Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan.
Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido, Japan
Appl Environ Microbiol. 2018 Aug 17;84(17). doi: 10.1128/AEM.00824-18. Print 2018 Sep 1.
Bifidobacteria are a major component of the intestinal microbiota in humans, particularly breast-fed infants. Therefore, elucidation of the mechanisms by which these bacteria colonize the intestine is desired. One approach is transposon mutagenesis, a technique currently attracting much attention because, in combination with next-generation sequencing, it enables exhaustive identification of genes that contribute to microbial fitness. We now describe a transposon mutagenesis system for subsp. 105-A (JCM 31944) based on IS, a native IS family insertion sequence. To build this system, xylose-inducible or constitutive bifidobacterial promoters were tested to drive the expression of full-length or a truncated form at the N terminus of the IS transposase. An artificial transposon plasmid, pBFS12, in which IS terminal inverted repeats are separated by a 3-bp spacer, was also constructed to mimic the transposition intermediate of IS elements. The introduction of this plasmid into a strain expressing transposase resulted in the insertion of the plasmid with an efficiency of >10 CFU/μg DNA. The plasmid targets random 3- to 4-bp sequences, but with a preference for noncoding regions. This mutagenesis system also worked at least in NCC2705. Characterization of a transposon insertion mutant revealed that a putative α-glucosidase mediates palatinose and trehalose assimilation, demonstrating the suitability of transposon mutagenesis for loss-of-function analysis. We anticipate that this approach will accelerate functional genomic studies of subsp. Several hundred species of bacteria colonize the mammalian intestine. However, the genes that enable such bacteria to colonize and thrive in the intestine remain largely unexplored. Transposon mutagenesis, combined with next-generation sequencing, is a promising tool to comprehensively identify these genes but has so far been applied only to a small number of intestinal bacterial species. In this study, a transposon mutagenesis system was established for subsp. , a representative health-promoting species. The system enables the identification of genes that promote colonization and survival in the intestine and should help illuminate the physiology of this species.
双歧杆菌是人类肠道微生物群的主要组成部分，特别是母乳喂养的婴儿。因此，阐明这些细菌在肠道中定植的机制是很有必要的。一种方法是转座子诱变，这是一种目前备受关注的技术，因为它与下一代测序相结合，可以彻底鉴定出对微生物适应性有贡献的基因。我们现在描述了一种基于 IS 的 subsp. 105-A (JCM 31944) 的转座子诱变系统，IS 是一种天然的 IS 家族插入序列。为了构建这个系统，我们测试了木糖诱导型或组成型双歧杆菌启动子，以驱动 IS 转座酶全长或 N 端截短形式的表达。还构建了一个人工转座子质粒 pBFS12，其中 IS 末端反向重复序列由 3 个碱基对的间隔区隔开，以模拟 IS 元件的转座中间物。将该质粒引入表达转座酶的菌株中，导致质粒以>10 CFU/μg DNA 的效率插入。该质粒靶向 3 到 4 个碱基对的随机序列，但偏爱非编码区。这种诱变系统在至少 NCC2705 中也有效。转座子插入突变体的特征表明，一种假定的α-葡萄糖苷酶介导了棉子糖和海藻糖的同化，证明了转座子诱变在功能丧失分析中的适用性。我们预计，这种方法将加速 subsp. 的功能基因组研究。 有几百种细菌定植在哺乳动物的肠道中。然而，使这些细菌能够在肠道中定植和茁壮成长的基因在很大程度上仍未被探索。转座子诱变与下一代测序相结合，是一种很有前途的全面鉴定这些基因的工具，但迄今为止仅应用于少数几种肠道细菌。在这项研究中，建立了一个转座子诱变系统，用于代表促进健康的 subsp. 。该系统能够鉴定促进在肠道中定植和存活的基因，应该有助于阐明该物种的生理学。
