Beloin Christophe, Dorman Charles J
Department of Microbiology, Moyne Institute of Preventive Medicine, University of Dublin, Dublin2, Ireland.
Mol Microbiol. 2003 Feb;47(3):825-38. doi: 10.1046/j.1365-2958.2003.03347.x.
The H-NS nucleoid structuring protein has been shown previously to play a negative role in controlling virulence gene expression in Shigella flexneri by repressing transcription of the virF and virB regulatory genes and the VirF-dependent icsA structural gene under non-permissive growth conditions. Here, we show that H-NS also acts at the promoters of the VirB-dependent structural genes in the regulatory cascade. H-NS protein binds to the promoter regions in vivo and in vitro. The promoters were shown physically and by in silico analysis to contain regions of DNA curvature, a feature of H-NS binding sites. H-NS binding sites were determined by DNase I footprinting at the icsB and the virA promoters. The locations of these sites were consistent with a role for H-NS as a transcription repressor. The VirB-dependent structural gene promoters were found to respond directly to the H-NS repressor, revealing a level of control that is additional to that exerted by the H-NS-dependent virB activator gene. Moreover, the promoters were sensitive to the level of VirB protein in the cell, requiring a threshold level of VirB to be reached before becoming active. A model is discussed in which the levels of expression of the structural genes reflect the outcome of competition between the countervailing regulatory activities of the H-NS and VirB proteins.
先前的研究表明，在非允许生长条件下，H-NS类核结构蛋白通过抑制virF和virB调控基因以及VirF依赖性icsA结构基因的转录，在控制福氏志贺氏菌毒力基因表达方面发挥负向作用。在此，我们表明H-NS在调控级联反应中也作用于VirB依赖性结构基因的启动子。H-NS蛋白在体内和体外均与启动子区域结合。通过物理方法和计算机分析表明，这些启动子含有DNA弯曲区域，这是H-NS结合位点的一个特征。通过DNase I足迹法确定了icsB和virA启动子处的H-NS结合位点。这些位点的位置与H-NS作为转录抑制因子的作用一致。发现VirB依赖性结构基因启动子直接响应H-NS抑制因子，揭示了一种除H-NS依赖性virB激活基因所发挥作用之外的调控水平。此外，这些启动子对细胞中VirB蛋白的水平敏感，在激活之前需要达到一定的VirB阈值水平。文中讨论了一个模型，其中结构基因的表达水平反映了H-NS和VirB蛋白相互对抗的调控活性之间竞争的结果。
