Functional analysis of a potassium sensing kinase in Bacillus subtilis

描述（由申请人提供）：多细胞群落的形成需要广泛的细胞内交流，尽管我们已经了解了许多涉及分化和生物膜形成的复杂调节网络，但对启动细菌多细胞性的信号知之甚少。最近，一种引发枯草芽孢杆菌多细胞化的新机制被描述，令人惊讶的是，该信号机制涉及钾泄漏激活的组氨酸激酶。在响应天然产物如制霉素和表面素引起的钾泄漏时，组氨酸激酶KinC启动信号转导级联，最终形成生物膜。新的信号机制的发现为对钾敏感组氨酸激酶进行严格的功能分析提供了机会，并确定其他基因是否以及如何参与感知钾泄漏。我建议解决三个具体目标：1)评估多环萜在信号传感中的作用，2)评估钾摄取在信号转导机制中的作用，以及3)确定参与信号传感的关键KinC残基，并确定是否有辅助因子参与。这项研究旨在为未来能够感知细胞内离子浓度变化的蛋白激酶的研究奠定基础，并使我能够熟练地在各种细菌背景下使用先进的遗传和分子技术。对这种新的信号传导机制的研究将有助于推进我们对大型感觉系统的理解，并将对其他研究领域，包括癌症研究和传染病研究产生重要影响。公共卫生相关性：许多细菌和哺乳动物的组氨酸激酶是同源的，由于结构和功能的相似性，有可能研究模型细菌系统并获得对哺乳动物系统的重要见解。通过增加我们对细菌中组氨酸激酶活性的分子基础的理解，有可能设计出更有效的策略来控制或调节导致细胞癌变的有缺陷的哺乳动物蛋白激酶和/或开发出更有效的方法来对抗细菌生物膜引起的感染。

DESCRIPTION (provided by applicant): The formation of multicellular communities requires extensive intracellular communication and although much has been learned about the intricate regulatory network involved in differentiation and biofilm formation, relatively little is known about the signals responsible for initiating bacterial multicellularity. Recently, a novel mechanism for triggering multicellularity in Bacillus subtilis was described and surprisingly, the signaling mechanism involves a histidine kinase activated by potassium leakage. In response to potassium leakage caused by natural products, such as nystatin and surfactin, the histidine kinase KinC initiates a signal transduction cascade that culminates in biofilm formation. Discovery of the novel signaling mechanism provides an opportunity to perform a rigorous functional analysis of the potassium sensing histidine kinase and to determine whether and how other genes are involved in sensing potassium leakage. I propose to address three specific aims: I) evaluate the role of polycyclic terpenoids in signal sensing, II) assess the role of potassium uptake in this signal transduction mechanism, and III) identify key KinC residues involved in signal sensing and determine if there is a cofactor involved. The proposed research is designed to both lay the foundation for future studies of protein kinases capable of sensing changes in intracellular ion concentration and allow me to become proficient using advanced genetic and molecular techniques in a variety of bacterial backgrounds. Studies of this new signaling mechanism will help advance our understanding of large sensory systems and will also have important implications in other research fields, including cancer research and infectious disease research. PUBLIC HEALTH RELEVANCE: Many bacterial and mammalian histidine kinases are homologous and due to structural and functional similarities, it is possible to study model bacterial systems and gain important insight into mammalian systems. By increasing our understanding of the molecular basis for histidine kinase activity in bacteria, it may be possible to design more effective strategies to control or modulate defective mammalian protein kinases that cause cells to become cancerous and/or develop more effective ways to fight infections caused by bacterial biofilms.