Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria.
Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria.
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DOI：
10.1186/1471-2199-7-47
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发表时间：
2006-12-15
影响因子：
--
通讯作者：
Winter N
中科院分区：
文献类型：
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作者：
Vultos TD;Méderlé I;Abadie V;Pimentel M;Moniz-Pereira J;Gicquel B;Reyrat JM;Winter N
分枝杆菌噬菌体Ms6整合到耻垢分枝杆菌和M. tRNAala基因的3'端为牛BCG染色体。同源重组发生在位于T环中的噬菌体attP核心和attB位点之间。来自Ms6的整合熟练载体是有用的遗传工具，但它们在BCG染色体中的插入位点仍然不清楚。本研究的主要目的是鉴定M.斯美拉和卡介苗。然后，我们的目标是修饰Ms6衍生载体中的attP位点，以将整合切换到其他tRNAala基因座。这为重组M.牛BCG菌株表达几个插入不同tRNAala基因的报告基因。这三个tRNAala基因在M.斯美拉和卡介苗。然而，在含有attB位点的tRNAalaU和tRNAalaV的T环中，在两个物种之间观察到单个碱基差异。我们观察到tRNAalaU基因是Ms6衍生的整合型载体在M.在BCG中，tRNAalaV基因被用作靶标。在BCG tRNAalaU T环中没有发生整合，尽管与26个碱基的Ms6 attP核心仅有一个碱基的差异。我们突变了attP核心，使其与M.斯美拉和卡介苗。七个碱基的T环的修饰降低了整合效率，确定该位点为可能的链交换位点。最后，构建了两个Ms6载体，以将两个报告基因整合到同一BCG染色体的tRNAalaU和tRNAalaV T环中。Ms6的7bp T环attP位点的小变化使得可以使用另一个attB位点，尽管整合效率较低。这些对BCG tRNAala基因的分子研究使得有可能为在同一BCG菌株中定点插入几个基因创造有价值的工具。这些工具将有助于开发新的多价疫苗和遗传研究。
Mycobacteriophage Ms6 integrates into Mycobacterium smegmatis and M. bovis BCG chromosome at the 3' end of tRNAala genes. Homologous recombination occurs between the phage attP core and the attB site located in the T-loop. Integration-proficient vectors derived from Ms6 are useful genetic tools, but their insertion sites in the BCG chromosome remain poorly defined. The primary objective of this study was to identify Ms6 target genes in M. smegmatis and BCG. We then aimed to modify the attP site in Ms6-derived vectors, to switch integration to other tRNAala loci. This provided the basis for the development of recombinant M. bovis BCG strains expressing several reporter genes inserted into different tRNAala genes. The three tRNAala genes are highly conserved in M. smegmatis and BCG. However, in the T-loop of tRNAalaU and tRNAalaV containing the attB site, a single base difference was observed between the two species. We observed that the tRNAalaU gene was the only site into which Ms6-derived integration-proficient vectors integrated in M. smegmatis, whereas in BCG, the tRNAalaV gene was used as the target. No integration occurred in the BCG tRNAalaU T-loop, despite a difference of only one base from the 26-base Ms6 attP core. We mutated the attP core to give a perfect match with the other tRNAala T-loops from M. smegmatis and BCG. Modification of the seven-base T-loop decreased integration efficiency, identifying this site as a possible site of strand exchange. Finally, two Ms6 vectors were constructed to integrate two reporter genes into the tRNAalaU and tRNAalaV T-loops of the same BCG chromosome. Small changes in the 7 bp T-loop attP site of Ms6 made it possible to use another attB site, albeit with a lower integration efficiency. These molecular studies on BCG tRNAala genes made it possible to create valuable tools for the site-directed insertion of several genes in the same BCG strain. These tools will be useful for the development of novel multivalent vaccines and genetic studies.