Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA.
Appl Environ Microbiol. 2013 Nov;79(22):7006-12. doi: 10.1128/AEM.01800-13. Epub 2013 Sep 6.
We recently reported that the Thermotogales acquired the ability to synthesize vitamin B12 by acquisition of genes from two distantly related lineages, Archaea and Firmicutes (K. S. Swithers et al., Genome Biol. Evol. 4:730-739, 2012). Ancestral state reconstruction suggested that the cobinamide salvage gene cluster was present in the Thermotogales' most recent common ancestor. We also predicted that Thermotoga lettingae could not synthesize B12 de novo but could use the cobinamide salvage pathway to synthesize B12. In this study, these hypotheses were tested, and we found that Tt. lettingae did not synthesize B12 de novo but salvaged cobinamide. The growth rate of Tt. lettingae increased with the addition of B12 or cobinamide to its medium. It synthesized B12 when the medium was supplemented with cobinamide, and no B12 was detected in cells grown on cobinamide-deficient medium. Upstream of the cobinamide salvage genes is a putative B12 riboswitch. In other organisms, B12 riboswitches allow for higher transcriptional activity in the absence of B12. When Tt. lettingae was grown with no B12, the salvage genes were upregulated compared to cells grown with B12 or cobinamide. Another gene cluster with a putative B12 riboswitch upstream is the btuFCD ABC transporter, and it showed a transcription pattern similar to that of the cobinamide salvage genes. The BtuF proteins from species that can and cannot salvage cobinamides were shown in vitro to bind both B12 and cobinamide. These results suggest that Thermotogales species can use the BtuFCD transporter to import both B12 and cobinamide, even if they cannot salvage cobinamide.
我们最近报道称，Thermotogales 通过从两个远缘谱系（古菌和厚壁菌门）获取基因，获得了合成维生素 B12 的能力（K. S. Swithers 等人，Genome Biol. Evol. 4:730-739, 2012）。祖先状态重建表明 cobinamide salvage 基因簇存在于 Thermotogales 的最近共同祖先中。我们还预测 Thermotoga lettingae 不能从头合成 B12，但可以使用 cobinamide salvage 途径合成 B12。在这项研究中，这些假设得到了检验，我们发现 Tt. lettingae 不能从头合成 B12，但可以 salvage cobinamide。当培养基中添加 B12 或 cobinamide 时，Tt. lettingae 的生长速度增加。当培养基中补充 cobinamide 时，它合成了 B12，而在缺乏 cobinamide 的培养基中生长的细胞中则没有检测到 B12。 cobinamide salvage 基因的上游是一个假定的 B12 核糖开关。在其他生物体中，B12 核糖开关允许在没有 B12 的情况下提高转录活性。当 Tt. lettingae 在没有 B12 的情况下生长时，与在有 B12 或 cobinamide 的情况下生长的细胞相比， salvage 基因上调。上游有一个假定的 B12 核糖开关的另一个基因簇是 btuFCD ABC 转运蛋白，其转录模式与 cobinamide salvage 基因相似。来自可以和不能 salvage cobinamides 的物种的 BtuF 蛋白在体外被证明可以结合 B12 和 cobinamide。这些结果表明，Thermotogales 物种可以使用 BtuFCD 转运蛋白来导入 B12 和 cobinamide，即使它们不能 salvage cobinamide。
