Metal Ion Homeostasis in Bacillus subtilis

描述(申请人提供)：这项研究的长期目标是了解细菌细胞如何维持金属离子稳态。必需的金属离子必须从环境中获得，运输到细胞内，并运输到正确的细胞内目的地。当金属离子被限制时，高亲和力吸收机制被诱导。当过量时，细胞诱导合成外排、解毒或储存机制。从宿主获得足够的金属离子的能力对细菌病原体至关重要，金属摄取系统在体内高度表达，暴露在表面的运输蛋白通常是宿主免疫反应的靶标。金属离子稳态失调也是人类疾病的主要原因，包括贫血、门克斯病、威尔逊病和其他几种神经退行性疾病。 对金属离子有效性变化的遗传反应由金属调节蛋白协调。在革兰氏阳性菌中，枯草芽孢杆菌的金属特异性调节剂控制着对锰(MntR)、铁(Fur)和锌(Zur)的吸收，而其他调节剂则调节金属离子过量时的外排机制(CueR、ChurA、ArsR、YdeT)。我们将测量金属离子动态平衡改变的野生型和突变体的生长所需的金属离子含量和配额。我们将验证这一假设，即金属离子在细胞壁上的吸附在金属的吸收和储存中都起着作用，我们将描述铁和锌的吸收途径。我们将验证一些假设，即MRGA和DPS在铁的储存和解毒中发挥作用，以及含锌的核糖体蛋白提供了一个主要的细胞内锌储存，在锌限制下可以被动员。遗传和生化方法将通过金属配体的定点突变、功能突变的体内分析和结构确定方法来加深我们对MntR、Fur、Zur、CueR和ArsR家族成员对金属敏感的理解。

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand how bacterial cells maintain metal ion homeostasis. Essential metal ions must be obtained from the environment, transported into the cell, and trafficked to the correct intracellular destinations. When metal ions are limiting, high affinity uptake mechanisms are induced. When in excess, the cell induces the synthesis of efflux, detoxication, or storage mechanisms. The ability to obtain sufficient metal ions from the host is crucial to bacterial pathogens, metal uptake systems are highly expressed in vivo, and surface-exposed transport proteins are often targets for the host immune response. Disorders of metal ion homeostasis are also a major cause of human disease including anemias, Menkes', and Wilson's diseases, and several other neurodegenerative diseases. The genetic responses to changes in metal ion availability are coordinated by metalloregulatory proteins. In the Gram-positive bacterium Bacillus subtilis metal-specific regulators control the uptake of Mn (MntR), Fe (Fur), and Zn (Zur), and others regulate efflux mechanisms induced when metal ions are in excess (CueR, CzrA, ArsR, YdeT). We will measure the metal ion content and quotas needed for growth for both wild-type and mutants altered in metal ion homeostasis. We will test the hypothesis that adsorption of metal ions to the cell wall plays a role in both uptake and storage of metals and we will characterize pathways for Fe and Zn uptake. We will test the hypotheses that MrgA and Dps function in Fe storage and detoxification and that Zn-containing ribosomal proteins provide a major intracellular Zn store that can be mobilized under Zn limitation. Genetic and biochemical approaches will be used to further our understanding of metal-sensing by MntR, Fur, Zur, CueR, and ArsR family members by site-directed mutagenesis of metal ligands, in vivo analyses of altered function mutants, and structure-determination methods.