Age of the exopolysaccharide of sinR was significantly CPI-203 site larger than those of the other two strains (Figures 2D and S1D). Furthermore, exopolysaccharide production was normalized to the levels of cells in the biofilms and expressed as the exopolysaccharide per cell ratio. The ratio of the exopolysaccharide per cell of sinR was significantly higher than those of the other strains (RO5190591 biological activity Figure 2E).Results Quantitative analysis of protein and carbohydrates in biofilm of the sinR mutantWe measured the amounts of protein and carbohydrate in biofilms on saliva-coated coverglasses using BCA protein assay kit and the phenol-sulfuric acid method. There was no significant difference in the amounts of protein per colony formation unit (CFU) between wild type, sinR mutant strain (sinR), and sinR+complemented strain (sinR-C) (Figure 1A). In contrast, the biofilm formed by sinR contained significantly larger amounts of carbohydrate per CFU than biofilms formed by the wild type and sinR+-C (Figure 1B).Scanning electron microscopy (SEM) of the biofilm produced by the sinR mutantWe examined the surface structure of the sinR biofilm on the saliva-coated coverglasses using SEM. The EPS-like structure of wild type and sinR-C strain biofilms exhibited a flattened shape (Figure 3A and 3C) in contrast to that of sinR, which was mesh-like (Figure 3B).Physical strength of biofilm of sinR mutantTo analyze the influence of the mutation of the sinR gene on the stability of biofilms, we compared the mutant’s ability to resist brief ultrasonication and found that it was significantly higher resistant to sonic disruption than the other two strains (Figure 4).DiscussionMicroorganisms synthesize the EPS present in their biofilms [10]. The EPS that protects organisms against biocides, and host immune defenses is widely recognized as one of the main reasons that biofilms cause a number of problems, such as intractability of infection and failure of treatment [11]. In B. subtilis, sinR controls the biosynthesis of the EPS [13]. Furthermore, P. gingivalis possesses PGN_0088 as one of the orthologs of sinR of B. subtilis. In our present study, we muted PGN_0088 (sinR) and investigated the role of this gene in the formation of biofilms formed by P. gingivalis strain ATCC 33277. The amount of carbohydrate in P. gingivalis biofilms was reduced by the expression of SinR (Figures 1 and 2). Furthermore, the mature biofilm of sinR mutant formed by using the flow-cell model described in our previous publication [16] contained significantly more carbohydrate than that of wild type. In B. subtilis, SinR acts on the epsA operon as a transcriptional regulator and depresses the biosynthesis of exopolysaccharide in biofilms [17]. P. gingivalis has at least three sugar macromolecules on its surface as follows: lipopolysaccharide (LPS), anionic cell surface polysaccharide (APS), and capsular polysaccharide (CPS). APS functions to anchor arginine-specific gingipain A (RgpA) on the bacterial outer membrane and is distinct from LPS and CPS [18,19]. Acting as a transcription factor, SinR could participate in the regulation of the expression of some of these polysaccharides. In B. subtitlis SinR also controls the yqxM-sipW-tasA operon whose products participate in the biosynthesis of a secreted protein, TasA [13]. In the present study, the SinR of P. gingivalis decreased overall levels of carbohydrate but not that of proteins (Figure 1). An important group of biofilm matrix-associatedFigure 1. Quanti.Age of the exopolysaccharide of sinR was significantly larger than those of the other two strains (Figures 2D and S1D). Furthermore, exopolysaccharide production was normalized to the levels of cells in the biofilms and expressed as the exopolysaccharide per cell ratio. The ratio of the exopolysaccharide per cell of sinR was significantly higher than those of the other strains (Figure 2E).Results Quantitative analysis of protein and carbohydrates in biofilm of the sinR mutantWe measured the amounts of protein and carbohydrate in biofilms on saliva-coated coverglasses using BCA protein assay kit and the phenol-sulfuric acid method. There was no significant difference in the amounts of protein per colony formation unit (CFU) between wild type, sinR mutant strain (sinR), and sinR+complemented strain (sinR-C) (Figure 1A). In contrast, the biofilm formed by sinR contained significantly larger amounts of carbohydrate per CFU than biofilms formed by the wild type and sinR+-C (Figure 1B).Scanning electron microscopy (SEM) of the biofilm produced by the sinR mutantWe examined the surface structure of the sinR biofilm on the saliva-coated coverglasses using SEM. The EPS-like structure of wild type and sinR-C strain biofilms exhibited a flattened shape (Figure 3A and 3C) in contrast to that of sinR, which was mesh-like (Figure 3B).Physical strength of biofilm of sinR mutantTo analyze the influence of the mutation of the sinR gene on the stability of biofilms, we compared the mutant’s ability to resist brief ultrasonication and found that it was significantly higher resistant to sonic disruption than the other two strains (Figure 4).DiscussionMicroorganisms synthesize the EPS present in their biofilms [10]. The EPS that protects organisms against biocides, and host immune defenses is widely recognized as one of the main reasons that biofilms cause a number of problems, such as intractability of infection and failure of treatment [11]. In B. subtilis, sinR controls the biosynthesis of the EPS [13]. Furthermore, P. gingivalis possesses PGN_0088 as one of the orthologs of sinR of B. subtilis. In our present study, we muted PGN_0088 (sinR) and investigated the role of this gene in the formation of biofilms formed by P. gingivalis strain ATCC 33277. The amount of carbohydrate in P. gingivalis biofilms was reduced by the expression of SinR (Figures 1 and 2). Furthermore, the mature biofilm of sinR mutant formed by using the flow-cell model described in our previous publication [16] contained significantly more carbohydrate than that of wild type. In B. subtilis, SinR acts on the epsA operon as a transcriptional regulator and depresses the biosynthesis of exopolysaccharide in biofilms [17]. P. gingivalis has at least three sugar macromolecules on its surface as follows: lipopolysaccharide (LPS), anionic cell surface polysaccharide (APS), and capsular polysaccharide (CPS). APS functions to anchor arginine-specific gingipain A (RgpA) on the bacterial outer membrane and is distinct from LPS and CPS [18,19]. Acting as a transcription factor, SinR could participate in the regulation of the expression of some of these polysaccharides. In B. subtitlis SinR also controls the yqxM-sipW-tasA operon whose products participate in the biosynthesis of a secreted protein, TasA [13]. In the present study, the SinR of P. gingivalis decreased overall levels of carbohydrate but not that of proteins (Figure 1). An important group of biofilm matrix-associatedFigure 1. Quanti.
