好氧及兼性厌氧γ-变形菌纲细菌的生物汞甲基化特性和机制

Methyl mercury (MeHg) is a neurotoxin that is more toxic than inorganic mercury. The methylation process in nature is mainly caused by microbial metabolism. At present, numerous studies have been conducted in depth about the anaerobic mercury methylation of Sulfate-Reducing Bacteria (SRB), and have clearly pointed out the methylation mechanism in genomics and proteomics (such as hgcA/B gene cluster and the folate branched metabolic pathway of acetyl Co-A). Supported by the National Natural Science Foundation of China, a number of aerobic and/or facultative anaerobic mercuric methylation bacteria were isolated from the hydro-fluctuation belt soil of the Three Gorges Reservoir. Among them, 5 strains were identified as gamma Proteobacteria. However, the hgcA/B gene cluster in these 5 strains could not be found by analyzing their full genomic sequence, and what caused their mercuric methylation is unclear. It is worthy to make further studies to investigate bio-methylation characteristics and mechanism of aerobic and/or facultative anaerobic γ-Proteobacteria. Thus, this project will be carried out by applying the coupling techniques of double stable isotopes labeling and Multi-Omics (i.e genome, transcriptome and proteomics) to find out the difference of gene expression and the key functional protein/enzyme in the mercuric bio-methylation of these special functional bacterial strains under the aerobic and/or facultative anaerobic conditions. The results will elucidate the biological mechanisms and environmental response of mercuric bio-methylation in aerobic and/or facultative anaerobic microorganisms. It could provide a further clarifying the release characteristics and environmental effects of mercuric bio-methylation, and a further theoretical basis on the biogeochemical cycle of mercury.

甲基汞(MeHg)是一种神经毒素，毒性较无机汞更强。自然界中无机汞的甲基化过程主要由微生物代谢完成。目前，国内外学者对δ-变形菌纲中硫酸盐还原菌的厌氧汞甲基化研究较为深入，明确了其hgcA/B基因簇、乙酰辅酶A叶酸分支途径等汞甲基化机制。在国家自然基金资助下，本项目组从三峡库区消落带土壤中，分离获得数株具好氧/兼性厌氧汞甲基化能力的菌株。经鉴定其中5株为γ-变形菌纲细菌，通过基因组全序列分析，未发现有hgcA/B基因存在，其汞甲基化机制何在？值得深究。为此，本项目拟从这5株细菌的汞生物甲基化特性研究出发，应用稳定同位素标记技术、微生物基因组学、转录组学、蛋白质组学等研究技术相结合，以期发现汞甲基化基因表达差异，进而找出关键的功能基因或蛋白质(酶)，阐明好氧/兼性厌氧微生物甲基汞产生的生物学机制和环境响应规律。为进一步明确自然界甲基汞的产生及环境效应，深入研究汞生物地球化学循环提供理论依据。

汞生物甲基化过程及其产生的环境问题，是汞环境生物地球化学循环的热点研究领域，但已有研究尚有一定局限性：（1）试验在高汞浓度条件下进行，这与自然条件不符；（2）许多研究围绕δ-变形菌纲厌氧菌展开，如硫酸盐还原菌等；（3）将汞生物甲基化与去甲基化作用割裂开来, 往往只关注甲基汞净增量，而忽略了生物汞甲基化和去甲基化作用的整体动态响应。本研究重点在前期项目基础上，即从三峡库区消落带土壤中分离筛选获得的γ-变形杆菌为供试材料，以硫酸盐还原菌为对照，开展了好氧/兼性好氧细菌汞生物甲基化与去甲基化作用特性探究试验，主要试验结果如下：.（1）.完全厌氧条件下：SRB1、SRB2及TGRB3均能够良好生长；单位细菌最大汞甲基化量和最大甲基化率均表现为：SRB1 > SRB2 > B3，而汞甲基化速率（km）则表现为：SRB1 > B3 > SRB2；完全好氧条件下：SRB1、SRB2 完全无法生存，更无法进行汞生物甲基化、去甲基化等功能；而各好氧菌株均表现出一定甲基化能力：最大汞甲基化量表现为：TGRB2 ≥ TGRB3 > TGRB4，而km表现为：TGRB2 ≈ TGRB3 > TGRB4。试验结果表明，所有供试γ-变形杆菌可能存在维持菌体汞和甲基汞动态平衡的吸收转化机制；.（2）.以TGRB5为供试菌，在不同汞浓度条件下进行比较蛋白组分析，共鉴定出1302个蛋白质，通过生信分析，筛选出96个存在显著差异的蛋白质，其中的硫解酶、乙酰转移酶、孔蛋白、2Fe-2S相关蛋白、琥珀酰相关蛋白等重要蛋白质；.（3）.采用基于质粒pK18mob SacB的同源重组技术，对菌株TGRB4进行SH417目标基因敲除，初步探究了野生型γ-变形杆菌目标基因敲除试验条件；.（4）.检测了菌株TGRB3在不同汞环境中的转录组变化，发现汞应激响应及转运相关功能基因发生了上调，如过氧化氢酶（KatG）、金属金属转运体的钠汞转运蛋白(YfeH)等。.本试验结果为进一步研究好氧/兼性厌氧微生物对汞与甲基汞迁移转化机制提供基础理论依据和供试材料。