Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48109-1078, USA.
Infect Immun. 2010 Oct;78(10):4122-33. doi: 10.1128/IAI.00566-10. Epub 2010 Aug 2.
ToxR-dependent recruitment of TcpP to the toxT promoter facilitates toxT transcription in Vibrio cholerae, initiating a regulatory cascade that culminates in cholera toxin expression and secretion. Although TcpP usually requires ToxR to activate the toxT promoter, TcpP overexpression can circumvent the requirement for ToxR in this process. To define nucleotides critical for TcpP-dependent promoter recognition and activation, a series of toxT promoter derivatives with single-base-pair transversions spanning the TcpP-binding site were generated and used as plasmid-borne toxT-lacZ fusions, as DNA mobility shift targets, and as allelic replacements of the chromosomal toxT promoter. When present in ΔtoxR V. cholerae overexpressing TcpP, several transversions affecting nucleotides within two direct repeats present in the TcpP-binding region (TGTAA-N(6)-TGTAA) caused defects in TcpP-dependent toxT-lacZ fusion activation and toxin production. Electrophoretic mobility shift assays demonstrated that these same transversions reduced the affinity of the toxT promoter for TcpP. The presence of ToxR suppressed transcription activation defects associated with most, but not all, transversions. Particularly, the central thymine nucleotide of both pentameric repeats was essential for efficient toxT activation, even in the presence of ToxR. These results suggest that the toxT promoter recognition function provided by ToxR can facilitate the interaction of TcpP with the toxT promoter but is insufficient for promoter activation when the TcpP-binding site has been severely compromised by mutation. Thus, the interaction of TcpP with nucleotides of the direct repeat sequences appears to be a prerequisite for toxT promoter activation.
ToxR 依赖性募集 TcpP 到 toxT 启动子有助于霍乱弧菌中转录 toxT，启动一个调节级联反应，最终导致霍乱毒素的表达和分泌。尽管 TcpP 通常需要 ToxR 来激活 toxT 启动子，但 TcpP 的过表达可以规避这个过程中对 ToxR 的需求。为了确定 TcpP 依赖性启动子识别和激活所必需的核苷酸，我们生成了一系列跨越 TcpP 结合位点的单个碱基对颠换的 toxT 启动子衍生物，并将其用作质粒携带的 toxT-lacZ 融合、DNA 迁移率变动靶标，以及染色体 toxT 启动子的等位基因替换。当在过度表达 TcpP 的 ΔtoxR 霍乱弧菌中存在时，几个影响 TcpP 结合区域（TGTAA-N(6)-TGTAA）中两个直接重复序列内核苷酸的颠换导致 TcpP 依赖性 toxT-lacZ 融合激活和毒素产生缺陷。电泳迁移率变动分析表明，这些相同的颠换降低了 toxT 启动子与 TcpP 的亲和力。ToxR 的存在抑制了与大多数但不是所有颠换相关的转录激活缺陷。特别是，两个五聚体重复的中央胸腺嘧啶核苷酸对于有效的 toxT 激活是必需的，即使在存在 ToxR 的情况下也是如此。这些结果表明，ToxR 提供的 toxT 启动子识别功能可以促进 TcpP 与 toxT 启动子的相互作用，但当 TcpP 结合位点因突变而严重受损时，启动子激活是不够的。因此，TcpP 与直接重复序列核苷酸的相互作用似乎是 toxT 启动子激活的前提条件。
