In Vivo Domain-Based Functional Analysis of the Major Sporulation Sensor Kinase, KinA, in Bacillus subtilis
In Vivo Domain-Based Functional Analysis of the Major Sporulation Sensor Kinase, KinA, in Bacillus subtilis
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DOI：
10.1128/jb.00503-09
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发表时间：
2009-09-01
影响因子：
3.2
通讯作者：
Fujita, Masaya
中科院分区：
文献类型：
--
作者：
Eswaramoorthy, Prahathees;Guo, Tao;Fujita, Masaya
传感器组氨酸激酶被细菌广泛用于检测和响应环境信号。在枯草芽孢杆菌中，KinA是一种主要的激酶，其向磷酸化中继提供磷酸盐输入，磷酸化中继在饥饿时通过磷酸化Spo 0A转录因子激活孢子形成途径。KinA在其氨基末端传感器结构域中包含三个PAS结构域，其似乎参与感测饥饿时产生的未鉴定的孢子形成信号。先前的生物化学研究表明，KinA作为一种功能酶形成同源二聚体，并且最氨基末端的PAS结构域（PAS-A）在感知信号以激活ATP依赖性自磷酸化反应至组氨酸残基中起重要作用。为了分析体内激酶的结构和功能，我们使用了一种菌株，其中KinA的合成是在异丙基-β-D-硫代半乳糖苷（IPTG）诱导型启动子的控制下。结合基于结构域的缺失分析的体内功能研究表明，细胞溶质KinA形成一个同源寡聚体作为一个活跃的形式下营养丰富和营养缺乏的条件下，通过其氨基和羧基末端结构域独立。此外，我们发现，PAS-A结构域被删除的突变体仍然能够在野生型水平诱导孢子形成，而不管营养的可用性，这表明PAS-BC结构域足以维持激酶活性。基于这些结果，我们提出氨基末端传感器结构域的主要作用是形成稳定的复合物作为功能性激酶，但可能不用于结合未鉴定的孢子形成信号。
Sensor histidine kinases are widely used by bacteria to detect and respond to environmental signals. In Bacillus subtilis, KinA is a major kinase providing phosphate input to the phosphorelay that activates the sporulation pathway upon starvation via the phosphorylated Spo0A transcription factor. KinA contains three PAS domains in its amino-terminal sensor domain, which appear to be involved in the sensing of an unidentified sporulation signal(s) produced upon starvation. Prior biochemical studies have suggested that KinA forms a homodimer as a functional enzyme and that the most amino-terminal PAS domain (PAS-A) plays an important role in sensing the signal(s) to activate an ATP-dependent autophosphorylation reaction to a histidine residue. To analyze the structure and function of the kinase in vivo, we have used a strain in which the synthesis of KinA is under the control of an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter. In vivo functional studies in combination with domain-based deletion analysis show that the cytosolic KinA forms a homo-oligomer as an active form under both nutrient-rich and nutrient-depleted conditions via its amino- and carboxyl-terminal domains independently. Furthermore, we found that a mutant in which the PAS-A domain was deleted was still able to induce sporulation at a wild-type level irrespective of nutrient availability, suggesting that PAS-BC domains are sufficient to maintain the kinase activity. Based on these results, we propose that the primary role of the amino-terminal sensor domain is to form a stable complex as a functional kinase, but possibly not for the binding of an unidentified sporulation signal(s).