School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, 2052, NSW, Australia.
J Proteome Res. 2010 Feb 5;9(2):653-63. doi: 10.1021/pr9005102.
Methanococcoides burtonii is a cold-adapted methanogenic archaeon from Ace Lake in Antarctica. Methanol and methylamines are the only substrates it can use for carbon and energy. We carried out quantitative proteomics using iTRAQ of M. burtonii cells grown on different substrates (methanol in defined media or trimethylamine in complex media), using techniques that enriched for secreted and membrane proteins in addition to cytoplasmic proteins. By integrating proteomic data with the complete, manually annotated genome sequence of M. burtonii, we were able to gain new insight into methylotrophic metabolism and the effects of methanol on the cell. Metabolic processing of methanol and methylamines is initiated by methyltransferases specific for each substrate, with multiple paralogs for each of the methyltransferases (similar to other members of the Methanosarcinaceae). In M. burtonii, most methyltransferases appear to have distinct roles in the metabolism of methylated substrates, although two methylamine methyltransferases appear to be nonfunctional. One set of methyltransferases for trimethylamine catabolism appears to be membrane associated, potentially providing a mechanism to directly couple trimethylamine uptake to demethylation. Important roles were highlighted for citrate synthase, glutamine synthetase, acetyl-CoA decarbonylase/synthase, and pyruvate synthase in carbon and nitrogen metabolism during growth on methanol. M. burtonii had only a marginal response to the provision of exogenous amino acids (from yeast extract), indicating that it is predisposed to the endogenous synthesis of amino acids. Growth on methanol appeared to cause oxidative stress in the cell, possibly through the formation of reactive nonoxygen species and formaldehyde, and the oxidative inactivation of corrinoid proteins, with the cell responding by elevating the synthesis of universal stress (Usp) proteins, several nucleic acid binding proteins, and a serpin. In addition, changes in levels of cell envelope proteins were linked to counteracting the disruptive solvent effects of methanol on cell membranes. This is the first global proteomic study to examine the effects of different carbon sources on the growth of an obligately methylotrophic methanogen.
伯顿甲烷球菌是一种来自南极洲艾斯湖的耐冷产甲烷古菌。甲醇和甲基胺是其唯一可用于碳和能量的底物。我们使用 iTRAQ 对在不同底物（定义培养基中的甲醇或复杂培养基中的三甲胺）上生长的 M. burtonii 细胞进行了定量蛋白质组学研究，除了细胞质蛋白外，该方法还富集了分泌蛋白和膜蛋白。通过将蛋白质组学数据与 M. burtonii 的完整、人工注释基因组序列整合，我们能够深入了解甲基营养代谢以及甲醇对细胞的影响。甲醇和甲基胺的代谢加工是由每种底物特有的甲基转移酶启动的，每种甲基转移酶都有多个基因（类似于 Methanosarcinaceae 的其他成员）。在 M. burtonii 中，大多数甲基转移酶似乎在甲基化底物的代谢中具有独特的作用，尽管两个甲基胺甲基转移酶似乎不起作用。一组用于三甲胺分解代谢的甲基转移酶似乎与膜相关，可能为直接将三甲胺摄取与去甲基化偶联提供了一种机制。在甲醇生长过程中，柠檬酸合酶、谷氨酰胺合成酶、乙酰辅酶 A 脱羧酶/合成酶和丙酮酸合酶在碳氮代谢中发挥了重要作用。M. burtonii 对外源氨基酸（酵母提取物）的提供仅有轻微反应，表明它倾向于内源性合成氨基酸。在甲醇上生长似乎会导致细胞氧化应激，可能是通过形成反应性非氧物种和甲醛以及钴胺素蛋白的氧化失活，细胞通过提高普遍应激（Usp）蛋白、几种核酸结合蛋白和丝氨酸蛋白酶的合成来做出响应。此外，细胞包膜蛋白水平的变化与对抗甲醇对细胞膜的破坏性溶剂效应有关。这是首次对不同碳源对专性甲基营养型产甲烷菌生长的影响进行的全面蛋白质组学研究。
