Molecular mechanisms regulating cell cycle progression in Caulobacter crescentus

描述（由申请人提供）：生物学中的一个基本问题是一个细胞如何分裂产生两个具有不同身份的后代。像真核细胞一样，细菌经历复杂的生命周期，经常产生具有不同形状和特性的子细胞。在这项工作中，Caulobacter crescentus被用来作为一个模型系统的不对称耦合到分裂周期的研究。在这种细菌中，每次分裂都会产生两个形态上不同的子细胞，一个是具有附着在表面上的柄的非运动细胞，另一个是具有鞭毛的运动蜂群细胞，该鞭毛推动它在水中运动。有柄细胞立即开始新一轮的染色体复制和分裂，但群集细胞无法启动DMA复制，直到它分化成有柄细胞。这个复杂的细胞分裂周期是由一个双组分信号转导蛋白网络协调的。反应调节因子CtrA是一种转录因子，其控制许多细胞周期调节基因的表达，但也通过与复制起点结合来阻断DNA复制。CtrA活性是细胞活力所必需的，但它必须在有柄细胞中暂时消除以允许染色体复制的起始。CtrA活性间接地受到基本反应调节剂DivK的反对。磷酸化的DivK导致CtrA活性降低，这最终允许有柄细胞中的染色体复制。因此，DivK的磷酸化对于生存力是必需的。已知DivK被组氨酸激酶（HK）DivJ激活，但由于DivJ是可分配的，DivK必须被柄杆菌属中的另一种HK或小分子磷酸供体磷酸化。我们提出了一个全基因组的方法，以确定其他香港可能参与DivK磷酸化。具体而言，我们的目标是删除59个非必需HK中的每一个的基因，并结合divJ缺失。这种方法产生了一个可能的候选人，我们暂时命名为DivM，其他候选人仍然必须排除。我们还旨在表征缺乏DivJ和DivM的细胞的末端表型，确定DivM在细胞周期中的位置和活性，并阐明从DivM到DivK的磷酸化途径。公共卫生相关性：这里提出的工作是不可或缺的，以实现一个完整的理解的监管级联导致细胞周期的进展和分化柄杆菌。对调控级联的理解可能具有深远的影响，因为在柄杆菌中发现的许多机制在农业，生物战，生物传感和生物工程中具有重要作用的其他物种中是保守的。此外，阐明细菌细胞周期进程中涉及的基本机制将产生对原核细胞生物学的关键见解，这反过来将有助于确定抗菌药物发现的新靶点。

DESCRIPTION (provided by applicant): A fundamental question in biology is how one cell divides to yield two progeny with different identities. Like eukaryotic cells, bacteria undergo complex life cycles and often produce daughter cells with distinct shapes and properties. In this work, Caulobacter crescentus is used as a model system for the study of asymmetry coupled to the division cycle. In this bacterium, each division produces two morphologically distinct daughter cells, a non-motile cell with a stalk that attaches to surfaces and a motile swarmer cell with a flagellum that propels it through the water. The stalked cell immediately begins a new round of chromosome replication and division, but the swarmer cell is unable to initiate DMA replication until it differentiates into a stalked cell. This complex cell division cycle is orchestrated by a network of two-component signal transduction proteins. The response regulator CtrA is a transcription factor that controls the expression of many cell cycle- regulated genes but also blocks DNA replication by binding to the origin of replication. CtrA activity is required for cell viability but it must be temporarily eliminated in stalked cells to permit the initiation of chromosome replication. CtrA activity is indirectly opposed by the essential response regulator DivK. Phosphorylated DivK results in a decrease in CtrA activity, which ultimately allows chromosome replication in the stalked cell. Thus, phosphorylation of DivK is essential for viability. DivK is known to be activated by the histidine kinase (HK) DivJ but because DivJ is dispensible, DivK must be phosphorylated by another HK or small molecule phosphodonor in Caulobacter. We propose a whole-genome approach to identify other HKs that could participate in DivK phosphorylation. Specifically, we aim to delete the gene for each of the 59 non-essential HKs in combination with a divJ deletion. This approach has yielded a likely candidate which we have tentatively named DivM other candidates must still be ruled out. We also aim to characterize the terminal phenotype of cells lacking both DivJ and DivM, determine the location and activity of DivM during the cell cycle, and elucidate the phosphorylation pathway from DivM to DivK. PUBLIC HEALTH RELEVANCE: The work proposed here is integral to achieving a complete understanding of the regulatory cascade leading to cell-cycle progression and differentiation in Caulobacter. Comprehension of the regulatory cascade could have far-reaching implications because many of the mechanisms discovered in Caulobacter are conserved among other species with important roles in agriculture, biowarfare, biosensing, and bioengineering. In addition, elucidating the basic mechanisms involved in bacterial cell cycle progression will generate key insights into prokaryotic cell biology, which will in turn help to identify new targets for antibacterial drug discovery.