Collectively, these molecules seem to act coordinately to regulate the development of mature biofilms. Methods Bacterial strains and media The P. gingivalis strains used in this study are shown in Table 4. P. gingivalis cells were inoculated from blood agar plates and grown anaerobically (85% N2, 10% H2, 5% CO2) at 37°C in trypticase soy broth supplemented with 1 mg/ml of yeast extract, 1 μg/ml of menadione and 5 μg/ml of hemin (TSB). At stationary phase, the cells were harvested by centrifugation at 6,000 × g for 7 minutes, resuspended in pre-reduced 10 mM phosphate

buffer containing 0.15 M sodium chloride (PBS; pH 7.4) and then used in the assays. When necessary, the following antibiotics were used at the concentrations shown in parentheses: chloramphenicol (20 μg/ml), erythromycin (10 μg/ml), and tetracycline Combretastatin A4 cost (1 μg/ml). To observe initial attachment and ARN-509 order organization of biofilms, P. gingivalis cells were anaerobically incubated in pre-reduced PBS without a nutrition source [19]. In order to monitor an increase in biovolume due to cell division as well as exopolysaccharide accumulation, bacterial cells were cultured in TSB medium diluted with PBS (dTSB; TSB/PBS ratio, 1:2) [47]. Table 4 P. gingivalis strains used in this study Strain Genotype Relevant properties Reference 33277 Wild type Wild type

ATCC KDP150 fimA::erm Long fimbria (FimA)- deficient [20] MPG67 mfa1::erm Short fimbria (Mfa1)- deficient [18] MPG4167 fimA::erm mfa1::tetQ Long and short

fimbria-deficient [18] KDP129 kgp::cat Kgp-null [20] KDP133 rgpA::tetQ rgpB::erm Rgp-null [20] KDP136 rgpA::erm rgpB::tetQ kgp::cat Rgp/Kgp-null [20] Autoaggregation assay An autoaggregation assay was essentially performed as described previously [48]. Briefly, 1 ml of P. gingivalis suspension (4 × 108 cells) was transferred into a UV-cuvette then incubated at 37°C with stirring. Autoaggregation was monitored by measuring the decrease in optical density at A 550 (OD550) using a UV-visible recording Benzatropine spectrophotometer (UV-265FW; Shimadzu Co. Kyoto, Japan). During the incubation, dA/dt was continuously calculated and recorded by subtraction of At, the absorbance at time t min, from At+, at time (t + 1) min. The maximum value of – dA/dt in this curve was used as the autoaggregation activity [48]. The data LY2874455 concentration represent the mean ± standard error of three separate experiments with each strain in duplicate. Saliva Saliva stimulated by mastication of paraffin balls was collected in a sterile centrifuge tube on ice from healthy donors and pooled, as described previously [49]. Dithiothreitol (Sigma-Aldrich, St. Louis, MO) was added to a 2.5 mM final concentration, then the saliva was gently stirred on ice for 10 minutes and centrifuged at 3,000 × g for 20 minutes at 4°C.