基于Rhodococcus sp. strain p52二噁英降解质粒接合转移的二噁英污染的基因强化修复机制研究

Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans, generally termed as dioxins, have attracted considerable attention over recent decades because of their persistence, bioaccumulation, toxicity and global dispersion. Removal of dioxins by microorganisms is an important method of remediation. Because dioxins are recalcitrant, bioaugmentation is a preferred strategy during remediation. However bioaugmentation efforts often fail because of a rapid depletion of the introduced bacterial strains, which exhibit low fitness and survival in contaminated environments during competition with indigenous bacteria. An alternative approach is to use genetic bioaugmentation, which introduces bacteria harboring conjugative, catabolic plasmids, which encode enzymes that degrade the target contaminants. In this way, an enhanced degradation potential can be achieved by the plasmid-mediated horizontal gene transfer (HGT) of the corresponding dioxin-catabolic genes to indigenous microorganisms in polluted sites. Therefore genetic bioaugmentation has an advantage over conventional bioaugmentation, which relies on the survival of exogenous microorganisms. By now, there are a few studies on conjugative transfer of dioxin-catabolic plasmids, and even less addressed strategy of genetic bioaugmentation. We previously isolated a dioxin-degrader, Rhodococcus sp. strain p52. The strain has two distinct gene clusters (dfdA and dbfA) involved in initial dihydroxylation of dioxins which located on two mega plasmids, pDF01 and pDF02, respectively. In our previous study, we confirmed conjugative transfer of the dioxin-catabolic plasmids pDF01 and pDF02 harbored by strain p52 to a group of tested recipients. Moreover, we also detected their transfer to activated sludge bacteria by a mating test, which revealed the potential for application of pDF01 and pDF02 for genetic bioaugmentation. The proposed research will focus on the mechanism of genetic bioaugmentation with catabolic plasmids pDF01 and pDF02 harbored by strain p52. We will study the substantial and interrelated effects of biotic and abiotic factors on conjugative transfer, stable maintenance, and catabolic genes expression of pDF01 and pDF02. Efforts will be made to examine the effect of biotic factors, such as the properties of pDF01 and pDF02, the donors effect (of inherent host or derivate transconjugant hosts), and the indigenous bacterial community (composition and structure). Attentions will be paid to influencing abiotic factors such as selection pressure, supplemental carbon source, and environmental conditions (e.g., temperature and moisture in soil). The proposed study will provide insight into genetic bioaugmentation with the catabolic plasmids, and therefore contribute to its application in dioxins remediation.

二噁英是国内外广泛关注的持久性有机污染物，生物修复是去除环境中二噁英污染的重要途径。生物修复过程中，借助于降解质粒的接合转移而使修复系统中土著菌群获得降解能力的基因强化方式具有突出优势。国际上关于二噁英降解质粒接合转移的报道较少，缺乏通过基因强化去除环境中二噁英的研究。本项目拟以红球菌（Rhodococcus sp. strain p52）携带的天然二噁英降解质粒pDF01、pDF02为研究对象，在二噁英污染的基因强化修复过程中，考察生物因素（包括降解质粒的分子遗传学特征、供体菌差异、土著菌群组成与结构等）与非生物因素（包括选择压力、外加有机碳源、物理性环境因子）的作用与影响，分析降解质粒接合转移的机制，研究接合子中降解质粒稳定保持与执行降解功能的机制，获得对基于降解质粒接合转移的二噁英污染的基因强化修复机制的认识。本研究可为通过基因强化修复环境中二噁英污染的实际应用提供理论依据。

生物强化是生物修复二噁英污染的重要手段，基于降解质粒接合转移的基因（生物）强化可克服传统的生物强化的应用劣势。为探明基因强化修复二噁英污染的机制，项目以红球菌p52携带的二噁英降解质粒pDF01、pDF02为研究对象，完成了以下研究：（1）质粒的分子遗传学特征。利用生物信息学手段，分析了质粒复制系统与不相容组；将pDF01和pDF02的可移动性系统归入MOBF2家族；pDF01、pDF02的接合配对形成系统分别属于MPFG和MPFT；质粒的T4CP属于T4CPT类型。（2）质粒与宿主细胞的相互作用。通过竞争实验及生长曲线法证明pDF01产生适合度代价，pDF02产生适合度优势；pDF01与pDF02共存对宿主适合度不产生明确影响。进化实验显示进化后宿主细胞适合度增加，质粒保持能力改善，细胞发生了补偿性适应；转录组及RT-PCR分析表明细胞通过下调部分代谢基因而改善质粒对宿主适合度的不利影响。（3）质粒供体菌与受体菌群的影响。接合子及其质粒再转移的继代接合子中降解质粒具有不同水平的接合转移能力及降解功能。活性污泥菌群中降解质粒的主要宿主菌群属于核心微生物组中的Gamma-Proteobacteria和Actinobacteria，但丰度不占优势。（4）选择压力。不同污染物作为选择压力对质粒接合转移有不同促进作用；高选择压力可提高接合子比率。选择压力使菌群细胞ROS水平上升、胞外LDH水平提高，同时质粒上参与接合转移的基因virB4、virD4、traA表达活性上调，表明选择压力胁迫可促进质粒接合转移。（5）非生物因素的影响。有机碳源（葡萄糖、乙酸钠、正十六烷、蔗糖）、吸附载体可使活性污泥反应器中接合子比率提高。温度及土壤含水率对土壤中质粒接合转移有影响，而纳米材料（如nZVI）有一定促进作用。本研究可丰富对基因强化修复二噁英污染的理论认识，为其实际应用打下基础。