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

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. A five-chip study using total RNA recovered from three separate cultures of Ferroglobus placidus DSM 10642 grown with 0.5 mM phenol (experimental condition) and two separate cultures of Ferroglobus placidus DSM 10642 grown on 1 mM benzoate (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.

本研究以嗜铁铁球菌（*Ferroglobus placidus*）为实验对象，旨在探究稳定分子苯在严格厌氧环境下的代谢机制，重点研究了与三价铁（Fe(III)）还原偶联的厌氧苯氧化过程。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℃条件下通过该过程耦联能量生成以维持生长。苯的消耗与Fe(III)还原的化学计量比，以及[¹⁴C]-苯向[¹⁴C]-二氧化碳的转化结果，均与苯完全氧化为二氧化碳并将电子传递至Fe(III)的过程一致。在苯代谢过程中，仅苯甲酸（benzoate）会少量积累，而非苯酚（phenol）或甲苯（toluene）；且可通过[¹⁴C]-苯生成[¹⁴C]-苯甲酸。对基因转录水平的分析显示，相较于以乙酸盐（acetate）为碳源生长的菌体，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在苯培养条件下并未出现表达上调。本研究还鉴定出一种推定的羧化酶（carboxylase）编码基因，该基因在苯培养的菌体中表达量高于苯甲酸培养的菌体。上述结果表明，苯通过羧化反应转化为苯甲酸，且苯酚并非嗜铁铁球菌苯代谢途径中的关键中间产物。本研究首次证实，纯培养条件下的微生物可在严格厌氧环境中以苯为唯一碳源生长，且该菌株的苯降解途径具有明确的厌氧代谢通路支撑，相关证据充分。因此，嗜铁铁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。本研究开展五芯片转录组实验，实验所用总RNA分别提取自3组以0.5 mM苯酚为唯一碳源培养的嗜铁铁球菌DSM 10642菌株（实验组），以及2组以1 mM苯甲酸为唯一碳源培养的该菌株（对照组）。每张芯片可检测嗜铁铁球菌DSM 10642的2613个基因的表达水平，每个基因对应9组45-60-mer探针对（PM/MM），且具备三倍技术重复冗余。