PEP-CTERM蛋白质参与Aquincola tertiaricarbonis RN12 菌胶团形成机制研究

Activated sludge process has been widely used for the treatment of municipal sewage and industrial waste water. The floc-forming bacteria play a key role in this process. Aquincola tertiaricarbonis RN12 is a floc-forming bacterium isolated from the nuisance brown precipitates in a water supply factory located in Xishui County, Hubei Province, China. In this project, we will use the RN12 as a model to explore the mechanism of floc-forming. We will construct in-frame deletion mutant of pepA(RN12) gene in RN12 strain to testify its important role in the floc-forming. We will also use the pepA(RN12) gene to recover the in-frame deletion mutant of the rpoN gene in another floc-forming bacterium Zoogloea resiniphila MMB to explore the common function of pepA gene(s). We will use GC-MS method to investigate that the PepA(RN12) protein just take part in the assemble of exopolysaccharides other than the biosynthesis. Futhermore, NanoLC-MS/MS will be used to demonstrate the relationship of this protein and exopolysaccharides. The location of this protein in the cell will be studied by the western blot analysis of the extractions of the cell components, flow cytometry and immunofluorescence. This project is helpful to reveal the molecular mechanism of microbial community formation in activated sludge process, and to provide theoretical basis for scientific and economic wastewater treatment.

近几十年来，活性污泥法广泛应用于工业废水和城市污水的处理中，菌胶团形成菌在活性污泥法中占有重要位置。本项目以一株从湖北浠水县自来水厂中黑褐色絮状物质中分离的一株形成菌胶团的解叔丁醇水居菌（Aquincola tertiaricarbonis RN12）为模板研究菌胶团形成机理。本项目通过将pepA(RN12)基因进行敲除回补，同时将该菌株与另一株产菌胶团的喜树脂动胶菌（Zoogloea resiniphila MMB）中的rpoN敲除菌株进行交叉回补探究该基因参与菌胶团形成是否具有普遍性；通过乙醇沉淀、GC-MS和NanoLC-MS/MS方法探究 PepA(RN12)蛋白质与多糖的关系；通过组分提取后的western blot检测、流式细胞法和免疫荧光法探究该蛋白质的在细胞中的定位。本项目有助于揭示活性污泥法中微生物形成菌教团的分子机理，为实现废水处理的科学化和经济化提供理论依据。

近年来，我国乃至世界水体污染和自来水水质问题层出不穷，活性污泥法目前已广泛应用于城市污水和工业废水的处理中。活性污泥法的关键即是通过形成的污泥絮体在重力作用下以沉淀的方式实现泥水分离，最终完成水质净化。然而，活性污泥菌胶团形成的分子机理研究还不够。本研究以一株分离到的菌胶团形成菌解叔丁醇水居菌RN12（Aquincola tertiaricarbonis RN12）为研究对象，通过三代基因组测序技术完成RN12菌株的完成图，通过转录组测序发现RpoN因子调控810个基因的转录，其中的pepA（RN12）基因的转录水平受到明显下调，且通过引物延伸实验和实时定量PCR实验得到验证，回补实验表明在RN12菌株中的60多个PEP-CTERM蛋白质中只有PepA（RN12）蛋白质可以使RN12ΔrpoN突变株恢复菌胶团性状；随后，通过乙醇沉淀法结合苯酚硫酸法定量测定结合型胞外多糖和溶解型胞外多糖发现PepA（RN12）蛋白质参与将RN12菌株的胞外多糖结合在细胞上；最后，通过AlphaFold2软件预测的PepA（RN12）蛋白质三维结构模型中该蛋白质成筒状结构，具有典型外膜蛋白的特点，通过免疫荧光法发现该蛋白质定位于细胞表面，通过Western blot和UPLC-MS/MS共同发现该蛋白质存在3个条带，预示其可能存在蛋白质的翻译后修饰或加工过程，还需要进一步研究。本项目有助于揭示活性污泥法中微生物形成菌胶团的分子机理，有助于对活性污泥法的操作过程进行调整和改善，达到改善活性污泥法处理污水废水的效率，为实现废水处理的科学化和经济化提供理论依据。