Fermentative nitrogen-fixing bacteria have not yet been examined in detail in thermal environments. In the present study, we isolated the thermophilic fermentative bacterium, strain YA01 from a hot spring. This strain grew at temperatures up to 78°C. A phylogenetic analysis based on its 16S rRNA gene sequence indicated that strain YA01 belonged to the genus Caldicellulosiruptor, which are fermentative bacteria in the phylum Firmicutes, with 97.7-98.0% sequence identity to its closest relatives. Strain YA01 clearly exhibited N2-dependent growth at 70°C. We also confirmed N2-dependent growth in the relatives of strain YA01, Caldicellulosiruptor hydrothermalis 108 and Caldicellulosiruptor kronotskyensis 2002. The nitrogenase activities of these three strains were examined using the acetylene reduction assay. Similar activities were detected for all tested strains, and were slightly suppressed by the addition of ammonium. A genome analysis revealed that strain YA01, as well as other Caldicellulosiruptor, possessed a gene set for nitrogen fixation, but lacked the nifN gene, which encodes a nitrogenase iron-molybdenum cofactor biosynthesis protein that is commonly detected in nitrogen-fixing bacteria. The amino acid sequences of nitrogenase encoded by nifH, nifD, and nifK shared 92-98% similarity in Caldicellulosiruptor. A phylogenetic tree of concatenated NifHDK sequences showed that NifHDK of Caldicellulosiruptor was in the deepest clade. To the best of our knowledge, this is the first study to demonstrate the nitrogen-fixing ability of fermentative bacteria at 70°C. Caldicellulosiruptor may have retained an ancient nitrogen-fixing enzyme system.

中文翻译：

---

Caldicellulosiruptor 嗜热发酵细菌的固氮能力和固氮相关基因。

尚未在热环境中详细检查发酵固氮细菌。在本研究中，我们从一个温泉中分离出了嗜热发酵菌 YA01 菌株。该菌株在高达 78°C 的温度下生长。基于其16S rRNA基因序列的系统发育分析表明，菌株YA01属于Caldicellulosiruptor属，是厚壁菌门中的发酵细菌，与其近亲具有97.7-98.0%的序列同一性。菌株 YA01 在 70°C 下明显表现出 N2 依赖性生长。我们还证实了菌株 YA01、Caldicellulosiruptor hydrothermalis 108 和 Caldicellulosiruptor kronotskyensis 2002 的亲属中的 N2 依赖性生长。使用乙炔还原测定法检查了这三种菌株的固氮酶活性。所有测试菌株都检测到类似的活性，并且通过添加铵而略微抑制。基因组分析显示，菌株 YA01 以及其他 Caldicellulosiruptor 具有固氮基因集，但缺乏 nifN 基因，该基因编码固氮酶铁钼辅助因子生物合成蛋白，通常在固氮细菌中检测到。由 nifH、nifD 和 nifK 编码的固氮酶的氨基酸序列在 Caldicellulosiruptor 中具有 92-98% 的相似性。连接的 NifHDK 序列的系统发育树表明 Caldicellulosiruptor 的 NifHDK 位于最深的进化枝中。据我们所知，这是第一项证明发酵细菌在 70°C 下固氮能力的研究。Caldicellulosiruptor 可能保留了古老的固氮酶系统。