Anaerobic Oxidation of Benzene by the Hyperthermophilic Archaeon Ferroglobus placidus (Benzene vs. Phenol). Ferroglobus placidus DSM 10642

Anaerobic benzene oxidation coupled to the reduction of Fe(III) was studied in Ferroglobus placidus in order to learn more about how such a stable molecule could be metabolized under strict anaerobic conditions. F. placidus conserved energy to support growth at 85°C in a medium with benzene provided as the sole electron donor and Fe(III) as the sole electron acceptor. The stoichiometry of benzene loss and Fe(III) reduction, as well as the conversion of [14C]-benzene to [14C]-carbon dioxide, was consistent with complete oxidation of benzene to carbon dioxide with electron transfer to Fe(III). Benzoate, but not phenol or toluene, accumulated at low levels during benzene metabolism and [14C]-benzoate was produced from [14C]-benzene. Analysis of gene transcript levels revealed increased expression of genes encoding enzymes for anaerobic benzoate degradation during growth on benzene versus growth on acetate, but genes involved in phenol degradation were not up-regulated during growth on benzene. A gene for a putative carboxylase that was more highly expressed in benzene- versus benzoate-grown cells was identified. These results suggest that benzene is carboxylated to benzoate and that phenol is not an important intermediate in the benzene metabolism of F. placidus. This is the first demonstration of a microorganism in pure culture that can grow on benzene under strict anaerobic conditions and for which there is strong evidence for degradation of benzene via clearly defined anaerobic metabolic pathways. Thus, F. placidus provides a much needed pure culture model for further studies on the anaerobic activation of benzene in microorganisms. Overall design: A six-chip study using total RNA recovered from three separate cultures of Ferroglobus placidus DSM 10642 grown with 1 mM benzene (experimental condition) and three separate cultures of Ferroglobus placidus DSM 10642 grown on 0.5 mM phenol (control condition). Each chip measures the expression level of 2,613 genes from Ferroglobus placidus DSM 10642 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

本研究以嗜铁火球菌（Ferroglobus placidus）为研究对象，针对与Fe(III)（三价铁）还原偶联的厌氧苯（benzene）氧化过程展开考察，以期深入解析这类稳定难降解分子在严格厌氧环境下的代谢机制。
该菌株可在以苯为唯一电子供体、Fe(III)为唯一电子受体的培养基中，于85℃条件下获取能量以支持生长。
苯的消耗与Fe(III)还原的化学计量关系，以及[¹⁴C]-苯（[¹⁴C]-benzene）向[¹⁴C]-二氧化碳（[¹⁴C]-carbon dioxide）的转化结果，均与苯被完全氧化为二氧化碳且电子传递至Fe(III)的过程相符。在苯代谢过程中，苯甲酸（benzoate）会低水平积累，而苯酚（phenol）与甲苯（toluene）则无明显累积；同时可检测到[¹⁴C]-苯转化生成[¹⁴C]-苯甲酸。
基因转录水平分析显示，相较于以乙酸盐（acetate）为碳源的培养条件，以苯为碳源生长时，编码厌氧苯甲酸降解相关酶的基因表达量显著上调，但参与苯酚降解的基因并未出现表达上调。本研究还鉴定出一种假定的羧化酶编码基因，该基因在以苯为碳源的培养细胞中的表达量显著高于以苯甲酸为碳源的细胞。
上述结果表明，苯可通过羧化作用转化为苯甲酸，且苯酚并非嗜铁火球菌苯代谢途径中的关键中间产物。本研究首次在纯培养体系中证实，嗜铁火球菌可在严格厌氧条件下以苯为唯一碳源生长，且其苯降解过程存在明确的厌氧代谢通路，具备坚实的实验证据支撑。因此，嗜铁火球菌为后续开展微生物厌氧活化苯的相关研究提供了亟需的纯培养模型体系。
实验设计概况：本研究采用6张基因芯片开展转录组分析，所用总RNA（total RNA）提取自6组独立培养的嗜铁火球菌DSM 10642菌株：其中3组以1 mM苯为碳源（实验组），另外3组以0.5 mM苯酚为碳源（对照组）。每张芯片可检测嗜铁火球菌DSM 10642的2613个基因的表达水平，每个基因对应9组45~60个碱基的探针对（PM/MM），并设置3倍技术重复以保障实验重复性。