Institute of Medical Microbiology, Immunology and Parasitology, University of Bonn, Bonn, Germany.
PLoS One. 2012;7(1):e30403. doi: 10.1371/journal.pone.0030403. Epub 2012 Jan 20.
The YycFG two-component regulatory system (TCS) of Staphylococcus aureus represents the only essential TCS that is almost ubiquitously distributed in gram-positive bacteria with a low G+C-content. YycG (WalK/VicK) is a sensor histidine-kinase and YycF (WalR/VicR) is the cognate response regulator. Both proteins play an important role in the biosynthesis of the cell envelope and mutations in these proteins have been involved in development of vancomycin and daptomycin resistance.
METHODOLOGY/PRINCIPAL FINDINGS: Here we present high yield expression and purification of the full-length YycG and YycF proteins as well as of the auxiliary proteins YycH and YycI of Staphylococcus aureus. Activity tests of the YycG kinase and a mutated version, that harbours an Y306N exchange in its cytoplasmic PAS domain, in a detergent-micelle-model and a phosholipid-liposome-model showed kinase activity (autophosphorylation and phosphoryl group transfer to YycF) only in the presence of elevated concentrations of alkali salts. A direct comparison of the activity of the kinases in the liposome-model indicated a higher activity of the mutated YycG kinase. Further experiments indicated that YycG responds to fluidity changes in its microenvironment.
CONCLUSIONS/SIGNIFICANCE: The combination of high yield expression, purification and activity testing of membrane and membrane-associated proteins provides an excellent experimental basis for further protein-protein interaction studies and for identification of all signals received by the YycFGHI system.
金黄色葡萄球菌的 YycFG 双组分调控系统（TCS）是唯一一种几乎普遍存在于低 GC 含量革兰氏阳性菌中的必需 TCS。YycG（WalK/VicK）是一种传感器组氨酸激酶，而 YycF（WalR/VicR）是其同源响应调节剂。这两种蛋白质在细胞壁生物合成中都起着重要作用，这些蛋白质的突变与万古霉素和达托霉素耐药性的产生有关。
方法/主要发现：在此，我们展示了金黄色葡萄球菌全长 YycG 和 YycF 蛋白以及辅助蛋白 YycH 和 YycI 的高产量表达和纯化。在去污剂胶束模型和磷脂双层模型中，对 YycG 激酶及其在细胞质 PAS 结构域中含有 Y306N 交换的突变体进行了活性测试，结果表明，只有在高浓度碱盐存在的情况下，激酶才具有活性（自身磷酸化和磷酸基团转移到 YycF）。在脂质体模型中对激酶活性的直接比较表明，突变的 YycG 激酶具有更高的活性。进一步的实验表明，YycG 对其微环境中的流动性变化有反应。
结论/意义：膜和膜相关蛋白的高产表达、纯化和活性测试的结合，为进一步的蛋白质-蛋白质相互作用研究以及鉴定 YycFGHI 系统接收到的所有信号提供了极好的实验基础。
