The thermophilic hydrogenotrophic methanogen, Methanothermobacter sp. CaT2, which possesses an extracellular sugar layer, commonly aggregates by itself or with other microorganisms. To elucidate the molecular mechanisms responsible for this aggregation, the aggregation-defective mutant, CLA160, was isolated. Optical and electron microscopy observations revealed that the mutant exhibited a significant reduction in aggregation. Genomic sequencing showed that CLA160 has a single point mutation, causing a nonsense mutation in MTCT_1020, which encodes a hypothetical protein. Motif and domain analyses indicated that the hypothetical protein bears two membrane-spanning segments at the N- and C-terminal regions and a large middle repeat-containing region. The results of a bioinformatic analysis suggested that the first middle region (RII) of the protein or the whole structure is responsible for the function of the product of MTCT_1020 in the aggregation of CaT2. A treatment with proteinase K suppressed sedimentation in CaT2, indicating a reduction in aggregation, with almost no effect on sedimentation in CLA160. The addition of Ca2+ or Mg2+ ions enhanced sedimentation in CaT2, whereas a DNase treatment had no effect on sedimentation in either strain. These results suggest that the hypothetical protein encoded by MTCT_1020 plays a key role as a membrane-bound adhesion protein in the aggregation of CaT2, which is enhanced by the addition of Ca2+ or Mg2+ ions.

中文翻译：

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Methanothermobacter sp.的聚集缺陷突变体。CaT2 揭示了独特的蛋白质依赖性聚集

嗜热氢营养产甲烷菌 Methanothermobacter sp。CaT2 具有细胞外糖层，通常单独或与其他微生物聚集。为了阐明导致这种聚集的分子机制，分离了聚集缺陷突变体 CLA160。光学和电子显微镜观察显示突变体表现出聚集的显着减少。基因组测序显示 CLA160 具有单点突变，导致 MTCT_1020 发生无义突变，MTCT_1020 编码假设蛋白质。基序和域分析表明，假设的蛋白质在 N 和 C 末端区域具有两个跨膜片段以及一个大的中间重复区域。生物信息学分析的结果表明，蛋白质的第一个中间区域 (RII) 或整个结构负责 MTCT_1020 产物在 CaT2 聚集中的功能。用蛋白酶 K 处理抑制了 CaT2 中的沉淀，表明聚集减少，对 CLA160 中的沉淀几乎没有影响。Ca2+ 或 Mg2+ 离子的添加​​增强了 CaT2 中的沉降，而 DNase 处理对任一菌株的沉降都没有影响。这些结果表明，由 MTCT_1020 编码的假设蛋白在 CaT2 的聚集中作为膜结合粘附蛋白起关键作用，CaT2 的聚集可通过添加 Ca2+ 或 Mg2+ 离子而增强。用蛋白酶 K 处理抑制了 CaT2 中的沉淀，表明聚集减少，对 CLA160 中的沉淀几乎没有影响。Ca2+ 或 Mg2+ 离子的添加​​增强了 CaT2 中的沉降，而 DNase 处理对任一菌株的沉降都没有影响。这些结果表明，由 MTCT_1020 编码的假设蛋白质在 CaT2 的聚集中作为膜结合粘附蛋白起关键作用，CaT2 的聚集可通过添加 Ca2+ 或 Mg2+ 离子而增强。用蛋白酶 K 处理抑制了 CaT2 中的沉淀，表明聚集减少，对 CLA160 中的沉淀几乎没有影响。Ca2+ 或 Mg2+ 离子的添加​​增强了 CaT2 中的沉降，而 DNase 处理对任一菌株的沉降都没有影响。这些结果表明，由 MTCT_1020 编码的假设蛋白在 CaT2 的聚集中作为膜结合粘附蛋白起关键作用，CaT2 的聚集可通过添加 Ca2+ 或 Mg2+ 离子而增强。