Role of polyphosphate kinase in biofilm formation by Porphyromonas gingivalis

Abstract

In order to assess the role of polyphosphate kinase (PPK) in the physiology of Porphyromonas gingivalis, a ppk gene mutant, CW120, was constructed and characterized. P. gingivalis was demonstrated to synthesize short-chain polyphosphate (polyP) but not long-chain polyP. CW120 failed to survive in the stationary phase as well as the parental cell did, and it was attenuated in biofilm formation on polyvinylchloride and glass surfaces. Furthermore, the complementation by insertion of an intact copy of the ppk gene into the mutant CW120 restored its biofilm formation and stationary-phase survival. These results suggest that PPK may be important for incorporation of these organisms into subgingival plaque in the human oral cavity.

FIG. 1.

Construction of the ppk-deficient mutant CW120. A 2.1-kb ppk fragment amplified by PCR from P. gingivalis 381 was ligated into the pCR2.1-TOPO vector. The 2.1-kb fragment was then isolated and inserted into pUC19 following cleavage of plasmid TOPO/ppk and pUC19 with EcoRI, producing plasmid pCW110. A 2.1-kb ermF-ermAM BamHI-digested cassette from plasmid prtT:Em was next inserted into the ppk gene at the BglII site of pCW110. The resulting plasmid, pCW111, was linearized with PvuII and electroporated into P. gingivalis 381. The resulting mutant, CW120, was identified by Southern blot analysis of the genomic DNA of P. gingivalis (bottom left). The chromosomal DNA of strains 381 (lanes 1 and 3) and CW120 (lanes 2 and 4) was digested with BspEI (lanes 1 and 2) or StuI (lanes 3 and 4), respectively, and probed with a 787-bp fragment of the ppk gene digested with PstI. BEI, BspEI; BI, BamHI; EI, EcoRI; BII, BglII; PI, PstI; PvII, PvuII; SI, StuI; Em, erythromycin cassette.

FIG. 2.

Short-chain polyP assay of P. gingivalis 381, ppk-deficient mutant CW120, and complemented strain CW120C. P. gingivalis 381 (♦), CW120 (•), and CW120C (▴) were grown to mid-log phase in TSB medium with hemin and menadione. The cells were pelleted, resuspended, and incubated anaerobically in MOPS defined medium containing 0.1 mM P_i, 0.01% BSA, and 4 mg of glucose per ml. The samples were collected at 0, 1, 2, 3, and 4 h. Short-chain polyP was extracted and analyzed as described in the text. The results are averages of quadruple samples and their standard deviations are shown.

FIG. 3.

Biofilm formation assay in PVC plates. P. gingivalis strains were incubated overnight in TSB medium diluted with PBS (TSB/PBS ratio, 1:2) with supplementation of hemin, vitamin K in the wells of 96-well PVC microtiter dishes (100 μl/well). The resulting biofilms were analyzed as described previously (24). Biofilm formation was calculated as follows: (OD₅₇₀ for the biofilm)/(OD₅₇₀ of total cell growth). The data are averages of triplicate assays with the standard errors of the means. T75, 75 phosphate polymer of polyP (100 μg/ml); PS, polyvinyl sulfate (100 μg/ml).

FIG. 4.

Confocal images of P. gingivalis 381 and CW120 biofilms in a flow cell. (A) Strain 381 at 4 h; (B) CW120 at 4 h; (C) strain 381 at 18 h; (D) CW120 at 18 h.

FIG. 5.

Strategy for complementation of the ppk-deficient mutant CW120. (A) A 2.4-kb ppk gene fragment, including the 400-bp upstream flanking region, amplified by PCR from P. gingivalis 381 was ligated into the pCR 2.1-TOPO vector. A 2.2-kb tetA(Q) cassette digested with SstI from pKFT2 was blunted and inserted into the ppk gene at the StuI site in the same transcription direction as the ppk gene. The portion of the ppk gene downstream from tetA(Q) was cut out with SnaB I and HindIII. An intact copy of the ppk gene was ligated downstream of tetA(Q) at the SnaB I and HindIII sites of pPPK:tQ. The resulting plasmid, designated pCW112, was linearized with HindIII and electroporated into the ppk-null mutant CW120. (B) The predicted integration of the resulting plasmid pCW112 into CW120. (C) Southern blot analysis of the genomic DNA of P. gingivalis wild-type 381, ppk mutant CW120, and the ppk-complemented strain CW120C. The chromosomal DNA of 381(lanes 1 and 4), CW120 (lanes 2 and 5), and CW120C (lanes 3 and 6) was digested with StuI (lanes 1, 2, and 3) and PstI (lanes 4, 5, and 6), respectively, and probed with a 2.4-kb ppk fragment.