are shown in Fig.5.

The C-terminal amino acid residues of RagA also exhibitsimilarity to TonB-dependent receptor proteins. A C-terminalphenylalanine residue is highly conserved in TonB-dependentreceptors, as are hydrophobic amino acids at positions 23, 25,27, and 29 from the C terminus (taking the C-terminal phe-nylalanine residue as 21). These features are thought to beimportant in outer membrane protein assembly and sorting(40). An additional feature identified by protein motif searchesof RagA is a conserved hydrophobic hexapeptide, LPxTGT(amino acid residues 798 to 803), which is involved in theanchoring of the protein to the cell wall (36). Hydropathy plotanalysis also indicated a hydrophobic region at the N terminus,which is typical of a signal sequence, and a hydrophobic regionat the C terminus due to the presence of the conserved TonB-linked C-terminal motif (not shown). From database searches,sequence analysis, and hydropathy plot analysis of the gene,RagA bears all the features typical of a TonB-linked outermembrane receptor.

Nucleic acid and amino acid sequence searches of the in-complete ORF, orf3, did not yield significant homology withany known sequence. However, a protein motif search didreveal the presence of a lipoprotein signal sequence, with thesignal peptidase II cleavage site located before the cysteineresidue at amino acid position 26 (Fig. 4).

Northern analysis of ragA and ragB: presence of a ragoperon. Transcription of ragA and ragB in P. gingivalis W50 wasexamined by Northern blot analysis of total RNA from expo-nentially grown cells (Fig. 6). A single mRNA transcript ofapproximately 4.7 kb hybridized to both the ragA and ragBprobes, indicating that ragA and ragB are cotranscribed as apolycistronic message. This result agrees with the earlier find-ing that an E. coli consensus promoter sequence could not befound for ragB and also with the finding that a 55-kDa-antigen-expressing clone could be isolated only from a P. gingivalislibrary with insert sizes of between 5 and 12 kb. The finding



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FIG. 4. Nucleotide and amino acid sequences of the coding regions of the P. gingivalis W50 insert in pPM1. Sequences shown are those of IS1126, ragA, ragB, andthe incomplete orf3. The putative RBS, the E. coli 235 and 210 promoter sites, and the methionine ATG start codons are shown in boldface. Restriction enzyme andprimer sites are shown above the sequence and underlined. The 12-bp inverted repeats of IS1126 are depicted in boldface. In the RagA sequence the TonB boxes andconserved TonB C terminus are labelled and underlined, and the conserved hexapeptide motif involved in membrane anchoring is boxed. The signal peptidase IIcleavage sites are indicated by arrows at the cysteine residues in the RagB and Orf3 deduced amino acid sequences.



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that ragA and ragB form a single transcriptional unit stronglysuggests that the products of these genes are functionally linked.

Distribution analysis of ragA, ragB, and orf3 among P. gin-givalis laboratory strains. Previous immunochemical investiga-

tions had indicated that RagB (the 55-kDa antigen) is ex-pressed in only a proportion of P. gingivalis strains (31). Todetermine whether this was reflected in a restricted distri-bution of the ragAB genomic locus, Southern hybridization

FIG. 4—Continued.



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experiments were performed under stringent conditions. Ge-nomic DNAs from laboratory strains of P. gingivalis, P. asac-charolytica ATCC 25260, and E. coli XL-1 Blue were restrictedand probed with a 32P-labelled DNA fragment. Analysis of the

ragA, ragB, and orf3 distributions was performed as follows:chromosomal DNA was restricted with ClaI and probed withan internal 1.6-kb PCR-amplified fragment of ragA (Fig. 7A),chromosomal DNA was restricted with HindIII and probed

FIG. 4—Continued.



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with a 1.5-kb PCR-amplified product of ragB (Fig. 7B), andchromosomal DNA was restricted with EcoRV and probedwith a 470-bp EcoRV-restricted fragment of orf3 (Fig. 7C).

The results indicate that ragA and ragB are codistributed assingle copies and show a restricted distribution pattern in thestrains examined: ragA and ragB are found in strain W50 andthe phenotypic variants W50Be1 and W50Br1 as well as W83,LB13D-3, and WPH35, but both are absent from 381, 11834,23A4, and WPH34. The codistribution of ragA and ragB isconsistent with their transcription as a single unit. This patternof distribution also complements the immunochemical findingsof Millar et al. (31), where 9 of 15 P. gingivalis strains examinedwere positive in Western blot analysis with the monoclonalantibody DRU55.5. The results from the Western and South-ern analyses are exactly correlated except in the case of strainLB13D-3, which contains the ragAB locus but does not expressthe epitope recognized by DRU55.5.

In contrast, orf3 is present as a single copy in all P. gingivalisstrains examined. Strains W50, W50Be1, W50Br1, W83, LB13D-

3, 23A4, and WPH35 give the expected 470-bp band size inEcoRV digests of chromosomal DNA, whereas the remainingstrains, 381, 11834, and WPH34, which are ragAB negative, give ahigher-molecular-size band of greater than 1.5 kb. Thus, thecoding sequences of ragA and ragB are found in a restrictednumber of laboratory strains, while orf3 is found in all P. gin-givalis strains examined, although some polymorphism in therestriction profile at the orf3 locus is evident and this partiallycorrelates with the presence or absence of the ragAB operon.

Distribution analysis of the rag locus among clinical iso-lates. In order to determine if the restricted distribution pat-tern of ragA and ragB observed among laboratory P. gingivalisstrains is reflected in vivo, PCR analysis with primer ragA.2or ragB.2 (Table 1) was performed on 38 P. gingivalis clinicalisolates. Chromosomal DNAs from these isolates were used astemplates in the PCRs. The 38 isolates were obtained from 17patients with chronic adult periodontitis. One to three sites perpatient were sampled, and one to three isolates were obtainedfrom each site (3).

FIG. 4—Continued.



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The expected 509- and 436-bp amplicons of ragA and ragB,respectively, were amplified from 17 of the 38 isolates, corre-sponding to 5 of the 17 patients. ragA and ragB were codistrib-uted in all positive isolates. These results confirm the Southernblot analysis of laboratory strains and also demonstrate thatthe restricted distribution pattern is not an artifact of repeatedsubculture of laboratory strains but is also reflected in freshisolates.

Analysis of the ragAB locus in clinical samples. The findingthat the ragAB locus is present in only a restricted proportionof P. gingivalis clinical isolates while the immune response toRagB is significantly elevated in periodontal disease patientsmay indicate that strains which harbor this locus are moreassociated with destructive periodontal disease than ragAB-negative strains or, alternatively, that RagB may be an impor-tant antigen expressed by another bacterial species in the peri-odontal microflora. In order to address these possibilities, weundertook a preliminary investigation to determine by PCR

the presence of this locus in subgingival plaque samples fromperiodontally healthy individuals (n 5 33) and periodontaldisease patients (n 5 28).

PCR of the P. gingivalis 16S RNA gene was performed onthe samples to establish the presence or absence of this organ-ism (Fig. 8). On the basis of the 16S RNA PCR, P. gingivaliswas present in 5 of 67 (7.5%) subgingival samples from the 33periodontally healthy individuals and in 7 of 30 (23%) samplesfrom shallow pockets (periodontal pocket depths of ,4 mm)and 55 of 72 (76%) deep pockets (pocket depths of 5 to 10mm) from periodontal disease patients (n 5 28). To establishthe presence of the ragAB locus in the samples, PCR wasperformed with primers to amplify ragB only (Fig. 8), sinceragB and ragA were shown to be codistributed in both P. gin-givalis laboratory strains and clinical isolates. ragB was notdetected in samples which were positive for P. gingivalis 16SRNA from periodontally healthy individuals or in P. gingivalis-positive samples from shallow pockets in periodontal disease

FIG. 5. Multiple alignments of TonB boxes with the highest similarity scores compared to P. gingivalis RagA. The receptors are SusC (starch utilization), FepA(ferric enterochelin), BfeA (ferrichrome-iron), BfrA (exogenous ferric siderophore), IrgA (iron-regulated outer membrane), CirA (colicin 1), HemR (hemin-regulatedprotein), Tla (TonB linked adhesion), and FyuA (yersiniabactin siderophore). Abbreviations for bacteria: pg, P. gingivalis; bt, B. thetaiotamicron; pa, Pseudomonasaeruginosa; ec, E. coli; bp, Bordetella pertussis; bb, Bordetella bronchiseptica; vc, V. cholerae; and ye, Y. enterocolitica. Sequences are aligned in descending order ofrelatedness. Boldface amino acid residues correspond to residues conserved throughout the alignment, with asterisks indicating the consensus sequence, while theunderlined residues are conserved in more than 50% of the sequences. Amino acid sequence positions are shown at the beginning and end of each sequence. For boxIIIthe percentage of similarity is calculated with reference to RagA.



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patients. However, ragB was detected in 20 of 55 (36%) ofP. gingivalis-positive samples from deep pockets in the peri-odontal disease patients.

The results of the PCR analysis of the clinical samples areshown in Fig. 9. Statistical analysis of these results revealed apositive correlation between detection of P. gingivalis and in-creasing pocket depth (P 5 0.01) and a stronger associationbetween P. gingivalis ragB-positive samples and increasingpocket depth (P 5 0.001). Overall, these data suggest thatP. gingivalis strains harboring the ragAB locus are associatedmore with sites of periodontal destruction than with sites fromperiodontally healthy individuals or shallow pockets in peri-odontal disease patients.

PCR of ragB was also performed on the P. gingivalis-negativesamples from periodontal disease patients, and 8 of 40 sampleswere found to be ragB positive. These samples were subse-quently reanalyzed for the presence of P. gingivalis by PCRwith primers to amplify the 1.7-kb catalytic domain of theP. gingivalis-specific rgpA protease gene (3) and were con-firmed to be negative. These results suggest that the ragABlocus may be present in the genome of another member(s) ofthe periodontal microbial community.

Together these findings suggest that the elevated immuneresponse to RagB in periodontal disease patients may be dueto the association of P. gingivalis RagAB-positive strains withsites of destructive periodontal disease and also to the pres-ence of a RagB homolog in another bacterial species in theperiodontal microflora.

DISCUSSION

The exact role that P. gingivalis plays in the initiation andprogression of periodontitis remains unclear. It is not yet es-tablished whether P. gingivalis is a minor component of theindigenous oral flora and behaves as an opportunistic patho-gen, with all strains having equal virulence potential, or wheth-er virulent and avirulent strains exist within this species. DNAfingerprinting and multilocus enzyme electrophoresis studieshave shown a diverse range of genotypes among P. gingivalisstrains, but no distinct correlation between a specific genotypeand disease has been made (23, 25, 30).

The present investigation was prompted by the analysis ofthe specific immune response of periodontal disease case serato surface antigens of P. gingivalis W50. The results demon-strate that the 55-kDa outer membrane antigen identified inthe earlier investigations is encoded by a gene (ragB) which iscotranscribed with an upstream gene (ragA). Since the pre-dicted size of RagA is approximately 115 kDa, it is possiblethat this antigen corresponds to the abundant antigen with thismolecular mass in P. gingivalis W50 outer membranes whichwas also identified in the initial case-control investigation (11).On the basis of the signal peptidase II cleavage site motif at theN terminus, the 55-kDa outer membrane antigen, RagB, is alipoprotein, while RagA is homologous to the family of TonB-linked outer membrane receptors which are involved in therecognition and active transport of specific external ligands bya wide range of gram-negative species (7). These receptorsinteract with TonB in the periplasm at distinct regions knownas the TonB boxes (boxI, boxII, and boxIII), which are locatednear the N-terminal amino acid sequences of the receptorproteins. The codistribution and cotranscription of ragA andragB strongly suggest that they are functionally linked, and theproducts may form a complex on the outer surface of P. gin-givalis that is involved in a TonB-linked process.

The genetic arrangement at the ragAB locus is very analo-gous to those for the lactoferrin and transferrin binding sys-tems in Neisseria and Haemophilus species, respectively. In theseorganisms a TonB-linked outer membrane receptor is also co-transcribed with an outer membrane lipoprotein, and the re-sulting surface complex is involved in the active transport oflactoferrin and transferrin iron sources (10, 15, 26). However,multiple alignments between RagB and the analogous trans-ferrin and lactoferrin binding lipoproteins, Tbp2 and Lbp2, didnot identify any significant homology between these proteins.Furthermore, database searches with RagA revealed only alow homology with TonB receptors involved in iron uptake butan extremely high similarity to SusC, a TonB-linked receptorinvolved in maltose uptake in B. thetaiotamicron. It is possible,therefore, that the putative RagAB surface complex of P. gin-givalis is involved in either the uptake or recognition of aspecific carbohydrate or glycoprotein. Given the asaccharolyticnature of P. gingivalis, it is likely that carbohydrate uptakewould be linked to an anabolic process.

The significantly lower G1C content of the ragAB locus thanof the rest of the P. gingivalis chromosome and its restricteddistribution in laboratory strains and clinical isolates is consis-tent with its acquisition from a foreign source via horizontalgene transfer. The ragB PCR analysis of subgingival samplesdemonstrated that this locus can be detected in the absenceof P. gingivalis. The other bacterial species which harbor theragAB locus in these subgingival samples may be the source ofthis locus in P. gingivalis or, equally likely, may have acquiredthis locus by a similar horizontal gene transfer event. Severalother characteristics indicate that this locus may correspond toone of a rapidly growing family of virulence-associated chro-mosomal loci termed pathogenicity islands (PAIs).

PAIs are chromosomal elements encoding virulence deter-minants which are present in pathogenic strains but are absentor rarely found in nonpathogenic strains of the same species.These genetic elements have been reported to range in sizefrom a 1.6-kb single gene to large 35- to 190-kb multigeneelements. Frequently observed characteristics of PAIs includea G1C content different from that of the DNA of the host bac-terium, mobility or the presence of mobile genetic elements,and the capability of horizontal gene transfer (17). Acquisitionof PAIs enhances the genetic flexibility of bacterial species andcontributes to the spread and evolution of virulence. PAIs have

FIG. 6. Northern analysis of RagA and RagB expression in exponentiallygrown P. gingivalis W50. Lane 1, P. gingivalis W50 RNA probed with a PCR-am-plified 1.6-kb ragA probe; lane 2, P. gingivalis W50 RNA probed with a PCR-amplified 1.5-kb ragB probe. The arrows indicate the position of the ragABmRNA transcript of approximately 4.7 kb and the positions of the 23S and 16SmRNAs.



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been found in a range of gram-negative organisms and includethe hemolysin gene cluster (hly) of uropathogenic E. coli 536,the cag (cytotoxin-associated antigen genes A to T) gene clus-ter of Helicobacter pylori, and the hms, fyuA, and irp genes in-volved in iron uptake and storage in Yersinia species (8, 9, 29, 32).

PAIs are found associated with mobile elements such asintegrases, transposons, and insertion sequence elements. TheragAB locus is flanked by two potentially mobile elements:IS1126W50 upstream of ragA and a region with sequence sim-ilarity to transposons downstream of ragB. Southern blot anal-ysis of IS1126 has demonstrated that this element is found inall P. gingivalis strains examined to date and is present inmultiple copies, ranging from 6 to 12, depending on the strain(27). The 1.3-kb copy of IS1126W50 upstream of ragA encodes

a transposase of approximately 41 kDa, indicating that this copyof IS1126W50 is capable of transposition. Unlike IS1126W50,the region downstream of ragB with transposon sequence sim-ilarity appears to be noncoding, which may be indicative of aprevious integration event between P. gingivalis and anotherorganism.

Database analysis of the region downstream of ragB revealedlocalized similarity to PGIS2 (41). This putative transposasebears significant homology to several mobile elements, includ-ing the predicted product of H repeats in E. coli, the productof the orfH region of Salmonella enterica, and the putativetransposase IS1358 of V. cholerae. The H-repeat elements inE. coli are associated with recombination hot spots (Rhs ele-ments). These elements have no known function but have

FIG. 7. Southern blot analysis of the distributions of ragA, ragB, and orf3 in laboratory P. gingivalis strains. (A) P. gingivalis chromosomal DNA was digested withClaI (C) and probed with a 32P-labelled 1.6-kb PCR-amplified product of ragA. (B) P. gingivalis chromosomal DNA was digested with HindIII (H) and probed witha 32P-labelled 1.6-kb amplified product of ragB. (C) P. gingivalis chromosomal DNA was digested with EcoRV (E) and probed with a 0.47-kb 32P-labelled EcoRV-restricted fragment of orf3. Lanes 1 to 10, P. gingivalis W50, W50Be1, W50Br1, W83, LB13D-3, 381, 11834, 23A4, WPH35, and WPH34, respectively; lane 11, E. coliXL-Blue; lane 12, P. asaccharolytica ATCC 25260.



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recently been incorporated into the E. coli genome and arefound in many but not all wild-type E. coli isolates (42). TheIS1358 element in V. cholerae is found in the lipopolysaccha-ride rfb gene cluster and is associated with novel lipopolysac-charide genes which, on the basis of G1C analysis, are con-sidered to have arisen from a foreign source. IS1358 is thoughtto be responsible for the generation of a new serotype, 0139, bymediating the incorporation of these novel O-antigen genesinto the V. cholerae rfb gene cluster (39). It is conceivable thatPGIS2, which has been shown to be mobile in P. gingivalis (41),and related mobile elements are capable of horizontal genetransfer and that this transfer facilitates a greater genetic flex-ibility in the recipient bacterial species.

PAIs carry virulence-associated genes and have a restricteddistribution pattern which correlates with the presence in andabsence from virulent and avirulent strains of the same species,respectively. As outlined above, the sequence homologies ofRagA and RagB suggest that they may have a role in recogni-tion and transport via a TonB-mediated mechanism. In thisrespect, the ragAB locus may be analogous to fyuA, a TonB-dependent receptor in the high-pathogenicity island of Yersiniaenterocolitica which is absent from low-pathogenicity Y. entero-colitica strains (8). Loss of the high-pathogenicity island inYersinia pestis does not abolish the capacity to cause diseasebut prevents the most fulminant expression of bacterial patho-genicity (33). There are few reliable measures of the virulencepotentials of different P. gingivalis strains in relation to peri-odontal disease. However, small-animal models of soft tissuedestruction have been employed to examine the effects of gene

inactivation or growth conditions on the virulence of this or-ganism (20, 32). Although few strain-to-strain comparativedata are available, the ragAB locus is present in the commonstrains W50 and W83, which are highly invasive in small-animalmodels, and in WPH35 (unpublished data) and is absent from381 and 33277, which produce nonspreading localized lesionsat the site of inoculation (32).

In the absence of definitive methods for predicting the vir-ulence potentials of P. gingivalis strains which harbor the ragABlocus, in the present study we used PCR of subgingival plaquesamples to determine whether ragAB-positive P. gingivalisstrains are associated more frequently with sites of periodontaldestruction than with periodontally healthy sites. Surprisingly,given the relatively small sample size, ragAB-positive P. gingi-valis strains were significantly more associated with diseasedsites than with healthy sites. Larger studies of the ragAB-pos-itive status of P. gingivalis from sites of well-defined clinicalconditions are required to confirm this association, since theremay be significant diagnostic implications. Furthermore, on thebasis of the data presented in the present report, analysis of thefunction of RagA and RagB is warranted, and these studies areunder way through the construction and phenotypic analysis ofisogenic mutants.

In summary, the 55-kDa immunodominant surface antigenof P. gingivalis W50 is the product of a polycistronic messagefrom a locus we have termed ragAB. The antigen gene iscotranscribed with a second gene, the product of which resem-bles TonB-linked receptors from other gram-negative bacteria,in particular SusC from B. thetaiotamicron. RagA and RagB

FIG. 8. PCR analysis of subgingival paper point samples from periodontal disease patients to detect the presence of P. gingivalis (A) and ragB (B). The primers usedwere P. gingivalis 16S RNA primers to amplify a 404-bp product and ragB.2 primers to amplify a 435-bp product. Lanes 1, 1-kb ladder; lanes 2 to 17, subgingival paperpoint PCRs; lanes 18, negative control (distilled H2O as template); lanes 19, positive control (200 ng of P. gingivalis W50 chromosomal DNA as template). Lanes 12,15, and 17 represent three of the eight samples which are PCR positive for ragB but PCR negative for P. gingivalis. Ten microliters from a total volume of 100 ml ofthe PCR mixtures was run on a 1% agarose gel.



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may therefore cooperate at the surface of P. gingivalis W50in the transport or recognition of an external ligand. On thebasis of a G1C content different from that of the remainderof the host chromosome, the presence of mobility elementsat this locus, a restricted distribution in laboratory strainsand clinical isolates, and the presence of this locus in clinicalsamples which did not contain P. gingivalis, we propose thatthe ragAB operon has arisen by horizontal gene transferfrom a foreign source which may also be a component of theperiodontal microflora. The greater prevalence of ragAB-positive P. gingivalis strains at sites of periodontal destruc-tion than at periodontally healthy sites suggests that RagABmay confer a selective advantage to strains which harborthese genes and hence that the ragAB locus may correspondto a novel PAI of this species.

ACKNOWLEDGMENTS

We are grateful to Catherine Williams and Francis Hughes for helpwith the clinical sampling.

This work was supported in part by the Research Advisory Com-mittee of St. Bartholomew’s and The Royal London School of Medi-cine and Dentistry and by the Medical Research Council (grantsG9711648PB and PG9318173).

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A 55-Kilodalton Immunodominant Antigen of Porphyromonas ... · DNA sequence data were analyzed by...

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