cis-Acting Elements That Control Expression of the Master Virulence Regulatory Gene atxA in Bacillus anthracis
cis-Acting Elements That Control Expression of the Master Virulence Regulatory Gene atxA in Bacillus anthracis
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DOI：
10.1128/jb.00776-12
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发表时间：
2012-08-01
影响因子：
3.2
通讯作者：
Koehler, Theresa M.
中科院分区：
文献类型：
--
作者：
Dale, Jennifer L.;Raynor, Malik J.;Koehler, Theresa M.
炭疽芽孢杆菌炭疽毒素蛋白和荚膜生物合成操纵子结构基因的转录受到AtxA（一种具有独特性质的转录调节因子）的正向调节。与 atxA 在毒力因子表达中的作用一致，炭疽芽孢杆菌 atxA 无效突变体在炭疽小鼠模型中是无毒力的。在培养中，多种信号影响 atxA 转录物水平，并且 atxA 表达的时间和稳态水平对于最佳毒素和胶囊合成至关重要。尽管 atxA 转录的控制明显复杂，但只有一种反式作用蛋白，即过渡态调节蛋白 AbrB，已被证明可以直接与 atxA 启动子相互作用。在这里，我们采用 atxA 控制区的 5' 和 3' 删除分析和定点诱变来证明 atxA 从主要起始位点 P1 的转录依赖于看家 sigma 因子 SigA 的共有序列和 RNA 聚合酶的富含 A+T 的上游元件。我们还表明，额外的反式作用蛋白特异性结合位于相对于 P1 的 -13 和 +36 之间的 atxA 启动子序列，并对转录产生负面影响。删除该区域可使启动子活性增加高达 15 倍。该区域内 9 bp 回文序列的定点诱变可防止反式作用蛋白的结合，从而将启动子活性提高 7 倍，并导致 AtxA 和炭疽毒素产量相应增加。值得注意的是，在培养过程中产生升高水平的 AtxA 和毒素蛋白的 atxA 启动子突变体在炭疽鼠模型中的毒力不受影响。
Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule is positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In culture, multiple signals impact atxA transcript levels, and the timing and steady-state level of atxA expression are critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to interact directly with the atxA promoter. Here we employ 5' and 3' deletion analysis and site-directed mutagenesis of the atxA control region to demonstrate that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA and an A+T-rich upstream element for RNA polymerase. We also show that an additional trans-acting protein(s) binds specifically to atxA promoter sequences located between -13 and +36 relative to P1 and negatively impacts transcription. Deletion of this region increases promoter activity up to 15-fold. Site-directed mutagenesis of a 9-bp palindromic sequence within the region prevents binding of the trans-acting protein(s), increasing promoter activity 7-fold and resulting in a corresponding increase in AtxA and anthrax toxin production. Notably, an atxA promoter mutant that produced elevated levels of AtxA and toxin proteins during culture was unaffected for virulence in a murine model for anthrax.