SEM observation of biofilms formed by <i>P. gingivalis</i> strains.

<p><i>P. gingivalis</i> wild type (A), <i>sinR</i> mutant (<i>sinR</i>, B) and <i>sinR</i>⁺-complemented (<i>sinR</i>-C, C) strains formed biofilms that developed on the coverglasses were observed with a SEM.</p

Bacterial strains, plasmids, and primers used in this study.

<p>Bacterial strains, plasmids, and primers used in this study.</p

Scheme of construction of the <i>sinR</i> mutants.

<p>To yield strain ODP001 (<i>sinR</i> mutant; Δ<i>sinR::ermF</i>), the linearized DNA fragment including <i>ermF</i> (red) was introduced into chromosomal DNA of <i>P. gingivalis</i> ATCC 33277 by electroporation.</p

Strength of biofilms formed by <i>P. gingivalis</i> strains.

<p>Standardized cultures of <i>P. gingivalis</i> were inoculated into dGAM in saliva-coated 12-well polystyrene plate and incubated statically at 37°C for 60 h, and the resulting biofilms were sonicated for 1 s. Immediately after sonication, supernatants containing floating cells were removed by aspiration and the biofilm remnants were gently washed with PBS. <i>P. gingivalis</i> genomic DNA was isolated from the biofilms, and the numbers of <i>P. gingivalis</i> cells were determined using real-time PCR. Relative amounts of bacterial cell numbers were calculated based on the number of wild type cells without sonication and assigned a value of 1.0. The percentages shown indicate the amount of remaining biofilm after sonic disruption. Duplicate experiments were independently repeated three times with each strain. Standard error bars are shown. Statistical analysis was performed using Welch's <i>t</i> test. *<i>P</i><0.001 in comparison with the wild type strain.</p

CLSM observation of biofilms formed by <i>P. gingivalis</i> strains.

<p><i>P. gingivalis</i> strains were stained with DAPI (blue) and incubated in PBS for 24 h. After washing, exopolysaccharide was stained with FITC-labeled concanavalin A and wheat germ agglutinin (green). <i>P. gingivalis</i> cells (A) and exopolysaccharides (B) of biofilms that developed on the coverglasses were observed with a CLSM equipped with a 40× objective. Scale bars represent 50 µm. Optical sections were obtained along the z-axis at 0.7-µm intervals, and images of the x-y and x-z planes were reconstructed with imaging software as described by Kuboniwa <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056017#pone.0056017-Kuboniwa1" target="_blank">[19]</a>. Fluorescent images were quantified using Imaris software and the average of total cell biovolume per field (C) and that of total exopolysaccharide biovolume per field (D) were calculated. Furthermore, exopolysaccharide levels are expressed as the ratio of exopolysaccharide/cells (FITC/DAPI) fluorescence (E). The experiment was repeated independently three times. Data are presented as average of 8 fields per sample along with the standard errors of the mean. Statistical analysis was performed using a Welch's t test. *<i>P</i><0.001 in comparison with the wild type strain.</p

Quantification of components of biofilms formed by <i>P. gingivalis</i> strains.

<p><i>P. gingivalis</i> strains were incubated in PBS for 24 h. After washing, the amounts of protein (A) and carbohydrate (B) per CFU were determined using the colorimetric methods described in the Methods section. Statistical analysis was performed using a Welch's t test. *<i>P</i><0.001 in comparison with the wild type strain.</p