唾液链球菌 VicR 活性受反义 RNA 调控。
Activity of Streptococcus mutans VicR Is Modulated by Antisense RNA.
机构信息
1 State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, Department of Preventive Dentistry, West China Hospital of Stomatology, Sichuan University, Chengdu, China.
2 The Forsyth Institute, Cambridge, MA, USA.
出版信息
J Dent Res. 2018 Dec;97(13):1477-1484. doi: 10.1177/0022034518781765. Epub 2018 Jul 3.
The VicRK 2-component system of Streptococcus mutans regulates genes associated with cell wall biogenesis and biofilm formation. A putative RNase III-encoding gene ( rnc) is located downstream from the vicRKX operon. The goals of this study were to investigate the potential role of VicR in the regulation of adjacent downstream genes and evaluate transcription levels of vicR during planktonic and biofilm growth. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate whether vicRKX and adjacent downstream genes were cotranscribed. Binding of purified recombinant VicR protein to promoter regions of vicR, rnc, and syfA genes was confirmed by electrophoretic mobility shift assay and by chromatin immunoprecipitation analyses. VicR antisense (AS vicR) RNA was detected by Northern blotting and qRT-PCR assays. AS vicR overexpression mutants were constructed, and the biofilm biomass was determined by crystal violet microtiter assay. Adjacent downstream genes rnc, smc, syfA, smu.1511, and syfB were cotranscribed with vicRKX. The predicted promoter regions of vicR, rnc, and syfA genes were directly regulated by VicR. An AS vicR RNA transcript was detected upstream of the rnc gene. Expression of the AS vicR RNA transcript was elevated in planktonic cultures and repressed during biofilm growth. In addition, Western blot data showed that expression of the VicR protein decreased by 35% in planktonic as compared with biofilm cultures. Furthermore, we show that overexpression of AS vicR led to a reduction in biofilm formation. The downstream genes rnc, smc, syfA, smu.1511, and syfB are cotranscribed with vicRKX. VicR is autophosphorylated, and rnc and syfA are directly regulated by VicR. Expression of VicR protein correlated inversely with different levels of AS vicR RNA transcript and growth conditions. The biofilm biomass decreased in the AS vicR overexpression mutant. These data suggest a role for the AS vicR RNA transcript in posttranscriptional regulation of VicR protein production in S. mutans.
变形链球菌 VicRK 二组分系统调节与细胞壁生物发生和生物膜形成相关的基因。一个假定的 RNase III 编码基因（rnc）位于 vicRKX 操纵子的下游。本研究的目的是研究 VicR 在调节相邻下游基因中的潜在作用，并评估浮游和生物膜生长过程中 vicR 的转录水平。定量实时聚合酶链反应（qRT-PCR）用于研究 vicRKX 和相邻下游基因是否共转录。电泳迁移率变动分析和染色质免疫沉淀分析证实了纯化重组 VicR 蛋白与 vicR、rnc 和 syfA 基因启动子区域的结合。通过 Northern 印迹和 qRT-PCR 分析检测到 VicR 反义（AS vicR）RNA。构建了 AS vicR 过表达突变体，并通过结晶紫微量滴定法测定生物膜生物量。rnc、smc、syfA、smu.1511 和 syfB 等相邻下游基因与 vicRKX 共转录。vicR、rnc 和 syfA 基因的预测启动子区域直接受 VicR 调控。在 rnc 基因上游检测到 AS vicR RNA 转录本。在浮游培养物中检测到 AS vicR RNA 转录本的表达升高，而在生物膜生长过程中则受到抑制。此外，Western blot 数据表明，与生物膜培养物相比，浮游培养物中 VicR 蛋白的表达降低了 35%。此外，我们还表明，AS vicR 的过表达导致生物膜形成减少。rnc、smc、syfA、smu.1511 和 syfB 等下游基因与 vicRKX 共转录。VicR 是自身磷酸化的，rnc 和 syfA 直接受 VicR 调节。VicR 蛋白的表达与不同水平的 AS vicR RNA 转录本和生长条件呈负相关。AS vicR 过表达突变体中的生物膜生物量减少。这些数据表明，AS vicR RNA 转录本在变形链球菌中 VicR 蛋白产生的转录后调控中起作用。
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