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Porphyromonas gingivalis, a gram-negative anaerobic bacterium, has been recognized as one of the major etiologic agents of human periodontal diseases. It is known that this organism produces a variety of potential virulence factors, and proteases have been implicated as important virulence factors of this organism (2, 13, 16, 19, 20, 31, 32). Two of these enzymes, Arg-gingipain A (RgpA) and Lys-gingipain (Kgp) (16, 21-23, 31), have been shown to contain highly homologous adhesin domains. These conserved sequences are also present in the large hemagglutinin HagA of P. gingivalis (7, 11). Recent data suggests that the precursor of Kgp comprises at least four domains: the signal peptide domain, the amino-terminal propeptide domain, the catalytic protease domain, and the carboxyl-terminal hemagglutinin domain (2, 12, 21, 22). Kgp has been implicated in the destruction of periodontal tissues and the disruption of host defense mechanisms due not only to its catalytic activity but also to its involvement in hemagglutination, heme accumulation, and hemoglobin binding by P. gingivalis (2, 3, 5, 8-10, 12-14, 18, 19, 21-29).

Previously, a pigmentless variant of P. gingivalis W50, W50/BE1, was isolated following chemostat culture (4, 15). This strain was altered in protease, collagenase, and hemagglutination activities and proved to be avirulent in a mouse abscess model system. However, the molecular basis for the spontaneous generation of pigmentless P. gingivalis mutants has not yet been determined. In addition, recent results (17, 19, 26) have implicated the kgp gene in the pigmentation phenotype. To gain further insight into this phenomenon, several spontaneously generated pigmentless mutants of strain 381 were isolated and analyzed. Our results suggest that one mechanism for the spontaneous development of pigmentless mutants is the generation of deletions of portions of the hagA and kgp genes following homologous recombination between the tandemly arranged hagA and kgp genes.

Isolation and identification of spontaneous pigmentless P. gingivalis mutants. P. gingivalis 381 and its variants MT10, G102, and WK, which are protease-deficient mutants with insertionally inactivated rgpA (31), rgpB (30), and prtT genes (29), respectively, were grown anaerobically (5% CO₂, 10% H₂, 80% N₂) in enriched TSB medium (containing, per liter, 40 g of tryptic soy broth [TSB; Difco, Detroit, Mich.], 5 g of yeast extract [Difco], 0.5 g of cysteine, 10 mg of hemin, and 1 mg of vitamin K₁) and maintained on tryptic soy blood agar (TSA) plates (containing, per liter, TSB plus 15 g of agar and 50 ml of sheep blood) (29). For growth of the antibiotic resistance strains, antibiotics were added to the media at the following concentrations: 50 µg/ml for gentamicin and 10 µg/ml for erythromycin (29). A few pigmentless colonies of WK, MT10, and G102 developed spontaneously on the TSA plates streaked with these strains. Each colony was passaged twice on TSA plates to isolate stable white colonies and named WK-W, G102-W, and MT10-W. These pigmentless colonies remained white on TSA plates for at least 3 to 4 weeks and then gradually became light brown in color. The shape, size, and odor of these white colonies were similar to those features of the corresponding black-pigmented colonies. The frequency of appearance of white colonies varied with each strain and was approximately 10⁶. However, no such colonies were obtained on the TSA plates from the parental strain, 381, under identical conditions. This suggested that protease deficiency may increase the rate of appearance of the pigmentless colonies. Additional studies will be necessary to determine if this increased rate is a direct or an indirect effect.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Hemoglobin binding assay of P. gingivalis strains. The cell suspensions in PBS were incubated anaerobically with bovine hemoglobin at pH 7.4 and 37°C for 30 to 60 min and centrifuged. The absorbance of the suspensions at 415 nm was then measured. The decrease in absorbance was used to calculate hemoglobin binding. Shown are hemoglobin binding results with strains 381 (), WK (), and WK-W () (A); with strains 381 (), G102 (), and G102-W () (B); and with strains 381 (), MT10 (), and MT10-W () (C). Bars represent the standard deviations of results of duplicate samples.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Genomic maps of the proposed orientations of the hagA and kgp genes of P. gingivalis 381. The major restriction sites of hagA and kgp are indicated. Restriction sites: K, KpnI (nucleotides 3760, 5116, 6484, 7852, 10606, 12589, and 16087); B, BamHI (nucleotides 3309, 3672, 12484, and 15999); N, NcoI (nucleotides 4840, 6208, 7576, 8944, 17170, and 18643); S, SmaI (nucleotides 4583, 5951, 7319, 8687, 16913, 19245, and 19554); V, Van91I (nucleotides 1741 and 20629). Solid boxes on the map show the probes N (for the Kgp N-terminal protease domain), C (for the Kgp adhesin domain), hagAN (for the exact HagA N-terminal region), and kgpC (for the exact Kgp C-terminal region). Boxes with hatching labeled "hagA" and "kgp" represent the adhesin regions of the hagA and kgp genes, respectively. The stippled box represents the N-terminal domain and protease domain of Kgp. The boxes above the restriction enzyme map with hatching, vertical lines, and shading represent the proposed minimal deleted regions of WK-W, MT10-W, and G102-W, respectively.

Molecular basis for deletions of the kgp gene. One possible explanation for the spontaneous deletion of portions of the kgp gene is that a recombinational rearrangement occurred. This may result from the presence of highly homologous related sequences in the adhesin domains of rgpA, hagA, and kgp (7, 11, 16, 21, 31). In order for this to occur, the kgp gene should be closely linked to one of these homologous genes. After searching the sequence database at The Institute for Genomic Research (28a) for P. gingivalis W83 with the kgp and hagA sequences, we identified three fragments sequenced from P. gingivalis W83 (contigs 20, 40, and 81) carrying portions of both genes (1). An examination of these fragments suggested that the hagA gene is located upstream of the kgp gene in strain W83. In addition, the hagA gene of strain 381 contains four adhesin domains (1), compared to three homologous domains for W83. Approximately 3 kb separates the hagA and kgp genes on the strain W83 chromosome. Therefore, in order to confirm this arrangement for strain 381, Southern blot analysis was carried out. Chromosomal DNA was isolated from P. gingivalis 381 cells with a Puregene DNA isolation kit by following the protocol of the supplier (Gentra System, Inc., Minneapolis, Minn.). The DNA was digested with several restriction enzymes, separated on 0.8% agarose gels, and transferred to Hybond-N⁺ nylon membrane (29). Probe N (nucleotides 12484 to 13457), the PCR product amplified with PR1 and PR2, was digested with BamHI and purified with a QIAEX II kit (Qiagen, Hilden, Germany). A 1.4-kb (nucleotides 17170 to 18643) NcoI fragment of the kgp adhesin region was used as probe C (Fig. 2). The labeling of the probes as well as hybridization and detection by the ECL system was performed as recommended by the supplier (Amersham International plc., Amersham, United Kingdom). Southern blot analysis revealed that both probes N (Fig. 3C, lane 1 and C (Fig. 3D, lane 1) hybridized with the same 8.2-kb (nucleotides 8944 to 17170) NcoI fragment, which included portions of both the hagA and kgp genes. This result is consistent with hagA being upstream of the kgp gene (Fig. 2). In order to further confirm this tandem arrangement, a portion of the intervening region (nucleotides 12290 to 12650) between the hagA and kgp genes was amplified by PCR, isolated, and sequenced (data not shown), and the results confirmed the postulated sequence. Nucleotide sequence analyses revealed that the four repeat adhesin regions of the hagA gene of P. gingivalis 381 are approximately 98% identical with the C-terminal adhesin region of its kgp gene. These homologous regions provided a good molecular basis for homologous recombination.

View larger version (73K): [in this window] [in a new window]   FIG. 3.   Southern blot analysis of genomic DNAs of P. gingivalis strains. (A to E) Southern blots of strain 381 (lanes 1), WK (lanes 2), WK-W (lanes 3), G102 (lanes 4), G102-W (lanes 5), MT10 (lanes 6), and MT10-W (lanes 7). The chromosomal DNAs of the P. gingivalis strains were digested with the following restriction enzymes and hybridized with the following probes: KpnI and probe N (A), KpnI and probe C (B), NcoI and probe N (C), NcoI and probe C (D), and Van91I and probe hagAN (E).