Identification of amino acid residues of Salmonella SlyA that are critical for transcriptional regulation.
Identification of amino acid residues of Salmonella SlyA that are critical for transcriptional regulation.
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DOI：
10.1099/mic.0.29259-0
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发表时间：
2007-02
期刊：
影响因子：
1.5
通讯作者：
N. Okada;Yorie Oi;Mayuko Takeda-Shitaka;Kazuhiko Kanou;Hideaki Umeyama;Takeshi Haneda;T. Miki;Sachiko Hosoya;H. Danbara
中科院分区：
文献类型：
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作者：
N. Okada;Yorie Oi;Mayuko Takeda-Shitaka;Kazuhiko Kanou;Hideaki Umeyama;Takeshi Haneda;T. Miki;Sachiko Hosoya;H. Danbara
沙门氏菌致病岛2（SPI-2）编码的III型分泌系统是肠道沙门氏菌在细胞内生存和复制所必需的。SPI-2基因的表达依赖于SPI-2区域编码的双组分调控系统SsrA（SpiR）/SsrB。本文表明，SlyA通过与ssrA启动子结合来调节传感激酶SsrA的转录，表明SlyA直接参与SPI-2基因表达的调节。构建了SlyA二聚体与DNA复合物的结构模型。SlyA的模型表明其结构与其他马尔R家族蛋白非常相似。基于该模型，对位于DNA结合所需的翼状螺旋区域和SlyA蛋白二聚化所需的N-末端和C-末端区域的α-螺旋中的残基进行定点诱变，以鉴定对SlyA功能至关重要的残基。9个突变体的SlyA单取代不能激活ssrA在体内转录。这些突变体SlyA蛋白揭示了翼形螺旋区域内的残基Leu-63、瓦尔-64、Arg-65、Leu-67、Leu-70、Arg-86和Lys-88是DNA结合所需的，并且N-末端和C-末端区域的α-螺旋内的残基Leu-12和Leu-126是有效二聚体形成所需的。携带表达在这些氨基酸位置含有突变的SlyA衍生物的质粒的沙门氏菌SlyA突变菌株在感染的HeLa细胞中没有表现出恢复的SlyA功能，从而证实了SlyA蛋白的结构和功能关系。
The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2) is essential for the intracellular survival and replication of Salmonella enterica. The expression of SPI-2 genes is dependent on a two-component regulatory system, SsrA (SpiR)/SsrB, encoded in the SPI-2 region. This paper shows that SlyA regulates transcription of the sensor kinase SsrA by binding to the ssrA promoter, indicating that SlyA is directly involved in the regulation of SPI-2 gene expression. A structure model of the SlyA dimer in complex with DNA was constructed. The model of SlyA indicated that its structure is very similar to that of other MarR family proteins. Based on this model, site-directed mutagenesis of residues located in the winged-helix region required for DNA binding and in the alpha-helices of the N-terminal and C-terminal regions required for dimerization of the SlyA protein was performed to identify the residues that are critical for SlyA function. Nine mutants of SlyA with single substitutions were unable to activate ssrA transcription in vivo. These mutant SlyA proteins revealed that the residues Leu-63, Val-64, Arg-65, Leu-67, Leu-70, Arg-86 and Lys-88 within the winged-helix region are required for DNA binding, and residues Leu-12 and Leu-126 within the alpha-helices of the N-terminal and C-terminal regions are required for efficient dimer formation. A Salmonella slyA mutant strain carrying a plasmid expressing SlyA derivatives containing mutations at these amino acid positions did not exhibit restored SlyA function in infected HeLa cells, thereby confirming the structural and functional relationships of the SlyA protein.