Bacterial iron acquisition

细菌获取铁

• 批准号: RGPIN-2022-04568
• 负责人: Murphy, Michael
• 金额: $ 2.91万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761041
• 关键词: Bacterial; iron; acquisition

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.

铁是大多数生命形式所必需的。它是细胞维持反应的辅因子，例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。金属如铁的这种特殊反应性需要严格控制细胞的摄取和处理，以避免毒副作用。该研究项目旨在了解微生物中铁稳态的细胞机制。来自许多门的细菌使用Fet系统在微需氧或低pH条件下获得铁。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和环境细菌中。该系统的核心由铁转运蛋白FetM和周质含铜蛋白FetP组成。编码FetMP的基因是编码另外5种蛋白质（FetABCDE）的更大簇的一部分。In C.空肠，FetA-E是在铁限制下细胞培养物中生长所需的。铁摄取功能所需的FetP的铜位点和FetP二聚体与FetM的周质结构域相互作用。我们假设FetP是一种氧化还原酶，将铁传递给FetM，以便运输到细胞中。FetA-F的功能仍然未知。FetA是预测的跨膜蛋白，具有由DUF 2318结构域和YHS结构域组成的大的周质区。DUF 2318结构域被预测为具有铁硫簇，并且我们假设FetA-F是通过FetP的作用来控制铁的氧化态的氧化还原系统。 使用大肠以大肠杆菌为模型系统，具体的目的是：1.确定FetP和FetM之间的相互作用界面，并确定它们之间的相互作用是否是金属依赖的。我们将使用定点突变来探测界面和结构生物学来构建复合物的结构模型以获得功能性见解。将在存在过渡金属的情况下进行结合研究，重点关注三价铁和亚铁氧化态的铁。 2.研究FetA在FetMP铁摄取中的作用。我们已经表达了含有两个周质结构域的FetA（DUF 2318/YHS）的可溶性构建体。我们将设计表达系统以产生具有铁硫簇的该结构域，并表征氧化还原和性质。我们将测试该结构域的周质定位是否依赖于达特分泌系统。 酸性条件降低了铁载体对铁的亲和力，还原的氧有利于亚铁的氧化态。这项工作的结果将告知细菌如何能够获得铁这些条件下常见的性质。这种理解对于拓宽细菌可用于合成生物学的条件至关重要。