Non-canonical functions of two-component signaling proteins in the cell cycle of Caulobacter crescentus

两组分信号转导是一种几乎所有细菌都用来组织细胞内事件和对环境变化做出反应的机制。在一个双组分的信号通路中，上游的组氨酸激酶蛋白感知信号，并在保守的组氨酸残基上自动磷酸化。然后，磷酸基被传递到下游的反应调节蛋白，该蛋白产生细胞反应，如基因表达、新陈代谢或运动性的变化。虽然这种线性范式在大多数情况下是正确的，但一些具有重要细胞作用的双组分蛋白质似乎通过替代机制或在分支网络中发挥作用。该项目的重点是两种蛋白质，DivK和DivL，这两种蛋白质对水生细菌新月弯杆菌的细胞周期进程至关重要。其目的是确定反应调节因子DivK如何抑制组氨酸激酶Cck A的活性，该活性在一个单独的两组分途径中发挥作用。在正确的时间抑制Cck A的活性对于Caulbacter细胞开始染色体复制是必要的。另一个目的是确定一种基本蛋白质DivL的功能，它与组氨酸激酶同源，但似乎通过磷酸转移以外的机制发挥作用。特别是，DivL被认为参与了蛋白质与蛋白质之间的重要相互作用，而这些相互作用是由ATP的结合和水解所调节的。建议进行实验，以测量导致不同细胞表型的DivL突变体的ATP结合和水解，并确定与DivL相互作用的新蛋白，以介导其对细胞分裂周期的影响。这项研究将扩大组氨酸激酶和反应调节器使用的生化活动的范围，以相互影响和细胞事件。Broader Impact该项目将为每年10名学生提供微生物学暑期强化实验室课程。这门课与许多实验课的不同之处在于，学生做的实验结果事先是不知道的。除了微生物学和分子生物学的基本技术外，他们还学习如何设计和排除实验故障，以便他们的结果是可解释的和可靠的。在过去的几年里，每个学生都删除了新月形杆菌中一个以前没有研究过的基因，并对产生的突变菌株进行了表征。在接下来的几年里，学生们将做与这项研究的科学目标密切相关的项目，例如产生divL的特定突变并确定它们对细胞的影响，或者进行基因筛选以识别与divL或divK相互作用的蛋白质。学生学习新的科学知识是如何产生的，他们获得了为独立研究项目做准备的经验。鼓励来自代表性不足群体的妇女和学生在申请人的实验室开展研究项目。

Two-component signal transduction is a mechanism used by nearly all bacteria to organize intracellular events and respond to changes in the environment. In a two-component signaling pathway, the upstream histidine kinase protein senses a signal and autophosphorylates on a conserved histidine residue. The phosphoryl group is then passed to a downstream response regulator protein, which generates a cellular response, such as a change in gene expression, metabolism, or motility. Although this linear paradigm is correct in most cases, some two-component proteins with important cellular roles appear to function by alternative mechanisms, or in branched networks. The project focuses on two proteins, DivK and DivL, that are essential for cell cycle progression in the aquatic bacterium Caulobacter crescentus. The aim is to determine how the response regulator DivK suppresses the activity of a histidine kinase, CckA, which functions in a separate two-component pathway. Suppression of CckA activity at the correct time is necessary for Caulobacter cells to begin chromosome replication. The other aim is to establish the function of an essential protein, DivL, which is homologous to histidine kinases, but which appears to act by a mechanism other than phosphoryl transfer. In particular, it is proposed that DivL participates in essential protein-protein interactions which are modulated by binding and hydrolysis of ATP. Experiments are proposed to measure the ATP binding and hydrolysis by DivL mutants that cause distinct cellular phenotypes, and to identify new proteins that interact with DivL to mediate its effects on the cell division cycle. This research will expand the repertoire of biochemical activities used by histidine kinases and response regulators to influence each other and cellular events.Broader Impacts This project will provide an intensive summer lab course in microbiology for 10 students per year. This course is different from many lab classes in that the students do experiments whose results are not known beforehand. In addition to fundamental techniques in microbiology and molecular biology, they learn how to design and troubleshoot experiments so that their results are interpretable and reliable. In past years, each student has deleted a previously unstudied gene in Caulobacter crescentus and characterized the resulting mutant strain. In upcoming years, the students will do projects closely related to the scientific goals of this research, such as generating specific mutations in divL and determining their cellular effects, or performing genetic screens to identify proteins that interact with DivL or DivK. Students learn how new scientific knowledge is generated, and they gain experience that prepares them for independent research projects. Women and students from underrepresented groups are encouraged to undertake research projects in the applicant's laboratory.