Mariner-based transposon mutagenesis for Bacteroides species
Mariner-based transposon mutagenesis for Bacteroides species
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DOI：
10.1002/jobm.201200763
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发表时间：
2014-06-01
影响因子：
3.1
通讯作者：
Kuwahara, Tomomi
中科院分区：
文献类型：
--
作者：
Ichimura, Minoru;Uchida, Keiko;Kuwahara, Tomomi
拟杆菌是人类肠道菌群中最主要的类群之一。最近对知菌小鼠的宏基因组分析和研究表明，拟杆菌与宿主的上皮功能、肠道免疫系统和全身代谢密切相关。mariner家族转座子显示相对低的靶位点特异性，并且具有从原核生物到真核生物的宿主。因此，使用mariner家族转座子的随机诱变有望鉴定人-拟杆菌共生的关键分子。在本研究中，我们构建了质粒pMI 07，以通过电转化将含有红霉素抗性标记（ermF）和Himar 1转座酶识别的反向重复序列（ITRs）的基因盒（ermF/ITR）传递到拟杆菌。pMI 07成功地将ermF/ITR递送到拟杆菌基因组中，并且在B中每微克pMI 07产生数千个插入突变体。Thetaiotaomicron，B.脆弱B卵圆形，以及B，尽管程度较轻。普通人B中ermF/ITR插入位点的分析。多形核和B。vulgatus揭示了该盒靶向二核苷酸TA并以无偏的方式整合到基因组中。本文报道的数据将为拟杆菌属物种的转座子诱变提供有用的信息，这将使其独特的表型的基因负责的鉴定。
Bacteroides is one of the most predominant groups of human gut microbiota. Recent metagenomic analyses and studies on gnotobiotic mice demonstrated the tight association of Bacteroides with epithelial function, the gut immune system and systemic metabolism in the host. The mariner family transposon shows relatively low target site specificity and has hosts ranging from prokaryotes to eukaryotes. Thereby, random mutagenesis using the mariner family transposon is expected to identify key molecules for human-Bacteroides symbiosis. In this study, we constructed the plasmid pMI07 to deliver the gene cassette (ermF/ITR), which harbors the erythromycin resistant marker (ermF) and the inverted repeat sequences (ITRs) recognized by Himar1 transposase, to Bacteroides via electrotransformation. pMI07 successfully delivered ermF/ITR to the Bacteroides genomes and generated thousands of insertion mutants/mu g of pMI07 in B. thetaiotaomicron, B. fragilis, B. ovatus, and also, although to a lesser extent, B. vulgatus. Analyses of the ermF/ITR insertion sites in B. thetaiotaomicron and B. vulgatus revealed that the cassette targeted the dinucleotide TA and integrated into the genomes in an unbiased manner. The data reported here will provide useful information for transposon mutagenesis in Bacteroides species, which will enable identification of the genes responsible for their unique phenotypes.