Regulation of Bacterial Heme Metabolism

描述（由申请人提供）：血红素及其生物合成的调控对正常细胞功能很重要，并有助于细菌在不同环境中的成功生存，包括那些与高等真核生物相关的病原和共生相互作用。血红素现在被认为是一种调节分子，它允许细胞感知和适应环境信号以维持体内平衡。提出的工作的广泛目标是了解血红素如何介导基因表达的控制，重点是铁代谢，并协调血红素的这种作用及其在细胞中的毒性。血红素生物合成途径在铁螯合酶催化的反应中将铁插入到原卟啉环中达到高潮。我们从日本慢生根瘤菌中发现了一种叫做Irr（铁反应调节器）的调节蛋白，它直接与铁螯合酶相互作用。这种新机制允许Irr在血红素合成位点局部响应血红素。Irr是一种条件稳定的蛋白，在铁限制下起作用，但在铁反应中以血红素依赖的方式降解。Irr最初被描述为血红素生物合成的调节剂，但新的证据表明它是铁依赖基因的全局调节剂。因此，本研究的一个主要目的是解决日本刺槐通过血红素依赖的方式感知铁，调节铁稳态和代谢的假设。此外，Irr的同系物在大多数？因此，所提出的工作可以作为理解病原菌中血红素和铁代谢的模型，这些病原菌在实验上远不如日本芽孢杆菌容易处理。提出了三个具体目标。1. 确定Irr识别靶基因并控制其表达的机制。Irr强烈调控编码铁转运蛋白的基因和许多其他铁相关基因，是一个正调控因子和负调控因子。我们对Irr的激活特别感兴趣，因为细菌铁转运的阳性控制并不常见。2. 阐明Irr感知血红素状态的机制。Irr与铁螯合酶相互作用，后者为Irr提供调控输入。我们将从生物化学和遗传学上描述这种相互作用，并阐明Irr随后的抑制和降解。阐明铁对血红素利用基因的不依赖机制。数据表明，从头合成的血红素与外源获得的血红素控制不同。我们将确定这种调节机制，并确定ir依赖性和非依赖性代谢是如何整合的。公共卫生相关性：虽然从宿主的角度来看，有益细菌和致病细菌入侵高等生物会导致非常不同的结果，但在分子水平上，发病机制和共生关系的各个方面可能非常相似。共生细菌日本根瘤菌在实验上比相关病原体更容易处理，因此是一个很好的模型系统。我们正在研究日本芽孢杆菌中细菌血红素和铁代谢的新机制，以了解细菌用于感染和适应的分子策略。

DESCRIPTION (provided by applicant): Heme and the regulation of its biosynthesis are important for normal cell function, and contribute to the success of bacteria to diverse environments, including those associated with pathogenic and symbiotic interactions with higher eukaryotes. Heme is now known to be a regulatory molecule that allows cells to sense and adapt to environmental cues to maintain homeostasis. The broad objective of the proposed work is to understand how heme mediates the control of gene expression, with an emphasis on iron metabolism, and to reconcile this role for heme with its toxicity in cells. The heme biosynthetic pathway culminates with the insertion of iron into a protoporphyrin ring by a reaction catalyzed by ferrochelatase. We discovered a regulatory protein called Irr (iron response regulator) from the bacterium Bradyrhizobium japonicum that interacts directly with ferrochelatase. This novel mechanism allows Irr to respond to heme locally at the site of heme synthesis. Irr is a conditionally stable protein that functions under iron limitation, but degrades in response to iron in a heme-dependent manner. Irr was initially described as a regulator of heme biosynthesis, but new evidence reveals that it is a global regulator of iron-dependent genes. Thus, a major objective of this proposal is to address the hypothesis that B. japonicum senses iron through the status of heme in an Irr-dependent manner to regulate iron homeostasis and metabolism. Furthermore, Irr homologs are found in most ?-Proteobacteria, and thus the proposed work serves as a model for understanding heme and iron metabolism in pathogenic bacteria that are experimentally much less tractable than B. japonicum. Three specific aims are proposed. 1. Determine the mechanism by which Irr recognizes target genes and controls their expression. Irr strongly regulates genes encoding ferric iron transporters and many other iron-related genes, and is both a positive and negative regulator. We are particularly interested in activation by Irr since positive control of bacterial iron transport is uncommon. 2. Elucidate the mechanism by which Irr senses the status of heme. Irr interacts with ferrochelatase, which provides the regulatory input to Irr. We will characterize this interaction both biochemically and genetically, and elucidate the subsequent inhibition and degradation of Irr. 3. Elucidate the regulation of heme utilization genes by iron by an Irr-independent mechanism. Data suggest that heme synthesized de novo is controlled differently than that acquired exogenously. We will identify this regulatory mechanism, and establish how Irr-dependent and -independent metabolism is integrated. PUBLIC HEALTH RELEVANCE: Although invasion of higher organisms by beneficial and disease-causing bacteria results in very different outcomes from the host perspective, aspects of pathogenesis and symbiosis can be remarkably similar at the molecular level. The symbiotic bacterium Bradyrhizobium japonicum is experimentally more tractable than related pathogens, and is therefore a good model system. We are studying novel mechanisms of bacterial heme and iron metabolism in B. japonicum towards the end of understanding molecular strategies that bacteria employ for infection and adaptation.