Haine Valérie, Sinon Audrey, Van Steen Frédéric, Rousseau Stéphanie, Dozot Marie, Lestrate Pascal, Lambert Christophe, Letesson Jean-Jacques, De Bolle Xavier
Unité de Recherche en Biologie Moléculaire, Facultés Universitaires Notre Dame de la Paix, 61 rue de Bruxelles, B-5000 Namur, Belgium.
Infect Immun. 2005 Sep;73(9):5578-86. doi: 10.1128/IAI.73.9.5578-5586.2005.
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.
为了鉴定参与调控布鲁氏菌毒力基因的转录调节因子，我们构建了一个包含88个突变体的文库，这些突变体分别来自AraC、ArsR、Crp、DeoR、GntR、IclR、LysR、MerR、RpiR和TetR家族的调节因子。这个文库被命名为LiMuR（调节因子突变体文库）。我们开发了一种方法，可以在一只动物中同时检测多个突变体，以鉴定那些无法存活的突变体。这种方法称为质粒标记诱变方法，用于在小鼠感染模型中检测感染1周后突变体的残余毒力。我们鉴定出10个减毒突变体，其中6个和3个分别属于GntR和LysR家族，并单独确认它们在小鼠体内的复制速率较低。在这10个突变体中，只有gntR10和arsR6在细胞模型中表现出减毒。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因子。作为第一个例子，我们分析了LiMuR突变体中virB操纵子的表达。我们使用针对VirB蛋白的多克隆抗血清对全细胞提取物进行Western印迹分析，以检测VirB蛋白的产生。与野生型菌株相比，有4个突变体产生少量的VirB蛋白，1个突变体过度表达VirB蛋白。在这5个突变体中，使用与lacZ融合的virB启动子进行报告基因分析表明，有3个突变体在转录水平上调控virB。LiMuR是一种资源，将为直接鉴定参与调控感兴趣基因的调节因子提供便利。
