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Tet-Trap a genetic approach to the identification of bacterial RNA thermometers: application to Pseudomonas aeruginosa

机译：Tet-Trap一种鉴定细菌RNA温度计的遗传方法：应用于铜绿假单胞菌

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Modulation of mRNA translatability either by trans-acting factors (proteins or sRNAs) or by in cis-acting riboregulators is widespread in bacteria and controls relevant phenotypic traits. Unfortunately, global identification of post-transcriptionally regulated genes is complicated by poor structural and functional conservation of regulatory elements and by the limitations of proteomic approaches in protein quantification. We devised a genetic system for the identification of post-transcriptionally regulated genes and we applied this system to search for Pseudomonas aeruginosa RNA thermometers, a class of regulatory RNA that modulates gene translation in response to temperature changes. As P. aeruginosa is able to thrive in a broad range of environmental conditions, genes differentially expressed at 37°C versus lower temperatures may be involved in infection and survival in the human host. We prepared a plasmid vector library with translational fusions of P. aeruginosa DNA fragments (PaDNA) inserted upstream of TIP2, a short peptide able to inactivate the Tet repressor (TetR) upon expression. The library was assayed in a streptomycin-resistant merodiploid rpsL+/rpsL31 Escherichia coli strain in which the dominant rpsL+ allele, which confers streptomycin sensitivity, was repressed by TetR. PaDNA fragments conferring thermosensitive streptomycin resistance (i.e., expressing PaDNA–TIP2 fusions at 37°C, but not at 28°C) were sequenced. We identified four new putative thermosensors. Two of them were validated with conventional reporter systems in E. coli and P. aeruginosa. Interestingly, one regulates the expression of ptxS, a gene implicated in P. aeruginosa pathogenesis.
机译：反式作用因子（蛋白质或sRNA）或顺式作用核糖调节剂对mRNA可翻译性的调节在细菌中很普遍，并控制相关的表型性状。不幸的是，转录后调节基因的全球鉴定由于调节元件的结构和功能保守性差以及蛋白质组学方法在蛋白质定量中的局限性而变得复杂。我们设计了一个用于识别转录后调控基因的遗传系统，并将其应用于搜索铜绿假单胞菌RNA温度计，这是一类调控RNA，可响应温度变化而调节基因翻译。由于铜绿假单胞菌能够在广泛的环境条件下生长，因此在37℃与较低温度下差异表达的基因可能与人类宿主的感染和存活有关。我们准备了一个质粒载体文库，该文库具有插入TIP2上游的铜绿假单胞菌DNA片段（PaDNA）的翻译融合，TIP2是一种短肽，能够在表达时失活Tet阻遏物（TetR）。该文库在抗链霉素的类金属倍体rpsL + / rpsL31大肠杆菌菌株中进行了分析，在该菌株中，赋予TetR抑制链霉素敏感性的显性rpsL + 等位基因。对赋予热敏性链霉素抗性的PaDNA片段（即在37°C而不是28°C时表达PaDNA–TIP2融合体）进行测序。我们确定了四个新的假定热传感器。其中两个已在大肠杆菌和铜绿假单胞菌中使用常规报告系统进行了验证。有趣的是，人们调节了与铜绿假单胞菌发病机理有关的基因ptxS的表达。

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