Biochemical characterization of the first essential two-component signal transduction system from Staphylococcus aureus and Streptococcus pneumoniae
Biochemical characterization of the first essential two-component signal transduction system from Staphylococcus aureus and Streptococcus pneumoniae
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DOI：
10.1159/000071077
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发表时间：
2003-01-01
影响因子：
1.2
通讯作者：
Wallis, NG
中科院分区：
文献类型：
--
作者：
Clausen, VA;Bae, WH;Wallis, NG
yycFG 双组分信号转导系统 (TCSTS) 已被证明对于多种革兰氏阳性菌的生存至关重要。然而，该基因对的功能仍然未知。有趣的是，虽然这两种成分对金黄色葡萄球菌和枯草芽孢杆菌都是必需的，但只有反应调节剂 (yycF) 对肺炎链球菌是必需的。为了进一步研究这种重要的 TCSTS，在生化水平上对肺炎链球菌和金黄色葡萄球菌截短的 YycG 组氨酸激酶和全长 YycF 反应调节蛋白进行了表征。来自两种生物体的重组蛋白在大肠杆菌中表达并纯化。 YycG 自磷酸化活性被铵激活。金黄色葡萄球菌YycG自磷酸化的表观K-m (ATP)为130μM，肺炎链球菌为3.0μM。每个都有类似的 k(cat) 值，分别为 0.036 和 0.024 min(-1)。还研究了同源磷酸转移，表明反应过程中磷酸化 YycG 中间体的水平不同。在其同源 YycF 存在的情况下，无法检测到肺炎链球菌 YycG 磷酸化中间体，而同时检测到磷酸化金黄色葡萄球菌 YycG 和 YycF。此外，两个物种之间还存在非同源磷酸转移。这些研究在生化水平上彻底比较了两个物种的基本 YycFG TCSTS，并建立了测定这些抗菌靶点活性的方法。版权所有 (C) 2003 S. Karger AG，巴塞尔。
The yycFG two-component signal transduction system (TCSTS) has been shown to be essential to the viability of several gram-positive bacteria. However, the function of the gene pair remains unknown. Interestingly, while both components are essential to Staphylococcus aureus and Bacillus subtilis, only the response regulator (yycF) is essential to Streptococcus pneumoniae. To study this essential TCSTS further, the S. pneumoniae and S. aureustruncated YycG histidine kinase and full-length YycF response regulator proteins were characterized at a biochemical level. The recombinant proteins from both organisms were expressed in Escherichia coli and purified. The YycG autophosphorylation activities were activated by ammonium. The apparent K-m (ATP) of S. aureus YycG autophosphorylation was 130 muM and S. pneumoniae was 3.0 muM. Each had similar k(cat) values of 0.036 and 0.024 min(-1), respectively. Cognate phosphotransfer was also investigated indicating different levels of the phosphorylated YycG intermediates during the reaction. The S. pneumoniae YycG phosphorylated intermediate was not detectable in the presence of its cognate YycF, while phosphorylated S. aureus YycG and YycF were detected concurrently. In addition, noncognate phosphotransfer was demonstrated between the two species. These studies thoroughly compare the essential YycFG TCSTS from the two species at the biochemical level and also establish methods for assaying the activities of these antibacterial targets. Copyright (C) 2003 S. Karger AG, Basel.