Department of Microbiology, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, the Netherlands.
BMC Genomics. 2010 May 12;11:299. doi: 10.1186/1471-2164-11-299.
Anaerobic ammonium-oxidizing (anammox) bacteria perform a key step in global nitrogen cycling. These bacteria make use of an organelle to oxidize ammonia anaerobically to nitrogen (N2) and so contribute approximately 50% of the nitrogen in the atmosphere. It is currently unknown which proteins constitute the organellar proteome and how anammox bacteria are able to specifically target organellar and cell-envelope proteins to their correct final destinations. Experimental approaches are complicated by the absence of pure cultures and genetic accessibility. However, the genome of the anammox bacterium Candidatus "Kuenenia stuttgartiensis" has recently been sequenced. Here, we make use of these genome data to predict the organellar sub-proteome and address the molecular basis of protein sorting in anammox bacteria.
Two training sets representing organellar (30 proteins) and cell envelope (59 proteins) proteins were constructed based on previous experimental evidence and comparative genomics. Random forest (RF) classifiers trained on these two sets could differentiate between organellar and cell envelope proteins with ~89% accuracy using 400 features consisting of frequencies of two adjacent amino acid combinations. A physicochemically distinct organellar sub-proteome containing 562 proteins was predicted with the best RF classifier. This set included almost all catabolic and respiratory factors encoded in the genome. Apparently, the cytoplasmic membrane performs no catabolic functions. We predict that the Tat-translocation system is located exclusively in the organellar membrane, whereas the Sec-translocation system is located on both the organellar and cytoplasmic membranes. Canonical signal peptides were predicted and validated experimentally, but a specific (N- or C-terminal) signal that could be used for protein targeting to the organelle remained elusive.
A physicochemically distinct organellar sub-proteome was predicted from the genome of the anammox bacterium K. stuttgartiensis. This result provides strong in silico support for the existing experimental evidence for the existence of an organelle in this bacterium, and is an important step forward in unravelling a geochemically relevant case of cytoplasmic differentiation in bacteria. The predicted dual location of the Sec-translocation system and the apparent absence of a specific N- or C-terminal signal in the organellar proteins suggests that additional chaperones may be necessary that act on an as-yet unknown property of the targeted proteins.
厌氧氨氧化（anammox）细菌在全球氮循环中起着关键作用。这些细菌利用一种细胞器将氨厌氧氧化为氮气（N2），因此贡献了大气中约 50%的氮。目前尚不清楚哪些蛋白质构成了细胞器蛋白质组，以及 anammox 细菌如何能够将细胞器和细胞包膜蛋白特异性地靶向到它们的正确最终目的地。由于缺乏纯培养物和遗传可及性，实验方法变得复杂。然而，anammox 细菌“Kuenenia stuttgartiensis”的基因组最近已经被测序。在这里，我们利用这些基因组数据来预测细胞器亚蛋白质组，并解决 anammox 细菌中蛋白质分拣的分子基础。
根据先前的实验证据和比较基因组学，构建了两个代表细胞器（30 种蛋白质）和细胞包膜（59 种蛋白质）的训练集。基于这两个集合的随机森林（RF）分类器可以使用由两个相邻氨基酸组合的频率组成的 400 个特征，以约 89%的准确率区分细胞器和细胞包膜蛋白。使用最佳 RF 分类器预测到一个具有独特物理化学性质的细胞器亚蛋白质组，其中包含 562 种蛋白质。这组蛋白质几乎包含了基因组中所有的分解代谢和呼吸因子。显然，细胞质膜不具有分解代谢功能。我们预测 Tat 易位系统仅位于细胞器膜上，而 Sec 易位系统位于细胞器和细胞质膜上。预测到了经典信号肽，并进行了实验验证，但仍未能找到可用于将蛋白质靶向细胞器的特定（N-或 C-末端）信号。
从 anammox 细菌 K. stuttgartiensis 的基因组中预测到了具有独特物理化学性质的细胞器亚蛋白质组。这一结果为该细菌中细胞器存在的现有实验证据提供了强有力的计算支持，并且是揭示细菌细胞质分化这一具有地球化学意义的案例的重要一步。预测的 Sec 易位系统的双重位置以及细胞器蛋白中明显缺乏特定的 N-或 C-末端信号表明，可能需要额外的伴侣蛋白来作用于目标蛋白的未知特性。
