Identification of genes controlled by the essential YycG/YycF two-component system of Staphylococcus aureus
Identification of genes controlled by the essential YycG/YycF two-component system of Staphylococcus aureus
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DOI：
10.1128/jb.186.4.1175-1181.2004
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发表时间：
2004-02-01
影响因子：
3.2
通讯作者：
Msadek, T
中科院分区：
文献类型：
--
作者：
Dubrac, S;Msadek, T
最初在枯草芽孢杆菌中鉴定的YycG/YycF必需双组分系统（TCS）是非常高度保守的，并且似乎对低G +C革兰氏阳性细菌具有特异性，包括几种病原体，如金黄色葡萄球菌。通过对S.金黄色葡萄球菌细胞中，yyc操纵子由异丙基-β-D-硫代半乳糖苷（IPTG）诱导型启动子控制，我们已经证明该系统在金黄色葡萄球菌中是必需的。金黄色葡萄球菌在37 ℃生长期间，YycG/YycF调节系统的饥饿导致细胞死亡。在先前对B的YycG/YycF TCS的研究中，在枯草芽孢杆菌中，我们定义了一个潜在的YycF共有识别序列，由两个六核苷酸直接重复序列组成，由五个核苷酸分开[5 '-TGT（A/T）A（A/T/C）-N-5，-TGT（A/T）A（A/T/C）-3']。对S.金黄色葡萄球菌基因组表明，该序列之前可能有12个基因，其中5个涉及毒力。进行了体外方法，以确定这些基因中的哪些是由YycF控制的。对S.金黄色葡萄球菌在大肠杆菌中过量生产并纯化。在体外显示YycG激酶的自磷酸化和磷酸转移到YycF。凝胶迁移率变化和DNA酶I足迹分析被用来显示直接结合在体外纯化的YycF的ssaA基因的启动子区，编码的主要抗原，以前建议由YycF控制。YycF也被证明能特异性地结合到两个基因的启动子区域，编码IsaA抗原和LytM肽聚糖水解酶，与该系统在控制毒力和细胞壁代谢中的拟议作用一致。
The YycG/YycF essential two-component system (TCS), originally identified in Bacillus subtilis, is very highly conserved and appears to be specific to low-G+C gram-positive bacteria, including several pathogens such as Staphylococcus aureus. By studying growth of S. aureus cells where the yyc operon is controlled by an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter, we have shown that this system is essential in S. aureus during growth at 37degreesC and that starvation for the YycG/YycF regulatory system leads to cell death. During a previous study of the YycG/YycF TCS of B. subtilis, we defined a potential YycF consensus recognition sequence, consisting of two hexanucleotide direct repeats, separated by five nucleotides [5'-TGT(A/T)A(A/T/C)-N-5,-TGT (A/T)A(A/T/C)-3']. A detailed DNA motif analysis of the S. aureus genome indicates that there are potentially 12 genes preceded by this sequence, 5 of which are involved in virulence. An in vitro approach was undertaken to determine which of these genes are controlled by YycF. The YycG and YycF proteins of S. aureus were overproduced in Escherichia coli and purified. Autophosphorylation of the YycG kinase and phosphotransfer to YycF were shown in vitro. Gel mobility shift and DNase I footprinting assays were used to show direct binding in vitro of purified YycF to the promoter region of the ssaA gene, encoding a major antigen and previously suggested to be controlled by YycF. YycF was also shown to bind specifically to the promoter regions of two genes, encoding the IsaA antigen and the LytM peptidoglycan hydrolase, in agreement with the proposed role of this system in controlling virulence and cell wall metabolism.