Tobe T, Yoshikawa M, Mizuno T, Sasakawa C
Department of Bacteriology, University of Tokyo, Japan.
J Bacteriol. 1993 Oct;175(19):6142-9. doi: 10.1128/jb.175.19.6142-6149.1993.
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 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.
弗氏志贺氏菌230 kb大质粒编码的侵袭基因的表达受virB基因控制，而virB基因本身又被另一个调节因子virF激活。侵袭基因的转录受温度调节，因为它们在37℃生长的细菌中被激活，而在30℃时不被激活。最近，我们已经表明侵袭基因的温度调节表达是由virB转录的热激活介导的（T. Tobe、S. Nagai、B. Adler、M. Yoshikawa和C. Sasakawa，《分子微生物学》5:887 - 893，1991）。还表明，使virR（hns）的产物H - NS失活的突变会解除对virB转录的抑制。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处。缺失分析表明，virF的转录激活需要一个从转录起始位点向上游延伸110 bp的DNA片段。通过使用含有能够激活virB转录的malE'-'virF融合基因的弗氏志贺氏菌粗提物进行蛋白质结合试验，显示两种蛋白质，一种70 kDa（MalE'-'VirF融合蛋白）和另一种16 kDa（H - NS），特异性结合到virB启动子区域。DNA足迹分析表明，VirF融合蛋白和H - NS蛋白分别结合到从 - 17至 - 117的上游序列和virB转录起始的 - 20至 + 20序列。在体外转录试验中，VirF融合蛋白被证明可激活virB转录，而H - NS蛋白则抑制它。只有当使用负超螺旋DNA作为模板时才能看到virB的激活。在体内研究中，通过向培养基中添加拓扑异构酶抑制剂新生霉素，virB转录显著降低，而通过使hns突变，virB转录增加。这些体外和体内研究表明，virB的转录通过VirF以DNA拓扑结构依赖的方式结合到virB启动子的上游序列而被激活，并被H - NS结合到virB转录起始位点直接抑制。
