Regulation of Bacterial Heme Metabolism

描述(由申请人提供)：血红素及其生物合成的调节对于正常的细胞功能是重要的，并有助于细菌在不同环境中的成功，包括那些与高等真核生物的致病和共生相互作用相关的环境。现在已知的是，血红素是一种调节分子，允许细胞感知并适应环境线索，以维持体内平衡。这项拟议工作的广泛目标是了解血红素如何调节基因表达的控制，重点是铁代谢，并协调血红素的这一作用与其在细胞中的毒性。血红素生物合成途径的最终结果是在铁络合酶的催化下将铁插入原卟啉环中。我们发现了一种名为IRR(铁反应调节蛋白)的调节蛋白，它来自于日本慢生根瘤菌，它直接与铁络合酶相互作用。这种新的机制允许irr在血红素合成的位置局部地对血红素做出反应。IRR是一种条件稳定的蛋白质，在铁限制下发挥作用，但在铁的反应中以依赖于血红素的方式降解。IRR最初被描述为血红素生物合成的调节因子，但新的证据表明，它是铁依赖基因的全球调节因子。因此，这一建议的一个主要目的是解决这样的假设，即日本血吸虫通过不依赖于血红素的状态来感知铁，以调节铁的动态平衡和代谢。此外，在大多数变形杆菌中都发现了irr同源物，因此拟议的工作为理解病原菌中的血红素和铁代谢提供了一个模型，在实验上，病原菌比日本变形杆菌难驯化得多。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，既是一个正性调节因子，也是一个负调节因子。我们对IRR的激活特别感兴趣，因为对细菌铁运输的积极控制是不常见的。2.阐明IRR感知血红素状态的机制。IRR与铁络合酶相互作用，为IRR提供调控输入。我们将从生物化学和遗传学两方面描述这种相互作用，并阐明IRR随后的抑制和降解。3.阐明了铁对血红素利用基因的调控是通过一种非独立的机制实现的。数据表明，从新合成的血红素受到的控制与通过外源获得的不同。我们将确定这种调节机制，并确定非依赖和非依赖新陈代谢是如何整合的。公共卫生相关性：尽管有益细菌和致病细菌对高等生物的入侵从宿主的角度来看会导致非常不同的结果，但在分子水平上，致病机理和共生方面可能非常相似。在实验上，共生细菌慢生根瘤菌比相关病原菌更容易驯化，因此是一个很好的模式系统。我们正在研究细菌中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.