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首页> 外文期刊>Infection and immunity >Systematic Targeted Mutagenesis of Brucella melitensis 16M Reveals a Major Role for GntR Regulators in the Control of Virulence
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Systematic Targeted Mutagenesis of Brucella melitensis 16M Reveals a Major Role for GntR Regulators in the Control of Virulence

机译：布鲁氏菌16M的系统化的靶向诱变揭示了GntR调节剂在毒力控制中的主要作用。

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In order to identify transcriptional regulators involved in virulence gene control in Brucella melitensis, we generated a collection of 88 mutants in the AraC, ArsR, Crp, DeoR, GntR, IclR, LysR, MerR, RpiR, and TetR families of regulators. This collection was named LiMuR (library of mutants for regulators). We developed a method to test several mutants simultaneously in one animal in order to identify those unable to survive. This method, called the plasmid-tagged mutagenesis method, was used to test the residual virulence of mutants after 1 week in a mouse model of infection. Ten attenuated mutants, of which six and three belong to the GntR and LysR families, respectively, were identified and individually confirmed to replicate at lower rates in mice. Among these 10 mutants, only gntR10 and arsR6 are attenuated in cellular models. The LiMuR also allows simple screenings to identify regulators of a particular gene or operon. As a first example, we analyzed the expression of the virB operon in the LiMuR mutants. We carried out Western blottings of whole-cell extracts to analyze the production of VirB proteins using polyclonal antisera against VirB proteins. Four mutants produced small amounts of VirB proteins, and one mutant overexpressed VirB proteins compared to the wild-type strain. In these five mutants, reporter analysis using the virB promoter fused to lacZ showed that three mutants control virB at the transcriptional level. The LiMuR is a resource that will provide straightforward identification of regulators involved in the control of genes of interest.
机译：为了鉴定出布鲁氏菌中毒力基因控制的转录调控因子，我们在AraC，ArsR，Crp，DeoR，GntR，IclR，LysR，MerR，RpiR和TetR系列监管机构。该集合被命名为LiMuR（用于 r 调节器的 mu tant的 li 库）。我们开发了一种在一只动物中同时测试多个突变体的方法，以鉴定那些无法存活的突变体。该方法称为质粒标记诱变法，用于在小鼠感染模型中1周后测试突变体的残留毒力。鉴定了十个减毒的突变体，其中六个和三个分别属于GntR和LysR家族，并被单独确认以较低的速率在小鼠中复制。在这10个突变体中，只有 gntR10 和 arsR6 在细胞模型中被减毒。 LiMuR还允许进行简单的筛选，以鉴定特定基因或操纵子的调节子。作为第一个例子，我们分析了LiMuR突变体中 virB 操纵子的表达。我们进行了全细胞提取物的蛋白质印迹分析，以使用针对VirB蛋白的多克隆抗血清分析VirB蛋白的产生。与野生型菌株相比，四个突变体产生少量的VirB蛋白，而一个突变体过表达的VirB蛋白。在这五个突变体中，使用与 lacZ 融合的 virB 启动子进行的报告基因分析表明，三个突变体在转录水平上控制 virB 。 LiMuR是一种资源，可以直接识别参与目标基因控制的调节子。

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