Transposition of Tn4351 in Porphyromonas gingivalis.

Genetic analysis of Porphyromonas gingivalis, an obligately anaerobic gram-negative bacterium, has been hindered by the apparent lack of naturally occurring bacteriophages, transposable elements, and plasmids. Plasmid R751::*omega 4 has previously been used as a suicide vector to demonstrate transposition of Tn4351 in B. uniformis. The erythromycin resistance gene on Tn4351 functions in Bacteroides and Porphyromonas. Erythromycin-resistant transconjugants were obtained at a mean frequency of 1.6 x 10(-7) from matings between Escherichia coli HB101 containing R751::*omega 4 and P. gingivalis 33277. Southern blot hybridization analysis indicated that about half of the erythromycin-resistant P. gingivalis transconjugants contained simple insertions of Tn4351 and half contained both Tn4351 and R751 sequences. The presence of R751 sequences in some P. gingivalis transconjugants most likely occurred from Tn4351-mediated cointegration of R751, since we were unable to detect autonomous plasmid in these P. gingivalis transconjugants. The P. gingivalis-Tn4351 DNA junction fragments from different transconjugants varied in size. These results are consistent with transposition of Tn4351 and with insertion at several different locations in the P. gingivalis chromosome. Tn4351 may be useful as a mutagen to isolate well-defined mutants of P. gingivalis.

Correlation of haemagglutination activity with trypsin-like protease activity of Porphyromonas gingivalis.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with various forms of periodontal disease. Several characteristics of P. gingivalis are thought to contribute to its pathogenicity; these include haemagglutination and trypsin-like protease activity. Previous studies suggest an association between haemagglutination and trypsin-like protease activity of P. gingivalis. To investigate this, two complementary quantitative experimental approaches were taken. Five independent mutants of P. gingivalis deficient in trypsin-like protease activity were shown to exhibit reduced haemagglutination activity. In addition, enhancers (cysteine and dithiothreitol) and inhibitors (N-ethylmaleimide, N-p-tosyl-L-lysine-chloromethyl ketone, and phenylmethylsulphonyl fluoride) of trypsin-like protease activity were shown, respectively, to significantly enhance and inhibit haemagglutination activity of washed, wild-type P. gingivalis cells (p less than 0.05, paired t-test). Statistical analysis indicated a strong correlation between haemagglutination and trypsin-like protease activity (r = 0.85, p less than 0.001, Spearman rank correlation). The effect of the protease enhancers and inhibitors on haemagglutination activity was specific for P. gingivalis, as they did not significantly change the haemagglutination activity of Fusobacterium nucleatum. These results suggest that the proteolytic site of the trypsin-like protease participates in haemagglutination activity of P. gingivalis.

Association of proteases of Porphyromonas (Bacteroides) gingivalis with its adhesion to Actinomyces viscosus.

P. gingivalis adheres to A. viscosus on mineral surfaces mimicking teeth. To study whether P. gingivalis proteases contribute to its binding, mutants of P. gingivalis deficient in proteases were compared with their parent strain and a P. gingivalis-type strain for their adherence to A. viscosus on saliva-coated hydroxyapatite by manipulating a radio-isotope binding assay. Adherence of P. gingivalis 2561 to A. viscosus was studied by tests of the effects of incubation temperature and known inhibitors or promoters of proteases. Controls were handled by the assay run in PBS buffer at 22 degrees C. Two mutants deficient in trypsin-like protease were found to be deficient in adherence (% attachment relative to control: 3.2 +/- 0.1% and 11.2 +/- 0.4%), while a collagenase-deficient mutant had an adherence score (51.6 +/- 8.4) closer to that of the parent strain (75.6 +/- 7.2%). Heating P. gingivalis at 70 degrees C decreased its subsequent adherence at 22 degrees C by 80%. Adherence decreased by 60% when the assay was run at 4 degrees C, but increased by 70% at 37 degrees C. Reducing agents (dithiothreitol, cysteine, and mercaptoethanol) enhanced P. gingivalis adherence by 50 to 60%. Protease inhibitors (BZMD, SBTI, TPCK, TLCK, CMPS, PMSF) decreased adherence by 10 to 50%. Also, Hg2+ and Zn2+ decreased adherence by 30 to 50%, and arginine decreased it by 50%. Most of these effects on P. gingivalis adherence were statistically significant (p less than 0.05). Analysis of these data suggests that P. gingivalis proteases may contribute to the cohesion of P. gingivalis and A. viscosus.