Protein-based stable isotope probing (Protein-SIP) reveals active species within anoxic mixed cultures
Protein-based stable isotope probing (Protein-SIP) reveals active species within anoxic mixed cultures
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DOI：
10.1038/ismej.2008.64
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发表时间：
2008-11-01
期刊：
影响因子：
11
通讯作者：
Vogt, Carsten
中科院分区：
文献类型：
--
作者：
Jehmlich, Nico;Schmidt, Frank;Vogt, Carsten
由于方法学的限制，将特定的代谢活动与微生物群落中的某些物种联系起来仍然是一个挑战。我们开发了一种利用[C-13(7)]-甲苯进行蛋白质代谢标记的方法来分析单一细菌物种在联合体中的特定代谢活性。随后用2D凝胶电泳法(2-DE)和质谱仪(MS)对标记的蛋白质进行分析，以确定它们的特性以及它们的C-13含量作为宿主生物体功能和活性的指示器。为了建立这种方法，我们分析了芳香委陵菌EbN1菌株蛋白质中C-13碳原子的代谢掺入情况。该菌株能够在硝酸盐还原条件下代谢甲苯，并在纯培养或混合培养体系中与葡萄糖酸盐消耗丰富培养物生长。首先，分别以非标记甲苯和标记[C-13(7)]-甲苯为碳源培养EbN1菌株，并对其蛋白质进行双向凝胶电泳。MALDI-MS/MS从38个蛋白质中鉴定出60个独特的蛋白质，其中C-13掺入水平为92.3+/-0.8%。随后，我们将菌株EbN1与不在甲苯而是在葡萄糖酸上生长的富集菌UFZ-1混合，并添加非标记甲苯、[C-13(7)]-甲苯和/或非标记葡萄糖酸作为碳源。蛋白质的同位素标记物在2-DE后用MS进行分析，作为该联合体的活性物种对同位素标记的甲苯的代谢转化的定量指标。C-13的掺入只存在于EbN1菌株的蛋白质中，含量为82.6+/-2.3%，由19种蛋白质计算得出的平均值，表明该方法适用于在混合培养中鉴定具有特定性质的代谢活性物种。
It is still a challenge to link specific metabolic activities to certain species in a microbial community because of methodological limitations. We developed a method to analyze the specific metabolic activity of a single bacterial species within a consortium making use of [C-13(7)]-toluene for metabolic labelling of proteins. Labelled proteins were subsequently analyzed by 2D gel electrophoresis (2-DE) and mass spectrometry (MS) to characterize their identity as well as their C-13 content as an indicator for function and activity of the host organism. To establish this method, we analyzed the metabolic incorporation of C-13 carbon atoms into proteins of Aromatoleum aromaticum strain EbN1. This strain is capable of metabolizing toluene under nitrate-reducing conditions and was grown in either pure culture or in a mixed consortium with a gluconate-consuming enrichment culture. First, strain EbN1 was grown with non-labelled toluene or labelled [C-13(7)]-toluene as carbon sources, respectively, and their proteins were subjected to 2-DE. In total, 60 unique proteins were identified by MALDI-MS/MS. From 38 proteins, the levels of C-13 incorporation were determined as 92.3 +/- 0.8%. Subsequently, we mixed strain EbN1 and the enrichment culture UFZ-1, which does not grow on toluene but on gluconate, and added non-labelled toluene, [C-13(7)]-toluene and/or non-labelled gluconate as carbon sources. The isotope labelling of proteins was analyzed after 2-DE by MS as a quantitative indicator for metabolic transformation of isotopic-labelled toluene by the active species of the consortium. Incorporation of C-13 was exclusively found in proteins from strain EbN1 at a content of 82.6 +/- 2.3%, as an average calculated from 19 proteins, demonstrating the suitability of the method used to identify metabolic active species with specific properties within a mixed culture.