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

双组分信号转导是几乎所有细菌用来组织细胞内事件和响应环境变化的机制。在双组分信号通路中，上游组氨酸激酶蛋白感知信号并在保守的组氨酸残基上进行自磷酸化。然后，磷酸化基团传递给下游反应调节蛋白，产生细胞反应，如基因表达、代谢或运动的变化。尽管这种线性模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分支网络发挥作用。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形茎杆菌的细胞周期进程至关重要。目的是确定反应调节因子DivK如何抑制组氨酸激酶CckA的活性，CckA在单独的双组分途径中起作用。在正确的时间抑制CckA活性对于茎状杆菌细胞开始染色体复制是必要的。另一个目的是确定一种必需蛋白DivL的功能，它与组氨酸激酶同源，但似乎通过磷酸化转移以外的机制起作用。特别是，有人提出，DivL参与必需的蛋白质-蛋白质相互作用，这是由ATP的结合和水解调节的。我们提出了实验来测量引起不同细胞表型的DivL突变体的ATP结合和水解，并鉴定与DivL相互作用介导其对细胞分裂周期影响的新蛋白。本研究将扩大组氨酸激酶和反应调节因子用于相互影响和细胞事件的生化活动的范围。本项目每年将为10名学生提供强化的暑期微生物学实验课程。这门课不同于许多实验课，因为学生做的实验事先不知道结果。除了在微生物学和分子生物学的基本技术，他们学习如何设计和故障排除实验，使他们的结果是可解释的和可靠的。在过去的几年里，每个学生都删除了一个以前未研究的新月形茎状杆菌基因，并描述了由此产生的突变菌株。在接下来的几年里，学生们将做与本研究的科学目标密切相关的项目，比如在divL中产生特定的突变并确定它们的细胞效应，或者进行基因筛选以识别与divL或DivK相互作用的蛋白质。学生们学习新的科学知识是如何产生的，并获得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女性和学生在申请人的实验室进行研究项目。

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.