铜胁迫下丝足虫类原生动物对土壤耐铜微生物种群及抗性调控机理研究

Protozoa are one of the major biotic factors driving the composition and dynamics of bacterial communities in soil. As the abundant free-living soil protozoa, Cercozoa are quantitatively important in soil food webs. During the interaction process, copper-mediated bacterial killing mechanisms in protozoa create a strong selective pressure on acquisition and maintenance of copper resistance determinants in bacteria. However, compared to the pressure from anthropologic copper contamination, the selective impacts of protozoan predation on copper-resistant bacterial communities and their copper resistance determinants in soil are largely unknown, especially the effects at the aggregate level. To investigate the correlation between protozoan grazing and copper-resistant bacterial communities, the DNA- and RNA-based structure of Cercozoa, total bacterial communities and copper-resistant bacterial communities will be compared in the aggregate fractions from agriculture soils exposed to long-term copper contamination gradients. The contributions from copper contamination and protozoan grazing to the occurrence and succession of copper-resistant bacterial communities in soil will be implicated spatially at the aggregate level. Microcosm studies will be carried out to monitor the dynamics of copper-resistant bacterial communities under the influence of predation by the representative Cercozoa. The proposed project will greatly extend our knowledge on the development and evolution of metal resistant bacteria, as well as the widespread presence of metal resistance determinants in pristine environments.

原生动物是调控土壤微生物种群最主要的生物因子，其中丝足虫类属优势类群。重金属介导的原生动物捕食机制是微生物维持/获取铜抗性重要的选择压力，但以原生动物捕食为主的生物因子对耐铜细菌种群及抗铜基因多样性调控的研究尚鲜有报道，在土壤团聚体微尺度环境中如何体现也不清楚。因此，本项目以梯度铜污染农田土壤团聚体为研究对象，通过野外原位试验，在DNA和RNA水平探究铜污染、丝足虫类原生动物多样性、与耐铜细菌群落间的互作关系，揭示丝足虫类原生动物捕食（生物因子）和环境铜污染程度（非生物因子）对耐铜细菌群落演替的相对贡献；同时通过培养试验模拟不同程度铜污染环境，探究在原生动物捕食作用的影响下，耐铜细菌群落抗性发展的动态变化过程。研究从新的视角审视环境中耐铜细菌群落的演替和进化机制，成果将为原生动物与细菌的互作、以及重金属抗性基因的选择机制研究提供重要的理论依据。

原生动物是影响土壤微生物多样性和群落组成的关键驱动力。本项目以梯度铜污染农田土壤团聚体为研究对象，结合野外调查研究和微宇宙培养试验，探究了铜污染胁迫下土壤团聚体中微生物群落的分布特征及驱动因子，揭示了原生动物影响耐铜细菌群落和铜抗性基因的微生态调控机制。研究取得的主要结果如下：1）细菌、耐铜细菌和原生动物群落在团聚体中分布不均，α-多样性和群落组成受团聚体粒径影响显著。总铜、有效铜、土壤pH、C/N是影响团聚体细菌和原生动物群落的主要环境因子，其中铜污染对细菌和原生动物群落的影响随团聚体粒径增大而降低。由于小粒径团聚体中铜敏感细菌和原生动物类群占比较高，导致铜污染对小粒径团聚体微生物的遗留效应更强。2）铜污染改变团聚体粒径效应。铜污染减弱了由团聚体粒径引起的细菌群落间的差异，但不影响吞噬类原生动物群落在团聚体中的分布。铜污染胁迫下团聚体细菌与原生动物群落的α-多样性呈显著正相关，且相关性随团聚体粒径增加而增强；原生动物-细菌共现网络拓扑参数及鲁棒性分析进一步证明了大粒径团聚体中原生动物在维持网络稳定性方面的作用更为重要。3）原生动物捕食促进耐铜细菌群落的富集。细菌和耐铜细菌群落共现网络分析及结构方程模型结果表明，原生动物通过调控铜敏感及抗性细菌生态簇，进而促进细菌抗铜基因的富集；而铜污染主要通过影响基因移动元件调控细菌抗铜基因丰度。微宇宙试验进一步证明铜胁迫条件下原生动物捕食显著促进细菌抗铜基因的富集，为原生动物捕食调控耐铜细菌群落提供了直接证据。综上所述，本项目在土壤微域尺度下深入探究了铜胁迫下生物及非生物因素对耐铜细菌群落的影响及驱动机制，研究结果为深入认识重金属污染胁迫下原生动物介导的土壤食物网结构和功能提供了重要的理论依据。