Regulation of bacterial manganese metabolism

说明(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，有助于细菌成功地适应它们所处的不同环境。锰可能是一种限制性营养素，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慢生根瘤菌如何感知和获取锰，维持动态平衡，并在细胞水平上协调锰依赖的活动。初步证据使我们检验了这样一种假设，即日本血吸虫通过与在大肠杆菌和其他模式生物中发现的显著不同的新机制来适应细胞内的锰水平。日本芽胞杆菌属于α-变形杆菌，这是一个多样化的分类群，包括许多成员，这些成员在共生或致病的背景下与真核宿主形成密切的或细胞内的联系。日本芽孢杆菌是研究实验分析困难的相关病原菌的模式生物。提出了三个具体目标。(1)阐明维持体内锰稳态的机制。我们鉴定了一个高亲和力的外膜通道，这是摄取Mn2+所必需的。外膜对二价离子的选择性是史无前例的，揭示了一种以前没有被认识到的细菌获取营养的策略。我们将阐明该通道在锰稳态中的作用。此外，日本血吸虫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，以达到了解这种有机体如何维持内稳态的目标。(2)阐明日本血吸虫Mur蛋白对锰的响应转录调控。B.Japan onicum已经改造了一种全球细菌铁反应转录抑制因子，以发挥Mn2+反应调节因子的功能。我们发现了Mur蛋白的新方面。首先，它与Mn2+结合的位置不同于Fur家族蛋白推测的金属结合部位，解决这个问题应该有助于了解它是如何适应成为Mn2+传感器的。其次，去金属形式的Mur是活性的，并且控制着不同于负载Mn2+的蛋白的调节。我们将分析这一新颖的功能。(3)确定铁、锰代谢整合的分子基础。初步证据表明，日本双歧杆菌在转录水平上感知铁和锰的同时缺乏并对其做出反应，这在性质上不同于每种金属的单独缺乏。我们将首先关注与支链氨基酸降解有关的两个操纵子，以深入了解这一全球反应。

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.