喜温硫杆菌硫代谢双组份调节系统基础研究

Acidithiobacillus caldus plays important role in bioleaching, biodesulfuring,and treatment of heavy metal pollutions. Sulfur metabolism of it is sophisticated and is important for the above applications. We have built the picture of the sulfur metabolism of the strain, and found the two-component signal transduction system (TCSTS) controlling the two SOX systems, which play central roles in sulfur metabolism. In this research, the TCSTC will be studied for the compositions, signals,and the regulation dynamics. The gene knockout and luciferase reporter gene will be used to construct the engineered strain, with the TCSTS being on-line controllable and on-line monitorable. An experimental system for on-line controling the input signals and monitoring the outputs of the gene expressions will be built, and be used in investigation of the relationships between the signal inputs and system outputs (gene expression shown by the fluorescence). The mathematicals models will be built and used in prediction of the gene expressions controlled by the TCSTS. The relationships between the TCSTS controlled SOX systems and the other sulfur oxidation systems will also be investigated. The sulfur metabolism will be controlled by controlling the gene expressions of the sox gene clusters. This research is noval and interdisciplinary.

极端嗜酸性喜温硫杆菌是在生物冶金、生物脱硫和重金属污染治理等方面有重要应用。研究其硫氧化系统提高其硫氧化能力对上述应用至关重要。该菌具有多个硫氧化系统，极为复杂。我们前期构建了该菌硫氧化模式图，发现SOX硫氧化系统在该菌中发挥重要作用，并发现一个双组分信号传导系统控制着两个sox基因簇。本项目将以该双组分系统为研究中心，研究其组成、信号源、调控特性，及其在硫代谢中的作用。本项目将使用基因敲除、荧光素酶报告基因融合表达或基因替换等方法，构建双组分调控效果可被在线监测的工程菌株，然后建立双组分环境信号可控、报告基因可在线监测的实验系统，动态研究信号输入与被控基因表达之间关系，建立系统数学模型，并用于被控基因表达控制。本项目还将研究双组分控制的SOX硫氧化系统与其它硫氧化系统的关系，通过对sox基因簇的控制达到对整个硫代谢途径控制或干预的目的。技术创新与多学科交叉是本项目特点。

本课主要研究了嗜酸性喜温硫杆菌A. caldus中与硫代谢相关的两套双组分调控系统。通过体内体外实验证实了Sox I的转录调控为σ54依赖的双组分调控系统TspS-TspR介导的转录起始机制，验证了该调控系统所需的关键元件组成。通过基因无痕敲除技术成功构建了调控S4I途径的双组分调控系统RsrS-RsrR的敲除菌株；通过体内和体外的实验验证了RsrS-RsrR双组分调控系统对S4I途径的正向调控功能；提出了RsrS-RsrR调控S4I途径的基本模型。提出了A. caldus中调控途径之间存在cross-talk。建立了以eGFP报告基因为基础的荧光检测技术；建立了A. caldus中调控研究完善的技术平台，包括胞内胞外核酸蛋白相互作用的关键技术等；开启了A. caldus中调控研究的新思路和新方向。利用硫代谢双组分调控系统感应外界含硫化合物的特性，利用分子生物学技术将荧光蛋白与双组分调控系统组合起来，利用荧光光电检测系统构建感应环境含硫化合物的荧光监测装置，用于对环境的探测。