TRANSCRIPTIONAL CONTROL OF THE INVASION REGULATORY GENE VIRB OF SHIGELLA-FLEXNERI - ACTIVATION BY VIRF AND REPRESSION BY H-NS
TRANSCRIPTIONAL CONTROL OF THE INVASION REGULATORY GENE VIRB OF SHIGELLA-FLEXNERI - ACTIVATION BY VIRF AND REPRESSION BY H-NS
复制标题
DOI：
10.1128/jb.175.19.6142-6149.1993
复制
发表时间：
1993-10-01
影响因子：
3.2
通讯作者：
SASAKAWA, C
中科院分区：
文献类型：
--
作者：
TOBE, T;YOSHIKAWA, M;SASAKAWA, C
由福氏志贺氏菌的 230 kb 大质粒编码的入侵基因的表达由 virB 基因控制，该基因本身由另一个调节因子 virF 激活。入侵基因的转录受到温度调节，因为它们在 37 摄氏度（而非 30 摄氏度）生长的细菌中被激活。最近，我们已经证明入侵基因的热调节表达是由virB转录的热激活介导的(T.Tobe、S.Nagai、B.Adler、M.Yoshikawa和C.Sasakawa，Mol.Microbiol.5：887-893，1991)。研究还表明，使 virR (hns) 产物 H-NS 失活的突变会抑制 virB 的转录。为了阐明 virB 激活的分子机制，我们确定了转录起始位点的位置，发现它位于 virB 编码序列 5' 端上游 54 bp。删除分析显示，virF 的转录激活需要一个 110 bp 的 DNA 片段延伸到转录起始位点的上游。通过对含有 malE'-'virF 融合基因（能够激活 virB 转录）的弗氏链霉菌粗提物进行蛋白质结合测定，两种蛋白质，一种为 70 kDa（MalE'-'VirF 融合），另一种为 16 kDa（H-NS），被证明与 virB 启动子区域特异性结合。 DNA足迹分析表明，VirF融合蛋白和H-NS蛋白分别与从-17到-117的上游序列和从-20到+20的序列结合，其中virB转录开始。在体外转录测定中，VirF 融合蛋白被证明可以激活 virB 转录，而 H-NS 蛋白则阻断它。仅当使用负超螺旋 DNA 作为模板时，才能观察到 virB 激活。在体内研究中，通过在培养基中添加新生霉素（一种促旋酶抑制剂）可显着降低 virB 转录，而通过突变 hns 可增加 virB 转录。这些体外和体内研究表明，virB 的转录是通过 VirF 以 DNA 拓扑依赖性方式与 virB 启动子上游序列结合而激活的，并通过 H-NS 与 virB 转录起始位点的结合直接抑制。
Expression of invasion genes encoded by the large 230-kb plasmid of Shigella flexneri is controlled by the virB gene, which is itself activated by another regulator, virF. Transcription of the invasion genes is temperature regulated, since they are activated in bacteria grown at 37 but not at 30-degrees-C. Recently, we have shown that the thermoregulated expression of invasion genes is mediated by thermal activation of virB transcription (T. Tobe, S. Nagai, B. Adler, M. Yoshikawa, and C. Sasakawa, Mol. Microbiol. 5:887-893, 1991). It has also been shown that a mutation that inactivates H-NS, the product of virR (hns), derepresses transcription of virB. To elucidate the molecular mechanisms underlying virB activation, we determined the location of the transcription start site and found it to be 54 bp upstream of the 5' end of the virB coding sequence. Deletion analysis revealed that transcriptional activation by virF requires a DNA segment of 110 bp extending upstream of the transcription start site. By using a protein binding assay with crude extracts of S. flexneri harboring the malE'-'virF fusion gene, which was able to activate virB transcription, two protein species, one of 70 kDa (MalE'-'VirF fusion) and another of 16 kDa (H-NS), were shown to bind specifically to the virB promoter region. DNA footprinting analysis indicated that the VirF fusion and H-NS proteins bound to the upstream sequence spanning from -17 to -117 and to the sequence from -20 to +20, in which virB transcription starts, respectively. In an in vitro transcription assay, the VirF fusion protein was shown to activate virB transcription while the H-NS protein blocked it. virB activation was seen only when negatively supercoiled DNA was used as a template. In in vivo studies, virB transcription was significantly decreased by adding novobiocin, a gyrase inhibitor, into the culture medium while virB transcription was increased by mutating hns. These in vitro and in vivo studies indicated that transcription of virB is activated through VirF binding to the upstream sequence of the virB promoter in a DNA-topology-dependent manner and is directly repressed by H-NS binding to the virB transcription start site.