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Journal of Bacteriology, September 2006, p. 6376-6386, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00731-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The RgpB C-Terminal Domain Has a Role in Attachment of RgpB to the Outer Membrane and Belongs to a Novel C-Terminal-Domain Family Found in Porphyromonas gingivalis

Christine A. Seers, Nada Slakeski, Paul D. Veith, Todd Nikolof, Yu-Yen Chen, Stuart G. Dashper, and Eric C. Reynolds*

Cooperative Research Centre for Oral Health Science, School of Dental Science, University of Melbourne, 720 Swanston Street, Victoria, Australia

Received 22 May 2006/ Accepted 9 June 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Porphyromonas gingivalis produces outer membrane-attached proteins that include the virulence-associated proteinases RgpA and RgpB (Arg-gingipains) and Kgp (Lys-gingipain). We analyzed the P. gingivalis outer membrane proteome and identified numerous proteins with C-terminal domains similar in sequence to those of RgpB, RgpA, and Kgp, indicating that these domains may have a common function. Using RgpB as a model to investigate the role of the C-terminal domain, we expressed RgpB as a full-length zymogen (recombinant RgpB [rRgpB]), with a catalytic Cys244Ala mutation [rRgpB(C244A)], or with the C-terminal 72 amino acids deleted (rRgpB435) in an Arg-gingipain P. gingivalis mutant (YH522AB) and an Arg- and Lys-gingipain mutant (YH522KAB). rRgpB was catalytically active and located predominantly attached to the outer membrane of both background strains. rRgpB(C244A) was inactive and outer membrane attached, with a typical attachment profile for both background strains according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in YH522KAB, the prodomain was not removed. Thus, in vivo, RgpB export and membrane attachment are independent of the proteolytic activity of RgpA, RgpB, or Kgp. However, for maturation involving proteolytic processing of RgpB, the proteolytic activity of RgpB, RgpA, or Kgp is required. The C-terminally-truncated rRgpB435 was not attached to the outer membrane and was located as largely inactive, discrete 71-kDa and 48-kDa isoforms in the culture supernatant and the periplasm. These results suggest that the C-terminal domain is essential for outer membrane attachment and may be involved in a coordinated process of export and attachment to the cell surface.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Porphyromonas gingivalis is a gram-negative anaerobe that is found predominantly in subgingival dental plaques associated with the destruction of the tooth's supporting tissues (14, 52; for reviews, see references 24, 26, and 32). P. gingivalis produces three major cysteine proteinases (gingipains), two of which, RgpAcat and RgpB (which are almost identical in sequence), are specific for Arg-Xaa cleavage (39, 49, 51) and one of which, Kgpcat, is specific for Lys-Xaa cleavage (34, 38, 50). The proteinases are encoded by rgpA, rgpB, and kgp and are produced as zymogens with a Sec-type leader peptide followed by a prodomain, the catalytic domain, and, in the cases of RgpA and Kgp, several sequence-related adhesin domains (50). The RgpA polyprotein is proteolytically processed to produce RgpAcat and several adhesins that are designated RgpAA1, RgpAA2, RgpAA3, and RgpAA4, while the Kgp polyprotein is processed to produce Kgpcat and the adhesins KgpA1, KgpA2, KgpA3, KgpA4, and KgpA5 (32). The RgpA and Kgp proteinase and adhesin domains occur together as a surface-associated complex (32, 50), with relatively little of the complex found free in the extracellular milieu.

RgpB is produced as a 736-amino-acid (aa) precursor with a Sec-type signal sequence that is predicted to be cleaved after Ala24 and a prodomain cleaved after Arg229 (49). The mature RgpB is predominantly associated with the outer membrane and is predicted to be 507 aa (56 kDa) at most, but it migrates through a polyacrylamide gel as a series of closely spaced bands with a molecular mass of 80 to 90 kDa (49). This membrane-associated RgpB is recognized by monoclonal antibody (MAb) 1B5, which has recently been shown to recognize a phosphorylated branched mannan proposed to be part of the cell envelope of P. gingivalis (37). A soluble RgpB of 48 to 50 kDa has also been isolated from P. gingivalis culture supernatants (40), but this protein is not recognized by MAb 1B5 (12) and is C-terminally truncated at Ser435 (mature enzyme numbering) (15). This has led to the suggestion that the carbohydrate modification recognized by MAb 1B5 is possibly in the C-terminal domain and that this domain may have a role in the outer membrane attachment of the enzyme (59).

It has been reported that the Rgp and Kgp proteinases have roles in the maturation of each other; RgpA maturation is dependent on the presence of RgpB (5), and Kgp is dependent on RgpA/B gingipains for full function (3-5, 47). Mutants lacking RgpA/B activity display aberrant Kgp domain processing with ragged N termini (59).

The RgpB C-terminal domain has been shown to have sequence similarity with the C-terminal domains of other P. gingivalis outer membrane proteins that were identified using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and peptide mass fingerprinting (PMF) (59). These proteins include P27 and P59, which are of unknown function; HagA; and the adhesins RgpAA4 and KgpA5 (59). RgpAA4, P27, and P59, which have no significant sequence identity except in their C-terminal domains, were also found to be immunoreactive with MAb 1B5 (59), which supports the concept that the C-terminal domains of this class of P. gingivalis outer membrane proteins are sites of glycosylation and cell attachment.

In this study, we have investigated the function of the C-terminal domain using RgpB as a representative protein and assessed the role of RgpA/B and Kgp proteolytic activity in the maturation of RgpB.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and culture conditions. Bacterial strains and plasmids used in this study are listed in Table 1. P. gingivalis strains were maintained by weekly passage on 10% (vol/vol) defibrinated, lysed horse blood in blood agar base no. 2 (HBA) (Oxoid, Basingstoke, England) incubated at 37°C in an anaerobic atmosphere of 5% H2, 80% N2, and 15% CO2. P. gingivalis strains were grown in batch culture in brain heart infusion broth (Oxoid) supplemented with 5 mg ml–1 cysteine, 5 µg ml–1 hemin, and 5 µg ml–1 menadione or grown in a BioFlo C30 bench-top chemostat (New Brunswick Scientific, Edison, NJ) at 37°C (pH 7.3 ± 0.1) with a working volume of 365 ml. Supplemented medium was added via a peristaltic pump at a flow rate of 36 ml h–1, and the culture was continuously gassed with N2-CO2 (95:5, vol/vol) (13). At steady state, the culture had a redox potential of –250 mV and a cell density of 5.0 x 109 cells ml–1. Escherichia coli cells were grown aerobically at 37°C in Luria-Bertani (LB) broth and on LB agar (45). P. gingivalis growth media were supplemented with 1 µg ml–1 tetracycline, 10 µg ml–1 erythromycin, 20 µg ml–1 chloramphenicol, or 5 µg ml–1 ampicillin when appropriate, and E. coli growth media were supplemented with 100 µg ml–1 ampicillin. Antibiotics were obtained from Sigma (St. Louis, MO). Chemically competent E. coli JM109 cells were purchased from Promega (Madison, WI), and QIAGEN-EZ cells were purchased from QIAGEN (Clifton Hill, Victoria, Australia).


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  		TABLE 1. Bacterial strains and plasmids

 
Preparation of P. gingivalis culture fractions. To prepare P. gingivalis culture fractions, exponentially growing batch cultures containing 2.5 x 109 cells ml–1 or a continuous culture of 5.0 x 109 cells ml–1 was used.

(i) Culture supernatant and whole-cell lysate fractions. P. gingivalis cells were harvested by centrifugation of a culture sample (7,000 x g at 4°C for 20 min), and the supernatant was retained as the culture supernatant. The supernatant proteins were precipitated by the addition of trichloroacetic acid to 10% (wt/vol), washed in acetone, and suspended in TC150 buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 20 mM cysteine-HCl, pH 8.0) to 1/10 the initial volume. A sample of the pelleted cells was washed and then lysed in 1% (vol/vol) Triton X-114 in 50 mM Tris-HCl (pH 8.0) and was designated the whole-cell lysate fraction.

(ii) Periplasmic and cytoplasmic fractions. Periplasmic fractions were prepared from P. gingivalis cells (40 ml of culture) by using a standard osmotic shock technique (7). Briefly, the pellet of P. gingivalis cells was suspended in 8 ml of 1-mg/ml lysozyme and 30 mM Tris-HCl (pH 8.0) and incubated at 37°C for 30 min. The cells were centrifuged as described above, and the pellet was suspended in 8 ml of SET buffer at 4°C (500 mM sucrose, 1 mM EDTA, 30 mM Tris-HCl, pH 8.0) and incubated at 4°C for 10 min. The cells were then pelleted by centrifugation, suspended in 8 ml of 5 mM MgCl2, and incubated at 4°C for 10 min. The cells were pelleted, and the supernatant was retained as the periplasmic fraction. The supernatant proteins were precipitated by the addition of trichloroacetic acid to 10% (wt/vol). The precipitated protein was washed with acetone and then suspended in 1/10 of the original volume using 50 mM Tris-HCl (pH 8.0). The pelleted cells were washed and then lysed in 1% (vol/vol) Triton X-114 in 50 mM Tris-HCl (pH 8.0), and after centrifugation, the supernatant was collected as the cytoplasmic fraction.

(iii) Outer membrane fractions. Outer membrane fractions were prepared from P. gingivalis cells following the addition of tosyl-L-lysine-chloromethyl ketone (TLCK; Sigma) to 5 mM. The cells were washed in magnesium buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM MgCl2, 1 mM TLCK), pelleted again by centrifugation, and then resuspended in magnesium buffer. The cell suspension was passed three times through a chilled French pressure cell (Newport Scientific Inc., Jessup, MD) at 120 MPa, with the addition of 1 mM TLCK prior to each pass. Undisrupted cells were cultured by centrifugation (5,110 x g at 4°C for 25 min), and the supernatant was centrifuged to pellet the membrane (43,900 x g at 4°C for 25 min). The membrane pellet was suspended in 20 ml 1% N-lauryl sarcosine with the addition of 1 mM TLCK and allowed to incubate for 1 h at 37°C. Upon the completion of incubation, the sample was centrifuged (43,900 x g at 4°C for 25 min), and the pellet was collected as the outer membrane fraction.

Protein electrophoresis. For sodium dodecyl sulfate (SDS)-PAGE, 12% (wt/vol) or 4 to 20% (wt/vol) polyacrylamide gels (Invitrogen, Carlsbad, CA) were used in Tris-glycine buffer with low-molecular-weight markers (Amersham Biosciences) as standards. Prior to electrophoresis, proteins were denatured by heating in the presence of 1% (wt/vol) sodium dodecyl sulfate and 1 mM dithiothreitol. Gels were stained using Coomassie blue R-250 (Bio-Rad, Hercules, CA) or transferred onto a polyvinylidene difluoride membrane (Bio-Rad) as previously described (33). 2D-PAGE was performed as described previously (60).

In-gel digestion of proteins and PMF. Protein bands from SDS-PAGE and protein spots from 2D-PAGE were excised and subjected to in-gel trypsin digestion as described previously (59, 60). Gel pieces from SDS-PAGE and 2D-PAGE were washed in 50 mM NH4HCO3-ethanol (1:1, vol/vol), reduced, alkylated with dithiothreitol and iodoacetamide, respectively, and digested with sequencing-grade modified trypsin (10 ng µl–1) (Promega) overnight at 37°C (31). Each peptide extract (0.5 µl) was then analyzed by mass spectrometry using an Ultraflex TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) in positive-ion and reflectron mode. A saturated solution of 4-hydroxy-{alpha}-cyanocinnamic acid was prepared in 97:3 (vol/vol) acetone-0.1% aqueous trifluoroacetic acid (TFA). A thin layer was prepared by pipetting and immediately transferring 2 µl of this solution onto 600-µm anchors of an AnchorChip target plate (Bruker Daltronics). The sample (0.5 µl) was deposited onto the thin layers with 2.5 µl of 0.1% (vol/vol) TFA and allowed to adsorb for 5 min, after which time the sample solution was removed, and the thin layers were washed once with 10 µl of ice-cold 0.1% (vol/vol) TFA for 1 min. Spectra were calibrated by external calibration using a standard peptide mix (Bruker Daltonics). Proteins were identified by PMF against the P. gingivalis database (available at The Institute for Genomic Research [TIGR] website [http://www.tigr.org]) using an in-house Mascot search engine (Matrix Science Ltd., London, United Kingdom) and BioTools 2.2 software (Bruker Daltonics) and by comparison to tryptic peptide mass lists generated by using General Protein Mass Analysis for Windows software (Lighthouse Data, Odense, Denmark).

NanoLC mass spectrometry. Peptide extracts were acidified with TFA to 0.1% and centrifuged before online nano-liquid chromatography (NanoLC) tandem mass spectrometry (MS/MS) analyses. An UltiMate NanoLC system (Dionex Pty. Ltd., Lane Cove, NSW, Australia) was used with a µ-precolumn of PepMap C18 (300-µm internal diameter [i.d.] by 5 mm; Dionex) and an analytical column of PepMap C18 (75-µm i.d. by 15 cm; Dionex). Buffer A consisted of 0.1% (vol/vol) formic acid in H2O, and buffer B consisted of a solution containing 80% (vol/vol) acetonitrile, 20% H2O, and 0.1% (vol/vol) formic acid. Each peptide extract (5 µl) was loaded onto the µ-precolumn in buffer A at a flow rate of 30 µl min–1 for 5 min to desalt, after which time it was separated on an analytical column at a flow rate of 0.3 µl min–1. The peptides were eluted directly into an EsquireHCT ion trap mass spectrometer equipped with a nanoelectrospray source (Bruker Daltonics) via a tip-end-coated fused silica needle with a 10-µm i.d. (New Objective Inc., Woburn, MA) using a linear gradient of 0 to 60% acetonitrile over 40 min at a flow rate of 0.3 µl min–1. The ion trap operated in the positive-ion mode at mass spectrometer scan speeds of 8,100 m/z per s and 26,000 m/z per s for MS/MS analysis. The capillary voltage was set to 1,500 V, and the drying gas (N2) was set to 3 liters min–1 and 150°C. Automatic MS/MS was employed over a precursor ion mass range of 300 to 2,200 m/z using the SmartFrag option. Data analysis and MS/MS database searching were performed using DataAnalysis 3.1, BioTools 2.2, and Mascot software.

Bioinformatics. Nucleotide sequences were analyzed using program suites available at the Australian National Genomic Information Service (Australian Genomic Information Centre, University of Sydney, Australia). Protein and peptide sequences were compared to the P. gingivalis W83 genome sequence annotated open reading frames (ORFs) available at the website of TIGR (http://www.tigr.org) and to other protein sequences available at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and the Los Alamos Oral Pathogens Database (http://www.oralgen.lanl.gov) using the BLAST algorithm (6). Protein secondary structure and solvent exposure predictions (43, 44) were determined using algorithms accessed at the Columbia University Bioinformatics Center website (http://cubic.bioc.columbia.edu).

DNA manipulations and generation of P. gingivalis mutant strains. Plasmids used in and generated during this study were propagated within E. coli JM109 cells, unless otherwise stated. P. gingivalis cells were transformed using electroporation (33). P. gingivalis YH522 (61) was chosen for the generation of gingipain-deficient mutants because it is readily genetically manipulated and is amenable to plasmid transformation. Mutants were generated by allelic exchange with linear DNAs bearing the strain W50 rgpA, rgpB, or kgp gene disrupted with antibiotic resistance markers (Table 1). The Arg-gingipain-deficient strain YH522AB was created according to methodology previously described for the construction of the P. gingivalis W50AB mutant (59). To create an Arg-gingipain/Lys-gingipain mutant, kgp of YH522AB was disrupted with the Bacteroides fragilis cephalosporinase-encoding gene cepA (41). To do this, plasmid pLys (50) was digested with MfeI and end filled using dATP, dTTP, and T4 DNA polymerase, and cepA, isolated as a 1.3-kbp MscI-EcoRV fragment from plasmid pEC474 (a kind gift from C. Jeffrey Smith, East Carolina University, NC), was then inserted into the linearized pLys, creating plasmid pLysCepA. pLysCepA was linearized with XbaI and then electroporated into P. gingivalis YH522AB for allelic exchange with kgp. Mutants (YH522KAB) were selected using HBA plates supplemented with 5 µg ml–1 ampicillin.

All gene disruptions were confirmed by Southern blot analysis essentially as described previously by Veith et al. (59), except that for YH522KAB, the chromosomal DNA was probed with the cepA 1.3-kbp gene fragment and the 3.3-kbp BamHI fragment from pLys.

Creation of RgpB mutations and recombinant bacterial strains. To produce RgpB truncated at Ser435 of the mature protein (Ser664 in the zymogen), a truncated rgpB ({Delta}rgpB435) was generated by PCR amplification of a P. gingivalis W50 genomic DNA template using the forward oligonucleotide used to generate the full-length rgpB gene that was previously cloned into pBH1.1 (33), 5'-GCGCGCTCTAGAGGACAGTATCTGCAACCGTCG-3', and the reverse oligonucleotide S-trunc (5'-TGGCTACGTCGGCTTAAGATGTACC-3'). The PCR product was cloned into pDRIVE (QIAGEN) and transformed into QIAGEN-EZ cells (QIAGEN) according to the manufacturer's instructions. The integrity of the insert DNA of the resultant plasmid construct, pRgpB435-4, was verified by nucleotide sequencing. Following this, {Delta}rgpB435 was excised from pRgpB435-4 using EcoRI and ligated into the unique EcoRI site of the shuttle plasmid pYH411, generating plasmid pYH435. To produce an inactive recombinant RgpB (rRgpB), the rgpB codon for the catalytic residue Cys244 (mature enzyme numbering) was mutated to encode Ala244. To do this, we employed the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), the pBH1.1 template, and the oligonucleotides 5'-CCGTTTATTTTCGACGTAGCTGCTGTGAATGGCGATTT-3' and 5'-GGAAATCGCCATTCACAGCAGCTACGTCGAAAATAAAC-3', generating pBC244A. The mutant gene, rgpB(C244A), was fully sequenced to verify that the only change was to the Cys244 codon and then excised from pBC244A using EcoRI and ligated into the EcoRI site of pYH411, generating plasmid pRgpBC244A. To produce a wild-type recombinant RgpB sequence, rgpB was excised from pBH1.1 using EcoRI and ligated into the EcoRI site of pYH411, generating pRgpB. pRgpB, pYH435, pRgpBC244A, and pYH411 were each transformed into YH522AB and YH522KAB with selection of recombinants on HBA plates supplemented with 10 µg ml–1 erythromycin.

Arg-gingipain activity. P. gingivalis strains were grown in batch culture to late logarithmic phase and harvested at a cell density of 2.5 x 109 cells ml–1 by centrifugation (7,000 x g at 4°C for 20 min). The supernatant was retained, the cell pellet was washed in TC150 buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 20 mM cysteine-HCl, pH 8.0), the centrifugation was repeated, and the cell pellet was finally suspended in fresh TC150 buffer to 1/10 the initial culture volume. A sample of the culture supernatant was subjected to centrifugation (40,000 x g at 4°C for 30 min) to sediment outer membrane vesicles. Arg-gingipain activity was determined by monitoring the change in absorbance at 410 nm of a sample (supernatant or cell suspension) with a solution containing 1 mM N{alpha}-benzoyl-DL-arginine-p-nitroanilide (BApNA; Sigma), 15% (vol/vol) isopropanol, 200 mM Tris-HCl, 50 mM NaCl, and 10 mM L-cysteine-HCl (pH 8.0) in a 1-cm-path-length disposable cuvette using a Hewlett Packard 8452A diode array spectrophotometer with an 89090A temperature controller set to 37°C and a molar extinction coefficient of 8,800 M–1 cm–1.

Western blots. After electrotransfer of proteins, polyvinylidene difluoride membranes were blocked using 5% (wt/vol) skim milk powder in TNT buffer (25 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% Triton X-100). The membranes were probed using preadsorbed anti-RgpAcat antibodies diluted at 1:200 in TNT buffer for at least 1 h. Following washes in TNT buffer, the secondary antibody, goat anti-mouse horseradish peroxidase-conjugated immunoglobulin G (Sigma), diluted 1:200 in TNT buffer was applied and incubated for at least 1 h. Following three washes in TNT buffer, the immunoreactive proteins were visualized using color development by reaction with the substrate 4-chloro-naphthol (Bio-Rad).

Preparation of preadsorbed anti-RgpAcat antibodies. A recombinant RgpAcat-His-tagged protein (28) was used to raise mouse polyclonal antibodies that were immunoreactive with both RgpAcat and RgpB. In order to remove antibodies specific for the adhesin-like sequences present in the C terminus of RgpAcat that would cross-react with the RgpA, HagA, and Kgp adhesin domains, the antiserum was preadsorbed to a sonicate of strain ECR5 lacking both RgpA and RgpB (Table 1). Briefly, 200 ml of ECR5 culture was grown to log phase, the cells were harvested by centrifugation (6,000 x g at 4°C for 20 min), the cell pellet was washed in 20 ml of TC150 buffer and centrifuged as described above, and the washed pellet was suspended in 20 ml TC150. The cells were lysed by sonication, the cell debris was pelleted by centrifugation (6,000 x g at 4°C for 20 min), and the supernatant was retained. The sonicate protein concentration was measured using the Bradford protein assay reagent (Bio-Rad) with bovine serum albumin as a standard (9). Sonicate material was added to sera to give a final sonicate concentration of 100 µg protein ml–1. The serum-sonicate mixture was incubated on ice for 1 h, with occasional mixing, after which the immunoprecipitate was pelleted by centrifugation (14,000 x g at 4°C for 20 min). The supernatant was retained.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Survey of P. gingivalis ORFs with C-terminal-domain similarity to RgpB. To determine if there were more P. gingivalis proteins with C-terminal-domain sequence similarity to RgpB than previously identified, the last 80 amino acid residues of RgpB were used in a BLAST search of the P. gingivalis W83 predicted proteins. When sequence similarity was found with the predicted C-terminal domain of an ORF, a BLAST search was conducted with the C-terminal 80 amino acid residues of this ORF. This process was repeated until no new C-terminal-domain paralogues were discovered. In total, 34 ORFs that potentially formed a C-terminal-domain sequence-related family (CTD family) were found (Table 2). Most of these ORFs have typical P. gingivalis type I cleavable signal peptides, indicating that they would be exported to the periplasm, the outer membrane, or beyond. Several of these ORFs corresponded to known outer membrane-associated proteins, so an investigation of the P. gingivalis outer membrane proteome was conducted to determine if other members of this CTD family could be identified.


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  		TABLE 2. C-terminal domain family proteins with sequence similarity to the RgpB C terminus identified by a BLAST search of the P. gingivalis W83 predicted ORFs

 
The P. gingivalis outer membrane proteome. Proteins from an outer membrane preparation of P. gingivalis ATCC 33277 were analyzed using mass spectrometry, and each protein was identified by sequencing a minimum of one unique peptide. In total, 65 proteins were identified from the outer membrane preparation, and of these proteins, 18 were found to belong to the proposed CTD family (Table 3). Combining our data with reports in the literature regarding experimentally identified P. gingivalis surface-associated proteins allowed us to create an alignment of the C-terminal domains of 24 proteins that belong to the proposed CTD family (Fig. 1). This alignment reveals that the C-terminal domain can be divided into five main motifs, motifs A to E. The most N-terminal motif (motif A) commences with alternating hydrophobic and polar/charged residues followed by a short stretch of polar/charged residues and then a stretch of hydrophobic residues and then another stretch of polar/charged residues. Motif B shows high sequence conservation between all the proteins with an hxhYDMpGbhVhxh motif, where "h" represents a hydrophobic residue (I, L, A, G, V, M, P, and F), "p" represents a polar residue (W, Y, N, Q, T, S, and C), "b" represents a basic residue (H, K, and R), and "x" is any amino acid. Percent identities are as follows: tyrosine, 75%; aspartic acid, 83%; methionine, 50%; glycine, 100%; and valine, 58%. Motif C, like motif A, has alternating hydrophobic polar/charged residues. Motif D has an xGhYhhbVhhx motif, where percent identities are as follows: glycine, 92%; tyrosine, 88%; and valine, 50%. Motif E, located at the C-terminal end of each protein, usually has the charged basic residue lysine (92%) followed by three hydrophobic residues. PredictProtein (http://cubic.bioc.columbia.edu) predicted that most of the C-terminal domains consist of alternating ß-strands and loops, with most of these strands (89%) having an expected prediction accuracy of >82%. Helices were rarely predicted for these sequences. Twenty-two of the C-terminal-domain sequences are predicted to contain seven to nine ß-strands. The prediction of residue solvent exposure is ambiguous, with less than 25% having an expected average correlation greater than 0.69, with these being dispersed over the length of each C-terminal-domain sequence.


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  		TABLE 3. Identification of P. gingivalis CTD family proteins isolated from an ATCC 33277 outer membrane preparation using PMF and NanoLC mass spectrometry analysis of tryptic peptides

 

Figure 1
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  		FIG. 1. Alignment of the C termini of experimentally identified P. gingivalis proteins with homology to the RgpB C terminus. The sequences were initially aligned using Clustal V (22), with minor manual adjustment. Each sequence is described using TIGR ORF (http://www.tigr.org) annotation; RgpB is PG0506. Residues are grouped based on chemical character. Neutral nonpolar (hydrophobic) residues are A, V, I, L, G, M, P, and F; neutral polar residues are S, T, Y, W, N, Q, and C; acidic residues are D and E; and basic residues are K, H, and R. Motifs of interest are noted with the letters A to E above the alignment (see the text). A consensus is noted below the alignment, where x is any residue, h is hydrophobic residues, p is polar residues, and b is basic residues; individual residues with ≥50% conservation are specified.

 
Generation of recombinant P. gingivalis expressing rRgpB. To investigate the role of both the RgpB C-terminal domain and gingipain activity in the maturation of RgpB, gingipain-null mutants of P. gingivalis strain YH522 were created. Mutants YH522AB and YH522KAB were constructed by insertional inactivation of rgpA with cat, rgpB with tetQ, and kgp with cepA (Table 1). The mutants displayed no Arg-gingipain or Arg- and Lys-gingipain activities, respectively (data not shown), and were used as hosts for analysis of recombinant rgpB genes.

The production of rRgpB from a plasmid-borne copy of rgpB has been demonstrated previously (33), but the vector used in that study relied on tetracycline as the selective antibiotic. Tetracycline has since been shown to inhibit gingipain activity (23), so we chose to express rgpB from an alternative vector, pYH411, that uses erythromycin as the selective agent. The control rRgpB was produced from rgpB excised from pBH1.1 and ligated into pYH411, creating pLC1. Transformation of YH522AB with pLC1 created strain ECR7, and transformation of strain YH522KAB with pLC1 created strain OPG44 (Table 1). The negative control strains were YH522AB and YH522KAB transformed with pYH411 to produce strains ECR5 and OPG37, respectively (Table 1). Wild-type YH522 was also transformed with pYH411 (strain ECR55) to compare the level of Arg-gingipain activity of this strain with that of recombinant RgpB strains. ECR7 and OPG44 had the same level of Arg-gingipain activity as ECR55 (where both RgpAcat and RgpB are being produced from chromosomal genes) (Table 4). This contrasts with the less-than-50% activity level observed when only RgpB is being produced from the expression of a chromosomal rgpB (5, 55) and confirms the successful expression of recombinant rgpB.


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  		TABLE 4. Arg-specific proteolytic activity of P. gingivalis mutant strains

 
Effect of a Cys244->Ala244 mutation on rRgpB maturation. To determine if gingipain activity is necessary for RgpB maturation, site-directed mutagenesis was used to alter the rgpB codon for the catalytic residue Cys244 to Ala244 (mature enzyme numbering) (Cys473 in the zymogen). This recombinant gene, rgpB(C244A), was ligated into pYH411 and expressed in both YH522AB and YH522KAB (producing strains OPG40 and OPG41, respectively). Neither strain was able to hydrolyze BApNA, confirming that we had altered a catalytic residue. The outer membrane fraction of each strain was isolated and subjected to SDS-PAGE and immunoblot analysis using the preadsorbed mouse polyclonal anti-rRgpAcat antibodies as probes (Fig. 2A). OPG40 and OPG41 both exhibited a typical membrane-associated RgpB profile, with numerous bands of elevated molecular mass (80 to 90 kDa) present, similar to those seen with the ECR55 wild-type control membrane-associated RgpB (Fig. 2A, lane 7) and rRgpB produced in ECR7 (Fig. 2A, lane 2) and OPG44 (Fig. 2A, lane 1). However, when rRgpB(C244A) was produced in YH522KAB (OPG41), there was a relatively large amount of immunoreactive protein greater than 94 kDa compared with wild-type RgpB produced in ECR55 and rRgpB produced in ECR7 and OPG44. When rRgpB(C244A) was isolated from a 2D-PAGE gel of outer membranes of OPG41 and subjected to PMF, peptides that were derived from the prodomain were identified, while no C-terminal-domain peptides were found. Thus, the observed increase in mass was attributed to the presence of the prodomain for rRgpB(C244A) expressed in YH522KAB. Prodomain peptides could not be detected in the RgpB proteins expressed and membrane attached in ECR55, OGP44, and ECR7.


Figure 2
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  		FIG. 2. Cellular localization of recombinant RgpB isoforms. (A) Western blots of outer membrane fractions separated by SDS-PAGE and probed with anti-rRgpAcat antibodies. Lane 1, OPG44 (YH522KAB::rRgpB); lane 2, ECR7 (YH522AB::rRgpB); lane 3, OPG41 [YH522KAB::rRgpB(C244A)]; lane 4, OPG40 [YH522AB::rRgpB(C244A)]; lane 5, OPG37 (YH522KAB::pYH411); lane 6, ECR5 (YH522AB::pYH411); lane 7, ECR55 (YH522::pYH411); lane 8, ECR104 (YH522AB::rRgpB435). (B) Western blots of whole-cell lysate fractions and culture supernatant fractions separated by SDS-PAGE and probed with anti-RgpAcat antibodies. Lanes 1 to 3, whole-cell lysate fractions. Lane 1, ECR7 (YH522AB::rRgpB); lane 2, ECR104 (YH522AB::rRgpB435); lane 3, ECR5 (YH522AB::pYH411). Lanes 4 to 6, culture supernatant fractions. Lane 4, ECR7 (YH522AB::rRgpB); lane 5, ECR104 (YH522AB::rRgpB435); lane 6, ECR5 (YH522AB::pYH411). Lanes 7 to 9, culture supernatant fractions after centrifugation at 40,000 x g. Lane 7, ECR7 (YH522AB::rRgpB); lane 8, ECR104 (YH522AB::rRgpB435); lane 9, ECR5 (YH522AB::pYH411). (C) Coomassie blue-stained SDS-PAGE gel of periplasmic fractions. Bands were identified using mass spectrometry. Lane 1, molecular mass markers; lane 2, ECR5 (YH522AB::pYH411); lane 3, ECR7 (YH522AB::rRgpB); lane 4, ECR104 (YH522AB::rRgpB435). The arrows indicate rRgpB435 in ECR104 (lane 4) and rRgpB in ECR7 (lane 3). Bands labeled 1 to 6 were shown to contain peptidase M16 (PG0196) (1), CPG70 carboxypeptidase precursor (PG0232) (2), prolyl oligopeptidase (PG1004) (3), conserved hypothetical protein (PG0491) (4), TPR domain protein (PG0449) (5), and TPR domain protein (PG1385) (6). (D) Western blots of whole-cell lysate fractions of YH522KAB recombinants separated by SDS-PAGE and probed with anti-RgpAcat antibodies. Lane 1, ECR123 (YH522KAB::rRgpB435); lane 2, OPG37 (YH522KAB::pYH411); lane 3, OPG44 (YH522KAB::rRgpB). The amount of protein loaded onto the SDS-PAGE gels was standardized with the equivalent of protein from 6.0 x 108 cells loaded onto each lane in A, B, and D and the equivalent of protein from 4.0 x 109 cells loaded onto each lane in C. The identity of RgpB in the bands of the Western blots (A, B, and D) was confirmed by mass spectrometric analysis of Coomassie blue-stained replicate gels. Similarly, the identity of RgpAcat in lane 7 of A was confirmed by the presence of RgpAcat-specific peptides in the 45-kDa band of a replicate gel as described previously (59).

 
Truncation of RgpB and proteolytic activity. A 3'-truncated rgpB gene, {Delta}rgpB435, was designed to produce a C-terminally-truncated zymogen, rRgpB435, of 664 aa, predicted to be 435 aa in length (aa 230 to 664 as encoded by {Delta}rgpB435) upon maturation. {Delta}rgpB435 was ligated into pYH411 (creating pYH435) and used to transform YH522AB and YH522KAB, generating strains ECR104 and ECR123, respectively. ECR104 and ECR123 whole cells had no significant level of Arg-specific proteolytic activity compared with the rRgpB-producing positive controls ECR7 and OPG44 (Table 4). Arg-specific proteolytic activity in ECR104 culture supernatants was detectable but very low compared with that of the rRgpB-producing strain ECR7, and this ECR104 culture supernatant activity remained the same after centrifugation to remove vesicles, indicating that the active enzyme was not vesicle associated. In contrast, the ECR7 supernatant Arg-specific proteolytic activity was reduced by 52% after centrifugation under the same conditions.

Localization of truncated rRgpB435. P. gingivalis culture fractions of ECR7 (rRgpB), ECR104 (rRgpB435), and ECR5 (negative control) were subjected to SDS-PAGE and Western blotting using anti-rRgpAcat antibodies and SDS-PAGE or 2D-PAGE with proteins identified using mass spectrometry. Analysis of outer membrane fractions (Fig. 2A) and whole-cell and culture supernatant fractions (Fig. 2B) revealed that unlike full-length rRgpB, where the majority of the protein is found as the 80- to 90-kDa membrane-attached isoform in YH522AB (ECR7), the truncated rRgpB435 expressed in the same YH522AB host (ECR104) was not membrane attached. The rRgpB435 protein was located in the culture supernatant (Fig. 2B) and periplasm (Fig. 2C) of ECR104 as 71-kDa and 48-kDa isoforms. The identity of the 71-kDa and 48-kDa proteins in the supernatant and periplasm as RgpB was confirmed using mass spectrometry. For the 71-kDa isoform, six peptides corresponding to the prodomain were identified using mass spectrometry. The full-length rRgpB precursor minus the leader sequence (78 kDa) could also be detected in the periplasmic fraction of ECR7 expressing the full-length rRgpB; however, this precursor was less abundant relative to the 71-kDa precursor isoform of rRgpB435 (Fig. 2C). The periplasmic fractions were confirmed to be enriched for periplasmic proteins, as mass spectrometric analysis of the major SDS-PAGE bands of the fractions (Fig. 2C) revealed proteins predicted to be located outside of the cytoplasm based on their leader sequences using PSORTb (18). The major proteins of the fraction predicted to be located in the periplasm were peptidase M16 (PG0196), CPG70 carboxypeptidase precursor containing the prodomain (PG0232), prolyl oligopeptidase (PG1004), conserved hypothetical protein (PG0491), and two TPR (tetratricopeptide repeat) domain proteins (PG0449 and PG1385) (Fig. 2C).

From the localization analysis of ECR104 for rRgpB435, it could be determined that the protein was distributed between the culture supernatant and periplasm, with no detectable amount attached to the outer membrane. The majority of the rRgpB435 protein was catalytically inactive and was present as 71-kDa and 48-kDa isoforms. The predicted molecular mass of the unprocessed zymogen (minus the leader sequence) produced by {Delta}rgpB435 is 71 kDa, which is expected to become 48 kDa after the removal of the prodomain. It was interesting that the whole-cell lysate fraction of ECR104 exhibited novel bands that were immunoreactive with anti-RgpAcat antibodies at 60 kDa and 56 kDa as well as with the 71-kDa and 48-kDa isoforms (Fig. 2B). The 60-kDa and 56-kDa bands were confirmed to be derived from rRgpB435 by mass spectrometry. The mass spectrometric analysis of all the proteins (71 kDa, 60 kDa, 56 kDa, and 48 kDa) derived from rRgpB435 in the ECR104 cell lysate fraction (Fig. 2B) showed that each protein exhibited a tryptic C-terminal peptide terminating at Lys651 (numbering from the initial Met residue), indicating little or no C-terminal truncation. This suggested that the 60-, 56-, and 48-kDa bands arose by N-terminal truncation of the 71-kDa precursor form. This was confirmed by mass spectrometry, where the most N-terminal tryptic peptide recovered from the digest of each 60-, 56-, and 48-kDa isoform was consistent with N-terminal truncation of the prodomain. Peptides that commenced at amino acid Leu37 for the 71-kDa protein, Gly165 for the 56-kDa and 60-kDa proteins, and, surprisingly, Asn208 for the 48-kDa protein (the 48-kDa mature, active wild-type RgpB is processed at Arg229 to remove the prodomain) were identified. Western blot analysis of lysed ECR123 whole cells producing rRgpB435 but not Kgp predominantly showed the 71-kDa band with only a small amount of the 48-kDa rRgpB435 isoform (Fig. 2D). These results suggest that Kgpcat was largely responsible for the N-terminal truncation of rRgpB435 and that the 60-kDa and 56-kDa isoforms were most likely produced by cleavage of the 71-kDa precursor by Kgpcat upon cell lysis, as these isoforms could not be located in any other cell fraction or the culture supernatant.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have identified a novel C-terminal-domain protein family within P. gingivalis comprising 34 possible members, 24 of which have been shown in this and previous studies to be surface associated. Zhang et al. (62) previously demonstrated the production of a further eight proteins in P. gingivalis that belong to the CTD family (Table 2). Although the precise cellular locations of these proteins identified by Zhang et al. (62) were not determined, the presence of a putative type I cleavable signal peptide in many of these proteins suggests that they would be exported out of the cytoplasm, so it would not be unreasonable to predict them to also be surface associated. The only predicted CTD family proteins identified by a BLAST search that have not so far been shown to be produced by P. gingivalis are PG0111, a protein putatively involved in capsular biosynthesis, and PG1969, a hypothetical protein. The presence of a domain that is conserved between so many P. gingivalis outer membrane proteins, as shown in this study, indicates that it is an important feature for this species that is likely to play a role in secretion and/or attachment, as the functions of these proteins are diverse and there are no other regions of high sequence conservation.

RgpAA4 (PG2024), P27 (PG1795), and P59 (PG2102), as well as RgpB, were previously identified as P. gingivalis outer membrane proteins and shown to be recognized by MAb 1B5 (59). MAb 1B5 has recently been shown to recognize a phosphorylated branched mannan proposed to be part of the cell envelope of P. gingivalis (37). Like membrane-associated RgpB, RgpAA4, P27, and P59 also migrate with nondiscrete, higher molecular weights in 2D-PAGE. This has led to the suggestion that they have a common posttranslational modification that allows them to be attached to the outer membrane (59). As soluble RgpB, found as a discrete 48-kDa enzyme in the P. gingivalis culture supernatant (40), is not recognized by MAb 1B5 (12) and lacks the C-terminal domain since it is C-terminally truncated at Ser435 (15), the membrane attachment site has been suggested to be the C-terminal domain. In the study reported previously by Veith et al. (59), no peptides from the C-terminal domains of membrane-associated RgpB, RgpAA4, P27, and P59 were identified using mass spectrometry, and the same result was found for the proteins analyzed during the course of this study. The most likely explanation is that the glycan-modified C-terminal peptide did not ionize during the mass spectrometric procedure or that the heavily modified peptide was not recovered or was lost during sample preparation and therefore was not detected. It is interesting that the unmodified C-terminal-domain tryptic peptide could be detected using mass spectrometry when the full-length rRgpB precursor found in the periplasm of ECR7 was analyzed, consistent with the modification preventing detection.

Database BLAST searches using the C-terminal domains identified in this study revealed similarities with the C-terminal domains of numerous predicted proteins of other members of the Bacteroidetes. These species include Rhodothermus marinus (25), Cytophaga hutchinsonii (19), a Microscilla species, (16), Zobellia galactanovorans (8), Flavobacterium columnare, and Prevotella intermedia, a species that is also found in subgingival dental plaque. Interestingly, C-terminal-domain similarity can also be found with predicted proteins of the photosynthetic sulfur bacterium Chlorobium phaeobacteroides, showing that the CTD family is not limited to the phylum Bacteroidetes. An alignment of the C-terminal domains of predicted proteins of several of these species revealed a consensus with similarity to that found here. The common features of the C-terminal domains found within the proteins of these other species and that of the P. gingivalis CTD are well-conserved DxxG and GxY motifs and the carboxyl region with a conserved K followed by hydrophobic residues. These other species have a highly conserved YPNP motif within the equivalent of P. gingivalis motif A of the CTD. Examination of the P. gingivalis alignment (Fig. 1) shows that several proteins have a YPNP-like sequence in motif A, but in general, it is not highly conserved. Notably, the CTD is not found within the predicted proteins of the sequenced genomes of other Bacteroidetes members that are considered to be more closely related to P. gingivalis, such as Bacteroides fragilis and Bacteroides thetaiotamicron.

To provide evidence for the role of the Arg-specific catalytic activity and conserved C-terminal domain in the processing and attachment of RgpB to the outer membrane, we created a series of gingipain-deficient mutant strains that produced recombinant RgpB as the full-length zymogen, with a Cys244->Ala244 mutation, or with a C-terminal-domain truncation. In the absence of any gingipain activity, P. gingivalis YH522KAB was able to export full-length rRgpB(C244A) and attach it to the outer membrane, indicating that gingipain proteolytic activity is not essential for the activation of any of the proteins involved in export and membrane attachment. It is possible that in vivo, RgpB has an autocatalytic function, as the full-length zymogen was processed in the YH522KAB background strain to a fully active, membrane-attached isoform. Mikolajczyk et al. (30) previously demonstrated autocatalytic activity in vitro with recombinant RgpB. However, rRgpB(C244A), although inactive, was still outer membrane attached, with a typical attachment profile by SDS-PAGE for both YH522AB and YH522KAB background strains, except that in YH522KAB, the prodomain was not removed. Thus, although RgpB export and membrane attachment are independent of the proteolytic activity of RgpA, RgpB, or Kgp, the maturation of the enzyme, involving proteolytic processing to remove the prodomain, does require the proteolytic activity of one of these enzymes.

The analysis of the cells producing the C-terminally-truncated rRgpB435 indicated that unlike its full-length counterpart, the protein was not detected attached to the outer membrane but was found as 71-kDa and 48-kDa isoforms in the culture supernatant and the periplasm, demonstrating that the C-terminal domain is essential for attachment. The abundance of the 71-kDa and 48-kDa isoforms of rRgpB435 in the culture supernatant of ECR104, the very low enzyme activity, and the presence of a prodomain suggest that the rRgpB435 protein, although secreted from the cell, had not been processed correctly and was misfolded. The lack of the C-terminal domain may have produced instability and aggregation of rRgpB435 in the periplasm, perhaps explaining its accumulation in this cellular compartment. Alternatively, the C-terminal domain may also have a role in export, and without this domain, rRgpB435 could not be secreted via the normal pathway and therefore accumulated in the periplasm. The accumulation of rRgpB435 in the periplasm may then have resulted in nonspecific export (21).

The mechanism(s) used by P. gingivalis to translocate proteins across the outer membrane is as yet undefined. A possible mechanism for export of the CTD family proteins such as RgpB is via a process related to autotransport. The length of the CTD at 70 to 80 amino acids and the predicted ß-strand structures within the domain suggest that the CTD family proteins may be similar to the recently described type Vc YadA-like autotransporter subfamily of trimeric autotransporters (11, 21, 42). Trimeric autotransporters include the Haemophilus influenzae Hia adhesin and the Neisseria meningitidis NhhA proteins (53), and they have been predicted to occur in a wide array of species (11). As it has been demonstrated that the C-terminal domains of the trimeric autotransporters form trimers in the outer membrane (42, 53), it has been hypothesized that these 70- to 80-amino-acid domains oligomerize to form a ß-barrel pore, similar to that of the outer membrane transporter TolC, through which the unfolded passenger domains (N-terminal domains) are translocated to the cell surface (11). Like conventional autotransporters, trimeric autotransporters are translocated to the periplasm via the Sec apparatus, but unlike conventional autotransporters that are usually cleaved at the outer membrane, trimeric autotransporters are not cleaved at the bacterial cell surface.

Other periplasmic and/or outer membrane molecules may also have a role in the translocation of the trimeric autotransporters across the outer membrane, possibly via facilitating oligomerization and pore formation and/or by acting as chaperones (11, 21). The CTD family proteins of P. gingivalis may function in a fashion similar to that of the trimeric autotransporters except that, at least for some of the CTD family proteins, they are also modified by covalent attachment to glycans (59) associated with the cell envelope (37), presumably to ensure secure anchorage at the cell surface. The secretion and attachment of the CTD family proteins to the outer membrane may therefore be coordinated and orchestrated by a number of different proteins, with the C-terminal domain having a central role in both secretion and attachment.

A recent report implicated PorT, a protein associated with the P. gingivalis periplasm, in the translocation of the gingipain and HagA adhesin precursors, which belong to the CTD family. A PorT mutant accumulated unprocessed gingipains and HagA within the periplasm (46). A putative homologue of PorT was identified within C. hutchinsonii and Prevotella intermedia (46), two of the species that have proteins with C-terminal domains similar to those of the CTD family. It is tempting to speculate, therefore, that there may be an interaction between PorT and the C-terminal domains of our proposed CTD family of proteins that facilitates their outer membrane export and attachment. PorT may be an essential accessory protein involved in the periplasmic transit and assembly of the CTD oligomers into the outer membrane (21).

Other proteins that may have a role in the coordination of export and attachment of the CTD family proteins are those of the bcp-recA-vimA, vimE, vimF, and porR loci (1, 2, 17, 35, 48, 56-58). Inactivation of recA affects the phenotypic expression and distribution of the gingipains, as does inactivation of vimA, vimE, vimF, and porR. High-molecular-mass soluble gingipains were found in the culture supernatant fraction of the vimA mutant, and RgpB was identified to be in the proenzyme form by mass spectrometry (35). The vimA, vimE, and vimF mutants also display aberrant polysaccharide biosynthesis (56). In fact, Shoji et al. (48) recently reported that the vimA and porR mutants produced no MAb 1B5-reactive material; thus, the altered distribution of RgpB observed in these mutants may relate to the inability to appropriately glycosylate and therefore attach the protein at the cell surface. This suggests that unlike the trimeric autotransporters, the P. gingivalis C-terminal domain per se does not secure attachment at the cell surface and that glycosylation is the event that secures attachment of the P. gingivalis CTD family proteins. Considered together, these results suggest that secretion of RgpB and attachment to cell envelope glycans are coordinated and are an important step for proper processing and folding to occur to produce a fully functional enzyme. When recombinant truncated Kgp polyproteins lacking C-terminal adhesins including the C-terminal domain were produced in P. gingivalis W83, there was an altered distribution of the enzyme (Kgpcat) between membrane-associated and secreted forms, with significant amounts of soluble Kgpcat being released into the culture media (54). The association of Kgpcat with the outer membrane fraction can be explained by the propensity of this enzyme to bind noncovalently to cell surface adhesins that contain the C-terminal-domain attachment site (49, 54). Therefore, those results (54) are not inconsistent with the findings of this current study.

In conclusion, the C-terminal domains of numerous P. gingivalis outer membrane-associated proteins have primary sequence and predicted secondary structure similarity to the RgpB C-terminal domain, suggesting that they have a common functional role. We propose that these domains form part of a new protein domain family that we have designated the CTD family. The inability of P. gingivalis to attach rRgpB with a truncated C terminus to the outer membrane suggests that the C-terminal domain of RgpB and other CTD family members is essential for attachment and may be involved in a coordinated process of export and attachment of the proteins to the cell surface.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the excellent technical assistance of Lucy Chroscicki, Caroline Moore, Dina Chen, Rita Paolini, and Patricia Lissel.

This work was supported by grants from the National Health and Medical Research Council and the National Institutes of Health (United States) and a SPIRT studentship (to T.N.).


   FOOTNOTES
 
* Corresponding author. Mailing address: Cooperative Research Centre for Oral Health Science, School of Dental Science, University of Melbourne, 720 Swanston Street, Victoria, Australia. Phone: 61-3-9341-1547. Fax: 61-3-9341-1596. E-mail: e.reynolds{at}unimelb.edu.au. Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, September 2006, p. 6376-6386, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00731-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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