喜温硫杆菌（Acidithiobacillus caldus）胞内硫代谢基础研究

Acidithiobacillus caldus plays a key role in bioleaching. Study of its sulfur metabolic system is important in order to increase its sulfur metabolic speed for above applications. A. caldus has multiple and sophisticated sulfur metabolic systems. We have built the model of the sulfur metabolism of the strain, focusing on the sulfur metabolism in the periplasmic space. In this research, the intracellular sulfur metabolic system will be thoroughly investigated. A gene cluster containing the genes of dsrE/tusA/hdr has been found in A. caldus. A similar gene cluster has been found playing the roles of sulfur tracfficking and oxidation in tetrathionate and thiosulfate metabolism. As the result, we expect there is a new sulfur metabolic pathway for tetrathionate and thiosulfate oxidation in A. caldus. Further more, according to the theory of evolution a sulfur trafficking and oxidation pathway for intracellular sulfite oxidation is expected existed in A. caldus. In this research, sulfur isotope labeling, sulfur transfer/trafficking protein identification, sulfur intermetabolite analysis, sulfur metabolic gene knockout or overexpression, transcriptome analysis, metabolic regulation analysis will be done in order to prove our hypothesis that intracellular sulfur is in protein binding form in catabolism in A. caldus. This research will explore the mechanism of intracellular sulfur metabolism of A. caldus, and will build an improved and more perfect sulfur metabolic model for this strain, which will be benefit both for knowledge development and for guiding practice.

极端嗜酸性喜温硫杆菌是生物冶金关键菌种。为提高该菌硫氧化能力，需要研究其硫氧化系统。该菌具有多个硫氧化系统，极为复杂。我们前期着重研究了该菌处于周质空间的SOX硫氧化系统，构建了该菌硫氧化模式图，本项目将研究该菌胞内硫氧化系统。在该菌中发现了含有dsrE/tusA/hdr的基因簇，类似的基因簇被发现具有连四硫酸盐/硫代硫酸盐结合和氧化作用。根据已有知识，我们对喜温硫杆菌胞内蛋白结合的连四硫酸盐/硫代硫酸盐代谢途径进行了新的预测。更进一步，根据进化规律，我们推测喜温硫杆菌胞内亚硫酸盐代谢也存在转运蛋白结合的亚硫酸盐氧化机制。本项目将利用硫同位素标记、硫结合蛋白鉴定、硫代谢中间物分析、硫代谢基因敲除与过表达、转录组分析、调控机制分析等方法，证明我们的推测即胞内硫以蛋白结合的形式进行代谢，揭示喜温硫杆菌胞内硫代谢机制，构建更完善的硫代谢模型，为相关理论发展和实践提供指导。

本项目以温泉分离的嗜酸性喜温硫杆菌（Acidithiobacillus caldus MTH, A. caldus MTH）为模式菌株，聚焦于其细胞质内的硫代谢途径研究。该菌严格好养的自养菌，具有复杂的硫代谢网络。本项目首先利用生物信息学方法分析了 A. caldus MTH中的胞内硫代谢相关基因，发现了一个胞内硫代谢基因簇——dsrE2A-tusA-rhd-hdr，并分析了这些基因的基本特征、保守位点和进化关系。关键位点突变实验确定了两个重要小分子硫载体DsrE2A和TusA的硫结合位点。利用基因无痕敲除技术和体内过表达实验，并结合生长特征分析、代谢物检测实验和转录组学等方法，深入研究了DsrE系统在A. caldus MTH在联四硫酸盐代谢中的功能以及胞内硫代谢网络调节的动态规律。此外，利用体内过表达实验初步探索了A. caldus MTH中亚硫酸的代谢。综合而言，本项目的研究成果补充完善了 A. caldus MTH的硫氧化代谢模型，为该类嗜酸自养菌的硫代谢提供了重要的理论数据，并为其在工业上的应用奠定了重要的理论基础。