Systematic targeted mutagenesis of Brucella melitensis 16M reveals a major role for GntR regulators in the control of virulence
Systematic targeted mutagenesis of Brucella melitensis 16M reveals a major role for GntR regulators in the control of virulence
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DOI：
10.1128/iai.73.9.5578-5586.2005
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发表时间：
2005-09-01
影响因子：
3.1
通讯作者：
De Bolle, X
中科院分区：
文献类型：
--
作者：
Haine, V;Sinon, A;De Bolle, X
为了鉴定参与梅氏布鲁氏菌毒力基因控制的转录调控因子，我们收集了88个突变体，包括AraC、ArsR、Crp、Deolk、GntR、IclR、LysR、MerR、RpiR和TOR调控因子家族。这个集合被命名为LiMuR （library of mutants for regulators）。我们开发了一种方法，可以在一只动物身上同时测试几个突变体，以确定那些无法存活的突变体。这种方法被称为质粒标记诱变法，用于在小鼠感染模型中测试突变体在1周后的残余毒力。鉴定出10个减毒突变体，其中6个和3个分别属于GntR和LysR家族，并分别确认在小鼠中以较低的速率复制。在这10个突变体中，只有gntR10和arsR6在细胞模型中被减弱。LiMuR还允许简单的筛选来识别特定基因或操纵子的调节因子。作为第一个例子，我们分析了virB操纵子在LiMuR突变体中的表达。我们对全细胞提取物进行Western blotting，使用针对VirB蛋白的多克隆抗血清分析VirB蛋白的产生。四个突变体产生少量的VirB蛋白，与野生型菌株相比，一个突变体过度表达VirB蛋白。在这五个突变体中，使用融合到lacZ的virB启动子的报告分析显示，三个突变体在转录水平上控制virB。LiMuR是一种资源，它将提供直接识别参与感兴趣基因控制的调节因子。
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, Deolk, GntR, IclR, LysR, MerR, RpiR, and TOR 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 I 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.