The sinR Ortholog PGN_0088 Encodes a Transcriptional Regulator That Inhibits Polysaccharide Synthesis in Porphyromonas gingivalis ATCC 33277 Biofilms

Biofilm-forming cells are distinct from well characterized planktonic cells and aggregate in the extracellular matrix, the so-called extracellular polymeric substances (EPS). The sinR gene of Bacillus subtilis encodes a transcriptional regulator that is known to be involved in the biosynthesis of EPS in biofilms. Porphyromonas gingivalis inhabits the subgingival and extraradicular biofilm of humans and is one of the primary pathogens that cause progressive marginal and refractory apical periodontitis. Furthermore, P. gingivalis possesses PGN_0088, which encodes a putative ortholog of B. subtilis sinR. Here, we investigated the role of PGN_0088 (sinR) on biofilm formation. P. gingivalis strains formed biofilms on saliva-coated glass surfaces in phosphate buffered saline. Quantitative analysis indicated that the biofilm of the sinR null mutant consisted of dense exopolysaccharide. Microscopic observations showed that the increased levels of exopolysaccharide produced by the sinR mutant changed the morphology of the EPS to a mesh-liked structure. Furthermore, physical analyses suggested that the enrichment of exopolysaccharide in the EPS enhanced the resistance of the biofilm to hydrodynamic shear force. The results presented here demonstrate sinR plays important roles in the ability of P. gingivalis strain ATCC 33277 to act as a negative mediator of exopolysaccharide accumulation and is indirectly associated with the structure of the EPS and the force of its adhesion to surfaces.


Introduction
Bacteria adhere widely to surfaces of diverse composition in the environment. These biofilms cause problems in a number of activities, such as agriculture, industry, and healthcare [1]. In the dental field, oral biofilms are defined to consist of multiple bacterial species and to cause opportunistic infection, resulting in dental caries and periodontal disease [2,3]. Porphyromonas gingivalis, a Gram-negative oral anaerobe, is distributed throughout subgingival and extraradicular biofilms and is one of the major pathogens in severe forms of marginal periodontitis and refractory periapical periodontitis [4,5]. Subgingival biofilms localize to the gingival sulcus, pathologically called the periodontal pocket, with thicknesses that range from tens to hundreds of microns [6]. An extraradicular area is located outside the apex field of a root canal over the apical foramen, and biofilms of 30 to 40 mm thickness are known to occupy the extraradicular area of patients with refractory periapical periodontitis [7].
Generally, the properties of bacteria in biofilms are markedly different from those in their planktonic state [8]. These changes occur in response to a variety of environmental signals and are reflected in the new phenotypic characteristics of biofilm-forming cells [9]. In the biofilm, cells aggregate in the EPS that they generate [10]. In most microorganisms, the EPS occupies more than 90% of the dry mass, forms the scaffold for the 3-dimensional (3D) architecture of the biofilm, and is responsible for adhesion to surfaces and for cohesion in the biofilm [11]. The EPS protects organisms against desiccation, oxidizing or charged biocides, some antibiotics and metallic cations, ultraviolet radiation, many protozoan grazers, and host immune defenses [11]. Therefore, deciphering the mechanism of matrix production might be lead to the development of a novel method for controlling the formation of biofilms.
In biofilms harboring the spore-forming bacterium Bacillus subtilis, the EPS consists of an exopolysaccharide, which is specified by the epsA-O operon and a secreted protein TasA, which is encoded by the yqxM-sipW-tasA operon [12]. The epsA-O and yqxM-sipW-tasA operons are controlled by the repressor protein SinR [13][14][15]. In contrast, using microarray analysis, we revealed that the number of genes differentially regulated by more than 1.5fold was highest at the later stage of biofilm formation by P. gingivalis strain ATCC 33277 (312/2,090 genes) [16]. Among them, PGN_0088 (one of the orthologs of sinR) was the most highly down-regulated (3.59-fold) gene. PGN_0088 and it's ortholog of B. subtilis, sinR, are 41.4% identical (http://www.kegg. jp/ssdb-bin/ssdb_best?org_gene = pgn:PGN_0088).
PGN_0088 (sinR) is listed in GenBank as a putative transcriptional regulator (http://www.ncbi.nlm.nih.gov/gene/6330436) and its actual biological function remains to be defined. Here, we demonstrated that the sinR ortholog PGN_0088 inhibits polysaccharide synthesis and infects the structure of EPS in P. gingivalis ATCC 33277 biofilms. We further showed that the mutation of sinR induced resistance to physical disruption, owing to the high exopolysaccharide per cell ratio.

Quantitative analysis of protein and carbohydrates in biofilm of the sinR mutant
We 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).

Confocal laser scanning microscope (CLSM) observation of biofilms formed by the sinR mutant
We evaluated the structure of the sinR mutant biofilm on the saliva-coated coverglasses using CLSM. The image of 49,6diamino-2-phenylindole (DAPI)-labeled cells of sinR could not be distinguished from wild type or sinR-C (Figure 2A). In contrast, only the image of fluorescein isothiocyanate (FITC)-labeled exopolysaccharide of sinR showed a mesh-like structure ( Figure 2B). Quantitation of the images did not detect a significant difference in the cell biovolumes among the examined three strains ( Figure 2C), whereas the biovolume and average substratum coverage 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).

Scanning electron microscopy (SEM) of the biofilm produced by the sinR mutant
We 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 mutant
To 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).

Discussion
Microorganisms 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-O 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-associated proteins are those that polymerize into fibers variously known as pili or fimbriae [20,21]. P. gingivalis produces long (FimA) and short (Mfa) fimbriae [19]. In our previous study, expression of fimbriae-associated genes during the development of biofilms was elevated in the early stage but remained unchanged during the later stages [16]. Furthermore, expression of sinR was down-regulated only in Figure 2. CLSM observation of biofilms formed by P. gingivalis strains. P. gingivalis 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). P. gingivalis cells (A) and exopolysaccharides (B) of biofilms that developed on the coverglasses were observed with a CLSM equipped with a 406 objective. Scale bars represent 50 mm. Optical sections were obtained along the z-axis at 0.7-mm intervals, and images of the x-y and x-z planes were reconstructed with imaging software as described by Kuboniwa et al. [19]. 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. *P,0.001 in comparison with the wild type strain. doi:10.1371/journal.pone.0056017.g002 the late stage of biofilm formation. In the present study our focus was on the transcriptional behavior of sinR, and studies on protein expression will be performed next. Moreover, in our present study, we only measured the total amount of protein. Thus, it is remain unresolved if the SinR protein influences the production of fimbriae. Further work on the influence of SinR on the expression of individual proteins containing fimbriae is necessary to define the targets of its activity.
Our present study demonstrates that SinR has an inhibitory effect on synthesis of exopolysaccharide in P. gingivalis biofilms. Therefore, we also determined the influence of carbohydrate levels on the morphological and physicochemical properties of biofilms formed by P. gingivalis. The EPS of bacterial biofilms comprises exopolysaccharides, proteins, lipids, nucleic acids, lipoteichoic acids, and lipopolysaccharides [11,[22][23][24][25]. The individual com-ponents of the EPS vary dynamically according to local environmental conditions [11,25,26]. Studies of diverse bacterial species have revealed that change in the quantity of any of these components influences the 3D-structure of EPS. For example, biofilms of a fimbriae-deficient strain (flp-1-disrupted mutant) of the periodontal pathogen Aggregatibacter actinomycetemcomitans forms microcolonies in looser formation, and fibrils of fimbriae are not observed [27]. Furthermore, its adhesion to the surface was significantly blocked by sodium metaperiodate or DNase I treatment but not by proteases. This mutant secretes carbohydrates and DNA instead of fimbriae to coalescent on a surface [27]. Friedman and Kolter screened for transposon insertion mutants of Pseudomonas aeruginosa PA14 that were unable to form pellicles that represent one type of biofilm formed at the air-liquid interface in static cultures [28]. They identified 7 flanking genes  (pel) that contribute to the formation of the pellicle, and revealed that the products of these genes are involved in the construction of the EPS [28]. In B. subtilis, the structures of the biofilms formed by the eps (required for production of exopolysaccharide) mutant and tasA (forms amyloid fibers) mutant were flat. In contrast, the biofilms produced by the sinR (inhibitor of eps and tasA) mutant were extremely wrinkly [19,21]. The CLSM ( Figure 2B) and SEM ( Figure 3) images acquired in the present study show that the mutation of sinR induces morphological changes of the EPS from a laminar to a mesh-like structure. Thus, the SinR produced by P. gingivalis ATCC 33277 might be indirectly involved in the 3Dconformation of the EPS in biofilms by controlling the expression of genes associated with the EPS components.
Xylella fastidiosa, a bacterium responsible for Pierce's disease in grapevines, possesses both type I and type IV pili at the same cell pole. De La Fuente et al. [29] evaluated the attachment of the bacteria to a glass substratum using a microfluidic flow chamber in conjunction with pilus-defective mutants. The adhesion force required to disperse X. fastidiosa mutant possessing only type I pili was significantly higher, whereas that of mutant cells possessing only type IV pili was significantly lower than those of wild type cells [29]. In contrast, Kuboniwa et al. [19] revealed that the exopolysaccharide per cell ratio of biofilms formed by a fimA mutant was significantly higher than that of wild type and that the mutant formed a tough and cohesive biofilm. Furthermore, the exopolysaccharide per cell ratio of the biofilm formed by an arginine-specific gingipain A and B (RgpA and RgpB, respectively) double-mutant was significantly smaller than that of wild type, and the biofilm of the mutant was fragile. Hence, in P. gingivalis, the exopolysaccharide per cell ratio might correlate with resistance to physical disruption.
We also show here that the sinR mutant formed carbohydraterich and stout biofilms (Figure 4). The exopolysaccharide of the P. gingivalis biofilm could contribute to the adhesion force to the surface; however, further studies are required to demonstrate that this is the case. Mounting evidence has accumulated over the past 20 years that supports a role for P. gingivalis in periodontal disease and infection and as a potential risk factor for several systemic diseases, including diabetes, preterm birth, heart disease, and atherosclerosis [30][31][32][33][34]. Dispersal of bacteria from the biofilm at the periodontal pocket or extraradicular area facilitates spread throughout the body via the bloodstream and initiates or detrimentally influences these systemic conditions [32]. Many factors trigger bacterial Dispersal from biofilms, including alterations in the availability of nutrients (such as carbon sources), oxygen depletion, low levels of nitric oxide, changes in temperature, and high or low levels of iron [35]. Furthermore, bacteria possess regulatory systems to drive differential gene expression in response to these changing conditions [35]. Acquisition of resistance to physical disruption by deletion of sinR might be associated with tolerance against the dispersal of biofilm. In the future, uncovering the relationship between sinR and biofilm dispersal will lead to the development of a method to block the diffusion of P. gingivalis from its biofilm.

Conclusions
Our investigations here on the role of sinR on the production during the formation of biofilms by P. gingivalis strain ATCC 33277 show that the gene is a negative mediator of exopolysaccharide synthesis and influences with the 3D-structure of the EPS. Furthermore, the biofilm produced by the sinR mutant exhibited resistance to physical disruption, owing to the high exopolysaccharide per cell ratio. These findings suggest that research focused on defining the detailed mechanisms of biofilm formation by P. gingivalis will contribute to the development of more effective methods for preventing pathologies associated with bacterial biofilms.

Construction of bacterial strains and plasmids
A P. gingivalis PGN_0088 (sinR) deletion mutant was constructed according to the method of Yamaguchi et al [37] as follows: sinRupstream and sinR-downstream DNA regions were amplified using the polymerase chain reaction (PCR) of strain ATCC 33277 chromosomal DNA with the primer pair SUF and SUR for the sinR-upstream region and with the primer pair SDF and SDR for the sinR-downstream region. The DNA primers and plasmids used in this study are listed in Table 1. The amplified DNA fragments were digested with KpnI and BamHI to generate the sinR-upstream region and with BamHI and NotI to generate the sinR-downstream region, which was then inserted into KpnI-NotI-digested pBlue-scriptH II SK(2) (Stratagene, La Jolla, CA.) to yield pOD001. The 1.1-kb BamHI-digested ermF DNA cartridge was acquired from pKD355 [38] using BamHI-digestion and inserted into the BamHI site of pOD001, resulting in pOD002 (DsinR::ermF). The BssHIIlinearized pOD002 DNA fragment was introduced into P. gingivalis ATCC 33277 by electroporation ( Figure 5) using a Gene-Pulser Xcell Microbial System (Bio-Rad Laboratories, Richmond, CA.) set at 25 mF, 400 V, and 2.5 kV to yield strain ODP001 (sinR mutant; DsinR::ermF). To construct the sinR + -complementing strain (Figure 6), the 0.7-kb DNA fragment containing the sinR gene region was amplified from ATCC 33277 chromosomal DNA using PCR primed by SCF and SCR. The amplified DNA fragment was cloned into the pGEMH-T Easy vector (Promega, Madison, WI.), resulting in pOD003. The sinR region DNA obtained by NotI and BamHI digestion was inserted into NotI-BamHI-digested pTCB [39] to yield pOD004 (sinR + ). The pOD004 plasmid DNA was introduced into the sinR mutant by conjugation with E. coli S17-1 [40] harboring pOD004 as a donor strain, resulting in strain ODP002 (sinR + -complemented strain; DsinR::ermF/sinR + ). Ampicillin, erythromycin, gentamycin or tetracycline was used to select colonies, which harbored these antibiotic-resistant gene cassettes.

DNA probes and Southern blot hybridization
A DNA fragment (0.8 kb) comprising the sinR gene was amplified using PCR from 33277 chromosomal DNA with the primer pair SSF and SSR. An ermF DNA fragment (0.8 kb) was from pKD355 [38] using PCR with the primer pair EMF and EMR [37]. These fragments were labeled with the AlkPhos Direct system for chemiluminescence (GE Healthcare, Amersham, UK). Southern blotting was performed using a nylon membrane and hybrids were detected using the CDP-star reagent. The results of this analysis confirmed that the ermF DNA cartridge was inserted into the PGN_0088 locus of ODP001.

Analysis of biofilm formation
Quantitative analysis. Biofilms were formed on a chambered coverglass system (Lab-Tek TM Chambered Coverglass; Nalge Nunc International, Rochester, NY.). Human saliva, centrifuged at 2,0006 g for 15 min and then filter-sterilized using a syringe filter with a pore size of 0.22 mm (Millex-GP Filter Unit, SLGP033RB; Millipore, Billerica, MA) [16], was poured on wells of the coverglass and incubated overnight. P. gingivalis cells were washed and suspended in phosphate-buffered saline (PBS) at an optical density at 550 nm (OD 550 ) of 1.0, then cultured in salivacoated wells of the coverglass for 24 h.
For quantitative analysis, biofilms were washed and resuspended in 1 mL PBS. Protein and carbohydrate concentrations of biofilm suspensions were determined using the BCA protein assay kit (Thermo Scientific, Rockford, IL.) according to the manufacturer's protocols and the phenol-sulfuric acid method as described previously [41]. Next, the numbers of colony forming units (CFUs) were calculated by determining the copy numbers of the P. gingivalis ATCC 33277 16S rRNA gene in biofilms essentially according to the method of Kuboniwa et al. [19,42]. The amounts of protein and carbohydrate per cell were calculated by dividing their concentrations by CFU equivalents.
CLSM observation. Biofilms were formed on the chambered coverglass described above. Briefly, P. gingivalis cells were stained with DAPI (50 mg/ml; Molecular Probes, Eugene, OR.), washed, suspended in PBS at an OD 550 of 1.0, and cultured in salivacoated wells of the coverglass for 24 h. The resulting biofilms were washed and the exopolysaccharide was labelled with Concanavalin-A-FITC conjugate and Wheat germ agglutinin-FITC as described previously [19]. After washing, images were obtained using CLSM (LSM-510; Carl Zeiss, München-Hallbergmoos, Germany) with reflected laser light at 405 and 488 nm and then analyzed as described above. Eight images per fields per a sample were acquired. The experiment was independently repeated three times.
SEM observation and quantitative analysis. Biofilms were formed as described above for the quantitative analyses. The resulting biofilms were washed, treated, and observed using SEM as described by Yamaguchi et al. [37] and Asahi et al. [43]. Figure 6. Scheme of construction of the sinR + -complementing strain. The 0.7-kb DNA fragment (aqua) containing the sinR gene region was PCR-amplified from ATCC 33277 chromosomal DNA with SCF and SCR primers. The amplified DNA fragment was cloned into the pGEMH-T Easy vector, resulting in pOD003. The sinR region DNA obtained by NotI and BamHI digestion was inserted into NotI-BamHI-digested pTCB [38] to yield pOD004 (sinR + ). doi:10.1371/journal.pone.0056017.g006 Sonic disruption assay. This assay was performed essentially according to the method of Kuboniwa et al. [19]. Briefly, 24well tissue culture plates (Becton Dickinson Labware, Franklin Lakes, NJ) were coated with human saliva. P. gingivalis cells (1.5610 9 CFU/well) were statically incubated in diluted GAM (dGAM; GAM/PBS ratio, 1:4) for 60 h at 37uC, and the resulting biofilms were sonicated for 1 second at output level 1 (output power, 25 W; oscillating frequency, 28 kHz; tip diameter, 2.5 mm) with a Handy Ultrasonic Disruptor (UR-20P, Tomy Seiko, Tokyo, Japan). Genomic DNA was isolated from biofilms formed by P. gingivalis and the CFU value (see above) was determined using real-time PCR, as described previously [19,42]. The data represent the mean 6 standard error of the mean of three separate experiments performed in duplicate for each strain.

Statistical analysis
The significance of intergroup differences of all data was analyzed using Welch's t tests. A P value,0.001 was considered to indicate statistical significance. Figure S1 Quantification of mean thickness and average substratum coverage from CLSM observation. Fluorescent images of CLSM (Figures 2A and 2B) were quantified using Imaris software and the mean thickness of cells (A) and that of exopolysaccharide (B), and average substratum coverage of cells (C) and that of exopolysaccharide (D) per field were calculated. 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. *P,0.001 in comparison with the wild type strain. (TIF)