恶臭假单胞菌B6-2中联苯降解相关外排泵基因簇ttgABC和ttgGHI的生理功能与调控

Efflux pumps are membrane proteins that can mediate the efflux of intracellular substances. They widely exist in almost all organisms, and are very important for controlling the mass balance between the inside and outside of a cell. They are considered playing crucial roles in pathogenesis, multi-drug resistance, microbial environmental adaptability, quorum sensing, and other important physiological processes. Researches that focus on the physiological functions and regulation mechanisms of efflux pumps are important for elucidating the interactions between microorganisms and their environments. These researches are of potentially important scientific values, because they can be used in the development of effective bioremediation strains and industrial strains, as well as in the development of drug targets. Pseudomonas putida B6-2 (DSM28064) is a typical PAHs degrader, which has been reported to be excellent in term of performance degradation efficiency, substrate spectrum, and robustness. It is found that the strain B6-2 contains gene clusters ttgABC and ttgGHI that are related to efflux pumps based on our previous research. It is also found that the expression of the 2 pumps is affected by the degradation of PAHs, and there is cross talk of the 2 clusters in regulation level. In this proposal, systematically investigation will be conducted on the efflux pump genes and regulators in the 2 clusters. Genetic engineering methods, EMSA, and instruments for investigating macromolecular interactions (such as ITC, and BLI), will be used in this proposal. It is expected that the researches of this proposal can elucidate the roles of the efflux pumps in the degradation of PAHs, and clarify the cross talk mechanism of the efflux pumps.

外排泵介导细胞内物质的泵出，几乎存在于一切生物体中，是细胞控制内外物质平衡的关键元件，与微生物致病性、多药抗性、环境适应性、群体感应等重要生理过程密切相关。外排泵多样的生理功能和调控机制的研究，对研究微生物与环境的互作具有重要科学价值，在开发高效抗逆菌株以及新的药物靶点等方面具有潜在的应用价值。恶臭假单胞菌B6-2（DSM28064）是典型的环境有机污染物降解菌，具有优良的PAHs降解和抗逆特性。前期研究发现该菌的外排泵基因簇ttgABC和ttgGHI与联苯降解效率以及鲁棒性有关，并且它们的表达有交互作用(Cross talk)。本课题拟针对这两个外排泵基因簇，使用同位素标记、基因敲除回补、DNase I足迹、凝胶阻滞分析（EMSA）、等温滴定微量热（ITC）、生物膜层反射光干涉技术（BLI）等研究手段，系统深入地开展研究，从而揭示它们在该菌降解生理条件下的独特功能，阐明其交互作用机制。

在许多降解菌的基因组中常常含有外排泵基因。但是，它们在降解过程中的作用尚未明确。恶臭假单胞菌B6-2（Pseudomonas putida B6-2）是课题组前期筛选到的一株高效的多环芳烃降解菌，测序发现其基因组中有两个RND（Resistance-Nodulation-Division）型外排泵基因簇ttgABC和ttgGHI。本课题项目按照任务书要求研究了TtgABC和TtgGHI两个外排泵在PAHs降解菌总的生理功能和调控分子机制。研究发现TtgABC主要和抗生素的抗性相关，TtgGHI和菌株的有机化合物耐受相关能够接触PAHs降解时产生的氧化压力和代谢毒性。在调控机制方面，发现TtgABC和TtgGHI具有交叉调控的现象，并且鉴定了这种交叉调控主要由TtgW所介导，TtgW的表达纯化和表征的结果表明这个蛋白是一个特化的调控蛋白。本研究的相关结果已经发表SCI论文7篇，包括Appl Environ Microbiol、ACS Synth Biol、J Hazard Mater、Angew Chem Int Ed等。本项目的研究成果丰富了人们对于外排泵的生理功能的认知，丰富了人们对微生物基因表达调控的认识，为提高菌株的抗逆性提供了元件，对于细菌耐药的传播和转移也提供了新的视角。