Regulation of bacterial manganese metabolism

描述（由申请人提供）：锰及其获取和随后代谢的调节对正常细胞功能很重要，并有助于细菌成功适应它们所处的多样化环境。锰可能是一种限制性营养物质，因此依赖锰的过程必须与金属的可用性相协调。我们感兴趣的是了解日本慢生根瘤菌如何感知和获取锰，维持体内平衡，并在细胞水平上协调锰依赖活动。初步的证据使我们能够验证日本芽孢杆菌适应细胞锰水平的新机制与大肠杆菌和其他模式生物的机制有很大不同的假设。B. japonicum属于α -变形菌门，这是一个多样化的分类群，包括许多成员，在共生或致病的情况下与真核宿主形成密切或细胞内的联系。日本芽孢杆菌可以作为一种模式生物来研究相关的难以进行实验分析的病原体。提出了三个具体目标。(1)阐明维持锰稳态的机制。我们发现了一个高亲和力的外膜通道，这是Mn2+摄取所必需的。外膜对二价离子的选择性是前所未有的，揭示了一种细菌的营养获取策略，以前没有被认识到。我们将阐明该通道在锰稳态中的作用。此外，日本芽孢杆菌耐受异常高的外部锰浓度，这对其他细菌是有毒的。我们将确定这种耐受性的基础，以了解这种生物体是如何维持体内平衡的。(2)阐明日本芽孢杆菌Mur蛋白对锰反应的转录调控。B. japonicum已经适应了一个全球性的细菌铁反应转录抑制因子作为Mn2+反应调节因子。我们发现了Mur蛋白的新方面。首先，它结合Mn2+的位点与Fur家族蛋白的金属结合位点不同，解决这个问题应该能让我们了解它是如何适应成为Mn2+传感器的。其次，脱金属形式的Mur具有活性，并且控制着与Mn2+负载蛋白不同的调控机制。我们将分析这个新函数。(3)确定铁锰代谢整合的分子基础。初步证据表明，B. japonicum在转录水平上感知并响应铁和锰的同时缺乏症，这与每种金属的单独缺乏症有质的不同。我们将首先关注参与支链氨基酸降解的两个操纵子，以深入了解这种全球反应。

DESCRIPTION (provided by applicant): Manganese and the regulation of its acquisition and subsequent metabolism are important for normal cell function, and contribute to the success of bacteria in adapting to the diverse environments in which they occupy. Manganese can be a limiting nutrient, and thus manganese-dependent processes must be coordinated with availability of the metal. We are interested in understanding how Bradyrhizobium japonicum senses and acquires manganese, maintains homeostasis, and coordinates manganese-dependent activities with cellular levels. Preliminary evidence lead us to test the hypothesis that B. japonicum adapts to cellular manganese levels by novel mechanisms that differ substantially from that found in E. coli and other model organisms. B. japonicum belongs to the alpha-Proteobacteria, a diverse taxonomic group that includes numerous members that form close or intracellular associations with eukaryotic hosts in a symbiotic or pathogenic context. B. japonicum serves as a model organism to study related pathogens that are refractory to experimental analysis. Three specific aims are proposed. (1) Elucidate the mechanisms that maintain manganese homeostasis. We identified a high affinity outer membrane channel necessary for Mn2+ uptake. Outer membrane selectivity for a divalent ion is unprecedented and reveals a bacterial strategy for nutrient acquisition not previously appreciated. We will elucidate the role of this channel in manganese homeostasis. Moreover, B. japonicum tolerates an unusually high external manganese concentration that is toxic to other bacteria. We will determine the basis of this tolerance towards the goal of understanding how this organism maintains homeostasis. (2) Elucidate manganese-responsive transcriptional control via the B. japonicum Mur protein. B. japonicum has adapted a global bacterial iron-responsive transcriptional repressor to function as a Mn2+ responsive regulator. We uncovered novel aspects of the Mur protein. Firstly, it binds Mn2+ at a site dissimilar to the presumed metal binding sites of Fur family proteins, and resolving this issue should give insight as to how it has adapted to be a Mn2+ sensor. Secondly, the demetallated form of Mur is active and controls a different regulon than the Mn2+-loaded protein. We will analyze this novel function. (3) Determine the molecular basis for the integration of iron and manganese metabolism. Preliminary evidence shows that B. japonicum senses and responds to a simultaneous deficiency in both iron and manganese at the level of transcription that is qualitatively different from individual deficiencies in each metal. We will focus initially on two operons involved in branched chain amino acid degradation to gain insight into this global response.