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首页> 外文期刊>Infection and immunity >Isolation of Acid-Inducible Genes of Mycobacterium tuberculosis with the Use of Recombinase-Based In Vivo Expression Technology
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Isolation of Acid-Inducible Genes of Mycobacterium tuberculosis with the Use of Recombinase-Based In Vivo Expression Technology

机译：基于重组酶的体内表达技术分离结核分枝杆菌的酸诱导基因

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A better understanding of mycobacterial gene regulation under certain stress conditions (e.g., low pH) may provide insight into mechanisms of adaptation during infection. To identify mycobacterial promoters induced at low pH, we adapted the recombinase-based in vivo expression technology (RIVET) promoter trap system for use with mycobacteria. Our results show that the TnpR recombinase of transposon γδ is active in Mycobacterium smegmatis and Mycobacterium tuberculosis. We developed a method to perform sequential double selection with mycobacteria by using RIVET, with a kanamycin preselection and a sucrose postselection. A library of M. tuberculosis DNA inserted upstream of tnpR was created, and using the double selection, we identified two promoters which are upregulated at low pH. The promoter regions drive the expression of a gene encoding a putative lipase, lipF (Rv3487c), as well as a PE-PGRS gene, Rv0834c, in a pH-dependent manner in both M. smegmatis and M. tuberculosis. The acid inducibility of lipF and Rv0834c was independent of the stress response sigma factor, SigF, as acid induction of the two genes in an M. tuberculosis sigF mutant strain was similar to that in the wild-type strain. No induction of lipF or Rv0834c was observed during infection of J774 murine macrophages, an observation which is in agreement with previous reports on the failure of phagosomes containing M. tuberculosis to acidify.
机译：对某些压力条件（例如低pH）下分枝杆菌基因调控的更好理解可能有助于洞察感染过程中的适应机制。为了鉴定在低pH值下诱导的分枝杆菌启动子，我们将基于重组酶的体内表达技术（RIVET）启动子捕获系统与分枝杆菌一起使用。我们的结果表明，转座子γδ的TnpR重组酶在耻垢分枝杆菌和结核分枝杆菌中具有活性。我们开发了一种通过使用RIVET，卡那霉素预选择和蔗糖后选择对分枝杆菌进行顺序双重选择的方法。 M库。创建了在 tnpR 上游插入的结核 DNA，并使用双重选择，我们发现了两个在低pH下均被上调的启动子。启动子区域驱动编码假定的脂肪酶 lipF （ Rv3487c ）以及PE-PGRS基因 Rv0834c 的表达。 ，在两个 M中均以pH依赖性方式存在。包皮垢和 M。结核病。 lipF 和 Rv0834c 的酸诱导能力与应力响应sigma因子SigF无关，因为它诱导了 M中两个基因的酸诱导。结核病sigF 突变株与野生型相似。在感染J774鼠巨噬细胞期间未观察到 lipF 或 Rv0834c 的诱导，这一观察结果与先前有关含 M吞噬体失败的报道相符。结核酸化。

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