CYCLE REGULATION IN THE SYMBIONT SINORHIZOBIUM MELILOTI

描述（由申请人提供）：我的实验室研究细菌利用的调节网络，以一种能够产生分化细胞类型并对环境条件敏感的方式影响细胞周期事件的有序进展。这一提议的潜在假设是，细菌细胞周期控制的机械知识对于理解这些网络如何响应不同的生活方式压力而进化是必不可少的。我们更广泛的目标是了解宿主入侵和慢性定植如何影响典型细菌细胞周期。为了达到这个目的，我们研究了sinorhizbium meliloti，因为它可以作为一种自由生活的细菌在土壤根际定植，并作为共生体侵入豆科植物的根，建立慢性细胞内感染。我们通过检测四种双组分传感器组氨酸激酶的功能开始了我们的研究：SmCbrA、SmCbrB、SmDivJ和SmPleC（作为一组被称为“hk”）。据预测，这些hk位于信号转导通路的顶端，对调节S. meliloti细胞周期进程和不对称细胞分裂至关重要。我实验室的初步数据表明，每个HK基因的破坏会产生细胞周期缺陷，这在形态上反映在无菌丝状生长中。我们的流式细胞术结果进一步表明，每个HK突变体都不能正确地协调DNA复制起始与细胞分裂。因此，我们的初步数据暗示hk在细胞周期控制中，我们将通过遗传学，细胞生物学和生化实验的结合进一步分析它们在S. meliloti细胞周期中的机制作用。为了评估基因活性和蛋白质定位作为细胞周期进程的功能，我们正在生成由SmdnaE编码的DNA聚合酶III全酶（Pol III）的温度敏感条件等位基因，该等位基因允许DNA复制延伸，但在37(C)的限制性温度下阻断DNA复制起始。我们假设HKs在SmDivK上游发挥作用，调节其磷酸化状态，已知这影响其细胞定位。我们正在使用荧光显微镜来评估HK突变对SmDivK- gfp定位的影响，以衡量它们对SmDivK~P水平的潜在影响。我们将进一步探讨SmDivK是否是HK酶活性的直接靶点，通过进行体外实验，从而检测组氨酸激酶的自磷酸化，以及SmDivK的磷酸化和去磷酸化。我们的目标是确定在细胞周期进程中HK功能的精确时间。我们将分离条件冷敏感HK等位基因，以便在不允许的温度下快速消耗HK功能，从而确定其在细胞周期进程中的精确时间需求。我们将量化各种细胞周期事件，包括DNA复制起始、SmDivK-GFP定位、细胞分裂装置的形成和不对称细胞分裂。综上所述，我们的研究将使我们能够构建一个HK时间和细胞周期机制控制的模型。

DESCRIPTION (provided by applicant): My lab studies a regulatory network utilized by bacteria to effect an orderly progression of cell cycle events in a manner that is capable of producing differentiated cell types and is sensitive to environmental conditions. The underlying assumption of this proposal is that a mechanistic knowledge of bacterial cell cycle controls is essential to understanding how these networks evolve in response to distinct lifestyle pressures. Our broader goal is to understand how host invasion and chronic colonization impinge upon the canonical bacterial cell cycle. To this end, we study Sinorhizibium meliloti because it can colonize the soil rhizosphere as a free-living bacterium and invade the roots of leguminous plants as a symbiont to establish a chronic intracellular infection. We began our studies by examining the function of four two-component sensor histidine kinases: SmCbrA, SmCbrB, SmDivJ and SmPleC (as a group referred to as "HKs"). These HKs are predicted to be positioned at the top of a signal transduction pathway central to regulating S. meliloti cell cycle progression and asymmetric cell division. Preliminary data from my lab demonstrates that disruption of each HK gene produces a cell cycle defect that is reflected morphologically in aseptal filamentous growth. Our flow cytometry results further demonstrate that each HK mutant is unable to properly coordinate DNA replication initiation with cytokinesis. Thus, our preliminary data implicate HKs in cell cycle control and we will further dissect their mechanistic role in the S. meliloti cell cycle through a combination of genetic, cell biological and biochemical experiments. In order to assess gene activity and protein localization as a function of cell cycle progression, we are generating a temperature-sensitive conditional allele of DNA polymerase III holoenzyme (Pol III), encoded by SmdnaE, that will allow DNA replication elongation but block DNA replication initiation at the restrictive temperature of 37(C. We hypothesize HKs function upstream of SmDivK to regulate its phosphorylation status, which is known to impact its cellular localization. We are performing fluorescence microscopy to assess the effect HK mutations have on SmDivK-GFP localization as a measure of their potential impact on the level of SmDivK~P. We will further explore whether SmDivK is a direct target of HK enzymatic activity by performing in vitro assays and thereby examine histidine kinase autophosphorylation, as well as SmDivK phosphorylation and dephosphorylation. We aim to determine the precise timing of HK function during cell cycle progression. We will isolate conditional cold-sensitive HK alleles in order to rapidly deplete HK function at the non-permissive temperature and thereby pinpoint its precise temporal requirement during cell cycle progression. We will quantify a variety of cell cycle events, including DNA replication initiation, SmDivK-GFP localization, formation of the cell division apparatus, and asymmetric cytokinesis. Taken together, our studies will allow us to construct a model for HK timing and mechanistic control over the cell cycle. PUBLIC HEALTH RELEVANCE: The cell cycle is a fundamental process required for growth, reproduction, and developmental differentiation in all living organisms. Moreover, it is becoming clear that modification of the bacterial cell cycle occurs during chronic host colonization in a variety of species, including our model organism Sinorhizobium meliloti. Insights gained from our analysis of cell cycle regulation in S. meliloti will provide a strong basis for dissecting modes of cell cycle control in other host-associated bacteria, and can thereby be applied towards development of novel targets for antimicrobial therapeutics at a time when resistance to ¿-lactams and aminoglycosides is found in a variety of potent pathogens.