CYCLE REGULATION IN THE SYMBIONT SINORHIZOBIUM MELILOTI

描述(申请人提供)：我的实验室研究细菌利用的调控网络，以一种能够产生分化细胞类型并对环境条件敏感的方式，实现细胞周期事件的有序进行。这一建议的基本假设是，细菌细胞周期控制的机械知识对于理解这些网络如何演变以应对不同的生活方式压力是必不可少的。我们更广泛的目标是了解宿主入侵和长期定居如何影响规范的细菌细胞周期。为此，我们研究了紫花苜蓿，因为它可以作为一种自由生活的细菌定居在土壤根际，并作为共生体入侵豆科植物的根，以建立一种慢性的细胞内感染。我们首先研究了四种双组分感受性组氨酸蛋白激酶：SmCbrA、SmCbrB、SmDivJ和SmPleC(统称为HKS)的功能。这些HKs被预测位于调控苜蓿链霉菌细胞周期进程和不对称细胞分裂的信号转导通路的顶端。来自我的实验室的初步数据表明，每个HK基因的破坏都会产生细胞周期缺陷，这在形态上反映在无间隔的丝状生长中。我们的流式细胞术结果进一步证明，每个HK突变体都不能正确地协调DNA复制启动和胞质分裂。因此，我们的初步数据表明HKS参与了细胞周期调控，我们将通过遗传学、细胞生物学和生化实验的组合进一步剖析它们在苜蓿链霉菌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展的函数，我们正在产生一个温度敏感的DNA聚合酶III全酶(POL III)的条件等位基因，由Smdna E编码，它将允许DNA复制延长，但在37(C)的限制性温度下阻止DNA复制启动。我们假设HKS在SmDivK上游发挥作用，调节其磷酸化状态，已知这会影响其细胞定位。我们正在利用荧光显微镜来评估HK突变对SmDivK-GFP定位的影响，以此来衡量它们对SmDivK~P水平的潜在影响。我们将通过体外实验进一步探讨SmDivK是否是HK酶活性的直接靶标，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。我们的目标是确定细胞周期进程中HK功能的准确时间。我们将分离有条件的冷敏感HK等位基因，以便在不允许的温度下快速耗尽HK功能，从而准确地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复制启动、SmDivK-GFP定位、细胞分裂装置的形成和不对称胞质分裂。综上所述，我们的研究将使我们能够构建一个模型，用于香港对细胞周期的时间和机制控制。 公共卫生相关性：细胞周期是所有生物生长、繁殖和发育分化所必需的基本过程。此外，越来越明显的是，细菌细胞周期的改变发生在各种物种的慢性宿主定植期间，包括我们的模式生物苜蓿中华根瘤菌。我们从对苜蓿链霉菌细胞周期调控的分析中获得的见解将为剖析其他宿主相关细菌的细胞周期控制模式提供强有力的基础，从而可以在多种有效病原体对内酰胺类和氨基糖苷类抗生素耐药的情况下，应用于开发新的抗菌治疗靶点。

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.