Supplementary Material for: The Regulator Gene <b><i>rnc</i></b> Is Closely Involved in Biofilm Formation in <b><i>Streptococcus mutans</i></b>

<i>Streptococcus mutans</i> is an important factor in the etiology and pathogenesis of dental caries, largely owing to its ability to form a stable biofilm. Previous animal studies have indicated that <i>rnc</i> could decrease the amount of sulcal caries, and that the downregulation of cariogenicity might be due to its capacity to disrupt biofilm formation. However, the biofunctions by which <i>rnc</i> is involved in biofilm formation remain to be elucidated. In this study, we further investigate the role of <i>rnc</i> based on the study of mature biofilm. Scanning electron microscopy and the crystal violet assay were used to detect the biofilm forming ability. The production and distribution of exopolysaccharides within biofilm was analyzed by exopolysaccharide staining. Gel permeation chromatography was used to perform molecular weight assessment. Its adhesion force was measured by atomic force microscopy. The expression of biofilm formation-associated genes was analyzed at the mRNA level by qPCR. Here, we found that <i>rnc</i> could occur and function in biofilm formation by assembling well-structured, exopolysaccharide-encased, stable biofilms in <i>S. mutans</i>. The weakened biofilm forming ability of <i>rnc</i>-deficient strains was associated with the reduction of exopolysaccharide production and bacterial adhesion. Over all, these data illustrate an interesting situation in which an unappreciated regulatory gene acquired for virulence, <i>rnc</i>, most likely has been coopted as a potential regulator of biofilm formation in <i>S. mutans</i>. Further characterization of <i>rnc</i> may lead to the identification of a possible pathogenic biofilm-specific treatment for dental caries.

变形链球菌（Streptococcus mutans）是龋病病因学与发病机制中的关键致病因子，这主要归因于其形成稳定生物膜（biofilm）的能力。既往动物实验显示，rnc可降低窝沟龋的发生量，其致龋性下调可能源于其干扰生物膜形成的功能，但rnc参与生物膜形成的具体生物学机制仍有待阐明。本研究以成熟生物膜为研究基础，进一步探究了rnc的生物学作用：采用扫描电子显微镜与结晶紫染色试验检测菌株的生物膜形成能力，通过胞外多糖（exopolysaccharides）染色分析生物膜内胞外多糖的产生与分布特征，利用凝胶渗透色谱法开展分子量测定，采用原子力显微镜测定菌体粘附力，通过实时定量聚合酶链反应（qPCR）在信使RNA（mRNA）水平分析生物膜形成相关基因的表达情况。本研究发现，在变形链球菌中，rnc可通过组装结构完整、被胞外多糖包裹的稳定生物膜参与生物膜的形成与功能发挥；rnc缺陷菌株的生物膜形成能力减弱，这与其胞外多糖产量降低以及细菌粘附力下降密切相关。综上，本研究结果揭示了一个有趣的现象：原本作为毒力相关基因被发现的rnc，很可能已被招募作为变形链球菌生物膜形成的潜在调控因子，对rnc的进一步功能表征有望为龋病开发出靶向致病性生物膜的特异性治疗手段。