Faculty of Food and Agricultural Sciences, Fukushima University, Fukushima, Japan.
Manufacturing Division, Yamasa Corporation, Choshi, Japan.
Appl Environ Microbiol. 2023 Mar 29;89(3):e0200522. doi: 10.1128/aem.02005-22. Epub 2023 Feb 21.
Studies on the microorganisms used in food production are of interest because microbial genotypes are reflected in food qualities such as taste, flavor, and yield. However, several microbes are nonmodel organisms, and their analysis is often limited by the lack of genetic tools. Tetragenococcus halophilus, a halophilic lactic acid bacterium used in soy sauce fermentation starter culture, is one such microorganism. The lack of DNA transformation techniques for makes gene complementation and disruption assays difficult. Here, we report that the endogenous insertion sequence IS, belonging to the IS family, is translocated at an extremely high frequency in and causes insertional mutations at various loci. We developed a method named argeting spontaneous nsertional utations enomes (TIMING), which combines high-frequency insertional mutations and efficient PCR screening, enabling the isolation of gene mutants of interest from a library. The method provides a reverse genetics and strain improvement tool, does not require the introduction of exogenous DNA constructs, and enables the analysis of nonmodel microorganisms lacking DNA transformation techniques. Our results highlight the important role of insertion sequences as a source of spontaneous mutagenesis and genetic diversity in bacteria. Genetic and strain improvement tools to manipulate a gene of interest are required for the nontransformable lactic acid bacterium Tetragenococcus halophilus. Here, we demonstrate that an endogenous transposable element, IS, is transposed into the host genome at an extremely high frequency. A genotype-based and non-genetically engineered screening system was constructed to isolate knockout mutants using this transposable element. The method described enables a better understanding of the genotype-phenotype relationship and serves as a tool to develop food-grade-appropriate mutants of .
用于食品生产的微生物的研究很有意义，因为微生物的基因型反映在食品的质量上，如味道、风味和产量。然而，有几种微生物是非模式生物，它们的分析往往受到缺乏遗传工具的限制。在酱油发酵起始培养物中使用的嗜盐乳酸菌四链球菌就是这样一种微生物。由于缺乏 DNA 转化技术，使得基因互补和敲除实验变得困难。在这里，我们报告说，属于 IS 家族的内源性插入序列 IS 在 中以极高的频率易位，并在不同的基因座引起插入突变。我们开发了一种名为靶向自发插入突变基因组（TIMING）的方法，该方法结合了高频插入突变和高效 PCR 筛选，能够从文库中分离出感兴趣的基因突变体。该方法提供了一种反向遗传学和菌株改良工具，不需要引入外源 DNA 构建体，并能够分析缺乏 DNA 转化技术的非模式微生物。我们的结果强调了插入序列作为细菌自发突变和遗传多样性的来源的重要作用。需要遗传和菌株改良工具来操纵非可转化的乳酸杆菌 Tetragenococcus halophilus 中的目的基因。在这里，我们证明了一种内源性可移动元件 IS 以极高的频率易位到宿主基因组中。我们构建了一种基于基因型而非基因工程的筛选系统，利用这种可移动元件分离敲除突变体。所描述的方法有助于更好地理解基因型-表型关系，并可作为开发食品级适当突变体的工具。
