Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Tsuruoka-shi, Yamagata 997-0017, Japan.
NODAI Genome Research Center, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan.
FEMS Microbiol Lett. 2019 Feb 1;366(4). doi: 10.1093/femsle/fnz032.
Bacillus subtilis (natto) is generally regarded as a safe bacterium and used as a host for the production of several materials. However, genetic engineering of B. subtilis (natto) is not well established because of poor DNA delivery methods and the lack of a standard strain for the aim. Here, we developed a genetic delivery tool in B. subtilis (natto) using the pLS20 conjugational plasmid (65 kbp). Transmission of pLS20 from B. subtilis 168 to wild-type B. subtilis (natto) did not occur via established mating protocols. We isolated B. subtilis (natto) mutants showing dramatically increased recipient activity. Whole-genome sequence analyses revealed three common alterations: mutations in the restriction endonuclease gene and in the methyl-accepting chemotaxis protein gene, and a 43-kbp deletion at the genome replication termination locus. A representative strain named NEST116 was generated as the first B. subtilis (natto) strain suitable for exploring pLS20-based genetic engineering.
纳豆芽孢杆菌通常被认为是一种安全的细菌，被用作生产多种材料的宿主。然而，由于 DNA 传递方法不佳和缺乏标准菌株，纳豆芽孢杆菌的基因工程尚未得到很好的建立。在这里，我们使用 pLS20 接合质粒（65 kbp）在纳豆芽孢杆菌中开发了一种遗传传递工具。pLS20 不能通过已建立的交配协议从枯草芽孢杆菌 168 传递到野生型纳豆芽孢杆菌。我们分离出显示出显着增加的受体活性的纳豆芽孢杆菌突变体。全基因组序列分析揭示了三个常见的改变：限制内切酶基因和甲基接受趋化蛋白基因的突变，以及基因组复制终止位点的 43 kbp 缺失。一个名为 NEST116 的代表性菌株被生成，作为第一个适合探索基于 pLS20 的遗传工程的纳豆芽孢杆菌菌株。
