A Proteomic View at the Biochemistry of Syntrophic Butyrate Oxidation in Syntrophomonas wolfei

In syntrophic conversion of butyrate to methane and CO2, butyrate is oxidized to acetate by secondary fermenting bacteria such as Syntrophomonas wolfei in close cooperation with methanogenic partner organisms, e.g., Methanospirillum hungatei. This process involves an energetically unfavourable shift of electrons from the level of butyryl-CoA oxidation to the substantially lower redox potential of proton and/or CO2 reduction, in order to transfer these electrons to the methanogenic partner via hydrogen and/or formate. In the present study, all prominent membrane-bound and soluble proteins expressed in S. wolfei specifically during syntrophic growth with butyrate, in comparison to pure-culture growth with crotonate, were examined by one- and two-dimensional gel electrophoresis, and identified by peptide fingerprinting-mass spectrometry. A membrane-bound, externally oriented, quinone-linked formate dehydrogenase complex was expressed at high level specifically during syntrophic butyrate oxidation, comprising a selenocystein-linked catalytic subunit with a membrane-translocation pathway signal (TAT), a membrane-bound iron-sulfur subunit, and a membrane-bound cytochrome. Soluble hydrogenases were expressed at high levels specifically during growth with crotonate. The results were confirmed by native protein gel electrophoresis, by formate dehydrogenase and hydrogenase-activity staining, and by analysis of formate dehydrogenase and hydrogenase activities in intact cells and cell extracts. Furthermore, constitutive expression of a membrane-bound, internally oriented iron-sulfur oxidoreductase (DUF224) was confirmed, together with expression of soluble electron-transfer flavoproteins (EtfAB) and two previously identified butyryl-CoA dehydrogenases. Our findings allow to depict an electron flow scheme for syntrophic butyrate oxidation in S. wolfei. Electrons derived from butyryl-CoA are transferred through a membrane-bound EtfAB:quinone oxidoreductase (DUF224) to a menaquinone cycle and further via a b-type cytochrome to an externally oriented formate dehydrogenase. Hence, an ATP hydrolysis-driven proton-motive force across the cytoplasmatic membrane would provide the energy input for the electron potential shift necessary for formate formation.在丁酸（butyrate）向甲烷（methane）与二氧化碳（CO₂）的互营转化过程中，次级发酵细菌（secondary fermenting bacteria）如沃氏互营单胞菌（Syntrophomonas wolfei，后文简称S. wolfei）会与产甲烷共生伙伴菌（methanogenic partner organisms，如洪氏产甲烷螺菌（Methanospirillum hungatei））紧密协作，将丁酸氧化为乙酸（acetate）。该过程涉及一次能量不利的电子转移：电子从丁酰辅酶A（butyryl-CoA）的氧化能级，转移至质子（proton）和/或二氧化碳还原（CO₂ reduction）所需的显著更低的氧化还原电位（redox potential），从而通过氢气（hydrogen）和/或甲酸（formate）将电子传递给产甲烷共生伙伴菌。 本研究采用一维凝胶电泳（one-dimensional gel electrophoresis）与二维凝胶电泳（two-dimensional gel electrophoresis）技术，对S. wolfei在以丁酸为底物的互营生长（相较于以巴豆酸盐（crotonate）为底物的纯培养生长（pure-culture growth））阶段特异性表达的所有主要膜结合蛋白（membrane-bound proteins）与可溶性蛋白（soluble proteins）进行了检测，并通过肽指纹图谱质谱法（peptide fingerprinting-mass spectrometry）完成了蛋白鉴定。 研究发现，一种膜结合、胞外定向且与醌偶联的甲酸脱氢酶复合物（quinone-linked formate dehydrogenase complex），在丁酸互营氧化阶段特异性高表达。该复合物包含带有膜转运通路信号（TAT）的硒代半胱氨酸（selenocystein）偶联催化亚基、膜结合铁硫亚基（iron-sulfur subunit）以及膜结合细胞色素（cytochrome）。而可溶性氢化酶（hydrogenases）则仅在以巴豆酸盐为底物的生长阶段高表达。 上述结果通过非变性蛋白凝胶电泳（native protein gel electrophoresis）、甲酸脱氢酶与氢化酶活性染色（activity staining），以及对完整细胞（intact cells）和细胞提取物（cell extracts）中甲酸脱氢酶与氢化酶活性的分析得到了验证。此外，本研究证实了膜结合、胞内定向的铁硫氧化还原酶（iron-sulfur oxidoreductase, DUF224）的组成型表达（constitutive expression），同时检测到可溶性电子传递黄素蛋白（EtfAB）以及两种此前已被鉴定的丁酰辅酶A脱氢酶（butyryl-CoA dehydrogenases）的表达。 基于本研究结果，我们得以构建S. wolfei中丁酸互营氧化的电子流动模型：源自丁酰辅酶A的电子通过膜结合的EtfAB-醌氧化还原酶（DUF224）传递至甲基萘醌循环（menaquinone cycle），并进一步通过b型细胞色素传递至胞外定向的甲酸脱氢酶。因此，跨细胞质膜（cytoplasmatic membrane）的ATP水解（ATP hydrolysis）驱动的质子动力势（proton-motive force），可为甲酸形成所需的电子电位偏移提供能量输入。