Regulatory elements of the Staphylococcus aureus protein A (Spa) promoter
Regulatory elements of the Staphylococcus aureus protein A (Spa) promoter
复制标题
DOI：
10.1128/jb.186.12.3738-3748.2004
复制
发表时间：
2004-06-01
影响因子：
3.2
通讯作者：
Stewart, GC
中科院分区：
文献类型：
--
作者：
Gao, JX;Stewart, GC
金黄色葡萄球菌蛋白A（SPA）是金黄色葡萄球菌的重要毒力因子。spa决定簇的转录发生在指数生长期，当细胞进入指数后生长期时被抑制。已经发现spa表达的调节是复杂的，调节涉及多个因素，包括Agr、SarA、SarS、SarT、Rot和MgrA。我们对这些因子如何作用于spa启动子以调节spa表达的理解是不完整的。为了确定SPA启动子内的调控位点，对缺乏一种或多种调控因子的宿主菌株中的启动子的缺失衍生物进行了分析，并揭示了SPA调控的几个关键特征。通过引物延伸确定了spa的转录起始位点。将spa启动子序列亚克隆在无启动子的氯霉素乙酰转移酶报告基因的前面。构建了具有相同3'端的不同长度的spa截短体，并将所得质粒转导到具有不同调控遗传背景的菌株中。我们的研究结果确定了上游启动子序列所需的Agr系统的SPA表达调控。确定了SarS活性（spa表达的激活剂）和SarA活性（spa表达的阻遏物）的顺式元件。SPA启动子上的良好表征的SarA共有序列被发现不足以用于SPA启动子的SarA阻遏。完全抑制需要存在的第二个共识网站相邻的SarS结合位点。发现核心启动子序列的直接上游序列刺激转录。
Staphylococcal protein A (Spa) is an important virulence factor of Staphylococcus aureus. Transcription of the spa determinant occurs during the exponential growth phase and is repressed when the cells enter the postexponential growth phase. Regulation of spa expression has been found to be complicated, with regulation involving multiple factors, including Agr, SarA, SarS, SarT, Rot, and MgrA. Our understanding of how these factors work on the spa promoter to regulate spa expression is incomplete. To identify regulatory sites within the spa promoter, analysis of deletion derivatives of the promoter in host strains deficient in one or more of the regulatory factors was undertaken, and several critical features of spa regulation were revealed. The transcriptional start sites of spa were determined by primer extension. The spa promoter sequences were subcloned in front of a promoterless chloramphenicol acetyltransferase reporter gene. Various lengths of spa truncations with the same 3' end were constructed, and the resultant plasmids were transduced into strains with different regulatory genetic backgrounds. Our results identified upstream promoter sequences necessary for Agr system regulation of spa expression. The cis elements for SarS activity, an activator of spa expression, and for SarA activity, a repressor of spa expression, were identified. The well-characterized SarA consensus sequence on the spa promoter was found to be insufficient for SarA repression of the spa promoter. Full repression required the presence of a second consensus site adjacent to the SarS binding site. Sequences directly upstream of the core promoter sequence were found to stimulate transcription.