Department of Microbiology, School of Pharmacy, Kitasato University, Tokyo, Japan.
Yakult Central Institute for Microbiological Research, Tokyo, Japan.
J Appl Microbiol. 2010 Aug;109(2):657-666. doi: 10.1111/j.1365-2672.2010.04690.x. Epub 2010 Feb 23.
Despite the fact that the entire genome sequence of probiotic Lactobacillus casei has recently been available, their mechanisms of beneficial effects are poorly clarified, probably because of the lack of an efficient mutagenesis system. The aim of this study was to establish a practical random mutagenesis system of L. casei using the Tn5 transposome complexes.
We optimized the conditions for transformation using a plasmid pUCYIT356-1-Not2 and then transposition reaction using Tn5 transposome system for L. casei ATCC 27139. Tn5 insertion library of this strain being consisted of 9408 mutants was constructed by repeating the mutagenesis procedure. To examine the utility of this mutagenesis system, we screened a panel of insertion mutants for nutrient requirements. Six auxotrophic mutants were isolated and their Tn5 insertion sites were determined by inverse PCR, which demonstrated that insertions occur randomly throughout the whole bacterial genome.
Tn5 transposome system functioned efficiently to generate transposon insertion mutants of L. casei and enabled to construct useful L. casei Tn5 insertion library at optimized conditions for transformation and transposition.
The availability of this system facilitates the study of the mechanisms of beneficial effects of L. casei for human health.
尽管益生菌干酪乳杆菌的全基因组序列最近已经公布，但它们的有益效果机制仍不清楚，这可能是因为缺乏有效的诱变系统。本研究旨在利用 Tn5 转座体复合物建立一种实用的干酪乳杆菌随机诱变系统。
我们使用质粒 pUCYIT356-1-Not2 优化了转化条件，然后使用 Tn5 转座体系统对干酪乳杆菌 ATCC 27139 进行转位反应。通过重复诱变程序，构建了该菌株的 Tn5 插入文库，由 9408 个突变体组成。为了检验这种诱变系统的实用性，我们筛选了一组营养需求的插入突变体。分离出 6 个营养缺陷型突变体，并通过反向 PCR 确定 Tn5 插入位点，这表明插入发生在整个细菌基因组中。
Tn5 转座体系统有效地生成了干酪乳杆菌的转座子插入突变体，并在优化的转化和转位条件下，能够构建有用的干酪乳杆菌 Tn5 插入文库。
该系统的可用性为研究干酪乳杆菌对人类健康的有益效果机制提供了便利。
