Uhl M A, Miller J F
Department of Microbiology and Immunology, School of Medicine, University of California, Los Angeles 90024.
Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):1163-7. doi: 10.1073/pnas.91.3.1163.
Expression of adhesins, toxins, and other virulence factors of Bordetella pertussis is under control of the BvgA and BvgS proteins, members of a bacterial two-component signal transduction family. BvgA bears sequence similarity to regulator components, whereas BvgS shows similarity to both sensor and regulator components. BvgA and the cytoplasmic portion of BvgS ('BvgS) were overexpressed and purified. 'BvgS autophosphorylated with the gamma-phosphate from [gamma-32P]ATP and phosphorylated BvgA. Kinetic analysis indicated that BvgA receives its phosphate from 'BvgS. Mutations in the transmitter, receiver, and C-terminal domains of BvgS were tested for activation of a BvgAS-dependent fhaB::lacZ reporter fusion in vivo and for autophosphorylation and phosphotransfer to BvgA in vitro. All mutations abolished activation of the fhaB::lacZ fusion. A point mutation in the transmitter (H729Q) prevented autophosphorylation of 'BvgS. In contrast to other characterized sensor proteins, autophosphorylation also required sequences in the 'BvgS receiver and C-terminal domains. A 'BvgS receiver point mutation (D1023N) had the novel phenotype of being able to autophosphorylate but unable to transfer the phosphate to BvgA. Autophosphorylation activity of the D1023N mutant protein was kinetically and chemically indistinguishable from wild-type 'BvgS despite an uncoupling of phosphotransfer from autophosphorylation. 'BvgS was shown to contain primarily amidyl phosphate and BvgA an acyl phosphate linkage. We present a model for a phosphorelay controlling virulence gene expression in B. pertussis.
百日咳博德特氏菌黏附素、毒素及其他毒力因子的表达受细菌双组分信号转导家族成员BvgA和BvgS蛋白的调控。BvgA与调节成分具有序列相似性，而BvgS与传感和调节成分均有相似性。BvgA和BvgS的胞质部分（“BvgS”）经过量表达和纯化。“BvgS”利用[γ-32P]ATP的γ-磷酸进行自身磷酸化，并使BvgA磷酸化。动力学分析表明，BvgA从“BvgS”获得磷酸基团。对BvgS的信号转导结构域、接收结构域和C末端结构域中的突变进行了测试，以检测其在体内对BvgAS依赖性fhaB::lacZ报告基因融合体的激活情况，以及在体外的自身磷酸化和向BvgA的磷酸转移情况。所有突变均消除了fhaB::lacZ融合体的激活。信号转导结构域中的一个点突变（H729Q）阻止了“BvgS”的自身磷酸化。与其他已鉴定的传感蛋白不同，自身磷酸化还需要“BvgS”接收结构域和C末端结构域中的序列。“BvgS”接收结构域的一个点突变（D1023N）具有能够自身磷酸化但无法将磷酸基团转移至BvgA的新表型。尽管磷酸转移与自身磷酸化解偶联，但D1023N突变蛋白的自身磷酸化活性在动力学和化学性质上与野生型“BvgS”无法区分。结果表明，“BvgS”主要含有氨基磷酸，而BvgA含有酰基磷酸键。我们提出了一个用于控制百日咳博德特氏菌毒力基因表达的磷酸中继模型。
