Parsot C, Mekalanos J J
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
J Bacteriol. 1992 Aug;174(16):5211-8. doi: 10.1128/jb.174.16.5211-5218.1992.
The Vibrio cholerae acfA, B, C, and D genes are involved in the synthesis of a colonization factor; their expression is under the control of ToxR, the cholera toxin transcriptional activator. By a combination of Southern blot analysis, cloning, and nucleotide sequence analysis, we determined that the acf genes are clustered on a 5-kb region, the acfA and acfD genes are transcribed divergently, and the translation start sites of the two genes are separated by only 173 bp. Expression from the acfA and acfD promoters in V. cholerae was studied by using acfA:phoA translational and acfD-lacZ transcriptional fusions; when carried by the chromosome, the acfA-acfD intergenic region flanked by the two reporter genes was found to contain the cis-acting element(s) necessary for the environmental regulation of the two promoters. However, this regulation was almost completely abolished when the same construction was carried by a low-copy-number plasmid. These results suggested that differences in DNA topology between the plasmid versus the chromosomal constructs might influence the expression of the acfA and acfD promoters. Support for this conclusion was obtained by showing that ToxR-dependent but not basal expression of both promoters was strongly inhibited by nalidixic acid and novobiocin, two DNA gyrase inhibitors, suggesting that the activation of these promoters is affected by changes in DNA supercoiling. Expression of the acfA and acfD promoters was also investigated in the heterologous host Escherichia coli harboring plasmids expressing either ToxR or ToxT, two transcriptional activators of the V. cholerae virulence genes. ToxR activated the acfD promoter 2.5-fold but inhibited the acfA promoter 2-fold. In contrast, the expression of the acfA promoter was activated 10-fold and that of the acfD promoter was activated 3-fold by ToxT, supporting the previously proposed cascade model for organization of the ToxR regulon.
霍乱弧菌的acfA、B、C和D基因参与一种定植因子的合成；它们的表达受霍乱毒素转录激活因子ToxR的控制。通过Southern印迹分析、克隆和核苷酸序列分析相结合的方法，我们确定acf基因聚集在一个5kb的区域，acfA和acfD基因转录方向相反，且两个基因的翻译起始位点仅相隔173bp。通过使用acfA:phoA翻译融合和acfD-lacZ转录融合研究了霍乱弧菌中acfA和acfD启动子的表达；当由染色体携带时，发现两个报告基因侧翼的acfA-acfD基因间区域包含两个启动子环境调节所需的顺式作用元件。然而，当相同构建体由低拷贝数质粒携带时，这种调节几乎完全被消除。这些结果表明质粒与染色体构建体之间DNA拓扑结构的差异可能影响acfA和acfD启动子的表达。通过显示两种DNA回旋酶抑制剂萘啶酸和新生霉素强烈抑制两个启动子的ToxR依赖性但非基础表达，获得了对这一结论的支持，这表明这些启动子的激活受DNA超螺旋变化的影响。还在携带表达ToxR或ToxT（霍乱弧菌毒力基因的两种转录激活因子）的质粒的异源宿主大肠杆菌中研究了acfA和acfD启动子的表达。ToxR激活acfD启动子2.5倍，但抑制acfA启动子2倍。相反，ToxT激活acfA启动子10倍，激活acfD启动子3倍，支持了先前提出的ToxR调控子组织的级联模型。
