Bacterial iron acquisition

铁是大多数生命形式所必需的。它是细胞维持反应的辅助因子，如介导电子传递链的单电子转移反应或脱氧核糖核苷酸的形成。这种特殊的金属反应性，如铁，需要严格控制细胞的吸收和处理，以避免毒副作用。该研究项目旨在了解微生物体内铁稳态的细胞机制。来自许多门的细菌使用Fet系统在嗜微气或低pH条件下获取铁。研究得最好的Fet蛋白来自大肠杆菌和空肠弯曲杆菌。该系统在自然界中广泛存在于动物相关细菌和环境细菌中。该系统的核心由铁转运蛋白fem和质周含铜蛋白FetP组成。编码FetMP的基因是一个更大的编码另外5种蛋白质（FetABCDE）的基因簇的一部分。在C. jejuni中，FetA-E是在铁限制下细胞培养生长所必需的。FetP的铜位点是铁摄取功能所必需的，并且FetP二聚体与FetP的质周结构域相互作用。我们假设FetP是一种氧化还原酶，它将铁传递给FetM并将其运输到细胞中。FetA-F的功能尚不清楚。FetA是一种预测的跨膜蛋白，具有由DUF2318结构域和YHS结构域组成的大的质周区。DUF2318结构域被预测含有铁硫簇，我们假设FetA-F是一个氧化还原系统，通过FetP的作用控制铁的氧化状态。以大肠杆菌为模型系统，具体目的是：1。我们将确定FetP和fem之间的相互作用界面，并确定这些蛋白质之间的相互作用是否依赖于金属。我们将使用位点定向诱变来探测界面和结构生物学，以建立复合体的结构模型，以获得功能洞察力。结合研究将在过渡金属的存在下进行，重点是铁和亚铁氧化态的铁。2.我们将通过FetMP表征FetA在铁摄取中的作用。我们表达了一个含有两个FetA质周结构域的可溶性结构体（DUF2318/YHS）。我们将设计表达系统，用铁硫簇产生这个结构域，并表征氧化还原和性质。我们将测试该区域的质周定位是否依赖于TAT分泌系统。酸性条件降低了铁载体对铁的亲和力，而还原的氧有利于亚铁的氧化态。这项工作的结果将告诉我们细菌是如何获得铁的，这些条件在自然界中很常见。这种理解对于拓宽细菌用于合成生物学的条件至关重要。

Iron is essential for most forms of life. It is a cofactor for cell sustaining reactions such as mediating single electron transfer reactions of the electron transport chain or the formation of deoxyribonucleotides. This special reactivity of metals such as iron necessitates tight control of uptake and handling by the cell to avoid toxic side effects. The research program is directed at understanding the cellular mechanisms of iron homeostasis in microbes. The Fet system is used by bacteria from many phyla to acquire iron under microaerophilic or low pH conditions. The best studied Fet proteins are from Escherichia coli and Campylobacter jejuni. The system is found broadly in nature in both animal associated and environmental bacteria. The core of the system is composed of FetM, an iron-transporter, and FetP, a periplasmic copper-containing protein. The genes encoding FetMP are part of a larger cluster that encodes 5 additional proteins (FetABCDE). In C. jejuni, the FetA-E are required for growth in cell culture under iron restriction. The copper-sites of FetP are required for iron-uptake function and the FetP dimer interacts with a periplasmic domain of FetM. We hypothesize that FetP is an oxidoreductase that delivers iron to FetM for transport into the cell. The functions of FetA-F are remain unknown. FetA is a predicted transmembrane protein with a large periplasmic region composed of a DUF2318 domain and YHS domain. The DUF2318 domain is predicted to harbour an iron sulfur cluster and we hypothesize the FetA-F is a redox system to control the oxidation state of iron through the action of FetP. Using E. coli as a model system, the specific aims are: 1.We will identify interaction interface between FetP and FetM and determine if the interaction between these proteins is metal dependent. We will use site-directed mutagenesis to probe the interface and structural biology to build a structural model of the complex to gain functional insight. Binding studies will be performed in the presence of transition metals with a focus on iron in the ferric and ferrous oxidation states. 2.We will characterize the role FetA in iron-uptake by FetMP. We have expressed a soluble construct containing two periplasmic domains of FetA (DUF2318/YHS). We will engineer the expression system to produce this domain with an iron sulfur cluster and characterize the redox and properties. We will test if the periplasmic localization of this domain is dependent on the TAT secretion system. Acidic conditions reduce the affinity of siderophores for iron and reduced oxygen favours the ferrous iron oxidation state. The results of this work will inform on how bacteria are able to acquire iron these conditions commonly found nature. This understanding is fundamental to broadening the conditions under which bacteria can be used for synthetic biology.